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Abstract

Detailed chemical constituents of essential oil from the Pterocephalus hookeri leaves and its antimicrobial activities were investigated in this study. The essential oil, obtained by hydrodistillation, was characterized by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry analyses. Among the 90 identified compounds, hexadecanoic acid (21.27%), phytol (8.03%), furfural (7.08%), oleic acid (5.25%), and phytone (4.56%) were the major components. In the antimicrobial assay, the essential oil showed strong inhibitory activities against Escherichia coli, Candida albicans, and Staphylococcus aureus with minimum inhibitory concentration values of 31.3, 62.5, and 125 µg/mL, respectively. To our knowledge, this is the first report concerning chemical composition and antimicrobial activities of the essential oil from Pterocephalus hookeri.

Keywords

essential oil GC-FID GC-MS antimicrobial activity

Essential oils, naturally occurring substances, are increasingly of interest for use as novel drugs because of their highly efficient, biodegradable nature and potential for commercial application.¹ Many plant-derived essential oils are known to exhibit antimicrobial activity against a wide range of bacteria and fungi.^2
-4 Essential oils have potential for use as new natural antimicrobial alternatives. The genus Pterocephalus, belonging to the family Dipsacaceae, comprises approximately 30 species widely distributed from Mediterranean to central Asia and tropical Africa.⁵ Two species, Pterocephalus hookeri (C. B. Clarke) Höeck and Pterocephalus bretschneideri (Batal.) E. Pritz, grow mainly in China. Pterocephalus hookeri, known as “Bang-zi-du-wu” in the Tibetan language, is recorded in the Tibetan medicine book as “Si-Bu-Yi-Dian”⁶ and the 2015 edition of the Chinese Pharmacopoeia. It has been clinically used to treat diseases including common cold, rheumatoid arthritis, enteritis, dysentery, and other conditions as a traditional medicine.^7,8 Previous phytochemical investigations have indicated that the main effective components of Pterocephalus hookeri are iridoid glycosides, triterpenoid saponins, and phenolic acids.^7

-10 In addition, the ethanol and aqueous extracts of Pterocephalus hookeri have been shown to possess readily detectable anti-inflammatory activity.¹¹ However, research on the chemical composition and antimicrobial activities, to our knowledge, of the essential oil of Pterocephalus hookeri has not yet been reported. We here report the chemical composition of the hydrodistilled essential oil from Pterocephalus hookeri leaves and evaluate their antimicrobial properties.

Table 1

Chemical Composition of the Essential Oil From Pterocephalus hookeri.

Compounds	RI^a	RI^b	%	Compounds	RI^a	RI^b	%
3-Methyl-2-butenal	784	790	0.09	α-Cubebene	1380	1378	0.25
Hexanal	802	800	0.16	β-Damascenone	1388	1388	1.20
Furfural	834	835	7.08	(E)-Jasmone	1391	1393	1.19
Isopropyl butyrate	844	843	1.60	n-Tetradecane	1400	1400	0.34
(E)−2-Hexenal	851	853	0.60	α-Cedrene	1417	1410	0.20
(Z)−3-Hexen-1-ol	854	857	0.82	α-Santalene	1422	1420	0.04
Norsabinane	859	856	0.09	4,11-Dimethyl-tetradecane	1459	1462	0.23
Hexyl alcohol	866	867	0.11	α-Curcumene	1486	1483	0.20
α-Angelica lactone	868	869	0.23	(E)-β-Ionone	1489	1485	0.16
2-Cyclopentene-1,4-dione	885	880	0.10	n-Pentadecane	1500	1500	0.33
(2E,4E)−2,4-Hexadienal	910	911	0.05	α-Muurolene	1505	1504	0.11
2-Acetylfuran	913	914	0.43	Cuparene	1513	1512	0.19
3-Ethylpyridine	957	959	0.40	(z)-Calamenene	1531	1531	0.44
Benzaldehyde	961	961	1.15	Dihydroactinidiolide	1538	1535	0.12
5-Methylfurfural	964	966	0.71	Lauric acid	1569	1570	2.37
3-Ethenylpyridine	966	968	1.28	Caryophyllene oxide	1591	1589	0.17
Hexanoic acid	978	981	0.26	n-Hexadecane	1600	1600	0.23
trans-Dehydroxylinalool oxide	995	994	0.49	Cedrol	1609	1608	0.15
(2E,4E)−2,4-Heptadienal	1012	1007	0.17	Cadalin	1681	1674	0.23
Benzyl alcohol	1035	1033	0.15	n-Heptadecane	1700	1700	0.37
Benzeneacetaldehyde	1045	1049	0.61	Myristic acid	1763	1765	1.91
Acetophenone	1068	1065	0.29	Benzyl Benzoate	1769	1770	0.21
cis-Linalol oxide (furanoid)	1076	1083	1.57	Phenanthrene	1781	1784	0.14
trans-linalool oxide (furanoid)	1091	1094	0.88	n-Octadecane	1800	1800	0.10
Linalool	1101	1104	1.54	Hexadecanal	1817	1818	0.16
Phenylethyl alcohol	1115	1114	0.53	Neophytadiene	1839	1840	0.19
Lilac aldehyde A	1145	1145	0.29	Phytone	1846	1845	4.56
Camphor	1149	1144	1.14	Pentadecylic acid	1861	1862	0.44
Lilac aldehyde C	1155	1160	0.61	n-Nonadecane	1900	1900	0.20
Borneol	1170	1172	0.54	(E)−11-Hexadecenoic acid	1922	1915	0.43
2-Methylacetophenone	1187	1174	0.22	Methyl palmitate	1927	1926	1.44
Anethofuran	1189	1191	0.16	(E)−9-Hexadecenoic acid	1940	1942	0.27
α-Terpineol	1194	1187	1.03	Isophytol	1948	1950	1.16
n-Dodecane	1200	1200	0.10	Palmitic acid	1970	1975	21.27
Carvomenthenal	1219	1217	1.86	Manool oxide	1983	1982	1.38
Citronellol	1222	1221	1.60	n-Icosane	2000	2000	0.13
Nerol	1230	1229	0.13	1-Octadecanol	2084	2081	1.36
Cuminaldehyde	1247	1240	0.36	Linoleic acid	2096	2104	0.58
Geraniol	1256	1258	0.25	Phytol	2115	2122	8.03
4-Phenyl-2-butanol	1261	1261	0.18	Methyl octadecanoate	2123	2127	0.93
Perillyl alcohol	1293	1295	0.27	Elaidic acid	2136	2140	1.70
n-Tridecane	1300	1300	0.17	Oleic Acid	2144	2141	5.25
1,1,6-Trimethyl-1,2-dihydronaphthalene	1357	1355	0.54	Oleic anhydride	2152	2157	1.46
1,1,6-Trimethyl-1,2-dihydronaphthalene	1357	1355	0.54	Ethyl linolenate	2170	2169	0.29
Chavibetol	1359	1362	0.38	Phytol acetate	2223	2225	2.51
Capric acid	1364	1374	0.32
	Total						96.16

^aRI: Retention index determined relative to n-alkanes (C7-C30) on the HP-5ms column.

^bRI: literature retention indices^12
-14.

Results and Discussion

Essential Oil Composition

The yield of leaf essential oil obtained by water distillation was 0.33% (w/w relative to dry material weight). Composition analysis by gas chromatography-flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) identified 90 volatile compounds, accounting for 96.16% of the total extracted oil, which were identified by matching retention times of available authentic standards, retention indices (RIs), and mass spectra in the NIST 17 database (Table 1). The essential oil was mainly composed of 15 oxygenated monoterpenes (10.62%), 6 sesquiterpenes (0.92%), 5 diterpenes (15.32%), 11 carboxylic acids (34.8%), 16 benzenoid aromatics (5.82%), 11 alkanes (2.29%), 3 aliphatic alcohols (2.29%), 6 hourseterocycle compounds (10.13%), and 17 carbonyl compounds (13.97%). As shown in Table 1, the major compounds are hexadecanoic acid (21.27%), phytol (8.03%), furfural (7.08%), oleic acid (5.25%), and phytone (4.56%). Hexadecanoic acid is also the main compound in the essential oil of Pterocephalus canus (7.90%). Linalool (1.10%), α-terpineol (0.30%), geraniol (0.20%), β-damascenone (0.20%), and caryophyllene oxide (1.10%) have been also reported in the essential oil of Pterocephalus canus.¹⁵ In Pterocephalus nestorianus, the content of furfural in the essential oil was below 0.10%, but that of lauric acid was more than 50.35%.¹⁶ Some n-alkanes have been reported in the essential oil of Pterocephalus nestorianus,⁵ which are also found in our study. These differences may be related to the site of collection, soil composition, or solar radiation.^17,18

Antimicrobial Activity

The in vitro antimicrobial activities of essential oil against 6 pathogenic microorganisms (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus fumigatus, and Candida albicans) were evaluated using the disc diffusion and broth microdilution methods. The disc diameters of the zone of inhibition and the minimum inhibitory concentration (MIC) of the essential oil for the tested microorganisms are shown in Table 2. The essential oil is effective against S. aureus, E. coli, and C. albicans with inhibition zones of 19.2, 20.9, and 20.7 mm, respectively. In the broth microdilution assay, the essential oil showed the highest sensitivity to E. coli with MIC and minimal bactericidal concentration (MBC) values of 31.3 and 62.5 µg/mL, followed by C. albicans (MIC and MBC values of 62.5 and 125 µg/mL), and S. aureus (MIC and MBC values of 125 and 250 µg/mL). The antimicrobial activities of phytol and furfural were confirmed against E. coli, C. albicans, and S. aureus.^19
-21 These main components could be, at least in part, responsible for the antimicrobial activities of the essential oil from Pterocephalus hookeri leaves. Other minor constituents such as benzaldehyde and α-terpineol were reported to have different degrees of antibacterial activities.^22,23 Synergistic effects of these minor compounds in the oil should also be considered.

Table 2

Zones of Growth Inhibition (mm), MICs, and MBCs of Essential Oil From Pterocephalus hookeri Against the Growth of Microorganisms.^a

Microorganisms	Diameter of zones of inhibition		MICs(μg/mL)	MBCs(μg/mL)
Microorganisms	Essential oil	Antibiotic^b	MICs(μg/mL)	MBCs(μg/mL)
Staphylococcus aureus	19.2 ± 0.6	23.3 ± 0.2	125	250
Bacillus cereus	18.3 ± 0.4	24.5 ± 0.4	250	500
Escherichia coli	20.2 ± 0.6	21.9 ± 0.4	31.3	62.5
Pseudomonas aeruginosa	10.9 ± 0.5	24.2 ± 0.7	NT^c	NT^c
Candida albicans	20.7 ± 0.7	23.8 ± 0.2	62.5	125
Aspergillus fumigatus	17.4 ± 0.5	23.2 ± 0.2	250	250

Abbreviations: MBC, minimal bactericidal concentration; MIC, minimum inhibitory concentration.

^aResults were mean ± SD of triplicate values.

^bAntibiotics used were ampicillin for Gram-positive bacteria, gentamicin for Gram-negative bacteria, amphotericin for fungi.

^cNT: not tested.

Conclusion

This study first reports that the essential oil extracted from Pterocephalus hookeri, with main components of hexadecanoic acid, phytol, furfural, oleic acid, and phytone, possesses pronounced antimicrobial activity against E. coli, C. albicans, and S. aureus. The results indicate that the essential oil of Pterocephalus hookeri might be suitable for use as a natural antimicrobial agent. However, further studies should focus on the synergism, mechanism of action, and bioavailability of different essential oil components.

Materials and Methods

Plant Material

The fresh leaves of Pterocephalus hookeri in the present study were collected from Shangri-La (27°82′N, 99°71′E), Yunnan Province, China, in October 2019. The plant was authenticated by one of the authors (Shen Huang) and the voucher specimens (No. 0136) were deposited at the Department of Plant Resources, School of Food and Bioengineering, Zhengzhou University of Light Industry, China.

Extraction of the Essential Oil

The collected leaves were dried in the shade at room temperature and powdered. The essential oil was extracted by hydrodistillation of powdered leaves (150 g) for 3 hours in a Clevenger-type apparatus. The essential oil was dried using anhydrous sodium sulfate and stored in a dark glass vial at 4 °C until analysis.

Identification of the Chemical Components of the Essential Oil

The components of the essential oil were determined by using an Agilent GC-MS (8890A-5977B, USA) equipped with a fused silica capillary column HP-5ms (60 m × 0.25 mm × 0.25 μm). The injector temperature was 280 ℃. The oven temperature was initially held at 50 ℃ for 2 minutes and increased to 140 ℃ at the rate of 2 ℃/min and held for 10 minutes, then increased to 180 ℃ with a rate of 3 ℃/min, held for 2 minutes, finally the temperature was increased to 270 ℃ with a rate of 1.5 ℃/min, held for 10 minutes. Carrier gas was helium at 1.0 mL/min. One-microliter aliquot of oil was injected into the column using a 20:1 split injection. The mass spectrometer was operated in electron-impact ionization mode with 70 eV energy. The ion source temperature was 230 ℃ and the scanning range of ion mass was from 35 to 550 amu. The GC analysis was conducted on a 6890N apparatus (Agilent Technologies, Santa Clara, California, USA) equipped with a flame ionization detector (FID) and an electronic pressure control injector. GC-FID analysis was conducted under the same experimental conditions using the same column as described for the GC-MS. Relative percentages of components were calculated based on the peak areas using the normalization method without correction factors. The components of essential oil were identified by matching retention times of available authentic standards, RIs, and mass spectra in the NIST 17 database. RIs were calculated for all components using a homologous series of n-alkane mixtures (C7-C30) injected under conditions similar to those of the samples and computer matched with the NIST libraries.

Antimicrobial Activity Assays

The microbial strains used in this investigation were S. aureus ATCC 25923, B. cereus ATCC 10987, E. coli ATCC 25922, and P. aeruginosa ATCC 15542. The fungal strain used in this study was C. albicans ATCC 10231 and A. fumigatus ATCC 1022. All strains were maintained on an agar slant at 4 °C. The bacterial strains were cultured in a Muller-Hinton broth at 37 °C for 24 hours. The fungal strains were cultured on Sabouraud Dextrose Agar (SDA) at 28 °C for 120 hours before testing. The disc diffusion method²⁴ was used to determine the antimicrobial activities of the essential oils. Petri plates were prepared by pouring 20 mL Muller Hinton Agar (MHA) or SDA and the solution was allowed to solidify. The plates were then dried, and 0.1 mL of the standardized inoculum containing 10⁶-10⁷ colony-forming units/mL of the bacterial suspension was poured, uniformly spread, and allowed to dry for 5 minutes. The oil was prepared in sterile dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL, of which 100 µL was added to the respective wells. The control well received only 100 µL DMSO. The reference antibiotics used were ampicillin for Gram-positive bacteria, gentamicin for Gram-negative bacteria, and amphotericin for fungi. The plates were left at room temperature to allow diffusion and then incubated at 37 °C for 24 hours for bacterial growth or at 28 °C for 48 hours for fungal growth. The antimicrobial activity was evaluated by measuring the diameter of the zones of inhibition against the test organisms. The experiments were repeated in triplicate and the results are expressed as average values. The MIC was determined using the broth microdilution method using 96-well microplates. The inoculum of the microbial strains was prepared from 24 to 48 hours broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. Serial concentrations (500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.9, 1.95, 0.98, and 0.49 µg/mL) of essential oil were prepared. One hundred microliters from culture broth was mixed with 100 µL of different concentration oils in the corresponding well and plates were incubated either at 37 °C for 24 hours for antibacterial activity or 28 °C for antifungal activity. The lowest concentration of the tested oil showing no microbial growth was defined as the MIC. MBC values were determined by taking a part of the liquid from each well that showed no growth and incubating on agar plates at 37 °C for another 24 hours. The lowest concentration that disclosed no visible growth of bacteria or fungi was confirmed as MBC.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (No. 81903507), and Doctoral Scientific Research Foundation of Zhengzhou University of Light Industry (0123-13501050063).

ORCID iD

Peng-fei Yang

References

Jiang

Wang

Song

et al. GC×GC-TOFMS analysis of essential oils composition from leaves, twigs and seeds of Cinnamomum camphora L. Presl and their insecticidal and repellent activities. Molecules. 2016;21(4):423.doi:10.3390/molecules21040423

http://www.ncbi.nlm.nih.gov/pubmed/27043503

Seow

YX.

Yeo

CR.

Chung

HL.

Yuk

H-G

. Plant essential oils as active antimicrobial agents. Crit Rev Food Sci Nutr. 2014;54(5):625-644.doi:10.1080/10408398.2011.599504

http://www.ncbi.nlm.nih.gov/pubmed/24261536

Boren

Crown

Multidrug

Pan-Antibiotic Resistance . The role of antimicrobial and synergistic essential oils: a review. Nat Prod Commun. 2020;15(10):1-19.

Jia

Zhu

. Chemical composition and antimicrobial activities of essential oil from Wedelia urticifolia growing wild in Hunan Province, China. Nat Prod Res. 2019;33(18):2685-2688.doi:10.1080/14786419.2018.1460830

http://www.ncbi.nlm.nih.gov/pubmed/29668311

Akhgar

MR.

Safavinia

. Essential oil composition of Pterocephalus gedrosiacus . Chem Nat Compd. 2016;52(3):514-515.doi:10.1007/s10600-016-1693-5

Zhang

J-J.

Lin

J-W.

Fang

W-S.

G-H

. Anti-inflammatory and analgesic effects of ethanol and aqueous extracts of Pterocephalus hookeri (C.B. Clarke) Höeck. J Ethnopharmacol. 2009;123(3):510-514.doi:10.1016/j.jep.2009.01.039

http://www.ncbi.nlm.nih.gov/pubmed/19443147

Y-C.

Guo

C-X.

Zhu

Y-Z.

Y-M.

Guo

F-J.

Zhu

G-F

. Four new bis-iridoids isolated from the traditional Tibetan herb Pterocephalus hookeri . Fitoterapia. 2014;98:104-109.doi:10.1016/j.fitote.2014.07.015

http://www.ncbi.nlm.nih.gov/pubmed/25065705

Yin

Guo

Zhu

. Secoiridoid/iridoid subtype bis-iridoids from Pterocephalus hookeri . Magn Reson Chem. 2014;52(11):734-738.doi:10.1002/mrc.4116

http://www.ncbi.nlm.nih.gov/pubmed/25104576

Shen

X-F.

Zeng

J-C.

Tang

Zhang

Meng

X-L

. The anti-arthritic activity of total glycosides from Pterocephalus hookeri, a traditional Tibetan herbal medicine. Pharm Biol. 2017;55(1):560-570.doi:10.1080/13880209.2016.1263869

http://www.ncbi.nlm.nih.gov/pubmed/27937009

10.

Tang

Wen

Wang

et al. Simultaneous determination of ten major compounds including iridoid glycosides and phenolic acids in Pterocephalus hookeri by UPLC-PDA. Chin J Chin Mater Med. 1993;42(7):1234-1237.

11.

Guan

XL.

Yan

YN.

Wei

TM.

Ren

ZH.

Song

. Anti-inflammatory effects and acute yoxicity test of Pterocephalus hookeri . J Beijing Univ Tradi Chin Med. 2004;27(2):71-73.

12.

Adams

. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Allured Publishing Corporation; 2007.

13.

Essien

Ogunwande

IA.

Flamini

Cioni

PL.

Kinola

. Analysis of the essential oil of Dialium guineense wild. J Essent Oil Res. 2007;19(6):545-547.doi:10.1080/10412905.2007.9699327

14.

Afsharypuor

Ranjbar

Mazaheri

Shakibaei

Aslani

. Essential oil constituents of seeds and fresh leaves of garden lettuce (Lactuca sativa L.) grown in Isfahan, Iran. Res J Pharma. 2018;5(3):1-5.

15.

Vahedi

Nasrabadi

Lari

Halimi

. Volatile constituents and antimicrobial activities of Pterocephalus canus . J Med Plants Res. 2011;5(23):5646-5648.

16.

Abdullah

FO.

Hussain

FHS.

Mannucci

et al. Composition, antifungal and antiproliferative activities of the hydrodistilled oils from leaves and flower heads of Pterocephalus nestorianus N ábělek. Chem Biodivers. 2017;14(7):e1700009. doi:10.1002/cbdv.201700009

http://www.ncbi.nlm.nih.gov/pubmed/28217981

17.

Paolini

Barboni

Desjobert

J-M.

Djabou

Muselli

Costa

. Chemical composition, intraspecies variation and seasonal variation in essential oils of Calendula arvensis L. Biochem Syst Ecol. 2010;38(5):865-874.doi:10.1016/j.bse.2010.07.009

18.

Morelli

Ferarrese

Munhoz

CL.

Alberton

. Antimicrobial activity of essential oil and growth of Ocimum basilicum (L.) inoculated with mycorrhiza and humic substances applied to soil. Genet Mol Res. 2017;16(3):1-11.doi:10.4238/gmr16039710

19.

Pejin

Savic

Sokovic

et al. Further in vitro evaluation of antiradical and antimicrobial activities of phytol. Nat Prod Res. 2014;28(6):372-376.doi:10.1080/14786419.2013.869692

http://www.ncbi.nlm.nih.gov/pubmed/24422895

20.

Ghaneian

MT.

Ehrampoush

MH.

Jebali

Hekmatimoghaddam

Mahmoudi

. Antimicrobial activity, toxicity and stability of phytol as a novel surface disinfectant. Environ Heal Eng Manage J. 2015;2(1):13-16.

21.

Chai

W-M.

Liu

Y-H

et al. Antityrosinase and antimicrobial activities of furfuryl alcohol, furfural and furoic acid. Int J Biol Macromol. 2013;57:151-155.doi:10.1016/j.ijbiomac.2013.02.019

http://www.ncbi.nlm.nih.gov/pubmed/23466497

22.

Ullah

Khan

AL.

Ali

et al. Benzaldehyde as an insecticidal, antimicrobial, and antioxidant compound produced by Photorhabdus temperata M1021. J Microbiol. 2015;53(2):127-133.doi:10.1007/s12275-015-4632-4

http://www.ncbi.nlm.nih.gov/pubmed/25626368

23.

Shi

Yin

et al. Antibacterial activity of α-terpineol may induce morphostructural alterations in Escherichia coli . Braz J Microbiol. 2014;45(4):1409-1413.doi:10.1590/S1517-83822014000400035

http://www.ncbi.nlm.nih.gov/pubmed/25763048

24.

CTT.

Thai

TH.

Hien

et al. Chemical composition and antimicrobial activity of the leaf and twig essential oils of Magnolia hypolampra growing in Na Hang nature reserve, Tuyen Quang province of Vietnam. Nat Prod Commun. 2019;14(6):1-9.

Chemical Composition and Antimicrobial Activities of the Essential Oil From the Leaves of Pterocephalus hookeri