Efficiently prepared ephedrine alkaloids-free Ephedra Herb extract: a putative marker and antiproliferative effects

Article abstract of DOI:10.1007/s11418-016-0977-1

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (麻黄, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104?%. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.

Full text of DOI:10.1007/s11418-016-0977-1

J Nat Med
DOI 10.1007/s11418-016-0977-1
ORIGINAL PAPER
Efficiently prepared ephedrine alkaloids-free Ephedra Herb
extract: a putative marker and antiproliferative effects
Naohiro Oshima1,2 Tadatoshi Yamashita3 Sumiko Hyuga4 Masashi Hyuga1
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Hiroyuki Kamakura1 Morio Yoshimura5 Takuro Maruyama1
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Takashi Hakamatsuka1 Yoshiaki Amakura5 Toshihiko Hanawa4
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Yukihiro Goda1
Received: 30 October 2015 / Accepted: 7 February 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Ephedrine alkaloids (EAs) have been considered
the main pharmacologically active substances in Ephedra
Herb (麻黄, Mao; EH) since they were first identified by Prof.
N. Nagai, and are known to induce palpitation, hypertension,
insomnia, and dysuria as side effects. Therefore, the admin-
istration of drugs containing EH to patients with
cardiovascular-related diseases is severely contraindicated.
While our previous studies suggest that some of the effects of
EH may not be due to EAs, considering their side effects
would be expedient to develop a new EAs-free EH extract
(EFE). Here, weestablisheda preparationmethodforEFEand
revealed its chemical composition, including the content of
herbacetin, a flavonoid aglycon present in EH and a potential
putative marker for EFE quality control. In addition, we
showed the antiproliferative effects of EFE against the H1975
non-small cell lung cancer (NSCLC) cell line. EFE was
preparedfromEHextractusingtheionexchangeresinSK-1B.
LC/Orbitrap MS analysis revealed the removal of EAs,
6-methoxykynurenic acid, and 6-hydroxykynurenic acidfrom
the original extract. Quantitative analysis of herbacetin using
LC/MS in acid-hydrolyzed EFE showed that its content was
0.104 %. Although several alkaloidal constituents were
removed from EH extract, the antiproliferative effect of EFE
against H1975 cells was comparable to that of EH extract.
These results indicate that EFE retained the anticancer effect
of EH and demonstrated its potential for future development
as a new herbal medicine with reduced side effects.
Keywords Ephedra Herb · Ephedrine alkaloids-free
Ephedra Herb extract · Chemical composition · Herbacetin
· Antiproliferative effect
Abbreviations
EH
EFE Ephedrine alkaloids-free Ephedra Herb extract
EA Ephedrine alkaloid
Ephedra Herb
Electronic supplementary material The online version of this
article (doi:10.1007/s11418-016-0977-1) contains supplementary
material, which is available to authorized users.
& Yukihiro Goda
goda@nihs.go.jp
1
National Institute of Health Sciences, 1-18-1 Kamiyoga,
Setagaya-ku, Tokyo 158-8501, Japan
Introduction
2
Present Address: Department of Pharmaceutical Sciences,
International University of Health and Welfare, 2600-1,
Kitakanemaru, Ohtawara city, Tochigi 324-8501, Japan
Ephedra Herb (EH) is officially defined as the terrestrial
stem of Ephedra sinica Stapf, Ephedra intermedia Schrenk
et C. A. Meyer, or Ephedra equisetina Bunge (Ephe-
draceae) in the Japanese Pharmacopoeia 16th edition
(JP16) [1]. EH is a component of Kampo (Japanese tradi-
tional herbal medicine) formulae for the treatment of
headaches, bronchial asthma, nasal inflammation, and the
common cold, and is reported to have anti-inflammatory
[2], antitussive [3], and anti-influenza activities [4].
3
TOKIWA Phytochemical Co., Ltd., 158 Kinoko, Sakura-shi,
Chiba 285-0801, Japan
4
Department of Clinical Research, Oriental Medicine
Research Center of Kitasato University, 5-9-1 Shirokane,
Minato-ku, Tokyo 108-8642, Japan
5
Department of Pharmacognosy, College of Pharmaceutical
Sciences, Matsuyama University, 4-2 Bunkyo-cho,
Matsuyama, Ehime 790-8578, Japan
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Ephedrine alkaloids (EAs) were isolated as principal
ingredients in EH by Prof. Nagayoshi Nagai in 1885 [5].
Miura [6] showed that ephedrine has mydriatic action in
the rabbits. Then, Amatsu and Kubota [7] reported that
ephedrine raised the blood pressure by contraction of the
peripheral vessels following intravenous (i.v.) injection in
dogs. Chen and Schmidt [8] found that ephedrine showed
circulatory stimulatory effects when it was orally admin-
istered. Furthermore, MacDermot [9] revealed that the
injection of ephedrine into patients with bronchial asthma
showed beneficial effects. EAs have considerable phar-
macological activities, and are believed to be the principal
active ingredients in EH. The content of EAs in EH are
regulated in the JP16. However, EAs are known to induce
palpitation, hypertension, insomnia, and dysuria as major
side effects. Therefore, the administration of EAs-con-
taining drugs to patients with cardiovascular-related
diseases is severely contraindicated.
authentic EAs used were: ephedrine, purchased from
Dainippon Pharma Co., Ltd.; methylephedrine and pseu-
doephedrine, from Alps Pharmaceutical Ind., Co., Ltd.; and
norephedrine from Tokyo Chemical Industry Co. Ltd.
6-Methoxykynurenic acid was purchased from Chemicia
Scientific, LLC. trans-Cinnamic acid was purchased from
Wako Co., while herbacetin 7-O-neohesperidoside and
herbacetin 7-O-glucoside were isolated from EH [15].
Herbacetin and apigenin were purchased from ChromaDex
Co. and Wako Co., respectively.
General procedures
Unless otherwise noted, the following instruments and
1
conditions were used. The H-NMR, 13C-NMR, and 2D-
NMR spectra were recorded using an ECA-800 or ECA-600
spectrometer (JEOL), and chemical shifts were expressed in
δ (ppm) with tetramethylsilane (TMS) as the reference
standard. The LC/Orbitrap MS analysis was performed using
an LC-20A UFLC system (Shimadzu) equipped with the
LTQ Orbitrap XL mass spectrometer (Thermo Fisher Sci-
entific). The UPLC/MS analysis was performed using a
Xevo TQD UPLC/MS system (Waters). The LC/MS analy-
sis was performed using an LC-20A UFLC system equipped
with an LCMS-2020 mass spectrometer (Shimadzu).
Previously, we found that EH extract impaired hepatocyte
growth factor (HGF)-induced cancer cell motility, likely by
suppressing the HGF-c-Met signaling pathway [10], since
dysregulation of this pathway promotes tumor formation,
growth, progression, metastasis, and therapeutic resistance
[11, 12]. Therefore, EH may have applications in cancer
therapy as a novel c-Met inhibitor. Recently, we revealed
that herbacetin, a flavonoid aglycon in EH, inhibited HGF/c-
Met/Akt signal and HGF-induced motility of human MDA-
MB-231 breast cancer cells [13]. In addition, we found that
herbacetin had analgesic effects in the formalin test [14].
These results indicate that some of the pharmacological
effects of EH may not be due to EAs and, therefore, the
prospect of preparing an EAs-free EH extract (EFE) as a new
and potentially safer natural medicine without the side
effects associated with EAs appealed to us.
Selection of optimal cation exchange resin for EFE
preparation
Sample preparation
EH (10 g) was extracted with hot water (100 ml) for 1 h at
95 °C. After filtration, the extract volume was adjusted to
100 ml with water and 2 ml was exposed to each cation
exchange resin (2 ml) shown in Table 1, stirred for 10 s,
and then the solution was left to stand for 1 h. The
supernatant (0.5 ml) volume was adjusted to 5 ml with
MeOH and subsequently used as the sample solution for
the HPLC analysis.
Therefore, to achieve the aim of this present study,
which was the production of a clinically useful EH extract
with none of the side effects associated with EAs, we
developed an efficient method for preparing EFE from EH
extract. Furthermore, we clarified the chemical composi-
tion of the EFE and analyzed the herbacetin content as a
candidate marker using LC–MS because EFE contains no
EAs, which are markers for the quantitative assay of EH
stipulated by the JP16. In addition, we examined its
antiproliferative effects against the H1975 non-small cell
lung cancer (NSCLC) cell line.
Quantitative analyses of ephedrine alkaloids
The HPLC analysis was performed using the HITACHI
HPLC system (pump, L-2130; degasser, L-2130; auto-
sampler, L-2200; column oven, L-2300; and diode array
detector, L-2450). The analytical conditions were as fol-
lows [1]: column, SHISEIDO AG 120 (4.6 mm i.
d. 9 150 mm; 5 μm, Shiseido, Tokyo); mobile phase,
sodium lauryl sulfate (5 g) dissolved in acetonitrile
(MeCN, 350 ml), followed by the addition of water
(650 ml) and phosphoric acid (1 ml); flow rate, 0.8 ml/min;
column oven temperature, 45 °C; injection volume, 10 μl;
and monitoring wavelength, 210 nm.
Materials and methods
Materials and reagents
EH (JP16 grade) originally produced from E. sinica was
purchased from Uchida Wakanyaku Co., Ltd. The
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Analysis of LC/Orbitrap MS
Table 1 Cation exchange resins
Resins
Class
Manufacturers
The LC/Orbitrap MS analytical conditions were as follows:
column, Inertsil ODS-3 (2.1 mm i.d. 9 150 mm, 5 μm; GL
Sciences); mobile phase, 0.1 % formic acid (HCOOH) in
water (A)–0.1 % HCOOH in MeOH (B) in a gradient mode
of 5 % B (0–10 min) → 75 % B (70 min) → 100 % B
A
B
C
D
E
F
WK10
Weak
Weak
Weak
Weak
Strong
Strong
Strong
Strong
Strong
Strong
Strong
Strong
Strong
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Mitsubishi Chemical Co., Japan
Organo Co., Japan
WK11
WK20
WK40L
SK104
SK110
SK1B
(80 min)
→ 100 % B (90 min) → 5 % B
(90.01 min) → 5 % B (95 min); injection volume, 1 μl;
flow rate, 0.2 ml/min; column oven temperature, 40 °C;
and photodiode array (PDA) (200–400 nm). Furthermore,
the MS conditions were: interface, electrospray ionization
(ESI) positive/negative; source voltage, 4.0 kV; capillary
voltage, 10 V; source temperature, 300 °C; sheath and
auxiliary gas flow rates, 50 and 25, respectively; scan
range, m/z 50–2000; and mass resolution, 30,000 full
width.
G
H
I
UBK530
UBK12
PK216
IR120B
FPC3500
1060H
J
K
L
M
Organo Co., Japan
Organo Co., Japan
Preparation of EFE
Analysis of UPLC/MS
EH (10 kg) was added to water (100 l), extracted at 95 °C
for 1 h, and the extract was filtered through the SK-1B ion
exchange resin (10 l, Mitsubishi Chemical Co.), which was
treated with 1 M HCl (30 l) and water (100 l) prior to use,
at a flow rate of 0.5 l/min, and then the resin was washed
with water (10 l). The unadsorbed fraction (110 l) was
adjusted to pH 5 with 5 % aqueous sodium bicarbonate
(NaHCO3aq., 6 l). The solution was evaporated under
reduced pressure to obtain EFE (1.2 kg, yield 12.0 %).
The UPLC analytical conditions were: column, Inertsil
ODS-3 (2.1 mm i.d. 9 150 mm, 5 μm; GL Sciences);
mobile phase, 0.1 % HCOOH in water (A)–0.1 % HCOOH
in MeOH (B) in a gradient mode of 5 % B (0 min) → 50 %
B (40 min) → 100 % B (50 min) → 100 % B
(55 min) → 5 % B (55.1 min) → 5 % B (60 min); injection
volume, 1 μl; flow rate, 0.2 ml/min; and PDA (200–
400 nm). In addition, the MS conditions were: interface,
ESI positive/negative; capillary voltage, 4.5 kV; source and
desolvation temperatures, 150 and 400 °C, respectively;
desolvation gas flow, 800 l/h; cone voltage, 50 V; cone gas
flow, 50 l/h; and scan range, m/z 100–1200.
Analysis of the chemical composition of EFE
Preparation of sample solution
Synthesis of 6-hydroxykynurenic acid (2)
EH (200 g) was added to water (2000 ml), extracted at 95 °C
for 1 h, filtered, and then the residue was washed with water
(200 ml). The extract was centrifuged at 1800 9 g for 10 min,
and then half of the supernatant was concentrated under
reduced pressure to obtain the EH extract (14.1 g), while the
other half was passed through the SK-1B ion exchange resin
(100 ml), which was treated with 1 M HCl (30 ml) and water
(100 ml) prior to use, and then the resin was washed with
water (100 ml). The unadsorbed fraction (1100 ml) was
adjusted to pH 5 using 5 % NaHCO3aq. (60 ml), and then the
solution was evaporated under reduced pressure to obtain
EFE (11.8 g). Each extract batch was adjusted to a concen-
tration of 5 mg/ml with 50 % aqueous MeOH (MeOHaq.),
filtered using a 0.45-μm filter, and then subsequently used as
the sample solutions.
6-Methoxykynurenic acid (48.4 mg) was dissolved in
ethylene glycol (5 ml) and potassium hydroxide (KOH,
1 g) was added, followed by refluxing for 4.5 h. The
reaction mixture was carefully neutralized with HCl under
cooling conditions, separated using LH-20, and preparative
TLC was performed to obtain 2 (10.7 mg).
1
6-Hydroxykynurenic acid (2): White crystal, H-NMR
(800 MHz, DMSO-d6): δH 6.35 (1H, s, H-3), 7.31 (1H, d,
J = 3.2 Hz, H-5), 7.05 (1H, dd, J = 8.8, 3.2 Hz, H-7), 7.77
(1H, d, J = 8.8 Hz, H-8), 9.73 (1H, br s), 11.2 (1H, s). 13C-
NMR (200 MHz, DMSO-d6): δC 146.9 (C-2), 106.5 (C-3),
163.7 (C-4), 127.4 (C-4a), 107.5 (C-5), 153.8 (C-6), 122.1
(C-7), 121.3 (C-8), 123.5 (C-8a), 166.8 (COOH). ESI-MS:
m/z 206 [M+H]+.
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Quantitative analysis of herbacetin 7-O-
neohesperidoside
graded concentrations of 0.018, 0.045, 0.09, 0.18, 0.45, 0.9,
and 1.8 μg/ml. Furthermore, apigenin was used as the
internal standard at a final concentration of 100 µg/ml.
Calibration curve
Quantitative analysis
The standard stock solution was prepared by accurately
weighing an adequate amount of herbacetin 7-O-neohes-
peridoside and dissolving it in 50 % MeOH. The working
standard solutions were prepared by diluting the stock
solution with 50 % MeOH to give six graded concentra-
tions of 0.1, 0.5, 1.0, 5.0, and 50.0 µg/ml. Each standard
solution was analyzed in sextuplicate, and the regression
equation was calculated in the form y = Ax + B.
The chromatographic analytical conditions were: column,
Xbridge C18 (2.1 mm i.d. 9 100 mm, 3.5 μm; Waters);
mobile phase, 0.1 % HCOOH in water (A)–MeCN (B) in a
gradient mode of
0 % B (0 min) → 40% B
(50 min) → 100 % B (60 min) → 100 % B (70 min) → 0 %
B (70.01 min) → 0 % B (75 min); injection volume, 1 μl;
and flow rate, 0.2 ml/min. The MS analytical conditions
were: interface, ESI negative; nebulizer and drying gas
flow, 1.5 and 10 l/min, respectively; CDL and heat block
temperature, 250 and 200 °C, respectively; detector and
interface voltage, 120 and 4.5/−4.5 kV, respectively;
interface current, 0.6 μA; and MS range, m/z 301 (SIM).
The EFE sample was analyzed in sextuplicate.
Sample preparation
The EFE sample for the LC/MS analysis was prepared by
adjusting the concentration to 1 mg/ml with 50 % MeOH
and filtering with a 0.45-μm filter.
Quantitative analysis
Antiproliferative effect
The chromatographic analytical conditions were: column,
Inertsil ODS-3 (150 mm 9 2.1 mm i.d., 5 μm; GL Sciences);
mobile phase, 0.1 % HCOOH in water (A)–MeCN (B) in a
gradient mode of 0 % B (0 min) → 40 % B (50 min) → 100 %
B (60 min) → 100 % B (70 min) → 0 % B (70.01 min) → 0 %
B (75 min); injection volume, 1 μl; and flow rate, 0.2 ml/min.
The MS conditions were: interface, ESI negative; nebulizer
gas flow, 1.5 l/min; drying gas flow, 10 l/min; curved des-
olvation line (CDL) and heat block temperature, 250 and
200 °C, respectively; detector and interface voltage, 120 and
4.5/−4.5 kV, respectively; interface current, 0.6 μA; and MS
range, m/z 609 (selective ion monitoring, SIM). The EFE
sample was analyzed in sextuplicate.
The H1975 NSCLC cell line was obtained from the
American Type Culture Collection (ATCC, Manassas, VA,
USA). The cells were suspended at a density of 2 9 103
cells in 100 μl Roswell Park Memorial Institute (RPMI)
1640 medium (Invitrogen Co.) containing 10 % fetal calf
serum (FCS, Sigma-Aldrich), with or without 50–200 μg/
ml of EH extract or EFE in each well of a 96-well plate and
incubated at 37 °C for 72 h. To each well was added 10 μl
of Cell Counting Kit-8 solution (Dojindo Co.), and after a
2-h incubation at 37 °C, the absorbance of the formazan
generated in each well was measured at 450 nm using an
iMark plate reader (Bio-Rad Laboratories, Inc.). The IC50
value was calculated using a four-parameter logistic model
(Prism 5.0, GraphPad software).
Quantitative analysis of herbacetin
Acid hydrolysis of EFE
Results and discussion
HCl (6 M, 5.0 ml) was correctly added to EFE (5.0 mg) and
the reaction mixture was heated at 70 °C for 6 h, followed
by separation on a CHP 20P column (ϕ 2 9 20 cm). After
washing with water, the MeOH eluted fraction was evap-
orated under reduced pressure, the residue was dissolved in
MeOH, and the volume was adjusted to 5.0 ml. Then,
4.5 ml was added to 0.5 ml of apigenin solution (1 mg/ml,
MeOH), followed by filtration with a 0.45-μm filter.
We used cation exchange resins to remove EAs from the EH
extract and efficiently prepared the EFE. The prototype EFEs
were formulated using the 13 cation exchange resins shown
in Table 1, and the residual EAs in each prototype were
quantitatively determined. The results revealed that the
residual ratios of EAs in the resins C-, F–K-, and M-treated
prototypes were less than 1 % (Fig. 1) and, therefore, we
focused on resin G (SK1B) considering cost efficiency, in
preparing the EFE for practical clinical use. By using resin G,
the yield of the EFE prepared for subsequent experiments
from the EH extract was high (1.2 kg; y. 12.0 % from EH).
To determine the chemical composition of the prepared
EFE, we analyzed the EH extract and EFE using LC/
Calibration curve
The calibration curve for herbacetin was constructed sim-
ilarly to that for herbacetin 7-O-neohesperidoside, with
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Fig. 1 Residual ratio of
ephedrine alkaloids (EAs)
exposed to each cation
exchange resin (%). Amounts of
ephedrine and pseudoephedrine
in Ephedra Herb (EH)
corresponding to 100 % of the
vertical axis were 74.0 and
22.1 mg, respectively
Orbitrap MS (Fig. 2). The chromatograms of both were
very similar, but the peaks around retention times 6–10 min
and peaks 1 and 2 of the EH extract disappeared from that
of EFE, while the height of peak 3 was slightly decreased.
This observation was also supported by the LC/Orbitrap
MS analysis of the adsorbed fraction on the cation
exchange resin: the peaks mentioned above were detected
in TIC or PDA (254 nm) chromatograms (Fig. 1S). The
peaks that disappeared around retention times 6–10 min
were identified as ephedrine/pseudoephedrine, methyle-
identified peak 2 as 6-hydroxykynurenic acid (2). Since
peak 3 was hard to identify based on MS data, we separated
the EH extract to identify it. Briefly, EH (100 g) was
extracted with hot water (1 l) for 1.5 h. The extract (13.6 g)
was fractionated by HP-20, CHP-20P, and silica gel col-
umn chromatography to get the fraction rich in peak 3. The
NMR spectral signals of the fraction were observed at δH
7.75 (1H, d, J = 16.0 Hz), δH 6.45 (1H, d, J = 16.0 Hz),
indicating a trans-double bond; δH 7.55 (2H, m), δH 7.40
(3H, m), indicating monosubstituted benzene; and δC
169.5, indicating carbonyl carbon of carboxylic acid, which
speculated that the compound was trans-cinnamic acid (3).
The UPLC/MS analysis of the authentic standard trans-
cinnamic acid sample gave a peak with the same retention
time and MS as those of peak 3 (Fig. 4S). Therefore, we
identified peak 3 as trans-cinnamic acid (3, Fig. 4).
For the prepared EFE to satisfy quality standards for
clinical use, a new stipulated quality control marker
compound was required because EAs, the previously
defined marker compounds for the quantitative assay of
EH, are not present in EFE. Thus, we focused on herba-
cetin 7-O-neohesperidoside that was previously isolated
from EH [15] and its aglycon, herbacetin, which have
anticancer effects [13], and quantitatively determined
their contents in EFE. First, we quantitatively analyzed
herbacetin 7-O-neohesperidoside using the absolute cali-
bration method. The quasi-molecular ion, m/z 609 [M–
H]−, was detected using the SIM mode to obtain good
linearity, accuracy, and precision in the concentration
range of 1–50 μg/mL (Table 2). Subsequently, we ana-
lyzed herbacetin 7-O-neohesperidoside in EFE in
sextuplicate using this condition (Fig. 5SA). The result
revealed that the herbacetin 7-O-neohesperidoside content
in EFE was 0.094 0.009 %.
phedrine/pseudomethylephedrine,
and
norephedrine/
pseudonorephedrine by comparing their quasi-molecular
ion peaks (m/z 166.12, 180.14, and 152.11) and retention
times with those of authentic standard compounds (Fig. 3).
Next, we examined the chromatograms to identify peaks 1–
3. Previously, Starratt and Caveney [16] isolated 6-meth-
oxykynurenic acid (1) and 6-hydroxykynurenic acid (2)
from the cation exchange resin-treated fraction of Ephedra
pachyclada subsp. sinica. The accurate determination of
the masses of peaks 1 and 2 using Orbitrap MS analysis
suggest that their molecular formulae were C11H9O4N and
C10H7O4N, respectively. Therefore, we speculated that
these peaks were those of 1 and 2. Furthermore, the UPLC/
MS analysis of the authentic sample of 1 showed a reten-
tion time and MS consistent with peak 1 (Fig. 2S).
Therefore, we identified peak 1 as 6-methoxykynurenic
acid (1, Fig. 4). Next, to identify peak 2, we synthesized 2
from the authentic sample of 1 using Kuo et al.’s method
[17]. The product obtained was isolated and analyzed using
NMR. The signal at δH 4.00 attributed to the 6-methoxy
group in 1 had disappeared from the NMR spectrum of 2,
indicating that it was a demethylated form of 1. The
retention time and MS of 2 in the UPLC/MS analysis were
the same as those of peak 2 (Fig. 3S). Therefore, we
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J Nat Med
Fig. 2 LC/Orbitrap MS
analyses of a Ephedra Herb
(EH) extract and b ephedrine
alkaloids-free EH extract
(EFE): a photodiode array
(PDA, 254 nm) and b total ion
chromatogram (TIC). Peak 1
6-methoxykynurenic acid; peak
2 6-hydroxykynurenic acid;
peak 3 trans-cinnamic acid
Fig. 3 Extracted ion chromatograms of a Ephedra Herb (EH) extract
and b ephedrine alkaloids-free EH extract (EFE) samples. m/z 166.12
[M+H]+, dl-ephedrine/dl-pseudoephedrine; m/z 180.14 [M+H]+, dl-
methylephedrine/dl-pseudomethylephedrine; m/z 152.12 [M+H]+, dl-
norephedrine/dl-pseudonorephedrine
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J Nat Med
prevented the proliferation of H1975 cells concentration-
dependently (Fig. 5). The IC50 values of the EH extract
and EFE were 88 and 76 μg/ml, respectively. These
results revealed that the biological activity of EFE was
retained and, therefore, the constituents that were
removed during the manufacturing process did not affect
its antiproliferative effect. Previously, we reported that
EH impedes the HGF-induced motility of cancer cells by
the inhibiting c-Met tyrosine kinase activity [10]. In
addition, Tang et al. [18] reported that SU11274, a
c-Met inhibitor, induced apoptosis of H1975 cells and
inhibited their tumorigenesis in vivo. These reports
suggest that EFE, as well as EH extract, suppressed the
growth of H1975 cells through the inhibition of c-Met.
Interestingly, EFE inhibited HGF-induced phosphoryla-
tion of c-Met and c-Met tyrosine kinase activity [19].
Therefore, EFE is expected to become a useful carci-
nostatic agent against cancer cells expressing c-Met.
Furthermore, we have recently clarified that EFE shows
analgesic and anti-influenza activity [19], suggesting that
EFE has considerable therapeutic effects.
Fig. 4 Structures of compounds 1, 6-methoxykynurenic acid; 2,
6-hydroxykynurenic acid; 3, trans-cinnamic acid; 4, herbacetin
Then, the total amount of herbacetin contained in the
acid-hydrolyzed EFE was quantitatively determined using
the internal reference method with apigenin (m/z 269 [M–
H]−) as an internal standard. The quasi-molecular ion, m/
z 301 [M–H]−, was detected using SIM to obtain good
linearity, accuracy, and precision in the concentration
range of 0.018–1.8 μg/ml (Fig. 5SB, Table 2). HCl (6 M)
was added to the EFE sample, which was then stirred at
70 °C until the peaks corresponding to herbacetin 7-O-
neohesperidoside and herbacetin 7-O-glucoside, which we
previously isolated from EH, disappeared [14] in the LC/
MS. Then, we analyzed the herbacetin content of the HCl-
It has been reported that therapeutic regimens using
Kampo medicines reduce the drug cost for patients com-
pared with therapy using modern medicines [20–22].
Therefore, the relatively low cost of Kampo prescriptions is
expected to have a considerable positive economic impact
on cancer therapy, which frequently involves the use of
expensive patented medicines. In the present study, EFE
showed antiproliferative effects against cancer cells.
Therefore, the clinical use of EFE derived from EH could
not only expand the application range of EH, but could also
contribute to reducing or, at least, limiting the use of
expense anticancer therapies. Although EFE originated
from EH, it is now a distinct medicine from EH. Therefore,
drug approval for the clinical use of EFE as a new material
will be required under the present drug regulation guide-
lines of Japan. However, no newly produced compounds
were detected following the process used for EFE pro-
duction from EH extract, which has been widely prescribed
hydrolyzed EFE and found that it was 0.104
0.002 %.
These data showed that the sum of the herbacetin glyco-
sides present in EFE was about 0.1 %, which appeared to
be adequate for quantitative evaluation using HPLC. Her-
bacetin is a c-Met inhibitor found in EFE and is
commercially available. These results suggest that it may
be a suitable quality control marker compound for EFE.
Finally, to confirm whether the pharmacological effect
was retained in the prepared EFE, we examined the
effect of the EH extract and EFE on the growth of
H1975 cells, and the result showed that they both
Table 2 Quantitative analysis of herbacetin 7-O-neohesperidoside and herbacetin
Compound
Monitoring
ion (m/z)
Retention
time (min)
Linear range Regression equation
(μg/ml)
R2
Concentration Accuracy Precision
(%)
(μg/ml)
(%)
Herbacetin 7-O-
neohesperidoside
609
301
31
30
1–50
y = 15413x + 3746
0.9997
1
−4.28
15.7
5
−22.6
−7.22
−1.20
2.84
16.2
9.87
1.22
8.21
2.64
50
Herbacetin
0.018–1.8
y = 0.4416x − 0.0122 0.9999 0.018
0.18
1.8
8.43
123

J Nat Med
agent against c-Met-expressing cancer cells without the
adverse effects associated with EAs.
Compliance with ethical standards
Funding This research is partially supported by the Research on
Development of New Drugs project of the Japan Agency for Medical
Research and Development (AMED).
Conflict of interest We have applied for a patent under the regu-
lations of the Patent Cooperation Treaty (PCT).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
Fig. 5 Ephedra Herb (EH) extract and ephedrine alkaloids-free EH
extract (EFE) prevented proliferation of the H1975 non-small cell
lung cancer (NSCLC) cell line
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