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In vitro cultures of Morus alba for enhancing production of phytoestrogens

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Vibhu Bakshi
Abstract:
Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high performance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic activity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.

TABLE OF CONTENTS

ACKNOWLEDGEMENT……………………………………………………………….iii LIST OF TABLES……………………………………………………………………….vii LIST OF FIGURES…………………………………………………………………..…viii

1. INTRODUCTION 1.1 Secondary Metabolites…..………………………………………..…………...1 1.2 Importance of Secondary Metabolites……...…………………………………2 1.3 What are Phytoestrogens...…………………………………………………….3 1.4 Phenyl Propanoid Pathway ………………………………………..………….4 1.5 Common Phytoestrogens …………………………………………………….8 1.6 Discovery of Phytoestrogens………………………………………………..10 1.7 Structural Resemblance of Phytoestrogens with Estradiol and Its Importance………...…………………………………………………………10 1.8 Tissue Culture ……………………………………………………………….12 1.9 Background for Mulberry …………………………………………………...14 1.10 Goals and Objectives ……………………………………………………....18

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2. METHODS AND MATERIALS 2.1 Plant Tissue Culture………………………………………………………….20 2.1.1 Plant Material and Preparation of Explants for Tissue Culture …...………20 2.1.2 Initiation and Maintenance of Callus …..……...………………………….20 2.2 Estrogenic Activity Determination or Analysis……………………………..22 2.2.1 Isolation of Estrogenic Fractions from Callus Extracts..……..……………22 2.2.2 Preparation of Plant Extracts for Estrogenic Activity Analysis……..……..22 2.2.3 Estrogenic Activity Assays ………………………………………………..22 2.3 Fractionation and Isolation of Phytoestrogens……………………………….25 2.3.1 Solid Phase Extraction...………………………………………………..….25 2.3.2 High Performance Liquid Chromatography (HPLC) ……….………….....25 2.3.3 Spectrophotometry….……………………………………………………...26 2.3.4 Electrospray Ionization Mass Spectrometry (ESI) .…………………….....26 2.4 Statistical Analysis …………………………………………………………..27

3. RESULTS 3.1 Results of Male and Female M. alba Callus Cultures…………..........…...…28 3.2 Results for the Estrogenic Activities of Callus and Adult Plant Tissues Extracts and its fraction obtained through SPE and HPLC……………….…30

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3.3 Spectrophotometry Analysis of MCE – HPLC15 and comparing it with Biochanin A…....……………………………………………………….…....47 3.4 Electrospray Mass Spectrometry (ESI–MS) of MCE – HPLC15 and Comparing it with Biochanin A…………......………………………..……...48 3.5 Results for Plant Tissue Extracts on Liquid Chromatography Mass Spectrometry (LC-MS)….……………………………..…………………….54

4. DISCUSSION …………………….……………………………………………58 4.1 Callus Culture Establishment………………………………………………59 4.2 Estrogenic Activity of Callus Extracts……………………………………..62 4.3 Chromatographic Techniques………………………………………………66 4.4 Significance of the Study……………………………………………….….75

REFERENCES………………..……………………………………………………….77

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LIST OF TABLES

Table 1. Composition of Murashige and Skoog media assayed for establishing callus cultures of white mulberry……………………………………….………………………29

Table 2. Estrogenic activities of the callus proliferating media in both male and female species of Morus alba……………………………………………………..……………..31

Table 3. Comparison of estrogenic activities of callus with adult plant tissues…………………………………………………………………………..………..34

Table 4. Comparison of estrogenic activities of different fractions obtained with solid phase extraction…………………………………………………………………………35

Table 5. Estrogenic activities and the retention times of the estrogenic active fractions obtained from the HPLC fractionation of MCE, MPE, FCE and FPE……………………………………………………………………………………….37

Table 6. Peaks obtained in the LC – ESI – MS negative ion chromatograms with the suspected compounds from on the studies of Wang et al., 2005………………………...57

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LIST OF FIGURES

Figure 1: Schematic representation of biosynthetic pathways leading……………….….7

Figure 2. Comparison of the chemical structures of the phytoestrogens………………..11

Figure 3. Foliage and inflorescences of the Morus species…..……………………...…..15

Figure 4. In vitro protocol for the establishment of white mulberry…………….………21

Figure 5. Estrogen-responsive transcriptional system in S. cerevisiae………….………23

Figure 6. Estrogenic activity of the three different proliferation media MS1, MS2 and MS 7……………………………………………………………………….……………31

Figure 7. Estrogenic activities of the established calli in March, July and September..………………………………………………………………………..……32

Figure 8. Comparison of estrogenic activities of MCE, FCE, MPE and FPE and their solid phase (SP), F20 fractions (F20), and F80 fractions (F80)….……………………..33

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Figure 9. Comparison of estrogenic activities of MCE, FCE, MPE and FPE and their fractionation after F80 and HPLC techniques……..……………………………………37

Figure10. Reverse phase HPLC profiles of (A) MCE – F80, (B) FCE – F80, (C) MPE – F80and (D) FPE – F80…….………………………………………………..…….…39-41

Figure 11. Reverse phase HPLC profiles of the standards estradiol (E), genistein (G) and biochanin A (B)..……………………………………………………………..………..41

Figure 12. Estrogenic activities of (A) MCE – HPLC fractions, (B) FCE – HPLC, (C) MPE – HPLC, and (D) FPE – HPLC fractions…………………………….………..45-46

Figure 13. Spectrophotometrical analyses of MCE – HPLC15 (A) and biochanin A (B)…..……………………………………………………………………..…..…………48

Figure 14. ESI – MS chromatogram of MCE – HPLC15 in full scan mode….…………50

Figure 15. ESI – MS/MS of the peak obtained at 284 m/z………………………………51

Figure 16. ESI – MS chromatogram of biochanin A in full scan mode...............……….52

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Figure 17. ESI – MS/MS of the peak obtained at 284 m/z...…………………..……….53

Figure 18. LC – MS in visible mode. (A) FCE – HPLC9 and (B) MCE – HPLC …………………………………………………………………………………….…55-56

Figure 19. LC – MS negative ion ESI – MS chromatograms of the MCE – HPLC16, FCE – HPLC9………………………….…………………………………………………….57

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CHAPTER 1 INTRODUCTION

Metabolism comprises different pathways for the survival of all cells. Secondary products occur in special, differentiated cells that are not required for the primary growth of the organism. Metabolism can be defined as the chemical processes occurring within a living cell or organism that are necessary for the maintenance of life. It could also be described as a large group of enzyme-controlled and regulated chemical reactions that produce energy in the form of ATP, substances needed for the growth and development of tissues, and help the organism survive in different conditions. In metabolism some substances, carbohydrates and lipids, are catabolised to yield energy for vital processes while other substances, necessary for life, are synthesized. Depending on the biosynthetic origin, general occurrence and biochemical role, compounds produced during metabolism are called either primary or secondary metabolites. Primary metabolites are essential for all life forms and include carbohydrates, lipids, proteins and nucleic acids.

1.1 Plant Secondary Metabolites Secondary metabolites often accumulate in specialized plant cells in smaller quantities than primary metabolites (Pichersky and Gang, 2002). Plant secondary

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compounds are usually classified according to their structures (Harborne, 1999). Phenolics, terpenes and alkaloids are three large families generally considered. The production of plant secondary metabolites in plant tissues varies with species, environmental conditions and internal hormonal levels (Bourgaud et al., 2001). Although secondary plant products are very common, this does not mean that every plant can produce every product. Some compounds are restricted to a single species, others to related groups. But they are nearly always found only in certain specific plant organs, often in just one type of cell (and there again only in a certain compartment). Also, secondary metabolites are often generated only during a specific developmental period of the plant. The chemical structure of secondary plant products is without exception more complex than that of primary products. The complexity of products becomes comprehensible when recognizing that many, though by far not all, of them are derived from amino acids or nucleotides. Most of the compounds found in plants belong to rather few families of substances. Only small chemical modifications such as methylations, hydroxylations, intercalations with metal ions, etc. lead to a wide spectrum of functionally different substances.

1.2 Importance of Secondary Metabolites for Plant Life Due to their multitude of biological effects, plant secondary metabolites have been used for centuries in traditional medicine. Currently, valuable secondary metabolites are extracted from plants and used as pharmaceuticals, cosmetics, fragrances, food additives pigments, flavoring and aromatic compounds and pesticides (Bourgaud et al.,

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2001). Good reasons exist for the use of some secondary compounds or, even better, groups of chemically similar compounds as features of classification. But what is true for other morphological features, is also true for these: the presence of a chemical substance in a plant is adaptive although this may not always be as clear as in the case of flower pigments, lignin or cutin. Among the numerous secondary metabolites, isoflavonoids, together with alkaloids and terpenoids are most commonly researched under in vitro conditions. The reason for this may be seen in their multidirectional biological activity (Dutta et al., 2007). Although plants possess a huge diversity of secondary metabolites, structures, functions, and medical benefits for most of these phytochemicals are not yet known.

1.3 What are Phytoestrogens? Phytoestrogens are a diverse group of naturally occurring compounds that exert estrogenic activities in plants. Phytoestrogens have structural similarity with estradiol. There have been reports of more than 300 plant species that contain estrogenic compounds. The phenolic ring is essential for binding to estrogen receptors (ERs). Phytoestrogens commonly occur in glycosylated forms and importantly the bioavailability of glyco-conjugates differs from that of the unsubstitued aglycones. Phytoestrogens are generally classified in three main chemical groups of compounds: isoflavones (genistein, daidzein), lignans, and coumestans. Isoflavones are found in a variety of plants including fruits and vegetables, but they are predominantly found in legumes such as beans, peas, lentils and soybeans. Lignans are widely found in cereals,

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fruits and vegetables. The third class of Phytoestrogens are coumestans, produced in legume sprouts after germination (Luczkiewicz and Glod, 2003). Isoflavones, being recognized as phytoalexins, play a key role in defense mechanisms in plants of the family Fabaceae. Isoflavones are, in addition, compounds with broad health-promoting activity. Isoflavones are known to have anti-inflammatory, antifungal and anti-free-radical activities that are typical for the whole group of isoflavonoids. They also feature compounds that inhibit estrogen beta receptors in mammals (Dixon et al., 2002).

1.4 Phenylpropanoid Pathway Phenylpropanoid-derived isoflavonoids are known to act as primary defense compounds and also signal as plant-microbe interactions. A large number of isoflavonoids are also being used as pharmacological and nutraceutical properties, including chemoprevention and osteoporosis (Alekel et al., 2000; Uesugi et al., 2001) and other postmenopausal disorders (MerzDemlow et al., 2000), antioxidants that improve cardiovascular health (Lichtenstein, 1998; Setchell and Cassidy, 1999; Heim et al., 2002), and it also reduces the risk of breast and prostate cancers in humans (Adlercreutz, 1998; Lamartiniere, 2000). Due to the human beneficial effects of isoflavonoids, these compounds are being researched comprehensively for a better understanding of its synthesis. Efficient engineering methods are being devised for the exploitation of isoflavonoids. Flavanones are immediate precursors for flavonoid biosynthesis and can occur as monomers, dimmers, and higher oligomers. Flavanones

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such as naringenin and liquiritigenin undergo migration at the B-ring from 2- to the 3- position by hydroxylation at the 2-position to yield isoflavanones. Isoflavone synthase (IFS) an enzyme, cytochrome P450, NADPH-dependent, dehydrates liquiritigenin or naringenin to yield 2-hydroxyisoflavone, spontaneously or enzymatically. Dehydration of 2-hydroxyisoflavone is catalyzed by 2-hydroxyisoflavanone dehydratase (IFD), which forms genistein and daidzein, isoflavonoids (Dixon, 1999; Akashi et al., 2005). The isoflavonoids can further be metabolized to yield phytoalexins, examples medicarpin, or rotenoids, example 9-demethylmunduserone (Akashi et al., 2005). Subsequent species and tissue-specific enzymatic conversions (e.g. glycosylation, o-methylation, and prenylation) create a wide array of structurally diverse group of flavonoids, and isoflavonoids. Phenylpropanoids are natural products derived from the amino acid L- phenylalanine. Phenylpropanoid pathway is conserved in all plant species. The pathway consists of plant phenolics that are responsible for cell wall structural roles, have different wood, and establishing flower color. Phenylalanine deaminates by L-phenylalaninelyase (PAL) as seen in Figure 1 to form cinnamic acid, cinnamate 4-hydroxylase (C 4 H), and then converted to p-coumaric acid. Hydroxycinnamic acids, example sinapic acid, and the monolignols, such as coniferyl alcohol contain C 6 C 3 phenylpropane skeleton. Complex phenylpropanoids are formed by condensation of p-coumaryl coenzyme A with a unit obtained from acetate from malonyl coenzyme A, 4-coumarate:coenzyme A ligase (4CL). Triamine derivatives can then branch out into different pathways such as flavonoids,

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isoflavonoids, lignins/monolignols, coumarins, benzoic acids and stilbenes (Dixon and Sumner, 2002). In most species,

in flavonoid pathway, chalcone synthase condenses p-coumaroyl- CoA

with 3 molecules of malonyl-CoA to generate 4, 2', 4', 6'-tetrahydroxychalcone

(naringenin chalcone). Naringenin chalcone

can be further metabolized to (2S)-5, 7, 4'- trihydroxyflavanone

(naringenin) by chalcone isomerase to form

the primary C15 flavonoid skeleton. In species

that synthesize isoflavones, the enzyme isoflavone synthase

(IFS), another cytochrome P450 monooxygenase, acts as the key metabolic

entry point for the formation of all isoflavonoid compounds

(Akashi et al., 1999; Steele et al., 1999; Jung et al., 2000).

IFS mediates, the intramolecular aryl migration of both liquiritigenin

and naringenin to form 2-hydroxyisoflavanones. Issoflavones are unstable in ambient temperatures and therefore 2-hydroxyisoflavonones in presence of a dehydratase (isoflavone dehydratase, IFD) converts into genistein and daidzein. However, this may not be the only route for their synthesis in plants. Even though phenyl propanoid pathway is the oldest pathway in plants, there are a lot of steps which still have to be elucidated at the molecular level. Very little is known about the flux control, cross talk between the enzymes such as CHS, CHI, IFS and IFD, the association of biosynthetic pathways, enzymes in metabolic channels or regulatory mechanisms in response to different stress conditions (Dakora and Phillips, 1996; Dixon and Sumner, 2003). Plant phenolics contain hydroxyl group attached to the aromatic phenyl ring. Al plant species do not show the presence of all classes, for example CHIs are ubiquitous in the plant kingdom and convert naringenin chalcone to naringenin.

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Figure 1. Schematic representation of biosynthetic pathways leading to flavonoid and isoflavonoid natural products including genistein, daidzein, and biochanin A. CHI, chalcone isomerase; CHR, chalcone reductase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; FS, flavone synthase; F3′H, flavonoid 3′ -hydroxylase; FLS, flavonol synthase; HI4′OMT, 2,7,4′- trihydroxyisoflavanone 4′ -O-methyltransferase; I3′H, isoflavone 3′ -hydroxylase; IFS, 2- hydroxyisoflavanone synthase (from Deavours and Dixon 2005).

In contrast, the other type II of CHIs, appear to be legume specific converting isoliquiritigenin to liquiritigenin (Jez et al., 2000). Members of these classes with specific substitution patterns may be peculiar to certain genera or species. Mostly the isoflavonoids are restricted to the subfamily Papilionoideae of the Fabaceae (Dixon et al., 2002).

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Isoflavonoids are derived from the 3-phenylchrome-4-one (3-phenyl-1,4- benzopyrone) structure. Chromone is a derivative of benzopyran with a substituted keto group on the pyran ring. Isoflavonids are synthesized by the phenylpropanoid pathway in which phenylalanine is used to produce 4-coumaroyl-CoA. This can combine with malonyl-CoA to yield a group of compounds called chalcones, which contain two phenyl rings. The most common isoflavonoids are genistein and daidzein. Genistein undergoes methylation to form compounds like biochanin A and formononetin. Genistein is notable for its estrogenic qualities. Biochanin A is also known for weak estrogenic activities. In particular, biochanin A (5,7-dihydroxy-4´-methoxyisoflavone) is a member of a class of isoflavonoid compounds characterized by estrogenic activity in humans and animals. It commonly occurs in many species of vascular plants, mostly belonging to the legumes (Fabaceae), and many species of grasses (Poaceae). Biochanin A has also been isolated from fruit trees and shrubs, mainly from the genus Prunus. Studies by Liggins et al., in 2000 showed biochanin A to be abundant in red clover (Trifolium pratense), broom dyers (Genista tinctoria), soybean (Glycine max) and plum (Prunus spinosa).

1.5 Commonly Found Phytoestrogens Many phytoestrogens have been identified in vascular plants: formononentine, genistein, daidzein, angolensin, phaseolin, pisatin, glyceolin, rygenin, kiewiton, coumestrol, medicarpin, prunetin, biochanin A and pterocarpin. Some of the isoflavone compounds, particularly phaseolin, glyceolin, kiewiton, coumestrol, medicarpin and pisatin, act as phytoalexins which, in many plant species, produce compounds that defend

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from the invasion of viral, bacterial and fungal pathogens. Earlier studies have shown that genistein, daidzein, ekwol, biochanin A and formononentine are the most known and biologically active compounds with the greatest estrogenic activity (Bruxelles and Roberts, 2001). Isoflavones are estrogenic because they have the hydroxyl and methyl groups resembling estradiol. The number and distribution of hydroxyl and methyl groups on rings A and B of isoflavonoids bears analogous cyclical structural resemblance to estradiol and also determines the strength of estrogenic activity. Estradiol is a female endogenous steroidal hormone. Diethylostilbestrol is a common synthetic analogue. Estrogenic compounds can compete with steroidal hormones as an agonist or antagonist (Mikisieck 1995; Pearce et al., 2003). Some plants are also known to contain steroidal estrogens. The most common phytoestrogens found in plants are cholesterol, and stigmasterol. The common phytosterols such as β-sitosterol, campesterol, and stigmasterol do not bind to human estrogen receptors (ER) and do not exert estrogenicity in female rats (Baker et al., 1999). Earlier studies have shown that isoflavones, commonly found in fodder, undergo biotransformation in animal organisms. Formononetin, which has weak estrogenic activity (found in red clover) possesses a hydroxyl group that undergoes demethylation and reduction into a compound with higher estrogenic activity.

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1.6 Discovery of Phytoestrogens Plants were originally shown to have estrogenic activity in the 1920s. One of the most dramatic examples of their effects on animals was seen in the 1940s when sheep grazing on clover became infertile in Western Australia (Bennets et al., 1946). It was discovered that the plant chemicals responsible for sheep infertility were coumestans. By 1975, several hundred plants had been discovered to produce estrogenic compounds (Chang et al., 1975; Drane et al., 1975). Between 1954 and 1974, reproductive problems were observed in cattle, guinea pigs, rabbits, cheetahs, and mice (Bradbury and White, 1954; Wright 1960; Leavitt 1963; Adams 1995). Since then, some compounds that had caused concerns about reproductive or related risk of phytoestrogens in humans have been discovered with beneficial aspects.

1.7 Structural Resemblance of Phytoestrogens with Estradiol and Its Significance Phytoestrogens act as estrogens in different in vivo and in vitro assay systems by binding to estrogen receptors (ER) (Pearce et al., 2003; Miksicek, 1995). Genistein, Daidzein and Daidzin, as shown in Figure 2, have ring structures similar to estradiol. The hydroxyl groups present in rings A and C of Estradiol correspond to hydroxyl groups on Genistein, Daidzein and Coumestrol at approximately the same positions (Kudou et al., 1991). These hydroxyl groups are located in such a position as to enable binding of the named chemicals to ER proteins to form ligand-binding complexes, thereby, activating transcription of target genes (Suetsugi et al., 2003).

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Figure 2. Comparison of the chemical structures of the phytoestrogens Genistein, Daidzein, and Daidzin with 17ß-estradiol, the female sexual hormone and ligand for estrogen receptors (adapted from Zhao et al., 2002).

Some of the phenolic phytoestrogens are believed to act as plant antibacterial and antifungal agents, as well as insect deterrents (Rivera-Vargas et al., 1993). Most animal research on phytoestrogens has focused on their potential protective effects against age related diseases, such as cardiovascular diseases, osteoporosis, and hormone-induced cancer (Barnes, 1998; Le Bail et al., 1998). Isoflavones are known to possess therapeutic and preventive properties. Compounds such as genistein, daidzein and their derivatives have proven to inhibit the formation of tumors (Danzo, 1998). Phytoestrogens have also been associated with antiestrogenic activities by altering the ER action in breast and other tissues, thus reducing the risk of cancers (Ingram et al., 1997; Nikov et al., 2000). Isoflavones have been shown to act both as attractants and as animal deterrants. A

C

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The biological activities of isoflavones range from properties that suggest important functions in the plant-environment interactions to pharmacological properties in animal cells that may or may not reflect corresponding functions or activities in plants (Dixon, 1999). Natural roles of isoflavones are in both positive and negative plant microbial interactions. For example, isoflavones function in establishing of symbiotic relationships between plant and rhizobial bacteria which is a positive plant microbial interaction (Pueppke et al., 1996). Conversely, production of isoflavonoids results in phenolics that repel the rhizosphere microbes (Weisskopf et al., 2006). A correlation was found between high levels of estrogenic activity and the formation of functional gynoecium in female flowers of mulberry, and of vestigial gynoecium in mulberry male flowers (Maier et al., 1997). The occurrence of endogenous phytoestrogens may suggest a possible pattern or strategy in the reproduction of these dioecious species.

1.8 Tissue Culture and Its Importance Tissue culture opens up an extensive area of biotechnological research into the pot ential use of in vitro cultures to produce highly valuable secondary metabolites, including compounds of medical applications (Verpoorte et al., 2000, 2003; Kieran et al.,1997). In vitro plant tissue cultures are used for: 1) for micropropagation of a large number of genetically uniform and pathogen-free, economically important clones in a limited time and space (Bhau and Wakhlu, 2003; Zobayed and Saxena, 2003); 2) an alternative to whole plants for production of secondary products for biochemical and

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developmental studies (Brisson et al., 1988; Seo et al., 1993); and 3) a source of useful novel phytochemicals that are not produced or have not been detected in differentiated plant tissues (Federici et al., 2003). Phytochemical research has provided valuable information about the presence of flavonoid compounds in plant tissue cultures. Therefore, in vitro plant tissue cultures are considered an attractive source of biologically active compounds and several approaches have been used to increase their accumulation in cultures. Thus, plant cultures can become new sources for beneficial phytochemicals, independent of season and climate conditions (Luczkiewicz et al., 2003). Plant cell cultures have been used to produce novel useful compounds, especially secondary metabolites involved in drug development (Staba et al., 1982). For example, Vinblastine and Vincristine, alkaloids obtained from periwinkle, Catharanthus roseus, are used as anticancer agents (Garnier et al., 1996; Datta and Srivastava, 1997). Diosgenin, a steroid saponin of fenugreek, Trigonella feonum graecum, has anti-fungal and anti-bacterial, and medicinal properties to treat diabetes, high cholesterol, wounds, inflammation and gastrointestinal ailments. Recent studies have shown that diosgenin may possess anticarcinogenic properties as well (Jayadev et al., 2004). Generally, plants such as Hypericium perforantum and Arnica montana do not withstand large field cultures due to pathogen sensitivity (anthracnose), which has led scientists to consider plant cell tissue and organ cultures as an alternative way to produce useful secondary metabolites (Bourgaud et al., 2001). Therefore, cultivation of plant tissues on synthetic

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media offers an efficient alternative to the traditional cultivation in the fields or greenhouses for the production of metabolites of interest. Examples of plant tissue cultures are callus, cell suspension and protoplast cultures. Traditionally, plant tissue explants were used to induce callus, but they are also useful for obtaining cultures of differentiated tissues, such as hairy root cultures, known for the production of secondary compounds in some plant species (Yoojeong et al., 2002). Callus produces an undifferentiated mass of parenchymatic cells that can be subcultured indefinitely to regenerate whole plants under proper hormonal conditions. The induction and growth of callus tissue is dependent on the composition of the medium, mostly on the concentration of the plant growth hormones (auxins and cytokinins), and on plant genotypes (Gonzalez et al., 2001). When friable callus is grown in liquid medium on a shaker, it becomes a cell suspension culture that can be used for production of secondary metabolites of interest. For example, Hypericin and Pseudohypericin have been isolated from Hypericum perforantum in cell suspension cultures (Bais et al., 2002).

1.9 Importance of Mulberry for the Study Osage-orange, Maclura pomifera, red mulberry, Morus rubra, and white mulberry, M. alba (Figure 3) belong to the Moraceae family of angiosperms containing 55 genera and 1,000 species. Both Maclura and Morus genera are dioecious with greenish-yellow unisexual flowers grouped in inflorescences (Harrar and Harrar, 1962; Smith and Perino, 1981).

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Osage-orange is the only species of its genus and a native to the Red River Valley between Texas, Oklahoma and Arkansas (Harrar and Harrar, 1962). Mulberry species are perennial woody plants of considerable economic importance because of their foliage, which constitutes the food for the mulberry silkworms, Bombyx mori, and because the males are widely used in landscape as “fruitless mulberry.”

Male Female Figure 3. Foliage and inflorescences of the Morus alba. A. M. alba male; B. M. alba female.

There are several species of mulberry worldwide. In the USA, red mulberry and white mulberry are distributed from the northeastern areas to Florida in the south to Texas in the west. Maclura and Morus both contain phytoestrogens that activate the estrogen receptor in a transgenic yeast system (Maier et al., 1995; 1997) and thus could produce phytochemicals of interest for medical research in prevention and or treatment of cancers. The Moraceae family of dioecious flowering plants is native to warm, temperate, and subtropical regions of Asia, Africa, North America, and southern Europe. Mulberry trees grow fast initially and have a slow growth later. Their fruits are multiple compound, 2–3 cm long. In several species, mulberries begin as white to pale yellow with pink edges, becoming red during ripening and dark purple to black when fully ripened.

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Since in vitro plant tissue cultures have proven to be attractive sources of biologically active compounds that can be independent of the season and climate conditions (Luczkiewicz and Glod, 2003), one aim of this study was to establish in vitro tissue cultures. Mulberry trees are conventionally propogated by grafting and cutting (Honda 1972). Studies have been done on the establishment of callus culture, regeneration of plants, micropropogation, cryopreservation of germplasm, synthetic seed, and the formation of secondary metabolites in cell cultures. Morus species, in addition to producing many secondary metabolites, are a rich source of prenylchalcone and prenylated 2-arylbenzofuran. Morin (2,3´,4,4´,6-pentahydroxybenzophenone) is a constituent of the heartwood of M. alba (Haley and Bassin 1951; Spada et al 1956). Quercitin, rutin, and quercitin-3-triglucoside (Moracetin) were obtained from an aqueous methanolic extract of mulberry leaves (Naito 1968). Later four prenylated flavonoids, Mulberrin, Mulberrochromene, Cyclomulberrin, and Cyclomulberrochromene were isolated along with betulinic acid from the stem and root bark of M. alba, (Deshpande et al., 1968). Beta–tocopherol was isolated from root bark (Kang et al., 1999). Mulberry constituents are comprised of two molecules of prenylphenols (Nomura 1988). Leaves of M. alba, when infected with Fusarium solani f. spp. Mori, produced a natural Diels- Alder-type adduct Chalcomoracin, known as a phytoalexin and as 1-deoxy-nojirimycin found in leaves, root and bark (Nomura 1988). Mulberry is a crop of economic importance in the sericulture industry. Previous studies indicate that estrogen / estrogen-like compounds occur during the development of

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female reproductive structures in Osage-orange and mulberry (Maier et al., 1997) and quaking aspen, Populus tremuloides Michx. (Khaleel, 2003). Bacillus thuringiensis has been reported on the adaxial as well as the abaxial surfaces of the leaf (Ohba, 2007). Mulberry supplies leaves to raise silkworms. Mulberry leaves, bark, and branches have been used in Chinese medicine to treat fever, protect the liver, improve eyesight, strengthen the joints, facilitate discharge of urine and lower blood pressure. It is only recently that their mechanism action has been related to their antioxidant activity. The chemical composition of mulberry leaves includes rutin, quercetin, isoquercitin and other flavonoids. In most mulberry-growing countries, especially in China and India, production is focused on enhancing the foliage. The leaves, the sole food source of the silkworm (Bombyx mori L.), the cocoon of which is used to make silk, have a large ecological and economical importance. In Europe, mulberry is appreciated more for their fruits than for their foliage. However, almost all the parts of the tree are used for pharmacological studies all over the world. The leaves have been shown to possess diuretic, hypoglycemic, and hypotensive activities, whereas the root bark of mulberry has been used for antiinflammatory, antitussive, and antipyretic purposes. Mulberry fruits can be used as a warming agent, as a remedy for dysentery, and as a tonic, sedative, laxative, odontalgic, anthelmintic, expectorant, and emetic (Ercisli and Orhan et al., 2006). In Italy, the berries of Morus nigra and Morus alba are consumed fresh or in the form of various confectionary products such as jam, marmalade, frozen desserts, pulp, juice, paste, ice cream, and wine (Ercisli and Orhan et al., 2006).

Full document contains 105 pages
Abstract: Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high performance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin A has been known to have an antiestrogenic activity in mammals. Isoflavonoid compounds have been characterized as strong protein tyrosine kinase inhibitors in variety of animal cells. Isoflavones are structurally similar to estradiol, and display agonistic and antagonistic interactions with the estrogen receptor. Isoflavones possess therapeutic and preventive properties such as being used for postmenopausal osteoporosis, breast cancer, and inhibition of tumors.