Chemical composition of P. dhana essential oil
The essential oil of P. dhana fruits was obtained by hydrodistillation with a yield of 0.76%v/w on dry weight basis. The chemical composition of the P. dhana essential oil is presented in Table 1. In total, 42 volatile compounds were identified, representing 99.27% of the oil. Trans-piperitol (50.9%), o-cymene (11.4%), β-pinene (11.0%), γ-terpinene (9.2%), and limonene (4.9%) were the major volatile compounds of the P. dhana essential oil. Trans-arbusculone (2.4%), cis-4-caranone (2.0%), β-cubebene (1.5%), yomogi alcohol (1.4%), and isobornyl acetate (1.3%) were detected as minor components. These results showed a high content of monoterpene hydrocarbons over 97.7% in P. dhana essential oils.
Table 1
Volatile compounds of P. dhana essential oil by GC-MS
Compound | RI* | RI** | % area | Compound | RI* | RI** | % area |
heptanal | 911 | 901 | t*** | pinocarvone | 1161 | 1160 | t |
tricyclene | 928 | 921 | t | borneol | 1167 | 1165 | t |
artemisia triene | 931 | 923 | t | neoiso-isopulegol | 1168 | 1167 | t |
α-thujene | 934 | 924 | 0.05 | terpinen-4-ol | 1179 | 1174 | 0.06 |
α-pinene | 940 | 932 | 0.90 | α-terpineol | 1193 | 1186 | 0.07 |
camphene | 954 | 946 | 0.10 | cis-4-caranone | 1200 | 1200 | 2.03 |
benzadehyde | 966 | 952 | t | decanal | 1207 | 1201 | 0.22 |
sabinene | 979 | 969 | 0.10 | trans-piperitol | 1217 | 1207 | 50.91 |
β-pinene | 983 | 979 | 11.02 | isobornyl acetate | 1285 | 1283 | 1.26 |
cis-meta-mentha-2,8-diene | 991 | 987 | t | β-cubebene | 1390 | 1388 | 1.49 |
myrcene | 996 | 990 | 0.20 | decyl acetate | 1410 | 1407 | 0.55 |
dehydroxy-trans-linalool oxide | 1002 | 992 | t | E-caryophyllene | 1417 | 1417 | 0.08 |
yomogi alcohol | 1007 | 999 | 1.35 | δ-amorphene | 1519 | 1511 | 0.14 |
δ-2-carene | 1014 | 1001 | t | α-calacorene | 1539 | 1539 | t |
α-terpinene | 1020 | 1014 | t | maaliol | 1578 | 1566 | t |
o-cymene | 1028 | 1022 | 11.37 | 2-ethylbutyric acid, octyl ester | 1581 | 1581 | 0.77 |
limonene | 1031 | 1024 | 4.92 | cis-dihydro-mayurone | 1604 | 1595 | t |
γ-terpinene | 1061 | 1054 | 9.20 | guaiol | 1615 | 1600 | t |
trans-arbusculone | 1073 | 1066 | 2.39 | muurola-4,10(14)-dien-1-β-ol | 1624 | 1630 | t |
terpinolene | 1091 | 1086 | t | α-muurolol | 1643 | 1646 | t |
linalool | 1103 | 1095 | 0.07 | pogostol | 1650 | 1651 | 0.36 |
*Calculated retention indices obtained on a DB-5 column. |
**Retention indices on a DB-5 column obtained from Adams.[54] |
*** trace amount < 0.05 |
The results agreed with previous study on the chemical composition of essential oils extracted from Peucedanum genus reporting monoterpenes and sesquiterpenes as major constituents. Aggarwal et al.18 reported that β-caryophyllene oxide was identified as main components in the P. dhana essential oil. The major chemical compounds of the essential oil obtained from P. longifolium were monoterpene hydrocarbons, with myrcene as the dominant compound13. Alavi et al.10 identified 31 volatile compounds in the essential oil obtained from P. ruthenicum M. Bieb., and the major compounds were 1,8-cineole and camphor. In addition, volatile components of the essential oils extracted from P. japonicum11, P. austriacum19, P. cervaria, and P. alsaticum20 have also been reported, with similar main components including α-pinene and β-phellandrene. Meanwhile sabinene was the main constituent of P. ostruthium essential oil21. Moreover, the major components in the essential oil obtained from P. paniculatum L. were β-cyclolavandulyl and β-isocyclolavandulyl esters14. Essential oil of P. officinale presented limonene and α-pinene as main components22. It is noted that it is possible to detect a diversity of terpenoids, especially monoterpenes in plant species of the Peucedanum genus. However, different factors including plant age, cultivation, climatic and micro-environmental conditions, seasons, harvest time, chemotypic variation, and geography play important roles in the variations of essential composition of plants23.
Antimicrobial activity
The antimicrobial activity of the P. dhana essential oil was evaluated against seven pathogenic microorganisms, using the disc diffusion and the broth microdilution methods. The zone of inhibition diameter, MIC, and MMC of the P. dhana essential oil and chloramphenicol for the tested microorganisms are shown in Table 2. Both Gram-positive and Gram-negative bacteria were susceptible to the essential oil of P. dhana fruits. The MIC of the bacterial pathogens ranged from 62.50 µg/mL to 250 µg/mL, whereas the MIC for fungus was 125 µg/mL. The P. dhana essential oil was effective in inhibiting the growth of all tested microbial strains. The data obtained from the disc diffusion method indicated that P. aeruginosa ATCC 27853 was the most significantly sensitive (40.80 mm) to 1 mg/mL of P. dhana essential oil compared to those obtained from other tested pathogens ranging between 10.70–19.60 mm. In agreement with the results from the disc diffusion method, the results of the MIC determination indicated that the P. dhana essential oil had the lowest MIC value (62.50 µg/mL) for all Gram-negative bacterial pathogens, including P. aeruginosa ATCC 27853, E. coli ATCC 25922, and E. aerogenes ATCC 13048. Meanwhile Gram-positive bacteria and fungus showed higher MIC values, ranging from 125 µg/mL to 500 µg/mL. As can be seen in Table 2, the lowest MMC value was 250 µg/mL for P. aeruginosa ATCC 27853, while the highest MMC value was 1,000 µg/mL for S. aureus ATCC 25923. In addition, the zone of inhibition diameter of the P. dhana essential oil for all microbial strains was similar to those obtained from chloramphenicol that was used as a positive control.
Table 2
Antimicrobial activity of P. dhana essential oil and chloramphenicol
Microorganism | Chloramphenicol | P. dhana essential oil |
MIC (µg/mL) | MMC (µg/mL) | Zone of inhibition diameter (mm) | MIC (µg/mL) | MMC (µg/mL) | Zone of inhibition diameter (mm) |
Gram-positive bacteria | | | | | | |
S. aureus ATCC 25923 | 125.00c | 500.00b | 19.10 ± 0.01c | 250.00c | 1000.00c | 18.20 ± 0.20b |
S. epidermidis ATCC 12228 | 125.00c | 500.00b | 19.50 ± 0.01c | 125.00b | 500.00b | 19.25 ± 0.25b |
B. subtilis ATCC 6051 | 62.50b | 500.00b | 20.40 ± 0.01b | 125.00b | 1000.00c | 19.60 ± 0.31b |
Gram-negative bacteria | | | | | | |
E. coli ATCC 25922 | 62.50b | 500.00b | 10.10 ± 0.00e | 62.50a | 500.00b | 12.90 ± 0.20c |
P. aeruginosa ATCC 27853 | 31.25a | 250.00a | 40.30 ± 0.01a | 62.50a | 250.00a | 40.80 ± 0.20a |
E. aerogenes ATCC 13048 | 62.50b | 500.00b | 19.20 ± 0.03c | 62.50a | 500.00b | 18.70 ± 0.30b |
Fungus | | | | | | |
C. albicans ATCC 10231 | 62.50b | 500.00b | 17.50 ± 0.20d | 125.00b | 500.00b | 10.70 ± 0.20d |
The data are mean ± standard deviation. Different letters indicate significant differences (p < 0.05) |
Regarding the antimicrobial activity, P. dhana essential oil was able to inhibit the growth of microbial pathogens, especially the Gram-negative bacteria with MIC value of 62.50 µg/mL. The antibacterial potential against pathogenic bacteria of several species from the genus Peucedanum has been reported. Ilić et al.13 demonstrated that essential oils extracted from P. longifolium fruits exhibited slight antibacterial activity against the tested bacterial strains in vitro. According to Pirbalouti et al.24, essential oil extract from P. membranacea Boiss exhibited a significant antibacterial activity against the Proteus vulgaris strain. The essential oil of P. ruthenicum M. Bieb. fruits showed antibacterial activities against S. aureus, S. epidermidis, and B. cereus10. Essential oils from several species, including P. ferulaefolium25, P. ruthenicum26, P. japonicum11, and P. alsaticum, and P. cervaria27, had antibacterial properties. However, there is no report on the antifungal activity from essential oil extracted from Peucedanum species. The data obtained in this work show a growth reduction potential for bacterial pathogens. Moreover, the essential oil of P. dhana fruits possesses antifungal activity against C. albicans. The antimicrobial activity of essential oil is mainly associated with the presence of active components, including monoterpenes, sesquiterpenes, and their derivatives, as reported by Burt3. Essential oils of Spanish Mentha rotundifolia28 and Ocimum canum29 were rich in piperitol and presented weak antibacterial activity29. Thus, the strong antimicrobial activity of P. dhana oil could be explained by the synergetic effect of major and minor compounds30–32. These active compounds could enhance the antibacterial property of trans-piperitol. It was found that the mode of action of P. dhana oil could depend on the hydrophobicity of their functional groups, which enables them to partition the lipids of the bacterial cell membrane disturbing the cell wall and the cytoplasmic membrane, leading to lysis and leakage of intracellular compounds33. In addition, Moleyar and Narasimtram34 described that the antimicrobial activity of volatile compounds in essential oil resulted from the combined effect of direct and indirect vapor absorption on pathogenic strains through the aqueous medium. The vapor absorption on microbial pathogens was detected by their membrane permeability. The absorption into aqueous media is examined by solubility, volatility, and stability of their volatile components. However, there is a limited detailed information about the antimicrobial activity mechanism of major volatile compounds. Thus, the antimicrobial potential of pure compounds presented in P. dhana oil is also required.
Antioxidant activity and total phenolic content
The antioxidant activity of P. dhana essential oil was investigated using the DPPH and ABTS-scavenging assays. Results of the antioxidant activity of P. dhana essential oil and trolox are shown as IC50 (Table 3). The IC50 of P. dhana essential oil was 9.13 ± 0.11 mg/mL and 9.36 ± 0.21 mg/mL using the DPPH and ABTS assays, respectively, which was significantly higher than those from trolox with DPPH (0.66 ± 0.04 mg/mL) and ABTS (0.78 ± 0.04 mg/mL) assays. The total phenolic content of the P. dhana essential oil was 183.05 ± 0.21 mg GAE/100 g dw (Table 3).
Table 3
Total phenolic content and antioxidant activities of P. dhana essential oil and trolox
Sample | yield | Total phenolic content | IC50 (mg/mL) |
(%w/w) | (mg GAE/100 g dry weight) | DPPH | ABTS |
P. dhana essential oil | 0.76 ± 0.21 | 183.05 ± 0.21 | 9.13 ± 0.11a | 9.36 ± 0.21a |
Trolox | - | - | 0.66 ± 0.04b | 0.78 ± 0.04b |
The data are mean ± standard deviation. Different letters indicate significant differences (p < 0.05) |
The antioxidant activity and total phenolic content of the P. dhana essential oil have not yet been reported. However, there are few reports about the antioxidant activity and total phenolic content of essential oils obtained from Peucedanum species. Normally, essential oils were evaluated as a weak antioxidant35. Our study agreed with previous studies. Tepe et al.36 studied the antioxidant properties of P. longifolium and P. palimbioides essential oils. Their results revealed that the inhibition percentage of free radical DPPH of P. longifolium and P. palimbioides essential oils was 8.59–41.87% and 10.67–47.26%, respectively, while the standard compounds – butylated hydroxytoluene and butylated hydroxyanisole had strong inhibition percentage of free radical DPPH of 93.85% and 94.98%, respectively. Matejić et al.37 reported that the total phenolic contents of the crude extracts obtained from the aerial parts of P. aegopodioides, P. alsaticum, P. officinale, and P. longifolium varied from 52.18 mg GAE/g to 118.32 mg GAE/g of extract. In another study, the total phenolic contents of polyphenolic extract and hydroalcoholic extract from the aerial parts of P. pastinacifolium Boiss. & Hausskn was 117.1 ± 6.2 mg GAE/g and 44.3 ± 1.7 mg GAE/g of extract, respectively38. The antioxidant activity of the essential oil of P. dhana might be due to the occurrence of major monoterpenic constituents like β-pinene, γ-terpinene, and limonene. Beta-pinene, which is the major compound in many essential oils such as turpentine oils, exhibited outstanding antioxidant activity39. Previous studies have also confirmed that γ-terpinene40 and limonene41 possessed good antioxidant activity. Moreover, the antioxidant activity of essential oil may be correlated to the volatile compounds listed in Table 1. Although the phenol compounds are evaluated mainly as the antioxidant compounds due to their oxidation and redox properties, terpenes have more potential in inhibiting free radicals. Their strong antioxidant activity is due to the activated methylene group42. Moreover, the phenolic content of P. dhana essential oil differed from those obtained from other species, which may be attributed to environmental and genetic factors43.
Cytotoxicity
Due to the cytotoxicity of essential oils, they have been applied as potential antitumor agents44,45. Therefore, the cytotoxicity of essential oil of P. dhana fruits was evaluated in both cancer and normal cell lines using MTT assay by comparing the IC50 values to those of doxorubicin (Table 4). The P. dhana essential oil had cytotoxicity against all tested cancer cell lines, with IC50 values ≤ 60 µg/mL, and demonstrated significantly low dose-dependent cytotoxicity in all cell lines compared to those obtained from the positive control, doxorubicin. The IC50 values obtained from P. dhana essential oil were 56.63 ± 0.11 µg/mL, 51.67 ± 0.23 µg/mL, 18.24 ± 0.11 µg/mL, and 961.36 ± 0.11 µg/mL for Hela, A549, SW480, and 3T3L1 cell lines, respectively. Meanwhile, the IC50 values obtained from doxorubicin were 0.55 ± 0.21 µg/mL, 0.46 ± 0.16 µg/mL, 0.34 ± 0.13 µg/mL, and 876.34 ± 0.16 µg/mL for Hela, A549, SW480, and 3T3L1 cell lines, respectively. Cell lines of SW480 were more sensitive to P. dhana essential oil than the other cancer cell lines. These IC50 values, which are less than those obtained from antimicrobial activity tests of P. dhana oil, suggest that the P. dhana oil has moderate to strong cytotoxic effect, depending on the cell lines. This result agreed with the previous studies on essential oils of various plant species46.
Table 4
Cytotoxicity of P. dhana essential oil and doxorubicin
Cell line | IC50 (µg/mL) |
P. dhana essential oil | doxorubicin |
cervical cancer (Hela) | 56.63 ± 0.11c | 0.55 ± 0.21c |
human lung adenocarcinoma (A549) | 51.67 ± 0.23b | 0.46 ± 0.16b |
human colonic adenocarcinoma (SW480) | 10.24 ± 0.11a | 0.34 ± 0.13a |
murine fibroblast (3T3L1) | 961.36 ± 0.11d | 876.34 ± 0.16d |
The data are mean ± standard deviation. Different letters indicate significant differences (p < 0.05) |
The study of Sylvestre et al.2 reported the cytotoxic effect of essential oils. Based on their study, IC50 values between 10–50 µg/mL indicate strong cytotoxic activity, while IC50 values between 50–100 µg/mL, 100–200 µg/mL, and 200–300 µg/mL were indicate moderate, weak, and very weak cytotoxic properties, respectively. In addition, IC50 values higher than 300 µg/mL indicates no cytotoxicity. Considering the cytotoxic activity on the four tested cells based on the MTT assay, P. dhana oil showed a moderate cytotoxic activity against the Hela, and A549 cells. However, a strong cytotoxic activity of P. dhana oil was demonstrated in SW480 cells. The doxorubicin, used as a positive control in this study, had a strong cytotoxic effect on cancer cells, which is considered as one broad spectrum of antitumor antibiotics of the most extensively used agents in chemotherapy regimens of cancer patients. The molecular mechanism of the cytotoxicity of P. dhana oil may appear according to its lipophilic property which accumulated in the cell membrane. The lipophilic property may increase its permeability reducing membrane-bound enzymes, metabolism activity, and induction of apoptosis47. In addition, β-pinene, α-pinene, and γ-terpinene have been considered as the major components responsible for some of the cytotoxic activities48,49. However, the cytotoxic effect of other components that are present at lower concentrations in the P. dhana oil essential oil must be considered. Examples of such components are limonene50, β-cubebene51, α-terpineol52, and camphene53, which are previously known to have cytotoxic effects against different cell lines. Considering an investigation about the cytotoxic effect of Peucedanum spp. on tumor cells, Yeong et al.54 reported that the essential oil of P. japonicum Thunb revealed a cytotoxic activity against A549 cells with IC50 of 0.04192% v/v. The obtained results in this study also present the importance of P. dhana as an alternative herbal source of essential oil that can be used as a cytotoxic agent against cancer cells, especially in human colonic adenocarcinoma.