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

References

(1) J. L. Jungnickel, J. W. Forbes, Quantitative measurement of hydrogen types

by integrated nuclear magnetic resonance intensities. Anal. Chem., 35, 938-

942 (1963).

(2) R. Watanabe, C. Sugai, T. Yamazaki, R. Matsushima, H. Uchida, M.

Matsumiya, A. Takatsu, T. Suzuki, Quantitative nuclear magnetic

resonance spectroscopy based on pulcon methodology: Application to

quantification of invaluable marine toxin, okadaic acid. Toxins, 8, 294-302

(2016).

(3) S. K. Chauthe, R. J. Sharma, F. Aqil, R. C. Gupta, I. P. Singh, Quantitative

NMR: An applicable method for quantitative analysis of medicinal plant

extracts and herbal products. Phytochem. Anal., 23, 689-696 (2012).

(4) F. Malz, H. Jancke, Validation of quantitative NMR. J. Pharm. Biomed.

Anal., 38, 813-823 (2005).

(5) G. F. Pauli, B. U. Jaki, D. C. Lankin, Quantitative 1HNMR: Development

and potential of a method for natural products analysis. J. Nat. Prod., 68,

133-149 (2005).

(6) S. Sun, M. Jin, X. Zhou, J. Ni, X. Jin, H. Liu, Y. Wang, The application of

quantitative 1H-NMR for the determination of orlistat in tablets. Molecules,

22, 1517 (2017).

(7) Z. Y. Li, E. Welbeck, R. F. Wang, Q. Liu, Y. B. Yang, G. X. Chou, K. S.

Bi, Z. T. Wang, A universal quantitative 1H nuclear magnetic resonance

(qNMR) method for assessing the purity of dammarane-type ginsenosides.

Phytochem. Anal., 26, 8-14 (2014).

94

(8) S. K. Bharti, R. Roy, Quantitative 1H NMR spectroscopy. Trends. in. Anal.

Chem., 35, 5-26 (2012).

(9) D. P. Hollis, Quantitative analysis of aspirin, phenacetin and caffeine

mixtures by nuclear magnetic resonance spectrometry. Anal. Chem., 35,

1682-1684 (1963).

(10) T. Godecke, J. G. Napolitano, M. F. Rodriguez-Brasco, S. N. Chen, B. U.

Jaki, D. C. Lankin, G. F. Pauli, Validation of a generic quantitative 1H NMR

method for natural products analysis. Phytochem. Anal., 24, 581-597

(2013).

(11) R. Tanaka, R. Inagaki, N. Sugimoto, H. Akiyama, A. Nagatsu, Application

of a quantitative 1H-NMR (1H-qNMR) method for the determination of

geniposidic acid and acetoside in Plantaginis semen. J. Nat. Med., 71, 315-

320 (2017).

(12) R. Tanaka, H. Shibata, N. Sugimoto, H. Akiyama, A. Nagatsu, Application

of a quantitative 1H-NMR method for the determination of paeonol in

Moutan cortex, Hachimijiogan and Keishibukuryogan. J. Nat. Med., 70,

797-802 (2016).

(13) T. Schonberger, Y. B. Monakhova, D. W. Lachenmeier, T. Kuballa, Guide

to NMR Method Development and Validation-Part1: Identification and

quantification. Eurolab Technical report, (2014). http://www.eurolab.org/

documents/EUROLAB%20Technical%20Report%20NMR%20Method%

20Development%20and%20Validation%20May%202014_final.pdf.

(14) D. Holmes, Basic Practical NMR Concepts: A Guide for the Modern

Laboratory. Michigan State University, (2013). https://www2.chemistry.

msu.edu/facilities/nmr/handouts/DH%20NMR%20Basics.pdf.

95

(15) J. Reich, Relaxation in NMR Spectroscopy. University of Wisconsin,

(2010). https://www.chem.wisc.edu/areas/reich/nmr/08-tech-01-relax.htm.

(16) H. Shu, S. Zhang, Q. Lei, J. Zhou, Y. Ji, B. Luo, L. Hong, F. Li, B. Liu, C.

Long, Ethnobotany of Acorus in China. Acta. Soc. Bot. Pol., 87, 3585

(2018).

(17) S. B. Rajput, M. B. Tonge, S. M. Karuppayil, An overview on traditional

uses and pharmacological profile of Acorus calamus Linn. (Sweet flag) and

other Acorus species. Phytomedicine, 21, 268-276 (2014).

(18) X. L. Feng, Y. Yu, D. P. Qin, H. Gao, X. S. Yao, Acorus Linnaeus: A review

of traditional uses, phytochemistry and neuropharmacology. RSC. Adv., 5,

5173 (2015).

(19) J. Sugimoto, Keys to herbaceous plants of Japan: II Monocotyledoneae. 230

(1973).

(20) H. Lim, S. Y. Lee, K. R. Lee, Y. S. Kim, H. P. Kim, The rhizomes of Acorus

gramineus and the constituents inhibit allergic response in vitro and in vivo.

Biomol. Ther., 20, 477-481 (2012).

(21) A. Ahlawat, M. Katoch, G. Ram, A. Ahuja, Genetic diversity in Acorus

calamus L. as revealed by RAPD markers and its relationship with β-

asarone content and ploidy level. Sci. Hortic., 124, 294-297 (2010).

(22) Ministry of Health and Sport, Myanmar Herbal Pharmacopoeia, 1, 1-6

(2013).

(23) D. Zuba, B. Byrska, Alpha- and beta-asarone in herbal medicinal products:

A case study. Forensic. Sci. Int., 223, e5-e9 (2012).

96

(24) K. M. Hanson, M. Gayton-Ely, L. A. Holland, P. S. Zehr, B. C. G.

Soderberg, Rapid assessment of β-asarone content of Acorus calamus by

micellar electrokinetic capillary chromatography. Electrophoresis, 26, 943-

946 (2005).

(25) C. H. Park, K. H. Kim, L. K. Lee, S. Y. Lee, S. U. Choi, J. H. Lee, K. R.

Lee, Phenolic constituents of Acorus gramineus. Arch. Pharm. Res., 34,

1289-1296 (2011).

(26) J. Y. Lee, J. Y. Lee, B. S. Yun, B. K. Hwang, Antifungal activity of beta-

asarone from rhizomes of Acorus gramineus. J. Agric. Food. Chem., 52,

776-780 (2004).

(27) S. Shailajan, S. Menon, G. Swar, D. Singh, S. Nair, Estimation and

quantitation of β-asarone from Acorus calamus rhizome and its

formulations using validated RP-HPLC method. Pharm. Methods., 6, 94-

99 (2015).

(28) H. L. Zuo, F. Q. Yang, X. M. Zhang, Z. N. Xia, Separation of cis- and trans-

asarone from Acorus tatarinowii by preparative gas chromatography. J.

Anal. Methods. Chem., 2012, 1-6 (2012).

(29) X. M. Bai, T. Liu, D. L. Liu, Simultaneous determination of α-asarone and

β-asarone in Acorus tatarinowii using excitation-emission matrix

fluorescence coupled with chemometric methods. Spectrochim. Acta. A.

Mol. Biomol. Spectrosc., 191, 195-202 (2018).

(30) B. L. Sun, W. W. Li, C. Zhu, W. S. Jin, F. Zeng, Y. H. Liu, X. L. Bu, J.

Zhu, X. Q. Yao, Y. J. Wang, Clinical research on Alzheimer’s disease:

Progress and perspectives. Neurosci. Bull., 34, 1111-1118 (2018).

97

(31) M. J. R. Howes, P. J. Houghton, Plants used in Chinese and Indian

traditional medicine for improvement of memory and cognitive function.

Pharm. Biochem. Beh., 75, 513-527 (2003).

(32) F. Ahmed, N. S. Chandra, A. Urooj, K. S. Rangappa, In vitro antioxidant

and anticholinesterase activity of Acorus calamus and Nardostachys

jatamansi rhizomes. J. Pharm. Res., 2, 830-833 (2009).

(33) B. Lee, B. Sur, S. G. Cho, M. Yeom, I. Shim, H. Lee, D. H. Hahm, Effect

of beta-asarone on impairment of spatial working memory and apoptosis in

the hippocampus of rats exposed to chronic corticosterone administration.

Biomol. Ther., 23, 571-581 (2015).

(34) Y. X. Yang, Y. T. Chen, X. J. Zhou, C. L. Hong, C. Y. Li, J. Y. Guo, Beta-

asarone, a major component of Acorus tatarinowii schoot, attenuates focal

cerebral ischemia induced by middle cerebral artery occlusion in rats. BMC.

Complement. Altern. Med., 13, 236-242 (2013).

(35) J. Liu, C. Li, G. Xing, M. Dong, Y. Geng, X. Li, J. Li, G. Wang, D. Zou,

Y. Niu, Beta-asarone attenuates neuronal apoptosis induced by beta-

amyloid in rat hippocampus. Yakugaku. Zasshi., 130, 737-746 (2010).

(36) P. K. Mukherjee, V. Kumar, M. Mal, P. J. Houghton, In vitro

acetylcholinesterase inhibitory activity of the essential oil from Acorus

calamus and its main constituents. Planta. Med., 73, 283-285 (2007).

(37) Y. B. Monakhova, B. W. K. Diehl, Practical guide for selection of 1H

qNMR acquisition and processing parameters confirmed by automated

spectra evaluation. Magn. Reason. Chem., 1-10 (2017).

98

(38) P. Parhoodeh, M. Rahmani, N. M. Hashim, M. A. Sukari, G. E. C. Lian,

Lignans and other constituents from aerial parts of Haplophyllum Villosum.

Molecules, 16, 2268-2273 (2011).

(39) Y. Lu, Y. Xue, J. Liu, G. Yao, D. Li, B. Sun, J. Zhang, Y. Liu, C. Qi, M.

Xiang, Z. Luo, G. Du, Y. Zhang, (±)-Acortatarinowins A-F, norlignan,

neolignan and lignan enantiomers from Acorus tatarinowii. J. Nat. Prod.,

78, 2205-2214 (2015).

(40) A. Shrivastava, V. P. Gupta, Methods for the determination of limit of

detection and limit of quantitation of the analytical methods. Chron. Young.

Sci., 2, 21-25 (2011).

(41) A. P. Murray, M. B. Faraoni, M. J. Castro, N. P. Alza, V. Cavallaro, Natural

AChE inhibitors from plants and their contribution to Alzheimer’s disease

therapy. Curr. Neuropharmacol., 11, 388-413 (2013).

(42) N. Tabet, Acetylcholinesterase inhibitors for Alzheimer’ disease: anti-

inflammatories in acetylcholine clothing. Age and Ageing., 35, 336-338

(2006).

(43) M. Pohanka, Cholinesterase, a target of pharmacology and toxicology.

Biomed. Pap. Med. Fac. Univ. Palacky. Olomouc. Czech. Repub., 155, 219-

230 (2011).

(44) T. H. Ferreira-Vieira, I. M. Guimaraes, F. R. Silva, F. M. Ribeiro,

Alzheimer’s disease: Targeting the cholinergic system. Curr.

Neuropharmacol., 14, 101-105 (2016).

(45) M. H. Oh, P. J. Houghton, W. K. Whang, J. H. Cho, Screening of Korean

herbal medicines used to improve cognitive function for anti-cholinesterase

activity. Phytomedicine, 11, 544–548 (2004).

99

(46) G. L. Ellman, K. D. Courtney, V. Andres, R. M. Featherstone, A new and

rapid colorimetric determination of acetylcholinesterase activity. Biochem.

Pharmacol., 7, 88-95 (1961).

(47) O. D. Lopina, Enzyme inhibitors and activators. Intech. Open., 243-257

(2017).

(48) V. Tougu, Acetylcholinesterase: Mechanism of catalysis and inhibition.

Curr. Med. Chem., 1, 155-170 (2001).

(49) M. D. Lopez, F. J. Campoy, M. J. P. Villalobos, E. M. Delgado, C. J. Vidal,

Acetylcholinesterase activity of electric eel is increased or decreased by

selected monoterpenoids and phenylpropanoids in a concentration-

dependent manner. Chem. Biol. Interact., 229, 36-43 (2015).

(50) L. L. Jiang, B. R. Sun, C. Zheng, G. L. Yang, The antitumor effects of

eudesmin in lung cancer by inducing apoptosis via mitochondria-mediated

pathway in the tumour cells. Pharm. Biol., 55, 2259-2263 (2017).

(51) J. S. Yang, C. M. Wang, C. H. Su, H. C. Ho, C. H. Chang, C. H. Chou, Y.

M. Hsu, Eudesmin attenuates Helicobacter pylori-induced epithelial

autophagy and apoptosis and leads to eradication of H. pylori infection.

Exp. Ther. Med., 15, 2388-2396 (2018).

(52) H. Liu, Z. Song, D. G. Liao, T. Y. Zhang, F. Liu, K. Zhuang, K. Luo, L.

Yang, J. He, J. P. Lei, Anticonvulsant and sedative effects of eudesmin

isolated from Acorus tatarinowii on mice and rats. Phytother. Res., 29, 996-

1003 (2015).

100

(53) C. Li, G. Xing, M. Dong, L. Zhou, J. Li, G. Wang, D. Zou, R. Wang, J. Liu,

Y. Niu, Beta-asarone protection against beta-amyloid-induced

neurotoxicity in PC12 cells via JNK signaling and modulation of Bcl-2

family proteins. Eur. J. Pharmacol., 635, 96-102 (2010).

(54) Z. Xue, Y. Guo, S. Zhang, L. Huang, Y. He, R. Fang, Y. Fang, Beta-asarone

attenuates amyloid beta-induced autophagy Akt/m TOR pathway in PC12

cells. Eur. J. Pharmacol., 741, 195-204 (2014).

(55) A. T. Cartus, S. Stegmuller, N. Simson, A. Wahl, S. Neef, H. Kelm, D.

Schrenk, Hepatic metabolism of carcinogenic β-asarone. Chem. Res.

Toxicol., 28, 1760-1773 (2015).

(56) Committee on herbal medicinal products, Public statement on the use of

herbal medicinal products containing asarone. European. Medicines.

Agency., 1-7 (2005) [ Doc Ref. EMEA/ HMPC/13921 5/2005]

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