Journal of Essential Oil Bearing Plants

ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: https://www.tandfonline.com/loi/teop20

Chemical Composition, Antimicrobial, Hemolytic,

and Antiproliferative Activity of Essential Oils from
Ephedra intermedia Schrenk & Mey

Jingli Ni, Behnam Mahdavi & Somayeh Ghezi

To cite this article: Jingli Ni, Behnam Mahdavi & Somayeh Ghezi (2019) Chemical Composition,
Antimicrobial, Hemolytic, and Antiproliferative Activity of Essential Oils from Ephedra
intermedia Schrenk & Mey, Journal of Essential Oil Bearing Plants, 22:6, 1562-1570, DOI:
10.1080/0972060X.2019.1707717

To link to this article: https://doi.org/10.1080/0972060X.2019.1707717

Published online: 27 Jan 2020.

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 TEOP 22 (6) 2019 pp 1562 - 1570 1562
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Chemical Composition, Antimicrobial, Hemolytic, and Antiproliferative

Activity of Essential Oils from Ephedra intermedia Schrenk & Mey

Jingli Ni 1, Behnam Mahdavi 2*, Somayeh Ghezi 2

Department of Traditional Chinese Medicine, Lishui Hospital of Traditional

1

Chinese Medicine, Lishui City, Zhejiang Province, China, 323000, China

2
Department of Chemistry, Faculty of Science, Hakim Sabzevari University, Sabzevar, Iran
Received 12 November 2019; accepted in revised form 18 December 2019

Abstract: We report the chemical composition and bioactivity of flowers essential oil of Ephedra
intermedia (EIO) for the first time. The essential oil was obtained using a Clevenger-type apparatus. The
essential oil composition was identified by chromatography methods. The minimum inhibitory concentration
method (MIC) was chosen to investigate antibacterial activity of EIO. Hemolytic activity and the cytotoxicity
effects of EIO on human cancer cells were also evaluated. The essential oil was dominated by 2-ethyl-pyrazine
(67.37 %), γ-elemene (9.21 %), benzyl acetate (9.10 %), 2-methyl-butyl acetate (5.28 %). EIO prevented the
growth of 6 out of 7 selected microorganisms. The highest activity was observed against Enterococcus faecalis.
Due to the low hemolytic rate (below 2.53 %) on humane red blood cells (hRBS); the safety of EIO as an
additive for different aspects can be considered. On the other hand, EIO presented anti-proliferative effect on
Hela, HCT116, and LnCap cells with mortality of 67 % to 81 %.

Key words: Ephedra intermedia; 2-ethyl-pyrazine; hemolytic activity; antiproliferative activity.

Introduction recreational drug 6. Six ephedrine alkaloids in-

Ephedra plants are known as one of the oldest cluding ephedrine, pseudoephedrine, methyl-
medicinal plants in human civilization 1. The ge- ephedrine, methylpseudo-ephedrine, norephe-
nus of Ephedra L. belongs to the Ephedraceae drine, and norpseudoephedrine are the major
family, within the Gnetales order 2. In terms of effective constituents of Ephedra 7. These com-
phylogenetic, the Ephedraceae plants belong to pounds exhibit both direct agonisms at the α and
the group of gymnosperms, which also includes α adrenergic receptors as well as indirect agonism
pines, firs, and larches 3. The Ephedra genus con- by intensification the release of norepinephrine
sists of approximately 50 species that are distrib- from presynaptic neurons 6. The antimicrobial
uted all over the world, particularly in coastal and potential of some Ephedra species, such as E.
subalpine areas 4. Up to the present, Ephedra has altissima, E. transitorai, E. nebrodensis, E. ma-
been used in Traditional Chinese Medicine to rem- jor and E. breana has been also reported 8.
edy different diseases such as allergies, bronchial Ephedra intermedia is a perennial shrub, that
asthma, chills, fever, colds, coughs, edema, flu, grows in grasslands, deserts, river valleys, flood
headaches, and nasal congestion 5. Furthermore, plains and at an elevation range of 800 to 4600 m
dietary supplements that consist of Ephedra have 9
. E. intermedia is native to Southwest Asia, the
been popularly used for muscular performance Middle East, Central Asia, and South Asia. The
improvement, body weight loss, and even as a stems of the plant are a pungent, bitter, warm herb
*Corresponding author (Behnam Mahdavi)
E-mail: < b.mahdavi@hsu.ac.ir, behnammahdavi@yahoo.com > © 2019, Har Krishan Bhalla & Sons
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1563
that dilates the bronchial vessels whilst stimulat- formed by a Shimadzu 17A on the fused-silica
ing the heart and central nervous system. The capillary column of CBP-5 (25 m × 0.32 mm i.d.;
stems are also antidote, diaphoretic, diuretic, vaso- film thickness 0.5 μm; from Shimadzu, Japan).
constrictor, and vasodilator. They are used to treat The conditions and parameters of analysis are
asthma, hay fever and allergic complaints 10. We following: carrier gas: Helium (rate of flow: 1.1
found a medicinal use of E. intermedia in the sam- mL min-1); injector temperature, 220°C; the ini-
pling location of the plant for this research. The tial temperature, 50°C for 3 min, raised at 3°C
locals use the plant in form of poultice to treat min-1 to 250°C and held for 10 min; detector tem-
inflamed areas in leg or hand injuries for humans perature, 250°C; split ratio, 1:20; and 1 μL from
or animals. Due to no report on chemical composi- the volatile oil in n-hexane, with ratio of 1:10,
tion and bioactivity of essential oil of E. inter- was injected into the instrument.
media (EIO) in our literature review; in this re- The essential oil was also subjected to Shimadzu
search, we focus on the identification of chemi- (QP2010SE). A RTX-5 was used as the capillary
cal compositions of the plant essential oil and column (30 m × 0.32 mm i.d.; 0.25 μm film thick-
evaluated of EIO bioactivity including antibacte- ness from Restek). Framework and conditions
rial, antiproliferative, and hemolytic activity. So including carrier gas, temperature program, and
far, there is no report on the evaluation of the two split ratio were the same to GC/FID analysis. 70
last bioactivities of an Ephedra species essential eV for the ionization voltage and 40-600 amu for
oil. the scan mass range were used for the mass de-
tector.
Materials and methods To identify individual components, retention
Plant materials time (in term of minute) was obtained from the
Aerial parts of Ephedra intermedia were col- GC/FID chromatogram. The homologous series
lected (about 5 kg) in October 2015 from its natu- of saturated hydrocarbon (C8 to C20 and C21 to C40)
ral habitat beside the road of Sabzevar- Afcheng were analyzed using the same column and condi-
with the latitude of N36°36‘85“and longitude of tions to measure the retention indices (I). The re-
E57°65‘36“ at an elevation of 1560-1575 m from tention indices data were given by the equation
sea level, Khorasan-Razavi, Iran. The Plant was for relative retention indices (RRI). The identity
identified by Dr. Tazari, a botanist at Hakim of components was assigned by comparing the
Sabzevari University. A voucher specimen of the GC retention indices and mass spectral fragmen-
plant with a number of 611 was deposited at the tation patterns with those of the known com-
Hakim Sabzevari University Herbarium (HSUH). pounds from the literature data 11 and the National
Institute for Standard and Technology (NIST)
Isolation of the essential oil database 12. Relative amounts of each component
First, to obtain more yield two hundred grams were reported in terms of percent peak area rela-
of the dried plant parts were macerated in water tive to the total peak area.
for 1 h. Then the mixture was subjected to
hydrodistillation using Clevenger apparatus. Af- Antimicrobial activity assay
ter 4 hours the obtained essential oil was dried Antibacterial activity
over anhydrous sodium sulfate and kept in a vial Microorganisms
at -18°C before further analysis. The hydrodis- Three Gram-positive bacteria of Staphylococ-
tillation was repeated for five times for determin- cus aureus ATCC 25923, S. coagulase, Entero-
ing average yield of essential oils; all the essen- coccus faecalis ATCC 14506; and four Gram-
tial oils were combained together. negative bacteria including Escherichia coli
ATCC 25922, Klebsiella pneumonia ATCC
Gas chromatography(GC-FID) and Gas chro- 13883, Proteus vulgaris ATCC 33420, Pseudomo-
matography/Mass Spectrometry (GC/MS) nas aeruginosa ATCC 27853 were used as the
analysis tested strains. All the microorganisms were ob-
GC/FID analysis of the essential oil was per- tained from the Microbiology laboratory culture
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1564
collection of Sabzevar Medical Science Univer- with phosphate buffer saline (PBS), and a sus-
sity. pension of hRBS in PBS (8 % v/v) was prepared.
For the next step, EIO with different concentra-
Minimum inhibitory concentration assay tions (0.005 to 2.5 mg/mL) was added to the
(MIC) above-mentioned suspension and incubated with
MIC assay was carried out according to the pre- agitation for 60 min at 37°C. Finally, after centri-
vious study with some modifications 13. Aliquots fuge the hemoglobin release was measured by
of culture media and EIO solutions (concentra- absorbance at 540 nm, using a plate reader
tions of 0.008-1.000 mg/mL) were pipetted into (Thermo Lab systems, Franklin, MA USA). So-
the wells of a 96-well plate and inoculated with dium dodecyl sulfate (SDS) 1 % and hRBCs in
microorganisms; positive of chloramphenicol was PBS with no essential oil (untreated) were used
used with same concentrations. The covered plates as the positive and negative controls, respectively.
were incubated at 37°C for 24 hours. The mini- Percentage hemolysis was calculated according
mum inhibitory concentration was assessed as the to the following equation:
minimum concentration that resulted in no visi-
Hemolysis% = [(AS-ANC)/ (APC-ANC)] × 100
ble growth.
Where As is the absorbance of the sample (con-
Antiproliferative assay tains EIO and hRBCs), ANC s is the absorbance of
Human cervical carcinoma cell line (HeLa the negative control (hRBCs in PBS), and APC
cells), human colon cancer cell line (HCT116), positive control (SDS and hRBCs).
and human prostate adenocarcinoma cells (LnCap
cells) were purchased from Pastor Institute (Iran). Results and discussion
In this study, cytotoxicity of EIO on HeLa and The yield of essential oil obtained by hydro-
LnCap cells proliferation was evaluated with MTT distillation was 0.43±0.04 % (w/w) for E.
(3-(4, 5-dimethylthi-azol-2-yl)-2, 5-diphenyltetra- intermedia. The chemical composition of the
zolium bromide) assay according to a previous essential oil of the aerial parts of E. intermedia is
study with some modifications 14. The cells were presented in Table 1. According to the results, 21
evenly distributed (5×103 cells/well) in 96-well compounds were identified in which eight com-
plates and incubated in a humidified incubator at pounds were accounted for 97.5 % of the oil com-
37°C with 5 % CO2 overnight. Then the cells were position. The oil was characterized by the pres-
treated with EIO with different concentrations ence of a heterocyclic compound of 2-ethyl-
(31.2-1000 μg/mL) and incubated for 24h. For pyrazine (67.4 %), ester of benzyl acetate (9.1
the next step, the medium in each well was re- %), and sesquiterpene of γ-elemene (9.2 %). How-
placed with 20 μl MTT (5 mg/mL in PBS) and ever, ester of 2-methyl-butyl acetate (5.3 %) and
incubated at 37°C for 4 h. The purple-blue phenolic compound of Z-isoeugenol (4.2 %) also
formazan crystals were dissolved in 100 μL Dim- identified in the oil.
ethyl sulfoxide (DMSO) and the absorbance was α-Terpineol has been reported as the main con-
measured at a wavelength of 510 and 630 nm stituent of E. sinica from different locations
(control wavelength) on a 96-well plate reader around the world 1,16,17. Hexadecanoic acid and
(Thermo Lab systems, Franklin, MA USA). Fi- 1,4-cineole were the main components For E.
nally, IC50 (concentration of oil that achieved a equisetina and E. intermedia of China respecti-
50 % of mortality) of oil was calculated. vely 18. The essential oils of E. Sinica from dif-
ferent locations of China were rich in tetramethyl-
Hemolysis assay pyrazine, linalool, α-terpineol, 4-terpineol 19.
The hemolytic activity of EIO was carried out Ehtesham-Gharaee et al. reported the chemical
according to the previous study with some modi- composition of essential oils from different
fications 15. First, the plasma was removed using Ephedara species of Iran. They found significant
a centrifuge. Then red blood cells were washed differences in the main constituents of various
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1565
Table 1. Chemical composition of the essential oils from aerial parts of Ephedra intermedia

No. Compound RT RI* RI** Area %

1 n-Hexanol 5.53 863 861 0.6

2 2-Methyl-butyl acetate 6.22 875 876 5.3
3 2-Methoxy-pyrazine 6.26 896 894 0.3
4 Ethyl pentanoate 6.41 901 903 1.4
5 2-Ethyl-pyrazine 6.56 912 913 67.4
6 Camphene 7.46 946 944 <0.1
7 ρ-Cymene 9.21 1020 1019 <0.1
8 Limonene 10.11 1024 1022 <0.1
9 1,8-Cineol 10.24 1026 1028 <0.1
10 Camphor 14.56 1141 1139 <0.1
11 Benzyl acetate 15.26 1157 1155 9.1
12 α-Terpineol 16.76 1186 1189 <0.1
13 Myrtenol 17.23 1194 1191 <0.1
14 Carvone 19.31 1239 1237 <0.1
15 Chavicol 19.66 1247 1244 <0.1
16 Eugenol 23.21 1356 1357 <0.1
17 Methyl eugenol 26.09 1403 1401 <0.1
18 Z-Isoeugenol 26.20 1406 1408 4.2
19 γ-Elemene 27.38 1434 1433 9.2
20 Germacrene A 30.49 1508 1506 <0.1
21 δ-Cadinene 31.14 1522 1525 <0.1

* Retention indices (I) from literatures

** Retention indices (I) on CBP-5 capillary column
plant species’ essential oils. E. foliate was rich in 2. According to the results, the oil showed the
limonene, ρ-cymene, and α-terpineol. The vola- highest activity against E. faecalis followed by
tile oil of E. major was dominated by citronellol S. aureus, E. coli, P. vulgaris, P. aeruginosa, and
and (3Z)-3-hexenyl benzoate. Carvone and car- K. pneumoni. EIO activity was less than the posi-
vacrol were the main compounds of E. sarco- tive control of chloramphenicol against all strains.
carpa. α-terpineol and myrcenol were Furthermore, the essential oil was not susceptible
recognaized as the main compounds of E. to S. coagulase. Previous studies have reported
distachya essential oil 20. The volatile oil of E. the antibacterial activity of extracts or essential
alata alenda was rich in linalool 8. Another study oil from Ephedra species. For example, E. alata
reported that the oil of the aerial parts of E. alenda essential oil prevented the strains of
distachya, E. fragilis, and E. major from Italy were Baccilus cereus, Enterococcus facealis, and Sal-
marked by ethyl benzoate, (E)-phytol, and eu- monella enterica 8. E. procera extract inhibited
genol respectively 21. Citronellol was the abun- the growth of P. vulgaris, P. aeruginosa, E.
dant compound in the essential oil of E. nebro- aerogenes, B. cereus, and S. aureus 23.
densis 2. The antimicrobial activity of the plants’ essen-
tial oil depends on their chemical constituents. E.
Antimicrobial activity intermedia essential oil dominated by 2-ethyl-
For this study, we chose E. coli, K. pneumoni, pyrazine and γ-elemene. The antibacterial acti-
E. faecalis, and S. aureus because they are known vity of these compounds or volatile oil that com-
as multi-drug resistant pathogens 22. Antibacte- prise them have been reported previously.
rial activity results of EIO are tabulated in Table Pyrazine derivatives, which are well known as
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1566
Table 2. Antimicrobial activity of Ephedra intermedia aerial parts
using minimum inhibition concentration (MIC) assays

Microorganisms Essential oil Positive controla

mg/mL mg/mL

Gram-positive
Staphylococcus aureus 0.25±0.00b 0.25±0.00
Staphylococcus coagulase NAc NTd
Enterococcus faecalis 0.21±0.07 0.21±0.07
Gram-negative
Escherichia coli 0. 25±0.00 0.21±0.07
Klebsiella pneumonia 0.50±0.00 0.12±0.00
Proteus vulgaris 0.33±0.14 0.17±0.07
Pseudomonas aeruginosa 0.33±0.14 0.21±0.07

a: Chloramphenicol; b: Values are presented as means ± SD (n =3)

c: non-active; d: not tested
volatile compounds in plant essential oil, exhib- 0.08 to 1.85 mg/mL 31.
ited antimicrobial activity against wide spectrum Obviously, the synergistic effects of the chemi-
of microorganisms 24,25. 2-Ethyl-pyrazine inhib- cal constituents of the plant essential oils contri-
ited the growth of Bacillus spp 26. Furthermore, bute to their biological activity such as antimi-
In our review of the literature, antimicrobial acti- crobial and antiproliferative activity 13. However,
vity of the essential oils from different plants, a few studies have reported the cytotoxicity acti-
which comprise elemenes as major or minor con- vity of the major compounds of E.intermedia es-
stituents, have been reported 22, 27-29. sential oil. For instance, peanut oil that comprises
pyrazine derivatives such as 2-ethyl-pyrazine, 2-
Antiproliferative activity methyl-pyrazine, and 2,5-dimethyl-pyrazine is
Results of the antiproliferative activity assay known as an anticancer food 32. β-Elemene, γ-
(shown in Fig. 1 as representative cell mortality elemene and α-elemene have a wide spectrum of
diagrams) expressed that the EIO exhibited a dose- antineoplastic activities 33,34. γ-Elemene and its
related activity on all selected cell lines in the derivatives exhibited antiproliferative activity
tested range of essential oil concentrations. The against leukemia and lymphoma cells 35. Curcuma
highest activity was obtained for HeLa (IC50= wenyujin is the most famous traditional Chinese
423.22±8.243 μg/mL) followed by HCT116 (IC50 remedies to treat cancer. γ-Elemene is the main
= 543.02±5.63 μg/mL). The LnCap cell line was compound of the plant essential oil 36.
the least sensitive to the treatment with EIO (IC 50
= 616.28±6.22 μg/mL). Hemolysis
In our literature review, a few studies have re- Hemolysis of hRBCs should be considered as a
ported the antiproliferative of Ephedra plant ex- problem of biomaterials. The hemolytic force of
tracts. Mellado et al evaluated the cytotoxicity of a natural product or drug can result in different
hexane, dichloromethane, and ethanolic extract mechanisms such as dissolving or increasing the
of E. chilensis. The extracts exhibited antiprolife- permeability of cell membranes to complete cell
rative activity against breast, colon, and prostate lysis 15. Based on the observed results (Fig. 1),
cancer cells with IC50 range of 0.03 to 5.24 μg / EIO presented almost imperceptible hemolytic
mL 30 . In another study, the cytotoxicity of dif- rates on hRBCs, the percentage of hemolysis
ferent extracts of E. aphylla against breast can- ranged from 0.25 % to 2.53 %. This result pro-
cer cell lines has been reported with IC50 range of posed the safety of EIO to human and its poten-
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1567

Fig. 1. Antiproliferative activity of Ephedra intermedia essential oil on the HeLa, HCT116,
and LnCap cell lines. The values are presented as means ± SD (error bars) n =3

Fig. 2. Hemolysis activity of Ephedra intermedia essential oil at different

concentrations. The values are presented as means ± SD (error bars) n =3
tial application is confirmed as a safe food pre- essential oil contains 2-ethyl-pyrazine, γ-elemene,
servative. According to previous reports, hemo- benzyl acetate, and 2-methyl-butyl acetate as the
lysis up to 5% is acceptable for materials 25,26. main components. The essential oil showed anti-
bacterial effect against S. aureus, E. faecalis, E.
Conclusions coli, K. pneumonia, P. vulgaris, and P. aeruginosa.
The present study highlighted the essential oil The cytotoxic assay showed that E. intermedia
constituents from the aerial parts of Ephedra essential oil inhibited the growth of HeLa, HCT
intermedia as well as it’s antibacterial, cytotoxic, 116, and LnCap cells. The safety of E. intermedia
and hemolytic activities for the first time. The essential oil was approved in hemolysis assay
 Jingli Ni et al., / TEOP 22 (6) 2019 1562 - 1570 1568
because the hemolysis value was in the standard Acknowledgment
range of the food or medicinal additives. The authors would like to thank the Department
of Chemistry of Hakim Sabzevari University for
Conflict of interest statement technical assistance.
We declare that we have no conflict of interest.

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