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

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 1    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 2    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 phthalidescontaining 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. 3    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.     4                  Senkyunolide I Senkyunolide H   Senkyunolide A                             Z-Ligustilide Neocnidilide   3- Butylidenephthalides           Senkyunolide J          Senkyunolide F 3-Butylphthalides Figure 1.2 The chemical structures of the identified phthalides compounds 5    H O H    O       Cnidilide Riligustilide     Z,Z’-6,8’,7,3’-diligustilide    Levistolide A            Tokinolide B   Senkyunolide P Figure 1.2 (Continued) The chemical structures of the identified phthalides compounds 6    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 7    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 (HPLCMS) [34, 38], and high performance liquid chromatography with ultraviolet detector (HPLC-UV) [35, 38-39]. However, some of the phthalides like zligustilide and senkyunolide A may decompose at high temperatures [40], GC 8    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]. 9    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 2.1 Liver Cancer Hepatocellular carcinoma or usually known as liver cancer is the most common primary tumor of the liver causing roughly a million deaths yearly worldwide [47]. It is different from metastatic liver cancer, which begins in another organ like breast or colon, and spreads to the liver. The available treatment for liver cancer patients is greatly dependent on the phase of the disease, presence 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 10    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[...]... 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... 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... 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... 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... 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,... 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 33    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. .. aggregation, antithrombotic and serotonergic activities Thus, the overall objective of this study was to explore the potential of chuangxiong extract in exhibiting cytotoxicity 17    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... 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],... [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... 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; ... 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. .. ºC in a humidified incubator containing 5% CO2 and 95% air Cells were maintained at a cell concentration between × 105 and × 106 cells/mL, and were subcultured by total medium replacement using... duplication and cell division The increase of sub-G1 phase from cell cycle analysis showed that IPE had a minor or insignificant influence on apoptosis induction in Hep-G2 cells In contrast,