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Article

Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam

by
Pham Thi Hong Minh
1,2,
Nguyen Thuong Tuan
2,3,
Nguyen Thi Hong Van
1,
Hoang Thi Bich
1 and
Do Tien Lam
1,2,*
1
Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10072, Vietnam
2
Faculty of Chemistry, Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10072, Vietnam
3
Institute of Life Sciences, Thai Nguyen University of Agriculture and Forestry, Quyet Thang, Thai Nguyen 24119, Vietnam
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(6), 2580; https://doi.org/10.3390/molecules28062580
Submission received: 12 January 2023 / Revised: 8 March 2023 / Accepted: 9 March 2023 / Published: 12 March 2023
(This article belongs to the Special Issue Pharmacological Evaluation and Phytochemical Analysis of Natural Products)
Browse Figure
Versions Notes

Abstract

:
The essential oils (EOs) of the aerial parts of four Asarum species (A. geophilum, A. yentunensis, A. splendens and A. cordifolium) were isolated by steam distillation and analyzed by the GC/MS method. The A. cordifolium EO contains 33 constituents with the main component being elemicine (77.20%). The A. geophilum EO was contains 49 constituents with the main components being determined as 9-epi-(E)-caryophyllene (18.43%), eudesm-7(11)-en-4-ol (13.41%), β-caryophyllene (8.05%) and phytol (7.23%). The A. yentunensis EO contains 26 constituents with the main components being safrole (64.74%) and sesquicineole (15.34%). The EO of A. splendens contains 41 constituents with the main components being 9-epi-(E)-caryophyllene (15.76%), eudesm-7(11)-en-4-ol (14.21%), β-caryophyllene (9.52%) and trans-bicyclogermacrene (7.50%). For antimicrobial activity, the A. yentunensis EO exhibited the highest inhibition activity against Staphylococcus aureus and the A. cordifolium EO against Bacillus subtillis (MIC values of 100 μg/mL). For antioxidant activity, the A. geophilum EO showed the highest potential with an SC (%) value of 63.34 ± 1.0%, corresponding to an SC50 value of 28.57 µg/mL. For anti-inflammatory activity, the A. splendens EO exhibited the highest potential with an IC50 value of 21.68 µg/mL, corresponding to an inhibition rate of NO production of 69.58 ± 1.3% and the percentage of cell life was 81.85 ± 0.9%.

1. Introduction

The Asarum genus belonging to the Aristolochiaceae family, including 100 species in the world, is distributed mainly in the Northern Hemisphere, East Asia from the Himalayas to China, Taiwan, Japan, South Korea, Sakhalin island, North America and Europe [1,2]. In Vietnam, there are eleven Asarum species, including A. balansae, A. blumei, A. caudigerum, A. glabrum, A. petelotii, A. reticulatum, A. wulingense, A. geophilum, A. yentunensis, A. splendens, and A. cordifolium distributed mainly in the North of Vietnam, except for A. wulingense which was discovered in Central Vietnam in Ha Tinh province [3]. Four Asarum species were used in traditional medicine A. geophilum, A. yentunensis, A. splendens, and A. cordifolium [3,4].
Plants of the genus Asarum are rich in EOs which are the components possessing biologically or pharmacologically active ingredients. Up to now, more than 155 compounds have been identified from the EO of genus Asarum, whose main characteristic components are phenylpropanoids (elemicin, safrole, asarone, methyleugenol, myristicin, eugenol, etc.), terpenoid derivatives and aromatic compounds. The higher content of methyleugenol, kakuol, myristicin, asarone, elemicin, eucarvone, sesamin, asarinin, etc., may be the main toxic ingredients when used in high doses [5,6,7].
The A. heterotropoides var. mandshuricum EO was found to have antibacterial, anti-phytopathogenic, antidepressant effects and fumigant toxicity. In detail, the A. heterotropoides EO showed antimicrobial effect towards five types of epidermal bacteria producing human body odor Corynebacterium jeikeium, Corynebacterium xerosis, Micrococcus luteus, Propionibacterium freudenreichii, and Staphylococcus epidermidis, with MIC values ranging from 10.1 to 46 mg/mL [8]; against three periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, in vitro and in vivo [9]; inhibited five pathogenic fungi infecting plants Alternaria humicola, Colletotrichum gloeosporioides, Rhizoctonia solani, Phytophthora cactorum and Fusarium solani with IC50 values < 0.42 μg/mL [10]; fumigant toxicity to Tetranychus urticae, at a fumigant concentration of 8 μg/mL; the mortality rate of mites was 72.6% in 24 h, and reached 100% after 48 h [11]. This essential oil also exhibited an inhibitory effect on the growth and development of Streptoccus sp., Shigella sp. and Salmonella typhi [12].
The A. sieboldii EO exhibited strong insecticidal activity against Sitophilus oryzae with an LC50 of 2.37 μg/mL; eucarvone and safrole were the most effective compounds, with LC50 values of 3.32 and 11.27 μg/mL [13]. For individual compounds in volatile oil, methyleugenol, β-asarone and myristicin showed significant insecticidal activity against the tobacco beetle Lasioderma serricorne, with LC50 values of 720, 60, and 540 ppm [14]. The A. sieboldii EO demonstrated very high antifungal activity against the Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Candida albican and Neolentinus lepideus [2,15].
The compounds elemicin and (N-Isobutyl-(2E,4Z,8Z,10E)-dodecatetraenamide isolated from the genus Asarum EOs noticed a significant anti-allergic activity by the inhibitory effect on enzyme 5-lipoxygenase with IC50 values of 6.0 μM and 0.16 μM [16].
The chemical composition of EOs of some Asarum species growing in Vietnam has been studied. The major constituent of the trunk and leaf EO of A. caudigerum was safrol (96.2%) [17], while the major constituents of the trunk and leaf EO of A. glabrum were safrol (42.24%), apiole (27.11%) and myristicin (6.13%) [18]. Elimicin was the major compound of A. balansae and A. cordifolium EO (71.53% and 84.38%, respectively), while E-methyl isoeugenol and myristicine were major compounds of A. yunnanense and A. petelotii EO (47.39% and 59.06%, respectively) [19]. The major constituent of the leaf EO of A. geophyllum was 9-epi-β-caryophyllene (27.16%), bicyclogermacrene (16.98%) and β-caryophyllene (11.91%) [17], while the major constituents of the roots and rhizomes EO of A. geophyllum were eudesm-7(11)-en-4-ol (16.94%), β-pinene (12.61%), aristolene (7.01%) and 9-epi-β-caryophyllene (7.88%) [20].
The aim of this study was to evaluate the chemical composition and antimicrobial, antioxidant, and anti-inflammatory activities of the aerial part essential oils of four Asarum species growing in Vietnam, including A. geophilum, A. yentunensis, A. splendens and A. cordifolium.

2. Results and Discussion

2.1. Chemical Composition of the Essential Oils

The EOs of the aerial parts (leaves and stems) from four Asarum species (A. geophilum, A. yentunensis, A. splendens and A. cordifolium) were obtained by steam distillation in a Clavenger apparatus for 7 h. The chemical composition of these EOs was analyzed by gas chromatography coupled with mass spectrometry (GC-MS) (Table 1).
For the A. cordifolium EO with a yield of 0.30% (w/w, based on fresh material), 33 compounds were identified, accounting for 98.78%, of which there were ten monoterpene hydrocarbons (7.58%), two oxygenated monoterpenes (0.43%), eleven sesquiterpene hydrocarbons (6.53%), five oxygenated sesquiterpenes (1.96%) and five derivatives of benzene (benzenoids) (82.03%). The main component of this EO was elemicin (77.20%) (Figure 1a, Table 1). This result was similar to the reference reported that elemicin presents with a high amount in the A. cordifolium EO (84.38%) [19]. Elemicin has been also previously found in EOs of other Asarum species: A. sieboldii (4.8–11.1%); A. himalaicum (13.1–42.2%), A. canadense (4.9%), and A. insigne (5.4%) [2,5]. Elemicin has been known to be the main ingredient that leads to the antibacterial and antifungal activities of some essential oils [2]. The EO of Daucus carota L. spp. carota (16.3% elemicin) was active against Campylobacte jejuni, Campylobacte coli, and Campylobacte lari [21]. The EO of Daucus carota subsp. halophilus (26.0% elemicin) was also shown to have antimicrobial activity with MIC values ranging from 0.16 to 0.32 µL/mL [22].
For the EO of A. geophilum, with a yield of 0.19% (w/w, based on fresh material), 49 compounds were identified, accounting for 93.05%. There were five monoterpene hydrocarbons (2.46%), seven oxygenated monoterpenes (5.41%), seven-teen sesquiterpene hydrocarbons (46.84%), eleven oxygenated sesquiterpenes (23.29%), one benzenoid (0.17%) and eight other constituents (14.88%). The main components were 9-epi-(E)-caryophyllene (18.43%), eudesm-7(11)-en-4-ol (13.41%), β-caryophyllene (8.05%), phytol (7.23%), cis-bicyclogermacrene (4.46%) and α-terpineol (4.07%) (Figure 1b, Table 1). This result was similar to the reference that reported that eudesm-7(11)-en-4-ol and β-caryophyllene are present in high amounts in the A. geophilum EO [20]. However, there was a significant difference of 9-epi-(E)-caryophyllene (18.43%) with a high amount in the studied A. geophilum EO and a low amount in the reference reported and vice versa, a high amount of β-pinene (12.61%) in the reference reported and low amount in the studied A. geophilum EO [20]. The components and contents of the A. geophilum EO were different, maybe due to differences in the geographical locations, soil and harvesting conditions.
For the EO of A. yentunensis, with a yield of 0.05% (w/w, based on fresh material), 26 compounds were identified, accounting for 96.30%. There were seven monoterpene hydrocarbons (3.21%), two oxygenated monoterpenes (3.50%), nine sesquiterpene hydrocarbons (6.00%), two oxygenated sesquiterpenes (15.75%) and six benzenoids (67.84%). The main components of this EO were safrole (64.74%), sesquicineole (15.34%), linalool (3.19%) and (Z)-β-farnesene (2.81%) (Figure 1c, Table 1). Safrole has been also known to be a main component of the EO of other Asarum species: A. caudigerum (96.2%) [17] and A. glabrum (42.24–46.60%) [19,23].
For the EO of A. splendens, with a yield of 0.11% (w/w, based on fresh material), 41 compounds were identified, accounting for 91.36%, including seven monoterpene hydrocarbons (7.01%), eight oxygenated monoterpenes (3.45%), twenty-five sesquiterpene hydrocarbons (59.06%), seven oxygenated sesquiterpenes (19.13%), one benzenoid (1.49%) and three others compounds (1.49%). The main components of this EO were 9-epi-(E)-caryophyllene (15.76%), eudesm-7(11)-en-4-ol (14.21%), β-caryophyllene (9.52%), trans-bicyclogermacrene (7.50%), β-pinene (4.71%) and cis-β-elemene (3.48%) (Figure 1d, Table 1). This result shows that the EO of A. cordifolium has sesquiterpene hydrocarbons (25/41 components, 59.06%) higher than other Asarum species [4].
The constituents: α-pinene (0.33–0.84%), β-pinene (0.91–4.71%) and (E)-nerolidol (0.32–0.79%) were present in the four species studied.
Three components α-pinene (0.33–0.84%), β-pinene (0.91–4.71%) and (E)-nerolidol (0.32–0.79%) are present in all four sudied Asarum species. In the EOs of A. geophilum and A. splendens, sesquiterpene hydrocarbons and oxygenated sesquiterpenes were dominant constituents: 17/49 components with 46.84% and 11/49 components with 23.29% for A. geophilum EO; 25/41 components with 59.06% and 7/41 components with 19.13% for A. splendens EO. Meanwhile, in the essential oils of A. yentunensis and A. cordifolium, benzenoids were dominant constituents: 6/26 components with 67.84% for A. yentunensis EO and 5/33 components with 82.03% for A. cordifolium EO; monoterpene hydrocarbons and sesquiterpene hydrocarbons were also present in this two EOs but with a quantity lower than that of A. geophilum and A. splendens EOs (7/26 components with 3.21% and 9/26 components with 6.0% for A. yentunensis EO; 10/33 components with 7.58% and 11/33 components with 6.53% for A. cordifolium EO.
Methyl ether (0.13%), geranial (0.18%), endo-isocamphanyl acetate (1.01%), geranyl acetate (0.51%), γ-muurolene (0.59%), ar-curcumene (0.63%), trans-muurola-4(14),5-diene (0.94%), β-bisabolene (1.65%), cuparene (0.16%), zonarene (0.24%), humulene epoxide II (0.14%) and (Z)-ligustilide (0.48%) were common components for the four Asarum EOs. However, exo-fenchol, α-terpinyl acetate (0.35%), viridiflorene (1.48%), elemol (0.41%), 4-epi-maaliol (0.67%), viridiflorol (2.16%), cubeban-11-ol (2.27%), rosifoliol (0.62%), neo-intermedeol (1.01%), n-tetradecanoic acid (0.18%), isophytol (0.12%), n-hexadecanoic acid (1.31%), phytol (7.23%), linoleic acid (2.45%) and linolenic acid (2.31%) were characteristic for the A. geophilum EO; while safrole (64.74%), (E)-β-farnesene (0.19%), γ-curcumene (0.24%), (E)-methyl isoeugenol (0.27%), (E,E)-α-farnesene (0.36%), sesquicineole (15.34%), myristicin (0.31%), β-sesquiphellandrene (0.16%) and isoelemicin (0.32%) were characteristic for the A. yentunensis EO; exo-fenchol, δ-selinene (0.17%), γ-amorphene (0.33%), α-bisabolene (1.18%), elemicine (77.20%), scapanol (0.33%), α-asarone (0.73%) and α-murolol (0.24%) were characteristic for the A. cordifolium EO.
Safrole [24], elemicin [21], 9-epi-(E)-caryophyllene [25] and eudesm-7(11)-en-4-ol [26] showed moderate antimicrobial, antioxidant and anti-inflammatory activity. The difference in chemical composition maybe led to the difference in biological activities of these four Asarum EOs.

2.2. Antimicrobial Activity of the EOs

The biological activity of the EOs of the aerial parts of A. geophilum, A. yentunensis, A. splendens and A. cordifolium were evaluated in terms of anti-microbial activity on eight strains of fungi, yeast, and bacteria (Table 2).
The results showed that the A. geophilum EO exhibited good inhibition activity on the Escherichia coli, Pseudomonas aeruginosa, Fusarium oxysporum and Saccharomyces cerevisiae with a MIC value of 200 μg/mL. The A. yentunensis EO demonstrated potent inhibition activity on the Escherichia coli, Aspergillus niger and Candida albicans with a MIC value of 200 μg/mL, and on the Staphylococcus aureus with a MIC value of 100 μg/mL. The A. splendens EO exhibited moderate inhibition activity on the Escherichia coli, Saccharomyces cerevisiae and Fusarium oxysporum with a MIC value of 200 μg/mL. The A. cordifolium EO exhibited potent inhibition activity on the Escherichia coli, Bacillus subtillis and Candida albicans with a MIC value of 200 μg/mL, and on the Bacillus subtillis with a MIC value of 100 μg/mL.
For the antibacterial activity against Escherichia coli; Pseudomonas aeruginosa, Bacillus subtillis and Staphylococcus aureus showed:
Escherichia coli is a Gram-negative, a large and diverse group of bacteria, found in the lower intestine of people and animals. Some kinds of Escherichia coli can make you sick and cause diarrhea, cause urinary tract infections, respiratory illness and pneumonia, etc. [27,28]. All four species Asarum EOs demonstrated moderate antibacterial activity with a MIC value of 200 μg/mL. This suggests the potential for the Asarum EOs to have antimicrobial activity against growing Escherichia coli.
Pseudomonas aeruginosa is a Gram-negative, aerobic, extremely versatile, antibiotic-resistant bacteria and causes infections in the blood, lungs, or other parts after surgery [29]. Only A. geophilum EO showed activity against Pseudomonas aeruginosa with a MIC value of 200 μg/mL. Despite the high contents and predominant composition of the EOs from A. geophilum and A. splendens being similar, the A. splendens EO showed no activity. This may be due to the presence of alcohols and fatty acids (14.88%) in the A. geophilum EO.
Bacillus subtillis is a Gram-positive, ubiquitous bacteria, it is not pathogenic and produces important commercial products (fermented products, sweeteners, flavor enhancers and animal feed additive) [30,31]. Only A. cordifolium EO exhibited potent antibacterial activity against Bacillus subtillis with a MIC value of 100 μg/mL. Elimicine presents with a high amount in the A. cordifolium EO (77.20%).
Staphylococcus aureus is a Gram-positive bacteria and has a wide variety of clinical manifestations. Infections caused by this pathogen are common both in community-acquired and hospital-acquired settings [32,33]. The A. yentunensis EO with the main components: safrole (64.74%) and sesquicineole (15.34%) demonstrated stronger inhibition activity than the A. splendens EO with the main components: 9-epi-(E)-caryophyllene (15.76%), eudesm-7(11)-en-4-ol (14.21%) and β-caryophyllene (9.52%), corresponding to MIC values of 100 and 200 μg/mL. However, high safrole content is potentially phytotoxic [5].
For the antifungal activity against Aspergillus niger, Fusarium oxysporum, Saccharomyces cerevisiae and Candida albicans:
Aspergillus niger is among the most common fungi, responsible for post-harvest decay of fresh fruit, fish products and meat products [34,35]. Only the A. yentunensis EO with high amounts of safrole (64.74%) and sesquicineol (15.34%) was likely responsible for these antifungal activities.
Fusarium oxysporum is the common soilborne fungi and the fungal communities in the rhizosphere of plants. All strains of Fusarium oxysporum are saprophytic and penetrate into the roots inducing either root rots or tracheomycosis [36]. The EOs of A. geophilum and A. splendens were rich in contents of sesquiterpene hydrocarbons and oxygenated sesquiterpenes, and exhibited good inhibition activity against Fusarium oxysporum with MIC values of 200 μg/mL.
Saccharomyces cerevisiae is a species of budding yeast, responsible for bread formation and alcohol production. It is useful in studying the cell cycle [37]. Only A. geophilum EO showed antifungal activity with a MIC value of 200 μg/mL and may have been related to compounds of alcohol and fatty acid.
Candida albicans is a yeast that lives on the human body in small amounts and is responsible for infections such as thrush and vaginal yeast infections, etc. [38]. The A. yentunensis EO and A. cordifolium EO showed demonstrated inhibition activity against Candida albicans with MIC values of 200 μg/mL. The main components: safrole and elemicine could have been responsible for these antifungal activities. However, it needs to be studied further for its antifungal activity.

2.3. Antioxidant Activity of the EOs

The results of in vitro antioxidant activity testing by the DPPH method of the four Asarum EOs with the positive control as ascorbic acid were shown in Table 3.
The obtained results showed that the A. geophilum EO displayed the best antioxidant activity with an SC (%) value of 63.34 ± 1.0%, corresponding to the SC50 value of 28.57 µg/mL. The A. cordifolium EO with an SC50 value of 57.86 ± 0.8% corresponded to an SC50 value of 39.62 µg/mL and the A. yentunensis EO with an SC50 value of 51.58 ± 0.5% corresponded to SC50 value of 50.24 µg/mL. The A. splendens EO did not show activity at the tested concentration. These results have shown the good antioxidant capacity of Asarum EOs.
The A. geophilum EO displayed better antioxidant activity (SC50 value of 28.57 µg/mL) than the A. splendens EO (SC50 value of over 100 µg/mL). The EOs of A. geophilum and A. splendens were of similar high amounts of the predominant composition of sesquiterpene hydrocarbons and oxygenated sesquiterpenes. This difference may be due to the different content of alcohols and fatty acids in these two species (14.88% of in the A. geophilum EO and 1.49% of the A. splendens EO).
Although the EOs of A. yentunensis and A. cordifolium were the similar benzenoids dominant constituents (67.84% for A. yentunensis EO and 82.03% for A. cordifolium EO) the A. cordifolium EO showed the better antioxidant activity (SC50 value of 39.62 µg/mL) than the A. yentunensis EO (SC50 value of 50.24 µg/mL). The main component of A. cordifolium EO (elemicine: 77.20%) and A. yentunensis EO (safrole 64.74%) are interesting ways to explain this antioxidant activity results.

2.4. Anti-Inflammatory Activity of the EOs

The results of testing the in vitro anti-inflammatory activity of the four Asarum EOs were evaluated through the inhibition of NO production by using LPS- on RAW 264.7 as shown in Table 4.
The results showed that the A. splendens EO exhibited the best anti-inflammatory activity through inhibition of NO production with an IC50 value of 21.68 µg/mL. The next are A. geophilum EO with an IC50 value of 40.35 µg/mL and A. yentunensis EO with an IC50 value of 49.87 µg/mL. Finally, the A. cordifolium EO illustrates lower anti-inflammatory activity at the tested concentration with an IC50 value of 66.37 µg/mL.
The anti-inflammatory activity of the EOs rich in contents of benzenoids from A. yentunensis and A. cordifolium was better than the EOs rich in contents of sesquiterpene hydrocarbons and oxygenated sesquiterpenes from the A. geophilum and A. splendens with IC50 values from 21.68 to 40.35 µg/mL and from 49.87 to 66.37 µg/mL, respectively.
For the anti-inflammatory activity of the A. splendens EO, the main components of 9-epi-(E)-caryophyllene (15.76%), eudesm-7(11)-en-4-ol (14.21%), β-caryophyllene (9.52%) and trans-bicyclogermacrene (7.50%) exhibited the best anti-inflammatory activity through the inhibition of NO production with an IC50 value of 21.68 µg/mL, produced NO inhibition of 69.58 ± 1.3% and a high cell survival rate of 81.85 ± 0.9%. It has the potential to be developed into future anti-inflammatory drugs because of their effectiveness and safety.
The anti-inflammatory activity of the A. geophilum EO with 9-epi-(E)-caryophyllene (18.43%), eudesm-7(11)-en-4-ol (13.41%) and β-caryophyllene (8.05%) as the main components demonstrated moderate anti-inflammatory activity with an IC50 value of 40.35 µg/mL (inhibition rate of NO production was 58.20 ± 0.4% and the cell survival rate was 78.05 ± 0.8%). In China, the species Asarum produces pungent aromatic roots that are used in traditional medicine as a remedy for pain and colds. The above results have contributed to elucidating this activity in traditional Chinese medicine [2,5].
For the anti-inflammatory activity of the A. yentunensis EO, the main components of safrole (64.74%) and sesquicineole (15.34%) displayed the anti-inflammatory activity with IC50 value of 49.87 µg/mL (inhibition rate of NO production was 53.14 ± 1.6% and the cell survival rate was 66.87 ± 1.5%). In particular, the low cell survival rate may be due to the toxicity of safrole. A. yentunensis is an endemic species for the flora of Vietnam. Therefore, the study has clarified the chemical composition and biological activity of this species.
For the anti-inflammatory activity of the A. cordifolium EO with elemicin (77.20%) as the main component, a lower anti-inflammatory activity was found with an IC50 value of 21.68 µg/mL; NO inhibition only reached 40.87 ± 0.6% and cell survival rate of 60.65 ± 1.4%. The low cell survival rate may be due to the toxicity of elemicin. That contributes to explaining the meaning of the Red Dao Minority Vietnam (Sapa town, Lao Cai province) use in folk medicine to treat back pain and wound infections.

3. Materials and Methods

3.1. Plant Materials

The fresh aerial parts of four Asarum species (Asarum geophilum, Asarum cordifolium, Asarum splendens, Asarum yentuense) were collected and identified by Dr. Nguyen Anh Tuan, Indochina Institute of Biological and Environmental sciences and Dr. Nguyen Quoc Binh, Vietnam National Museum of Nature, VAST. The herbarium specimen was deposited at the Institute of Natural Products Chemistry, VAST. Asarum geophilum Hemsl. was collected in Trung Khanh district, Cao Bang province in May 2020. Asarum cordifolium C. E. C. Fischer and Asarum splendens (F.Maek.) C.Y.Chen and C.S.Yang were collected in Sapa district, Lao Cai province in January 2021 and June 2020, respectively. Asarum yentuense N. Tuan and Sasamoto was collected in Uong Bi district, Quang Ninh province in June 2020.

3.2. Extraction the EOs

The fresh aerial parts of four Asarum species (500 g/a species/a time) were washed with water, allowed to dry at room temperature, minced, put in a round-bottom flask 3 L, added to 1000 mL of pure water and subjected to hydrodistillation for 7 h using a Clevenger type apparatus. The obtained EOs were dehydrated with anhydrous Na2SO4, kept in sealed glass vials and stored at −15 °C [39,40]. The experiments of each species were repeated three times to determine the EOs contents compared with fresh samples. Then they were pooled for component determination by GC-MS analysis and bioactivity was tested in vitro.

3.3. Analyzing Chemical Constituents of the Essential Oils

The chemical constituents of the EOs of four species from genus Asarum were determined by combining the GC-FID and GC–MS system with the standard library and the MASSFinder library of natural compounds: The GC-MS analysis was carried out with Agilent Technologies HP7890A GC equipped with a mass spectrum detector (MSD) Agilent Technologies HP5975C, and an HP5-MS column (60 m × 0.25 mm, film thickness 0.25 µm). The GC-FID analysis was carried out with the same conditions as those for the GC-MS analysis. MassFinder 4.0 software connected to the HPCH1607, W09N08 libraries and the NIST Chemistry WebBook was used to match mass spectra and retention indices [41]. The analysis was conducted at the Chemical Analysis Lab, Institute of Natural Products Chemistry, The Vietnam Academy of Science and Technology.

3.4. Antimicrobial Assays

The method of assessment of antimicrobial activity was conducted to evaluate the antibiotic activities of EO samples by the method of McKane and Kandel (1996) [42,43] on eight strains, including Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25923), Bacillus subtillis (ATCC 11774), Staphylococcus aureus subsp. aureus (ATCC 11632), Aspergillus niger (439), Fusarium oxysporum (M42), Candida albicans (ATCC 7754), and Saccharomyces cerevisiae (SH 20). The antimicrobial tests were conducted at the Department of Biologically active, Institute of Natural Products Chemistry, VAST.
Positive testing: Streptomycin for Bacteria Gr(+), Tetracyclin for Bacteria Gr(-) and Nystatin or Amphotericin B for Mold and Fungi. Antibiotics mixed in 100% DMSO with appropriate concentration.
Negative testing: Microbiological control does not mix with antibiotics and samples.
Breeding and preservation medium: Saboraud Dextrose Broth (SDB-Sigma) for yeasts and molds. Trypcase Soya Broth (TSB-Sigma) for bacteria.
Experimental medium: Eugon Broth (Difco, USA) for bacteria, Mycophil (Difco, USA) for fungi.
Conduct experiments: The control strains were activated and diluted according to McFarland standard 0.5 and then tested. The plates were incubated at 37 °C/24 h for bacteria and 30 °C/48 h for filamentous fungi and yeasts.
Calculate the result: Minimum inhibitory concentration (MIC-Minimum Inhibitor Concentration) of the sample: Samples were diluted on descending concentration scales, to calculate the minimum inhibitory concentration (MIC), which is the concentration at which the microorganism is almost completely inhibited.

3.5. Antioxidant Assays

Dissecting the ability to trap free radicals generated by using DPPH (1,1-diphenyl-2-picrylhydrazyl); Brand-Williams et al. 1995 [19], Shela et al., 2003 [44], Kumar et al., 2013 [45]) is a method that has been recognized for the rapid determination of antioxidant activity. The reagent was dissolved in dimethyl sulfoxide (DMSO 100%) and DPPH was mixed in 96% ethanol. The absorbance of DPPH at λ = 515 nm was determined after adding DPPH to the sample solution on a 96-well microplate. The results are expressed as the mean of at least three replicate trials ± Standard Deviation (p < 0.05).
Samples were reconstituted in 100% DMSO at a concentration of 4 mg/mL for the crude extract and 1 mg/mL for the purified sample. Use 5 mM ascorbic acid in 10% DMSO as a positive control. Samples were inoculated onto a 96-well microplate with DPPH solution to obtain final test sample concentrations from 200 μg/mL to 12.5 μg/mL (for crude extracts) and from 50 μg/mL to 3.1 μg/mL (purified sample). Incubate at 37 °C for 30 min and measure Optical Density (OD) at λ = 515 nm on a photometer (Infinite F50, Tecan, Switzerland).
The average value of the ability to neutralize free radicals (Scavenging capacity, SC%) at the individual sample concentrations was entered into the Excel data. The sample (reagent) was diluted to decreasing concentrations and repeated three times at each concentration. The DPPH-induced free radical scavenging efficiency of each sample was calculated based on the percent of free radical neutralization compared to the blank sample (Blank) and negative controls. Samples showing antioxidant activity in the DPPH system were subjected to further steps to find the SC50 value (μg/mL, μM/mL). The SC50 value is the concentration of the reagent at which 50% of the free radicals are neutralized, determined using the TableCurve v5.0 AISN Software (Jandel Scientific, San Rafael, CA, USA) using the SC% value and a range of similar reagent concentrations. The tests were conducted at the Department of Biologically active, Institute of Natural Products Chemistry, The Vietnam Academy of Science and Technology.

3.6. Anti-Inflammatory Assays

The anti-inflammatory activity in-vitro was investigated through the inhibition of NO on RAW264.7 cells (American Type Culture Collection, Manassas, VA, USA) of mice; it was carried out at the Institute of Natural Compound Chemistry, VAST [46]. The RAW264.7 cells (mouse macrophages) were cultured for 48 h in Dulbecco’s culture medium (DMEM-Dulbecco’s Modified Eagle Medium) at 37 °C, 5% CO2, 10% Fetal Bovine serum (FBS-Fetal Bovine Serums). The cell fluid was inoculated onto a 96-well microplate, density 2.5 × 105 cells/microplate. Cells were stimulated in 2 µL of the control sample (-) LPS (0.1 mg/mL) for 24 h, supplemented with drugs or reagents at different concentrations with cardamonin (+) as a control. The cell suspension was incubated with Griess reagent and NaNO2 at different concentrations to develop a calibration curve. Measuring the reaction mixture at λ = 570 nm. The higher the NO concentration, the higher the optical density, which was determined by the NaNO2 standard curve, compared to the % of the control sample as (-) LPS. The ability of the samples to inhibit NO production was determined according to the following as Formula (1):
%   Inhibitor = 100   −   The   concentration   of   NO   sample The   concentration   of   NOLPS × 100

4. Conclusions

The EOs of aerial parts from four Asarum species: A. geophilum, A. yentunensis, A. splendens and A. cordifolium were obtained by steam distillation and analyzed by GC-MS. The constituents as α-pinene (0.33–0.84%), β-pinene (0.91–4.71%) and (E)-nerolidol (0.32–0.79%) were present in all four studied EOs. Sesquiterpene hydrocarbons and oxygenated sesquiterpenes were dominant constituents of A. geophilum and A. splendens EOs in which 9-epi-(E)-caryophyllene and eudesm-7(11)-en-4-ol were the major components (18.43% and 13.41%, 15.76% and 14.21%, respectively. Benzenoids were dominant constituents of A. yentunensis and A. cordifolium EOs, but the major component of Asarum yentunensis EO was elemicin (77.20%) while safrole (64.74%) was a major component of Asarum cordifolium EO. Especially, the chemical composition of the EOs from A. yentunensis and A. splendens has been identified for the first time.
The A. geophilum EO exhibited good inhibition activity on the Escherichia coli, Pseudomonas aeruginosa, Fusarium oxysporum and Saccharomyces cerevisiae with MIC values of 200 μg/mL. The A. yentunensis EO exhibited good inhibition activity on Escherichia coli, Aspergillus niger and Candida albicans with MIC values of 200 μg/mL, and on the Staphylococcus aureus with MIC values of 100 μg/mL. The A. splendens EO exhibited good inhibition activity on the Escherichia coli, Saccharomyces cerevisiae and Fusarium oxysporum with MIC values of 200 μg/mL. The A. cordifolium EO exhibited good inhibition activity on the Escherichia coli, Bacillus subtillis and Candida albicans with MIC values of 200 μg/mL, and on the Bacillus subtillis with a MIC value of 100 μg/mL. The A. geophilum EO displayed the best antioxidant activity with an SC (%) value of 63.34 ± 1.0%, corresponding to an SC50 value of 28.57 µg/mL, following the A. cordifolium EO with an SC50 value of 57.86 ± 0.8% corresponding to SC50 value of 39.62 µg/mL. The A. splendens EO displayed the best anti-inflammatory activity with an IC50 value of 21.68 µg/mL (corresponding to the inhibition rate of NO production being 69.58 ± 1.3% and the percentage of cell life being 81.85 ± 0.9%).
For the first time, the in vitro antimicrobial, antioxidant and anti-inflammatory activity of the EOs from four Asarum species: A. geophilum, A. yentunensis, A. splendens and A. cordifolium have been studied. They are a very interesting medicinal plant that deserves to be studied for better applications in new therapeutic drugs. However, note that the EOs of A. cordifolium and A. yentunensis have high amounts of toxic elemicin and safrole, which are potentially toxic when used in overdose.

Author Contributions

Conceptualization and formal analysis D.T.L. and P.T.H.M.; investigation, methodology and validation, N.T.T.; H.T.B. and N.T.H.V.; supervision, writing-review and editing, D.T.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01–2019.317.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not applicable.

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Molecules 28 02580 g001 550
Figure 1. The GC-MS spectrums of the EOs of four Asarum species. (a) Asarum cordifolium, (b) Asarum geophilum, (c) Asarum yentunensis, (d) Asarum splendens.
Figure 1. The GC-MS spectrums of the EOs of four Asarum species. (a) Asarum cordifolium, (b) Asarum geophilum, (c) Asarum yentunensis, (d) Asarum splendens.
Molecules 28 02580 g001
Table
Table 1. Chemical composition (in%) of the EOs of four Asarum species.
Table 1. Chemical composition (in%) of the EOs of four Asarum species.
No.Chemical NameFormula A. geophilumA. yentunensisA. splendensA. cordifolium
a RITimeb RI%FIDTimeb RI%FIDTimeb RI%FIDTimeb RI%FID
1α-pineneC10H1693210.019380.3310.379390.8410.369390.8410.709390.56
2campheneC10H16946---10.879550.3910.869550.2811.219560.38
3β-pineneC10H1698611.369831.4511.749840.9111.749844.7112.119850.96
4myrceneC10H16988------11.959910.1512.329920.21
5α-phelandrenC10H16101012.1710090.13---12.5510100.1512.9310100.79
6δ-3-careneC10H161015---12.7710160.39---13.1510170.83
7o-cymeneC10H14102212.8210280.17---13.2010291.2213.6010301.80
8limoneneC10H16103012.9710330.18---13.3610340.5513.7510341.87
9β-phelandreneC10H161031------13.4110350.3313.8210360.31
10eucalyptolC10H18O1032------13.4810370.16---
11(Z)-β-ocimeneC10H161039---13.4910380.13---13.8810381.33
12(E)-β-ocimeneC10H161050---13.8710490.42---14.2610490.34
13terpinoleneC10H16109415.0210930.2015.4310940.13---
14linaloolC10H18O109515.2711010.1815.6711013.19---16.1011020.20
15exo-fencholC10H18O111815.9511200.13---------
16borneolC10H18O117117.8411740.1518.2511750.31------
17terpinen-4-olC10H18O118418.2011840.30---18.6111850.12---
18α-terpineolC10H18O119518.6511974.07---19.0411970.26---
19thymol methyl etherC11H16O1232------20.2712330.13---
20geranialC10H16O1271------21.6612730.18---
21bornyl acetateC12H20O2128921.9412930.23---22.3612941.0822.8512950.23
22safroleC10H10O21293---22.59130064.74------
23n-tridecaneC13H28130022.1412990.18---22.5612990.14---
24δ-elemeneC15H24134323.7213470.81---24.1613480.30---
25α-terpinyl acetateC12H20O2134924.0013550.35--- ---
26endo-isocamphanyl acetateC11H18O21350------24.4313561.01---
27geranyl acetateC12H20O21379------25.3513840.51---
28α-copaeneC15H24139425.0713880.42---25.5313890.4626.0013900.50
29cis-β-elemeneC15H24139625.5414022.75---25.9914043.4826.4514041.84
30methyl eugenolC11H14O21403---26.0914071.22---26.5814082.16
31β-caryophylleneC15H24143426.6014368.0527.0314370.2927.0714389.52
32γ-elemeneC15H24144026.8414440.68---------
33α-trans-bergamoteneC15H241443---27.3114460.6727.3214461.71---
34β-gurjuneneC15H24144627.0114491.66---27.4814514.06---
35aromadendreneC15H24144927.2014550.61---27.6614570.60---
36(Z)-β-farneseneC15H241451---27.7814612.8127.7914610.23---
37(E)-β-farneseneC15H241460---27.9214650.19------
38α-humuleneC15H24147127.6714703.8728.1114710.5528.1414723.72---
399-epi-(E)-caryophylleneC15H24147427.95147918.43---28.43148115.76---
40γ-curcumeneC15H241481---28.6614880.24------
41γ-muuroleneC15H241483------28.7114900.59---
42ar-curcumeneC15H241484------28.7514910.63---
43germacrene DC15H24149628.4914960.56---28.9514981.2129.4414991.00
44n-pentadecaneC15H32150028.5814991.10---29.0315000.87---
45(E)-methyl isoeugenolC11H14O21501---29.0515010.27------
46β-selineneC15H24150228.6915030.60---29.1615051.7129.6215050.17
47δ-selineneC15H241503---------29.6515070.17
48trans-muurola-4(14),5-dieneC15H241503------29.3215100.94---
49viridifloreneC15H24150528.9315111.48---------
50γ-amorpheneC15H241508---------29.8215120.33
51cis-bicyclogermacreneC15H24151028.9815124.46------29.9115150.73
52(E,E)-α-farneseneC15H241511---29.3915120.36------
53trans-bicyclogermacreneC15H241512---29.4215130.7329.4715157.50---
54β-bisaboleneC15H241514------29.5515181.65---
55α-bulneseneC15H241517------29.6415210.1430.1315220.20
56cupareneC15H221518------29.7415240.16---
57sesquicineoleC15H26O1521---29.80152615.34------
58γ-cadineneC15H241524------29.9115300.4930.4415330.27
59myristicinC11H14O21529---29.9515310.31------
60β-sesquiphellandreneC15H241531---30.0315340.16------
61δ-cadineneC15H24153329.6515350.60---30.1215370.7230.5815380.41
62zonareneC15H241537------30.2015400.24---
63α-bisaboleneC15H241544---------31.0015521.18
64selina-4(15),7(11)-dieneC15H24154530.1515521.02---30.6115531.18---
65selina-3,7(11)-dieneC15H24156030.3615590.41---30.8215611.46---
66elemolC15H26O156230.4315610.41---------
67elemicinC12H16O21565---------31.43156677.20
68(E)-nerolidolC15H26O156930.6415680.4731.0715690.4131.0815690.7931.6015720.32
69germacrene BC15H24157230.8615750.43---31.3315780.60---
70isoelemicinC12H16O21577---31.3315780.32------
714-epi-maaliolC15H26O157731.1715860.67---------
72scapanolC15H26O1580---------32.3215960.33
73spathulenolC15H24O159331.4415951.24---31.9015971.21---
74viridiflorolC15H26O159531.6516022.16---------
75caryophyllene oxideC15H24O1599------32.1216041.3132.6516070.23
76cubeban-11-olC15H26O160131.9116112.27---------
77rosifoliolC15H26O161532.1416200.62---------
78humulene epoxideIIC15H24O1620------32.8816310.14---
79γ-eudesmolC15H26O164632.9616480.29---33.5816561.31---
80α-asaroneC12H16O31650---------34.0916580.73
81α-muurololC15H26O1654---------34.1816610.24
82α-cadinolC15H26O166533.5816700.74---33.6416580.1634.5516740.84
83neo-intermedeolC15H26O167033.6716741.01---------
84β-asaroneC12H16O31678---34.3816840.98---34.8816860.14
85eudesm-7(11)-en-4-olC15H26O170934.90171813.41---35.40172114.21---
86(Z)-ligustilideC12H14O21741------36.2917550.48---
87n-tetradecanoic acidC14H28O175935.9717580.18---------
88isophytolC20H40O194640.8619490.12---------
89n-hexadecanoic acidC16H32O2195941.1719621.31---------
90phytolC20H40O212244.8121177.23---------
91linoleic acidC18H32O213245.3021392.45---------
92linolenic acidC18H32O214345.4421452.31---------
Monoterpene hydrocarbons 5 (2.46%)7 (3.21%)7 (7.01%)10 (7.58%)
Oxigenated monoterpenes 7 (5.41%)2 (3.5%)8 (3.45%)2 (0.43%)
Sesquiterpene hydrocarbons 17 (46.84%)9 (6.0%)25 (59.06%)11 (6.53%)
Oxigenated sesquiterpenes 11 (23.29%)2 (15.75%)7 (19.13%)5 (1.96%)
Derivatives of benzene (benzenoids) 1 (0.17%)6 (67.84%)1 (1.22%)5 (82.03%)
Others 8 (14.88%)-3 (1.49%)-
Total 93.05%96.30%91.36%98.78%
a RIs from NIST Chemistry WebBook. b RIs relative to n-alkanes (C7–C30) on the HP5-MS column.
Table
Table 2. Antimicrobial activity of Asarum EOs.
Table 2. Antimicrobial activity of Asarum EOs.
SampleMIC (μg/mL)
ECPABSSAANFOSCCA
A. geophilum EO200200---200200-
A. yentunensis EO200--100200--200
A. splendens EO200--200-200--
A. cordifolium EO200-100----200
EC: Escherichia coli, PA: Pseudomonas aeruginosa, BS: Bacillus subtillis, SA: Staphylococcus aureus, AN: Aspergillus niger, FO: Fusarium oxysporum, SC: Saccharomyces cerevisiae, CA: Candida albicans.
Table
Table 3. Antioxidant activity of genus Asarum EOs.
Table 3. Antioxidant activity of genus Asarum EOs.
No.SampleSC(%) ValuesSC50 Values (µg/mL)
Positive control: ascorbic acid81.55 ± 0.913.26
Negative control [DPPH/EtOH+ DMSO]0.0 ± 0.0-
1A. geophilum EO63.34 ± 1.028.57
2A. yentunensis EO51.58 ± 0.550.24
3A. splendens EO34.24 ± 1.4>100
4A. cordifolium EO57.86 ± 0.839.62
Table
Table 4. Anti-inflammatory activity of genus Asarum EOs.
Table 4. Anti-inflammatory activity of genus Asarum EOs.
No.Sample *The Percentages of Inhibition of NO Production (%)The Percentages of Cell Life (%)IC50 Values
Positive control: Cardamonin85.40 ± 0.771.80 ± 0.52.33 µM
Negative control100.00 ± 0.8100.99 ± 1.0-
LPS0.00 ± 0.0--
1A. geophilum EO58.20 ± 0.478.05 ± 0.840.35 µg/mL
2A. yentunensis EO53.14 ± 1.666.87 ± 1.549.87 µg/mL
3A. splendens EO69.58 ± 1.381.85 ± 0.921.68 µg/mL
4A. cordifolium EO40.87 ± 0.660.65 ± 1.466.37 µg/mL
* The highest concentration of the EO was 100 µg/mL and the refined compounds was 50 µM.
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Minh, P.T.H.; Tuan, N.T.; Van, N.T.H.; Bich, H.T.; Lam, D.T. Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam. Molecules 2023, 28, 2580. https://doi.org/10.3390/molecules28062580

AMA Style

Minh PTH, Tuan NT, Van NTH, Bich HT, Lam DT. Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam. Molecules. 2023; 28(6):2580. https://doi.org/10.3390/molecules28062580

Chicago/Turabian Style

Minh, Pham Thi Hong, Nguyen Thuong Tuan, Nguyen Thi Hong Van, Hoang Thi Bich, and Do Tien Lam. 2023. "Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam" Molecules 28, no. 6: 2580. https://doi.org/10.3390/molecules28062580

APA Style

Minh, P. T. H., Tuan, N. T., Van, N. T. H., Bich, H. T., & Lam, D. T. (2023). Chemical Composition and Biological Activities of Essential Oils of Four Asarum Species Growing in Vietnam. Molecules, 28(6), 2580. https://doi.org/10.3390/molecules28062580

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