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

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%.


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].

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). 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, antibioticresistant 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%).
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.

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 SC 50 value of 28.57 µg/mL. The A. cordifolium EO with an SC 50 value of 57.86 ± 0.8% corresponded to an SC 50 value of 39.62 µg/mL and the A. yentunensis EO with an SC 50 value of 51.58 ± 0.5% corresponded to SC 50 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 (SC 50 value of 28.57 µg/mL) than the A. splendens EO (SC 50 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).

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 IC 50 value of 21.68 µg/mL. The next are A. geophilum EO with an IC 50 value of 40.35 µg/mL and A. yentunensis EO with an IC 50 value of 49.87 µg/mL. Finally, the A. cordifolium EO illustrates lower anti-inflammatory activity at the tested concentration with an IC 50 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 IC 50 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 IC 50 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.
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 IC 50 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 IC 50 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.

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.

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 Na 2 SO 4 , 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.

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.

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]  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.

Antioxidant Assays
Dissecting the ability to trap free radicals generated by using DPPH (1,1-diphenyl-2picrylhydrazyl); 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 SC 50 value (µg/mL, µM/mL). The SC 50 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.

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% CO 2 , 10% Fetal Bovine serum (FBS-Fetal Bovine Serums). The cell fluid was inoculated onto a 96-well microplate, density 2.5 × 10 5 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 NaNO 2 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 NaNO 2 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): The concentration of NO sample The concentration of NOLPS ×100 (1)
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 SC 50 value of 28.57 µg/mL, following the A. cordifolium EO with an SC 50 value of 57.86 ± 0.8% corresponding to SC 50 value of 39.62 µg/mL. The A. splendens EO displayed the best antiinflammatory activity with an IC 50 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.