application of the 1h-qnmr method to quality assessments
TRANSCRIPT
Application of the 1H-qNMR method to quality assessments of herbal drugs, rhizomes of Acorus calamus and A. gramineus and the effects of their constituents on in vitro acetylcholinesterase activity
March 2019
Ph.D. Thesis Submitted
by
Nwe Haymar Lynn
OKAYAMA UNIVERSITY
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
(Doctor’s course)
1
Contents
Abbreviations ----------------------------------------------------------------------------- 5
List of figures and tables ----------------------------------------------------------------- 9
Chapter 1: Introduction --------------------------------------------------------------- 10
1.1. Overview of qNMR method ----------------------------------------------------- 10
1.1.1. Fundamental principle and development history
of qNMR method ---------------------------------------------------------- 11
1.1.2. Experimental parameters of qNMR ------------------------------------- 11
1.1.2.1. Pulse angle (excitation pulse) and pulse width value (pw) - 12
1.1.2.2. Acquisition time (Aq) ------------------------------------------- 12
1.1.2.3. Relaxation time (T1 and T2) ------------------------------------ 13
1.1.2.4. Relaxation delay (D1) ------------------------------------------- 13
1.2. Overview of Acorus species ------------------------------------------------------ 14
1.2.1. Traditional uses and pharmacological profiles of Acorus species --- 15
1.2.2. Chemical constituents of Acorus species ------------------------------- 16
1.2.3. β-asarone contents and ploidy levels ------------------------------------ 17
1.3. Objectives of the research -------------------------------------------------------- 18
Chapter 2: Application of the 1H-qNMR method on Acorus species --------- 19
2.1. Optimization of 1H-qNMR method ---------------------------------------------- 19
2.1.1. Selection of deuterated solvent based on the solubility of analyte -- 19
2.1.2. Selection of the internal standard ---------------------------------------- 22 2.1.3. Selection of important acquisition parameters for
the qNMR method --------------------------------------------------------- 23
2.1.3.1. Sample preparation for analyses of experimental
parameters -------------------------------------------------------- 24
2.1.3.2. Pulse angle and pulse width calibration (pw 90˚) ------------ 25
2
2.1.3.3. Inversion recovery analysis (T1) ------------------------------ 28
2.1.3.4. Relaxation delay (D1) ------------------------------------------- 29
2.1.3.5. Spectral width (SW) and acquisition time (Aq) ------------- 29
2.1.3.6. Number of scans (ns) ------------------------------------------- 30
2.1.3.7. Acquisition temperature ---------------------------------------- 30
2.2. Preparation of principal constituents from Acorus rhizomes ------------------ 31
2.2.1. Extraction and isolation of principal constituents --------------------- 31
2.2.2. Identification of known compounds ------------------------------------ 34
2.3. Validation of qNMR method ----------------------------------------------------- 35
2.3.1. Linearity, accuracy and precision analyses of β-asarone by qNMR -- 37
2.3.2. Limit of quantitation (LOQ) and limit of detection (LOD)
of qNMR method ---------------------------------------------------------- 38
2.4. Quantitative analyses by optimized qNMR method --------------------------- 39
2.4.1. Preparation of crude extracts --------------------------------------------- 40
2.4.2. Sample preparation for qNMR analyses ------------------------------- 42
2.4.3. Purity and stability analyses of isolated main asarones by qNMR -- 45
2.4.4. Content determinations of crude extracts by qNMR ------------------ 51
2.5. Comparison between qNMR and HPLC ---------------------------------------- 52
2.5.1. Quantitative analyses by HPLC-DAD ---------------------------------- 54
2.5.2. Comparative quantification of β-asarone (1) and α-asarone (2)
by HPLC and qNMR method ------------------------------------------- 58
Chapter 3: In vitro acetylcholinesterase activity assay -------------------------- 61
3.1. AChE inhibitory effects of crude extracts prepared from Acorus species --- 62
3.2. Correlation between the inhibitory effects of crude extracts
and β-asarone (1) contents in crude extracts ----------------------------------- 63
3.3. Contribution of the principal constituents on AChE inhibition activity---- 65
3
3.4. IC50 values of the main constituent, β-asarone and Acorus crude extract B-- 67
3.5. Concentration-dependence in the effects of eudesmin A (6) on the
in vitro AChE activity ------------------------------------------------------------ 69
Chapter 4: Experimental section ----------------------------------------------------- 73
4.1. Instruments ------------------------------------------------------------------------ 75
4.2. Plant materials -------------------------------------------------------------------- 76
4.3. Extraction and fractionation of Acorus species ------------------------------- 78
4.4. Physical properties of compounds from Acorus species -------------------- 85
4.5. Component analyses ------------------------------------------------------------- 86
4.5.1. Purity and content determinations by qNMR analyses --------------- 87
4.5.2. HPLC analyses ------------------------------------------------------------- 90
4.6. Bioactivity test (Acetylcholinesterase inhibition assay) ---------------------- 92
4.7. Statistical analysis ---------------------------------------------------------------- 92
References ------------------------------------------------------------------------------- 100
Paper and conference presentations -------------------------------------------------- 101
Acknowledgment --------------------------------------------------------------------- 102
4
Abbreviations
Abbreviations Full Name
A. calamus Acorus calamus
A. gramineus Acorus gramineus
ACh Acetylcholine
AChE Acetylcholinesterase
AD Alzheimer’s disease
ATCI Acetylthiocholine iodide
CC Column chromatography
CD Circular dichroism
CDCl3 Chloroform-d
CHCl3 Chloroform
DMSO Dimethyl sulphoxide
DMT Dimethyl terephthalate
DTNB 5,5’-Dithiobis (2-nitrobenzoic acid)
EtOAc Ethyl acetate
Ext. Extract
Fr. Fraction
GC Gas chromatography 1H-qNMR Proton specific quantitative NMR
HPLC High-performance liquid chromatography
I.S. Internal standard
LOD Limit of detection
LOQ Limit of quantitation
MEKC Micellar electrokinetic chromatography
MeOH Methanol
5
MOPS 3-(N-morpholino)propanesulfonic acid
ESI-MS Electrospray-ionization mass spectrometry
NMR Nuclear magnetic resonance
Prep. Preparative
SD Standard deviation
S/N Signal-to-noise ratio
TLC Thin layer chromatography
TNB Thiobis-2-nitrobenzoic acid
UV Ultraviolet
6
List of figures and tables
List of figures
Figure 1-1. The nuclear spin magnetization (orange arrow) before
and after application of a pulse ----------------------------------- 12
Figure 1-2. Relaxation mechanism of nuclear spin after 90˚ pulse
(T2-blue arrow, T1-red arrow) ----------------------------------- 13
Figure 1-3. Schematic diagram of a single pulse sequence of nuclear
spin magnetization ------------------------------------------------- 13
Figure 1-4. Acorus calamus plant ---------------------------------------------- 14
Figure 1-5. Chemical structures of reported compounds from
Acorus species ----------------------------------------------------- 16
Figure 2-1. 1H NMR spectrum of crude extract from A. calamus --------- 20
Figure 2-2. 1H NMR spectrum of dimethyl terephthalate (I.S.) ------------ 21
Figure 2-3. 1H NMR spectrum of crude extract with I.S. ------------------- 22
Figure 2-4. Schematic diagram of a single pulse sequence ----------------- 23
Figure 2-5. Flowchart of sample preparation for qNMR
parameter analyses ------------------------------------------------ 24
Figure 2-6. Conceptual scheme for pulse-width array experiment -------- 25
Figure 2-7. Conceptual scheme for inversion recovery method ------------ 27
Figure 2-8. Inversion recovery spectra of A. calamus crude extract ------- 27
Figure 2-9. Flowchart for isolation of constituents from Acorus rhizomes- 31
Figure 2-10. Structures of isolated asarone-related compounds (1-5) ------ 32
Figure 2-11. CD spectrum of eudesmin A (6) --------------------------------- 33
Figure 2-12. Structures of eudesmin A (6), (+)-acortatarinowin F and
(−)-acortatarinowin F --------------------------------------------- 34
7
Figure 2-13. Test of linearity: concentration and signal intensity
of β-asarone (1) ---------------------------------------------------- 35
Figure 2-14. 1H-qNMR spectra of β-asarone (1) with I.S.
in different molar ratios ------------------------------------------- 36
Figure 2-15. Signal-to-noise ratio of asaronaldehyde quantitative
signal in sample H -------------------------------------------------- 38
Figure 2-16. Flowchart of preparation of crude extracts from Acorus
rhizomes for analyses --------------------------------------------- 39
Figure 2-17. Sample preparation for purity analyses ------------------------- 42
Figure 2-18. Sample preparation of crude extracts for
content determinations -------------------------------------------- 42
Figure 2-19. 1H-qNMR spectra of β-asarone (1), α-asarone (2), and
asaronaldehyde (3) with I.S. ------------------------------------- 44
Figure 2-20. 1H-qNMR spectrum of A. calamus (sample E) ---------------- 47
Figure 2-21. 1H-qNMR spectra of the crude n-hexane extract from
samples A-H ------------------------------------------------------ 49
Figure 2-22. Signal intensity ratio between aldehyde and I.S.
with various concentrations of crude extract and I.S. ---------- 50
Figure 2-23. HPLC chromatograms of crude n-hexane extract from
Acorus rhizomes sample A-H ------------------------------------ 54
Figure 2-24. HPLC chromatograms showing the UV absorbance patterns of
β-asarone and α-asarone in crude extracts from Acorus rhizomes
sample A-H --------------------------------------------------------- 58
Figure 3-1. Synthesis and metabolism of acetylcholine --------------------- 60
Figure 3-2. AChE activities of each crude extract from Acorus species
(p ≤ 0.05) ----------------------------------------------------------- 62
8
Figure 3-3. AChE inhibitory effects of asarone-related compounds
in Acorus species -------------------------------------------------- 65
Figure 3-4. HPLC profile of 100% MeOH fr. obtained by silica gel
chromatography of extract B indicating the presence of
eudesmin A detected at 280 nm ---------------------------------- 67
Figure 3-5. Concentration-dependent effects of eudesmin A on
AChE activity (p ≤ 0.05) ------------------------------------------ 68
Figure 4-1. 1H NMR spectrum of β-asarone (1) ----------------------------- 79
Figure 4-2. 1H NMR spectrum of α-asarone (2) ----------------------------- 80
Figure 4-3. 1H NMR spectrum of asaronaldehyde (3) ---------------------- 81
Figure 4-4. 1H NMR spectrum of dihydroxyasarone (4) ------------------- 82
Figure 4-5. 1H NMR spectrum of acoraminol B (5) ------------------------ 83
Figure 4-6. 1H and 13C NMR spectra of eudesmin A (6) ------------------ 85
Figure 4-7. Calibration curves of (a) β-asarone, (b) α-asarone ------------ 89
Figure 4-8. Calibration curve of eudesmin A -------------------------------- 90
Figure 4-9. Reaction mechanism of AChE assay in vitro ------------------ 91
List of tables
Table 1-1. The morphological differences between the different parts of
A. calamus and A. gramineus plants ---------------------------- 14
Table 2-1. Solubility of A. calamus n-hexane extract
in various solvents ------------------------------------------------- 19
Table 2-2. Exponential data analysis of A. calamus ------------------------ 28
Table 2-3. S/N of the main component signal in different
scan number -------------------------------------------------------- 30
Table 2-4. ECD values of eudesmin A (6), (+)-acortatarinowin F
and (−)-acortatarinowin F ---------------------------------------- 34
9
Table 2-5. Molar ratio of β-asarone solutions with I.S. and recovery results
(for accuracy) ------------------------------------------------------ 37
Table 2-6. Molar ratio of β-asarone solution with I.S. and % RSD
value (for precision) ----------------------------------------------- 37
Table 2-7. Extract amount and yield (w/w %) of each Acorus rhizome-- 40
Table 2-8. Purity of main asarones estimated by qNMR ------------------ 45
Table 2-9. Stability of pure asarones estimated by qNMR --------------- 45
Table 2-10. Amounts of β-asarone (1), α-asarone (2), and asaronaldehyde
(3) in Acorus rhizomes estimated by qNMR method ---------- 51
Table 2-11. Comparison of the quantity of β-asarone (1) and α-asarone (2)
analyzed by either HPLC or qNMR method ------------------ 55
Table 3-1. AChE inhibition (%) of crude extracts and β-asarone (1)
contents in the extracts -------------------------------------------- 63
Table 3-2. AChE inhibitory effects of constituents isolated from
Acorus crude extracts --------------------------------------------- 64
Table 3-3. IC50 values of β-asarone, the crude extract B, and tacrine
on AChE inhibition ----------------------------------------------- 66
Table 3-4. Concentration-dependent effects of eudesmin A (6) on AChE ----------------------------------------------------------- 69
10
Chapter 1: Introduction
1.1. Overview of qNMR method
Quantitative NMR (1H-qNMR) method was firstly described in 1963 as an
analytical method based on the principle that the intensity of a proton signal directly
reflects the molar concentration of the compound [1-2]. The quantitative method
relies on the comparison of the signal intensities of an analyte with those of a
reference compound to obtain their molar ratio and the quantitative inaccuracy is
less than 2.0% for quantification [2-4]. Quantitative measurement has found
widespread applications because it has several advantages over chromatographic
methods; (i) non-destructive nature (ii) ease of sample preparations (iii) no need of
intensity calibrations (signal intensity is directly proportional to the number of
nuclei) and (iv) simultaneous determinations of analytes in a mixture by using a
reference standard. The most distinctive character of 1H-qNMR is to simultaneously
provide qualitative and quantitative information [5]. It has been reported in many
research areas including natural product chemistry as a powerful analytical tool for
content determinations and purity assessments. It has also been used to measure
materials in complex mixtures such as plant metabolites, marine toxins, food and
beverages, and pharmaceuticals [4, 6-7].
1.1.1. Fundamental principle and development history of qNMR method
The fundamental principle of qNMR method is that the signal intensity (integrated
signal area, Ix) in NMR spectrum is directly proportional to the number of the nuclei
(Nx) responsible for the corresponding resonance line:
Ix = Ks Nx,
where Ks is the spectrometer constant that equals for all resonance lines in NMR
spectrum [4,8].
11
The first quantitative measurement by using qNMR was developed in 1963 by
Jungnickel and Forbes for determination of intra-molecular proton ratios of 26 pure
organic compounds [1]. At the same time, Hollis determined the amounts of aspirin,
phenacetin, and caffeine in respective mixtures [9]. In natural product analysis, the
qNMR technique has been developed since 2004 [5]. The qNMR technique does not
rely on authentic, identical reference materials for quantification because the signal
intensity is directly proportional to the number of protons (fundamental principle).
It can use any highly pure reference compounds as an internal standard (I.S.) for
quantification but the proton signals of I.S. are required to clearly separate from the
analyte signals [4,10]. This characteristic of quantification technique of qNMR
without relying on authentic reference compounds makes qNMR suitable for
quantification of natural products. Because conventional LC (liquid
chromatography) techniques coupled to UV and MS detector depend on analyte
response factor and pure authentic materials are required for quantification [10]. On
the other hand, there is a limitation regarding the commercial availability of reliable
pure natural products. Therefore, qNMR method has been widely used in natural
product researches [2, 7, 11-12].
1.1.2. Experimental parameters of qNMR
There are several important experimental parameters that impact on the quantitative
results and they are described below.
1.1.2.1. Pulse angle (excitation pulse) and pulse width value (pw)
Pulse angle is the radio frequency (Rf) pulse applied per one cycle. This radio pulse
excites the nuclei which then emits Rf during the acquisition time, giving rise to an
NMR signal in the form of an exponentially decaying sine wave, termed free-
induction decay (FID). A pulse angle 90˚or lower can be used for quantification
12
method. Generally, 90˚pulse is used to provide more precise quantitative results [13-
14].
Pulse width is the amount of time when the pulse is applied to the sample in order
to flip all the spins into the XY plane (Figure 1-1) [14]. It depends on the
physicochemical properties of samples and varies from sample to sample [8]. The
value of pulse width should be uniform throughout the experiment.
Figure 1-1. The nuclear spin magnetization (orange arrow) before and after
application of a pulse [14].
1.1.2.2. Acquisition time (Aq)
Acquisition time is the time taken to acquire FID. Acquisition time should be
carefully adjusted because the acquisition time is related to the resolution of the
spectrum. The long acquisition time will acquire unnecessary noise and too short
acquisition time will lead to the generation of wiggles at the base of the signals [8,
14].
1.1.2.3. Relaxation time (T1 and T2)
There are two types of relaxation times such as spin-lattice relaxation (T1,
relaxation in z plane) and spin-spin relaxation (T2, relaxation in x-y plane) (Figure
1-2). T1 relaxation is responsible for gain and loss of magnetization in the z-direction. Then, T1 value should be considered for qNMR measurements because its
13
value is unique for each signal of every compound. T2 describes the decay of excited
nuclear spin on the x-y plane and its value is generally lower than T1 value [13,15].
Figure 1-2. Relaxation mechanism of nuclear spin after 90˚ pulse (T2-blue arrow,
T1-red arrow) [15].
1.1.2.4. Relaxation delay (D1)
Relaxation delay is the delay time of nuclear spin to go back to the equilibrium state
along the z-axis after acquiring FID. Nuclear spin relaxes according to a time
constant called T1 (spin-lattice relaxation time) and delay time should be enough for
getting full relaxation prior to the next pulse. When 90˚pulse is used to excite the
spin, delay time should be 5 times of T1 in order to have complete relaxation [14].
Figure 1-3. Schematic diagram of a single pulse sequence of nuclear spin
magnetization.
14
1.2. Overview of Acorus species
Acoraceae family members including the genus Acorus are rhizomatous herbs.
Acorus genus consists of about 40 species but 3 Acorus species called Acorus
calamus, A. gramineus and A. tatarinowii, are extensively investigated for research
and medicinal purposes [16-17]. Acorus plants also known as sweet flags occur in
the wild and are also cultivated for medicinal and culinary uses in some Asian
countries, especially India and Myanmar. A. calamus is native to Temperate India,
Southern Asia, Eastern Europe as well as A. gramineus is widely distributed in Japan,
China and Korea [18]. Table 1-1 shows the morphological differences between the
different parts of the A. calamus and A. gramineus plants [19].
Figure 1-4. Acorus calamus plant.
Parts of plant A. calamus A. gramineus
Rhizome thicken (8 ~12 mm) thin (2 ~ 8 mm)
Leave 50 ~ 80 cm length x 6 ~ 15 mm width (center skeleton is present)
5 ~ 50 cm length x 1 ~ 8 mm width (center skeleton is absent)
Flower (spike) 4 ~ 7 cm length x 6 ~ 10 mm width
5 ~ 10 cm length x 3 ~ 4 mm width
Table 1-1. The morphological differences between the different parts of A. calamus and A. gramineus plants [19]
Acorus calamus plant figure was taken
from National herbal garden,
Nay Pyi Taw, Myanmar.
15
1.2.1. Traditional uses and pharmacological profiles of Acorus species
Acorus species are well-known medicinal herbs which are used for CNS disorders,
respiratory system diseases, and gastrointestinal disorders. A. calamus has been used
as an emetic in dyspepsia, sedative, mental illness, nerve tonic by ancient Chinese
people [17] as well as used as an anesthetic for toothaches and headaches by Native
Americans [18]. A. tatarinowii is also an important traditional Chinese medicine and
used for CNS disease, epilepsy, and dementia. A. gramineus known as Japanese
sweet flag has been used for the treatment of cognitive deficit, stomachache, and as
an insecticide. Moreover, recent studies on biological activities have indicated that
the Acorus species have the anti-inflammatory, antidiabetic, antimicrobial and
anticonvulsant activities [18, 20].
1.2.2. Chemical constituents of Acorus species
Bioactive chemical constituents isolated from Acorus species are mainly
phenylpropanoids, sesquiterpenoids, and lignans (Figure 1-5). Other miscellaneous
components such as flavonoids, sterols, diterpenoids, amides, and alkaloids have
also been isolated from Acorus species [18]. The main constituents of Acorus species
have been identified as β-asarone and α-asarone which are attributed to the main
components responsible for their various pharmacological activities [18].
16
Phenylpropanoids Sesquiterpenoids
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
R1
R2
OCH3
H3CO
OCH3
Lignans
O
OCH3
OCH3OCH3
H3CO
R
O
OCH3
R2
OCH3
H3CO
R1
OH
Figure 1-5. Chemical structures of reported compounds from Acorus species [18].
α-asarone β-asarone
dihydroxyasarone: R1 = OH, R2 = OH acoraminol B: R1 = OCH3, R2 = OH
O
OR2 R1
18
O
5
1-hydroxyepiacorone: R1 = OH, R2 = H (1R, 8S) calamusin: R1 = OH, R2 = OH (1R, 8S)
(-)-acorenone:5S
galgravin: R = H ligraminol A, R = OCH3 (7S, 8S, 7’S, 8’R) 5-methoxygalbelgin, R = OCH3
(7S, 8S, 7’S, 8’S)
(-)-virolin: R1 = H, R2 = OCH3 polysyphorin: R1 = OCH3, R2 = OCH3
surinamensinols A: R1 = OCH3, R2 = H (7R) surinamensinols B: R1 = OCH3, R2 = H (7S)
17
1.2.3. β-asarone contents and ploidy levels
The content of β-asarone from Acorus species corresponds to the polyploidy level
of various Acorus karyotypes. There are four karyotypes; diploid, triploid, tetraploid
and hexaploid. Diploid varieties were characterized by the absence of β-asarone and
distributed in North America, and some parts of Asia. 3-19 % of β-asarone is
contained in triploid varieties whereas up to 90% of β-asarone is in tetraploid
varieties of India, Indonesia, and Taiwan [17, 21].
1.3. Objectives of the research
Acorus calamus and A. gramineus rhizomes are the most commonly used home
herbal remedies as well as food condiments in Asian countries including Myanmar.
Moreover, A. calamus rhizome is already listed in Myanmar herbal pharmacopeia,
published in 2013, as traditional medicine and has been analyzed by only TLC
method for its quality assessments [22]. It has been reported that most of
pharmacological effects and toxicities are directly related to their major
phenylpropanoids, β-asarone found in both species together with its isomer, α-
asarone [23-26]. Therefore, accurate quantification of β-asarone content in Acorus
herbal products is still required and researchers established the conventional
chromatographic methods such as high-performance liquid chromatography (HPLC),
gas chromatography (GC), micellar electrokinetic capillary chromatography
(MEKC), and fluorescence-coupled chemometric methods [24, 27-29]. However,
these reported methods have some drawbacks such as the necessity of standard
compounds with reliable purity, longer run times, and considerable volumes of
organic solvents. In addition, care must be taken to ensure reproducible results
because asarone compounds are volatile or unstable. This thesis deals with the study
18
of the applicability of 1H-qNMR method for quantification of β-asarone and asarone-
related compounds in Acorus herbal products.
Alzheimer’s disease (AD) is a degenerative neurological disorder associated with
memory loss and cognitive dysfunction, and AChE inhibitors are commonly
employed as primary treatments [30]. Acorus species have a long history of their
medicinal application to neuropharmacology as an anticonvulsant, neuroprotective,
and memory enhancing agent [18, 31-32]. Furthermore, β-asarone improves the
cognitive impairment induced by chronic stress, enhances the neurological outcomes
after cerebral ischemia, attenuates neuronal apoptosis in mice, and inhibits the
acetylcholinesterase activity in vitro [33-36]. Thus, the effects of crude extracts from
Acorus and their principal constituents towards the AChE enzyme were also
investigated in this study.
19
Chapter 2: Application of the 1H-qNMR method on Acorus species
In contrast to other chromatographic methods, careful selection of experimental
parameters to maximize the accuracy and precision of the quantitative results of
qNMR method will be required [8, 37]. In this study, the deuterated solvent showing
excellent solubility of crude extracts and suitable internal standard for measurements
were primarily investigated. Then, the critical acquisition parameters were
optimized with an analyte sample for quantification of main asarone compound in
Acorus products.
2.1. Optimization of 1H-qNMR method
2.1.1. Selection of deuterated solvent based on the solubility of analyte
The crude extract was prepared from A. calamus rhizome (sample A) for analyzing
the solubility. Pulverized rhizome sample A was dissolved in n-hexane, vortexed for
1 min and sonicated for 5 min. Then, the solution was centrifuged for 3 min, filtered
and concentrated to yield crude n-hexane extract. Each ca. 11.0 mg of crude extract
was taken and dissolved in 700 µL of CHCl3, DMSO and MeOH, respectively. As a
result, the crude extract was completely dissolved in CHCl3 and DMSO to give clear
solutions. Crude sample solution with MeOH gave turbidity, and only 82% of the
sample was dissolved (Table 2-1). DMSO-d6 was found unsuitable due to the water
resonance problem. Therefore, CDCl3 was selected as a deuterated solvent for
measurements.
Table 2-1. Solubility of A. calamus n-hexane extract in various solvents
Solubility of crude extract (mg/700 µL)
CHCl3 DMSO MeOH
11.0 mg 11.0 mg 9.0 mg
20
2.1.2. Selection of the internal standard
Suitable qNMR certified reference material was selected as an internal standard
(I.S.) in this study. Before selecting reference material, 1H NMR spectrum of Acorus
crude n-hexane extract was acquired (Figure 2-1), and it was inferred that the I.S.
signals should be located between the chemical shift ranges of δ 7.5 to δ 9.0.
Figure 2-1. 1H NMR spectrum of crude extract from A. calamus (600 MHz, CDCl3).
21
Among qNMR certified reference materials, dimethyl terephthalate (DMT) was
chosen as an internal standard (I.S.) due to its high solubility in chloroform-d
(CDCl3) and simplicity of the NMR spectrum showing only two singlet signals at δ
8.10 and δ 3.94 (Figure 2-2). These two singlet signals, where four aromatic protons
appear at δ 8.10 can be used as a reference quantitative signal because this signal
was located in the area of δ 7.5 to δ 9.0.
Figure 2-2. 1H NMR spectrum of dimethyl terephthalate (I.S.) (600 MHz, CDCl3).
22
Then, the compatibility between the signals of analyte from crude extract and those
from DMT (I.S.) was examined, and it was ascertained that four aromatic protons
signal at δ 8.10 could be separated from the analyte signals (Figure 2-3). Therefore,
DMT was used as an internal standard (I.S.) showing aromatic signal associated with
four protons at δ 8.10 (quantitative reference signal) for further experiments.
Figure 2-3. 1H NMR spectrum of crude extract with I.S. (600 MHz, CDCl3).
23
2.1.3. Selection of important acquisition parameters for the qNMR method
After optimizing deuterated solvent and internal standard (I.S.) appropriate for the
qNMR measurements, acquisition parameters were optimized to ensure accurate
quantification. In this study, acquisition parameters for quantitation were determined
by single pulse NMR experiment where the relaxation, excitation, and acquisition
states were optimized (Figure 2-4) [8]. Thus, we determined the important
parameters representing the three states such as pulse angle (excitation pulse), pulse
width value (P1 or pw 90˚), inversion recovery (T1) value, relaxation delay (D1)
value, acquisition time (Aq), and number of scans (ns) prior to the quantification.
Figure 2-4. Schematic diagram of a single pulse sequence [8, 37].
Excitation
Acquisition Relaxation
24
2.1.3.1. Sample preparation for analyses of experimental parameters
To optimize acquisition parameters, one representative crude extract sample of
A. calamus was prepared in CDCl3 containing I.S. and was used as indicated in the
following flowchart (Figure 2-5).
Figure 2-5. Flowchart of sample preparation for qNMR parameter analyses.
sample crude extract (6.0 mg)
Dimethyl terephthalate (I.S.)
(1.0 mg/100 µL with CDCl3)
sample crude extract (6.0 mg) +
I.S. (20 µL)
transferred to NMR tube
taken 20 µL (0.2 mg) with micro-syringe
key parameter analyses for qNMR
dissolved in 730 µL of CDCl3
vortexed for 1 min and sonicated for 1 min
25
2.1.3.2. Pulse angle and pulse width calibration (pw 90˚)
Pulse angle and pulse width value are significant parameters which represent the
excitation state in a single pulse program [8, 37]. Pulse angle 90˚ was used as an
excitation pulse in this study for maximizing peak intensities and acquiring better
accuracy although it was time-consuming.
Then, the calibration value of the pulse width 90˚ for Acorus crude extract was
examined by using the pulse width array experiment. Pulse width array analysis was
started from 2 to 40 µs to calculate the duration of pulse 90˚ (Figure 2-6).
Figure 2-6. Conceptual scheme for pulse-width array experiment.
However, it was difficult to look at the exact time of the 90˚pulse by comparing the
similar intense signals (90˚ and 91˚). Thus, we searched the duration of the null peak
(no signal) at 360˚pulse and it was divided by 4. As a result, the time taken for the
excitation pulse 90˚ of sample analyte was determined to be 9.9 µs.
Duration of the null peak at 360˚/4 = pulse duration of 90˚
39.6 µs / 4 = 9.9 µs (pulse width value of 90˚)
2 µs 10 µs
30 µs
90°
180°
270°
360°
Increasing pulse width
pulse width array starts from 2 to 40 µs
maximum signal intensity
null peak
null peak
26
2.1.3.3. Inversion recovery analysis (T1)
Spin-lattice relaxation (T1) value was measured by inversion recovery method. The
pulse sequence mechanism of inversion recovery method is based on the 5T1-π-τ-
π/2-FID pulse sequence. The mechanism involves a series of NMR spectra that are
measured with the incremented waiting period τ. Then, the degree of the signal
returned to its equilibrium value during the waiting period τ for each sequence is
measured. Figure 2-7 shows the conceptual scheme for inversion recovery method
[15]. The T1 value of Acorus crude extract was examined by using this inversion
recovery method (Figure 2-8) and T1 values of the internal standard proton, three
methoxy protons, and the solvent proton from the extract were found to be 3.5 s, 3.8
s, and 7.1 s, respectively (Table 2-2).
27
Figure 2-7. Conceptual scheme for inversion recovery method [15].
Figure 2-8. Inversion recovery spectra of A. calamus crude extract.
π/2
π/2
π/2
π/2
π/2
τ = 0
τ1
τ2 = T1In(2)
τ3
τ4
π x
y
z
increasing delay
28
Table 2-2. Exponential data analysis of A. calamus peak T1 error 1 3.585 0.03635 IS peak 2 7.088 0.08589 solvent peak 3 2.941 0.03068 4 2.335 0.02938 5 3.719 0.05755 6 3.82 0.06326 7 3.799 0.05055 8 3.8 0.03703 9 3.032 0.1436 10 3.401 0.07009 11 3.02 0.1224 12 3.298 0.03798 13 3.36 0.06406 14 2.812 0.1147 15 3.273 0.04813 16 2.736 0.1323 17 2.753 0.02026 18 2.713 0.02365 19 2.518 0.02115 20 2.513 0.03588 21 2.483 0.02078 22 2.563 0.03491 23 2.85 0.03917 24 2.738 0.02939 25 2.908 0.05098 26 1.507 0.2758 27 1.987 0.03695 28 1.409 0.09051 29 1.616 0.006487 30 1.766 0.3314 31 1.736 0.05591 32 1.677 0.02923 33 1.638 0.04224 34 1.636 0.07274 35 1.793 0.007824 36 1.822 0.05801 37 1.826 0.005264 38 2.034 0.09095 39 1.434 0.09069 40 2.746 0.02848
29
2.1.3.4. Relaxation delay (D1)
D1 value is an important parameter that reflects the relaxation state. Its value
depends on the pulse angle used in the pulse program and the longest T1 value of
analyte signals [7]. D1 value can be calculated as five times, seven times and ten
times of the longest T1 value to gain 99.3%, 99.9%, and 99.99% recovery,
respectively [37]. According to the previous inversion recovery analysis (T1)
[mentioned in section 2.1.3.3.], the longest T1 value was found to be 7.1 s. Then, D1
value was calculated as five times of T1 value, and the calculated value was
approximately 36 s. However, there are another T2 relaxation time (spin-spin
relaxation) that needs to consider for full relaxation of nuclear spin before starting
next pulse sequence. In generally, T2 value is always lower than T1 value. Therefore,
extra time 4 s as T2 relaxation time (spin-spin relaxation) was added to be on the
safe side because T1 values of the quantitative analyte signals are around 3.8 s.
Therefore, final D1 value was set to 40 s to ensure the full relaxation of nuclear spin.
2.1.3.5. Spectral width (SW) and acquisition time (Aq)
In this experiment, the spectral width 20 ppm (12019.2 Hz) was applied.
Acquisition time (Aq) is the time taken to acquire FID and depends on the number
of data point collected (NP), spectral width (SW) and magnetic field strength. In this
experiment, 600 magnetic field and spectral width 20 ppm were used, and the
collected data point was 96154 for sample analyte. Then, acquisition time was
calculated by the following equation to be 4.0 s.
Aq = NP/ (2 x SW x field strength)
= 96154/ (2 x 20 x 600) = 4.0 s
30
2.1.3.6. Number of scans (ns)
Number of scans is also an important parameter that affects the detection of the
qNMR signals and needs to be analyzed to ensure the S/N ratio is above 150 [8]. In
this study, scan number 8, 16 and 32 were acquired and calculated in terms of the
S/N ratio of the main component signal. As shown in Table 2-3, scan number 16
was found suitable for analytes.
Table 2-3. S/N of the main component signal in different scan number
2.1.3.7. Acquisition temperature
Acquisition temperature was constantly set to 25˚C throughout the study because it
influences on the reproducibility of the quantification [8].
Scan number
S/N of the overall spectrum
S/N of the main component signal
Total runtime
8 4536.8 480.8 5 min
16 10698.8 553.3 11 min
32 12743.5 710.6 23 min
31
2.2. Preparation of principal constituents from Acorus rhizomes
2.2.1. Extraction and isolation of principal constituents
Acorus rhizomes were used to isolate principal constituents. Pulverized rhizome
powder of Acorus was extracted with n-hexane by sonication to obtain a crude
extract, which was purified by column chromatography followed by preparative
HPLC to yield compound 1-6. Figure 2-9 shows the protocol for the isolation of the
compounds from Acorus rhizomes.
Figure 2-9. Flowchart for isolation of constituents from Acorus rhizomes.
extracted with n-hexane
n-hexane crude extract
CC. Column Chromatography
Silica gel
ODS gel
MCI CHP-20P gel
Bond-Elut C18 cartridge column
Preparative-HPLC
β-Asarone (1)
α-Asarone (2)
Asaronaldehyde (3)
Dihydroxyasarone (4)
Acoraminol B (5)
Eudesmin A (6)
Pulverized rhizome powder of Acorus
CC. Prep-HPLC
32
2.2.2. Identification of known compounds
As described in section 2.2.1., principal constituents 1-6 were isolated from Acorus
rhizomes and identified by their 1H NMR and ESI-MS spectral data. Among
compounds 1-6, β-asarone (1), α-asarone (2), asaronaldehyde (3), dihydroxyasarone
(4) and acoraminol B (5) were the known asarone-related compounds, as presented
in Figure 2-10 and their chemical structures were determined by comparing with the
reported spectral data [25, 28].
CH3
OCH3
OCH3
H3CO
OCH3
H3COOCH3
CHO1
3
61
3
6
1'
3'
H3COCH3
OCH3
H3CO
1
3
6
1'3' 2' 2'
H3COOCH3
H3COOCH3
CH3
HOOH
OCH3
CH3
HOOCH3
OCH3
36
36
1 2 3
4 5
Figure 2-10. Structures of isolated asarone-related compounds (1-5).
33
Compound (6) was identified as eudesmin A, hexahydrofuranofuran-type lignan
which was first isolated from Acorus species. 1H, 13C NMR spectral data, and ESI-
MS data of compound (6) were compared with those reported [38]. Then, the specific
rotation and CD spectrum were also acquired to determine the absolute configuration
of compound (6). Moreover, its UV absorbance was found at 207, 230 and 280 nm,
respectively. Figure 2-11 exhibited the CD spectrum that shows the positive cotton
effect at 207, 225 and 275 nm.
Figure 2-11. CD spectrum of eudesmin A (6).
After that, the absolute configuration of eudesmin A (6) was determined by the
comparison with reported known lignans, (+)-acortatarinowin F and (−)-
acortatarinowin F from Acorus tatarinowii [39]. Table 2-4 reveals that the ECD
values of eudesmin A (6) are correlated with those of (+)-acortatarinowin F. In
addition, specific rotation of eudesmin A was found to be +22.63 that corresponds
to that of (+)-acortatarinowin F. Therefore, the absolute configuration of eudesmin
A was elucidated as 2S, 6S, 3R, 7R.
-1-0.5
00.5
11.5
22.5
3
200 220 240 260 280 300 320 340
[θ] x
10-4
Wavelength
34
H3CO
OCH3
O
O
OCH3
OCH3H
H
2S
6S
3R7R
H3CO
OCH3
O
O
OCH3
OCH3H
H
2S
6S
3R7R
H3CO
O
O
OCH3
OCH3OCH3
OCH3
H3COH
H
2R
3S
6R
7S
Figure 2-12. Structures of eudesmin A (6), (+)-acortatarinowin F and (−)-acortatarinowin F.
Table 2-4. ECD values of eudesmin A (6), (+)-acortatarinowin F and (−)-acortatarinowin F Eudesmin A (6) [θ] (nm)
(+)-Acortatarinowin F [θ] (nm)
(−)-Acortatarinowin F [θ] (nm)
+2.27 x 104 (207) +2.25 x 104 (211) −2.93 x 104 (211)
+0.42 x 104 (225) +0.31 x 104 (228) −0.38 x 104 (228)
+0.21 x 104 (275) +0.29 x 104 (276) −0.31 x 104 (275)
Eudesmin A (6) (+)-Acortatarinowin F
(−)-Acortatarinowin F
35
2.3. Validation of qNMR method
2.3.1. Linearity, accuracy and precision analyses of β-asarone by qNMR
Linearity
Four solutions of the β-asarone (1) 0.25 – 3.0 mg were prepared with CDCl3
containing I.S. to examine the linearity of the method and β-asarone (1)
concentrations were corrected with its purity (94.6%) estimated by qNMR. Figure
2-13 showed the concentration versus the signal intensity of β-asarone (1) along with
a linear regression line of y = 5. 2264x + 0.3798 and yield regression coefficient of
0.9971. The qNMR spectra of β-asarone (1) with I.S. in different molar ratios were
shown in Figure 2-14.
Figure 2-13. Test of linearity: concentration and signal intensity of β-asarone (1).
y = 5.2264x + 0.3798R² = 0.9971
02468
10121416
0.00 0.50 1.00 1.50 2.00 2.50 3.00
sign
al in
tens
ity
Concentration (mg/700 µL)
36
Figure 2-14. 1H-qNMR spectra of β-asarone (1) with I.S. in different molar ratios.
3.0 mg
1.5 mg
0.5 mg
0.25 mg
37
Accuracy and precision
Accuracy means the closeness in agreement between the measured values and actual
values [8]. In this study, recovery analyses were carried out by comparing the molar
ratios calculated by weight and by qNMR at different molar concentrations of pure
β-asarone (1) (Table 2-5).
Table 2-5. Molar ratio of β-asarone solutions with I.S. and recovery results (for accuracy)
The precision of the qNMR method was also estimated by triplicate measurements
of the same sample ( β-asarone (1): 1.0 mg, I.S.: 0.2 mg) within 24 h (intraday
precision) and the relative standard deviation (RSD) value was within 0.86 %,
suggesting good precision (Table 2-6).
Table 2-6. Molar ratio of β-asarone solution with I.S. and % RSD value (for precision)
Weight of β-asarone (mg)
Weight of I.S. (mg)
Molar ratio prepared
Molar ratio by qNMR
Recovery (%)
0.25 0.2 1.01 1.04 102.9 (n=3)
0.5 0.2 2.02 1.99 99.0 (n=3)
1.5 0.2 6.03 6.04 100.1 (n=3)
3.0 0.2 12.2 11.12 91.1 (n=3)
weight of β-asarone (mg)
weight of I.S. (mg)
Molar ratio prepared (mM)
Molar ratio by qNMR (mM)
1.0 0.2 4.66 4.82 1.0 0.2 4.66 4.77 1.0 0.2 4.66 4.73
Average 4.78 SD 0.041
%RSD 0.86
38
2.3.2. Limit of quantitation (LOQ) and limit of detection (LOD) of qNMR method
LOQ and LOD exhibit the lowest concentrations of analytes that can be measured
by analytical methods. These values can be estimated by various methods including
visual evaluation, S/N (signal-to-noise ratio) measurement, and method based on SD
(standard deviation) of response and slope [10, 40]. In the qNMR method, LOQ can
be estimated based on S/N of a quantitative signal (10:1) and LOD is the 3.3 times
reducing of LOQ values [6, 10, 13]. Then, the estimation of LOD and LOQ of a
single analyte in the matrix is enough if common quantification method is used for
analytes from sample matrix [13]. Thus, in this study, LOD and LOQ of the
optimized qNMR method were estimated by measuring manually the signal to noise
magnitude of the smallest signal of asaronaldehyde found in sample matrixes
(Figure 2-15). The results demonstrated that LOQ of the asaronaldehyde by qNMR
method was about 38.7 µg (S/N 11:1) and LOD was 11.7 µg (LOQ/3.3).
Figure 2-15. Signal-to-noise ratio of asaronaldehyde quantitative signal in sample H.
N
S
39
2.4. Quantitative analyses by optimized qNMR method
2.4.1. Preparation of crude extracts
In this experiment, five samples of A. calamus and three samples of A. gramineus
rhizomes were subjected to qNMR, HPLC and bioactive analyses. These rhizome
samples were pulverized by a blender and extracted with n-hexane by the following
protocol (Figure 2-16). These extract samples were stored at −30˚C prior to analyses.
Figure 2-16. Flowchart of preparation of crude extracts from Acorus rhizomes for analyses.
each around 2.0 g powder of Acorus products
20 mL n-hexane
vortexed for 1 min, sonicated for 5 min
centrifuged at 3000 rpm for 3 min, filtered
x 4 times, concentrated in vacuo
crude n-hexane extracts (kept in the refrigerator at −30˚C)
1H-qNMR and HPLC analyses
40
Table 2-7 shows the amounts and yields percent of each rhizome extracts. Sample
A collected from Myanmar was found to provide the highest yield than other
samples. Additionally, the yields were found to be varied depending on the samples.
Table 2-7. Extract amount and yield (w/w %) of each Acorus rhizome
Sample Extract amount (mg/2.0 g)
Yield (w/w %)
A 137.5 6.9
B 95.7 4.8
C 83.4 4.2
D 29.3 1.5
E 43.9 2.2
F 53.1 2.7
G 43.1 2.2
H 26.3 1.3
Samples A-E are derived from A. calamus and samples F-H are from A. gramineus.
41
2.4.2. Sample preparation for qNMR analyses
Sample preparation is also essential for accurate quantification [8]. In this context,
the determination of purity and contents were carried out by the optimized qNMR
method. For these experiments, DMT (I.S.) was used as an internal standard and
dissolved in CDCl3 to prepare 1.0 mg/100 µL stock solution. Then, isolated asarone
derivatives, β-asarone (1), α-asarone (2), and asaronaldehyde (3) were separately
dissolved in 700 µL CDCl3 containing different amounts of I.S. for purity analyses
(Figure 2-17). For content determinations of crude extracts, ca. 6-9 mg of the
sample crude extracts and 0.2 mg of I.S. were dissolved in 700 µL CDCl3 and used
for the analyses (Figure 2-18).
42
Figure 2-17. Sample preparation for purity analyses.
Figure 2-18. Sample preparation of crude extracts for content determinations.
each crude n-hexane extract (6.2-8.9) mg
+ I.S. (0.2) mg
Quantitative analyses by qNMR
dissolved in 700 µL of CDCl3
vortexed for 1 min and sonicated for 1 min
transferred to NMR tube
β-Asarone (3.0) mg + I.S. (0.2) mg
α-Asarone (0.5) mg + I.S. (0.05) mg
Asaronaldehyde (0.8) mg + I.S. (0.1) mg
dissolved in 700 µL of CDCl3
Purity analyses by qNMR
transferred to NMR tube
vortexed for 1 min and sonicated for 1 min
43
2.4.3. Purity and stability analyses of isolated main asarones by qNMR
As mentioned in isolation study discussed in section 2.2.1., three major asarones, β-
asarone (1), α-asarone (2), and asaronaldehyde (3) were isolated from Acorus
rhizomes. These three isolated asarones were dissolved in CDCl3 containing I.S. as
shown in Figure 2-17 and 1H-qNMR spectra of each compound were acquired. In
Figure 2-19, all of the proton signals of each compound were found to be well
resolved from those of aromatic four protons of I.S. that appear at δ 8.10. Among
these signals, an H-6 singlet at δ 6.84 for β-asarone (1), an H-6 singlet at δ 6.94 for
α-asarone (2), and an aldehyde signal at δ 10.31 for asaronaldehyde (3) were used
to estimate the purities of the compounds because these singlet signals were clearly
separated from other concomitant constituent signals. Table 2-8 presents the purity
of each compound. The results uncover that the purity of α-asarone (2) is lower than
those of the other two compounds. It was assumed that the relatively low purity of
the α-asarone is due to its instability during measurements.
44
Figure 2-19. 1H-qNMR spectra of (a) β-asarone (1), (b) α-asarone (2), and (c) asaronaldehyde (3) with I.S.
(a)
(b)
(c)
OCH3
H3CO
H
H3CH
OCH3
3
6
OCH3
H3COOCH3
CH3
HH
3
6
CHOOCH3
OCH3
H3CO 3
6
β-asarone (1)
α-asarone (2)
asaronaldehyde (3)
45
Table 2-8. Purity of main asarones estimated by qNMR
Therefore, the stability analyses of these pure asarones were also performed after
storage at –30˚C for two days and it was found that the signal intensity of α-asarone
(2) reduced to 5.2% compared to that of the fresh solution. On the other hand, the
signal intensities of the other two compounds were found to be stable under the
conditions. (Table 2-9).
Table 2-9. Stability of pure asarones estimated by qNMR
Compound Purity (%) (mean ± SD) (n=3)
β-Asarone (1) 94.6 ± 0.18
α-Asarone (2) 88.7 ± 0.27
Asaronaldehyde (3) 96.8 ± 0.51
Compound Molar ratio prepared (mM)
Mean recovery (%) ± SD (n=3) of fresh solution
Mean recovery (%) ± SD (n=3) after 2days at
–30˚C β-Asarone (1) 4.66 102.49 ± 0.88 101.54 ± 0.23
α-Asarone (2) 8.30 93.93 ± 0.28 89.09 ± 0.23
Asaronaldehyde (3) 7.93 96.88 ± 0.53 96.96 ± 0.21
46
2.4.4. Content determinations of crude extracts by qNMR
For quantitative determinations of main asarones from Acorus samples, five
products of A. calamus and three products of A. gramineus were prepared as
previously described in sections 2.4.1 and 2.4.2. Then, qNMR spectra of each crude
extract were acquired. Figure 2-20 demonstrates the representative spectrum of
Acorus. From this spectrum, most of the signals of β-asarone (1), α-asarone (2) and
asaronaldehyde (3) were detected, and the signals from δ 5.5 to δ 11.0 were used as
quantitative signals. As some constituent signals appeared around the H-6 signal at
δ 6.84, the H-2’ signal at δ 5.77 of β-asarone (1) , the H-2’ signal at δ 6.10 of α-
asarone (2) and aldehyde proton signal at δ 10.31 of asaronaldehyde (3) were chosen
as the representative signals suitable for quantification because these signals were
well separated from the signals of I.S. and other constituents. The remaining spectra
of crude extracts were shown in Figure 2-21 and β-asarone was found to be the main
constituent in all Acorus products. Consequently, the amounts of β-asarone (1), α-
asarone (2), and asaronaldehyde (3) from each crude extract were estimated by the
integrations of these signals.
47
Figure 2-20. 1H-qNMR spectrum of A. calamus (sample E).
600MHz, CDCl3, 25˚C Quantitative signal region
OCH3
H3CO
H
H3CH
OCH3
3
61'
2'3'
OCH3
H3COOCH3
CH3
HH
3
61'2'
3'
CHOOCH3
OCH3
H3CO 3
6
β-asarone (1)
α-asarone (2)
asaronaldehyde (3)
48
Sample A
Sample B
Sample C
Sample D
asaronaldehyde
α-asarone
β-asarone I.S.
49
Figure 2-21. 1H-qNMR spectra of the crude n-hexane extract from samples A-H (600 MHz, in CDCl3, 25˚C).
Sample F
Sample G
Sample H
β-asarone
α-asarone
asaronaldehyde
I.S.
50
Unfortunately, the amount of asaronaldehyde (3) was found to be quite lower than
those of the other two compounds. Furthermore, I.S. concentrations were not
suitable for quantification of its content since the signal ratio between aldehyde
proton and I.S. was found to be 1:10 (Figure 2-22). The remarkable difference
between the concentrations of the I.S. and target analyte affects the accuracy of
analyte [8]. Therefore, more concentrated crude samples that contained the one-
tenth amount of I.S. relative to the other two compounds were used for quantification
of asaronaldehyde (3).
Figure 2-22. Signal intensity ratio between aldehyde and I.S. with various concentrations of crude extract and I.S.
1:10
1:3
Sample A (8.8mg) + I.S. (0.2 mg)
Sample A (20.1mg) + I.S. (0.02 mg)
0.13 0.59
51
Table 2-10 summarizes the estimated amounts of the three major asarone
constituents from Acorus extracts. The amounts of β-asarone (1) and α-asarone (2)
in five lots of A. calamus samples were 4.4 – 0.82 w/w% and 0.33 – 0.027 w/w%,
respectively, and those of asaronaldehyde (3) were 7.0 x 10-3 – 8.3 x 10-4 w/w%. The
constituents in A. gramineus samples were found to be somewhat lower than those
in A. calamus samples. The three lots of A. gramineus samples contained 1.2 – 0.91
w/w% of β-asarone (1), 0.23 – 0.023 w/w% of α-asarone (2), and 2.1 x 10-3 – 5.0 x
10-4 w/w% of asaronaldehyde (3), respectively. Likewise, there are differences of
the distributions found in the collected samples. The differences may originate from
the variation of several factors including collection times, growth climates, sources,
storage time and ploidy levels.
Table 2-10. Amounts of β-asarone (1), α-asarone (2), and asaronaldehyde (3) in Acorus rhizomes estimated by qNMR method
Data are expressed as means ± SDs based on triplicate measurements. Samples A-E are derived from A. calamus and samples F-H are from A. gramineus.
Sample β-Asarone (1) (w/w %)
mean ± SD
α-Asarone (2) (w/w %)
mean ± SD
Asaronaldehyde (3) (10-3 w/w %) mean ± SD
NMR NMR NMR A 4.4 ± 0.03
3.0 ± 0.01
2.5 ± 0.01
0.82 ± 0.01
1.3 ± 0.01
1.2 ± 0.01
1.1 ± 0.01
0.91 ± 0.01
0.065 ± 0.008 4.8 ± 0.4
B 0.12 ± 0.001 4.9 ± 0.1
C 0.083 ± 0.005 7.0 ± 0.1
D 0.027 ± 0.006 0.83 ± 0.15
E 0.33 ± 0.006 1.8 ± 0.1
F 0.22 ± 0.010 1.1 ± 0.2
G 0.23 ± 0.005 2.1 ± 0.1
H 0.023 ± 0.005 0.50 ± 0.11
52
2.5. Comparison between qNMR and HPLC
2.5.1. Quantitative analyses by HPLC-DAD
The amounts of asarones present in the eight samples of Acorus crude extracts were
estimated by using HPLC-DAD (Figure 2-23). The HPLC analysis was performed
by using the mobile solvent mixture of MeOH-H2O (50:50 v/v) with 0.1% (v/v)
formic acid and an ODS-A 302 column (150 x 4.6 mm I.D.). Although β-asarone
(1) and α-asarone (2) peaks were detected, asaronaldehyde (3) peak was not
identified under this condition. Therefore, β-asarone (1) and α-asarone (2) contents
were estimated from crude extract samples by using each respective calibration line
of HPLC method and compared with the quantitative results of qNMR.
53
β-asarone (1)
α-asarone (2)
Sample A
Sample B
Sample C
Sample D
Sign
al st
reng
th (a
bsor
banc
e un
it)
Retention time (min)
54
Figure 2-23. HPLC chromatograms of crude n-hexane extract from Acorus rhizomes sample A-H.
Sign
al st
reng
th (a
bsor
banc
e un
it) Sample F
Sample G
Sample H
Sample E
Retention time (min)
55
2.5.2. Comparative quantification of β-asarone (1) and α-asarone (2) by HPLC
and qNMR method
The quantities of β-asarone (1) and α-asarone (2) assessed by either HPLC or
qNMR method were shown in Table 2-11.
Table 2-11. Comparison of the quantity of β-asarone (1) and α-asarone (2) analyzed by either HPLC or qNMR methoda
Sample β-Asarone (1) (w/w %) mean ± SD (n=3)
α-Asarone (1) (w/w %) mean ± SD (n=3)
qNMRb HPLCc qNMRb HPLCc
A 4.4 ± 0.03 4.3 ± 0.13 0.065 ± 0.008 0.20 ± 0.009
B 3.0 ± 0.01 3.0 ± 0.07 0.12 ± 0.001 0.23 ± 0.006
C 2.5 ± 0.01 2.4 ± 0.08 0.083 ± 0.005 0.19 ± 0.004
D 0.82 ± 0.01 0.78 ± 0.02 0.027 ± 0.006 0.030 ± 0.003
E 1.3 ± 0.01 1.2 ± 0.01 0.33 ± 0.006 0.36 ± 0.02
F 1.2 ± 0.01 1.6 ± 0.02 0.22 ± 0.010 0.32 ± 0.015
G 1.1 ± 0.01 1.3 ± 0.02 0.23 ± 0.005 0.26 ± 0.014
H 0.91 ± 0.01 1.1 ± 0.02 0.023 ± 0.005 0.036 ± 0.002 aData are expressed as means ± SDs based on triplicate measurements. bEstimated by qNMR, cEstimated by HPLC. Samples A-E are derived from A. calamus and samples F-H are from A. gramineus.
Table 2-11 shows that the amounts of β-asarone (1) in Acorus products estimated
by qNMR can be correlated with those estimated by HPLC. On the other hand, no
correlation was found in the results of α-asarone (2) obtained by these two methods.
It was found that the values acquired by the HPLC were higher than those obtained
by qNMR. It was assumed that some impurities or minor constituents were coeluted
with α-asarone (2) leading to the UV characteristic spectrum of α-asarone (2) in
56
Acorus samples. Figure 2-24 displays the characteristic UV patterns of β-asarone
(1) and α-asarone (2) in Acorus crude extracts. Sample E and G clearly afforded UV
absorbance at 255 nm and 314 nm associated with α-asarone (2) while other samples
did not. Furthermore, the levels of α-asarone (2) from sample E and G (distinct UV
absorbance patterns) estimated by HPLC were in good agreement with those by
qNMR. Thus, it could be assumed that there were some constituents or impurities
coeluted with α-asarone (2) in some Acorus samples and HPLC-DAD method was
not reliable for quantification of α-asarone (2) in Acorus samples.
Conventional HPLC method showed only two peaks that were associated with β-
asarone (1) and α-asarone (2), but optimized qNMR method exhibited the signals
originated from other components in addition to those of the main two asarone
compounds. The quantitative results of α-asarone (2) estimated by qNMR cannot be
confirmed by comparing with those obtained by HPLC because the problem
regarding with overlapping peaks of α-asarone (2) in some Acorus products were
found in the HPLC analysis. On the other hand, the average closeness value between
the quantitative results of β-asarone (1) acquired by two methods was found to be
more than 95%. Thus, it can be concluded that optimized qNMR method was
accurate for quantification of β-asarone (1) in crude extracts from Acorus sample
products.
57
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
255 nm
304 nm
Sample (D)
Sample (C)
Sample (B)
Sample (A)
UV pattern of α-asarone
wavelength
abso
rban
ce
UV pattern of β-asarone
wavelength
abso
rban
ce
58
Figure 2-24. HPLC chromatograms showing the UV absorbance patterns of β-asarone and α-asarone in crude extracts from Acorus rhizomes sample A-H.
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
Sample (H)
Sample (G)
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
Sample (F)
240 260 280 300 320 340 360 380 400 240 260 280 300 320 340 360 380 400
Sample (E)
314 nm 255 nm 304 nm
255 nm
wavelength
abso
rban
ce
wavelength
abso
rban
ce
59
Summary of Chapter 2
Firstly, we optimized the qNMR parameters suitable for quantification of main
asarone content in Acorus products. Then, five asarone-related compounds and one
lignan were isolated from crude extracts of Acorus rhizomes. Consequently, isolated
three major asarones, β-asarone (1), α-asarone (2), and asaronaldehyde (3) were
analyzed in terms of their purities and stabilities by using the optimized qNMR
method. Finally, the quantity of β-asarone (1), α-asarone (2), and asaronaldehyde
(3) in Acorus products was estimated by qNMR and compared with that calculated
by HPLC.
As a result, 1H-qNMR method was found to usefully quantify β-asarone (1), α-
asarone (2), and asaronaldehyde (3) in the crude extracts of A. calamus and A.
gramineus. On the other hand, the conventional HPLC method was not reliable in
quantification of α-asarone (2) and unsuitable for detection of asaronaldehyde (3) in
the crude extracts. Therefore, it can be concluded that 1H-qNMR method is a reliable
alternative to the common HPLC and it can be used for quality control of Acorus
species and for purity assessment of asarone related compounds.
60
Chapter 3: In vitro acetylcholinesterase activity assay
H3C O
O
NCH3
CH3CH3
H3C OH
O
+HO
NCH3
CH3CH3
Figure 3-1. Synthesis and metabolism of acetylcholine [41-42]. Alzheimer’s disease (AD) is a degenerative neurological disorder related to the
deficiency of acetylcholine in the synapse [43-45]. Acetylcholine (ACh) is a major
neurotransmitter that plays a vital role in learning and memory, and level of ACh in
the synapse effects on memory [43]. Acetylcholine (ACh) is formed by the
condensation of acetyl CoA with choline in presynaptic neuron and the process is
catalyzed by choline acetyltransferase. Then, ACh is released from presynaptic
neuron and combined with acetylcholine receptor to release nerve impulses. After
signaling, ACh is released from the response and broken down by
acetylcholinesterase (AChE) to choline and acetate and is transported to a
H3C
O
S-CoA+
HON H3C
O
ON
CH3
CH3
CH3
CH3
CH3CH3
Acetylcholinesterase (AChE)
Acetylcholine (ACh) Acetyl CoA Choline
Acetic acid Choline
Acetylcholine synthesis
Acetylcholine metabolism
Choline acetyltransferase
Acetylcholine (ACh)
61
presynaptic neuron for continuing the recycle process (Figure 3-1). To attain the
level of ACh in the synapse, inhibition of acetylcholinesterase (AChE) which
catalyzes the degradation of ACh is a primary symptomatic treatment for
Alzheimer’s disease (AD).
AChE inhibition assay was carried out by using 96 well microplate with the slight
modification of the Ellman’s method [46]. Tacrine (AChE inhibitor) was used as a
positive control.
62
3.1. AChE inhibitory effects of crude extracts prepared from Acorus species
The AChE activities of the crude n-hexane extract from five samples of A. calamus
and three samples of A. gramineus were examined in vitro and compared with
negative and positive controls. The AChE activities were shown in Figure 3-2. It
was observed that the AChE activities of the crude extract samples (at 1 mg/mL
concentration) were lower than that of the negative control and they showed
statistical significance. Thus, it was confirmed that A. calamus and A. gramineus
extracts exhibit the inhibitory effect on AChE enzyme in vitro.
Figure 3-2. AChE activities of each crude extract from Acorus species (p ≤ 0.05).
-10
10
30
50
70
90
110
negativecontrol
tacrine A B C D E F G H
AC
hE a
ctiv
ity (%
)
A. calamus A. gramineus
** **
** ** **
** **
*
Samples concentration: 1 mg/mL Tacrine: positive control (11.75 µg/mL)
63
3.2. Correlation between the inhibitory effects of crude extracts and β-asarone
(1) contents in crude extracts
According to the previous studies on the quantification of main components in
Acorus rhizomes (section 2.4.4.), β-asarone (1) was the main constituent. As shown
in Table 3-1, all crude extracts inhibited the AChE enzyme in vitro to similar extents
(46 – 64 %), and β-asarone (1) contents in the crude extracts were found to be 46 -
64 w/w %. Moreover, the correlation coefficient between the inhibitory effects and
the levels of β-asarone (1) was estimated and showed a positive correlation
(correlation coefficient r,0.80). Therefore, the inhibitory effects on AChE of Acorus
crude extracts were mainly attributable to their main constituent, β-asarone (1).
Table 3-1. AChE inhibition (%) of crude extracts and β-asarone (1) contents in the extracts
Sample AChE inhibition (%) mean ± SD (n=3)
β-Asarone (1) contents in the extracts (w/w %) mean ± SD (n=3)
A 64 ± 4.8 64 ± 0.5
B 64 ± 5.4 64 ± 0.1
C 52 ± 3.8 60 ± 0.1
D 58 ± 2.8 61 ± 0.2
E 54 ± 2.1 57 ± 0.3
F 46 ± 3.9 46 ± 0.2
G 53 ± 5.1 53 ± 0.4
H 54 ± 4.6 62 ± 0.1
Data are expressed as mean inhibition percentages ± SDs for samples at 1 mg/mL based on triplicate experiments. Samples A-E are derived from A. calamus and samples F-H are from A. gramineus.
64
3.3. Contribution of the principal constituents on AChE inhibition activity
A contribution study was carried out with isolated six constituents (mentioned in
section 2.2.1.) and their AChE inhibitory activities in vitro were examined. Table 3-
2 shows that β-asarone (1) exerts a potent inhibitory effect on AChE than other
asarone-related compounds (compound 2-5) at its 10 µg/mL concentration.
Moreover, α-asarone (2), a geometric isomer of β-asarone (1), showed the lowest
inhibitory activity on AChE. Thus, it was confirmed that the inhibitory effects of
crude extracts were attributable principally to the major constituent, β-asarone (1)
as previously suggested in the study.
Table 3-2. AChE inhibitory effects of constituents isolated from Acorus crude extracts
Samples AChE inhibition (%)a
mean ± SD (n=3) β-Asarone (1) 76.5 ± 0.2
α-Asarone (2) 18.9 ± 10.2
Asaronaldehyde (3) 42.1 ± 8.2
Dihydroxyasarone (4) 32.7 ± 7.9
Acoraminol B (5) 27.3 ± 3.8
Eudesmin A (6) −16.2 ± 8.5 aData are expressed as mean inhibition percentages ± SDs for samples at 10 µg/mL, based on data obtained by triplicate experiments.
65
N
NH2
CH3
OCH3
OCH3
H3CO
OCH3
H3COOCH3
CHOH3C
OCH3
OCH3
H3CO
H3CO
H3COOCH3
CH3
OH
OH
H3CO
H3COOCH3
CH3
OCH3
OH
Figure 3-3. AChE inhibitory effects of asarone-related compounds in Acorus species.
β-Asarone (1) Asaronaldehyde (3) Dihydroxyasarone (4)
Acoraminol B (5) α-Asarone (2)
Inhibition % at 10 µg/mL concentration decrease
Tacrine (positive control)
66
3.4. IC50 values of the main constituent, β-asarone and Acorus crude extract B
IC50 values of the main constituent, β-asarone (1), one crude extract (sample B) and
positive control, tacrine were evaluated in this study. For IC50 analyses, at least five
DMSO solutions of each sample with varied concentrations were prepared and their
inhibitory effects on AChE were examined. The values of IC50 for the respective
sample were determined in (Table 3-3). It was indicated that the IC50 value of β-
asarone (1) was comparable to those of tacrine, positive control (commercially
available AChE inhibitor). On the other hand, IC50 values of crude extract B and
main constituent, β-asarone (1) showed a notable difference although the extract
contained a large amount of β-asarone (1). It can be assumed that there are some
constituents which retard the inhibitory effect of crude extract B on AChE.
Table 3-3. IC50
values of β-asarone, the crude extract B, and tacrine on AChE
inhibition
Samples IC50 of AChE (µg/mL)
Crude extract B 73.7
β-Asarone 0.61 (2.93 µM)
Tacrine (positive control) 0.14 (0.59 µM)
67
In the contribution study of constituents on AChE enzyme activity, eudesmin A (6)
showed the enhancing effect on AChE activity at its 10 µg/mL concentration (Table
3-2). Since the content of eudesmin A (6) in crude extract B was quite lower than
that of β-asarone (1), its actual level was also estimated by HPLC after partial
purification of extract B (Figure 3-4). The amount of eudesmin A (6) in the 1 mg/mL
extract of sample B was estimated to be 9.3 µg/mg, and it was close to the level that
showed the enhancement of the AChE activity in Table 3-2. Thus, the reason for the
significant difference in the IC50 values of crude extract B and β-asarone (1) is
attributable, in part, to the AChE-enhancing effect of eudesmin A (6).
Figure 3-4. HPLC profile of 100 % MeOH fr. obtained by silica gel chromatography of extract B indicating the presence of eudesmin A detected at 280 nm.
68
3.5. Concentration-dependence in the effects of eudesmin A (6) on the in vitro
AChE activity
A further study on the enhancing effects of eudesmin A (6) on the AChE activity
was made with different concentrations. The AChE activities of eudesmin A (6)
were found to be higher than that of the negative control, and statistical significance
was observed. Table 3-4 demonstrates the concentration-dependent inhibition of
eudesmin A (6).
Figure 3-5. Concentration-dependent effects of eudesmin A on AChE activity (p ≤ 0.05). It was found that a higher concentration of eudesmin A would enhance the AChE
enzymatic activity. However, the mechanism of the activation of AChE exerted by
eudesmin A (6) remains unclear, and it was speculated that eudesmin A (6) might
interact with allosteric activator site located on the peripheral anion active site of
AChE enzyme [47-48]. Enhancing effects of some monoterpenoids on AChE have
also been reported in previous research [49]. Therefore, enhancing effect of
-20
30
80
130
180
230
280
negativecontrol
tacrine23.5µg/mL
eudesmin A 10μg/mL
eudesmin A 100μg/mL
eudesmin A 1000μg/mL
AC
hEac
tivity
**
**
69
eudesmin A is the second report for the enhancing effect by natural compounds on
AChE enzyme.
Table 3-4. Concentration-dependent effects of eudesmin A (6) on AChE
Compound Concentration
(µg/mL)
AChE inhibition (%)
mean ± SD (n=3)
Eudesmin A
10 − 16 ± 8.5
100 − 64 ± 12
1000 − 3.4 x 102 ± 2.0
Data are expressed as the mean inhibition % ± SDs based on data obtained from triplicate experiments. Moreover, there have been no reports on the activities of eudesmin A yet, while
several pharmacological activities such as the antitumor effects on lung cancer,
attenuation of the Helicobacter pylori-induced epithelial autophagy, and
anticonvulsant and sedative effects have been reported for eudesmin, an isomer of
eudesmin A [50 - 52]. Thus, further studies on biological activities of eudesmin A
are needed.
70
Summary of Chapter 3
An in vitro bioactivity of the crude n-hexane extracts obtained from both species of
Acorus and their principal constituents was examined on AChE enzyme. It was
revealed that the crude extracts of A. calamus and A. gramineus rhizomes moderately
inhibited the AChE enzyme in vitro. Their principal constituent, β-asarone (1), was
mainly responsible for the inhibitory effects. Moreover, the notable difference
between the IC50 values of crude extract B and β-asarone (1) was observed. It was
attributable to the AChE enhancing effect of eudesmin A (6), a compound first
isolated from Acorus species.
71
Conclusion
In this research, the 1H-qNMR method was applied as a new method for the
estimation of purity and quantification of major asarone-related compounds. It was
concluded that optimized qNMR method enables simple simultaneous quantification
of β-asarone, α-asarone and asaronaldehyde by using DMT as an internal standard
(I.S.). It was also found that the optimized qNMR method has advantages over the
HPLC-DAD method. First, qNMR method is appeared to be more accurate and
easier to perform. Second, the qNMR method is time-saving than the HPLC-DAD
method. Third, qNMR method is highly selective in the determination of β-asarone
(1). Thus, the qNMR method could be used as a reliable alternative for quality
assessment of Acorus herbal products.
In bioactivity assay, it was found that crude n-hexane extracts of both A. calamus
and A. gramineus rhizomes inhibited AChE enzyme and the inhibition was caused
by their principal constituent, β-asarone. β-asarone showed more potent inhibitory
activity than other asarone-related compounds and its activity was found to be
comparable to that of tacrine (commercially available AChE inhibitor). It has been
reported that β-asarone attenuates the toxicity of amyloid-β protein and serve as a
promising preventive agent for AD [53-54]. Thus, β-asarone may be a valuable lead
compound for AD. On the other hand, β-asarone is a hepatic carcinogen and the
Committee on Herbal Medicinal Products (HMPC) of the European Medicines
Agency limits the exposure of β-asarone and α-asarone in herbal products to 115
µg/day [55-56]. Therefore, the quantitative measurement of the main asarone levels
in Acorus herbal products is essential for quality control and safety assessment for
the usage of Acorus herbal products. Finally, the present study has revealed that the
72
eudesmin A suppresses the inhibitory activity of Acorus crude extracts towards
AChE enzyme.
73
Chapter 4: Experimental section
4.1. Instruments
1H-NMR spectra were recorded on a Varian INOVA 600 AS instrument (600 MHz
for 1H, and 151 MHz for 13C), and signal chemical shifts were recorded as delta (δ)
values from tetramethylsilane, referenced to the residual solvent signals at δH:7.26,
δC:77 [CDCl3], δH:2.05, δC:29.8 [acetone-d6], δH:3.31, δC:49.8 [MeOH-d4],
respectively. Electrospray-ionization mass spectrometry (ESI-MS) measurements
were performed using an amaZon ETD/X instrument (Bruker). Optical rotation and
electronic circular dichroism (ECD) spectra were measured using a DIP-1000 digital
polarimeter and a J-720W spectrometer (JASCO), respectively. UV spectra were
measured by Shimadzu UV-1800. A model 680 microplate reader (Bio-Rad) was
used for conducting the AChE inhibition assay.
Column chromatography was performed by using YMC-gel-Sil, ODS-A gel, Bond-
Elut C18 cartridge column, MCI-CHP20P gel, Diaion HP-20, and Toyopearl HW-
40F, respectively. Sep-Pak C18 was used for purification after preparative HPLC.
The solvent was removed with a rotary evaporator at 40˚C.
74
HPLC analysis conditions were described in the following.
NP-HPLC
Column: YMC-Pack Sil A-003 (250 x 4.6 mm I.D)
Detector: L-7405 HITACHI (254 nm)
Pump: LC-10AT SHIMADZU
Temp: r.t
Flow rate: 1.5 mL/min
Mobile phase:
[N1] n-Hexane: EtOAc = 9: 1
[N2] n-Hexane: EtOAc: 0.1% HCOOH= 9: 1: 0.1%
RP-HPLC
Column: YMC-Pack ODS-A302 (150 x 4.6 mm I.D.)
Detector: L-7400 HITACHI (254, 280 nm)
Pump: L-7100 HITACHI
Temp: 40˚C
Flow rate: 1.0 mL/min
Mobile phase:
[R1] H2O: MeOH: HCOOH = 7: 3: 0.1%
[R2] H2O: MeOH: CH3CN: HCOOH = 7: 1.5: 1.5: 0.1%
[R3] H2O: MeOH: HCOOH = 5.5: 4.5: 0.1%
[R4] H2O: MeOH: HCOOH = 5: 5: 0.1%
[R5] H2O: MeOH: HCOOH = 4: 6: 0.1%
[R6] H2O: MeOH: HCOOH = 3: 7: 0.1%
[R7] H2O: MeOH: CH3CN: HCOOH = 4: 3: 3: 0.1%
[R8] 0.01M H3PO4: 0.01M KH2PO4: MeOH = 47.5: 47.5: 5
75
Prep NP-HPLC
Column: YMC-Pack Sil A024 (300 x 10 mm I.D.)
Detector: L-7405 HITACHI (254 nm)
Pump: LC-10AT SHIMADZU (3.0 mL/min)
Mobile phase:
[PN1] n-Hexane: EtOAc = 9: 1
[PN2] n-Hexane: EtOAc: MeOH = 7: 3: 0.1%
Prep RP-HPLC
Column: YMC-Pack Sil A324 (300 x 10 mm I.D.)
Detector: L-7400 HITACHI (254, 280, 354 nm)
Pump: L-7100 HITACHI (2.0 mL/min)
Temp: 40˚C
Mobile phase:
[PR1] H2O: MeOH: HCOOH = 4.5: 5.5: 0.1%
TLC conditions were showed in following.
Plate: Silica gel 60 F254 (Aluminium Sheet)
Mobile phase:
[T1] n-Hexane: EtOAc: AcOH = 3: 2: 1
76
4.2. Plant materials
Sample Species Lot No. Collected place
A A. calamus - National herbal garden, Nay Pyi Taw, Myanmar
B A. calamus #034816001 Tochimoto-tenkai-do, Osaka, Japan
C A. calamus #034815002 Tochimoto-tenkai-do, Osaka, Japan
D A. calamus #024415001 Tochimoto-tenkai-do, Osaka, Japan
E A. calamus #024417001 Tochimoto-tenkai-do, Osaka, Japan
F A. gramineus #T5KC5K16E Kinokuniya Pharmacy, Tokyo, Japan
G A. gramineus #T7DE7D17E Kinokuniya Pharmacy, Tokyo, Japan
H A. gramineus #46428202 Horie Shoyaku, Osaka, Japan
77
4.3. Extraction and fractionation of Acorus species
Pulverized rhizome sample A (4.2 g) in 40 mL of n-hexane was sonicated for 15
min at room temperature and filtered. This procedure was repeated for three times,
and the combined solution was concentrated in vacuo to yield a crude extract (225
mg). A portion (195 mg) of the extract A was separated on silica gel column
chromatography with the gradient elution of EtOAc in n-hexane (10:0, 9:1, 8:2, 5:5,
0:10, all v/v) and then washed with 100% MeOH. The n-hexane/EtOAc (9:1 v/v)
(32 mg) eluate was further purified via PN-1 to yield β-asarone (1) (19 mg). The
crude n-hexane extract (983.0 mg) from sample A (15 g) was similarly
chromatographed, and the 100% MeOH eluate was further purified on a Bond-Elut
C18 cartridge column eluted with increasing amounts of MeOH in H2O (3:7, 4:6,
5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, v/v) to yield dihydroxyasarone (4) (0.5 mg) from
30% MeOH fr. and acoraminol B (5) (1.0 mg) from 80% MeOH.
The crude n-hexane extract (1.0 g) from sample B (20 g) was also treated
analogously to yield compound 4 (0.7 mg). Pulverized rhizome powder (200 g) of
sample B was macerated in n-hexane for 24 hours three times to obtain 7.8 g of crude
extract and followed by silica gel column, analogously. From this extract, 100%
MeOH fraction was further purified by ODS-gel column with the gradient elution of
MeOH in H2O (3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, v/v) and followed by PN-2
of 60% MeOH fraction to yield a lignan, eudesmin A (6) (1.6 mg).
The n-hexane extract (144 mg) obtained from pulverized sample E (6.0 g) was
separated by silica gel column chromatography eluting with mixtures of n-
hexane/EtOAc (10:0, 9:1, 8:2, and 5:5, v/v) and 100% MeOH. The n-hexane/EtOAc
(8:2) eluate (20 mg) was further purified by PR-1 to yield α-asarone (2) (2.0 mg).
78
Pulverized sample C (103.6 g) was homogenized in MeOH for 20 min three times
to yield a MeOH extract (16.9 g). Most (16.3 g) of this extract was chromatographed
on silica gel and eluted with n-hexane/EtOAc mixtures (10:0, 9:1, 8:2, 5:5, and 0:10,
v/v). Then the n-hexane/EtOAc (5:5) eluate (90 mg) was chromatographed on MCI-
gel CHP-20P gel eluted with MeOH/H2O (5:5, 6:4, 7:3, and 10:0, v/v), and the
MeOH/H2O (7:3 v/v) eluate yielded asaronaldehyde (3) (5.1 mg).
79
4.4. Physical properties of compounds from Acorus species
β-Asarone (1) [28]
Pale-yellow oil
UV λmax (in CHCl3) nm (log ε): 307 (3.76)
ESI-MS m/z: 209 [M+H]+
1H-NMR (in CDCl3) δH: 1.84 (3H, dd, J=1.8, 6.6 Hz, H-3’), 3.81, 3.84, 3.90 (3H
each, s, 3 x -OCH3), 5.77 (1H, dd, J=6.6, 11.4 Hz, H-2’), 6.48 (1H, dd, J=1.8, 11.4
Hz, H-1’), 6.53 (1H, s, H-3), 6.84 (1H, s, H-6).
Figure 4-1. 1H NMR spectrum of β-asarone (1) (600 MHz, CDCl3)
OCH3
H3CO
H
H3CH
OCH3
80
α-Asarone (2) [28]
Pale-yellow oil
UV λmax (in CHCl3) nm (log ε): 314 (3.81)
ESI-MS m/z: 209 [M+H]+ 1H-NMR (in CDCl3,) δH: 1.89 (3H, dd, J=1.8, 6.6 Hz, H-3’), 3.81, 3.84, 3.88 (3H
each, s, 3 x -OCH3), 6.10 (1H, dd, J=6.6, 15.6 Hz, H-2’), 6.49 (1H, s, H-3), 6.65
(1H, dd, J=1.8, 15.6 Hz, H-1’), 6.94 (1H, s, H-6).
Figure 4-2. 1H NMR spectrum of α-asarone (2) (600 MHz, CDCl3)
OCH3
H3CO
OCH3
CH3
HH
81
Asaronaldehyde (3) [25]
Colorless needles
UV λmax (in CHCl3) nm (log ε): 275 (3.97), 340 (3.87)
ESI-MS m/z: 219 [M+Na]+ 1H-NMR (in CDCl3) δH: 3.88, 3.92, 3.97 (3H each, s, 3 x -OCH3), 6.49 (1H, s, H-3),
7.32 (1H, s, H-6), 10.31 (1H, s, -CHO).
Figure 4-3. 1H NMR spectrum of asaronaldehyde (3) (600 MHz, CDCl3)
CHO
OCH3
OCH3
H3CO
82
Dihydroxyasarone (4) [25]
Colorless oil
[α]20 D (c 0.7, MeOH) –16.1
UV λmax (in MeOH) nm (log ε): 234 (3.66), 290 (3.30)
ESI-MS m/z: 265 [M+Na]+
1H-NMR (in CD3OD) δH: 1.01 (3H, d, J=6 Hz), 3.79, 3.81, 3.84 (3H each, s, 3 x
-OCH3), 4.76 (1H, d, J=6.6 Hz, H-1’), 6.65 (1H, s, H-3), 7.03 (1H, s, H-6). The H-
2’ signal is overlapped with the solvent signal.
Figure 4-4. 1H NMR spectrum of dihydroxyasarone (4) (600 MHz, MeOH-d4)
CH3
OH
OH
OCH3
H3CO
OCH3
83
Acoraminol B (5) [25]
Colorless oil
[α]20 D (c 0.6, MeOH) –2.7
UV λmax (in MeOH) nm (log ε): 287 (2.74)
ESI-MS m/z: 255 [M−H]– 1H-NMR (in CD3COCD3-D2O, 9:1) δ: 0.95 (3H, d, J= 6.6 Hz, H-3’), 3.26 (3H, s,
-OCH3), 3.71, 3.76, 3.79 (3H each, s, 3 x -OCH3), 3.91 (1H, dq, J=4.2, 6.6 Hz,
H-2’), 4.93 (1H, d, J= 3.6 Hz, H-1’), 6.63 (1H, s, H-3), 7.07 (1H, s, H-6).
Figure 4-5. 1H NMR spectrum of acoraminol B (5) (600 MHz, Acetone d6-D2O, 9:1)
CH3
OCH3
OH
OCH3
H3CO
OCH3
84
Eudesmin A (6) [38-39]
Pale-yellow amorphous powder
[α]20 D (c 1.0, MeOH) +22.6
UV λmax (in MeOH) nm (log ε): 203 (4.60), 233 (3.92), 280 (3.47)
CD (MeOH): [θ]207 +2.27 x 104, [θ]225 +0.42 x 104
ESI-MS m/z: 386 [M+Na]+ 1H-NMR (in CD3OD) δ: 3.15 (2H, m, J=4.8 Hz, H-8, 8’), 3.82 (6H, s, 5, 5’-OCH3),
3.84 (6H, s, 3, 3’-OCH3), 3.87 (2H, dd, J=3.6, 9 Hz, H-9, 9’b ax), 4.25 (2H, dd,
J=7.2, 9 Hz, H-9, 9’a eq), 4.75 (2H, brd, J=4.2 Hz, H-7, 7’), 6.93 (4H, s, H-2, 2’, 6,
6’), 6.98 (2H, s, H-4, 4’). 13C-NMR (in CD3OD) δ: 55.4 (C-8, 8’), 56.5 (-OCH3 x 4), 72.7 (C-9, 9’), 87.3 (C-
7, 7’), 111.2 (C-6, 6’), 112.9 (C-4, 4’), 119.8 (C-2, 2’), 135.3 (C-1, 1’), 150.2 (C-5,
5’), 150.7 (C-3, 3’).
85
Figure 4-6. 1H and 13C NMR spectra of eudesmin A (6) (600 MHz, MeOH-d4)
H3CO
OCH3
O
O
OCH3
OCH3H
H
86
4.5. Component analyses
4.5.1. Purity and content determinations by qNMR analyses
qNMR spectra were recorded in the non-spinning mode at a probe temperature of
298.1K (25.0˚C) on a spectrometer using the following parameters:
Parameters Value
Excitation pulse 9.9 µs; 90˚ tip angle
Receiver gain 30 (autogain)
Operating frequency 599.76 MHz
Spectral Width (SW) 20 ppm
Relaxation Delay (D1) 40 s
Spin Off
VT 25˚ (Constant)
Apodization 0.3 Hz (exponential line broadening)
Digital resolution 0.2 Hz/pt
Number of scans (ns) 16 (S/N > 150)
Total runtime 11 min
Data processing Vnmrj qEstimate software
87
Signal chemical shifts were recorded as delta (δ) values from tetramethylsilane,
referenced to the residual solvent (CDCl3) signal at δ 7.26. Data were processed
using Vnmrj ver. 4.2 qEstimate software. Purity values were calculated by using the
equation (1) and content determinations by equation (2).
------------Eq (1)
------------------------------Eq (2)
Whereas, I: integral area H: number of protons MW: molecular weight MC: molar concentration P: purity x: analyte sample std: internal standard
Ix x MWx x Hx x CstdIstd x MWstd x Hstd x Cx
Px x P
std =
= Ix
Istd MCx x Hx
MCstd x Hstd
88
4.5.2. HPLC analyses
A published HPLC method [27] was slightly modified to quantify β-asarone (1) and
α-asarone (2) in crude extracts. Isolated pure β-asarone (1) and α-asarone (2) were
used for calibration lines as follows. β-Asarone was dissolved in MeOH to give a 1
mg/mL stock solution. Then, it was serially diluted to 0.5, 0.25, 0.1, and 0.05
mg/mL, and each standard solution (2 µL) was injected three times to establish a
calibration line. A calibration line for α-asarone (2) was also prepared analogously.
The concentrations of β-asarone (1) and α-asarone (2) were adjusted after the
estimation of its purity by qNMR. The regression line of β-asarone was y= 2036.5x
+ 3198.3, R² = 0.9993 and that of α-asarone was y = 2058.6x + 8764.5, R² = 0.9996
(Figure 4-7). Each Acorus rhizome extract in MeOH (1 mg/mL) was injected three
times and contents were quantified based on their calibration lines.
89
Figure 4-7. Calibration curves of (a) β-asarone, (b) α-asarone
y = 2036.5x + 3198.3R² = 0.9993
0
500000
1000000
1500000
2000000
2500000
0 200 400 600 800 1000
Peak
are
a
Concentrations (µg/mL)
β-asarone
y = 2058.6x + 8764.5R² = 0.9996
0200000400000600000800000
100000012000001400000160000018000002000000
0 200 400 600 800 1000
Peak
are
a
Concentration (µg/mL)
α-asarone
(a)
(b)
90
Eudesmin A was dissolved in distilled MeOH to get 1mg/mL, and serially diluted
with MeOH to give 0.5, 0.25, 0.125, and 0.05 mg/mL solutions. Each standard
solution (2 µL) was injected three times to establish a calibration line and gave the
regression line y = 314663x + 5746.7, R² = 0.9992 (Figure 4-8).
100% MeOH fraction of extract B (1mg/mL in MeOH) was injected three times by
using gradient elution profile with solvent A [H2O – MeOH 7:3 (v/v) containing 0.1
% HCOOH] and solvent B [H2O – MeOH 3:7 (v/v) containing 0.1 % HCOOH] was
used: 0% B (in A+B) at 0 – 5 min, 10% B at 5 – 10 min, 20% B at 10 – 25 min, 30%
B at 25 – 35 min, 50% B at 35 – 45 min, 70% B at 45 – 55 min, 80% B at 55 – 65
min, 90% B at 65 – 75 min, 100% B at 75 – 90 min.
Figure 4-8. Calibration curve of eudesmin A
y = 314663x + 5746.7R² = 0.9992
0
50000
100000
150000
200000
250000
300000
350000
0 0.2 0.4 0.6 0.8 1 1.2
Peak
are
a
concentration (mg/mL)
Eudesmin A
91
4.6. Bioactivity test (Acetylcholinesterase inhibition assay)
AChE inhibition was measured by using a slight modification of Ellman’s method
[46]. The reaction mechanism of AChE assay is based on the principle that the
substrate, acetylthiocholine iodide, is hydrolyzed by AChE enzyme to afford acetic
acid and thiocholine. The thiocholine reacts with Ellman’s reagent, DTNB, to
provide the yellow color reaction product TNB, which was detected with an
absorbance at 415nm (Figure 4-9).
SN
CH3
CH3CH3
O I
+ H2O AChEOH
O+
SN
CH3
CH3CH3 + 2H
SN
CH3
CH3CH3 +
S SHOOC
O2N
COOH
NO2
SS
NCH3
CH3CH3
HOOC
O2N+
COOH
NO2
S
Figure 4-9. Reaction mechanism of AChE assay in vitro
Based on the above-mentioned reaction mechanism, AChE inhibition assay was
performed as follows:
AChE (0.5 U/mL) (10 µL) was added to a mixture of 0.1 M MOPS buffer (pH 7.4)
(150 µL) and a test compound solution in DMSO (20 µL) in 96-well microplates,
and the solutions were preincubated at 25˚C for 10 min. The mixture was further
incubated for 30 min at 25˚C after adding ATCI (75 mM) (10 µL) and DTNB (10
Acetylthiocholine iodide Thiocholine
DTNB (5,5’-Dithiobis (2-nitrobenzoic acid))
TNB
92
mM) (10 µL) to a final volume of 200 µL per well, and then the absorbance at 415
nm was measured. In this experiment, tacrine (commercially available AChE
inhibitor) was used as a positive control.
Inhibition (%) was estimated using the following equation:
Inhibition % = [(CE-CB) - (SE-SB)]/(CE-CB) x 100 (%)
Whereas, CE = Control (with AChE),
CB = Control (without AChE),
SE = Test compound (with AChE),
SB = Test compound (without AChE)
4.7. Statistical analysis
Statistical evaluations were performed using Graph-pad prism version 8 (GraphPad
Software, Inc., San Diego, CA, USA). The data were subjected to analysis of
variance (ANOVA), and a significant difference between means was calculated by
Turkey’s t-test. If the P value is less than 0.05, the data were considered statistically
significant.
93
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101
Paper and Conference Presentations
1. Paper 1H Quantitative NMR analyses of β-asarone and related compounds for
quality control of Acorus rhizome herbal drugs in terms of the effects of their
constituents on in vitro acetylcholine esterase activity. Nwe Haymar Lynn,
Thein Zaw Linn, Cui Yanmei, Yuuki Shimozu, Shoko Taniguchi, and
Tsutomu Hatano. Bioscience, Biotechnology and Biochemistry, accepted
(2019) (IF 2017 1.255).
2. Conference Presentation
Constituents of Artemisia capillaris flowers (Part 2)
Nwe Haymar Lynn, Shoko Taniguchi, Kohei Mishima, Kaori Shimamura,
Yuuki Shimozu, Tsutomu Hatano
65th annual meeting of Japanese Society of Pharmacognosy (2018-09-16).
This thesis is derived in part from an article published in ‘Bioscience,
Biotechnology, and Biochemistry’ (2019) available online: http://www. tandfonline.
com /10.1080/ 09168451. 2019. 1569493.
102
Acknowledgment
This doctoral dissertation was completed with the support of several people. Firstly,
I would like to express my deepest gratitude to my major supervisor, Prof. Tsutomu
Hatano, for the valuable support of my Ph.D. study, especially his guidance,
patience, and valuable suggestions. My research would have been impossible
without his guidance. I would also like to thank Dr. Shoko Taniguchi who always
taught me and gave her precious time for discussions during my doctoral course. My
sincere thank goes to Dr. Yuuki Shimozu who often taught me experimental
techniques, gave encouragement, and open for discussions.
I am very indebted to Japanese Government (Ministry of Education, Culture, Sport
and Technology) for providing financial support for my study in Japan. I would like
to extend my gratitude to the Department of Natural Product Chemistry, and SC-
NMR laboratory, Okayama University for all facilities provided. I thank Dr. Thein
Zaw Linn, Former Director of Traditional medicine Department, Ministry of Health,
who support the plant material for my research.
Finally, my deepest thanks to my parents, sisters, and brother for encouraging
throughout my study.
2019.3
Nwe Haymar Lynn