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Research Journal of Pharmacognosy 5(2), 2018: 51-56
Received: 30 Oct 2017
Accepted: 10 Mar 2018
Published online: 13 Mar 2018
DOI: 10.22127/RJP.2018.58507 Original article
Preparation of Saussurea costus Traditional Oil and Investigation of Different
Parameters for Standardization
Sahar Bagheri1, Nastaran Ebadi1, Zahra Taghipour2, Azadeh Manayi3, Tayebeh Toliyat4, Mehran
Mirabzadeh Ardakani1*
1Department of Traditional Pharmacy, School of Traditional Medicine, Tehran University of Medical Sciences,
Tehran, Iran.
2School of Pharmacy, Tehran University of Medical Science, Tehran, Iran.
3Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
4Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Science, Tehran, Iran.
Abstract
Background and objective: Medicinal oils are one of the most common and special dosage forms in
oral and topical therapies of Persian medicine (PM). The oil of Saussurea costus (bitter qust) root is
prominent topical oil with different applications in PM. In this study, the oil of bitter qust was
prepared according to ancient Persian medical texts. Methods: To prepare traditional qust oil, 100 g
of the root was soaked in 600 mL aqueous ethanol 25% overnight. The supernatant was then filtered
and boiled in 800 g sesame oil until all water was evaporated. The essential oil of the root and volatile
components of its traditional oil were extracted using hydro-distillation method in a Clevenger-type
apparatus and were analyzed by gas chromatography-mass spectrometry (GC-MS) method. Total
phenolics, flavonoids, tannins and polysaccharides were determined by spectrophotometric methods to
evaluate the chemical parameters of traditional bitter qust oil. Results: The content of volatile
compounds in both investigated samples was determined (0.5% and 0.1% (v/w), respectively).
Dehydrocostus lactone and 1, 3-cyclooctadiene were two similar main compounds in the both
analyzed samples. Total phenolics (788.290±0.61 mg/L gallic acid equivalent (GAE)), flavonoids
(303.2±2.52 mg/L catechin equivalent (CE)), tannins (23.97±0.52 mg/L GAE) and polysaccharides
(9.240±0.13 mg/L dextrose equivalent (DE)) contents were determined. Conclusion: According to the
obtained data, dehydrocostus lactone could be used for determination and evaluation of traditional
bitter qust oil.
Keywords: bitter qust oil; costus root; Persian medicine; Saussurea costus; traditional oil
Citation: Bagheri S, Ebadi N, Taghipour Z, Manayi A, Toliyat T, Mirabzadeh Ardakani M. Preparation of
Saussurea costus traditional oil and investigation of different parameters for standardization. Res J Pharmacogn.
2018; 5(2): 51-56
Introduction
Oils are one of the oldest dosage forms in ancient
medical systems such as Persian medicine (PM)
[1,2]. Persian medicine is a prominent, popular
and historical medicinal system similar to other
various traditional systems of medicine like
Chinese medicine, Ayurveda and Homeopathy
[3]. PM encompasses two fundamental parts
disease prevention in the first step and treatment
of different disorders at the next stage [4].
Medicinal herbs are mainly recruited in PM to
treat diseases [5] while oils are one of the most
common preparations [6]. Various topical and
systematic applications of herbal oils have been
introduced in PM medical texts [5,7]. Traditional
Bagheri S. et al.
52 Res J Pharmacogn 5(2): 51-56
oils are called ''Adhan'' (singular form: “Dohn”)
in pharmaceutical books of PM and their
therapeutic usage and preparation procedures
have been explained in detail [8].
More than thirty species of plants have been
introduced in PM to prepare herbal oils. These
drugs were administered in various diseases
especially arthritis, sciatica and muscle aches [9].
Traditional oils are divided into two categories;
those which are taken directly from the oily parts
of the plants (sesame seed, olive fruits) and the
others obtained from extraction of non-oily parts
of the plants in oil vehicle that causes trapping of
hydrophobic and hydrophilic agents in the
vehicle [1,2,10,11]. The mentioned oils are both
categorized as fixed oil. Saussurea costus (Falc.)
Lipsch. (bitter qust ) oil is an example of the
second mentioned category that has different
therapeutic applications in the traditional
medicine of Iran and other countries such as
India and China [12].
Saussurea costus, is one of the main species of
the genus Sausssurea (Asteraceae family) [13].
The reported active ingredients of this well-
known medicinal plant are mainly terpenes, while
different amounts of flavonoids, anthraquinones,
alkaloids, tannins and inulin were reported in the
previous studies from the plant [14-16].
Sesquiterpene lactones, such as custonolide and
dehydrocostus lactone are the major components
of S. costus with several pharmacologic effects
including anti-inflammatory, antiulcer, anti-
cancer and hepatoprotective activities which were
demonstrated in the various experiments [14].
The present study has aimed to prepare
traditional bitter qust oil (TQO) based on
traditional methods of Persian medicine and
determine some active ingredients of the oil to
conduct the oil standardization. Further, chemical
composition of essential oil of the plants root was
analyzed, since relatively non-polar constituents
of the essential oil could trap in the fixed oil
during preparation of TQO. As it has been
mentioned before [1,2,11], other hydrophilic
compounds like phenol, tannins, flavonoids and
polysaccharides may be extracted in traditional
oils, therefore these compounds were detected in
the TQO.
Material and Methods
Plant material
Roots of S. costus were purchased from herbal
market (Tehran, Iran, 2016) and identified at the
Herbarium of Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
with the voucher number of PMP-240.
Chemicals
All chemical substances were of analytical grade
and obtained from Merck Company (Germany).
Preparation of TQO
To prepare TQO, 100 g of plant's root coarse
powder was soaked overnight in 600 mL aqueous
ethanol 25%. The supernatant was then filtered
using whatman filter paper (No.1, Sigma-
Aldrich, Germany) and boiled in 800 g sesame
oil (Omid Company, Iran) until all water was
evaporated [5,6].
Extraction and analysis of essential oils
The oil from the powdered roots of the plant (100
g) and TQO (100 mL) were extracted using
hydro-distillation method in a Clevenger type
apparatus for 4 h at room temperature. The
obtained volatile fractions were separately
collected and dried using sodium sulphate
anhydrous [17].
Gas chromatography-mass spectrometry (GC-
MS)
The volatile compounds were analyzed using an
Agilent Technologies Gas chromatography
device connected to Mass system (Agilent, USA)
with DB-5 fused silica column (30 m×0.25 mm
i.d., film thickness 0.25 µm). The oven
temperature was held at 50 ºC for 5 min and
raised to 280 °C at a rate of 10°C/min. Helium
was used as the carrier gas at a flow rate of 1
mL/min. The injector and detector temperatures
were 280 ºC. Ion source temperature was 150 °C
and scan mass range of m/z was 50-550.The
compounds were identified by comparison of
their mass spectra with the Wiley libraries and
retention indices with those reported in the
literature.
Methanol extraction of the TQO
The methanol extraction of TQO was needed for
further experiments. TQO (10 mL) was mixed
with methanol (10 mL) and after complete
mixing, the methanol phase was separated by
decantation funnel, the procedure was repeated
three times to optimize extraction. The obtained
methanol phase was then centrifuged for 8 min at
Saussurea costus traditional oil
53
4000 rpm (Megafuge 1.0, Germany) to remove
oil droplets and the remained methanol was
removed using rotary evaporator (Heidolph,
Germany). The extraction yield was 1.1% and the
concentration of 1 mg/mL in methanol was used
for analysis.
Quantification of total flavonoids content
Determination of total flavonoids was based on
complex creation by the AlCl3 in a methanol
medium [18]. For the measurement, sodium
nitrate (0.15 mL, 5% in water) and AlCl3 (0.15
mL, 10% in methanol) were added to 1 mL of the
methanol extract of TQO and standard solutions
of catechin (50, 75, 125, 150 µg/mL). Sodium
hydroxide 4% (2 mL) was then added to the
experimental system after 6 min and the volume
reached to 5 mL with distilled water. Eventually,
passing 15 min, the absorbance was measured at
510 nm by UV-visible spectrophotometer
(Camag, Korea).
Quantification of total polysaccharide content
Inulin is a primary storage polysaccharide in the
roots of Asteraceae members and some of
monocotyledons [16]. Saussurea costus is one of
the sources of inulin therefore, polysaccharide
determination is a useful test for the TQO. The
procedure was done according to a previous
study [19]. The methanol extract of TQO (1 mL)
was mixed with phenol 4% (0.5 mL) and sulfuric
acid 96% (2.5 mL) that breaks all the glycoside
linkages. After 5 min, absorbance of colored
aromatic complex that achieved between phenol
and the carbohydrate was measured at 490 nm
using UV-visible spectrophotometer and was
compared to absorbance of dextrose (repeated
unit in the structure of inulin) standard solutions
(0.1, 1, 10, 100 μg/mL).
Quantification of total phenolics and tannins
contents
Determination of total phenols and tannins was
performed using Folin-Ciocalteu reagent [20]. In
the first step, 0.1 mL of the methanol extract of
TQO and gallic acid standard solutions (50-200
µg/mL) were mixed with distilled water (0.4
mL), Folin-Ciocalteu (0.25 mL) and sodium
bicarbonate 20% (1.25 mL); after 40 min the
absorbance of the sample was measured at 725
nm. In the second step, the methanol extract of
TQO (1 mL) was vortexed with powdered
polyvinyl poly pyrrolidone (100 mg), and
distilled water (1 mL). The mixture was
refrigerated for 15 min at 4 °C. Then it was
centrifuged for 10 min at 3000 rpm to precipitate
tannins. The supernatant (0.2 mL) was mixed
with distilled water (0.3 mL), Folin-Ciocalteu
(0.25 mL), sodium bicarbonate (1.25 mL) and the
absorbance of sample was measured at 725 nm
after 40 min. The total tannins content was
estimated with subtracting the first and second
absorption values.
Statistical analysis
All tests were performed in triplicate and the data
were reported as mean±standard deviation (SD).
Results and Discussion
The TQO of the S. costus root was successfully
prepared from aqueous ethanol (25%) extract of
the plant regarding the Persian medicine
instructions. According to the Persian traditional
text, by evaporation of the aqueous phase, the
constituents of the extract would be trapped in
the sesame oil (vehicle) [1]. The prepared TQO
was subjected to essential oil extraction which
yielded 0.1% v/w volatile oil. The essential oil of
S. costus roots was subsequently extracted to
yield bright yellow color oil (0.5% v/w).
The data from GC-MS conduced to the
identification of more than 90% of the oils
chemical compounds. The essential oil of the
root contained thirty-one compounds of which,
ten chemicals were also identified in the TQO
(table 1). The predominant compounds in the root
oil of the plant were dehydrocostus lactone
(17.73%) and 1, 3-cyclooctadiene (16.10%). The
main constituents of the TQO were thymol
(14.44%), 1, 3-cyclooctadiene (14.34%) and
dehydrocostus lactone (6.77%). There were other
compounds identified just in TQO which were
derivatives of fatty acids and probably relevant to
the sesame oil (47.9%). Although, both
investigated oils contained monoterpenes and
sesquiterpenes, the essential oil of bitter qust
root (65.57%) was much more reach of
sesquiterpenes comparing to the TQO (12.94%);
however, percentage of monoterpenes in TQO
(14.88%) was higher than the oil of root plant
(4.28%). The other class of compounds identified
in the oils was hydrocarbons, 20.30% and
15.26% in root oil and TQO, respectively.
Saussurea costus traditional oil
1
Table1. Chemical composition of essential oils of Saussurea costus root and traditional qust oil (TQO)
Identified compounds
KI1
KI2
KIr
% in root oil
% in TQO
Thymol
1256
1249
1290
1.07
14.44
Carvacrol
1274
-
1298
0.70
-
beta-Elemen
1354
1345
1382
5.90
2.79
trans-Caryophyllene
1402
-
1409
4.37
-
alpha-Ionone
1417
1390
1426
1.67
0.44
trans-α-Bergamotene
1425
-
1434
0.53
-
alpha-Humulene
1433
-
1449
0.45
-
Geranylacetone
1447
-
1453
0.54
-
beta-Selinene
1478
1449
1481
3.15
1.81
alpha-Curcumene
1481
-
1485
2.93
-
alpha-Selinene
1482
1459
1494
1.43
0.82
Cetene
1487
1464
-
0.38
0.55
cis-gamma-Bisabolene
1494
-
1515
0.48
-
Elemol
1509
1525
1547
2.70
0.75
gamma-Eudesmol
1595
-
1630
1.01
-
beta-Eudesmol
1622
-
1649
1.14
-
alpha-Eudesmol
1630
-
1652
1.44
-
Elema-1,3,11(13)-trien-12-ol
1648
-
-
11.56
-
7-Tetradecyne
1658
-
-
1.61
-
1,3-Cyclooctadiene
1665
1658
-
16.10
14.34
Cyclododecene, 12-methyl-1-(1-propynyl)
1682
-
-
0.36
-
(3E,5E,8Z)-3,7,11-Trimethyl-1,3,5,8,10-dodecapentanene
1702
-
-
0.17
-
Z-alpha-trans-Bergamotol
1711
-
1693
0.30
-
(+)gamma-Costol
1753
-
-
1.18
-
Valerenol
1775
-
1699
5.28
-
(-)alpha-Costol
1783
-
3.74
-
2(3H)-Benzofuranone
1859
2006
-
1.41
0.37
Germacra-1(10),4,11(13)-trien
1918
-
-
0.21
-
(-)Isodiospyrin
1961
-
-
0.27
-
Dehydrocostus lactone
2028
2051
-
17.73
6.77
Costunolide
-
-
-
0.34
-
Monoterpenes
-
-
-
4.28
14.88
Sesquiterpenes
-
-
-
65.57
12.94
Hydrocarbons
-
-
-
20.30
15.26
Other compounds
-
-
-
-
47.90
Total
90.15
90.98
KI1: Kovats index of essential oil constituents of Saussurea costus root, KI2: Kovats index of volatile
constituent of traditional qust oil, KIr: Kovats index reported in databases
For determination of flavonoids in TQO, the
standard curve was plotted with different
concentrations of catechin (y=0.002x+0.0227,
R2=0.9975) and total flavonoids content of TQO
was calculated 303.2±2.52 mg CE/L.
The polysaccharide content of the TQO was
estimated 9.240±0.13 mg DE/L based on plotting
the standard curve with different concentrations
of dextrose (y=0.0122x+0.0231, R2=0.9994).
Determination of total phenolic and tannin
contents was done in two steps. The standard
curve was plotted with different concentrations of
gallic acid (y=0.0074x-0.0756, R2=0.9795) and
the amount of phenolics and tannins were
calculated as 788.290±0.61 and 23.97±0.52 mg
GAE/L, respectively.
One of the special truths about application of
medicinal plants in treatment of diseases is the
presence of many different active components
with various pharmacological effects that can
demonstrate synergistic effects, decreasing side
effects or other surprising actions. Saussurea
costus is a historical herbs that is well known
from 2500 years ago and has been used in various
ancient systems of medicine [13]. The roots and
root oil of S. costus have been sold out as an
important drug in the international herbal markets
[14]. It has been prescribed as neuroprotective,
anticonvulsant, anti-cancer, anti-ulcer, anti-
arthritic, hepatoprotective, anti-viral, anti-
inflammatory herb and for treatment of cough
and cold in old systems of medicines such as
India, China and Iran [14]. Terpenes, phenols,
flavonoids, tannins and inulin, that are present in
the S. costus root oil, demonstrate some
pharmacological activities such as anti-
54 Res J Pharmacogn 5(2): 51-56
Bagheri S. et al.
Saussurea costus traditional oil
55
inflammatory, analgesic, anti-cancer,
hepatoprotective, anti-ulcer, anti-bacterial and
anti-fungal effects which is conformed to its
traditional usage [15].
The plant has reputation in the treatment of
various diseases related to muscular and neural
organs [7], which are current complications and
challenges in the societies. The presented dosage
form of S. costus for muscular and neural
disorders is in oil form which has been prepared
according to traditional manuscripts [8].
According to the previous studies, the main
components in the essential oil of S. costus root
were sesquiterpenes [21], which were also
identified in TQO and the obtained root oil.
Sesquiterpene lactones are colorless, bitter,
relatively stable and lypophilic constituents that
are common in Asteraceae family but also occur
in some other flowering plants such as
Umbelliferae, Magnoliaceae, Lauraceae,
Winteraceae, Illiciaceae, Aristolochiaceae,
Menispermaceae, Curtiariaceae and Acanthaceae
[22]. These compounds have reputation as
antitumor and anti-inflammatory agents in vitro
and in vivo [14,23]. Dehydrocastus lactone and
costunolide are two sesquiterpenes introduced in
other studies as major active constituents of S.
costus essential oil [13,24]. Anti-tumor, anti-
inflammatory, imunomodulatory,
hepatoprotective, anti-ulcer and anti-viral
activities have been confirmed for dehydrocastus
lactone and costunolide [13,14].
A portion of essential oil and hydrophobic
constituents of S. costus root were imported in
the prepared oil, certainly. The hydrophilic
compounds consistent to the previous reports
[2,10,11] were also trapped in the sesame oil
through the Persian medicine procedure for
preparation of TQO, because in this process, the
aqueous extract of the plant enters in an oily
vehicle and the water is then eliminatedg from
the system. The hydrophilic and hydrophobic
contents of TQO were successfully determined in
the present experiment. The qualitative tests on
the traditional oil aren't practicable and
repeatable; so a methanol extract of the oil was
prepared. We suggested that dehydrocostus
lactone could be a suitable option for
identification and qualification of the bitter qust
preparations such as TQO. Although its amount
is little in the prepared oil, specification of this
chemical constituent in Asteraceae family and S.
costus and its important pharmacological
activities are distinguishing options for the
selection.
Acknowledgments
This study was supported by Tehran University
of Medical Sciences, Tehran, Iran.
Author contributions
Sahar Bagheri, Nastaran Ebadi, Zahra Taghipour
and Azadeh Manayi contributed to the design and
implementation of the research to the analysis of
the results and to the writing of the manuscript.
Tayebeh Toliyat and Mehran Mirabzadeh
Ardakani supervised the project.
Declaration of interest
The authors declare that there is no conflict of
interest. The authors alone are responsible for the
content of the paper.
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Abbreviations
PM: Persian medicine; TQO: Traditional bitter
qust oil; GAE: Gallic acid equivalent; CE:
Catechin equivalent; DE: Dextrose equivalen
