3 .1-l BIO.ACTIVE COMPOUNDS

ISOLATED FROM MISTLETOE (Scurulla oortiana (Korth.) Danser)

PARASITIZING TEA PLANT (Camellia sinensÍs L.)

by

CHANDRA KIRANA

Department of Horticulture, Viticulture and Oenology

Waite Agricultural lnstitute

The UniversitY of Adelaide

South Australia

Thesis submitted in part fulfilment of the requirements of the Degree of

Master of Agricultural Science

of

The University of Adelaide

Faculty of Agriculture and Natural Resource Sciences

October 1996 Table of contents

page no.

Declaration

Acknowledgements ii

List of Tables i¡i iv List of Figures vi Summary

Chapter 1. Introduction 1

Chapter 2. Literature Review 5

2.1. lmportant components of mistletoes 5

2.l.l.Pharmacologicallyactivelowmolecularweight 7 Proteins in mistletoes 2.l.2.Pharmacologicallyactivehighmolecularweight

9 Proteins in mistletoes

2.1 .3. Pharmacological ly active non-proteinaceous

comPounds in mistletoes 10

2.2.lmporlant components of the tea plant (camellia sinensis) 14

2.3. Chemical relationships between mistletoes and their host

plants 21

Chapter 3. lnvestigation of flavonoids in mistletoes 26

3.1. lntroduction 26

3.2. Materials and methods 28

3.2.1. Source of Plant materials 28 3.2.2. Extraction and isolation of flavonoid compounds 29

g.2.g. separation and identification of flavonoids present in

the butanol extracts 29

g.2.g.1. Thin Layer Chromatography 30

3.2.9.2. Acid Hydrolysis of flavonoid glycosides 31

3.2.3.3. Ultraviolet spectral analysis 33

9.2.9.4. High Performance Liquid Chromatography -

Electrospray-Mass Spectrometry 34

3.2.3.5. High Performance Liquid Chromatography 35

3.2.4. Quantification of phenolic compounds in mistletoes and

their hosts 36

9.2.4.1. Colou ri metric assays f o r total phenol ic compounds 36

9.2.4.2. Quantitation of individ ual f lavonoids

by HPLC 37

3.3. Results and discussion 37

3.3.1 . ldentification and characterisation of flavonoids

in mistletoes 37

9.9.2. Relationships between total flavonoid levels in

mistletoes and host Plants 54

Chapter 4. Antifungal activities of phenolic compounds found in mistletoe

and tea extract 59

4.1. lntroduction 59

.2Malerials and Methods 61

4.2.1. Source of plant materials and fungi 61 4.2.2. Extraction of phenolic compounds

4.2.3. Pre-AssaY

4.2.3.1. Stock culture

4.2.3.2. Antif ungal screening

4.2.4. Antifungal assaY

4.3. Results and discussion

Chapter 5. lnvestigation of alkaloid components of mistletoes

5.1. lntroduction

5.2.Materials and methods

5.2.1. Source of Plant materials

5.2.2. Extraction and isolation of alkaloid compounds

5.2.9. separation and identification of alkaloids present in

fractions of extract

5.2.3. 1 . Thin Layer Chromatography

5.2.3.2. lnfra Red spectral analysis

5.2.3.3. Gas Chromatography-Mass Spectrometry

5.2.3.4. High Performance Liquid Chromatography

5.2.4. Quantification of alkaloids in mistletoe and tea plant

5.3. Results and discussion

5.3.1. ldentification and characterisation of purine alkaloids

5.3.2. Total purine alkaloid levels in mistletoe and

in tea plant

Chapter 6. General Discussion and Conclusions

6.1. General discussion

6.2. Conclusions 6.3. Future Research 86

7. References 87

8. Appendices 98

Appendix 1 99

Appendix 2 112

Appendix 3 121

Appendix 4 125

Appendix 5 127

Appendix 6 128 DECLARATION This work contains no material which has been accepted for the award of any other degree or diploma in any University or tertiary institution and to the best of my knowledge and belief, contains no material previously published or written by any other person, except where due reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.

signed: date: 30 Seftembet ln6 ACKNOWLEDGEMENTS

Dr Max E' I would like to thank my supervisors, Dr Graham P' Jones and my Tate for their invaluable encouragement, advice and help throughout project.

guidance, help I would also like to thank Dr Andrew J. Markides for his and advice during my work using the microbiological assays'

I am most grateful to Dr lr. Martanto Martosupono at the Research lnstitute of Tea and Cinchona, Bandung, lndonesia, for his kindness in supplying the Plant material.

My thanks go to Mr Yoji Hayasaka at the Australian Wine Research lnstitute for helping with the mass spectral analyses and to Gael Fogafty and Margaret Cargill ( English consultation).

I am grateful to all the staff and students of the Department of Horticulture, Viticulture and Oenology, Waite Campus, University of Adelaide, particularly Ms Lis Williams, for their help and friendship during my studies.

I would Iike to thank AusAlD for the funding of my studies allowing me and my family to reside in Australia during this time and also the University of Brawijaya, Malang, lndonesia for allowing me study leave'

I owe special thanks to my husband, Eko and daughters, Anindita and Larissa for their love, understanding and patience throughout my studies.

Finally, I thank my father and mother for their continuous encouragement, caring love and prayers.

ii List of Tables

page no. Table number

Table 3.1 Extraction and isolation of flavonoids in S. oortiana grown on different hosts and their hosts' 32 35 Table 3.2 Mass Spectra'HPLC gradients 35 Table 3.3 Analytical HPLC gradients Table 3.4 Thin Layer Chromatographic properties of flavonols in mistletoes growing on different hosts 39 Table 3.5 The maximum wavelength (À,", nm) values of uv spectra 44 of flavonols found in S. oo¡tiana growing on different hosts Table 3.6 The bathochromic shifts of band I of flavonols found in S' oortianagrowing on different hosts and the standards in NaOMe 47 Table 3.7 The bathochromic shift of band ll of flavonols found in S' oortianaon different hosts and standards in NaOAc 47 Table 3.8 The bathochromic shift of band I in NaOAc with addition of H.BO. relative to MeOH spectrum of flavonols found in s. oo¡tianagrowing on different hosts 48 Table 3.9 The hypsochromic shift of band I of the AlCl. spectrum with that obtained in Alcl/Hcl of flavonols found in s.oorliana growing on different hosts 48 Table 3,10 HPLC retention times of flavonols in S. oo¡tiana (Korth.) Danser growing on different hosts 51 Table 4.1 Analysis of variance of the diameter ol Fusarium sp. al 1g'hday in the presence of an ethyl acetate extract of tea and S. oortiana grown on tea and citrus 64 Table S.1 HPLC retention times of purine alkaloids in S. ooriiana (Korth.) Danser grown on tea and C. srnensls 76

iii List of Figures page no.

Figure 2.1 Structure of selected compounds found in mistletoes 12 Figure 2.2 Structures of principal polyphenolic components found in tea leaves 20 Figure 3.1 Flavone and flavonol structures 27 Figure 3.2 Thin Layer Chromatogram of standards and flavonoids found in butanol extract of S. ooftiana (Korth.) Danser growing on different hosts 40 Figure 3.3 Thin Layer Chromatogram of standards and flavonoids found in S. oortiana (Korth.) Danser growing on different hosts and the respective hydrolysates 42 Figure 3.4 Electrospray positive ion mode mass spectra of the hydrolysate of flavonoids found in S. oortiana growing on different host plants 50 Figure 3.5 HPLC of flavonoids in butanol extract oI S. oortiana growing on different host plants 52 Figure 3.6 Total phenolic content of butanol of mistletoes and the hosts using Folin Ciolcateu's Reagent 56 Figure 3.7 Quercitrin content of butanol extracts of S. oortiana grown on different hosts and in the host plants 56 Figure 3.8 lsoquercitrin content of butanol extracts ol S. oo¡tiana grown on different hosts and in the host plants 57 Figure 3.9 Rutin content of butanol extracts of S. oortiana grown on different hosts and in the host plants 57 Figure 4.1 The effect of ethyl acetate extracts ol C. sinensis and S. oortiana grown on tea and citrus on the growth of Fusarium sP. 65 Figure 5.1 Structure of purine alkaloids 68 Figure 5.2 Thin Layer Chromatogram of purine alkaloids and found in different fractions of extracts of S.oortiana (Korth.) Danser growing on tea and C. srnensrs 74 Figure 5.3 Thin Layer Chromatogram of purine alkaloids found

IV in different fractions of extracts of S. oo¡tiana (Korth.) Danser growing on different host plants 75 Figure 5.4 Gas chromatogram and Mass spectra of theobromine in S. oo¡tiana (Korth.) Danser grown on tea 77 Figure 5.5. Content of caffeine obtained in different fractions of extract 79 Figure 5.6 Content of theobromine obtained in different fractions of extract 79 Figure 5.7 Caffeine content in mistletoe and tea plant in different fractions of extract 80 Figure 5.8 Theobromine content in mistletoe and tea plant in different fractions of extract 80

V SUMMARY

The primary aim of this thesis was to investigate non-proteinaceous low molecular weight flavonoid and alkaloid compounds in Scurulla oortiana (Korth.)

Danser grown on Cameltia sinensis. S. oo¡tiana (Korth.) Danser growing on

Citrus maxima and Persea americana have been used as control comparisons for the flavonoid and alkaloid compounds when this mistletoe parasitizes the tea plant. Three flavonols, quercitrin (quercetin 3-rhamnoside); isoquercitrin

(quercetin 3-glucoside) and rutin (quercetin 3-rhamnoglucoside) have been identified in S. oortiana (Korth.) Danser growing on different hosts. The identification and characterisation of these flavonoids was carried out using various chromatographic and spectrometric procedures. The high performance liquid chromatography patterns of phenolic components found in mistletoes were significantly different to those found in the host plants. The flavonoids found in

S. oo¡tiana (Korth.) Danser were found to be independent of the host plants.

The total content of individual flavonols found in butanol extracts of mistletoes and the hosts were quantitated by HPLC and, in general, the amount of individual flavonols found in the parasitizing mistletoes was always much higher than those found in the respective host plants. However, between

different mistletoes, the amount of individual flavonols varied substantially. The

HPLC patterns of individual flavonols of mistletoes were different of those of the

respective hosts, thus the flavonols with the highest concentration in the

mistletoes were not necessarily the highest in the mistletoe's host.

VI The activities of the phenolic compounds in an ethyl acetate extract of mistletoes grown either on tea or citrus were measured. They showed only weak activity against the growth of Fusarium sp. The activity of these extracts can be attributed to the present of quercetin derivatives in the extracts'

Two purine alkaloids, caffeine (1,9,7, trimethylxanthine) and theobromine

(3,7 dimethylxanthine), have been isolated from and identified in S' ooftiana

(Korth.) Danser parasitizing tea ptant, C. sinensis. The identification and characterization of these compounds was carried out using chromatographic, spectrophotometric and spectrometric proced u res.

The total individual purine alkaloids found in the mistletoe and the host were quantitated by HPLC. In contrast to the findings with flavonoids, the content of purine alkaloids in mistletoe was always less than those found in the host. The total caffeine found in C. srnensis host was 6.0% (dry weight) and theobromine O.4% (d¡y weight). The relative amount of caffeine in the parasitizing mistletoe was approximately 10% of that found in the host, whilst the amount of theobromine was approximately 71" ol that in the host tea plant. lt seems likely that both caffeine and theobromine in the mistletoe were derived from the host tea plant, although the possibility that signal molecules from the tea plant induced alklaoid synthesis in the parasite can not be excluded'

vii YOÃ

CHAPTER I

INTRODUCTION

Mistletoes are perennial evergreen hemiparasitic plants which attach to stems and branches of many species of trees and shrubs by using modified roots called haustoria (Lawrence,1951; samuelsson, 1974; Anderson and

phillipson, 1g82). Mistletoes belong to the family Loranthaceae. These parasitic

plants are widely distributed in the world; they can be found in the subtropical

regions but mostly occur in tropical areas (Samuelsson, 1974). Mistletoes have

extremely varied host plants including apple, ash, hawthorn, lime and acorn

(Anderson and Phillipson, 1982). Although some hardwood trees were said to

be resistant to parasitisation by mistletoes and others rarely infected by mistletoe

(Becker, 19BO), Nair and Krishnakumary (1990) reported lhal Dendrophthoe

falcata Ettingsh, which is a bushy, smooth, grey-barked mistletoe and one of the

seven species of mistletoes in lndia, may have more than 300 host plants.

Although many Loranthaceae decrease the quality of timber, fruit

production and growth of ornamental plants (Pancho, 1985) it is understood that

mistletoes growing on certain host trees have been used as medicinal herbs

from ancient times because of their demonstrated pharmacological activities. As

long ago as 1910 it was suggested that mistletoe extracts had hypotensive,

1 (Anderson and antihaemorrhagic, diuretic, antispasmodic and cardiac activities coloratum Phillipson, 1982). ln China an extracl ol Viscum album Linn' var' "Sokhisei", OHWI is used as a medicine to control hypertension and is called ,,Matsunomidori" whilst in Japan is commercially sold as a medicine to treat kaempferi diabetes. Both medicines contain extracts from the mistletoe Taxillus plant to Danser (Fukunaga et a1.,1989 a). The susceptibility or resistance of a be infected by mistletoes is therefore of practical interest.

Recently, mistletoes grown on specific host plants have been recognized as containing antitumor agents (samuelsson, '1974; Anderson and Phillipson' from 1g82; Khwaja et a1.,1986). ln central Java in lndonesia, extracts obtained mistletoes parasitizing tea plants are used as medicinal agents for combating tumors. According to Suharwadji et at. (1979) there are 5 species of mistletoe which parasitize tea plants (camellia sinensþ in lndonesia. They are scurrula

philipensis, Dendropthoe pentandra, Macrosolen cochinchinensis, M. tetragonus

and Lepeostegeres gemmiflorus. Although, little is known about the species of mistletoe which grow on tea plants or about the bioactive compounds in the

extracts made from these mistletoes, the extracts from mistletoe on tea plant are

extensively used to treat cancerous diseases to such an extent that mistletoes

are rarely found in the wild nowadays. Most of the material is cultivated in

plantations.

The tea plant is grown in about 30 countries but the beverage made from it

is consumed worldwide (Graham, 1992). From ancient times tea plant extract

has been known to possess some pharmacological activities. Tea extract has

already been identified as anti-cariogenic (Sakanaka et a1.,1989). Recently, it

2 has been shown that tea extract has antimutagenic and antioxidant propefties as

well as promoting anti-tumour activity (sigler and Ruch, 1993; Zhu and Xioa,

1ee1).

It is understood that most of mistletoes are xylem-tapping parasites and

derive no nutritional benefit from their host other than the small amounts of

organic carbon and nutrients. The fact that mistletoe leaves tend to have

transpiration rates several times higher than those of their host has been

reported (Raven, 1983, Schulze et a1.,1984). Mistletoes also accumulate large

amounts of calcium, potassium, phosphorus and smaller amounts of nitrogen

(Schulze et a\,1984; Schulze and Ehleringer' 1984).

ln terms of the chemical relationships between the mistletoe and host, mistletoes contain different compounds ü research has shown that different r depending upon the host plants on which they live (Anderson and Phillipson,

1982; Khwaja et al., 1986; Cordero et al., 1989). However, a number of

contradictory findings regarding chemical relationships between mistletoes and

their host plants have also been reported (Hull and Leonard, 1964 a & b; Becker,

1986). lndeed, it is apparent that there is no universal understanding about the

chemical constituents of mistletoes and their host plants.

As a means of better understanding the chemical relationships between

mistletoe parasitizing the host tea plant, the low molecular weight compounds

especially flavonoids and alkaloids found in both mistletoe and the host plant

have been studied. Tea plants (Camellia sinensis) are known to possess

ceftain polyphenolic compounds which are considered to have anticancer

3 activities. Also, s¡nce mistletoes in many instances are known to contain isolated bioactive compounds, the antifungal activity of the phenolic compounds

.*1. from mistletoe parasitizing tea plant has been examined.

I

i í t'

! I !

T I

I

4

T

I CHAPTER 2

LITERATURE REVIEW

2.1 IMPORTANT COMPONENTS OF M¡STLETOES genera, Mistletoes belong to the family Loranthaceae, which consists of 36

includes 1,300 species (Anderson and Phillipson,1982) and contains two

subfamilies, Loranthoideae and viscoideae (Lawrence, 1951; Rendle, 1952).

According to modern taxonomy, however, mistletoes are classified as flowering i hemiparastic plants and belonging to three families, Loranthaceae,

Eremolepidaceae and Viscaceae (Mitich, 1991). In fact, usage of the term

,,mistletoe" has recently become more complicated and varied depending on ü r¡û how their taxanomic groupings have been approached' For example, I phoradendron spp. have been described as xylem tapping mistletoes since from

a physiological point of view they are gaining their nutrients from xylem mistletoe

- host connections (Marshall and Ehleringer, 1990; Panvini and Eickmeier, 1993)

I whilst other species are classified on the grounds of them being epiphytic.

Scurulla oortiana (Korth.) Danser is one such xylem tapping mistletoe. lt has a

wide host range and grows upon both trees and shrubs (Backer and Van den

Brink, 1965). It has been noted that different countries apparently have different i "common" mistletoes. For example Viscum album, L. is found in Europe, I Phoradendron flavescens Nutt. in The United States of America (Anderson and

5 r The phillipson, 1982), and Hyphear tanakae in Japan (Fukunaga et al., 1988)' population of Korean mistletoe, viscum album var. coloratum, is the east Asian of the v. atbum and has distinct features compared to the European population

same species (Khwaja et a1.,1980; Becker, 1986)'

Currently there is an increasing interest in the relationship between these

parasites and their host plants as well as in the pharmacological activities of their

extracts, especially their antitumour activities. ln Europe a mistletoe extract, of which is fermented by Lactobacittus plantarum and sold under the trade name

lscador, has been recognized as an anticancer agent. lt is used by herbalists,

as well as therapists in private practice (Holtskog et a1.,1988). lt was repofted

by Bradley and Clover (1989) that lscador was able to increase the length of

su¡vival of lung cancer patients from the time of diagnosis up to four times that

commonly recorded. ln addition, it is known that mistletoe extract has an nl potentially important in the treatment of ip immuno-modulatory effect which is 'tü i cancer (Kovacs et â1., 1991). Khwaja et al. (1980) reported that low

concentrations (0.4-50 pg/ml) of mistletoe extract (V. album var.coloratum)

completely inhibited the growth of human tumour cell lines Hela, KB and

Leukemias, Molt 4, RPMI-1788 and CCRF-CEM. Khwaja et al' (1986)

suggested that extracts from mistletoe growing on different host plants be used

to treat different types of malignancies; the degree of cell growth inhibition varies

with the type of cell lines. They also suggested that Leukemia L1210 cell line

was the most suitable to test the anticancer activities of mistletoe extracts rn

vitro. The methods used to prepare mistletoe extracts also have an effect on the

I degree of cell growth inhibition repofted.

I

6 r Like other chlorophyllic plants, mistletoes contain compounds necessary for

normal metabolic functions such as carbohydrates (ranging from simple sugar to

polysaccharides), phenolic compounds, faüy acids, amino acids and amines'

Mistletoes may also contain compounds which have pharmacological activities

(Anderson and Phillipson, 1982). Different mistletoes may contain different

pharmacologically active compounds qualitatively as well as quantitatively

depending on the host plants on which they live (Anderson and Phillipson, 1982;

Khwaja et a1.,1986)

The pharmacologically active components found in mistletoes have been

identified as both low molecular weight compounds and certain proteins. The

nature of these compounds is discussed in the following sections.

2.1.1 Pharmacologically active low molecular weight proteins in

mistletoes

The proteinaceous fractions found in mistletoe extracts consist of both

small basic proteins and high molecular weight proteins (Samuelsson, 1974;

Anderson and Phillipson, 1982).

A mixture of small basic proteins was first isolated from European mistletoe

(V. album) by Winterfeld and Bijl in 1948 and given the name viscotoxin

(Samuelsson and Pettersson, 1970). ln mice, viscotoxins produce hypotension

and bradycardia in sublethal doses, vasoconstriction in arteries of skin and

skeletal muscle at higher doses when administered intra-arterially, and had high

toxicity when administered intraperitoneally (Samuelsson, 1974). The same

qualitative pharmacological activities as viscotoxins have been reported for

I phoratoxin which is a small basic protein isolated from the mistletoe

7 Phoradendron tomentosumsubsp. macrophyllum (Mellstrand and samuelsson,

1g7g). However, phoratoxin had lower activity than viscotoxin since it needed a dose about 10 times higher to produce the same effect (Rossel and

Samuelsson, 1966).

Viscotoxins comprise three groups which have different molecular weights and amino acid sequences. These are known as viscotoxin 42, viscotoxin 43,

and viscotoxin B and have molecular weights of 4833, 4856 and 5123

respectively (Samuelsson and Pettersson, 1970). Phoratoxin has a molecular

weight of 5000 (Mellstrand and samuelsson, 1973). The main features of

viscotoxins are that theY :

- consist of a sequence of 46 amino acid residues with lysine at both N and C

termini;

- have 6 half cystine residues which form three disulfide bridges; and

- have high isoelectric points (about pH 11) because of the large number of

lysine and arginine residues and also because most of the dibasic amino

acids are present as the corresponding amides (Samuelsson, 1974).

The compact nature of these proteins, with three disulfide bridges in a chain of

only 46 amino acids, causes viscotoxins to be very stable; heating at 100oC for

30 minutes has no influence on their toxic propefties (Samuelsson, 1974). The

amino acid sequences of the viscotoxins and phoratoxins are identical in 28 of

the 46 positions, including the 6 half cystine residues although their C terminal

sequences are different and the amino acid residues tryptophan and histidine are only found in phoratoxin. Any differences between viscotoxins and

phoratoxin sequences may be explained by:

I (1) a single base is replaced in the coding tripletfor differences in position 6, 9,

15, 18, 21,22,28,37,38,39 and 45

(2) two bases are replaced in the coding triplet for differences in position 19,24,

25,41,43,44, and 46

2.1.2 Pharmacologically active high molecular weight proteins in mistletoes

The high molecular weight proteins found in mistletoe extracts are lectins.

They comprise three different fractions according to their average molecular weights and sugar specificities. Lectin l, lectin ll, and lectin lll have molecular weights of 1 1SO0O, 60000 and 50000 respectively, and their sugar specificities are D-galactose binding to lectin l, D-galactose and N-acetyl D-galactosamine attaching to lectin ll, and lectin lll interacting with N-acetyl-D-galactosamine

(Franz et al., 1981 ; Ziska and Franz, 1981). Franz et al. (1981) found that all of these lectins reacted with human erythrocytes in the three blood groups A, B, and O. All these proteins are toxic but lectin I is highly toxic; lymph-node cells were killed within 6 hours by lectin I at a concentration of 25-80 pg/ml. lt was

reported that lectin I also inhibited protein synthesis in a lysate of rabbit

reticulocytes with an lD5g of 2.6 pg/ml (Stirpe et al., 1980; Franz et al., 1981).

Stirpe et al. (1980) reported that lectins inhibited protein synthesis in a cell free

system as well as in whole cells.

From three cytotoxic extracts of mistletoes known to possess antitumor

activity, Khwaja et al. (1986) found that mistletoe lectin was more cytotoxic (IDSO I the mistletoes 3.5 ng/ml) than viscotoxin or the alkaloid fractions isolated from extracts against Leukemia L1210. The concentration of lectins in mistletoe that a fresh decrease after fermentation. Khwaia et at. (1986) also showed the fermented extract of Korean mistletoe (lDso 0.1 ug/ml) was more active than

of lscador "Qu" was a better one (lD5g 9.6 ¡rg/ml), and that a fresh crude extract Ribereau- inhibitor of Leukemia L1210 cell growth than a fermented extract.

Gayon et al. (1986) found that the concentration of lectins was approximately They 100 ng/ml in fermented lscador and looo ng/ml in unfermented lscador' cell lines' stated that the effects of fermentation were different on various tumour

They found that the growth of Molt 4 cells was markedly less inhibited by fermented lscador than by the unfermented one whereas fermented lscador the inhibited the growth of HTC cells more than the unfermented extract. Using but it same fermented extract of lscador on Molt 4 cells gave the same effect, be needed a longer time than for HTC cells. The extreme toxicity of lectins may the reason why lscador has to be fermented. The species of mistletoe can also

influence the inhibition; Khwaja et al. (1986) indicated that an extract of Korean

mistletoe growing on an oak tree was more effective as an inhibitor of tumour

cell lines than European lscador and an extract of Californian mistletoe growing

on the same hosts

2.1.g Pharmacologically active non-proteinaceous compounds in

mistletoes A range of low molecular weight compounds have been identified in

mistletoes growing on different hosts. Mortimer (1957) isolated and identified

10 are four alkaloids from Loranthus sp. growing on Duboisia myoporoides' These nicotine (the structure is illustrated on figure 2.1. B(¡¡) ), hyoscine, anabasine' and isopelletierine. Khwaja et al. (1980) found as many as 10 unidentified alkaloid components which inhibit the growth of cancer cells in vitro in Korean mistletoe (V. atbum var coloratum). However, alkaloid fractions ll and lV were the most effective in inhibiting the growth of the leukemia cells (ED5O at 0.19 pg/ml and 0.4S pg/ml). ln addition to this class of compounds, Cordero et al.

(1ggg) isolated five quinolizidine alkaloids from Viscum cruciatum growing on

Lygos sphaerocarpa using alumina and silica gel column chromatography.

Three are tetracyclic derivatives of the sparteine group (the structure of

sparteine is illustrated in figure 2.1 B(i) p 8) viz (+)- retamine, (-)-lupanine, (-)-

anagyrine, and two tricyclic degradation products and (-)-cytisine' (-)'N-

methylcytisine. Quinolizidine alkaloids are very toxic and inhibitory to most

organisms while sparteine is known as an oxytoxic drug (stimulating uterine

contraction) (Wink, 1987).

Another group of compounds which has been found in mistletoes are the

cardiac glycosides. Boonsong and Wright (1961) isolated the same three cardiac

glycosides, strospeside, odoroside and neritaloside (figure 2.1. A) from three

different species of mistletoe, Phrygitanthus celastroides, Dendrophthoe falcata

and Amyema congener, growing on Nerium oleander.

11 H co

I R H B(Ð OH

( CH A I HCOH I ¡ H3COCH cHs B(ii) HOCH I

cHs

Figure 2.1. A. StructureS of cardiac glycosideS. Strospeside'R = OH; neritaloside:

B(ii) Nicotine. R = OAci Odoroside: R = H. B. Structure of alkaloids : B(i) sparteine,

ln addition, a var¡ety of flavonoids from different mistletoes parasitizing different host plants have been characterized. For example, Graziano et al-

(1967) isolated (+)-catechin, quercitrin, reynoutrin and avicularin from Argentine mistletoe (a folk medicine in Argentina) growing on five different host plants using paper and thin layer chromatographic methods. Ohta and Yagishita (1970) found three kinds of flavonoids in V. atbum vat. colorafum growing on Pyrus communis. They are rhamnazin-3-Omono-D-glucoside (flavoyadorinin-A), and two complex flavonoids named flavoyadorinin-B (7,3' di Gmethyl-luteolin-4'-G mono-D-glucoside) and homoflavoyadorinin-B (7,3' di'Omethyl-luteolin-4'-GD- glucoapioside). Fukunaga et al. (1987) isolated 2'-hydroxy'4' ,6'' dimethoxychalcone-4-o-glucoside; 2'-hydroxy-3,4',6'-tri-methoxychalcone-4-G glucoside; 2'-hydroxy,4',6'-dimethoxy-chalcone-4'ollapiosyl(1+2)l glucoside;

12 5,7-dimethoxyflavonone-4'-o-glucoside and 3',5,7-trimethoxyflavonone-4'-O glucoside from European mistletoe (the dried commercial product). Fukunaga ef

at. (1988) isolated 4 flavonoids from Japanese mistletoe (Hyphear tanakae) epiphyting lo Castanea crenata, Prunus mume, P. armeniaca var' anzu, and

Zetkova serrata. These are rhamnocitrin-3-Orhamnoside, kaempferol-3-G rhamnoside, [email protected] and quercetin-3-O-rhamnoside.

Kaempferol monoglucoside isolated from So/anostema argelhas been shown as an antispasmodic agent and used for the treatment of various colics and pains by the sudanese (Khalid et al., 1992). Moreover, Fukunaga et al. (1989 a) identified rhamnazin -g,4'-di-oglucoside, (2s)-homoeriodictyol'7'@- from [apiosyl(1+2) glucoside, flavoyadorinin B and homoflavoyadorinin B

Japanese mistletoes growing on twelve different host trees. Fukunaga et

a/.(1g6g b) also isolated hyperin and quercitrin from Taxillus yadoriki, avicularin,

quercetin, hyperin, quercitrin and taxillusin from T. kaempferi and chrysoeriol-4'-

Gglucoside from Korthatsetta japonica growing on different host plants.

According to Fukunaga et at. (1989b) hyperin and taxillusin possess anti-

microbial activity against Ktebsietla pneumonia while quercitrin, rhamnetin'3'(}.

rhamnoside, rhamnocetrin-3-Grhamnoside and homoflavoya-dorinin-8, which

were obtained from L yadoriki, H. tanakae and V. album var colorafum, cause a

temporary hypotensive response in normal rats when iniected intravenously

(Fukunaga et a\.1988).

The separation and identification of these flavonoids was done by standard

TLC techniques and by using UV, lR, proton NMR and carbon NMR spectral

data (Fukunaga et a1.,1987; Fukunaga et al., 1988 ; Fukunaga et al., 1989 a, b).

It has also been reported that flavonoids have been successfully separated by a

13 (Johnston column chromatographic method using Sephadex LH 20 in methanol et al., 1968).

The effectiveness of an extract ol Crataegus species used clinically in by treating heaft disease is largely due to the inhibition of phosphodiesterase hyperoside, vitexin, vitexin rhamnoside, and monoacetyl vitexin rhamnoside

(schuessler et at., 1992) whilst diosmin, a flavonoid extracted from citrus limon, is used clinically for the treatment of venous insufficiency (Codignola et al',

1 ee2).

ln addition to the flavonoids, a group of norditerpene lactones found in

tteostylus micranthus, one of New Zealand's large leafy mistletoes, showed

cytotoxic activity on a P-338 leukemia cell line (Bloor, 1991)

Further to the above, lipophilic compounds are often found in various

mistletoes. For example, oleanolic acid, p-amyrin acetate, phytosterol were

isolated from the n-hexane extracts whilst phytosterol-p-D-glucoside, betulinic

acid, and syringin from the chloroform extracts of Vicium, Taxillus and Hypear

species (Fukunaga et al., 1987, Fukunaga et al., 1988, Fukunaga et al', 1989 a

& b).

2.2 IMPORTANT COMPONENTS OF TEA PLANT (Camet ia sinenstq

Camettia sinensis belongs to the family Theaceae, sometimes also named

Ternstroemiaceae. The Theaceae is a family of some 30 genera and 500

species of shrubs and trees (Lawrence, 1951; de Wit, 1963). The plant is

distributed in tropical and subtropical regions. The genus Camellia includes more than 50 species, distributed in the warmer and particularly the

14 of mountainous, patts of eastern Asia. C. sinensis originated from the highlands

Assam (de Wit, 1963).

Economically, C. sinensis is the most important commercial species of tea plant and is used as a beverage all over the world. lt is the most widely consumed beverage other than water, especially in Eastern countries. lt has been reported that the worldwide consumption of tea as a beverage was

approximately 0.12 litre per capita per year (Graham, 1992)'

Tea as a beverage can be differentiated into two types of product, either

fermented or nonfermented. The type and yield of polyphenols present in the tea

leaves depends upon this fermentation. ln the fermented form (black tea), the

concentration of monomeric polyphenols is reduced and polymerised and

oxidised polyphenols increase during the fermentation process. On the other

hand the non-fermented form (green tea) effectively retains the chemical

composition of the green leaf (Samarasingham, 1990). Oolong tea is a partially

oxidized product (Graham, 1992).

Drinking tea is customary after every meal in Japan and its widespread use

has been the stimulus for scientific studies. A traditional Japanese saying that

"drinking tea makes the mouth clean" led Sakan aka et al., (1989) to study the

effects of Japanese Green Tea extract on the inhibition of the cariogenic

bacterium, Streptococcus mutans. Additionally, lt has been reported that green

tea extract inhibits cary formation in teeth (Otake et al., 1991;Yu et al., 1992)

and it also has been found that several polyphenols in the tea extract inhibit the

growth ol Streptococcus mutans, a cariogenic bacterium (Sakanaka ef a/., 1989)

and Clostridium difficite and C. pertringens (Ahn et al., 1991). Fukai ef a/. (1991) noted that tea polyphenols have antibacterial activity against several

15 phytopathogenic bacteria. ln addition to this, from ancient times tea plant extract green has been known to possess other pharmacological activities. ln China tea has been used as a crude medicine for 4000 years (Kada et al., 1985)'

Although tea has traditionally been used as a medicinal herb, only recently have ln scientific studies been initiated to study the pharmacological activities of tea. lndonesia, saturated tea extract is commonly used to treat diarrhoeal diseases and it has been reported that Japanese green tea leaves inhibited the growth of various bacteria causing diarrhoeal diseases (Toda et âl', 1989)' The pharmacological activities of compounds found in tea extract have been studied clinically and experimentally. lt has been repofted by Sakanaka et a/' (1989) that Japanese green tea has antimutagenic and anti-hepatotoxic effects,

antitumour activity, hypolipemic and antioxidative effects.

With respect to the compounds found in the tea plant, it is known that

Camettia sinensis contains caffeine, vitamin C, tannins and saponins (Sekine ef

a/., 1991). Liyanage et al. (1988) reported that the major fatty acids in tea are

palmitic, oleic, stearic, linolenic and linoleic acids. These last two fatty acids are

impoftant in imparting flavour to the tea. Caffeine, a stimulant, was reported to

make up as much as 2.5"/" - 4.5To of dry tea leaves, which is almost twice as

much of that substance as is found in roasted coffee beans (de Wit, 1963).

Graham (1gg2) reported that caffeine in tea leaves is present along with

methylxanthines, theobromine, theophylline and theanine. Theanine (L-glutamic

acid ethylamide) is a characteristic amino acid derivative in tea leaves (Furuya et

a/., 1990). tt was also noted that tea accumulates aluminium and manganese

(Graham, 1992).

16 Flavonoids are polyphenotic compounds naturally present in vegetables, fruits and in beverages such as tea and wine. lt was repofted that flavonoids in (Hertog regularly consumed foods may reduce the risk of coronary heart disease et al,199g). Cheng et al. (1987) isolated flavone and two flavonoid glycosides from "Bai-shui-cha", C sinensis L. which is used as a traditional folk beverage in china. They are apigenin, apigenin-5-oa-L-rhamnosyl-6-acetyl-B-D-glucoside

(camellianin A) and apigenin-S-Go-L-rhamnosyl-p-D-glucoside (camellianin B).

Also, from seeds of C. srnensis two flavonot glycosides, camelliaside A and

camelliaside B have been isolated. They were identified as kaemplerol'3-O'12'

oLp-D-galactopyranosyl 6-ota-L-rhamnopyranosyll-B-D-glucopyranoside and

kaempferol-g-oll2-of-D-xylopyranosyl6-Oa-L-rhamnopyranosyll-p-D-gluco-

pyranoside (Sekine et al., 1991). Moreover Finger et al. (1991) isolated two

flavonol triglycosides from commercial black tea, identified as quercetin glucosyl

rhamnosyl galactosides and kaempferol glucosyl rhamnosyl galactosides.

These compounds produce quercetin or kaempferol, glucose, rhamnose and

galactose at about 1:1:1:1 when hydrolyzed. The aglycones were characterized

by HPLC and UV spectra while monosaccharides were identified by GC-MS.

A number of more complex compounds have also been identified in tea

plants. lt was reported that camellin B, a dimeric hydrolyzable tannin, was

isolated from the flower buds of C. iaponica (Yoshida et al., 1989) and two

complex tannins, camelliatannins A and camelliatannins B, from fresh leaves of

the same plant were also identified (Hatano ef a/., 1991). ln addition Cho et al.

(1993) purified tannins from Korean Green Tea and identified them as (+)- catechin, (-)-epicatechin-3-o-gallate, (-)-epigallocatechin-3-o-gallate, (+)-

17 (The gallocathechin, (-)-epigallocathechin, and pro-cyanidin-beta-3-Ggallate on structures are illustrated in ligure 2.2). By using partition chromatography green tea silica gel as many as 7 compounds were isolated from dried ceylon leaves. They are l-gallocatechin (1.8%); d,l-gallocatechin (0.89%); l-epicatechin

(O.4g%); d,l-catechin (0.18%); l-gallocatechin gallate (5.5a%); A-gallocatechin gallate (O.72"/.) and l-epicatechingallate(1 .16%') (Roberts, 1962)' ln addition' 22 polyphenolic constituents of green tea leaves were identified by using paper chromatographic methods. These include the flavonols myricetin, isoquercitrin, kaempferol, quercetin, rutin and ellagic acid, chlorogenic acid and theogallin

(Roberts, 1962).

lndonesian green tea is unfermented C. srnensisvar. assamica- lt is very harsh and bitter compared with Japanese and Chinese tea (which is C. sinensrs var. sinensis). The polyphenol content in the assamica type (25-35 % of dry weight) is higher than in the sinensis type (10-17 "/o of dry weight) but the amino acid content is lower compared with sinensis (Samarasingham and Bambang,

1 e88).

Recently, tea extracts have been identified as having beneficial activities against various tumours in in vivo and rn vitro experiments, lt has been reported that green tea extract inhibited gastric cancer (Xu ef al., 1992),lung cancer (Luo

and Li, 1992; Xu et at., 1992i Katiyar et a1.,1993b), mammary cancer (Komori ef

a/., 1gg3), hepatic carcinogenesis (Klaunig, 1992), and skin tumours (Agarwal ef

al., 1992i Huang et a1.,1992; Katiyar et a1.,1992a; Katiyar et al., 1992 b; Katiyar

et al., 1993a). Wang et al. (1992) noted that the growth of pulmonary tumours

was decreased by 18 and 44/" after giving infusions of 0.63 and 1 .25 % green

tea, and that tumour numbers were also reduced by 36 and 60 % respectively.

18 leaves ln addition, oguni et at. (1988) reported that an extract of fresh green tea 180 cells at a dose of 400 mg/kg exhibited a growth inhibition of Mouse Sarcoma in mice of approximately 50% when administered orally.

Current research has indicated that the principal bioactive compounds found in tea extracts are polyphenols and the most important of these are derivatives of catechin (Kada et a1.,1985; Sakanaka et al., 1989; Cheng et al',

1991; Agarwal et al., 1992; Xu et at., 1992). Klaunig (1992) noted that catechin (1992) components of green tea show anticarcinogenic propedies. Huang et al. found that (-)-epigallocatechin gallate, (-)-epigallocatechin and (-)-epicatechin gallate are the most active catechins in green tea extracts. Moreover, Katiyar et

at. (1gg2a) reported that the major constituent of green tea which possesses antitumour and anticarcinogenic effects in rodent tumour bioassay systems is (-

are considered to be the )-epigallocatechin-3 gallate (EGCG). These compounds

pharmacologically active compounds in the extract of green tea.

19 OH o- OH 1H c-cHoH H2 OH OH OH

gallocatechin OH o O.t' CH I OH CHz o\ OH o OH

catechin gallate OH

OH o_ OH

c-cHoH H2 OH catechin

Figure 2.2 Structures of abundant polyphenolic components in tea leaves

Increasingly, studies of the components of tea extracts and their pharmacological activities have been initiated in order to better understand the medicinal values of this traditional beverage.

20 2.g CHEMICAL RELAT¡ONSHIPS BETWEEN MISTLETOES AND THEIR

HOST PLANTS

Mistletoes are important economically because of their destructive effect on both plantation and forest ptants, However, recently they have become equally important because of their pharmacological potential. The significant area of research interest lay in the chemical relationships between the mistletoe and its host especially the non-proteinaceous low molecular weight compounds.

Contradictory findings about the chemical relationship still remain since much

research (for example : Boonsong and wright, 1961 ; cordero et al., 1989; Bloor,

1gg1) has suggested that compounds present in the mistletoes depend on the

hosts parasitized while other research (for example: Graziano et al., 1967;

Tronchet, 1975; Becker 1986) found that there is no significant difference

between compounds found in the species of mistletoe growing on different host

plants. The section below describes these apparently contradictory findings with

regard to the low molecular weight compounds found in mistletoes and their

hosts.

The fact that low molecular weight compounds in mistletoes tend to

correlate with those found in their host plants was noted by Trautner (1952) who

reported that the major alkaloid found in a Loranthus sp. mistletoe was hyoscine,

a nicotine alkaloid, which was also the main alkaloid found in its host plant,

Duboisia myoporoides. Moftimer (1957) also found that two samples of

Loranthus sp. contained the same alkaloids as their host trees (D. myoporoides)

but in lesser proportions than in the host plant. ln addition, Boonsong and

Wright (1961) reported that three different species of mistletoe (Phrygilanthus

celastroides, Dendrophthoe falcata and Amyema congener) growing on Nerium

21 oleander contained three glycosides which were identified as strospeside, are the ner¡talos¡de and odoroside H. These three glycosides in the mistletoes the most polar of the twelve glycosides which can be isolated from the leaves of host N. oleander. They concluded that the presence of iust these three compounds could be due to the fact that only the polar glycosides present in the leaves are translocated in the oleander and that the haustoria of the mistletoes select the polar glycosides preferentially. To support these findings Cordero et

at. (1gg9) found that Viscum cruciatum contained five quinolizidine alkaloids: three tetracyclic derivatives of the sparteine group, (+) retamine, (-)-lupanine and

(-)-anagyrine; and two tetracyclic degradation products (-)-cytisine and (-)-N- methylcytisine. The structures and relative levels of these quinolizidine alkaloids are very close to those of the host, Lygos sphaerocarpa, which is known to contain retamine and sparteine. Moreover, Bloor (1991) suggested that

tteoustylus micranthus (Hook. f) Tieghem (Loranthaceae), had assimilated

norditerpene lactones since this class of compounds is present in the

podocarpus totara, an evergreen conifer, the host plant. This large leafy

mistletoe usually parasitises a wide range of trees and shrubs.

ln addition to the above obseruations pointing out the similarities of

compounds found in the host with those in the mistletoe, the same spec¡es

mistletoes growing on different host plants have been shown to contain different

low molecular weight components. For example Ohta and Yagishita (1970)

found three kinds of flavonoids, flavoyadorinin-A, flavoyadorinin-B and homo-

flavoyadorinin-B, in leaves of V. album var coloratum growing on Pyrus

communis, whereas in leaves of the same mistletoe growing on Zelkowa serrata

they found only flavoyadorinin-B and homo-flavoyadorinin-8. Fukunaga et al.

22 (1ggg a) also reported that flavoyadorinin A was not isolated from the Japanese mistletoe (V. album var. coloratum) growing on twelve ditferent host trees examined. Nair and Krishnakumary (1990) also repofied that D. falcata Ettingsh growing on 6 different host plants contained different flavonoid compounds, and that the flavonoid quercitrin was the major compound found in all cases.

However, they concluded that there was no influence of the host on the flavonoids of D. fatcata. Marshall and Ehleringer (1990) did suggest, however, that organic carbon dissolved in host xylem water was assimilated by the mistletoe. This was indicated by the high transpiration rates and low photosynthetic rates typical of mistletoe. They found that 62 x.2"/" of the carbon in phoradendron juniperum was obtained from the host, Juniperus osteosperma,

14CO2, and not from mistletoe autotrophic activities. Using Hull and Leonard

(1964 a&b) showed that Arceuthobium, dwart light green mistletoe, accumulated large amounts of photosynthate from its host but Phoradendron, a green leafy mistletoe, failed to exhibit the accumulation of compounds from its host. They suggested that Phoradendron is less dependent than Arceuthobium on their host plants for the organic compounds due to the different content of chlorophyll 14C between these two mistletoes. Using uniformly labelled t-phenylalanine

Kuroda and Higuchi (1976) also concluded that mistletoes synthezise lignin

independently. On the other hand it has been reported that there is no

significant transport of organic substances from the mistletoe to the host and

vice versa (Becker, 1986).

The concentrations of individual compound present in the same species of

mistletoe growing on ditferent host plants is variable. For example, Wagner et

23 a/. (1986) noted that phenylpropanes, syringin and syringenin-apiosylglucoside' which were isolated from V. album growing on four different species (Pinus sPP.,

Matus spp., euercus spp. and Titia cordata) varied in concentrations, with the highest content found in mistletoe growing on Pinus. The remarkably high content of flavonoids found in V. atbum L. var. coloratum growing on twelve different host plants has also been reported by Fukunaga et al. (1989a)'

Moreover, it was noted that the concentrations of quercitrin varied greatly among samples of the same mistletoe (T. yadorikî) growing on seven different host plants (Fukunaga et â1., 1989b). Graziano et al. (1967) reported that psittacanthus cuneifolius, an Argentinean mistletoe, produces four flavonoids.

These are (+)-catechin (0.5 - 1.Oy"), quercitrin (quercetin-3-rhamnoside)

(0.0g%), reynoutrin (quercetin-3-xyloside) (0.015%), and avicularin (quercetin-3- alpha-arabofuranoside) (0.01%) which are not found in the five ditferent host plants (Accacia arome, A. caven, Geoffroea decorticans, Celtis spinosa and

Jodina rhambifotia) on which the mistletoes grew. They concluded that these flavonoids are normal metabolites originated in the hemiparasite only and not in the host. Tronchet (1975) found that Viscum album growing on six different host plants (Titia cordata, Crataegus monogyna, Acer compestre, Populus italica, pyrus communis and Quercus sessiliflora) all produce the same monophenolic flavonoid derivatives, none of which are found in the hosts. The compounds found in mistletoe Hyphear tanakae, (phytosterol, triterpene fatty acid ester,

phytosterol glycoside, rhamnocitrin-3-Grhamnoside, rhamnetin-3-Grhamno-

side and quercetin-3-Grhamnoside) growing on four different host plants did not

vary with the species of the host plants (C. crenata, P. mume, P. armeniacavar.

anzu and Z.serrata) (Fukunaga ef a/.,1988). Furthermore, Fukunaga et al.

24 (1989 a & b) found that the constituents found in the four Japanese mistletoes

(V. album var. coloratum; T. yadorik't T. kaempferi and K' iaponica) were

independent of the host trees on which they are ephiphytic.

Although, much research has been done on the anatomical and

physiological aspects of mistletoe-host plant interactions to provide a

considerable knowledge base, the interelationships between the chemical

composition of the mistletoe and its host plant are still not well understood

(Glatzel, 1983; Becker, 1986; Marshall and Ehleringer, 1994; Rennenberg et al.,

1 ee4).

,'I ff lrg .i ,i

25 ! CHAPTER 3

INVESTIGATION OF FLAVONOID COMPOUNDS IN MISTLETOES

al

3.1 INTRODUCTION

Polyphenols are defined chemically as substances which possess an

aromatic ring bearing at least one hydroxyl substituent including functional 1989)' derivatives such as glycosides, esters and methyl ethers (Harborne,

Flavonoids, the most important single group of phenolic compounds, are a

class of naturally occuring compounds characterised by having two benzene

ring rings that are linked by a C. group. The substitution patterns in these two

1967 and and the linking c. fragment vary in the different flavonoids (Harborne,

Ribereau-Gayon, 1972). Flavonoids are reported to be almost completely d flavonoids are commonly found l^þ absent in bacteria, fungi and algae whilst simple î in Bryophytes (Harborne, 1989). Harborne (1988) noted that only about 4000

flavonoids have been identified in vascular plants which is only a small number

of those that are likely to be present in nature. This is because only a small

number of plant species have been properly characterized for their flavonoid

compounds.

The flavonoids also occur naturally in the form of glycosides. The sugar

residues are combined with the aglycones either by O-linkages, connected

between c1 of the sugar and a flavonoid hydroxyl (phenol) oxygen, or by c-

linkages, where the sugar is linked to one of the flavonoid groups unsubstituted

carbon atoms forming a C-C bond. Unlike the O-glycoside flavonoids these C- I

26

T glycoside flavonoids are resistant to both acid and enzymic hydrolysis

(Harborne, 1967).

The most important groups of flavonoids are anthocyanins, flavonols,

flavones, flavanols and flavonones (Hertog et a1.,1993). The flavonols and the

flavones are the most abundant groups and are sometimes known as

anthoxanthins or yellow flower pigments (Ribereau-Gayon, 1972')' The

difference between flavones and flavonols is the substitution pattern on the C

ring. The flavones have a ketone group at position 4, while the flavonols also

have an hydroxyl group attached to carbon 3 (Figure 3'1)'

R

OH 2 4 B 8 R OH luteolin 5 = R H apigenin ,..I = il 7 c 6' ,'\if J i 6 4 5

OH

flavones R1 R2 J OH OH H quercetin 2 4 H H kaempferol 8 B OH OH myricetin R2 H isorhamnetin 7 c 6 OCH3

6 4 5 OH oHo

flavonols

Figure 3.1 Chemical structures of some important flavones and i flavonols.

27 * The study of the ftavonoids is an area of active research because they

occur naturally in plant foods which are common dietary elements such as

vegetables, fruits and beverages (Hertog et al., 1993). They create some

practical problems in the food industry because they are closely connected with

colour, taste and flavour properties of food (Harborne, 1967). Recently, much

research has been carried out to show that certain flavonoids possess anti-

inflammatory, antiallergic, anticarcinogenic and antitumor properties (Cody et al.,

1gg6; Middleton and Kandaswami, 1994). Bracke et al. (1994) found that tumor-

bearing mice treated orally with the citrus flavonoid tangeretin always lived

longer than controls. They suggested that tangeretin may inhibit the processes

that shorten the life expectancy of tumor-bearing animals, but the mechanisms

are still not known. Whether flavonoids, in general, have antitumor properties or

are anticarcinogenic agents is not yet clear (Hertog et al., 1993).

I fl ìx 1

I 3.2 MATERIALS AND METHODS

3.2.1 Source of Plant materials

All of the plant materials used were collected from the Tea Plantation at

Gambung, Bandung, lndonesia and sent in an air-dried condition to the Waite

Campus, Adelaide University, Australia, The species included Scurulla oortiana

(Korth.) Danser grown on'. Citrus maxima, Persea americana, Camellia sinensis

and also leaves of those host plants.

Í I 9.2.2 Extraction and isolation of flavonoid compounds

; Dried leaf samples were ground in methanol using an Ultra-Turrax (T25,

28 r IKA - Laboftechnik, Germany) and extracted three times with methanol. Each extract was kept overnight at room temperature to allow cell components to precipitate. The extracts were then centrifuged (30009 for 15 min). The clear supernatants were combined and evaporated in vacuo using a rotary evaporator

until syrupy. This extract was then paftitioned between n-hexane and water to

give a hexane extract. The aqueous layer was further extracted with chloroform

to give a chloroform extract and then with butanol to give butanol and water

extracts successively. The weight of samples and volume of solvents used are

listed on table 3.1.

g.2.g Separation and identification of flavonoids present in the butanol

extracts

The concentrated butanol extract (approximately 500m9 of solids at a

concentration of 1g per 20ml) was subjected to column chromatography using

Sephadex LH 20 (Pharmacia Biotech, Sweden) and a column (40 x 3 cm)

(Pharmacia, Sweden) and eluted with methanol. ln this study, it was found that

Sephadex LH 20 column chromatography was very useful to initially purify the

crude samples. However, it appeared that separation of individual flavonoids

from the crude extracts was very difficult since all collected fractions were found

to contain the same flavonoid components when monitored by thin layer

chromatography (TLC).

ldentification of flavonoids was confirmed by TLC using several different

solvents and various detection methods. Acid hydrolysis was done to identify

the aglycons and the sugars. The individual flavonoid components were always

compared with authentic standards of the flavonoids. The standard compounds

29 used in this experiment were naringenin, quercetin, quercitrin, rutin (Sigma' usA), quercetin-3-oglucoside (Apin chemicals Ltd, uK). Mass spectrometry

(using chemical ionization or fast atom bombardment, electron impact and electrospray methods), ultraviolet spectral analysis and high-performance liquid chromatography (HPLC) were carried out to further characterize the compounds.

3.2.3.1 Thin Layer Chromatography (TLC)

Thin layer chromatography was done using Kieselgel 60 F254 plates, 250 pm thickness, (Merck). The solvent system used initially was ethyl acetate- formic acid-glacial acetic acid-water (100:11:11:27). lt was found to be preferable not to activate the Kieselgel 60 Fruo plates prior to use for separation of the flavonoids. Preheat treatments caused variation in R,s of compounds and also caused the solvent front to curue. Untreated plates produced more reliable

R,s than the heat treated ones. The R,s of the compounds were much higher when the plates were activated.

TLC was also carried out using different solvents systems including

n-Butanol - glacial Acetic acid - Water (4:1:5; top layer) (BAW) using Kieselgel

60 F2s4 plates and 15% aqueous acetic acid using Polyester Polyethyleneimine

cellulose Fruo, plates (1OOpm thickness, Sigma). The standard set of flavonoid

compounds were also chromatographed against the individual flavonoids found

in the mistletoes. Kieselgel 60 Fruo plates with a BAW (6:4:3) solvent system

30 was used to identify the sugar moieties after acid hydrolysis of the flavonoid glycosides.

Spot behaviour of the ftavonoids was observed under UV light at 254 nm and 365 nm before and after exposure to ammonia and then sprayed with

Natural Product Reagent (Wagner et. al., 1986) to tentatively identify them.

The individual flavonoid components found in mistletoes were isolated by preparative Kieselgel 60 F2s4 plates developed in ethyl acetate-formic acid-glacial acetic acid-water (100:1 1:11:27). Each observable compound was scraped from the sudace and eluted with methanol by centrifuging several times. The methanolic extract was evaporated to dryness using a Speed Vac Concentrator

(Savant SVC 1OO H). The individual components were then examined and

characterised by degradative, chromatographic and spectrometric procedures.

9.2.3.2 Acid hydrolysis of flavonoid glycosides

A sample of flavonoids was mixed with 6% aqueous hydrochloric acid (3

ml) using the minimum amount of methanol to effect complete solution. The

solution was heated on a steam bath for 120 min at 55o C and then cooled and

extracted thoroughly by shaking with diethylether. Evaporation of the aqueous

layer using a Speed Vac concentrator (SVC 100 H, Savant) yielded the sugar.

The ether layer was evaporated to yield the aglycones (which were subjected to

TLC). lf the amount of sample was very small, the hydrolysis solution was

evaporated to dryness under vacuum and the residue chromatographed by TLC

(Kieselgel 60 F2s4, Merck) (Mabry et. a1.,1970) to identify the hydrolysates.

31 various solvents. Table 3.1 Extraction of flavonoids found in S. oortiana grown on different hosts and their hosts using water sample methanol n-hexane chloroform n-butanol

V E name W V EVEVEV E (g) (mL) (g) (mL) (g) (mL) (g) (mL) (g) (mL) (g) 2.42 S. oortiana on citrus 50 1200 11.88 600 3.26 600 0.33 600 5.65 200

1 .89 C. maxima 40 1050 5.84 500 1 .37 440 O.71 600 1.25 170 2.o7 S. oortiana on tea 50 1200 12.08 600 0.44 600 1.29 600 7.90 200 1.o7 C. sinensis 20.4 1200 3.31 600 0.86 600 0.54 600 0.84 200 2'75 S. oortiana on persea so 1200 12.18 600 0.91 600 1.48 600 6'85 2oo

1 .88 P. americana 19.3 1000 7.09 600 1 .81 600 1 .08 600 2.12 200

volume of solvent used for extraction; E wei$ht of extract note : W = weight of dried plant materi al extracted; V = =

32 3.2.3.3 Ultraviolet spectral analysis - Ultraviolet spectral analyses was carried out using a recording Perkin

Elmer S UVA/IS Spectrophotometer. A number of treatments, described below,

were applied to the samples (MeOH, sodium methoxide, aluminium chloride,

hydrochloric acid, sodium acetate and boric acid) (Mabry et al., 1970)'

A small amount of the flavonoid compound in methanol was diluted with

methanol to adjust the optical density (oD) of the major absorption peak (200

and 450 nm) to a suitable level. This was used as the stock solution. All spectra

were measured at 60 nm per min scan speed in a 10 mm path length quartz

cuvette.

The stock solution of the flavonoid was firstly measured as a pure MeOH spectrum. This solution was made alkaline with three drops of sodium

methoxide (NaOMe) and again measured immediately and reported as the

NaOMe spectrum. Secondly, six drops of AlOlg (0.05%) was added to an aliquot

of fresh methanol stock solution and again measured immediately and recorded

as the Alçlg spectrum. This solution was then acidified with 3 drops of the stock

HCI solution was added to obtain the Alclg / Hcl spectrum. Thirdly, the

methanolic solution was saturated with anhydrous sodium acetate (NaOAc) and

shaken. The residual, undisolved sodium acetate was allowed to settle to the

bottom of the cuvette and the spectrum was measured after two min. and than

again after 5-10 min. The spectrum was measured once more after the addition

of anhydrous boric acid (H'BO.) to the saturated methanolic sodium acetate

solution to obtain the NaOAc/ H.BO. spectrum.

33 g.2.g.4 High performance liquid chromatography - Electrospray mass spectrometrY (HPLC' ES'MS)

Flavonoid compounds were identified from their mass spectra and retention - quadrupole times by comparison with standard compounds. An API-300 triple mass spectrometer equipped with an lon Spray ion source (PE Sciex) connected to a HPLC was used.

The compounds were separated on a C18 microbore reversed-phase column (spherisorb:25 cm x 1 mm, s5 oDS2) with an ABI 1408 solvent delivery system (Applied Biosystems) using the solvent gradient system shown in table g.2. The samples were injected with a Rheodyne model 8125 injector fitted with a 5 pl sample loop. The flow rate was 25 pl/min. The HPLC column was (15 connected to the Electrospray MS system using a fused silica capillary tube

cm x 100 pm i.d).

The mass spectra were measured in the positive ion mode under the

following conditions : an unit mass resolution of 5000 V of ES voltage, 30 V of

orifice voltage and scan range of 100 - 8OO mlz in 3 sec. The samples were

either infused into the ES ion source at a constant flow rate of 5 pl/min with a

medical syringe infusion pump (COLE-PALMER: 74900-00) or using the eluent

from the HPLC sYstem above.

34 Table 3.2 :The solvent gradient used for HPLC with solvent A 0'05%TFA/MeCN and solvent B 0.05% TFA/H.O' Time (mins) Solvent A (%) Solvent B (%)

95 1 5

15 90 10

25 90 10

g.2.g.5 High performance liquid chromatography (HPLC)

Each of partially purified flavonoid sample was dissolved in Hipersolv. pl MeOH and filtered through a 0.45¡rm membrane filter ( type HV, Millipore). 20

of this filtrate was iniected into the HPLC unit using a Waters WISP 710 B auto

sampler. A solvent gradient (Table 3.3) was used with a flow rate of 1.0 ml/min

with a Lichrospher 100 RP18 (5 pm) column (RT 250 - 4 C1B, Merck) at 35eC.

The compounds were detected by their absorbance at 280 nm (Waters 441

UV/Vis Absorbance Detector).

Table 3.3 The solvent gradient used for HPLC; solvent A 100% acetonitrile and

solvent B water PH 2.2. Time (mins) Solvent A Solvent B

(%) (%)

0 0 100 35 100 0 40 100 0

35 All the solvents were filtered through Millipore 0.45 ¡rm membrane filters and degassed prior to use. In addition to membrane filtering, the Milli-Q water

(Millipore Corporation) was carbon filtered to remove any organic impurities'

g.2.4 euantification of phenolic compounds found in mistletoes and their host plants

9.2.4.1 Colourimetric assays for total phenolic compounds Total phenolic compounds found in mistletoes and the hosts were measured using the Fotin - Ciocalteu method adapted from Waterman and Mole

(19g4). The butanol extract of mistletoes and the hosts (1 mg) was dissolved in

10 ml of methanol. One ml of this solution was added to a 100 ml volumetric flask containing 60 - 75 ml deionized water and mixed. Five ml of Folin -

Ciocalteu's reagent (Ajax Chemicals) was then added and mixed. Sodium carbonate solution (15 ml) was added after 1 min and before I min and the time recorded as time zero. The flask was again shaken to mix the contents. The volume was then made up to exactly 100 ml with deionized water. The contents of the flask were mixed thoroughly by inverting several times. The absorbance of the reactants was measured after 2 hours at 760 nm using a PYE Unicam PU

8600 UV/Vis Spectrophotometer. A standard curve was prepared using gallic acid at concentrations of 0.57; 1.32; 2.24 and 3.89 mg/L in methanol using the same procedure as above. The total phenolic content of samples was expressed as mg/L gallic acid equivalents (GAE).

36 9.2.4.2 Quantitation of individual flavonoids by HPLC

The amount of individual flavonols found in mistletoes and the host plants were measured by HPLC using the same conditions as used for flavonoid identification (sub chapter 3.2.3.5). Standard curves of quercitrin, isoquercitrin and rutin were used to quantify the total amount of these compounds found in the mistletoes and the hosts.

3.3 RESULTS AND DISCUSSION g.3.1 ldentification and characterization of flavonoids in mistletoes

growing on three f nvestigations of flavonoids in S. oo¡tiana (Korth.) Danser different host plants and their hosts, Citrus maxima, Persea americana, Camellia srnensls have been carried out by four independent methods. They were TLC using different solvents and various detection methods, UV spectral analysis using various treatments, high peÉormance liquid chromatography (HPLC), and

HPLC connected to electrospray-mass spectrometry. The identification and characterization of the flavonoids in mistletoes and their hosts were verified using standard compounds.

Three flavonoid components in the butanol extract of S. oortiana (Korth.)

Danser growing on Camellia, Citrus and Persea were separated and identified by

TLC using an ethyl acetate - glacial acetic acid - formic acid - water /

100:1 1:11:27 solvent system (EFAW throughout this section). They gave yellow

spots on the plates with Rf's of 0.81 ;0.73; 0.54. The intensity of the yellow color

of the spots was increased by spraying with Natural Product (NP) reagent.

These spots were dark purple under the UV light at 254 nm and became yellow

when exposured to NH. vapour and observed under UV light at 365 nm.

37 Correlation of the Rfs and colour reactions of the unknown compounds with the standard referenced compounds indicated that they were the flavonols quercetin-3-rhamnoside (quercitrin) (Rf 0.81); quercetin-3-glucoside (iso- quercitrin) (Rf 0.73) and quercetin-3-rhamnoglucoside (rutin) (Rf 0.54)

respectivety (Table 3.4; Figure 3.2). lt was noted that standard compounds run slightly faster than the respective compound found in the mistletoes in some

cases. Overspotting standard and unknown compounds failed to resolve them

and the difference in Rf's can be attributed to retardation of the flavonoid

components by other components in the crude extracts'

EFAW was found to be the most useful solvent system in this study for

screening and separating flavonoids from the crude extracts. Other solvents

(water, 1S% acetic acid and BAW) often separated the compounds poorly and

produced tailing sPots.

A mixture of compounds chromatographed on cellulose plates did not

separate satisfactorily using 15% acetic acid solvent system. The n-butanol :

acetic acid : water (BAW) system (4:1:5 upper phase) did not separate a mixture

of compounds on Kieselgel 60 Fr* satisfactorily, but the individual pure

compounds did run in this solvent and enabled R,s of the pure components to be

obtained.

The Rf values of the various components of the extracts determined using

the different solvents indicated that these compounds either have different

sugars or different number of sugars attached to the aglycone. ln this case the

higher the mobility (Rf) of the compound in EFAW and BAW indicates the fewer

number of sugars attached to the aglycone.

38 Table 3,4 Thin layer chromatographic propefties of flavonols and standards found in mistletoes grow¡ng on different hosts. name color observed under UV light Rf values

of sample UV* UV**+NH. NPA EFAW BAW

Quercetin dark purple yellow yellow 0.98 0.90

Quercitrin dark purple yellow yellow 0.81 0.73

lsoquercitrin dark purple yellow yellow o.73 0.64

Rutin dark purple yellow yellow 0.54 0.51

S. oo¡1iana on tea:

component 1 dark purple yellow yellow 0.81 0.71

component 2 dark purple yellow yellow o.73 0.61

component 3 dark purple yellow yellow 0.54 0.49

S.oortiana on citrus:

component 1 dark purple yellow yellow 0.81 0.71

component 2 dark purple yellow yellow o.73 0.61

component 3 dark purple yellow yellow 0.54 0.49

S.oortian on Persea:

component 1 dark purple yellow yellow 0.81 0.71

component 2 dark purple yellow yellow 0.73 0.61

component 3 dark purple yellow yellow 0.54 0.49

UV* viewed under UV at 254 nm; UV** viewed under UV at 365 nm after spraying with ammon¡a' NPA : Chromatogram sprayed with Natural Product Reagent Solvents: BAW: n butanol-acetic ac¡d-water, 4:1:5 (upper phase); EFAW: ethyl acetate-formic acid-acetic acid-water, 1 00: 1 1 :1 1 :27

39 Figure 3.2 Thin Layer Chromatogram of standards and flavonoids found in the butanol extract of S. oortiana (Korth.) Danser growing on different host plants chromatographed on Kieselgel 60 Fruo ptates developed in ethyl acetate : formic acid : acetic acid : water (100:11:11:27\. compounds were visualised with Natural Product Reagent and observed under UV light at 365 nm. Each sample has been applied in two separate lanes on the plate as a mirror image.

40 ã EB e Eg 8sg, å ar "=' Tea S. ooriana on slll lO'¡, ¡ Tea I S. oorianaon Persea

1ù Persea Citrus t S. oortionø on Citrus

a The inference above was confirmed when the acid hydrolysis of flavonol fraction (which on TLC gave 3 spots Rf: 0.81 ; O.73;0'54, in EFAW) produced quercetin (Rf: 0.98) as the aglycone by TLC when compared with the standard'

From this chromatogram it can also be seen that the hydrolysis was not complete, because traces of the original compounds still appeared in the hydrolysates (Figure 3.3). The aqueous phase of the hydrotysates when chromatographed on

Kieselgel 60 F254 and developed in BAW (3:3:1) showed that the first component

(solvent EFAW Rf 0.81) yielded rhamnose as the sugar, the second (Rf 0.73)

produced glucose and the third spot (Rf 0.54) produced rhamnose and glucose.

This was in accordance with controt hydrolysate of the standards. Quercitrin,

isoquercitrin and rutin yielded rhamnose, glucose, and rhamnose and glucose

(disaccharide) resPectivelY.

From figure 3.3, it can be seen that the hydrolysate of S. oo¡tiana on Citrus

produced one new white fluorescent spot (Rf =0.41) compared to its original

butanol extract. This compound did not produce a yellow color when sprayed

with NP reagent but showed a white fluorescence spot under UV light at 365 nm'

This can be explained by the fact that the hydrolysis process was not complete

since the quercitrin (quercetin-3-rhamnoside) was still present in large quantity

together with the component observed as a white spot, as well as residual

quercitrin. These spots were no longer detectable when the hydrolysis process

was continued to completion. This white spot could be due to the sugar (in this

case: glucose) having migrated to carbon-S of the ring A which made this

compound more polar than the rutin.

41 Figure 3.3 Thin Layer Chromatogram of standards and flavonoids found in S' oo¡tiana (Korth.) Danser growing on different hosts and the respective hydrolysates chromatographed on Kieselgel 60 Fruo developed in ethyl acetate : formic acid : acetic acid : water (100:1 1:11:27). Compounds were visualised with Natural Product Reagent and observed under uv light at 365 nm. Each sample has been applied in two separate lanes to the plate as a mirror image.

42 d EAo a 9õ ã F 9.ã ã "= g$ å

|}-{ S. oortiana on Tea S. oorianaon Tea (hYdrolYsate)

S. oorfiana on Persea S. oortianøon Persea (hydrolysaæ) S. oortiana on Citrus S. oortianaon Citrus (hydrolysate) Another possibility is that the conditions used to carry out the glycoside the hydrolysis also caused destruction of the flavonoid ring since after treatment

residue did not give a yellow colour when sprayed with NP reagent'

The UV spectral analyses of methanolic samples of the flavonols subjected

to five different treatments were used in this experiment to fufther identify these

compounds. These analysis are very useful to initially characterize the flavonoids

since the flavones and flavonols show two major absorbance peaks in methanol'

Band I is considered to be associated with absorbance involving the B ring

cinnamoyl system usually in the region 3OO - 380 nm while band ll, usually in the

region 24O - 280 nm, is considered to be associated with the A ring benzoyl

system (Mabry et a1.,1970).

The individual component of flavonoids found in the mistletoes, obtained

from preparative TLC plates, analysed using this method showed that the spectral information provided independent confirmation of the putative ü I characterisation of the compounds by TLC (see appendix 1). The fact that there

were slight differences in the absorbance maximums (Table 3.5) between the

compounds and the respective standards was found to be due to the fact that the

isolated individual components were not homogenous because they were later

seen to contain 2 or 3 compounds by HPLC.

The UV spectra in methanol, especially the position of band l, indicates the

type of flavonoids as well as its oxidation pattern (Mabry et al., 1970)' The

spectra of compounds found in mistletoes showed that band I was more

pronounced than band ll and appeared at longer wavelengths. This shows that

the compounds were flavonols which were oxygenated in the A and B rings.

43 { + __{- i*--4ã4-

Table 3.5. in oortiana growing on different hosts and the standards using various The Maximum wavelength (1. -". nm) values of UV spectra of flavonols found S. solvents. The samples were dissolved in methanol and subjected to various treatmenls.

name ol max ?t (nm)

compound treatments

MeOH NaOMe Alcl. ArcuHcl NaOAc NaOAc/H.BO,

271.6;378.7 260.2;362.9 tQuercilrin : 258.0;348.8 271.3:397.6 274.4;332.8:424.8 269.9; 349.4; 395.9 265.7;368.5 259.9;368.4 ln S. oortiana on lea 255.2;348.2 271.2;395.5 274.1;424.4 269.2',352.0; 395.0 266.3;360.7 260.1;368.3 ln S. oortiana on citrus 255.4;349.4 270.2;395.7 274.7;423.9 270.8;353.1; 395.8 269.8; 352.5; 394.5 268.9;390.8 262.1;375.3 ln S. oortiana on Persea 255.1; 350 270.8;396.7 274.5;427.5

5 Þ 261.2:381.2 'lsoquercitrin 256.0;358.0 272.8:330.3;414.1 274.0:432.0 268.8;407.6 272.6;379.1 272.7;390.1 261.6; 381.0 ln S. oortiana on tea 256.9;360.0 272.8:410.7 274.5:429.0 268.6;396.5 272.8;387.9 261.7; 380.0 ln S. oortiana on citrus 255.5;356.2 271.2:406.9 274.6;420.5 270.3;396.3 268.4;396.6 272.3;394.9 261.8; 381.4 ln S. oofliana on Persea 256.2;357.3 272.8;411.3 274.5;428.6

272.4;401.3 26'1.2:379.1 'Rutin 255.9:357.7 272.3;415.2 274.0:430.7 268.5;397.0 267.7;386.6 261.1:379.4 ln S. oorliana on tea 256.7;357.8 273.2;416.4 274.1;430.5 267.9;396.0 271.5;414.8 261.9; 388.9 ln S. oortiana on citrus 259.0;364.5 272.6;417.0 274.4:429.5 267.2;396.2 271.6;414.5 262.0;389.6 ln S. oorliana on Persea 260.0;364.8 272.9;418.0 274.7:429.9 266.9;396.0 nole:'standard comPounds The spectrum of samples treated with sodium methoxide provides The information on free 3- and/ or 4'-OH groups of the compounds detected'

addition of NaoMe to the methanolic solution produces a large bathochromic the shift of band I (40 - 65 nm) without a decrease of intensity and indicates

presence of a free 4'-OH (Mabry et al., 1970). Table 3'6 shows that the growing on bathochromic shifts of band I of the flavonols found in S. oortiana

different hosts and the standards were in a range of 46-59 nm. This indicates

that they have a free 4'-hydroxyl group. ln addition the NaOMe spectra of positions are flavonols which possess free hydroxyl groups at both the 3 and 4'- al', unstable and the absorbtion peaks degenerate in a few minutes (Mabry et

1g7O). The fact that absorbance peaks of NaOMe spectra of flavonols found in

mistletoes did not degrade after a few minutes indicated that they did not contain

a free g-OH group. lt was therefore assumed that the 3-position for these

ß"T compounds was glYcosYlated. u l It has been suggested by Mabry et at. (1970) that if a NaOAc spectrum in

which band ll exhibits a bathochromic shift of 5-20 nm relative to its MeOH

spectrum then it shows the presence of a free 7-OH group in the flavones and

flavonols (Table 3.7). In addition, the presence of a shoulder on the long

wavelength side of band I when NaOAc is added suggests that the compounds

posess a free 4' hydroxyl group and no free 3 OH group (see appendix 1)'

The effect of band I exhibiting a bathochromic shift of 12 - 30 nm when

NaOAc/HrBO. is added to methanolic solution of the compounds indicates that

those compounds contain a B ring bearing on an orthedihydroxyl group. This

was interpreted by suggesting the boric acid chelates the ortho4ihydroxyl groups I

45 r at position C3' - C4'in the presence of NaOAc (Mabry et al', 1970)' The

flavonols found )n S. oortiana grow¡ng on different hosts and standards exhibited

a bathochromic shift of 14 - 25 nm (Table 3.8).

The presence of an orthodihydroxyl system in the B ring of flavonols can

also be detected by a hypsochromic shift of band I in the spectrum of the

compounds in methanol to which A¡Cl3 is added compared with that obtained by

ll addition of Alclr/Hcl (Mabry et a1.,1970). Moreover, any shift in band I or band

of AlCl, treatment relative to the MeOH spectrum indicates the presence of free 5

OH group in the comPound.

The hypsochromic shift of band I observed in the compounds was about 24

- 35 nm (Table 3.9), That these compounds also contained a free 5 OH group

was indicated because the spectrum in MeOH was different to the AlCl.

spectrum.

it 'l ln conclusion, the UV qualitative analyses suggests that the flavonols found

in S. oortiana on different hosts possess free 4'-OH, s-OH and 7-OH groups; an

ortho-dihydroxyl system in B ring and are substituted in the 7 position of the A

ring. These conclusions are in accordance with the assumption that the major

components were quercitrin, isoquercitrin and rutin.

I I i

46 r oortiana Table 3.6 The bathochromic shifts of band I of flavonols found in S. of g*ing on different hosts and the standards in NaOMe on which the intensity the spectra were not decreased' (nm) Name of compound Bathochromic shifts of band I

*Quercitrin : 48.8 in S. oortiana on lea 47.3 tn S. oortiana on citrus 46.3 in S. ooftiana on Persea 46.7

*lsoquercitrin : 56.1 in S. oortiana on tea 50.7 in S. oortiana on citrus 50.7 in S. oortiana on Persea 54.0

57.5 "Rutin : in S. oortiana on tea 58.6 in S. oortiana on citrus 52.5 in S. ooriiana on a 53.2 *standard comPounds

Table 3.7 The bathochromic shift of band ll of flavonols found in s. oortiana on suggested the presence of a free fl different hosts and standards in NaOAc which il 7 OH group of those comPounds. j Name of compound Bathochromic shifts of band ll (nm) *Quercitrin : 13.6 in S. oortiana on tea 10.5 in S. oortiana on citrus 10.9 in S. oortiana on Persea 13.8

*lsoquercitrin : 16.6 in S. oortiana on tea 15.8 in S. oortiana on citrus 17.3 in S. oortiana on Persea 16.1

*Rutin : 16.5 in S. oortiana on tea 11.0 tn S. ooftiana on citrus 12.5 in S. oortiana on rsea 10.6 * standard compounds

I

47 r Table 3.g The bathochromic shift of band I in NaOAc with addition of H.BO. relative to MeOH spectrum of flavonols found in S. oortiana growing on ditferent hosts and standardi as an indication of ortho-dihydroxyl system in B ring'

Name of comPound Bathochromic shifts of band I (nm)

*Quercitrin : 14.1 in S. oortiana on tea 20.2 in S. oorliana on citrus 18.9 in S. oo¡tiana on Persea 25.3

*lsoquercitrin : 23.2 in S. oortiana on tea 21.0 in S. ooriiana on citrus 23.8 tn S. ooftiana on Persea 24.1

*Rutin : 21.4 in S. oo¡tiana on tea 21.6 in S. oortiana on citrus 24.4 in S. oortiana on rsea 24.8 *standard comPounds

Table 3.g The hypsochromic shift of band I of the AlCl. spectrum with that obtained in AlCl/HCl of flavonols found in S. oortiana growing on different hosts which indicated the ortho-dihydroxyl group in B ring.

Name of compound Hypsoochromic shifts of band I (nm) *Quercitrin : 28.9 in S. oortiana on tea 29.4 in S. ooftiana on citrus 28.1 in S. oortiana on persea 33.0

*lsoquercitrin : 24.4 in S. oo¡tiana on tea 32.5 in S. oortiana on citrus 24.2 in S. oortiana on persea 32.0

*Rutin : 33.7 in S. oorliana on tea 34.5 in S. oortiana on citrus 33.3 tn S. oortiana on rsea 33.9 * standard compounds

I

48 3.3.1.1 HPLC - MS AnalYses used to HPLC coupled to an electrospray mass spectrometer (ES-MS) was found in obtain a more rigorous identification and characterization of compounds misiletoes. standard compounds were used for comparison' The retention times of quercitrin, isoquercitrin and rutin were 21.09, 18'93 and 21'4 min' respectively. Mass spectral analyses performed in positive ion modes showed The the molecular weight of these flavonols were 449;465 and 611 respectively. to data of the flavonols from mistletoes gave similar signals and were matched those of standards (see appendix 2). The hydrolysate of flavonols in S' ooftiana

(Korth.) Danser grown on different hosts gave a very strong intense signal al mlz

303 amu which was the M + H ion of the aglycone, quercetin (Figure 3.4)' These data were again consistent with the hypothesis that the flavonoids found in the mistletoes were quercetin derivatives. To obtain further data on the homogenecity of these TLC purified compounds from mistletoes were examined by reverse phase HPLC using a gradient solvent system of acetonitrile - water (pH 2.2\ at flow rate 1ml/min and

UV detection at 280 nm. The retention times t, of these compounds found in mistletoes were then compared against the retention time of the standard compounds (table 3.10). Using these conditions, the more polar compounds

were eluted first.

The chromatograms of hydrolysates of butanol extracts of mistletoe

parasitising different hosts all showed the aglycone quercetin with t, 17 -24 min-

These data indicate that the butanol extracts of the mistletoe all contained

quercetin derivatives.

49 Figure 0.4.: Electrospray mass spectra (positive ion mode) of the hydolysates of fla-vonoids found in S. oortiana growing on tea (A); on citrus (B) and on Persea (C) which show the molecular weight of the aglvcone. quercetin (mlz3O3 amt¡\

A

I I

t.l.t tlt il.t ¡¡a rÉ rú Ita tt! 5

B

í { ¡I

ru

tta ¡ta !a

c

¡

r*a

la ata 50 Table 3.10 Danser growlng HPLC retention times of flavonols found in S. oortiana (Korth') on different hosts together with standards' corrected Name of comPound Retention t, internal time (t,) (min) standard (min) t, (min) *Quercetin 17.24 18.22 17.24

*Quercitrin 14.54 : 14.54 18.22 14.52 in S. oortiana on tea 14.54 18.24 in S. oortiana on citrus 14.54 18.23 14.53 14.55 in S. oo¡tiana on Persea 14.56 18.23

*lsoquercitrin 13.72 18.22 13.72 in S. oortiana on tea 13.73 18.23 13.72 in S. oo¡tiana on citrus 13.70 18.23 13.69 13.73 tn S. oortiana on Persea 13.74 18.23

13.27 "Rutin 13.27 18.22 in S. oortiana on tea 13.30 18.23 13.29 in S. ooriiana on citrus 13.34 18.25 13.32 13.29 in S. oortiana on Persea 13.30 18.24 standard

By using HpLC, the flavonols found in S. ooñiana growing on tea, citrus and

persea have been characterised as quercitrin, isoquercitrin and rutin.

51 z9 ' goDttr' r.lhulr ,or ¡ n¡f oo'l oo'c oo'o oo'o

I s I Ê ¡ ú ./II ot'o

,c t È¡ I oo'f

o3'f c I t s.lhut¡ rol x ìrl¡uqullr oo.) oo'c oo'z o0'o oo'o f I

I a F t E ! I ôo'z ! ¡ ã: *', I i. z I

o0' t

g oo't I

E r.tnulr fÎ x f,lDngÐtr 00 '' oo 't oo'1 oo'o o0.o

a Ë

I !F ol'o

! 5 ì oo 't

o¡' I

V: Luu ogz acueqrosqP le ,{qdBJôo¡eurorq3 plnbll acueuitopod-rlôlH ,{q (C) pasrad uo pue (g) srulp uo :(y) Eal uo 6utmo.¡ô eueuJoo 'S to Ìcellxe louelnq ul sprouo^Pll lo r.uB.¡6oleu¡oJtlC : 'g'e ernôrl The butanol extracts of mistletoe grown on different hosts showed HPLC (Figure additional peaks in the HPLC to the three identified flavonols by same mistletoe 3.5). These additional components were slightly different for the growing on different hosts. However, basically the chromatograms showed a peaks could similar pattern of phenolic compounds in all the mistletoes. These be other phenolic compounds present in the mistletoes which were not separated by TLC using the conditions applied.

Figure 3.5 shows that the phenolic constituents, the HPLC patterns of the mistletoes from different host plants are similar to one another and clearly different from the patterns in their respective hosts (see appendix 3). This demonstrates that the minor phenolic compounds found in S. oortiana extracts were independent of the phenolic composition of the host plant and provide a useful fingerprint for S. oortiana mistletoe.

Fukunaga et al. (1g89 a & b) also reported that there was no difference regarding the flavonoid components found in two genera of Japanese mistletoes,

Taxittus and Hyphear, parasitizing different hosts. ln fact they suggested that the aglycones of flavonoid glycosides are one of the valuable criteria to be used as a taxonomic marker in term of genera among mistletoes. Similarly, the flavonoid compounds found in S. oortiana growing on three ditferent host plants had the same flavonoid compounds. A similar finding was reported by Nair and

Krishnakumary (1990) who found that quercetin 3-Grhamnoside was the major

common flavonoid in D. fatcafa Ettings growing on 6 ditferent host plants. They

concluded that there was no influence of the host plants on the flavonoids ol D.

falcata. Similar results also have been reported by Dossaji et al (1983) showing

that apigenin mono and di-G-glycosides were the major flavonoid compounds

53 (Ulmus found tn phoradendron tomentosum growing on three ditferent host trees crassifolia Nutt., Prosopis glandutosaTorr., and Cettis laevigata (W¡lld.) and that there was no influence of the hosts.

g.g.2 Relationships between total flavonoid levels in mistletoes and host plants

The butanol extracts of mistletoe grown on different hosts were found to contain different total amount of phenolic compounds when measured in gallic acid equivalents (GAE) using the Folin Ciocalteu reagent (Figure 3.6). The total levels of phenolic compounds found in the mistetoe growing on tea, citrus and persea were approximately two times greater that the levels found in the hosts.

Mistletoe grown on tea had the highest concentration whist the tea plant contained the highest level of the hosts.

The total contents of individual flavonols found in butanol extracts of the mistletoes and the hosts, were quantified using HPLC. In general, the amounts of individual flavonols found in misteltoes were always much higher than those found in the respective host plants. For example, the amount of quercitrin found in the butanol extract of mistletoes was much higher than that found in the host plants (Figure 3.7). Mistletoe grown on tea possessed the highest level of quercitrin whereas the host plant had quercitrin levels in between those found in persea and citrus. Persea had the highest content of those among the hosts and citrus contained the lowest levels of quercetrin among the hosts. Similarly the content of quercitrin found in mistletoe grown on citrus was the lowest one among the mistletoes. From the present data it is apparent that quercitrin is the

major flavonoid found in this species of mistletoe.

54 The highest content of isoquercitrin was found in the mistletoe grown on persea while that found in mistletoe growing on tea and citrus were relatively the same (Figure 3.8). The content of isoquercitrin in persea was the highest among the hosts. ln addition, isoquercitrin found in tea was very low but it did not follow that the content found in mistletoe grown on tea was the lowest among the mistletoes.

The content of rutin found in mistletoe growing on tea was much higher than those found in mistletoe growing on citrus and persea while persea was

noted to contain the highest level of those among the hosts (Figure 3.9). Rutin

levels found in tea and citrus were quite similar but those found in its respective

mistletoes were significantly different.

The proposal that the flavonols have been synthesized by the mistletoe is

not suppoñed since the levels of the various individual flavonols were quite

different in the same mistletoe growing on different host plants. The fact that

these flavonols have been simply taken up from the hosts also was not reflected

from these data since the highest content of flavonols in the host was not always

followed by those found in the respective mistletoe, thus also discounting

selective uptake of flavonols as a mechanism. lt is possible, however, that the

hosts may have influenced the mistletoes in their synthesis of flavonoids. lt is

also possible that the mistletoes take up a range of flavonoids directly from the

hosts and modify them to be their own specific flavonoids which they then

accumulate. This is because the amounts found in the mistletoes were always

higher than those in the respective host plants.

55 Figure 3.6.

Total phenolic content (means of two values) (GAE mg/L) of butanol extract per 5 g dried leaves of midletoes and the hosts measured using Folin ciolcateu's Reagent at absorbance 760 nm

400

300

J 250 Et E 200 ul o 150 100

50

0 c. (ú c G' C an o d) o o) o = d) L c¡Q E oø) o(d o 9ö () o5 õ.9 o- eÞ o)Ë .E=al, Øs äo Ê E

Figure 3.7

Quercitrin content (mg) (means of three values) of butanol extract per 5 g dry leaves ol S. oortiana grown on different hos{s and thoæ found in the hosts

600

Þ) 400 E tr 300 E .J c) =ET

00

0 (ú (ú c U' .c. c o o o o Ø EE É= 8e (l) o Y' (t, o (1)-b o(Ú o- ä.) õg #e.E 'Ë .Eut

56 Figure 3.8

lsoquercitrin content (mg) (means of three values) of butanol extract per 5 g dry leaves ol s. oortiana grown on different hos and those found in the host Plants

45 40 35 o' E 30 Ê 25

a) o C'= 1 o o 1

5

0 (ú (ú c U' .c c o o f o) o ø oQ 8E o o o oó cl .yØ oÈ õ.E .E-fr'6 .E#8. .t!) E

Figure 3.9

Rutin content (mg) (means of three values) of butanol extract per 5 g dry feaves ol S. oortiana grown on different hoSs and those found in the host Plants

50

40 ctt E c =

10 ./ 0 (ú (ú c u, .c, c. o o o o oQ 2= fQø (¡) Io o o(Ú o- 9ä oÞ õ9 at #s..E E'6 = E É

Further studies of these relationships would profit from the use of tissue

culture experiments whereby mistletoes are cultured with and without the hosts.

57 The findings of this thesis on the relationships between the flavonoid content of mistletoe parasitizing particular host plants and that of their hosts support the observations of Fukunaga et al. (1989a) who noted that the quantitative content of flavonoid glycosides found in the Japanese mistletoe,

Hyphaer tanakae, grown on 12 different hosts were significantly different, whereas the qualitative aglycone content is a valuable taxonomic marker for the particular genera of mistletoe. Fukunaga et al. (1989b) also reported that the content of the flavonoid quercitrin found in T. yadonkr growing on seven different hosts varied significantlY.

58 CHAPTER 4

ANTIFUNGAL ACTIVITIES OF PHENOLIC COMPOUNDS

FOUND IN MISTLETOE AND TEA EXTRACTS

4.1 INTRODUCTION

Fungi are ubiquitous and are well adapted to consume a wide range of

substrates. Consequently they appear to be able to catabolise most organic

matter and can be especialty difficult to control. ln addition, fungal diseases

cause many problems for plants, humans and other animals. Plant diseases

caused by fungi can have unpleasant effects for the consumers. For example,

fungal contaminants in food can produce toxins which cause poisoning of

humans and animals. Fungi and other microorganisms can further modify the

production of foods such as yoghurt, cheese, wine and other stabilised food

products. There is a continuing need for new fungicides because of the

development of resistant strains of fungi to currently avaitable synthetic

fungicides. ln addition, fungicides have an important role in the preseruation of

woods, cloths, and other organic material bases. ln controlling fungi, it is

important to take into consideration both the beneficial and deleterious effects of

fungal growth upon agriculture and the environment.

Research has been carried out to investigate naturally occuring plant

products as antifungal and antimicrobial resources to replace currently utilised

synthetic products (Weidenborner et al., 1990; Vanden Berghe and Vlietinck,

1gg1; Weidenborner and Jha, 1993). ln particular, the naturally occurring

59 flavonoids have been proposed as atternatives to conventional fungicides because they are, for the most paft, harmless to animals, and humans'

Research by weidenborner et at. (1990) has shown that flavones and flavonones are active growth inhibitors of several fungi. They noted that flavones and flavonones inhibited the growth of Aspergillus glaucus up to 90% at

to a concentration of g X 104 M while A. flavus and A. petrakiiwere inhibited up

70% using the same concentration of flavonoids. Unsubstituted flavone and flavanone showed high activity against Fusarium as reported by Weidenborner

and Jha (1993). Wiedenborner ef a/. (1990) found that flavonol caused inhibition

of mycelial growth ol A. repens of up to 33% at a flavonoid concentration ol 2 x

A. 1O-4 M. On the other hand, this compound actually enhanced the growth of

chevaliers up to 29% using concentrations of 2 x 10{ M and I x 1O-4 M'

wiedenborner and Jha (1993) also noted that flavonol, in the same

concentrations as above, stimulated mycelial growth of Altenaria alternata by

19% and 22o/o respectively. Similarly, Weidenborner ef a/. (1990) reported that a

mixture of flavonols showed no higher efficacy against Fusarium and even

stimulated the fungal growth. They also reported that quercetin was totally

ineffective against the growth of all Aspergillus examined.

Fusarium species cause potato tuber dry rot which is one of the most

economically important diseases of stored potatoes around the world (Boyd,

1972). lt was found that the minimum inhibition concentration of thymol, a useful

standard for antifungal activity, against F. sambucinum was about 400 pg/mL at

20oC (Vaughn and Spencers, 1994).

60 Methods for detecting antifungal activity can be categorised into: (a) inhibition of radial growth on an agar medium in a petri plate and (b) growth in liquid culture which can be measured as increase in dry weight or in optical density at a given wavelength (Paxton, 1991).

4.2 MATERIALS AND METHODS

4.2.1 Source of materials and fungi

plant material used in this study was S. oortiana (Korth.) Danser grown on

tea and citrus and C. srnensis. The fungi used for screening were Fusarium sP., Trichoderma SP.,

Alternaria tenuis and Botrytis cinerea. Fusarium sp. was used for antifungal

assay. All fungi were obtained from the Undergraduate Teaching Unit, Waite

Campus, University of Adelaide, Australia.

4.2.2 Extraction of phenolic compounds

Dried leaf samples (20 g) were ground and extracted with methanol (250

mL) two times at room temperature. The methanolic extract was evaporated to

dryness in vacuuo using a rotary evaporator. This extract was then sequentially

partitioned into hexane (200 mL), chloroform (200 mL) and ethyl acetate (300

mL). The ethyl acetate extract was evaporated into dryness and used to assay

the anti fungal activity. The weights of the ethyl acetate extract obtained from S.

oorliana grown on tea, S. oo¡tiana on citrus and C. srnensis were 0.59, 1.11 and

0.76 g respectively.

4.2.3 Pre-assay

4.2.3.1 Stock Culture

Fungi were cultured on malt extract agar media (Merck, USA). 33.6 g of

61 malt extract agar was dissolved in 1oo0 mL of distilled water and autoclaved at

121e c for 15 min. and the cultures were incubated at 25e c.

4.2.3.2 Antifungal Screening

The dried ethyl acetate extract (200 mg) of tea or mistletoe grown on tea was dissolved in 0.5 mL MeOH to effect complete solution and then made up to

10 mL w1h distilled water to give a concentration of extract 20 mg/mL. Six mL of this solution, which comprises 120 mg of extract, was used to make up 240 mL of media at an extract concentration of 500 ppm. The media was autoclaved at i21e C for 15 min. 20ml of this media was used for each experiment involving the four different fungi. Three replicates were run for each treatment. The agar plate was inoculated with fungus at its centre and the culture kept on wet cotton wool in a container to retain the moisture and incubated at 25e C. The growth diameter of the fungi was measured every two days and compared to the control which was media without extract. The measurement was stopped after 7 days when all the cultures had completely covered the plates.

There were no differences found between either the treatments or controls in terms of the rate growth of the fungi. This is attributed to insufficient activities of the extracts to inhibit the growth of these fungi even at the high concentrations of extract used. From the preliminary screening, however, Fusarium sp. WaS chosen for an anti-fungal assay because its growth was slower than the other fungi. The concentration of extract was increased and the pour plate method was used instead of a lawn plate whereby extracts were placed in pre-formed wells as a means of better quantifying weak antifungal activities.

62 4.2.4 Antifungal assaY at Malt extract agar (2.4 S) was dissolved in 60 mL water and autoclaved

,l 121e C for 15 mins to make up 1 treatment, 3 replicates. The ethyl acetate

extract (30 and 60 mg) of tea and S. oortiana grown on tea, and citrus was first

dissolved in 0.5 mL acetone and made up 5 mL with distilled water. This ertract

was sterilised using a 0.45¡rm filter (Millex-HA Millipore). The filtered extract was

directly added to the autoclaved medium to give concentrations of 500 ppm and

lOOO ppm respectively. The solution was then poured in to 90 mm petri dish and

cooled. A one mm diameter of pre-cultured fungus was inoculated at the centre

of the plate and the diameter of the growth was examined every two days for a

total time of 10 days. The medium without any extract was used as control. The

growth diameters after 10 days were statistically analysed using analysis of

variance.

ü (ù Ì 4.3 RESULTS AND DISCUSSION

It was apparent that ethyl acetate extract of mistletoe grown on tea and

citrus as well as C. sinensis showed only weak activity against the growth of

Fusarium sp, although there was a significant difference between treatments

and control at the 1% level (Table 4.1). These extracts were found to retard the

growth of Fusarium sp rather than to inhibit it (Figure 4.1). The antifungal

activity of these crude extracts were significantly lower than the reference

standard, thymol. After 1O days, the growth of Fusarium sp was retarded by

about 11"/o and '15.0"/" when treated with 500 and 1000 ppm of extract of

mistletoe grown on tea when compared to the control, while those treated with

extract of mistletoe grown on citrus showed a 1O "/" and 1 1.0% retardation at the

63 same concentrat¡ons. The extract of misttetoe grown on Persea produced a

retardation of Fusarium sp. growth of 6 % and I % at the same extract

concentrations.

Table 4.1 Analysis of variance of the etfect of the ethyl acetate extracts of tea and S' oortianagrown on tea on the grourth diameter of Fusarium sp.and citrus after 10 days.

Sv Df SS MS F

treatment b 2.6 o.4 7.7*"

error 14 0.8 0.1

total 20 3.4

CV=3.3% ** = significant at 1% level ü rü I

Kramer et at. (1984) noted that there was no absolute generalization which

could be made about fungal inhibition by isoflavonoids and concluded that the

fungicidal property of isoflavonoids was specific to individual fungi, the particular

isoflavonoid and its concentration. They found that the growth of Fusarium

culmorum and Penicitlium digitatum were both inhibited as well as stimulated by

isoflavonoids isolated from soybean and chickpea depending on the compound

and the concentrations applied. This suggested that an explanation for the weak

antifungal activity against Fusarium species could reside in the nature of the

flavonoids present in the extracts.

I

64 r Fígure 4.1 . The effect of ethyl acetate extract oÍ C. sinensis and S. oortiana grown on tea and citrus on the growth of Fusarium sp.on different concentratíons

O ? .e F. -o € cc (g I q ñ CJ q No ôJ ñ ? N f\ l\. ñ aO

Þ

rO a rf o

o 4 o 6 !

2

.'t il 0 iri 'c A1 A2 81 82 C1C2T i treatments

ll{e : lrafue means of three followed by a common letter are not significantly ditferent at the 1% lev€tby DMRT. 'C = control; A = extraðt of S. oorTiana on tea; B = ãxtract oi C. srnensls; C = extr3ct ol S. ædiana grown on citrus; T = thymol; 1 = 500 ppm; 2 = 1000 ppm.

t T

I l

65 I of ooñiana The chromatogram (appendix 4) of the ethyl acetate e)dract s' they were grown on tea and citrus monitored at 280 nm established that querc¡trin and chemically similar and contained mainly quercetin derivatives, These isoquercitrin (retention times 14.54 min and 19.77 m¡n respectively)' mostly epicatechin were quite different from those in c. sinensis which contained

(retention time 12.09 min).

The fact that quercetin derivatives were less effective than other flavonoids different has been reported by Weidenborner et at. (1990) although they used (1989b) also conditions from the experiments conducted here. Fukunaga et al'

found that quercetin and quercitrin isolated from Japanese mistletoe, Taxillus

sp., were not effective against the growth of staphytococcus aureus, Escherichia

coli, Pseudomonas aureginosa and Ktebsietta pneumomae while hyperin and

taxillusin were active only againsl K. pneumoniae' attributed to the flavonol d ff the antifungal activity on Fusarium sp.can be !r¿ I compounds present in the extracts then it is reasonable to conclude that the

weak activity of these crude extracts shown in figure 4.1 can be attributed to the

quercetin derivatives, which predominate in these extracts. lt is therefore

concluded, on the basis of the present analytical and microbiological data, that

extracts of both the tea plant or the mistletoe grown on tea and citrus are likely to

be equally effective against lhe Fusarium spp. used in these experiments.

I f

66 r CHAPTER 5

MISTLETOE INVESTIGATION OF ALKALOID COMPONENTS OF

5.1 INTRODUCTION possess a diverse variety Alkaloids are a class of natural compounds which natural products of structures. Alkaloids have been referred to as a class of pharmacological which have (a) nitrogenous heterocyclic systems (b) significant to the plant activities (c) complex molecular structures and (d) are restricted found in both kingdom. This definition does not cover all alkaloids since they are The plants and animals and some of them have a simple chemical structure' repofied significant pharmacological activity of alkaloids has been extensively and Nowacki, but the role of alkaloids in plants is not yet fully understood (waller

1 978)

purine alkaloids possess a purine ring in the nucleus and include caffeine

(1,g,7- trimethylxanthine), theobromine (3,7 dimethyl-xanthine) (Figure 5'1) and

theophylline (1,3-dimethylxanthine). Alkaloids such as caffeine, theobromine

and other methylxanthines play a major role as stimulants in such beverages I and foods as coffee, tea, soft drinks, chocolate and cocoa (suzuki and waller, to 1988); Suzuki et al., 1992). More than 60 plant species have been found

contain caffeine, especially from the genera coffea, camellia, Paullina also been (guarana) , Theobroma (cacao) and tlex (paraguay tea). lt has

reported that caffeine was isolated from the flower buds and leaves of Valencia

oranges (Citrus srnensrs) (Stewart, 1985).

I

67 r l" l" ¡tsc\ 6 7 6 7 5 I 5 8 4 N 4 N 3 J

cHg cHs

Caffeine Theobromine

Figure 5.1 Structure of purine alkaloids: caffe¡ne (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanth ine).

purine derivatives, especially caffein e, are known to stimulate the central

neruous system, cause a rise in blood pressure, accelerate respiration and act

as diuretic agents (Henry, 1924). Theobromine and theophylline have similar effects. Caffeine and derivatives are often present in pharmaceutical

preparations in combination with other drugs. Suzuki and Waller (1988) noted

that caffeine is one of the most widely consumed drugs in the world.

Fufthermore, theophylline is currently being used for the treatment of

cardiorespiratory disorders such as bronchial asthma (Verpoorte and Svendsen,

1 e84).

The overall synthetic pathways of purine alkaloid formation in coffee and

tea plants are well understood, but little is known about the sites of purine

alkaloid synthesis in these plants (Suzuki and Waller, 1988). Purine alkaloids

which are present in considerable amounts in the seed coat of the tea seed have

I

68 by no funct¡on as nitrogen reserves but they may have an ecological significance 1988)' inhibiting the germination of tea seeds (Suzuki, 1985; Suzuki and Waller,

tt has been reported that the amount of caffeine and theobromine among genus Camellia is very variable. For example, the content of catfeine in Camellia sjnensis var. sinensrs was 2.8% dry weight whereas C. sinensis var. assamica was 2.4"/o. and C. taliensis was 2.5"/o (Nagata and Saka¡, 1 984). C. irrawadiensis which has been suggested as an intermediate between C. sinensis var.

assamica and C. tatiensis on the basis of morphological characteristics, was found to contain no caffeine and the content of theobromine exceeded 0.5% which is high when compared to the other members of the genus (Nagata and

Sakai, 1985).

Since some authors have reported that mistletoes contained alkaloids similar to those found in their hosts and because tea plants are widely known to contain alkaloids it was of interest to investigate the alkaloids in mistletoe growing on Camellia sinensis.

5.2 MATERIALS AND METHODS

5.2.1 Source of plant materials

plant materials used in this study were leaves oÍ Camellia srnensis and

Scurulla oortiana grown on tea which were collected from the Tea Plantation at

Gambung Bandung, lndonesia and sent in an air dried condition to the Waite

Campus, Adelaide University, Australia'

5.2.2 Extraction and isolation of alkaloid compounds

The extraction and isolation of alkaloids found in tea and mistletoe grown

on tea was adapted from Valka and Simanek (1988) as follows:

69 The dried leaf samples (5 g) were powdered in a mortar and extracted with 200 mL of methanol in a soxhlet for 75 min. The methanolic extract was evaporated into dryness in vacuo (tea extract = 1.94 g; mistletoe extract = 0.74 g). The dried methanolic extract was acidified with 100 mL o1 5"/" acetic acid and washed with

25 mL of diethyl ether three times. The aqueous phase was adjusted to pH I with solid sodium carbonate and extracted with diethyl ether (200 mL) to give

pH 12 fraction A (tea = 0.19 g; mistletoe =0.01 g) which was then adjusted to with the addition of sodium hydroxide pellets and further extracted with 200 mL diethyl ether to give fraction B (tea = 0.09 g; mistletoe = 0.012 9). The aqueous phase was then acidified to pH 5 with hydrochloric acid and solid potassium iodide was also added and then extracted with chloroform 200 mL to yield fraction C (tea = O.24 g; mistletoê = 0.20 g). Fractions A, B, and C were evaporated into dryness and further characterized.

5.2.g Separation and identification of alkaloids present in fractions of extract

5.2.3.1 Thin Layer Chromatography (TLC)

The TLC was done using precoated 60 Kieselgel Fr* plates, 250 pm

eC thickness (Merck). These plates were preheated in an oven at 100 overnight

prior to use.

Fractions A,B,C of tea and mistletoe grown on tea as well as those of

mistletoe grown on citrus and persea were applied to the plates and developed

in a toluene-ethylacetate-diethylamine (70.20:10) solvent system (Wagner et al.,

1984) to identify the alkaloids. Caffeine and theobromine (BDH, Merck) were

70 used as standard compounds for comparison. The spot appearance of alkaloids were observed under UV light at 254 nm. Additionally, the plates were sprayed with Dragendorff reagent or iodoplatinate reagent (lP) (Wagner ef a/., 1984) to characterize the sPots.

The individual alkaloid components found in mistletoe and tea plant were obtained by preparative Kieselgel 60 F.uo plates developed in toluene:ethyl- acetate:diethylamine (70:20:10). Each observable compound was scraped from the plate and eluted with chloroform by centifuging several times. The chloroform

extract was evaporated to dryness using a Speed Vac concentrator. lndividual

components were then characterised by degradative, chromatographic and

spectrometric procedu res.

5.2.3.2 lnfra Red Spectral Analysis

The individual compounds or isolates were made into a halide disc at a

concentration of approximately 5% with potassium chloride (KCl) and examined

using a g83G double beam Perkin Elmer Infrared Spectophotometer fitted with a

Harrick Praying Mantis Diffuse Reflectance Model 3SP attachment (Harrick

Scientific Corp., Ossining, NY). Data were collected in scan mode 1, filter noise

4, in the region 4000 - 650 cm't using an Acer 466 DXZ PC Clone (Acer Tech.

Corp., Taiwan ROC) connected to the spectrophotometer. The reflectance data

were convefted to absorbance units, substracted from a KCI blank and offset

using the GRAMS ll (Galactic lndustries Corp., Salem NH) data manipulation

software.

71 5.2.3.3 Gas Ghromatography - Mass Spectrometry (GG-MS)

purine alkaloids were identified from their mass spectra and retention time data by comparison with those of standard compounds. Mass spectral data were collected on a Finnigan Mat TSQ 70 Mass Spectrometer which was interfaced to a Varian Model 34OO Gas Chromatograph. A DB1 capillary column

30 m long, 0.32 mm dia., film thickness 0.25 pm (J & W Scientific ) was used.

The injector and transfer line temperatures were 22O and 300o C, respectively.

The column was temperature programmed at sO'C for 1 min, 1O'C/min to 300'C and held at this temperature for 1O min. The sample was injected under splitless mode (30 s). Electron impact positive mass spectra at 70 eV were acquired over the range of mlz 35 - 350 in 0.5 s.

5.2.9.4 High Performance Liquid Chromatography (HPLC)

Samples were dissolved in Hipersolv MeOH and filtered through a 0.45 pm membrane filter (type HV, Millipore). All conditions used were the same as those which were used for flavonoids investigation (sub chapter 3.5.5).

Berberine (Aldrich) was used as a internal standard.

5.2.4 Quantification of individual purine alkaloid in misteltoe and tea plant

Standard curves of caffeine and theobromine are used to quantify the

levels of these compounds found in mistletoe and tea plant by HPLC. The HPLC

conditions used were the same as those used for the analysis of the flavonoids

(sub chapter 3.5.5).

72 5.3 RESULTS AND DISCUSSION

5.3.1 ldentification and characterisation of purine alkaloids

Caffeine and theobromine were identified and separated from both mistletoe grown on tea and the hosts by TLC using 60 Kieselgel Fr* plates pretreated by heating to 6O"C for 4 hours and developed in toluene:ethylacetate:diethylamine (70:20:10) solvent system (Figure 5.2). Using the same extraction procedures and the same TLC conditions, these compounds were found to be absent in mistletoe grown on citrus and persea (Figure 5.3). lt has been reported that Citrus sinensis contains caffeine (Stewart, 1985); however in this work, caffeine was not identified in C. maximus or in the mistletoe growing on it.

Both caffeine and theobromine did not give a colour reaction when sprayed either with lP and or Dragendorff reagent but appeared as deep purple

spots under UV at 254 nm with Rf values of 0.43 and 0.19 respectively.

It is widely known, that caffeine and theobromine are found in most tea

plants (Nagata and Sakai, 1984; Nagta and Sakai, 1985; Suzuki et al., 1992).

From the current work it appears likely that the purine alkaloids present in S.

ooftiana have been derived from its host, C. sinensisvar. assamica.

The presence of host specific alklaoids in mistletoes parasitizing the host

plant has been reported earlier. Trautner (1952) and Mortimer (1957) both

found that nicotine alkaloid found in the mistletoe, Loranthus sp., was also the

main alkaloid found in its host, D. myoporoides. Cordero et al. (1989) also

isolated 5 quinolizidine alkaloids from V. cruciatum and suggested that they were

obtained from its host, L. sphaerocarpa.

73 Figure 5.2 Thin Layer Chromatogram of purine alkaloids and standards found in different fractions (A,B,C) of extracts oÍ S. oo¡tiana (Korth.) Danser growing on tea and C' sinensis chromatographed on Kieselgel 60 Fr* developed in toluene:ethylacetate: diethylamine (70:20:10). Compounds were observed under UV light at 254 nm. Each sample has been applied in two separated lanes as a mirror image.

74 caffeine tleobromine

rl(Îl Ë((l .3 cl Cll E- ù, C" vt .sõ q) ÈcpÈ !óa 4 "i foÈ U Figure 5.3 different fractions Thin Layer chromatogram of purine alkaloids found in the on different host (A,B,C) of the extracts ol s. oortiana (Korth.) Danser growing plants chromatographed on Kieselgel 60 Fr* developed in toluene:ethylacetate: light at 254 nm' diethylamine (70:20:10). compounds were obserued under uv

75 caffeine

theobromine

S. oortiana S. oortiana S. oortiana gfown on gfown on gIown on tßa cirus perseÍt The presence of caffeine in mistletoe grown on tea and in the tea plant itself was confirmed by infra red analysis (the spectra are presented in appendix 5), The analysis of theobromine in mistletoe and tea, however, showed inconsistent results which were attributed to the low purity of the samples. They were later shown to contain more than one compound by HPLC.

The retention times of components in the extracts were compared with the respective standards which were used to further characterize these compounds

(Table 5.1).

Table 5.1 HPLC retention times of purine alkaloids isolated from S. oottiana (Kotth,) Danser grown on tea and C. sinensis and reference standards. Name of Retention time t, (min) of internal corrected compounds (t,) (min) standard retention time * caffeine 12.12 44.45 12.12 in mistletoe 12.10 44.47 12.09 in tea 12.12 44.52 12.09

* theobromine 3.94 44.58 3.93 in mlstletoe 3.91 44.52 3.90 in tea 3.99 44.48 3.99 * standard

76 LL

(nu:e) z¡l 5 o a'tÊt I o'rct ó e ,Þ. I IE

I I'JEI C G o'ó¿l

e ¿a

I o ¿t t

g .sel (urur)eu¡ uoluatar g9 66.ìa ú6rCa 66r6¿ E9rE¡ ùtt 9l ú¡ ti¡ aat¿l 9t ¡¡ tù 3att 3& r¡

¿¿l ' 9g-3¡ 3la l1

0¿ç'ì V ßs-!r û3t tarr ¡r 'fuuauorlcads sspf{ - Áqderôo}puloJLlC spÐ Áq Ea} uo urvrolôlosueg ('Lluoy) eueruoo 'S: ur aururorqoaLll lo (g) etpadg sspf{ pue (V) ure:ôo¡euolqg '¡'9 atnô¡3 provided GC-MS of caffeine and theobromine found in mistletoe and tea of confirmatory data of their identities (Figure 5.5, appendix 6). Retention times in caffeine and theobromine were 16.3 and 16.8 min and their molecular weights positive ion mode were 194 and 180 respectively. lt can be seen in the both chromatograms (see appendix 6) that there are additional compounds in the mistletoe grown on tea and the host plant but the identities of these compounds have not been confirmed'

5.g.2 Total purine alkaloids levels in mistletoe and in tea plants

It can be seen from figures 5.6 and 5.7 that most of the caffeine and theobromine were extracted (fraction C) by chloroform at pH 5 using extraction

procedures adapted from (valka and simanek, 1988) while fractions A and B contained negligible levels.

The total amount of caffeine found in C. sinensis var. assamica was 6.0%

dry weight while that found in its mistletoe was 0.6% dry weight (Figure 5.8).

The content of caffeine has been reported to be as much as 2.5"/o ' 4-5% of dry

tea leaves (de Wit, 1963). The difference in the levels of caffeine found in this

study with other studies may be due to the species of tea used but could also be

due to differences in environment factors where and when the plants were

grown. lt has been reported by Waller and Nowacki (1978) that environmental

conditions affect the general growth of plants and therefore large variations in

alkaloid content per plant are to expected.

78 Figure 5.5 Caffeine levels found in the various solvent extracts

200 .+ c¡lfeino in le¡ cafeine in mistal¡oe

cl

c l@ c a o

0 A

frrctlo n note: fractíon A was extracted by diethylether at pH I fraction B was extracted by diethylether at pH 12 fraction C was extracted by chloroform at pH 5

Figure 5.6 Theobromine levels found in the var¡ous solvent extracts æ 6¡€br€úh h te¡, -r- ü'n€blmÛrr h rîitd.toc

c!

a lo o a o c

A s l^ctlon notg: fraction A was extracted by diethylether at pH I fraction B was extracted by diethylether at pH 12 fraction C was extracted by chlorotorm at pH 5

79 Figure 5.7

Caffeine content (means of three values) (mds g dry weight of leaves) in mistletoe and tea plant in different fractionsof extract

200 180

160 Etea 140 I nistletoe (t 120 E o 100 Ê 0) 80 65.38 a! o 52.5 60 40 24.82 5.33 20 2 0 A B c fraction

Figure 5.8

Theobromine content (means of three values) (mg per 5 g dry weight of leaves) in mistletoe and tea plant in different fractions of extract 17.s5 18

16

14

ct) '12 E I in tea 0, 10 .E tr in mistletoe E o I ¡¡ o o 6 3.85 4 0.064 1.33 2 0.013 0.1 0 A B c fraction

80 Total theobromine found in c. sinensis var. assamica was approximately was 0.03% dry weight (Figure O.4g "/"dry weight while that found in its mistletoe o/" 5.9). Thus the amount of caffeine in mistletoe was approx. 10 of that found in the host while the percentage of theobromine in mistletoe was approx- 7"/" or that in tea Plant.

That the content of these purine alkaloids found in mistletoe was less than the levels in the host agree with the findings of Mortimer (1957) who showed that parasitizing of alkaloids related to nicotine found in the mistletoe Loranthus sp.

Duboisia myoporoides were always less than the levels found in the host.

81 Ghapter 6

GENERAL DISCUSSION AND CONCLUSIONS

6.1 GENERAL DISCUSSION

There is a range of contradictory findings concerning the relationships and their between the constituent chemicals present in the parasitizing mistletoe

host plants. For example, Trautner (1952), Mortimer (1957), Boonsong and

Wright, (1961), Cordero et at. (1989), Bloor (1991) reported that compounds

present in the mistletoes depend on the hosts parasitized while other workers, (1976), Fukunaga Graziano et at. (1967), Tronchet (1975), Kuroda and Higuchi, were no significant et at. (1988), Fukunaga et at. (1989 a & b), found that there

differences between compounds found in the species of mistletoe growing on ü liD different host I Plants. The above papers described various classes of compounds from different

species of mistletoes; for example, Trautner (1952) and Mortimer (1957) both

found that nicotine alkaloids in Loranthus sp. were derived from the host D' alkaloids myoporoides; similarly cordero et al. (1989) found quinolizidine Lygos isolated from Visc um cruciatum were similar to those found in its host, polar sphaerocarpa, Boonsong and Wright (1961) found that the three most

cardiac glycosides, of the twelve glycosides present in the leaves of N. oleander,

were translocated into three different species of mistletoe, while Bloor (1991)

obserued that norditerpene lactones were assimilated by llleostylus micranthus

from its host plant Podocarpus totara. Conversely, in another set of reports it

82 r was observed that mistletoes contain compounds different from their hosts

(Graziano et at. (1967), Tronchet (1975), Fukunaga et al. (1988) and Fukunaga (a quite different et at. (19g9 a & b)). These groups reported that the flavonoids

class of compounds from the alkaloids, cardiac glycosides and norditerpene

lactones) found in various species of mistletoes were different from those found

in their hosts. ln another instance, Kuroda and Higuch¡ (1976) observed that the

lignin found in the misttetoe was also independent of the host. The work repofted in this thesis confirmed that the similarity and

dissimilarity in chemical constituents in the mistletoe or its host is a function of

the class of compound examined. Thus, the flavonoids, quercitrin; isoquercitrin

and rutin, were similar in S. oo¡tiana growing on three different hosts. Even

though these three flavonols were also present in the hosts, the pattern of the

phenolic constituents of mistletoes was clearly quite different from the pattern in

,.'I il the other hand, the purine alkaloids caffeine and ,n respective hosts. On IL Ì theobromine found in S. oo¡tiana were probably derived from the host tea plant

because these compounds were not found in the same species of mistletoe

growing on citrus and persea. However the possibility that alkaloid synthesis in

I the mistletoe was induced by factors from the host plant can not be excluded.

In the case of flavonoids, the content of the individual flavonols varied

between the same mistletoe growing on different host plants indicating that

although this class of compounds may have been synthesized by the mistletoe

the three different host plants appear to have influenced the actual content of

these compounds in the same mistletoe. The fact that the amount of individual

flavonols in the mistletoes was always much higher than the respective content

I in the host and that the highest flavonol content in the mistletoe was not always

83 r that these the correspondingly highest flavonol content in the host suggests From the compounds were not simply taken up by osmosis from the mistletoe' has its own examples studied so far, it seems that each species of mistletoe flavonoids from characteristic flavonoid composition, so that if they do take up to form an the host they subsequently metabolised the assimilated compounds

entirely new group. were Ehleringer et al. (1984) has suggested that xylem tapping mistletoes rates in the both water and nutrient parasites. They found that photosynthesis (1990) found, mistletoe leaf were higher than in the host leaf . stewart and Press

from their ultrastructure studies, that the haustoria contained very dense developed organelles such as dictyosomes, mitochondria, ribosomes and a well have an endoplasmic reticulum. From this it was proposed that the haustoria

important role in the regulation of metabolism of solutes taken from the host' up d obseruations it can be proposed that mistletoes selectively take tR From these r\ti i To compounds from the hosts and assimilate them for their own needs.

understand this relationship the nutrition of parasitic plants has been studied

using several approaches including mineral nutrition and water relations

between mistletoes and their hosts, but to date, there are no direct comparisons

of compounds found in mistletoe growing on the host and mistletoe cultured rn

vifro (Stewart and Press, 1990).

ln the context of nutrient uptake of mistletoes from their hosts, it has been

observed that Phoradendron juniperium derived more than 60% of the carbon

from its host, Junþerus osteosperma (Marshall and Ehleringer, 1990) while Pate

et al. (1991) obserued that 24"/" ol the dry matter carbon in Amyema sp. i (Australian mistletoe), was derived from the xylem sap of its host. These

84 T amounts of findings suggested that differing species of mistletoe derive different needs. carbon matter from their hosts because of different requirements for their

With respect to nitrogen containing compounds, Trautner (1952)' Mortimer

(19S7), Cordero et at. (1989) and the findings in this thesis show that mistletoes

contain alkaloids which are similar to those in their host. Stewart and Orebamjo

(1ggo) assumed that mistletoes were largely dependent on reduced nitrogen in

the host xylem fluid. By examining translocation of amino acid and amide from to host to parasite, they suggested that parasitic plants have a limited capacity

assimilate inorganic nitrogen sources. ln addition, Raven (1983) noted that

xylem tapping mistletoes derived their nitrogen mainly via nitrogen containing

organic compounds from their host plants. schulze et al. (1984) also suggest

that high rates of transpiration of mistletoes was important for the uptake of

sufficient nitrogen from host xylem to produce their vegetative and reproductive

organs. Schulze and Ehleringer (1984) noted that the high rates of transpiration

of misiletoe was as an indicator of a mechanism to passively acquire sufficient

nitrogen from a very dilute nitrogen source in the host xylem.

6.2 GoNCLUSIONS

The evidence presented in this thesis shows that the three identified

flavonols, quercitrin, isoquercitrin and rutin, found in S. ooftiana (Korth.) Danser

growing on three different hosts were independent of the concentration present

in their host plants. The content of individual flavonoids was also variable in

between S. oortiana growing on different host plants. Two purine alkaloids, I I caffeine and theobromine, found in S. oortiana (Koth.) Danser were consistent in

; their relative concentrations with being assimilated from their host plant, C.

t 85 always less than srnensrs. The content of purine alkaloids in S. ooftiana was those found in the host Plant

6.3 FUTURE RESEARCH It is proposed that additional and decisive information on the chemical gained by relationships between mistletoes and their host plants can be given both conductin g in vitrotissue culture experiments where the cultures are

radio labelled and stable isotope labelled compounds' The types of labelled existing compounds used to innoculate the culture media would be derived from an knowledge presented in this thesis. These experiments would also lead to from understanding of the mechanisms by which mistletoes take up compounds

their hosts.

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95 Ellagitannin dimers and related dimers and their antitumor activity. Chem' Pharm. Bull. 37 (1 1), 317 4-3176' effects of Yu, H., Oho, T., Tagomori, s., and Morioka, T. (1992). Anticariogenic green tea. Fikuoka tgaku ZasshiSS(4)' 174'180'

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96 L6 8. APPENDICES

98 Appendix 1. Appendix 1 consist ol12 ultra violet spectra of ftavonols found in S. ooftiana growing on different hosts and standards in (A) methanol and sodium methoxide; (B) aluminum chloride and hydrochloric acid; (C) sodium acetate and boric acid.

99 TQUERCITRIN

HO o McOH OH McOH -+ N¡Ot\fÊ .-.--

O- rhamnosyl OH n t , I A t I I I ¡ I I UV Speclral data (X.¡¡¿¡,nm) \ I I I MeOH 258.0,348.8 I I \ I I \ NaOMe 271.3,397.6 I I I \ t AlOlg 274.4,332.8, 424.8 \ T \ AlCl3/HCl 269.9,349.4 \

NaOAc 271.6t 378.7 NaOAc/H.BO. 260.2;362.9 note:'= standard

2QO 300 a@ø 4

M€OH + NaOAc- MoOH + NaOAc + H¡8q McOH + AICI¡- MGOH+ AIOr + HO...... - '

B c

I t I I I I I

I I I \ \ t \ \ \ \

22Ð DO loo \nm l, nm

100 .ISOQUERCITRIN

MeOH- McOH + NrOMc -

I^ OH I t I A t I I I ì O - glucosyl I ì I I oHo , I I \ I I I I I I I I UV Spectral data (l.¡¡¿¡,nm) I I I , t I I I I MeOH 256.0,358.0 I t I I I NaOMe 272.8;330.3; 414.1 t I AlCl3 274.0,432.0 AlCl3/HCl 268.8; 407.6 NaOAc 272.6;379.1 NaOAc/H.BO. 261.2;381.2 note: '= standard Lnm

'¡ McOH+AlO¡- MeOH + NaOAc- itl MoH+Atqt ìHo- McOH + NaOAc + H,8O. ì* l!, - I ì

B I I c I I I t v t I t I ì t \ I I f I I \ t \ I \ ì \ \ ì \\ I I

zút æo 100 110 ,lêo a4s ). nm lnm i

I 101 .RUTIN

OH McOH - McOH + N¡OMc --- H OH

O- rhamnoglucosyl A oHo ,r I I I I I UV Spectral data (À¡¡¿y,nm) I I t I I I MeOH 255.9,357.7 I I t I I I I NaOMe 272.3,415.2 t AlCl3 274.0,430.7 AlClg/HCl 268.5,397.0 NaOAc 272.4;401.3 n ¡.a iO ¡l{g NaOAc/HrBO. 261.2;379.1 \-r note: '= standard

# t I 'q I I I MoOH + NaOAc - ,l ¡t t M€OH + NaOAc + H,8O' I - il MeOH.AlOr- I å + + HCI ...... t McOH AtCtr I I I I c t t h B t t T t t rl \ I I I t ì I I I I t \ t \ I :t ..-:-.. :r ì¡ t \ t \

2.. taa <- .F¡a À.na 220 v0 10o ilo I l. nm

102 T Quercitrin in

Scurulla oortiana growing on tea McOH- MeOH + N¡OMc - lln UV Speclral data (Î.¡¿¡,nm) tl rl1l I A MeOH 255.2,348.2 t t 271.2,395.5 t I \ NaOMe I I \ I I t AlCl3 274.1,424.4 I I b \ t I AlClg/HC¡ 269.2,352.0,395.0 I t I t t NaOAc 265.7;368.5 t t NaOAc/H.BO, 259.9; 368.4

¡Oo 300 - 4Ao r.o

¡r il ¡l€OH- M.OH + NaOAc- n rt M€OH + NaOAc r H,BO¡ ¡t MGOH+AIO| _ McOH+^¡Clr+Hq- I - I I , I I ,I t ru I IrS ,i , I B l c I I I II I I I I I I I t

t 4 / I \ t \. t \ \ \ \

nm 2.ú l, 50(t 100 lnm

I

r 103 ISOOUERCITRIN In MÊOH..,...... McOH t N¡OMo growing I - Scurulla oortiana on tea I

Spectral data (l.¡¡¿¡,nm) I UV A I

MeOH 256.9; 360.0 t I NaOMe 272.8;410.7 t a

I AlClg 274.5:429.0 I I AlCl3/HCl 268.6; 390.5 NaOAc 272.7;390.1

NaOAc/HrBO. 261.6; 381.0

Lm

l l M¡OH ¡NeOAc- MoOH + NaOAc r HrBq I McOH+ AlOt- I MeOH+^¡O¡+HO- i I I - ì I I I I j I I I I I c I \ B , I I I I I I I I I t t\ I I t I t I I I t t I I ì \ I I \ I I \ \ I \ \ \ I I \ \ \ \

Lm L2t Joo 1.o 14o l. nn

I

104 N RUTTN In

oortiana growing on tea McOH- Scurulla MeOH + N¡Ol\fc -

UV Spectral data (I¡¡¿¡,nm)

MeOH 256.7,357.8 A

273.2,416.4 ¡ NaOMe t AlCl3 274.1,430.5 t t AlCl3/HCl 267.9,396.0 \ t \ NaOAc 267.7;386.6 t \\ I NaOAc/HrBO. 261 .2;381 .2 t

tnm

I \ I MoOH + NaOAc- I MoOH + NeOAc + Hr8O, , - McO¡l+A¡Ot -- MÊOH+A¡O¡ +HO_ I

c

B I t /1 t I I

t I 1 t \ I t I \

zzO 30o qoo 41o

l. nm I. nm

105 QUERCITRIN in

Scurulla oortiana growing on citrus McOH- McOH + N¡OMc I - I UV I Spectral data (I¡¿¡,nm) , MeOH 255.4,949.4 A NaOMe 270.2,395.7 \ \ AlCl3 274.7,429.9 I I I I \ I I AlCl3/HCl 270.A,3S3.1, g9S.B I I I I I NaOAc 2GG.3; 360.7 I I \ NaOAc/HrBO. 260.1;368.3 \

aee l.4- l- na

ùGOH+NClt M8OH r NaOAc- l'rcH+,t¡qr+HO- - MoOH + NaOAc + H!8O¡ -

I t \ I I ì I c I I t I I I I \ I I I I I t I I I \ t I t \ \

,!1C ..1ø 220 Lnn Joo .lo(ì l. nm

106 ISOQUERCITRIN in I

I MGOH- Scurulla oortiana growing on citrus I McOH + N¡OMç I rì - I I I I t , UV Spectral data (1.¡¡¿¡,nm) \ I

MeOH 255.5,356.2 I I I NaOMe 271.2,406.9 t ì I AlCl3 274.6,420.5 \ I I AlClg/HCl 270.3,396.3 \ \ NaOAc 272.8;387.9 NaOAc/H.BO. 261.7;380.0

l. nn

MgOH + NaOAc- MeOH + NaOAc + H,BO. McOH+AlGlt- - McOHTAtG¡ +HO---

I I I

I I t /tt I I I

I t \ I \ \ \

7Ð too lo0 4.40 l. *i I. nm

107 t I RUTIN ln I McOH- I McOH +N¡OMo I - Scurulla oortiana growing on citrus I I I A UV Spectral data (1"¡¿¡,nm)

MeOH 259.0; 364.5 I 272.6;417.0 I I NaOMe I I t I AlCl3 274.4;429.5

AlCl3/HCl 267.2;396.2 \ I NaOAc 271.5;414.8 NaOAc/H.BO. 261.9; 388.9

l.u

lvhOH + NaOAc- likoll+A¡Cl¡ i¡teOH + NaOAc + H¡8O! ll4tt +.1¡Cl¡ + BCl- - -

B

,I , I I ì l.rt\

2LO xt0 <

108 QUEHCITHIN in

Scurulla oortiana growing persea McOH- on + McOH NqOMc ---

UV Spectral data (À¡¿¡,nm) rì A MeOH 255.1, 350.8 I t t I I , t I NaOMe 270.8,396.7 I I AICIS 274.5,427.5 I t t AlCl3/HCl 269.8,352.5, 394.5 t t \ ¡ t NaOAc 268.9; 390.8 \ \ \ t t NaOAc/H.BO, 262.1; 375.3 I

l.nm

MaOH + NaOAc- n M€OH + NaOAc + H¡8O, I MGOH + A¡Gr M.OH+^lor +HO- - I -. I I I I B c

I71 I I t I , t , I t ì t

t

z¿.o loo 400 l. nm l" nm

109 ISOOUERCITHIN in Scurulla oortiana growing on persea McOH...... McOH + N¡OMc -

UV Spectral data (Î"¡¿¡,nm)

MeOH 256.2,357.3 I A NaOMe 272.8,411.3 AlCl3 274.5,428.6 AlCl3/HCl 268.4,396.6

NaOAc 272.3;394.9 NaOAc/HrBO. 261.8;381.4

.!oe Lnn 44e !r J rl It MoOH + NaOAc- it I M9OH + NaOAc + l{rBO¡ :l - tÌ it ir ir l1 Ir /t B c lt ii ¿l :ì /t /l ìr 1.. t ìr / \r tt ir ,l \ I i\ ,i lr I ,.\, ; I l ì - I ir I it I l\ \ t,\ ?' I I \I . -.¡- - ta

L nnr )20 so 1Ø 1

110 HUTIN In MG('H- Scurulla oortlana growing on persea MGOH r N|OM. -

UV Spectral data (I¡¿¡,nm) A

I MeOH 260.0,364.8 I I \ NaOMe 272.9;418.0 t I I\ I I AlCl3 274.7,429.9 I I 266.9,396.0 r AlCl3/HCl I 7\ t t I \ t NaOAc 271.6,414.5 I NaOAc/H.BO. 262.0;389.6

ltu

ll¡Oll+âlCt¡ llrOH + NaOAc - ltdl+^rc¡¡+¡G¡- tirOll + NeOAc + HPO. - -

B c

t

I I a

220 x,o 1oo a

111 Appendix 2. Appendix 2 consist of 9 chromatograms (A) and mass spectra (B) of flavonols found in S. ooftiana growing on different hosts and standards by High-

Pe rfo rmance Liqu id C h ro matog raphy- E lectrospray Mass Spectro m etry.

112 Chromatogram (A) and Mass Spectra (B) of quercitrin standard by High- Performance Liquid Chromatography-Mass Spectrometry

3 Þ A 2.2ø6

2.0€6

1.8e6

1.6€6 2 I ø 1.4e6 qo- j 1.206 ø 7 95 o 1.0e6 5.49

8.0e5 27.'17 24.13 6.0e5

4.0e5 14 tt 18.06 2.0€5

5 10 15 20 25 ]jme, min

60000 .2 B 54000

48000

42000

q o 36000

a 30000 o 24000

1 8000

1 2000

6000 229.2 345.0 449.0 153.0 387.0

140 210 280 350 420 490 m/2, amu

113 Chromatogram (A) and Mass Spectra (B) of quercitrin in S. oortiana (Korth.) Danser grown on tea by High-Performance Liquid Chromatography-Mass Spectrometry

20 I A

1.2e5

9.0e4

ð

3 20

'I 0 64 49 16.83 Qt

4 I 12 16 20 Time, min

30 2 B

2.5e5

ø 2. 0e5 (,

ø .5e5 o .0ê5

5.0e4 449.2 152.8 o zs7.q s44.8

140 210 280 350 420 490 m/2, amu

114 Chromatogram (A) and Mass Spectra (B) of quercitrin in S. oortiana (Kot1h.) Danser grown on citrus by High-Performance Liquid Chromatography-Mass Spectrometry

5 2.0es A

1.6e5

1.28s 5.43

8.0e4

10 15 20 25 Tme, min

1.5€5 B

1.2€5 q o. o 9.0e4 ø o 6.0e4

3.0e4 -¿ .2 4 zot.o 345.0 'I 40 210 280 3s0 420 490 m/2, amu

115 Chromatogram (A) and Mass Spectra (B) of quercitrin in S. oortiana (Korth.) Danser grown on persea by High-Performance Liquid Chromatography-Mass Spectrometry

5 2.0e5 A

1.605 q oè S1. 2ø5 5 43 ø g cq 0e4

4.0e4

5 10 15 20 25 i Time, min

.2 1.5€5 B

1.2e5 o oÉL j 9.0e4 cø o

3 44 .2 .22 2 .¿[ 201.0 345.0

140 210 280 350 420 490 m/2, amu

11o Chromatogram (A) and Mass Spectra (B) of isoquercitrin standard by High-

Pe rfo rmance Liqu id C h ro matog raphy- Mass Spectro met ry

1.2€6 A

I .1e6

1.0e6

9.0€5 5 2 , 8.0e5 o à 7.0€5 Ø .c 6.0e5 15 89 5.065

4.0€5

3.0eS 21.86 25.13 2.0€5 1.87 8.30 12.86

'I .0e5

5 10 15 20 2S Time, min

.2

1.0e5 B

9.0e4

8.0ê4

7.0ø4 6 o. o 6.0e4 i o 5.0e4

4.0€4

3.0e4

2.0e4 465.0

1.0e4 229.2 't 53.2 201.0 345.0 387.2

140 210 280 350 420 490 m/2. amu

117 Chromatogram (A) and Mass Spectra (B) of isoquercitrin in S. oortiana (Korth.) Danser grown on citrus by High-Performance Liquid Chromatography-Mass Spectrometry

3 4800 A

4000

ø (,o- 3200

Ø 2400 o 5.26 I 0

1 600

800

4 I 12 16 20 Time, min

.2 B 45000

6 36000 o q 27000 o

1 8000

9000 .2 .0 345.0 387.0 465.2

140 210 280 350 420 490 m/2, emu

118 Chromatogram (A) and Mass Spectra (B) of isoquercitrin in S. oortiana (Korth.) Danser grown on persea by High-Performance Liquid Chromatography-Mass Spectrometry

48000 5 A 40000

¿ 4 32000

24000

1 6000

8000

5 10 15 20 25 Time, min

.2 35000 B

2800 6 o 21 000 ø o 4000

7000 .2 34 .0 465.2 137.4 165.2 41 0

140 210 280 350 420 490 m/2, amu

119 Chromatogram (A) and Mass Spectra (B) of Rutin standard by High-Peñormance

Liquid Ch romatog raphy- Mass Spectrometry

1.2e6 2

1.1e6

1.0€6 5.49 9.0e5

8.0e5 6 cl o 7.0€5

ø 6.0€5 o 5.0e5

4.0€5

3.0e5 7.15 2.0es 10 87 't6 71 26.00 1.75 1.0e5

5 't0 'I 5 20 25 Îme, min

.2 45000

40000

35000

30000 6 oo- 25000 ø o 20000

1 5000

1 0000

.0 611 5 000 3A7.2 153.2 229.0 0 46 4

180 270 360 450 540 630 m/2, amu

120 Appendix 3. Appendix 3 consist of 3 chromatograms of flavonoids in butanol extract of S. oortiana (A) growing on different hosts and the hosts (B) by High-Performance Liquid Chromatography at absorbance 280 nm.

121 I

I zzt i

eu¡n uonuelo¡ 3.lnul¡. Tol x oo'Þ oo'e oo.¿ oo'T oo'o oo''o II ì, I B

I !Þ Ft¡ E{ oo'z X oõF5 .'øIJ

C 0

t+ oo'Þ !t

oo'9

I g þ

3u¡l¡ uoluo¡e¡ x s¡ltlu!¡. ToT oo'Þ oo't o0'z oo'l oo'o oo'o

rF F,¡l ,i I t I B l¡ ¡¡ I I ? a rl os'o I

C 0 o= ã 6 n

oo T

os'T

å a .V I (g) ew¡xew 'g uo 6u¡rrrro.r6 (y) eueryoo S lo lcellxa louelnq lo u,tu OgZ lB rueJôolpu,olllC ezl I

eu¡ll uol¡uo¡e,t r.lnul¡. Tol x I I oo'Þ oo'c oo'z o0'T oo o ¡ oo'o *

oz'o I'i I I

I F ¡t oÞ'o I

I

o o= 09'o ' =ø

I F E o8'o

oo't

I |J E oz'T I s g

eu¡[ uoNe¡o,l s¡ltìu!¡. ToT x oo'Þ oo't oo'z oo'f oo'o oo'o

I ? ¡¡

I F I B I P og'o Ë

I C= F t¡ 3É h€ oo'T

o9'r

I .V a it'[ '(g)s¡suau¡s ? uo 6u¡wrol6 (v) eueuoo s lo lcEJlxa louBtnq lo uru ogz lp rupJôolErloJqc Chromatogram at 280 nm of butanol extract ol S. oortiana(A) growing on P, americana (B).

I, A I I LOO

6.00

t¡ I

0 >t 8.4 oo Ëo6l

x g I 2. OO I a ß ..1 ¿ I I I ¿ a I ,l I o. oo o oo l. oo 2,OO 3.OO 4. OO j tot ;rinqtcs retentlon üme

a A B I

1.50

i É

t¡ I i r.oo t I

EIø.' i go CJ a à i .l x I t' s I

o.50 I I I I È á I

o. oo

o. oo t. oo 2.OO 3.OO 4. OO I x lol ninutcs

r€lenüon ilme

124 T Appendix 4. Chromatograms of phenolic compounds in ethyl acetate extract of C. srnensis (A); S. oortianagrowing on tea (B) and on citrus (C) by High-Performance Liquid Chromatography at absorbance 280 nm.

i

I

1 il

i,l

I

I ,l

I

,t ill T ,¡

i

(

I I I !'

T I i I

r 125

I I 9Zt I

surl¡ uolluel€J .at¡cl¡ tol r oo') oo.3 00. t 't oo 'o 00'o I !, Í t ,a ot'o o o'.o u,

gt a a ot'o : I

or.o

oo. t 3

I a : eurn uollusleJ ..tnult lot x oo.Ê oo.z oo't 'o oo.o I a ot'o : I

o).0 @ v,

ot.o !a ¡ oa.o

'¡

ol't

s I

er¡¡[ uollu€l8l r.lnúl¡ tat x oo'Þ oo't o0'o ) I E 7 a F ¡ oz'o f ¡ o 'o a,

a ot.o a t

ot'o

oo'Ì

V o?'1 ! ,(g) 'ruu OgZ le (C) snrllc pue pal uo urno.¡6 euellroo s puB (y)s¡suau¡s ? lo lcerlxa alplecB ¡Áq¡e ¡o ue.¡ôoler.uorrlc Appendix 5. lnfra-red spectra of caffeine standard (A); caffeine in S. oortiana growing on tea (B) and in C. sinensis (C).

A

ù t gÍ

a. IE ¡ I ¡l $¡t8 9 ¡ å Êi Ë

I

I J

I I

hrwffilætt

B Í. AR

! là Eã t

F¡ r ã¡¡r¡ ¡E

hrhr(æt)

c

!t $ I I

I 2

ãtr I ßF enEÍ

&

{

127 Appendix 6. Appendix 6 consist of 5 chromatograms (A) and mass spectra (B) of caffeine and theobromine found in S. oortiana growing on tea and C. sinensis by Gas

Ch romatog raphy- Mass Spectrometry.

128 Chromatogram (A) and Mass Spectra (B) of catfeine standard by Gas Ch romatog raphy-Mass Spectro metry.

¿l ttc A

aa aa l¿r aa ta aa 9a retention time (min)

B

¡tt. I

tl. r

ae. a

a tú1.¡

nr/z (amu)

129 chromatogram (A) and Mass spectra (B) of caffeine in c. sinensrs by Gas Ch ro matography- Mass Spectro metry.

¿l rtc +aa ,aa A

ac a.aa It.a! ¡: at ¡ar aa ¡r¡tC retention time (min) aa

B

32

¡2. I I

ta5. I

nt/z (amu)

130 Chromatogram (A) and Mass Speara (B) of caffeine in S. ooftiana (Korth.) Danser grown on tea by Gas Chromatography-Mass Spectrometry.

¡l rlc A ¡¿a

çâ ta ItrQâ l2 ttrt€ I6 ât FC 2lr retent¡on time (min)

B

lat. ¡

t a7.l

a l:7. ¡ la!. ¡

n/z (amu)

131 Chromatogram (A) and Mass Spectra (B) of theobromine standard by Gas Ch romatography-Mass Spectrometry. Rtc ,:l rE+ø6 A 1.59e

lrå€ Â, qA 8rAA lar ã8 L¿ ¡aâ l4¡84 L6tqâ l8 .qq zøtqÐ ¿¿tqq 21tsø retention time (min) rE+ø6 B

4

l09, O t5 I 57.0 I

92. O

t7 ¡12.1 o t37. t 94 o t.o I t5t.o

2 nVz (amu)

132 Chromatogram (A) and Mass Spectra (B) of theobromine in C. sinensis by Gas Ch romatog raphy-Mass Spectrometry.

¡Eiø5 ¿l ù/r r I ÈO A I .9ø5

RIC rE+o6 ,l to r.265

a

.ir Êrâq Êrqq I 2Âr ââ 22r )4t ¡¡ â¡ lzra? !4! ââ !6 ra9 tErâ8 ^À retention time (min) ¡E+o6 LAø. ø B

4

r09. I J3, ø É?.ø I

e2. r

42. O 7ø.O 137.O

¡í.t. O 94 tl rt t- 3 o r5¡.o

B m/z (amu)

133