Chemical Composition and Antimicrobial Activity of the Essential Oil From Euphorbia helioscopia L.

Abstract

The chemical composition and antimicrobial activity of the essential oil from the aerial parts of Euphorbia helioscopia L. were investigated by gas chromatography-mass spectrometry (GC-MS), GC, and microdilution methods. Thirty-five compounds, representing 83.51% of the total oil, were identified. 1,6-Dihydrocarveol (31.39%), carvone (16.79%), menthol (8.23%), and trans-dihydrocarvone (5.53%) were the principal constituents of the oil. The essential oil exhibited strong antimicrobial activity against strains of the bacteria, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Shigella dysenteriae, and a strain of the fungus Candida albicans with a minimal inhibitory concentration value of 31.25 μg/mL and minimum bactericidal concentration values of 31.25 μg/mL (S. aureus), 62.50 μg/mL (E. faecalis), >62.50 μg/mL (E. coli), >62.50 μg/mL (S. dysenteriae), and >62.50 μg/mL (C. albicans). These results indicated that the essential oil from the aerial parts of E. helioscopia could be used to control diseases caused by these microbes.

Euphorbia helioscopia L. (Euphorbiaceae) is distributed in most areas of Europe, northern Africa, and eastern Asia. ¹ It is widely employed for its activity against skin diseases, intestinal parasites, migraine, gonorrhea, and other conditions. ² The aerial parts of this plant, referred to as “Ze Qi” in China, are used as traditional medicine for the treatment of ascites, edema, tuberculosis, phlegm, cough, and ringworm. ^3,4 The ethanolic extract of this plant has been reported to possess strong antibacterial activity against Pseudomonas aeroginosa and Staphylococcus aureus. ⁵ The secondary metabolites of E. helioscopia are reported to include terpenoids, amino acids, flavonoids, tannins, and steroids, ^6,7 some of which exhibit obvious antimicrobial activity. ^{8 -10} However, there are few reports on the antimicrobial activity of the essential oil from E. helioscopia. In this study, the chemical composition of the essential oil from the aerial parts of E. helioscopia was investigated using gas chromatography-mass spectrometry (GC-MS) and GC. Then, the essential oil was evaluated for antimicrobial activity against strains of the bacteria, S aureus, Enterococcus faecalis, Escherichia coli, and Shigella dysenteriae, and a strain of the fungus Candida albicans by microdilution method.

Materials and Methods

Plant Material

The aerial parts of E. helioscopia were collected from Jinhua City (29°06′N, 119°39′E), Zhejiang Province, China, in 2016, and identified by Dr Kezhong Deng. The voucher specimen (No. 20160405) was deposited in the Key Laboratory of Innovation Drug and Efficient Energy-saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine.

Isolation of the Essential Oil

The plant material (800 g) was added to 8 times its volume of water and subjected to hydrodistillation for 6 hours using a Clevenger-type apparatus. The essential oil (0.8 mL, 0.10% v/w) was extracted by ether, dried over anhydrous sodium sulfate for 24 hours, and then stored in an airtight container at 4 °C until use.

GC-MS Analysis

The composition of the essential oil was identified by a GC-MS 7890A-5975C system (Agilent Technologies, USA) equipped with an HP-5MS fused silica capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness). The GC-MS conditions were as follows: carrier gas, helium, at a flow rate of 1.0 mL/min; scan mode, full; scan time, 1 second; mass scan range, 30-650 m/z; electron-impact ionization, 70 eV; split ratio, 40:1. The oven temperature was maintained at 80 °C for 2 minutes, increased to 100 °C at a rate of 2.5 °C/min, increased to 120 °C at 1 °C/min, and then increased from 120 °C to 260 °C at 4 °C/min. A total of 100 µL of essential oil was diluted in 10 mL of ethanol, and 1 µL of the solution was injected into the GC injector at a temperature of 250 °C. The GC analysis was carried out on a 6890N apparatus (Agilent Technologies, USA) equipped with a flame ionization detector (FID) and an electronic pressure control injector. GC-FID analysis was carried out under the same experimental conditions using the same column as described for the GC-MS. The composition of the extracts was determined from the percentages from peak areas using the normalization method without correction factors. ¹¹

Identification of Compounds

The essential oil compounds were identified by using the NIST 11 Mass Spectral Database for GC-MS and comparing the mass spectra with those reported in Adams (2007) and other literature. ¹² Furthermore, the retention index (relative to C7-C40 n-alkanes, under the same experimental conditions) of each compound was matched with literature values to verify each compound identity, with its total percentage composition calculated as the peak area. ^{13 -19}

Antimicrobial Activity

A microdilution method was used for the determination of the antimicrobial activity of the extracts, according to the National Committee for Clinical Laboratory Standards. The microbial strains were prepared from overnight broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. A volume of 100 µL of suspension containing 1.0 × 10⁸ colony-forming unit (CFU)/mL of bacteria or 1.0 × 10⁴ CFU/mL of fungal spores was added to a depth of 4 mm on Mueller-Hinton agar or Sabouraud dextrose agar, respectively, in sterilized Petri dishes. Negative controls were prepared using dimethyl sulfoxide (DMSO). Standard discs of ampicillin, ceftazidime, and fluconazole from Sigma-Aldrich, USA, were used as positive controls. Each disc was impregnated with 30 µL of sample of microbial strain and placed on the inoculated agar. The inoculated plates were maintained at 4 °C for 2 hours and incubated at 37 °C (24 hours) for bacterial strains and at 28 °C (48 hours) for the fungal strain. All microorganisms were completely insusceptible to the control discs imbued with DMSO. The standard microbial strains comprised 4 strains of the bacteria, E. coli (ATCC 8739), E. faecalis (ATCC 29212), S. aureus (ATCC 6538), and S. dysenteriae (CMCC 51252), and 1 strain of the fungus C. albicans (ATCC 10231), which were purchased from National Center for Medical Culture Collections (CMCC). All experiments were performed in triplicate.

Results and Discussion

Essential Oil Composition

The essential oil was obtained by hydrodistillation as a green liquid from the aerial parts of E. helioscopia. The yield of the essential oil was 0.10% (v/w) of the dry weight of the plant. Its chemical composition was analyzed using GC-FID and GC-MS. Thirty-five compounds, representing 83.51% of the total oil, were identified (Figure 1). The essential oil was dominated by terpenes (75.35%), followed by esters (3.18%), alkenes (2.38%), carboxylic acids (1.67%). and others (0.93%). 1,6-Dihydrocarveol (31.39%), carvone (16.79%), menthol (8.23%), and trans-dihydrocarvone (5.53%) were the top 4 constituents of the peak area, as shown in Figure 1 and Table 1. 1,6-Dihydrocarveol, carvone, and menthol have been reported in the essential oils from other plants of the family Euphorbiaceae. For instance, 1,6-dihydrocarveol is the main compound in the essential oil from Euphorbia fischeriana Steud. roots (9.36%). As in E. helioscopia, the content of carvone in the essential oil of E. fischeriana is less than 0.05%. ²⁰ Menthol is found in the essential oil of Phyllanthus reticulatus Poir. leaves (1.9%). ²¹ trans-Dihydrocarvone is a common compound in the essential oils from some plants of the family Lamiaceae, including Mentha dumetorum, Mentha longifolia, Salvia officinalis, Origanum vulgare, and Thymus citriodorus, and some in the family Apiaceae, including Cuminum cyminum and Anethum graveolens. ^{22 -28} The content of trans-dihydrocarvone in these 2 families is 0.03%-12.78% and 3.20%-31.11%. In addition, trans-dihydrocarvone (15.3%-51.2%) is the main compound in all developmental stages of Poiretia latifolia of the family Fabaceae from South America. ²⁹ In the present study, trans-dihydrocarvone was found in the family Euphorbiaceae for the first time.

Figure 1

Total ion chromatogram of the essential oil from the aerial parts of Euphorbia helioscopia.

Table 1

Essential Oil Composition From Euphorbia helioscopia.

S. no.	Compound	CAS	Formula	[M]+ (m/z)	RT	RI a	RI b	Area (%)
1	Hexanol	000111-27-3	C6H14O	102.17	3.28	878	874	0.04
2	Furfural diethyl acetal	013529-27-6	C9H14O3	170.21	7.83	1076	1078	0.05
3	Camphor	000076-22-2	C10H16O	152.23	10.33	1144	1144	0.16
4	trans-Menthone	000089-80-5	C10H18O	154.25	10.65	1152	1150	0.61
5	iso-Menthone	000491-07-6	C10H18O	154.25	11.10	1163	1163	0.97
6	Menthol	015356-70-4	C10H20O	156.27	11.44	1172	1170	8.23
7	α-Terpineol	000098-55-5	C10H18O	154.25	12.26	1192	1190	0.47
8	1,6-Dihydrocarveol	000619-01-2	C10H18O	154.25	12.49	1198	1196	31.39
9	trans-Dihydrocarvone	005948-04-9	C10H16O	152.23	12.59	1200	1200	5.53
10	Dihydrocarveol	038049-26-2	C10H18O	154.25	13.41	1214	1206	2.04
11	trans-Carveol	001197-07-5	C10H16O	152.23	13.61	1218	1220	3.51
12	cis-Carveol	001197-06-4	C10H16O	152.23	14.22	1228	1225	1.64
13	Methyl thymyl ether	001076-56-8	C11H16O	164.24	14.42	1232	1234	0.42
14	Pulegone	000089-82-7	C10H16O	152.23	14.70	1237	1237	0.31
15	Carvone	002244-16-8	C10H14O	150.22	14.97	1241	1244	16.79
16	Piperitone	000089-81-6	C10H16O	152.23	15.52	1251	1253	0.4
17	Thymol	000089-83-8	C10H14O	150.22	17.99	1294	1290	0.46
18	Ethyl nonanoate	000123-29-5	C11H22O2	186.29	18.15	1297	1294	0.22
19	Chavibetol	000501-19-9	C10H12O2	164.20	22.39	1354	1362	0.18
20	α-Copaene	003856-25-5	C15H24	204.35	23.70	1371	1376	0.19
21	β-Bourbonene	005208-59-3	C15H24	204.35	24.40	1380	1384	0.98
22	β-Caryophyllene	000087-44-5	C15H24	204.35	27.13	1416	1420	1.23
23	γ-Muurolene	30021-74-0	C15H24	204.35	29.27	1442	1445	0.11
24	Curcumene	000644-30-4	C15H22	220.33	32.55	1483	1483	0.33
25	α-Selinene	000473-13-2	C15H24	204.35	33.15	1491	1494	0.08
26	Ethyl laurate	000106-33-2	C14H28O2	228.37	38.81	1596	1593	0.25
27	Globulol	051371-47-2	C15H26O	222.37	41.02	1653	1620	0.67
28	Tetradecanoic acid	000544-63-8	C14H28O2	228.37	44.95	1765	1769	0.12
29	Ethyl tetradecanoate	000124-06-1	C16H32O2	256.42	45.93	1795	1793	0.26
30	Phytone	000502-69-2	C18H36O	268.48	47.41	1846	1847	1.68
31	Ethyl pentadecanoate	041114-00-5	C17H34O2	270.45	48.81	1895	1897	0.05
32	Methyl palmitate	000112-39-0	C17H34O2	270.45	49.66	1927	1926	0.1
33	Palmitic acid	000057-10-3	C16H32O2	256.42	50.63	1965	1964	1.58
34	Ethyl palmitate	000628-97-7	C18H36O2	284.47	51.43	1995	1996	2.29
35	Ethyl linoleate	007619–08-1	C20H36O2	308.50	55.46	2164	2163	0.17
	Total identified (%)							83.51
	Terpenes							75.35
	Esters							3.18
	Alkenes							2.38
	Carboxylic acids							1.67
	Others							0.93

RI, retention indices; RT, retention time.

^aRIs calculated from RTs in relation to those of a series C7-C40 of n-alkanes on an HP-5MS capillary column.

^bRIs from the literature. ^{12 -18}

Antimicrobial Activity

The antimicrobial activities of the essential oil against 4 strains of the bacteria S. aureus, E. faecalis, E. coli and S. dysenteriae, and a strain of the fungus C. albicans were determined using the microdilution method. The essential oil exhibited strong antimicrobial activities against these microbial strains with an MIC value of 31.25 µg/mL and MBC values of 31.25 µg/mL, 62.50 µg/mL, >62.50 µg/mL, >62.50 µg/mL, and >62.50 µg/mL, as shown in Table 2. ³⁰ In previous reports, the essential oil from the inflorescence of E. helioscopia exhibited antibacterial activity against Bacillus subtilis with an inhibition zone of 11 ± 0.25 mm and a MIC value of 17 mg/mL, but exhibited no antibacterial activity against Pseudomonas aeruginosa, Salmonella typhimurium, or S. aureus. ³¹ In the previous study, carvone was demonstrated to exhibit various levels of antimicrobial activity against the bacteria S. aureus (with MIC and MBC values of 0.3125-5 mg/mL and 1.25-10 mg/mL, respectively), S. typhimurium (MIC = 2.5 mg/mL, MBC = 5 mg/mL), E. coli (MIC = 2.5 mg/mL, MBC = 5 mg/mL), and Klebsiella pneumoniae (MIC = 10 mg/mL, MBC = 10 mg/mL). ³² Menthol has previously been shown to possess strong antibacterial activity against strains of nonpathogenic E. coli (102-104 colony-forming units [CFU]/mL) and enterohemorrhagic E. coli (MIC = 0.400 mg/mL, MBC = 0.400 mg/mL). ³³ However, there are few reports of the antimicrobial activity of 1,6-dihydrocarveol. In our study, the antibacterial activity of the oil against E. coli was stronger than 2 of its constituent, namely, carvone and menthol. We suggest that 1,6-dihydrocarveol in the essential oil could play an important role in inhibiting and killing E. coli.

Table 2

Antimicrobial Activity (MIC and MBC, μg/mL) of the Essential Oil From Euphorbia helioscopia.

Sample	Fungus		Bacteria strain
	Candida albicans		Escherichia coli		Enterococcus faecalis		Staphylococcus aureus		Shigella dysenteriae
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Essential oil	31.25	>62.50	31.25	>62.50	31.25	>62.50	31.25	31.25	31.25	>62.50
Fluconazole	125	/	nt	nt	nt	nt	nt	nt	nt	nt
Ampicillin	nt	nt	125	/	1	/	15.6	125	0.1	1
Ceftazidime	nt	nt	1	/	15.6	100	0.1	/	0.1	/

MIC: minimal inhibitory concentration; MBC: minimum bactericidal concentration; (/): not active; nt: not tested.

Conclusion

In our study, the essential oil from the aerial parts of E. helioscopia was found to include 35 compounds (representing 83.51% of the total oil), including 1,6-dihydrocarveol (31.39%), carvone (16.79%), menthol (8.23%), and trans-dihydrocarvone (5.53%), and showed strong antimicrobial activity against strains of 4 bacteria E. coli, E. faecalis, S. aureus, and S. dysenteriae, and a strain of the fungus C. albicans. Therefore, we suggest that the oil could be used as an eco-friendly antimicrobial agent to promote public health.