CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF LIGUSTICUM WALLICHII EXTRACT IN 3t3 l1 AND HEP g2 CELL LINES

There are more than 20 compounds that have been identified, isolated and classified according to their structures into three types, including alkaloids tetramethylpyrazine, phenols ferul

CHAPTER 1

LITERATURE REVIEW AND OVERALL HYPOTHESES

1 Ligusticum wallichii and Phthalides

1.1 Ligusticum wallichii

In the past decades, the use of bioactive compounds from natural

resources has been intensified These compounds naturally occur in low

quantities and contribute extra nutritional constituents in plants [1] They include secondary metabolites such as antibiotics, pigments, plant growth factors,

phenolic compounds and alkaloids [1-2]. Herbal plants have become the main focus in order to discover and develop new drugs, which have more

effectiveness and no side action like those modern drugs [3]

Chuanxiong (Tousenkyu in Japanese) is the rhizome of Ligusticum

wallichii Hort (family Umbelliferae) and is commonly used as crude drug in

traditional Chinese, Japanese and Korean folk medicines [4-6] Besides the

medicinal uses, it also can be used as a flavor ingredient in foods and beverages

In traditional Chinese medicine (TCM) practice, this herb has pungent and warm properties related with gallbladder, liver and pericardium meridians The main functions of chuangxiong are to dispel wind, relieve pain, and promote the blood

circulation as well as the flow of “qi”, an active principle forming part of living

thing in Chinese culture Therefore many practitioners prescribe it to treat

migraine, hypertension, stroke and various cardiovascular diseases, such as cardiac arrhythmias, angina pectoris and ischemic stroke [7-11]

Previous studies on chuangxiong’s bioactivity are mainly focused on vasodilatation, anti-platelet aggregation, anti-thrombotic, serotonergic activity and

anti-proliferative effects [10, 12-16] However, chuangxiong extract has also been

reported to protect hydrogen peroxide damage in endothelial cell (ECV304), which was probably associated with activating the extracellular signal-regulated kinases (ERK) pathway and promoting endothelial nitric oxide synthase (eNOS)

expression [17] On the other hand, chuangxiong extract exhibited inhibition of

the proliferation in hepatic stellate cells (HSC-T6), but no direct cytotoxicity on primary hepatocytes was reported [18].This inhibitory effect was associated with the induced apoptosis mechanisms through the activation of caspase 9 and 3, an increase in cytosolic cytochrome c release and down-regulation of Bcl-2 and Akt phosphorylation [18] Moreover, the cell cycle promoting proteins, cyclins D1, D2,

E, A and B1 were found down-regulated as the inhibitory proteins p21 and 27 were up-regulated [18]

In another bioactive study, chuangxiong extract was found to inhibit the

vascular smooth muscle cell (VSMC) proliferation by detaining G1 to S

progression [8] The increase of nitric oxide (NO) production after treatment with the extract has been suggested that inhibition of VSMC proliferation is closely associated with the increase of p21 expression, a cell cycle inhibitor, resulting in inhibition of cdk2 and pRb expressions that are required for cell cycle

progression [8] Therefore, the expression of p21induces G0/G1 cell cycle arrest and prevents cells to enter synthesis phase and go through proliferation

Due to the diverse beneficial effects of chuangxiong, several studies have been focused on screening and identifying the bioactive constituents [7-12] There are more than 20 compounds that have been identified, isolated and classified according to their structures into three types, including alkaloids

(tetramethylpyrazine), phenols (ferulic acid and coniferylferulate) and phthalides

(senkyunolide A, z-ligustilide and etc) [11] Tetramethylpyrazine is a principal

ingredient from chuangxiong and is used as a chemical indicator for quality control of chuangxiong However, it was not detected in several studies due to its low content in the herb [11, 19] Therefore, phthalides become the center of attention when identifying the biologically active compounds in chuanxiong

1.2 Phthalides

Phthalides are the secondary metabolites of plants and

phthalides-containing plants are extensively used in many traditional medicines Phthalide has a basic core structure, 1(3H)-isobenzofuranone that contains a benzene ring (ring A) fused with a γ-lactone (ring B) between carbon atoms 1 and 3 as shown

in Figure 1.1 [20] The structure of its derivatives either has the core structure replaced with one or more hydroxyl or alkyl groups at different sites or contains a reduced form with one, two or no double bonds in ring A

Figure 1.1 Chemical structure of phthalide [1(3H)-isobenzofuranone]

To date, there are about 137 natural phthalides that have been identified from more than 200 plant species and reported to be pharmacologically active

These species include Apocynaceae, Aristolochiaceae, Gramineae, Mysinaceae, Umbelliferae and others [20] The majority of identified bioactive natural

phthalides are obtained from the two genera Ligusticum and Angelica in the Umbelliferae family, for example, Ligusticum acuminatum, Ligusticum sinense, Ligusticum wallichii, Ligusticum jeholense and others Most of these species are

used as herbal medicines [11, 21-24] Phthalides are the predominant

compounds found in chuangxiong and have been suggested to be the bioactive

ingredient Senkyunolide A and z-ligustilide are the most abundant phthalides in

chuangxiong [25] Other phthalides including senkyunolide I, senkyunolide J, riligustilide, 3-butylidenephthalides and others were also reported [11, 19] The chemical structures of these identified compounds are illustrated in Figure 1.2

Senkyunolide J Senkyunolide F 3-Butylphthalides

Figure 1.2 The chemical structures of the identified phthalides compounds

1.3 Phthalides Extraction and Purification

Though many scientific studies have focused on phthalides, the extraction and quantification of phthalides in extracts still has challenges To my knowledge, there are no optimal extraction conditions that have been developed for

phthalides extract from chuangxiong The conventional extraction methods of phthalides from chuangxiong involve the use of organic solvents like methanol, petroleum ether, ethyl ether or chloroform with agitation at room temperature [8,

11, 17-18] However, it usually produces relatively low yields and inadequate quantities for biological activity screening Optimization process is important in order to achieve higher yields and better quality for phthalides extraction The factors that affect the efficiency of solvent extraction are: type of solvent, pH, temperature, extraction time, volume of solvent, and particle size in samples In the extraction process, these independent variables may interrelate and influence each other’s effects on the recovery yield [26] Therefore, an experimental design study is needed to determine all the parameters and the possible interactions between these independent variables in order to develop the optimal

experimental conditions for phthalides extraction from chuangxiong

Furthermore, there is a lack of studies on the purification steps of isolating phthalides from crude extracts A purification process for phthalides extract

involving liquid-liquid extraction had been reported in a recent study [27] These procedures are complex and laborious with long preparation time Therefore, it is necessary to find alternative ways to purify and concentrate the phthalides found

in chuangxiong extracts Among those well-documented methods for purification

like reverse osmosis, activated carbon adsorption/desorption or freeze drying,

resins have received enormous attention in recent years [28] Kronberg et al

discovered that results obtained from triple liquid-liquid extraction with ethyl ether

at ratio of 1:8 were similar to those on Amberlite XAD-4 [29] Amberlite XAD resins are commonly used, as they are non-polar and non-ionic adsorbents for stripping and concentrating organic compounds from liquid samples [30-32]

They absorb compounds that are non-polar and non-ionic primarily by van der Waal’s forces [28] Since these forces are weak, the absorbed compounds are

easily eluted from the resins with lower polarity solvents, for example, methanol Since most of the identified phthalides are relatively mid- to non-polar [33],

Amberlite XAD-4 resin may be the appropriate option for the purification purpose

1.4 Analysis and Determination of Phthalides

Many methods for the analyses of the phthalides in chuangxiong have been reported [34-39] These include gas chromatography with mass

spectrometry (GC-MS) [34], gas chromatography with flame ionization detector (GC-FID) [35], capillary electrophoresis with ultraviolet detector (CE-UV) [36], high performance capillary electrophoresis with ultraviolet detector (HPCE-UV) [37], high performance liquid chromatography with mass spectrometry (HPLC-MS) [34, 38], and high performance liquid chromatography with ultraviolet

detector (HPLC-UV) [35, 38-39] However, some of the phthalides like

z-ligustilide and senkyunolide A may decompose at high temperatures [40], GC

methods may not be suitable for the identification and quantification of these heat sensitive compounds HPLC-MS was deemed to be the most sensitive tool for the identification and quantification of phthalides from chuangxiong

Although UV detection should be sufficient for the quantitative

investigation of the known compounds, it does not offer sufficient and precise information about their molecular structures.There are many different types of qualitative chemical analysis that are used to identify unknown substances, for example, fourier transform infrared (FTIR) spectroscopy, nuclear magnetic

resonance (NMR) spectroscopy, X-ray crystallography Mass spectrometric detection is selected to solve this problem due to its high sensitivity and structural information can be derived from the mass spectra [41]

Among the ionization techniques, electro-spray ionization (ESI) and

atmospheric pressure chemical ionization (APCI) are the most frequently used In ESI, the formation of ions is caused by ion evaporation from charged droplets after the effluent sample is introduced into the atmospheric interface and moved across electric fields [42] Subsequently it will be heated up to assist further solvent evaporation from the charged droplets until it turns unstable upon

reaching its Rayleigh limit [43] The droplets will deform and emit charged jets in

a process called Coulomb fission [43] In contrast, ionization in APCI involves heating of an effluent sample at high temperatures, sprayed with high flow

nitrogen and the entire aerosol cloud is then disposed to a corona discharge that produces ions [43]

Unlike ESI, where the ionization occurs in the liquid phase, APCI produces ions in the gas phase Furthermore, it is considered as a less "soft" ionization technique than ESI since it can generate more fragment ions [44] APCI has the advantage over ESI since the mid to non-polar analytes that do not exist as

preformed ions in solutions can be readily ionized [45] Therefore, APCI is more extensively applicable than ESI to the analysis of non-polar compounds with low molecular weights [46]

2 Liver Cancer and Human Hepatocellular Carcinoma Cell Line

or absence of chronic liver disease and degree of hepatic dysfunction Surgical resection, percutaneous ethanol injection, chemotherapy, hormonal manipulation, radiotherapy, immunotherapy and liver transplant are designed to improve the survival rate of the patients and also should be the end-point of early detection plans [48] However, these treatments have their own limitations, for example, patients after chemotherapy treatment may encounter many side effects like fatigue, pain and organs dysfunction Liver transplant shows high survival rates

and the tumor recurrence rate is low, but the number of organ donors is much less compared to the patients [48] In contrast, this fatal disease can be

prevented through the avoidance of cancer-causing biologic, chemical, and physical agents and the selection of food diets that protect against cancer [49] Therefore, many natural products-derived compounds such as elliptinium,

taxanes, vinca alkaloids, podophyllotoxin are currently undergoing clinical trials,

animal studies or in vitro experiments for the use in cancer [50]

2.2 Human Hepatocellular Carcinoma (Hep-G2) Cell as In Vitro Model System

The immortalized human hepatoma cell lines, Hep-G2 has been well

characterized [51] and is commonly used as an in vitro model [52] These cells

have been extensively used in many cytotoxicity studies for the screening of potential hepatotoxic compounds [53-56] The well-differentiated Hep-G2 cells exhibit many genotypic and phenotypic properties of normal liver cells [57] Moreover, they can be cultured indefinitely for long-term studies to determine genotoxic and non-genotoxic carcinogens [56] Hep-G2 cells have low intensity

of phase I cytochrome P450 enzymes compared to primary hepatocytes [58], but

have normal levels of phase II enzymes (p<0.05) [59] Therefore, Hep-G2 cells represent a valuable in vitro model for hepatotoxicity studies to discover novel

therapeutic agents though they might underestimate the toxicity of particular compounds due to their low expression levels of phases I and II

biotransformation enzymes compared to the primary hepatocytes [58]

3 Type 2 Diabetes and 3T3-L1 Preadipocytes Cell Line

3.1 Type 2 Diabetes

Diabetes mellitus is defined as a group of metabolic diseases

characterized by chronic hyperglycemia due to a deficiency in insulin secretion, insulin action, or both [60] It affects almost 17 million people in the United States and more than 150 million people worldwide [61] It is currently considered as the third leading cause of disease death after cancer and cardiovascular diseases [62] Most of the diabetes cases are categorized into Type 1 and Type 2 diabetes [63-64] The former is caused by devastation of pancreatic beta cells, leading to

a shortage of insulin secretion [63] The latter, Type 2 diabetes, is a combination

of resistance to insulin response and an inadequate compensatory insulin

secretary action [63]

The amount of people diagnosed with Type 2 diabetes is growing rapidly

at a shocking rate This disease affects almost 6 % of the population in the

United States, Europe and most Western countries [65] The attack rate also reaches 6 % in China [66] The number of diabetic patients is estimated to reach about 366 million by the year 2030 due to obesity and inactive lifestyle [67] The resulting hyperglycemia may cause other long-term clinical problems, such as renal failure, retinopathy, neuropathy, and heart disease [60]

At the early stage of Type 2 diabetes, insulin resistance is initiated by genetics and lifestyle factors [68] However, if the β cells function normally, the insulin resistance will lead to a compensatory hyperinsulinemia condition that will maintain relatively normal glucose metabolism [69] In this compensated state,

one has either normal or impaired glucose tolerance, but no diabetes During the transition from the compensated state to diabetes, few pathophysiologic changes can be observed, for example, a significant decrease in the β cells’ function and insulin secretion (whether is due to genetic abnormalities or acquired defects)[69]

The glucose levels in plasma are normally maintained with the aid of

insulin [70] Insulin is a hormone produced by β cells of pancreas and is

necessary for anabolic and glucose homeostasis [71] It stimulates the cells in liver, muscle and fat tissue to utilize the glucose from blood, converts excess glucose into glycogen, which is stored in the liver and muscle [72] Insulin

resistance is defined as a condition of defects in insulin-signaling in the

responsive tissues [73] As a result, the normal levels of insulin are insufficient to induce the normal insulin response in fat, muscle and liver cells

The total expenditure of care among the diabetic patients and its

complications is very high and estimated to reach 300 million cases in 2025 [65] Although insulin therapy is available and can directly imitate the physiological control of glucose, the numerous daily injections of insulin sometimes can cause potentially life-threatening hypoglycemia [62] Therefore, great attention has been focused on the development of alternative medicinal foods like selection of

natural bioactive compounds with the ability to enhance glucose control and

lower the risk of diabetes complications [74-75]

3.2 Adipose Tissue and Adipogenesis

Adipose tissue is one kind of heterogeneous tissue that contains various cell types, with the highest percentage of adipocytes, which contain fat droplets The other cell types include undifferentiated preadipocytes, immune cells

(macrophages and leukocytes), lymph nodes, endothelial and smooth muscle cells, nerve fibers and a matrix of collagen and reticular fibers [76-78]

Two different types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), are identified based on their features [79] WAT is composed of unilocular, relatively large adipocytes that serve as energy store in the body [80] Conversely, BAT is composed of multilocular, relatively small adipocytes with many mitochondria [80] and functions to generate heat for body temperature regulation [81]

WAT is the predominant type of adipose tissue in human [82] and plays essential roles in energy homeostasis [83] Hence, it is necessary to understand the molecular and cellular mechanism of WAT progression This includes the proliferation of preadipocytes and their differentiation into mature adipocytes (adipogenesis) [84] Many studies have been carried out to achieve better

concepts on how the hormonal, cellular and molecular mechanisms influence adipogenesis [85] The adipogenesis process can be promoted by several factors such as insulin, insulin-like growth factor I (IGF-I), growth hormone [86-87],

transcription factors like fatty acid activated receptor, members of the

CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor (PPAR) families [88]

Increasing attention has been paid to the role of PPAR families since they demonstrated remedial benefits in treating different chronic diseases like

diabetes mellitus, atherosclerosis and cardiovascular diseases [89] The PPAR families are members of the nuclear hormone receptor family of transcription factors, a diverse group of proteins that regulate ligand-dependent transcriptional activities [90-91] They are involved in lipid metabolism, glucose homeostasis, adipocytes differentiation, inflammatory response and other biologic processes [92-93]

Three subtypes of PPAR that have been identified are -α, δ and γ [90-91] PPAR-α is involved in fatty acid catabolism and is mostly expressed in the liver, heart, muscle and kidney [94] PPAR- δ is suggested to be involved in basic lipid metabolism in brain [95] PPAR-γ is highly expressed in adipocytes and regulates adipocyte differentiation and glucose homeostasis [90-91] Therefore, several therapeutic agents that target insulin resistance are believed to stimulate the expression of PPAR-γ The most promising agents are the thiazolidinedione derivatives since they can reduce the glucose levels, and circulate insulin and free fatty acids at the same time [96] However, there are some drawbacks about these agents since they will cause weight gain and fluid retention, and increase risk for congestive heart failure and risk for bone fractures [97-98] Therefore, there is a need to search for a better therapeutic agent either from synthetic drugs or natural compounds

3.3 3T3-L1 Preadipocytes Cell Line as In Vitro Model System

There are several models available to study the molecular and cellular

events during adipogenesis The in vitro model includes some adipogenic cell

lines (clonal lines 3T3-L1, 3T3-F442A, Ob17, BFC-1, ST13 and A31T) and the primary culture of adipocyte precursors and pre-adipocytes [99] A 3T3-L1 cell line has been established from disaggregated Swiss mouse embryos and is capable of differentiating into cells resembling adipocytes [100-101] The cells show the morphology of fibroblasts and can be induced to form mature

adipocytes that accumulate oil droplets [102]

According to Keay and her colleague, 3T3-L1 cell line is widely used as in vitro model because these cells (i) initiate their phenotypic conversion when cultures become confluent and stop growth virtually; (ii) go through a distinctive morphological changes; (iii) produce new enzymes that stimulate each other; (iv) enhance the number of membrane receptors for insulin; (v) show variation in the hormonally regulated metabolism between normal and differentiated adipose cells; (vi) do not have endogenous viruses, and (vii) can experience

differentiation in culture without added chemical stimulation, although the

presence of serum factors, insulin, or caffeine analogs can accelerate the

process [103-105]

Thus, 3T3-L1 cells have been used to develop therapeutic approaches for the treatment and prevention of obesity since they are capable of undergoing

differentiation under specific culture conditions (presence of

1-methyl-3-isobutylxanthine (IBMX), dexamethasone and insulin) [106-107]

4 Overall Hypotheses, Objectives and Implications of This Study

4.1 Overall Hypotheses

It is understood that the recovery of phthalides varies according to the types of solvent used during extraction Hence, methanol is suggested to be an ideal solvent due to its greater affinity to phthalides compared to other solvents It

is hypothesized that the phthalides extract from chuangxiong influences human hepatocellular carcinoma (Hep-G2) cells proliferation through different

mechanisms of cell death or cell growth arrest On the other hand, this phthalides extract can also influence preadipocytes proliferation and differentiation to

adipocytes in a 3T3-L1 murine cell model

5.2 Overall Objectives

Chuangxiong has been extensively used as a traditional herbal medicine for many treatments in China, Japan and Korea However, most of the studies on chuangxiong are mainly focused on vasodilatation, antiplatelet aggregation, antithrombotic and serotonergic activities Thus, the overall objective of this study was to explore the potential of chuangxiong extract in exhibiting cytotoxicity

effects on Hep-G2 cells and the influence on 3T3-L1 adipocytes Therefore, investigation is needed through the pursuit of the following specific aims:

1 To determine the types of solvent for phthalides extraction from

chuangxiong that provide highest recovery yield

2 To develop an extraction and isolation methods from chuangxiong and to

obtain lyophilized powder for further in vitro experiments

3 To investigate the cytotoxicity effect of phthalides extracted from

chuangxiong on Hep-G2 cell line

4 To study the effect of phthalides extracted from chuangxiong in 3T3-L1 cell model as related to diabetes

5.3 Implications of This Study

At the end of this project, the findings would provide fundamental

knowledge about the optimal phthalides extraction conditions from chuangxiong

It also may be a reference for additional studies on the mechanisms of action on anti-proliferative effects on Hep-G2 cells Since the adipose tissue is the main target for anti-diabetic agents, an evaluation of phthalides extracts on 3T3-L1 adipocytes can further explore the use of chuangxiong for diabetes prevention or management

2 Introduction

Initially, the efficiency of several commonly used extraction solvents was examined under reflux condition The crude phthalides extracts (CPE) was

prepared according to the highest yield method for further in vitro experiments

Isolation was then employed to enhance the phthalides concentrations for the preparation of isolated phthalides extracts (IPE) from CPE Component analysis

of both extracts (CPE and IPE) was performed by HPLC-MS based on molecular weight and UV spectra confirmation

3 Materials and Methods

3.1 Selection of Extraction Solvents Used for Phthalides

Dried chuangxiong pieces were locally purchased and ground into fine powder, which was stored at 4 ºC until further extraction Extractions were carried out using five different solvents: 1 water; 2 methanol (Riverbank Chemicals, Singapore); 3 ethanol (Riverbank Chemicals, Singapore); 4 hexane (Prime Chem, Malaysia); and 5 ethyl acetate (Prime Chem, Malaysia) One gram of chuangxiong powder was subjected to 50 mL of different types of solvents

extraction under reflux condition (70 ºC) for 4 h The extracts were collected, transferred into a 50-mL volumetric flask and the total volume was corrected to

50 mL with the appropriate extraction solvent These aliquots were filtered

through a 0.45 µm PTFE syringe filter and the extraction yields of individual compounds extracted from chuangxiong were analyzed using HPLC

3.2 Preparation of Crude Phthalides Extracts from Chuangxiong

One gram of chuangxiong powder was subjected to 50 mL of methanol extraction under reflux condition (70 ºC) for 4 h The extract was collected and filtered through Whatman filter paper (No.4) The aliquots were then evaporated until dryness under vacuum, re-suspended with deionzed water and then

lyophilized The lyophilized residue was referred as crude phthalides extracts (CPE) as shown in Figure 2.1

3.3 Preparation of Isolated Phthalides Extracts from Chuangxiong

One gram of chuangxiong powder was subjected to 50 mL of methanol extraction under reflux condition (70 ºC) for 4 h The extract was collected and filtered through Whatman filter paper (No.4) An Amberlite XAD-4 resin (Sigma, Germany) column was used to purify the extracts as described by Popovich and his colleague [108] The filtrate was applied equally to the Amberlite XAD-4 resin column (surface area 725 m2/g, pore diameter 40 Å) with a bed volume of 100

cm3 each at a flow rate of 2 mL/min followed by 1 L of deionized water at 5 mL/min The sample was eluted by 2 L of methanol at 2 mL/min, and

concentrated by vacuum evaporation It was then re-suspended with deionzed water and lyophilized The lyophilized residue herein was referred as isolated phthalides extracts (IPE) as shown in Figure 2.1

Dried chuangxiong powder

Reflux with methanol, Discard residue

Methanolic extract

Removal of methanol under vacuum Dissolved in deionized water

Aqueous extract

CPE in powder form Eluted with methanol

Removal of methanol

Dissolved in deionized water

Lyophilization

Figure 2.1 Schematic diagram of methodology used in preparation of crude

phthalides extracts and isolated phthalides extracts from chuangxiong

3.4 Identification of Phthalides Compounds using High Performance Liquid

Chromatography-Mass Spectrum (HPLC-MS)

A Waters Alliance 2695 (Waters, USA) high performance liquid

chromatography (HPLC) system comprising of a vacuum degasser, binary pump, autosampler, thermostated column compartment and photodiode array detector was employed for acquiring chromatograms and UV spectra A Phenomenex reversed phase C-18 column (4.6mm x 250mm, 5µm diameter) was used for the separation of phthalides The mobile phase consisted of 0.5% acetic acid (JT Baker, USA) in deionized water (mobile phase A) and HPLC grade methanol (Tedia, USA) (mobile phase B), and the temperature of column was held at 30 ºC The gradient program was set as follows: 0-5 min 0% B, 5-10 min 0-20% B, 10-

15 min 20% B, 15-20 min 20-40% B, 20-25 min 40% B, 25-30 min 40-60% B,

30-35 min 60% B, 30-35-40 min 60-80% B, 40-45 min 80% B, 45-50 min 80-100% B and 50-65 min 100% B The flow rate was 0.5 mL/min with an injection volume of

20 µL and detection was at 294 nm wavelength

An AmaZon X quadrupole mass analyser equipped with atmospheric

pressure chemical ionization (APCI) was employed for MS detection The

conditions of mass spectrometry (MS) analysis were set as follow: corona

discharge current at -10.0 mA; nebulizer-gas pressure at 5 bars; dry gas flow rate

at 10.0 L/min; capillary voltage at +2.4 kV; capillary temperature at 280 ºC; and vaporizer temperature at 450 ºC The scanning mass spectra of sample were focused on the m/z range of 50-600 in positive ion mode

3.5 Statistical Analysis

Comparison of means was performed by independent sample t-test and one-way ANOVA with multiple comparison test of Duncan at 5 % confidence

level (p < 0.05) Statistical analyses were conducted using SPSS for Windows,

Version 13.0 (SPSS Institute Inc., Cary NC) Each result was expressed as mean values ± standard deviations of three separate experiments (unless stated

otherwise)

4 Results

4.1 Selection of Extraction Solvents Used for Phthalides

The representative HPLC-UV chromatogram (294 nm) of methanolic

extract of chuangxiong is shown in Figure 2.2 Two compounds were

unequivocally identified as ferulic acid (1) and z-ligustilide (6) with confirmation of

external standards Additional eight compounds were tentatively identified as senkyunolide J (2), senkyunolide I (3), senkyunolide H (4), senkyunolide A (5),

riligustilide or z,z’-6,8’,7,3’-diligustilide (7), tokinolide B (8), levistolide A (9) and

senkyunolide P (10), based on their MS data (Table 2.1) and the comparison of

UV spectra with the literature [11] Table 2.2 summarizes the extraction yields of individual compounds extracted from chuangxiong using five different types of solvents The results indicated that the highest yield of phthalides from

chuangxiong can be obtained by the use of methanol followed by ethanol,

hexane, ethyl acetate and water It is interesting to note that senkyunolide J was

not detected in all types of extract solvents in this study, except for methanol

Moreover, phthalides dimers (riligustilide / z,z’-6,8’,7,3’-diligustilide, tokinolide B,

levistolide A and senkyunolide P) were not detected in the water extract The remaining unknown peaks are suggested for further chemical characterization in

a future project

Figure 2.2 Representative HPLC chromatogram of chuangxiong extract detected

at 294nm The peaks were identified as ferulic acid (1), senkyunolide J (2),

senkyunolide I (3), senkyunolide H (4), senkyunolide A (5), z-ligustilide (6), riligustilide or z,z’-6,8’,7,3’-diligustilide (7), tokinolide B (8), levistolide A (9) and

10 

Table 2.1 Molecular weight confirmation of phthalides compounds in

Table 2.2 Comparison of the extraction yields of individual compounds extracted from chuangxiong using different

solvents All phthalides were expressed as an equivalent to z-ligustilide concentration in mg/g except for ferulic acid

Note: Values were reported as mean ± standard deviations (n=3) Means within the same row with different superscripts letter were

significantly different at p<0.05 level of significance, according to Duncan’s Multiple-Range Test

4.2 Comparison of Crude Phthalides Extracts and Isolated Phthalides Extracts

from Chuangxiong

The yields of lyophilized CPE powder and IPE powder from chuangxiong

powder were approximately 27.36 % w/w and 3.06 % w/w, respectively Table 2.3 reveals the phthalides content in CPE and IPE After the purification process, IPE shows significant increases of 172 to 365 % for each phthalides compound except for the Senkyunolide J The average recovery yields of total phthalides in CPE and IPE were 11.61 mg/g and 44.60 mg/g, respectively In CPE and IPE,

senkyunolide A was found to be the most abundant component and z-ligustilide

was determined as the major ingredient These two major ingredients consisted

of 77-78% of the total components determined in CPE and IPE samples

Table 2.3 Comparison of the recovery of individual compounds in crude

phthalides extracts and isolated phthalides extracts from chuangxiong All

phthalides were expressed as an equivalent to z-ligustilide concentration in mg/g

except for ferulic acid Asterisk (*) denotes a significant difference (p < 0.05)

compared to CPE value

5 Discussion

With the aim to study the effect of solvent extraction on the recovery yield

of phthalides from chuangxiong, several solvents were selected based on their different polarity, for example, water, methanol, ethanol, hexane and ethyl

acetate Polarity is defined as the ability of a molecule to go through interactions

of all kinds and the relative polarity is the sum of all possible interactions [109] The results showed that the highest extract yields of phthalides were obtained from extraction employing mid polar methanol as the extraction solvent Low extract yields were observed when the extraction was performed using the polar solvent such as water Although these phthalides compounds contain hydroxyl and carbonyl groups, the lactone ring and alkyl groups limit their solubility in polar solvents like water

On the other hand, a destruction mechanism would be initiated by the opening of the lactone ring from phthalides and thus would cause the low

extraction yield [110] This mechanism varies between water and alcohols Water will release the lactone ring by hydrolysis, whereas alcohols will react by

transesterification and the rate of hydrolysis is expected to be much faster than transesterification [111] In contrast, low polarity solvents as ethyl acetate and hexane were not able to significantly extract the phthalides from chuangxiong compared to methanol This may be due to the presence of hydroxyl and

carbonyl groups attached to the rings that limit their solubility in the low polar solvent [111]

Based the HPLC-MS results, a variety of phthalides compounds were identified in chuangxiong by comparing the molecular fragments and UV spectra

with the literature [11] According to Li et al [37], they had successfully identified

17 constituents from chuangxiong extract, and these included vanillin, coniferyl ferulate, neocnidilide, 3-butylidenephthalides, 3-butylphthalides, cnidilide and senkyunolide F However, only 10 compounds were identified in the present study This may be due to their relatively low contents in plants or being below the limit of detection (LOD) for the HPLC system

Various studies also focused on the major constituents (ferulic acid,

senkyunolide I, senkyunolide H, senkyunolide A, z-ligustilide and levistolide A) in

chuangxiong extract since they are generally regarded as the index or quality measurement of this plant [11, 38, 112] There is a study which reported that two new phthalides, senkyunolide R and senkyunolide S, were isolated after

confirmation with NMR spectroscopy and X-ray crystallographic analysis [113] Therefore, it is suggested that the remaining unknown peaks might be the

unidentified new phthalides compounds Consequently, further chemical

characterization is recommended in future studies

In this study, as the purified standards for all 8 phthalides were not

commercially available, the assessment of extraction efficiency was performed

by the comparison with available z-ligustilide standard Previous studies reported that the content of z-ligustilide was determined to be in the ranges of 1.745 –

12.611 mg/g [11, 38, 112] However, the chuangxiong sample used in this

experiment only contained about 4 mg/g of z-ligustilide Moreover, with a

consideration of the total content of all phthalides determined, the current

chuangxiong samples (11.68 ± 0.06 mg/g) had lower contents compared to the previous studies (19.67 – 34.93 mg/g) [11] This may be due to the differences in the cultivation environment and time of harvesting affecting the quality of

chuangxiong [11]

The methanolic extract (CPE) was selected for further in vitro assessment

based on its highest recovery yield However, due to its low bioactivity, a

purification step was suggested to concentrate the content With the aid of

Amberlite XAD-4 resin, the hydrophobic molecules were retained in the resin whereas the hydrophilic compounds were eluted as waste [28] Therefore, a more concentrated phthalides extract (IPE) was obtained at the end of the

process for further in vitro studies Although the purification involved the use of

Amberlite XAD-4 resin, further studies could be suggested to optimize the

parameters (ie size of column, volume of bed, flow rate, and etc) for the

chemical usage and experimental time Furthermore, a combination of

purification steps, for example, additional process involving solid phase

extraction, flash chromatography or others might help to improve the purity of phthalides extracts to maximize its bioactivity

Further investigations could be also suggested on the extraction

conditions since there is no any related study up-to-date The results of this preliminary study can help to design the optimal extraction conditions as well as the extraction methods (ie agitation, distillation, high pressure-supercritical extraction, sonication, microwave assisted extraction and etc) for phthalides

extract from chuangxiong To further study each individual phthalides, isolation

process could be first employed to separate them into single compound Once the individual phthalides is isolated, analytical techniques including LC-MS, NMR spectroscopy and X-ray diffraction methods could be used to confirm the

molecular structure

CHAPTER 3

EFFECTS OF PHTHALIDES ON HEP-G2 CELL LINE

1 Hypothesis

Phthalides containing extract from chuangxiong will reduce human

hepatocellular carcinoma (Hep-G2) cell proliferation through different

mechanisms of cell death (ie apoptosis or necrosis) or arrest at different phases

in cell cycle progression

2 Introduction

A recent study demonstrated that ethanolic extract from chuangxiong could inhibit the proliferation of Hep-G2 cells [114] However, there has been limited effort in relating the content from chuangxiong extract to its cytotoxicity in Hep-G2 cells Therefore, it is important to understand the anti-proliferative

mechanisms of chuangxiong extract in Hep-G2 Firstly, the effect of phthalides extract against Hep-G2 cells viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to examine its bioactivity To further assess the mechanisms that affect the cells proliferation after the

treatment, further investigations could be carried out using lactate

dehydrogenase (LDH) assay, cell cycle analysis and apoptosis assay

3 Materials and Methods

3.1 Cell Culture

Hep-G2 cell line was purchased from American Type Culture Collection (ATCC) (Manassas, USA) Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Caisson, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, USA), and penicillin / streptomycin (100 units/mL) (Gibco,

Canada) at 37 ºC in a humidified incubator containing 5% CO2 and 95% air Cells were maintained at a cell concentration between 2 × 105 and 1 × 106 cells/mL, and were subcultured by total medium replacement using 0.25% (w/v) trypsin – 0.53 mM ethylenediaminetetraacetic acid (EDTA) solution (Gibco, Canada) every

3 days depending on cell number Viable cells and cell number were determined with Guava ViaCount Reagent (Guava Technologies, USA)

3.2 Cell Viability Test (MTT assay)

A MTT assay was used to examine the cytotoxicity of the extracts as previously described by Mosmann [115] with minor modification Hep-G2 cells were seeded in a 96-wells plate (2500 cells/well) and allowed to attach overnight Controls consisted of cells and culture medium, but without test compounds The phthalides extracts were dissolved in DMEM with concentrations between 0.1 mg/mL to 1.0 mg/mL Cells were incubated for 72 h and replaced with MTT solution (0.5 mg/mL dissolved in DMEM) and incubated in the dark for 4 h A 100

µL sodium dodecyl sulfate (SDS) (Merck, Germany) acidified with 0.01 M HCl

(Merck, Germany) was added and incubated overnight to dissolve the formazan crystals The optical density of each plate was measured using a

spectrophotometric micro-plate reader (Thermo Multiskan Spectrum, Finland) at

570 nm absorbance with a reference wavelength of 650 nm Cell viability (%)

was calculated as [mean (absorbance of sample at 570 nm – absorbance of

reference sample at 650 nm) / mean absorbance of control] × 100%

3.3 Cell Toxicity Test (LDH Assay)

An LDH assay was employed as described by Popovich & Kitts [116] with minor modifications Hep-G2 cells were seeded in a 24-wells plate (5 × 104

cells/well) and allowed to attach overnight Phthalides extract in culture medium was added at its IC50 concentration which was determined from a 72 h MTT

assay as described in the previous section The untreated cells acted as control groups The cells were incubated at 37 ºC in a humidified incubator containing 5%

CO2 and 95% air for 24, 48, and 72 h The cell-free supernatant was transferred

to a 15-mL centrifuge tube and centrifuged at 400 × g for 10 min A buffer

solution of pH 7.4 was prepared with 50 mmol/L Tris buffer (NUMI, NUS

Singapore), 5 mmol/L EDTA (NUMI, NUS Singapore), and 150 µmol/L

nicotinamide adenine dinucleotide (NADH) (Sigma, Germany) Two milliliters of buffer solution were mixed well with 50 µL cell-free supernatant in a 24-wells

plate and incubated at 37 ºC for 10 min Subsequently, 200 µL of pre-warmed (37 ºC) pyruvate solution (Sigma, Germany) was added into each well and the

reaction rate was measured in the first three min by a spectrophotometric plate reader (Thermo Multiskan Spectrum, Finland) at 340 nm wavelength at 37

micro-ºC

3.4 Cell Cycle Analysis

Hep-G2 cells (5 × 104 cells/well) were seeded in a 24-well plate and

treated with phthalides extract at its IC50 as described previously and untreated cells acted as controls Cells were incubated at 37 °C in a humidified incubator containing 5% CO2 and 95% air for 24, 48, and 72 h After treatment for 24, 48, and 72 h, floating cells in suspension were collected and centrifuged for 10 min

(150 × g) The adherent cells were trypsinized, collected and centrifuged for 10 min (600 × g) The supernatant was discarded and pellet was washed twice with

phosphate buffered saline (PBS) (NUMI, Singapore) The pellets from floating cells and adherent cells were mixed, vortexed vigorously while 1 mL of ice-cold 70% ethanol was added slowly to fix the cells and stored overnight at 4 °C Pellets were obtained by removing the ethanol through centrifugation (10 min,

500 × g) and 1 mL of PBS containing 50 μg/mL propidium iodide (PI) (Sigma,

Germany) and 100 U/mL RNAse A (Applichem, USA) was added to each sample tubes and incubated for 30 min in the dark at room temperature Cell cycle was analyzed using Guava PCA flow cytometer with Cytosoft software (Guava

Technologies Inc, USA)

3.5 Statistical Analysis

Comparison of means was performed by independent sample t-test and

one-way ANOVA with Duncan’s multiple range test at 5 % probability level (p <

0.05) Statistical analyses were conducted using SPSS for Windows, Version 13.0 (SPSS Institute Inc., Cary NC) Each result was expressed as mean values

± standard deviations of three separate determinations (unless stated otherwise)

were determined from three separate experiments with three replicates per experiment and was found to be 0.27 ± 0.02 mg/mL

Figure 3.1 Dose-response relationship of crude phthalides extracts after 3 days incubations with Hep-G2 cells by MTT viability assay Values were expressed as mean ± standard deviations (percentage of untreated cells) of three separate experiments with three replicates

Figure 3.2 Dose-response relationship of isolated phthalides extracts after 3 days incubations with Hep-G2 cells by MTT viability assay Values were expressed as mean ± standard deviations (percentage of untreated cells) of three separate experiments with three replicates

4.2 LDH Release

The effect of IPE treatment on lactate dehydrogenase (LDH) release is presented in Figure 3.3 The Hep-G2 cells were treated for 24 - 72 h at the IC50

concentration of 272 µg/mL Treatment increased LDH release at all time points,

but this was not significant (p > 0.05) between 24, 48, and 72 h treatment

Figure 3.3 The effect of IPE on Hep-G2 cells LDH activity Cells were treated with IPE for 24, 48, and 72 h, respectively, at concetration of 272 µg/mL, the IC50

concentration determined from MTT assay Values were expressed in means ± standard deviations from three independent experiments Means with different

letters were significantly different (p<0.05)

92 94 96 98 100