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This is to certify that the

thesis entitled

BIOACTIVE CONSTITUENTS 0F CURCUHA LONGA, L.

presented by

Geoffrey Nicholas Roth

has been accepted towards fulfillment
of the requirements for

H. S . degree in Horticulture

 

 

 

 

Major professor

Date gl/Z 2/ i5

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

BIOACTIVE CONSTITUENTS OF C URCUIWA LONGA, L.

By

Geoffrey Nicholas Roth

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Horticulture

1996

ABSTRACT

BIOACTIVE CONSTITUENTS 0F CURCUAIA LONGA, L.

Geoffrey Nicholas Roth

Turmeric, the ground rhizome of the tropical monocot Curcuma Ionga Linn, has long
been noted for its therapeutic potential, especially in traditional Indian ayurverdic medicine.
For example, it possesses significant antioxidant and anti-inflammatory activities and is
reported to produce symptom relief in patients with external cancer lesions.

Bioassay-directed fractionation of ethyl acetate extract from turmeric rhizomes yielded
three main curcuminoid compounds, which displayed anticancer activity when tested for
inhibition of topoisomerase I and II. Curcumin III (3) was the most active curcuminoid,
inhibiting the topoisomerases at 25 ppm. Curcumin I (l) and curcumin 11(2) inhibited the
topoisomerases at 50 ppm. Fractionation of the volatile oil from the rhizomes afforded ar-
turmerone (4) which displayed mosquitocidal activity with an LDloo of 50 ppm on fourth
instar Aedes aegwtii larvae.

Bioassay-directed fractionation of hexane extract from the turmeric leaves yielded a

diterpene aldehyde, labda 8(17) lZ-diene-15,16 dial (5) with antifungal activity against

Candida albicans, at 1 ppm. Similarly, Candida kruseii and Candida parapsilosis showed
activity at 25 ppm and 25 ppm, respectively. In addition, 5 displayed mosquitocidal activity

on Aedes aegwti larvae with an mm of 10 ppm.

57°WW

ACKNOWLEDGEMENTS

Thanks to my major adviser, Dr. Muraleedharan G. Nair, for his help and
encouragement during this project and my guidance committee members, Dr. John Kelly, and
Dr. James Miller for their advice and assistance.

A special thanks to Dr. Amitabh Chandra for his overall assistance in the Bioactive
Natural Products Laboratory and his assistance in NW spectral interpretations. Dr. James
Nitao deserves thanks for his help with the insecticidal assays. I would also like to express
my appreciation to Dr. Long Lee and Kermit Johnson in the Max T. Rogers NMR facility at
Michigan State University for their help in obtaining NMR spectra on a very tight schedule.

I have been fortunate to work with many good people in this department and in the
BNPL, especially Mark Kelm, Jennifer Miles, Dr. Yu-Chen Chang, Di Zhang, Andy Erickson
and Haibo Wang.

A very special thanks to Abigail Holbrook, whose encouragement throughout this
project has been invaluable.

Finally, thanks to my parents for always being there for me, no matter what.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... .vii.
LIST OF FIGURES ....................................................................................................... viii
LIST OF SCHEMES ....................................................................................................... ix
LIST OF ABBREVIATIONS ............................................................................................. x
LIST OF APPENDICES .................................................................................................... xii
CHAPTER 1 - Introduction ................................................................................................... l
The Plant, Curcuma Ionga, Linn. .............................................................................. 1
History of turmeric ................................................................................................... 3
Microbial diseases and pests of turmeric ..................................................................... 6
Reported biological activity on turmeric compounds .................................................. 8
Reported chemical constituents of tumeric .............................................................. 21
CHAPTER II - Bioactive compounds from turmeric rhizomes ............................................. 28
Abstract ................................................................................................................... 28
Introduction ........................................................................................................... 29
Experimental ................................................. . ........................................................... 3O

Results and Discussion ........................................................................................... 40

CHAPTER III - A bioactive diterpene aldehyde from turmeric leaves ................................ 49
Abstract .................................................................................................................. 49
Introduction ............................................................................................................ 50
Experimental ....................................................................................................... 51
Results and Discussion ............................................................................................ 59
CHAPTER IV - Summary and Conclusions ......................................................................... 68
LITERATURE CITED ....................................................................................................... 72
APPENDICES ................................................................................................................... 84

LIST OF TABLES

Table 2.1 Preliminary anticancer bioassay results

for EtOAc and hexane extracts and compounds

recorded as zone of inhibition in mm extract ............................................................. 42

vii

LIST OF FIGURES

Figure l Alnustone, trans-1,7-diphenyl-l hepten-S-ol, and

trans, trans, 1-7, diphenyl -5-ol ............................................................................ 12
Figure 2 Diphenyl heptanoids from Curcuma comosa ...................................................... 16
Figure 3 Anti-AIDS boron-curcuminoid complexes ......................................................... 18
Figure 4 Non anti-AIDS boron complexes ....................................................................... 19
Figure 5 Compound 13 ................................................................................................... 20
Figure 6 Volatile oil components .................................................................................... 22
Figure 7 Curcuminoids .................................................................................................... 24
Figure 2.1 Procedure for extraction of C. Ionga rhizome ................................................ 33
Figure 2.2 For the extraction of volatile oil ..................................................................... 37
Figure 2.3 Structures of curcuminoids ................................................................................. 44
Figure 2.4 Structure of ar-turmerone .................................................................................. 46
Figure 2.5 EIMS fragmentation pattern of ar-turmerone ..................................................... 48
Figure 3.1 Structure of labda 8(17) 12-diene-15,l6 ........................................................... 63
Figure 3.2 COSY correlations for labda 8(17) l2-diene-15, l6 .......................................... 64
Figure 3.3 EIMS possible fragmentation pattern of labda 8(17) 12-diene-15, 16 ................ 65
Figure 3.4 Compound 6 .................................................................................................. 67

viii

LIST OF SCHENIES

Scheme 3.1 Extraction of leaves ......................................................................................... 54

LIST OF ABBREVIATIONS

BNPL ....................................................... Bioactive Natural Products Laboratory

CHCl3 ....................................................... Chloroform

CD ..................................................... Circular Dichroism

CDC], . ..................................................... Deuterated Chloroform

(CD,)2CO ..................................................... Deuterated Acetone

COSY ...................................................... Correlated Spectroscopy

DEPT ............................................. Distortionless Enhancement of Polarization
Transfer

DMSO ..................................................... Dimethyl Sulfoxide

EIMS ...................................................... Electron Impact Mass Spectrometry

EtOAc ....................................................... Ethyl Acetate

FABMS . ..................................................... Fast Atom Bombardment Mass

Spectrometry

NMR ...................................................... Nuclear Magnetic Resonance

PDA ....................................................... Potato Dextrose Agar

TLC . ...................................................... Thin Layer Chromatography

VLC ........................................................ Vacuum Liquid Chromatography

6 ......................................................... Chemical Shift

d ......................................................... Doublet

dd ........................................................ Doublet of a Doublet

J ........................................................ Coupling Constant

MeOH ....................................................... Methanol

m/z ....................................................... Mass-to-Charge ratio

MIC ...................................................... Minimum Inhibitory Concentration
Rel. Int ........................................................ Relative Intensity

Spp ....................................................... Species '

YPDA ...................................................... Yeast Potato Dextrose Agar

APPENDIX I

APPENDIX II

APPENDIX III

APPENDIXIV

APPENDIX V

APPENDD( VI

APPENDIX VII

APPENDD( VIII

APPENDIX IX

APPENDIX X

APPENDIX XI

APPENDIX XII

APPENDIX XIII

LIST OF APPENDICES

lHNMR spectrum of curcumin I .......................................................... 84
lHNMR spectrum of curcumin II ...................................................... 85
1I-INMR spectrum of curcumin III ..................................................... 86
lHNMR spectrum of ar-turmerone ..................................................... 87

13CNMR spectrum of ar-turmerone ................................................... 88
EIMS of ar-turmerone ...................................................................... 89
lI-INMR spectrum of labda 8 (17) 12-diene-15,16 dial .................... 9O
13CNMR spectrum of labda 8 (17) 12-diene-15, l6 dial ..................... 91
DEPT spectrum of labda 8 ( 17) 12-diene-15,16 dial ........................ 92
HMQC spectrum of labda 8 (l7) 12-diene-15, 16 dial ...................... 93

COSY spectrum of labda 3 (17) 12-diene-15,l6 dial ....................... 94

EIMS of labda 8 (17) 12-diene-15,l6 dial ........................................ 95

CD oflabda 8 (17) 12-diene-15,16 dial ........................................... 96

xii

CHAPTER I

Introduction

The Plant, Curcuma Ionga, Linn.

The tropical plant, Curcuma Ionga, L., with a long and distinguished human use in
Eastern civilization, is native to south and southeast tropical Asia and probably originated in
the slopes of hills in the tropical forests that grace the west coast of south India. It can be
grown in many tropical conditions, e. g. from sea level to 1500m elevation in the temperature
range of 20-30°C, in rainfall, or under irrigated conditions. It thrives in loose, friable loamy
or alluvial soils suitable for irrigation and good drainage (Govindarajan, 1980; Venill, 1940).
A member of the Zingiberacae family, akin to ginger, this attractive tropical monocot grows
to a height of 1 m with long green stems of parabolic cross section exiting a common rhizome
node, and ending in large, omately ovoid green leaves. The narrow, yellowish-white flowers
are present on cylindrical spikes bearing green-white bracts. Since most cultivated varieties
are sterile triploids, flowers and fiuits are known but rare (Govindarajan, 1980), and the seeds
have very low germination. However, this is not a problem since C. Ionga has been
propagated vegetatively by the rhizome for thousands of years.

The plant often is referred to by the name of its bright orange rhizome, “turmeric”.
The sanctity of turmeric color has traced back to the ancient sun-worshiping culture, which
began ceremonially using the cheap and easily grown tumeric as an alternative to saffron. The
direct English translation of turmeric is “ginger yellow”. Its name in the Malay Peninsula was

“kunyit”, the word for yellow. The genus “Curcuma” probably arises from the Arabic name

2

for turmeric, “kurkum” (Burkill, 1966; Brierley, 1994; Govindarajan, 1980). Of the
Curcuma genus, a few species produce the turmeric of commerce; mainly Curcuma Ionga L.
(syn. Curcuma domestica Valet), and to a smaller extent, Curcuma aromatica Salisb,
Curcuma amada Roxb, Curcuma zeodarr'a Rosb (In India and China) and Curcuma
xanthorrhiza Rosb (in Indonesia). The subterranial rhizome of C. Ionga is processed into
spice. The C. Ionga rhizome consists of a mother rhizome, the stem extension, and many
longer secondary rhizomes growing fiom the mother rhizome. The mother rhizome is known
as C. rotunda and the secondary rhizomes are known as C. Ionga. This often creates
confusion in the classification where the two forms are differentiated for trade (Govindarajan,
1980).

Over thirty varieties of turmeric (C. Ionga) are recognized now and are often
associated with a specific state in India. The state of Andhra Pradesh is known for
‘Amruthapani’, ‘Nizamabad’, ‘Cuddappa’, ‘Kasturi’, ‘Chaya’, ‘Kodur’, ‘Armoor’,
‘Duggirala’, and ‘Tekkurpeta’ varieties; Bengal for ‘Pattini’ and ‘Deshi’; Kerala for
‘Moovattupuzha’,‘Alleppey’, and ‘Wynad’; Maharashtra for ‘Rajpuri’, ‘Karhadi’ and
‘Waigan’; Tamil Nadu for ‘Chinnanadan’, ‘Salem’, ‘Karur’, ‘Erode’ and ‘Perianadan’; and
Orissa for ‘Behrampuri’, ‘Koraput’, and ‘Saveera’. Other varieties include.‘Lokhandi halad’
( used for dyeing ), ‘Kuchupudi’, ‘Sugandham’, ‘Erode’, ‘Gadhvi’, ‘Amoor (CLL 324),
‘Duggirala (B,9)’, ‘Duggirala (CLL 325-B,55)’, ‘Gorakpur (CLL 316)’, ‘Karhadi’, ‘Mydkur
(CLL 326-B,52)’, ‘Rajpore’, ‘Rajpore (CLL 327)’, ‘Tekkurpetta (CLL 327)’, ‘Vontimetta’,
‘Vontimetta (CLL 322)’, and ‘Miraj 26' (Shivashankar, 1976; Gopalan, 1976; Govindarajan,

1980). The world’s largest turmeric production is the state of Andhra Pradesh, India. In

3

1973, the 13,600 hectares yield was about 38,000 tonnes of dry turmeric at 2.79 tonnes
hectare". The state of Madras in India is the second largest producer, followed by the states
of Maharashtra, Orissa, Tamil Nadu, Bihar, Kerala, Assam, Mysore, West Bengal, and

Tripura (V ervai, 1971).

History of turmeric

In contrast to turmeric's limited use in Western countries as coloring agent for
mustard, pickles, and textiles (Survey, 1968), most turmeric is consumed in the countries of
origin, where its uses are unlimited. Almost every religious Hindu ceremony makes use of
turmeric in one form or another (Kurup, 1979). Addition of lime to turmeric produces a deep
brownish-red material known as ”kum-kum", which is the material used by Indian women to
make the auspicious mark on their foreheads (Govindarajan, 1980). Turmeric's use extends
back to the time of the Pharaohs, when it was an ingredient in balm of Gilead, and an
ingredient in preparation of Egyptian mummies (V errill, 1940). It was mentioned in ancient
Sumerian inscriptions, dating back further than 2000 BC. Turmeric was one of the principal
spices (the scriptural “calamus”) besides myrrh and sweet cinnamon which were considered
to compose the perfume compounded by Moses at Jehovah’s command in the Old Testament
Christian Bible (Verrill, 1940). It is mentioned in the Assyrian Herbal (551-479 B. C.), as a
medicine for external and internal use, and as a firmigant and beer condiment. Dioscorides
wrote about turmeric and called the spice “cyperis”. Theophrastus referred to it as
“cypeiros”, and noted its similarities to saffron (Miller, 1969). The plant also has a long

history of folk medicinal use in India and the Orient.

4

The plant has been used as a stimulant, tonic, stomach soother, fever alleviator, dropsy
cure, and for cleaning foul ulcers (Lindley, 1838). Turmeric paste is applied to facilitate
scabbing in chicken pox and small pox (Kuttan, 1985). The rhizome has been used as a blood
purifier, liver stimulant, and to remove catarrhal formations leading to jaundice of the liver
and gallbladder, which is possible since curcumin dissolves cholestrin, a component of bile
and gallstones (Harris, 1972; Vevai, 1971). The rhizome has been used for hazy vision, eye
inflammation, night blindness, subnorrnal temperature, body pain, rheumatism, to regain
consciousness, scabies, sores, infantile fistula ani, cough and to promote lactation. The flower
is used for sores in the throat, syphilis, dyspepsia, and cholera (Jain et al., 1991). Other
reported rhizome uses were as alterative, antiperiodic, antiseptic, carminative, tonic,
vermifuge, for anorexia, biliary disorders, coryza, diabetic wounds, hepatic disorders,
sinusitis, eosinophilia, and antifertility. Also it is suggested that burning turmeric cures head
colds (Kurup, 1979; Waring, 1982). Interestingly, it has been observed that turmeric given
to animals causes them to produce male offspring predominantly. In Africa, turmeric is used

to prevent pregnancy, and its use to control population may be feasible (Kuttan, 1994).

Turmeric in commerce

India is the largest producer and exporter of turmeric, providing 90% of the world
production, which is sold in powdered forms or as whole rhizomes (Shivashankar, 1976).
The mother rhizomes are known as ”bulbs”, and the primary and secondary branches are
known as ”fingers". In terms of export, west Asian countries prefer ‘bulbs’, while American

and European markets take ‘fingers’. In particular, the United States prefers Alleppey

5

turmeric from the state of Kerala, which is also one of the more expensive varieties of C.
longa because it has brighter color and better "non-fade" characteristics than other varieties
(Survey,1969 ;Govindarajan, 1980). The quality of color desired by the United States
market is understandable considering its applications in food coloring.

The United States is the largest user of spice oleoresins; approximately 29 different
oleoresins are demanded in the United States annually. Of these oleoresins, eight represent
90% of the volume of oleoresins used in the United States. Among these, turmeric oleoresin
is sixth in importance. Its main uses are in mustard paste, pickles and relishes (Govindarajan,
1980; Unterhalt, 1980). It also is used in the manufacture of mixes, soups, canned products,
and wrapped confectionary (Wilfred, 1980). The principal yellow pigment in turmeric is
curcumin; several national and international patents on the use of curcumin as a food coloring
are held by companies such as General Foods Corp. and McCormick & Co., Ltd. (Francis,
1986). The turmeric extract is a suitable replacement for FD&C Yellow #5. As such, it is
stable at low pH, and can be used to color dry beverage mixes, pudding mixes, confectionary
products, and foods where good clarity is needed (Andres, 1982). Its stability at low pH has,
however, caused some concern in quality control of the citnrs industry, where it was used to
adulterate orange juice (Petrus, 1984). Also, turmeric can be used in combination with
annatto extracts to impart yellow hues to cereal products. Food coloring powder (turmeric
extract, silica gel, and propylene glycol) and an oil-soluble liquid fi’om turmeric for food
coloring (turmeric extract and vegetable oil) are produced for this purpose (F reund, 1985).
Under the FDA regulation, certain color additives are certified or exempt from certification,

and turmeric and turmeric oleoresin are two additives that fall in this category (Hallagan,

6

1991). Many additives that are exempt fi'om certification fall under the category of natural
colors, as in the case of turmeric. Turmeric’s close match to FD&C yellow No. 5 and FDA
exemption has allowed it to be used in even more products such as canned beverages, dairy
products, sauces, cheeses, salad dressings, and margarine. In some color blends, it is

purposely utilized for its antioxidant properties (Lauro, 1991).

Microbial diseases and pests of turmeric

Interestingly, for all of its antimicrobial and insecticidal/insect repellant activities,
turmeric has plenty of pests and needs constant protection. (Anjaneyulu, 1968; Govindarajan,
1980; Rao, 1977; Vevai, 1971). The main insect pest of turmeric is the cigarette (or
“tobacco”) beetle, Lasr'odenna sem’come. Lasiodemra caused damage in the form of weight
loss in the turmeric rhizome. The infestation was identified by characteristic emergence of
holes visible on infested rhizomes. Dissection of these rhizomes revealed as few as three and
as many as thirty beetle larvae, each one in its own “cell”. Other pests include the common
shoot borer Dichocrocispunctrforalis, that causes damage to pseudostems and rhizomes, and
the leaf roller caterpillar (or “skipper”), Udaspesfolus. These two pests are controlled by
spraying 0.05% dimethoate or phosphamidon. Another pest, the scale insect ( or “stores”)
Aspidiotus hartii causes damage to the turmeric rhizome in the field and storage. It is
controlled by dipping rhizomes in 0.05% malation or dimethoate. (Regupathy, 1976;
Govindarajan, 1980). Other insect pests include the rhizome flies and fly maggots (Calobata
Spp., F onnosr'na flavipes), hairy caterpillars (Diacrisia obliqua), thrips (Panchaetothrr'ps

indica), lacewing bugs (Stephanitr’s opicus), scale insects (Aspidiotus curcumae), leaf beetles

7

(Lema praeusta and Lema signatipennis and Lema semi regularis), red pumpkin beetles
(Aulacophora intennedia), mealy bugs, and jassids (T ettigoniella ferruginea) (Vevai, 1971).
In addition, a dry rot of the rhizome was reported due to the association of Fusarium and an
unidentified nematode (Sarrna, 1974).

The most serious microbial disease is the leaf blotch T aphima maculans, which dries
up the leaves and affects the rhizome yield significantly. It has been controlled by application
of chemical pesticide. A leaf spot caused by Collectotricum capsici is a fungal disease that
also effects rhizome yield by drying up the affected leaves. It has been controlled by spraying
pesticide preventively or at onset of infection. Another disease (leaf soft rot) is caused by
Pythium grmninicolum, which passes from turmeric to its seed, and affects turmeric by rapid
and total loss of the crop, starting with leaf drying, softening of pseudostems, and rhizome
decay. It has been controlled by treating seed with pesticide prior to planting, or in the field
(Govindarajan, 1980). Other diseases include athracnose leaf spot (Colletotrichum
zingibefis), collar rot (Corticium rolfsii), leaf blotch (T aphrina deformans), rhizome rot, and
leaf rust (Puccinia curcumae) (Vevai, 1971).

To some degree, incidence of various pests and diseases of turmeric in India has been
contingent on the area in India where the turmeric is being produced. For instance, Andhra
Pradesh was plagued by shootborers, lacewing bugs, thrips, skippers, leaf spot, leaf blotch,
leaf soft rot and leaf rust; Assam by leaf blotch; and Bihar by the hairy caterpillar, leaf spot
and leaf blotch Other states including Delhi, Gujara, Haryana, Malabar, Tamil Nadu, Kerala,
Maharashtra, Salem, Sangli, Mysore, Orissa, Punjab, Uttar Pradesh and West Bengal have

also been plagued by a variety of pests and microbial diseases. These included leaf spot, leaf

8

blotch, leaf soft rot, leaf rust, skippers, shootborers, lacewing bugs, scales, thrips, rhizome
flies, leafbeetles, red pumpkin beetles, and hairy caterpillars.

Although they were not strictly considered pests, but rather are a nuisance to
processors and present potential health risks, certain microbes have contributed to a large
contamination in harvested turmeric rhizomes, and are worth mentioning. Turmeric has had
a large incidence of aerobic thermophiles and meSOphilic spores, in addition to a small
population of non-coagulase type Staphylococci and Closrridium perfiingens, although
Salmonella were absent (Krishnamurthy, 1971). Trade samples of turmeric bulbs contained
high amounts of coliforms, but the low incidence of coliforrns in general was not considered
a health risk (Krishnamurthy, 1973). Also, large amounts of yeast and mold were found in

turmeric powder (Mattada, 1974).

Bioactive Compounds of Turmeric

For hundreds of years, spices (especially turmeric) were used in Asia and around the
world to protect food from spoilage and mask the rancid taste of spoiled food. Antioxidant
and antimicrobial qualities of turmeric have been found responsible for this activity. About
0.5% of turmeric considerably reduced formation of peroxides in groundnut oil in accelerated
stability tests (Rimpler, 1970). Turmeric showed significant antioxidant activity when added
to olive, soybean, sesame and linseed oils (Rimpler, 1970) . Turmeric is used commonly with
many fish preparations. A dip treatment of turmeric on headless white shrimp did not develop
black melanoses and odor as observed in the control. Shrimps treated with the turmeric dip

were void of melanoses and gave an appealing and spicy aroma (Govindarajan, 1980;

8

blotch, leaf sofi rot, leaf rust, skippers, shootborers, lacewing bugs, scales, thrips, rhizome
flies, leafbeetles, red pumpkin beetles, and hairy caterpillars.

Although they were not strictly considered pests, but rather are a nuisance to
processors and present potential health risks, certain microbes have contributed to a large
contamination in harvested turmeric rhizomes, and are worth mentioning. Turmeric has had
a large incidence of aerobic thermophiles and mesophilic spores, in addition to a small
population of non-coagulase type Staphylococci and Clostridium perfringens, although
Salmonella were absent (Krishnamurthy, 1971). Trade samples of turmeric bulbs contained
high amounts of coliforms, but the low incidence of coliforrns in general was not considered
a health risk (Krishnamurthy, 1973). Also, large amounts of yeast and mold were found in

turmeric powder (Mattada, 1974).

Bioactive Compounds of Turmeric

For hundreds of years, spices (especially turmeric) were used in Asia and around the
world to protect food from spoilage and mask the rancid taste of spoiled food. Antioxidant
and antimicrobial qualities of turmeric have been found responsible for this activity. About
0.5% of turmeric considerably reduced formation of peroxides in groundnut oil in accelerated
stability tests (Rimpler, 1970). Turmeric showed significant antioxidant activity when added
to olive, soybean, sesame and linseed oils (Rimpler, 1970) . Turmeric is used commonly with
many fish preparations. A dip treatment of turmeric on headless white shrimp did not develop
black melanoses and odor as observed in the control. Shrimps treated with the turmeric dip

were void of melanoses and gave an appealing and spicy aroma (Govindarajan, 1980;

Hirahara, 1974).

Turmeric was a better antioxidant than BHA (butylated hydroxyanisole) (Kuttan,
1994). At least one component of turmeric that contributed to the antioxidant action was the
turmeric anti-oxidant protein (TAP), isolated from the aqueous extract of turmeric (Selvam,
1995). The TAP was heat stable. It showed a concentration-dependent inhibitory effect on
the promoter-induced lipid peroxidation. Inhibition at 50% was observed at a TAP
concentration of 50 pg/ml. Up to 50% of the initial activity of Ca2+ -ATPase fiom rat brain
was protected by the lipid-perorddant-induced inactivation by the TAP. This protective effect
was shown to be associated with binding of turmeric and -SH moieties (Selvam, 1995). In
addition, it was demonstrated that turmeric possibly lowered lipid peroxidation by promoting
high levels of activity of antioxidant enzymes superoxide dismutase, catalase and glutathione
- peroxidase in male rats (Reddy, 1994). Curcumin is an efl‘ective scavenger of reactive oxygen
species, and decreased formation of inflammatory agents such as prostaglandins and
leukotrienes (Huang, 1991; Reddy, 1994). The presence of curcumin in turmeric makes
turmeric an ideal dietary antioxidant (Reddy, 1994).

Turmeric has potential in cancer prevention. Feeding turmeric to mice prevented
tumor formation normally caused by benzopyrene, 3 - methyl cholanthrene, and 3'-methyl-4-
dimethylaminobenzene (Polasa, 1991; Reddy, 1994). Turmeric inhibited mutagenicity of
cigarette smoke condensates and tobacco extracts (Nagabhushan, 1987). Curcumins, the
yellow components of turmeric, inhibited cancer produced by a number of chemicals on skin,
stomach, and guinea pig pouch (Kuttan, 1994). Turmeric showed anticancer activity in vitro

using Dalton’s lymphoma cells, and against lymphocytes at a concentration of 4 rig-ml".

Hirahara, 1974).

Turmeric was a better antioxidant than BHA (butylated hydroxyanisole) (Kuttan,
1994). At least one component of turmeric that contributed to the antioxidant action was the
turmeric anti-oxidant protein (TAP), isolated from the aqueous extract of turmeric (Selvam,
1995). The TAP was heat stable. It showed a concentration-dependent inhibitory effect on
the promoter-induced lipid peroxidation. Inhibition at 50% was observed at a TAP
concentration of 50 ug/ml. Up to 50% of the initial activity of Ca2+ -ATPase from rat brain
was protected by the lipid-peroxidant-induced inactivation by the TAP. This protective effect
was shown to be associated with binding of turmeric and -SH moieties (Selvam, 1995). In
addition, it was demonstrated that turmeric possibly lowered lipid peroxidation by promoting
high levels of activity of antioxidant enzymes superoxide dismutase, catalase and glutathione
' peroxidase in male rats (Reddy, 1994). Curcumin is an efl‘ective scavenger of reactive oxygen
species, and decreased formation of inflammatory agents such as prostaglandins and
leukotrienes (Huang, 1991; Reddy, 1994). The presence of curcumin in turmeric makes
turmeric an ideal dietary antioxidant (Reddy, 1994).

Turmeric has potential in cancer prevention. Feeding turmeric to mice prevented
tumor formation normally caused by benzopyrene, 3 - methyl cholanthrene, and 3'-methyl-4-
dimethylaminobenzene (Polasa, 1991; Reddy, 1994). Turmeric inhibited mutagenicity of
cigarette smoke condensates and tobacco extracts (Nagabhushan, 1987). Curcumins, the
yellow components of turmeric, inhibited cancer produced by a number of chemicals on skin,
stomach, and guinea pig pouch (Kuttan, 1994). Turmeric showed anticancer activity in vitro

using Dalton’s lymphoma cells, and against lymphocytes at a concentration of 4 jig-ml“.

10

Also, turmeric extract inhibited cell growth in Chinese hamster ovary cells at 4 ppm. A
cytotoxic efl‘ect of turmeric was found within 30 minutes at 30° C (Kuttan, 1985). Ethanolic
extracts of turmeric relieved symptoms in patients with external cancerous lesions (Kuttan,
1987). The yellow coloring material of turmeric, the curcumin, was implicated for the
therapeutic potentials of turmeric (Kuttan, 1994). Curcumin had radical-scavenging
antioxidant activity against lipid peroxidation in various media, suppressed free-radical-
induced oxidation of methyl linoleate in solutions and aqueous emulsions, and was a
comparable as an antioxidant to isoeugenol (N oguchi, 1994). Also, turmeric was reported to
have higher antioxidative properties than BHT (butylated hydroxytoluene) as indicated by
the antioxidant index (AI) (Lee, 1982; Noguchi, 1994). Turmeric’s components also inhibited
lipid peroxidation induced by ascorbic acid and ferrous sulphate in erythrocyte membrane
(Salirnanth, 1986).

The anti-inflammatory activity of turmeric and curcumin was as good as aspirin or
ibuprofen, and was successfully used to treat arthritic patients (Kuttan, 1994). Two major
principles of anti-inflammatory drug activity are available; those inhibiting synthesis of
prostaglandins by interfering with the cyclooxygenase system (such as salicylic acid), and
glucocorticosteroids which inhibit the cyclooxygenase pathway and the lipoxygenase
pathways. Curcumin inhibited 5-lipoxygenase activity in rat peritoneal neutrophils and 12-
lipoxygenase and cyclooxygenase activities in human platelets (Ammon, 1993).

Curcumin, the anti-inflammatory agent in turmeric, has the caffeic acid moiety and is
largely responsible for the inhibition of lipid peroxidation. Two caffeic acid molecules joined

through a methylene bridge results in a bis-desmethoxy-curcumin. It has more anti-oxidant

l 1
activity than curcumin, ferulic acid, cafl‘eic acid, p-hydroxy cinnamic acid, O-hydroxy cinnamic
acid, cinnamic acid, or 3,4,5-trimethoxy cinnamic acid. Methylation of hydroxy groups
reduced its antioxidant character (Shanna, 1976). Sodium curcuminate, tetrahydro curcumin,
curcumin, phenyl butazone, and triethyl curcumin in decreasing order were found effective
in carrageenin-induced rat paw edema and cotton pellet granuloma tests. Ferulic acid and
diacetyl curcumin also were tested, but found devoid of anti-inflammatory activity.
Interestingly, another plant from the Zingiberaceae family, Curcuma xanthorrhiza, yielded
three non-phenolic diarylheptanoids, alnustone, trans -1,7-diphenyl-l-hepten-S-ol, and trans,
trans-1,7-diphenyl-1,3-heptadien-5-ol, with anti-inflammatory activity (Figure I). These
compounds were related to the curcuminoids, but only contained one carbonyl group, and
displayed significant anti-inflammatory effects in the assay of carrageenin-induced hind paw
edema in rats (Claeson, 1993). The activity of these compounds indicated that phenol groups
are not necessary for the anti-inflammatory activity. Curcumin inhibited TPA and arachidonic
acid induced epidermal inflammation in mice more than chlorogenic, caffeic, and ferulic acids.
Specifically, 3, 10, 30 or 100 uM of curcumin, in vitro, inhibited metabolism of arachidonic
acid to S-hydroxyeicosaterabaenoic acid (S-HETE) by 40, 60, 66, or 83%, respectively, and
to 2-HETE by 40, 51, 77 or 85% (IC ,0 = 5-10 uM) (Huang, 1991). However, sodium
curcuminate was not anti-pyretic or analgesic, and did not inhibit the arachidonic-acid-
dependent pathway of platelet aggregation. Therefore, it is not likely that the anti-
inflammatory activity of curcumin derivatives is mediated by inhibition of prostaglandin

synthetase enzyme. In any case, the anti-inflammatory activity of turmeric

12

Figure 1

O// O

O

alnustone
O / O
OH
trans-1 ,7-diphenyl-1-hepten-5-ol

O// O

OH

trans.trans-1 ,7-diphenyI-1,3-heptadien-5-ol

13

explains its effective use against pain and inflammation in Indian herbal medicine
(Mukhopadhyay, 1982).

Curcumin inhibited the response of blood neutrophils to superoxide anion
(Srivastava, 1989; Satoskar, 1986). Its scavenging effects on active oxygen radicals was
reported to be stronger than vitamin E, (Zhao, 1989) and it protected DNA from
peroxidative injury (Shalini, 1987). Turmeric has become increasingly important in diet for
preventing cancer and genotoxicity through its action as a potent antioxidant.
Antimodulatory efl‘ects of turmeric and curcumin on different levels of benzopyrene-induced
DNA (BP-DNA) adducts in rat liver have been studied by 32P-postlabelling experiments.
Turmeric at 0.1, 0.3 and 3% and curcumin at 0.03% levels in the diet significantly reduced
the levels of BP-DNA adducts (Mukundan, 1993). Turmeric may prove effective in treating
cancer, since it is very non-toxic to humans. About 50 g of turmeric per day was not toxic
to humans; it is nonmutagenic, non-carcinogenic and non-teratogenic. It was effective in
reducing animal tumors, and both turmeric and curcumin inhibited the fibrosis induced by
ethanol and carbon tetrachloride in animals. Turmeric showed excellent preventative activity
against carbon tetrachloride-induced liver injury in vivo and in vitro. It has been proposed
that turmeric may be usefirl for humans by preventing liver disease, including cancer, in people
who drink large amounts of alcohol (Kuttan, 1994; Kuttan, 1985; Kiso, 1983). Turmeric
showed no signs of toxicity (or spermatotoxic effects) in mice at doses of 3 g-kg", although
CNS stimulation was observed (Qureshi, 1992).

The properties of turmeric go beyond antioxidant and anti-inflammatory activities.

Curcumin inhibited platelet aggregation induced by arachidonate, adrenaline and collagen

l4

(Srivastava, 1994). Also, topical applications of curcumin inhibited TPA-induced tumors on
mouse skin better than chlorogenic acid, cafl‘eic acid or ferulic acid, at 10 umol
concentrations (Huang, 1988). Curcumin inhibited chemical carcinogenesis, (Kuttan, 1989)
and its components were noted for antimicrobial activities. An alcohol extract of turmeric
inhibited the growth of Sarcina, Gaflkya, Corynebacterium, Clostridium strains at 05-5
mgoml‘l concentration. Curcumin and essential oil of the rhizome had activities at 5-100 ppm
on these strains (Lutomski, 1974). In vitro study of antibacterial activity of turmeric
indicated that the sodium salt of curcumin was “antimicrobial at 1 ppm.” The essential oil
fi'action exhibited similar types of activity at high concentrations, with the whole oil being
more active than the purified compounds (Ramprasad, 1956; Chopra, 1961; Munasiri, 1987).

Curcumin, when illuminated (with unspecified wavelength light), exerts potent
phototoxic efi‘ects in micromolar amounts against gram-positive bacteria (Dahl, 1989).
Specifically, curcumin was phototoxic to Salmonella typhimurium and Escherichia coli
(T onnesen, 1987). Although turmeric showed inhibitory effects on the growth of intestinal
and pathogenic bacteria in vitro (Chopra 1956; Shankar, 1979), turmeric extracts were less
active against gram-positive and gram-negative bacteria than penicillin and streptomycin
(Basu, 1971). The antibacterial quality has been attributed to curcumin (Chopra, 1956;
Shankar, 1979). Turmeric showed antifirngal activity against Aspergillus parasiticus, which
produces a very potent mycotoxin, aflatoxin. This toxin is a major contaminant of food, and
produces severe liver diseases including cancer, and eventually may result in death. Poultry
are very susceptible to this toxin; an intake of 100 mg turmeric/day/bird is recommended as

a preventative measure against the fungus (Kuttan, 1994).

15

Turmeric possesses insecticidal activity. It was active as a repellant against the grain
borer (Jilani, 1990), the red flour beetle (J ilani, 1988), the housefly Musca domestica (Singh
1991), ants (Vrswanath, 1981), and T ripilium castaneum (Su, 1982). Also, growing
turmeric under coconut trees reportedly protected them fi'om white ants (Kuttan, 1994).
Alcoholic extracts of turmeric showed anti-protozoa] activity against Entamoeba histolytica
(Dhar, 1968; Gopalan, 1976). Turmeric showed activity against roundworrns, threadworrns,
and other intestinal parasites (Abbiw, 1990). It is antihelmenthic not only in the body, but
also when applied topically (Kuttan, 1994). Turmeric proved an effective cure for scabies,
and when used as a topical paste treatment on 814 people, cured the patients in 97% of the
cases within 3 to 15 days of treatment (Charies, 1992). Combinations of demethoxycurcumin
(curcumin II) and bisdemethoxycurcumin (curcumin III) were nematicidal against T oxocara
canis, but the curcuminoids were ineffective when applied independently (Kiuchi, 1993).
Interestingly, five di-phenylheptanoids (1-5, Figure 2) isolated from a related plant, Curcuma
comosa, demonstrated nematicidal activity against Caenorhabditis elegans. These
compounds are similar to curcumins, except for absence of methoxy and hydroxy substituents
in their aromatic rings (Jurgens, 1994).

The volatile oil from turmeric rhizomes possesses other bioactivities as well. The
main component, ar-turmerone, exhibited antivenom effects. Brazilian people used cut
rhizome slices against insect bite and for allergic reactions fi'om contact with caterpillars.
Aqueous extract of the rhizomes was considered to eliminate neuromuscular inhibition from
the neurotoxin of Naja naja siamensis (cobra) bite (Cherdchu, 197 8). Fractions consisting

of ar-turmerone removed hemorrhagic activity from Bothropsjararaca venom. This was

16

Figure 2

EQHHQ
KKKKK

17

shown by mixing 10 pg venom (equivalent to 10X the minimum hemorrhagic dose) with 100
pl of saline and ar-turmerone at pH=7, and injecting the solution intraderrnally into mice.
Also, in vitro studies indicated that hexane extracts from turmeric inhibited proliferation of
natural killer activity (NK) of human lymphocytes in a dose-dependent manner. An
inflammatory reaction is the result of cell population interactions, including lymphocytes
(Ferreira, 1992), so this indicated that anti-inflammatory properties of turmeric may have to
do with inhibition of lymphocyte activation. Ar—turmerone was as potent as the original
crude extract.

Turmeric reduced cholesterol in blood by reducing cholesterol uptake from the gut.
It inhibited serum LDL peroxidation, which can lead to atherosclerotic lesions, and hence
turmeric may prevent coronary and heart problems (Kuttan, 1994). Specifically, curcumin
increased HDL and decreased LDL. Turmeric is also an antidiabetic; the action may be due
to stimulation of pancreas cells, and increased insulin production (Kuttan, 1994). In addition,
Curcumin was found to contribute to lowering of blood sugar in diabetics (Rarnprasad, 1956;
Nagabhushan, 1987).

Curcumin was a modest inhibitor of HIV-1 and HIV-2 proteases (Sui, 1993). These
proteases are encoded by HIV viral genomes and are responsible for processing the
precursors produced fi'om gag and pol genes into proteins needed for replication and
production of mature viruses (F arrnerie, 1987). Inactivation of the HIV-1 protease yields
non-infectious virions (Kohl, 1988). The curcuminoids were assayed for anti-HIV protease
activities (Sui, 1993). The curcuminoids 1-4 and the boron-curcuminoid complexes 5—9

(Figure 3) exhibited activity against the HIV proteases (Sui, 1993): Curcumin-boron

18

Figure 3
oz“ 0 o—N
CH’OMOCH" CHGOMOCHS
I
I’hCO2 O’CPh H0 OH
I 2
0’" O a" O
CHaO MOCHS CHaO M 0 CH3
HO OH HO OH
3 4
o o
17‘ ,r o o
a \ I
0’ \ ’3‘
l ° '0
HO 5 OH HO OH
6
o o o
CVmWWQ

19

Figure 4

 

0H0
o o

12

PCHO

21
complexes exhibited higher activity than curcuminoids against the HIV proteases (Sui, 1993).
To insure that activity was not due to boron alone, and to investigate the importance of the
boron-curcuminoid complex in the inhibitory activity, compounds 11-12 (Figure 4) were
assayed against the HIV-1 and HIV-2 proteases (Sui, 1993). No activity for these
compounds was observed, indicating that the boron entity itself was not responsible for the
activity (Sui, 1993). However, the boron complex of curcuminoid (10) did not show activity
against HIV proteases (Sui, 1993). Compound 13 (Figure 5) was synthesized and assayed
against the HIV proteases. It displayed no activity against the proteases. Therefore, it
indicated the importance of the boron-curcuminoid complex in the inhibition of the proteases

(Sui, 1993).

Chemical composition of turmeric

Govindarajan (1980) reported that .the composition of the turmeric rhizome varies
with variety, growth conditions, maturity of rhizome and time of harvest, although there are
at least a few components that were consistently present. Turmeric contained 2.5-6%
curcuminoids and 3-5% essential oil, which is composed of 58% turmerones (Alexander,
1973) , 25% zingiberene, 1% phellandrene, 1% cineole, 0.6% sabinene, and 0.5% bomeol
(Shivashankar, 1976) (Figure 6). Different sources gave conflicting reports on these
ingredients, but a few of them are well documented. It is reported that next to ginger oil,
turmeric oil was the best source of zingiberene (Survey, 1969). It was proposed that ar-
turmerone is an artifact formed during the steam distillation process of the volatile oil, but

work by Govindarajan showed this to be false; he reported that ar-turmerone and turmerone

22
Figure 4

pit. 0‘

ar-turmerone turmerone

Q, OI
zingiberene phellandrene sabinene

0 $0,,

cineole bomeol

23

existed in the rhizome in the proportion of 2.5:1. Ar-turmerone was reported to have
antivenom activity (Ferreira,l992). However, the majority of bioactivity claims of turmeric’s
use in commerce are due to the presence of curcuminoids. The rhizomes of turmeric yield
three major phenolic pigments, curcumin I, curcumin II, and curcumin III (Figure 7).
Comparative studies reported that all three curcuminoids inhibited superoxide production and
tumor growth; curcumin III was the most active. Also it exhibited higher activity in
cytotoxicity assays (Anto, 1994). At least two other curcuminoids were identified from
turmeric, (Nakayama, 1992) curcumin IV and curcumin V (Figure 7). Another related
compound, dihydrocurcurnin, an unsymmetrical diary] heptanoid, was isolated from C. longa
(Ravindranath, 1980) (Figure 7). Only curcunrin I, II and 111 have reported activities.
Mayer reports that the structure of curcumin was elucidated in the early 1900s; in
1870 Daube isolated crystalline curcumin, and Perkin and Philps obtained curcumin by
precipitating lead curcuminate fi'om the alcohol extract of turmeric (Mayer, 1943). In 1897,
Ciarnician and Silber proposed that curcumin was a diferuloyl methane, and Mayer (1943)
vouches that this idea was confirmed by von Kostanecki and Lampe, who identified the
structure by studying degradative products of curcumin. Boiling curcumin with alkali caused
it to degrade to vanillic and ferulic acids. Curcumin reacted with alkali to give protocatechuic
acid, with permanganate to give vanillin, and with acetic anhydride to give acetyl-curcumin.
The structure of curcumin was elucidated to be diferuloyl methane in the enolic form (Mayer,
1943). Lampe (1919) synthesized curcumin by condensing carbomethoxy feruloyl chloride
with ethyl acetoacetate to give an ester. This ester was hydrolyzed to the diketone followed

by the condensation with another carbomethoxy feruloyl chloride to give the diferuloyl

24

Figure 7

 

dehydrocurcumin

25

compound (Mayer, 1943). This product was then hydrolyzed to curcumin. Pabon
(l964)improved a synthesis of curcumin from acetyl acetone and vanillin reported by Pavolini,
and obtained an 80% yield of curcumin.

Srinivasan (1952,1953) found that a characteristic reaction of curcumin with boric
acid resulted in a red color. Fluorescent orange color occurred with impure curcumin. These
impurities were later investigated. Using column chromatography with silica gel and benzene
mobile phase, he separated curcuminoids into three components. The first component eluted
was curcumin I (reddish-orange prisms), followed by amorphous orange material (curcumin
II) and a yellow plate-like compound (curcumin III). Characteristic reactions of the latter two
compounds with acid and alkali showed that all three compounds were related. The first
compound, curcumin I, had two methoxy groups. The second compound (curcumin II) had
one methoxy group. The third compound (curcumin 111) had no methoxy groups. These three
curcumins were obtained fi'om natural sources, the rhizome extracts, in good yield by
preparative thin layer chromatography (Roughly, 1973). NMR spectra of the curcuminoids
at low temperature indicated that in chloroform they existed in the enolic form (Roughly,
197 3). The relative amounts of the three curcuminoids from turmeric taken from various
harvests were determined by thin-layer chromatography and found in the following ratios;
60:30:10, 47:24:29, 49:29:22, and 42:24:34 for curcumin I, curcumin II and curcumin 111,
respectively (Perotti, 1975; Jentzsch, 1959; Krishnamurthy, 1976). It is not yet known if
these differences are due to cultivars.

Kelkar and Rao (1934) published analysis on steam-distilled turmeric oil which

showed that the oil was a mixture of sesquiterpene ketones and alcohols (Rao, 1934). The

26

major fraction of the oil, distilled at 158 to 165° C at 11 mm of Hg, was shown to be a
mixture of ketones. Rupe (1936) showed that turmeric aroma is largely due to the
sesquiterpene ketone ar-turmerone, which made up 40% of the volatile oil of turmeric.
Boiling point, optical rotation, derivative analysis and studies involving degradatiOn to known
compounds facilitated the characterization of ar-turmerone. Ar-turmerone was purified early
by Rupe and Gassrnan (Rupe, 1936), and more recently by Alexander and Rao (Anjaneyulu,
1968). A method for purifying ar-turmerone from turmeric oil obtained from turmeric
rhizome by hexane extraction (Khalique, 1968) involved treating the sesquiterpene ketone
portion of the oil (from fractional distillation) with chromium (IV) oxide in acetic acid at low
temperature and conversion to crystalline 2,4-dinitrophenyl hydrozone. Isolation of the
crystals, followed by exchange with m-nitrobenzaldehyde gave ar-turrnerone. Ar-turmerone
was one of the first natural sesquiterpene ketones to be characterized and assigned a structure
(Rupe, 1936; Govindarajan, 1980). Further purification by thin-layer chromatography gave
pure ar-turmerone with an optical rotation of +84 ° (Alexander, 1973). Ar-turmerone also
was synthesized by condensation of its degradation products, acetone and curcumone (Rupe,
193 6). As mentioned, other sesquiterpene ketones and alcohols and low-boiling terpenes
have been identified in the volatile oil of turmeric. With the exception of turmerone, however,
it was not possible to purify these components (Rupe, 1934; Rupe, 1936; Govindarajan,
- 1980). The structure of tumerone has not been identified conclusively, since the position of
the double bonds in the ring still are not known (Govindarajan, 1980). A synthesis has not

been reported for turmerone.

27

A significant amount of commercially important compounds have been realized from
turmeric rhizomes, some with excellent bioactivities. However, the studies have been
concentrated on antioxidative and anti-inflammatory activities. It is obvious that not all the
medicinal claims attributed to turmeric have been investigated, especially considering its use
in Indian folk medicine. New compounds continue to be discovered in turmeric, but few have
been studied for bioactivity. The Bioactive Natural Products Laboratory in the Department
of Horticulture at Michigan State University is involved actively in research on plants with
folklore medicinal value, by assaying plant natural products against some important human
and agricultural pests. Based on the research hypothesis that the tropical plant Curcuma
longa L. produces some bioactive secondary metabolites which are either novel or possess
novel bioactivities, we have studied turmeric to identify additional compounds with as yet

undescribed biological activities.

CHAPTER H

Bioactive Compounds From Turmeric Rhizomes

Abstract

Curcumin I (1), curcumin II (2) (demethoxy curcumin) and curcumin III (3)
(bisdemethoxy curcumin), the three main diaryl heptanoids fiom Curcuma longa, have
demonstrated anti-inflammatory and antioxidant activities. Curcumin III was the most active,
followed by curcumin II, and curcumin I. We have isolated these curcuminoids fi'om turmeric
rhizomes, and evaluated their biological activities. Curcunrin I, curcumin II and curcumin 111
showed topoisomerase I and II inhibition at 50, 50 and 25 ppm concentrations, respectively.

The volatile oil fi'om turmeric rhizomes yielded a sesquiterpene ketone, ar-turmerone.
We have isolated ar-turmerone fi'om the volatile oil, and evaluated it for novel bioactivity.

It displayed mosquitocidal activity on Aedes aegtptii with LDloo at 50 ppm concentration.

28

29

Introduction

In Indian folk medicine, turmeric has been used to combat a variety of ailments, such
as small pox (Kuttan, 1985), rheumatism, scabies, eye inflammation, body pain, sores, and
cough (Jain et al., 1991), to name just a few. Many of these reputed claims have been
substantiated. Turmeric has been used as a fever alleviator (Lindley, 1838), and it was
verified that it has anti-inflammatory activity similar to aspirin or ibuprofen (Kuttan, 1994).
Turmeric also has been used for centuries , especially in Asia, to prevent food spoilage, and
studies on the antioxidant and antimicrobial properties of turmeric verified this activity.
Curcumin, a main coloring agent present in turmeric, proved to be a better antioxidant than
BHA (butylated hydroxyanisole) or BHT (butylated hydroxytoluene) as rated on the
antioxidant index (Lee, 1982). Also, curcumin scavenges active” oxygen radicals better than
vitamin E (Zhao, 1989).

In Thai folk medicine, Curcuma spp. rhizomes were used as a cobra poison antidote
(Cherdchu, 1983). The local people claimed the plant to be effective orally for poisonous
snake bite. This activity was substantiated in rats, mice and dogs. (Tejasen, et al., 1969 a,
b; Tejasen, et al., 1970; Tejasen et al., 1978; Cherdchu, 1978). Ar-turmerone fi'om turmeric
abolished both the hemorrhagic activity of Bothrops jararaca venom and the lethal effect of
Crotalus durissus terrificus venom in mice (Ferreira, 1992).

Although the anti-inflammatory, antioxidative, and antivenom properties of turmeric

30

components have been substantiated, many medicinal and therapeutic claims attributed to
turmeric remain noninvestigated. In this chapter we report novel activity for some of the
turmeric metabolites isolated and characterized from the rhizomes. Three main curcuminoids
from turmeric, curcumin I, curcumin II and curcumin III, exhibited topoisomerase I and 11
inhibition, curcumin III exhibiting the best activity. In addition, the sesquiterpene ketone ar-

turmerone was found to display mosquitocidal activity.

Experimental

Plant Materials - Turmeric plants were grown in large plastic pots, using a mixture
of 50% loamy field and 50% bacto soils. Rhizomes were obtained fiom a grocer in Detroit,
Michigan Several rhizomes were planted 3 inches below the surface of the soil in each pot.
The pots were kept in the BNPL greenhouse at Michigan State University. The plants were
watered every two to three days, and fertilized with 20-20-20 Peters brand fertilizer. The
turmeric plants were harvested for the rhizomes every nine months. Rhizomes were
collected, washed with water, lyophilized, milled and kept in the -20 ° C freezer in ziploc bags.
Some of the fresh rhizomes were replanted for additional production.

Chemicalsand cell culture media.- YMG medium (yeast extract 4 gsL", maltose 10
g-L", glucose 4 g-L" and agar 12 g-L’l ), PDA (potato dextrose agar; potato infusion 200
g-L",.dextrose 20 goL", agar 15 g-L"), Emmons medium (neopeptone 10 g-L", glucose 20
g-L‘l and agar 15 g-L"), NG medium (NaCl 3.0 g-L", bacto peptone 2.5 g-L", cholesterol
1 ml-L‘l of 5 mg-ml'l stock solution, CaCl2 l mloL’l of 1 M stock solution, MgSO,, 1 nil-L‘l

of 1 M stock solution and potassium phosphate buffer 25 mch" of stock solution of KHZPO,

31

11.97 g-100ml‘I and K,I-IPO, 2.0 golOOml' ) and YPDA medium (yeast extract 20 girl. ,
peptone 10 g-L", dextrose 20 g-L’l and adenine sulphate 2ml-I;l of 0.5% stock solution)
were prepared were prepared as published (Nair et al., 1989) in our lab. The ingredients were
purchased from Difco Lab (Detroit, Michigan, USA). The anticancer standards,
carnptothecin and etoposide were purchased from Aldrich Chemical Company (Milwaukee,
Wisconsin, USA). Sterile saline was prepared by dissolving NaCl (8.5 g-L“) in an

appropriate volume of deionized water.

Isolation and identification of Curcumin I (1), curcumin II (2), and curcumin III (3)-
The extraction procedure for Curcuma Ionga rhizomes is summarized in Figure 2.1.
Lyophilized and pulverized turmeric rhizomes (100g) were extracted successively with
hexane, ethyl acetate, and methanol in an extraction column. Each solvent was used in 500
ml aliquots (3) over a 24-h period. Each extract was evaporated to dryness, yielding 1.11 g
of hexane extract, 4.75 g of ethyl acetate extract, and 5.50g of methanol extract. The ethyl

acetate extract exhibited activity on the topoisomerase inhibition assays. A TLC analysis of

32

32

Figure 2.1 Procedure for extraction of C. Ionga rhizome

 

 

(100 g)

Lyophilized turmeric rhizome

 

 

 

Extracted with hexane
lSOOmLX3l. 24h

 

l

GR-19-55
Yellow Oil (1.119)

 

 

 

 

 

 

Residue

 

 

 

Extracted with EtOAc
(500 mLX3). 24h

 

 

l

 

 

 

 

GR-19-598
Orange Solid (4.759)

 

 

 

Residue

 

 

Extracted with MeOH
(500 mLX3). 24h

 

 

l

 

 

l

 

GR-19-78
Green Gum (5.59)

 

 

I

Residue discarded

 

 

 

33

the EtOAc extract showed three yellow and UV flourescing bands as major components in
the extract. The EtOAc extract (1.6g), was purified by VLC using 71.6g silica gel. The
fi’actions collected were: hexane (100 ml) [fraction 1] and 4:1 hexane2CHCl3 (30an)
[Mon 2] and 1:1 hexane2CHCl3 (50 mL) [fraction 3]; 1:1 hexane2CHCl3 (50 ml) [fraction
4] and 1:1 hexanezCHCl3 (50 ml) [fiaction 5] and CHCl3 (50 mL) [fraction 6]; CHCl3 (50 ml)
[fi'action 7] and CHCl3 (50 ml) [fraction 8] and CHCl3 (50 ml) [fraction 9]; CHC] (50 ml)
[fraction 10] and CHCl3 (50 ml) [fiaction 11]; CHCl3 (50 ml) [fraction 12] and CHCl3 (50 ml)
[Mon 13] and CHCl3 (50 ml) [fiaction 14] and CHCl3 (50 ml) [fraction 15] and CHCl3 (35
ml) [fraction 16] and CHCl3 (50 ml) [fraction 17] and CHCl3 (100 mL) [fiaction 18]; CHCl3
(100 ml) [fraction 19] and 4:1 CHCl,:MeOH (50 mL) [fraction 20]; 4:1 CHCl32MeOH (50
ml) [fraction 21] and 1:1 CHCl,:MeOH (50 mL) [fraction 22]; 1:1 CHC] :MeOH (50 ml)
[fraction 23] and 1:1 CHCl3zMeOH (50 ml) [fraction 24] and 1:1 CHGI :MeOH (50 ml)
[fiaction 25] and MeOH (50 ml) [fraction 26] and MeOH (50 ml) [fraction 27] and MeOH
(50 ml) [fiaction 28] and MeOH (150 ml) [fiaction 29]. Based on TLC results, fiactions 1-3,
4-6, 7-9, 10-11, 12-18, 19-20, 21-22, and 23-29 were combined to form fractions 1-8. Based
on TLC, fi'action 7 and 8 were combined to form GR-19-126 (.914 g). Purification of GR-
19-126 (266.5 mg) by preparative TLC using a 30:1 CHCl,:MeOH mobile phase yielded
fractions A—E. Fractions B, C and D, the three curcuminoids, were collected separately.
Repeated purification of fractions B, C and D by TLC using a 9:1 CHC13/Acetic acid mobile
phase afforded pure compounds, curcumin I, curcumin II, and curcumin III, with yields of
79.0, 42.2, and 29.0 mg- these yields were used to calculate the % dry weights of these

compounds as 0.8, 0.4, and 0.3%, respectively. The presence of curcumin I (l), curcumin II

34

(2), and curcumin III (3) was confirmed by ‘HNMR studies (Appendices I, II, and III for
1H NMR spectra of the curcuminoids). Preparative silica gel TLC plates were purchased

from Analtech Inc. (Newark, Delaware, USA).

Spectra - NMR spectra were recorded at the Max T. Rogers NMR facility at
Michigan State University on Varian VXR 300 and 500 MHz spectrometers (Varian,
California, USA) at ambient temperature. Mass spectra were acquired at the Michigan State
University Mass Spectrometry Facility on a JEOL I-D(-110 double focusing mass
spectrometer (JEOL, Tokyo, Japan).

Compound (1).: C,,H,,o,; ’HNMR (c130,): 6 3.93 (6H, s, 2 0Me), 5.77 (1H, s, 1-
H), 6.46 (2H, d, J=16 Hz 3, 3'-H), 6.91 (2H, d, J=8.4 Hz, 9, 9'-H), 7.03 (2H, d, J= 2H2,
6—6'-H), 7.10 (2H, dd, J=2, 8 Hz, 10, 10' -H), 7.56 (2H, d, J=l6 Hz, 4, 4'-H). These
lHNMR assignments were identical to the published data for curcumin I, (Roughly, 1973).
No additional spectral data on compound 1 were obtained.

Compound (2).: Cal-11.0,; lHNMR (CDCl,): 6 3.95 (3H, s, 0Me), 5.89 (1H, s, 1-H),
6.48 (2H, d, J=16 Hz, 3, 3'-I-I), 6.86 (2H, d, J=8 Hz, 7'-9'-H), 6.93 (1H, d, J=8 Hz, 9-I-I),
7.05 (1H, d, J=2, 6-H), 7.12 (1H, dd, J=2, 8 Hz, 10-H), 7.47 (2H, d, J=8, 6'-10'-H), 7.59
(1H,. d, J=16Hz, 4-H), 7.61 (1H, d, J=l6 Hz, 4'-H). These lHNMR assignments were
identical to the published data for curcumin II (demethoxy diferuloyl methane) (Roughly,
1973).

Compound (3).: C,,II,,O,; lI-INMR ((CD,)2CO): 6 5.97 (II-I, s, 1-H), 6.68 (2H, d,

J=16 Hz, 3,3211), 6.89 (4H, d, J=8 Hz, 7, 7', 9, 9'-H), 7.56 (4H, d, J=8 Hz, 6, 6', 1o, 10'-H),

35

7.62 (2H, d, J=16 Hz, 4, 4'-H). The lI-INMR assignments were identical to the published
data for curcumin III (bis demethoxy diferuloyl methane) (Roughly, 1973). ‘

Ar-Turmerone (4): The isolation procedure for the compound is summarized in
Figure 2.2. A colorless oil was obtained from the ice accumulated on the condenser coil in
the lyophilizer when rhizomes were freeze dried. Purification of the colorless oil from the
rhizomes (464.4 mg) by TLC, using a 50:1 hexane: acetone mobile phase yielded fiactions
A-E. Fraction C (3.8 mg) exhibited mosquitocidal activity and was identified as ar-turmerone

from 1H and ”CNMR and EIMS spectral studies (Appendices IV, V, and VI ).

Compound4.: c,,H,,o ‘HNMR (CDC1,): 6 1.22 (3H, d, J=6.9 Hz H-12), 1.84 (3H,
s, H-13), 2.09 (3H, s, H-14), 2.29 (3H, s, H—l), 2.59 (1H, dd, J=8, 16 Hz, H—8), 2.69 (1H,
dd, J=6.6 ,16 Hz, H-8), 3.27 (1H, m, H-7), 6.00 (1H, s, H-10), 7.10 (4H, br s, H-2, 3, 4, 5).
13C (CDCl,): 6 20.55 (013), 20.83 (01), 21.84 (012), 27.49 (014), 35.13 ((2.7), 52.54
(08), 123.94 (C-10), 126.51 (C-4,5), 128.94 (C-2,3), 135.39 (015), 143.53 (011), 154.92

(06), 199.71 (C-9).E1MS: m/z (rel. Int); 216 (63), 201 (30), 119 (84), 91 (13), 83 (100).

Antimicrobial assay - Antifirngal and antibacterial assays of compounds 1,2,3 and 4
were conducted according to the reported procedure (Nair et al., 1989). Cultures of
Fusarium oxysporum (MSU-SM-1322), Fusarium moniliforme (MSU-SM-1323),
Gleasporum spp. and Rhizoctonia spp. were raised on potato dextrose agar (PDA) medium
in petri dishes. Cultures of Candida albicans and Aspergillusflavus (MSU strains) were

cultured on YMG medium in petri dishes. Cultures of Staphylococcus epidermidis (ATCC

36

Figure 2.2 For the Extraction of Volatile Oil

 

Fresh Turmeric Rhizomes (4.5 kg)

 

 

 

Lyophilized

] l

 

 

 

 

 

. . Condenser Coil
Drlfil 311$)” 1c Ice collected
. (3.2 kg)

 

 

 

 

 

Extracted with ether

 

 

 

 

 

 

 

 

 

 

600 mL X 3
Aqueous portion Ether extract
. Dried over
discarded M980,

Evaporated to dryness

 

 

colorless oil (559)

 

 

 

37

25923), Streptococcus aureus (MS strain), and Escherichia coli (ATCC 25922) cultures were

grown on Emmons medium in petri dishes.

Anticancer (topoisomerase inhibition) assay - Topoisomerase assays of compounds
1,2,3 and 4 were conducted according to the reported procedure of Chang, et al. (1995).
Mutant Saccharomyces cerevisae cell cultures IN 3 94, JN 3 94 H and IN 394 L, were
provided by Dr. John Nitiss at St. Jude Children’s Research Hospital (Nitiss et al., 1993).
JN3 94 is hypersensitive to topoisomerase I and II poisons because of deletions that
irradicated the RADS2 repair pathway. JN3 941.1 is isogeneic to IN 3 94 except that the top]
gene is deleted. The deletion of this gene causes a lack of response to topoisomerase I
poisons. IN 394,“, that contains the top2-5 gene, is resistant to topoisomerase II poisons,
but it responds to topoisomerase I poisons. Topoisomerase I and II are enzymes that alter the
DNA by catalyzing a three-step process. This involves cleavage of one strand of DNA by
topoisomerase I, or two strands of DNA by topoisomerase II, movement of a DNA segment
through this break, and then rescaling of the DNA strand (s) (Stryer, 1988). The mutant

yeasts were raised on YPDA medium (20 ml) in petri dishes.

The cell suspension for bacteria and mutant yeast and spore suspension for fungi were
prepared by suspending the bacteria or yeast or spores fiom a fully grown culture in a petri
dish in 10 ml of sterile saline solution, and transferring the suspension to a sterile culture
tube. The cell or spore suspension concentration was adjusted to 10‘ colony forming units

per milliliter (CFU/ml) to yield a stock solution. Serial dilutions of the stock culture were

38

made in sterile saline by innoculating the proper media with the approximate dilutions to
determine the required CFU/ml. Bioassays were conducted by lawning 100 pl of the desired
cell or spore suspension containing 10‘ CFU/ml on petri dishes of the corresponding medium.
The test compound was spotted carefirlly on the bioassay plates at varying concentrations,
along with 25 pl of DMSO alone as control. The plates were allowed to dry in a laminar flow

hood, and then incubated at 27° C for 72 h. The zone of inhibition was measured in mm.

Mosquitocidal and nematicidal assays - Aedes aegwtii mosquito larvae were used
to test for mosquitocidal activity of compounds 1,2,3 and 4, according to an established
procedure of Nair et al. (1989). A. aegyptii eggs were provided through the courtesy of Dr.
Alex Raikhel of the Department of Entomology at Mchigan State University. Approximately
200 mosquito eggs were placed in 500 ml of degassed, deionized water prepared by
sonication. About 5 mg of bovine liver powder was added to the water as a food source.
After four days, 4th instar mosquito larvae were transferred to 980 pl of the water in a test
tube. 20 pl of the test material at the desired concentration in DMSO was added to each
tube, which was shaken lightly to insure a homogeneous test solution. Each tube was covered
and left at room temperature. 20 pl of DMSO was used as the control. The larval mortality
was recorded at 2-, 4-, 6-, 24-, and 48-h intervals. The assay was conducted in triplicate.

Pcmagrellus redivivus and Caenorhabditis elegans were used to test for nematicidal
activity of compounds 1,2,3 and 4 according to the published procedure of Nair, et al. (1989).
A stock culture of nematodes in NG media in a scintillation vial was maintained at 14 day

integrals designed to provide uniform young nematodes. 10-20 nematodes in 48 pl of the

39

growth media were transferred to a 96-well tissue culture plate. 2 pl of 50% aqueous
DMSO solution of test material was added to this. 2 pl of 50% aqueous DMSO was used
as control. 'The experiment was completed in triplicate.

Gypsy moth, forest tent caterpillar, corn earworm and tobacco homworm assays-
Dry diet for each larval type was weighed out in a scintillation vial, one for each test
compound. Dry diet ingredients for the gypsy moth consisted of wheat germ (3 6g), casein
(7.5g), Wesson’s salt mix (2.4g), sorbic acid (0.6 g), methylparaben (0.3 g), and vitamin mix
Hofinan-LaRoche #26862 (3g). The dry diet mix used for each treatment weighed 0.845 g.
The corn earworm diet was obtained from North Carolina State, and the amount used for
each treatment was 0.94g. The tobacco homworrn diet also was obtained from North
Carolina State, and 0.95 g was used for each treatment. For each treatment, the test
compound was dissolved in DMSO, so that 20 pl of the resulting solution containing the test
compound added to the dry diet gave the desired concentration of the test compound. 20 pl
of DMSO served as the control. To this, agar solution was added to bring the total wet
weight to 5.0 g, and the mix was homogenized with a spatula. The mixture was poured
immediately (before jelling), equally into 15 micro beakers. The beakers were randomized
and allowed to cool'down for 3 h. One insect larvae of the type being tested was transferred
to each of the 15 micro beakers with a small paint bnrsh. The beakers were capped and
stored in a tray in an incubation chamber at the desired temperature for six days. At the end
of this period, each larvae was weighed. The average weight of each sample was compared
to the average weight of the control to determine bioactivity 0f the test compound. Data

were analyzed using Dunnet’s test; means of the treatments were compared with the control.

40

Results and Discussion

Rhizomes of C. longa were extracted sequentially with hexane, ethyl acetate and
methanol. Volatile oil was extracted from ice accumulated on condenser coils on the fi'eeze
dryer in which the rhizomes were lyophilized. Preliminary bioassays were performed on these
extracts to test for antimicrobial, anticancer, mosquitocidal, nematicidal, and gypsy moth,
forest tent caterpillar, tobacco homwonn and corn earworm growth inhibition.

The ethyl acetate extract inhibited the growth of Saccharomyces cerevisae mutants
JN394, IN 394 ,_, and JN394,“ at 250 ppm concentration (Table 2.1) The hexane extract,
an oily residue, demonstrated anticancer activity at 250 ppm. The volatile oil and hexane
extract demonstrated mosquitocidal activity at 250 ppm. Since a literature search showed that
the curcuminoids inhibited certain types of cancer, anticancer assays were directed on the
fractions obtained fi'om the EtOAc extract. Purification of the EtOAc fraction afforded
compounds 1, 2 and 3, the curcuminoids. Purification of the volatile oil yielded compound
4. The curcuminoids 1, 2 and 3 were evaluated for anticancer activity, and gave zones of
inhibition at 50, 50, and 25 ppm, respectively (Table 2.1). Mosquitocidal activity was then
evaluated for compounds 1-4. Compound 4 exhibited LDloo at 50 ppm on Aedes aegmti in
48 h. Compounds 1 and 2 exhibited minor activity, each with LDloo at 250 ppm in 48 h.
Compound 3 showed no mosquitocidal activity. The MeOH extract exhibited no anticancer
activity or mosquitocidal activity. In addition, none of the extracts or the volatile oil exhibited
activity against nematodes, or caused growth inhibition in the gypsy moth, forest tent

caterpillar, tobacco homwonn or corn earworms.

41

Table 2.1 Preliminary anticancer bioassay results for EtOAc and Hexane extracts and

compounds recorded as zone of inhibition in mm.

 

 

 

 

 

 

Test material JN 394 JN 394kl JN 3941!; II
Crude EtOAc 16.7 19.0 14.3

extract

Crude Hexane 12.7 20.0 23.7

extract

Curcumin I 13 16 15

curcumin II 14 12 15

curcumin III 15 18 13

 

 

 

 

 

 

 

42

The preliminary identity of the three curcuminoids was obtained by comparison
with the chromatographic data published earlier (Govindaraj an, 1980). The structures were
confirmed further by detailed spectral studies. The structures of 1-3 are shown as Figure
2.1. Since curcuminoids l and 3 are symmetrical, only halfof the signals were evident in their
lHNMR spectra.

In compound 1, the assignment of H-3 and H-4, as well as the H-3' and H-4' protons
as trans to each other, was based on the large coupling constant between them. The signals
for H—4 and H-4' appeared further downfield than H-3 and H-3', because these protons were
deshielded by the aromatic rings in the curcuminoids. The singlet at 5.77 ppm was assigned
to the H-1, because the methylene protons at C-1 in the curcunrinoids are enolyzed to the «-
Bcunsaturated carbonyl and appear as the singlet. The singlet at 3.93 ppm, integrated for six
protons, was assigned to the methoxy protons at the C-7 and C-7' positions.

Compound 2 lacked the symmetry evident in compounds 1 and 3. However, the
assignments of H-3, H-3', H-4, H-4‘ and H-1 were made by the same rationale as for
compound 1. Compound 2 showed two doublets at 6.86 and 7.47 ppm, respectively, with
a coupling constant 'of 8 Hz to each other. These doublets integrated for two protons each
and were assigned to H-6' and H-10', and H-7' and 119, respectively. The singlet at 3.95
ppm, integrated for three protons, was characteristic of a methoxy group at C-7.

For compound 3, the assignments of H-3, H-3', H-4, H-4' and H-1' were made by the
same reasoning as for compound 1. The lI-INMR spectrum of 3 gave two aromatic protons
at 6.89 and 7 .56 ppm, respectively, These protons were ortho to each other as evident from

ther 8Hz coupling constant. In Pople notation, this is characeristic of a AA’XX’ or AB

43

Figure 2.1 Curcuminoids

 

Curcumin l

 

Curcumin ll

 

Curcumin Ill

44

system, and is indicative of a para-disubstituted aromatic ring (Silverstein, 1991). Also, these
doublets integrated for four protons each. The protons at H-6, 6', 10, and 10' corresponded
to the doublet at 7.47 ppm, indicating they were deshielded further than the remaining
aromatic protons.

The three main curcuminoids from turmeric, curcumin I (l), curcumin II (2), and
arrcumin III (3), all were isolated previously rhizomes of Curcuma longa (Roughley, 1973).
The spectra of all three curcuminoids were confirmed by ’HNMR studies.

The structure of compound 4 is Figure 2.4. lHNMR and ‘3 CNMR spectra of
compound 4 revealed that it contained 20 protons and 15 carbons. The three singlet
integrated for three protons each at 1.84, 2.09, and 2.29 ppm, respectively, indicated that it
had three quaternary methyl groups. Also, the singlet at 2.29 ppm for 3 protons suggested
an aromatic methyl. group. The aromatic ring deshielded the methyl protons, hence, the
chemical shifts for these methyl protons were found further in the downfield region than a
regular aliphatic methyl group. The singlets at 2.09 and 1.84 ppm were typical of allylic
methylene protons and were assigned to C-14 and C-13, respectively. The protons at C-l4
were in close proximity to a carbonyl group and appeared further downfield. A doublet at
1.22 ppm integrated for three protons, indicative of a methyl group at C-12. A one-proton
singlet in the region at 6.00 ppm was assigned to C-10. Also, a broad singlet at 7.10 ppm
indicative of an AB system was due to H-2, H-3, H-4, and H-5 protons. The l3CNMR
spectrum of compound 4 supported the lHNMR assignments. The proton and carbon NMR
data and the proposed structure for compound 4 was confirmed further by the EIMS

spectrum. The molecular ion was evident with 63% intensity at m/z 216.

45

Figure 2.4 Ar-Turmerone Structure

 

46

The EIMS spectrum had a base peak at m/z 201, representing the loss of a methyl
group (M’ -15) by sigma-bond dissociation (or-cleavage ). A peak at m/z 119 represented
another sigma-bond dissociation which commonly occurs at tertiary carbons. This peak was
also particularly stable due to the aromatic ring and the resulting resonance. Additional
sigma-bond dissociation and the potential fragments generated from compound 4 are shown
in Figure 2.5. The lHNMR l3CNMR and EIMS spectral data of compound 4 were identical
to the published spectral data of compound 4, a sesquiterpene ketone from the rhizome of C
. longa (Rao, 1934), and identified compound 4 as ar-turmerone. Spectral data of this
compound were published recently (F erreira, 1992). The yield of pure ar-turmerone from
the rhizome in our research was much lower than previously reported value. Ar-turrnerone
is very volatile and hence it is possible that the rotatory evaporation and desiccation steps
evaporated most of the compound along with the solvent, resulting in an extremely low yield

of the compound.

Curcumin] (1), curcumin II (2) and curcumin 111(3) all showed anticancer activity,
with curcumin III being most active at 25 ppm concentration. Curcumin I (1) and curcumin
II (2) exhibited mosquitocidal activity on Aedes aegmtii at 250 ppm. Ar-turmerone (4)
displayed LDmo at 50 ppm against Aedes aegwtii.

Curcumin already is in use by the food industry as a safe coloring agent. This may
facilitate the acceptance of curcumin III as an efi‘ective chemotherapeutic cancer preventative:
In any case, curcumin is known to be a safe and nontoxic natural product, and may have

potential for use in chemotherapy. Also, ar-turmerone has proved to be an effective

47

Figure 2.5 EIMS Fragmentation of
Ar-Turmerone

O

mlz=2l6
= 63%

mlz=201

m/z=83
r. i a: 100% JCOEKH =30%

.

m/z=91 mlz=l 19
r.i. = 13% r-i- = 34%

48

mosquitocidal agent, and may be useful to control populations of A. aegyptii and manage it
as a disease vector. All of the activities reported in this chapter are novel. Further studies
need to be carried out to elucidate the mechanism of the activities associated with these three

curcuminoids and ar-turmerone.

CHAPTER HI

An Antifungal Terpenoid from the Leaves of Curcuma longa

Abstract

Curcuma spp. plants are an integral part of traditional and modern Indian Ayuverdic
medicine, and have provided some important antifirngal agents. Surprisingly, because of its
commercial importance in food industry, turmeric has not been investigated thoroughly for
medicinal or agricultural use. Therefore, our laboratory assayed turmeric extracts against
some important agricultural and human pathogens. Lyophilyzed Curcuma longa leaves were
extracted sequentially with hexane, ethyl acetate and methanol. Bioassay-directed
fractionation and purification by solvent partitioning and reverse phase preparative
chromatography of the hexane extract afforded an antifungal compound, 5. Detailed NMR
studies revealed that this compound is a diterpene dial. The structure was confirmed to be
labda-8(17),12-diene-15,l6—dial. It showed antifungal activities on several yeasts including
Candida albicans, Candida kruseii, and Candida parapsilosis, at 25, 25 and 1 ppm
concentrations, respectively. Also, it displayed mosquitocidal activity against Aedes aegwtii
with an LDloo at 10 ppm concentration. This is the first report of the isolation and
characterization of a diterpene from C. longa, and the biological activity for labda-8(17),12-

dime-15,16-dial.

49

50

Introduction

Plants fi'om the Zingiberaceae family play an important part in historical and
contemporary Ayurverdic Indian medicine (Jain, et al., 1991). Various members of this
family yield several antimicrobial agents (Bauer, 1988; Ghosh, 1980; Banerjee, 1976).
Recently, several fungi have become resistant to existing fungicides, and some of the
antimicrobial agents from plants in the Zingiberaceae family may be useful in managing these
fungal pathogens. An effective antifungal agent, the methyl ester of para-coumaric acid, was
isolated from rhizomes of the herbaceous Indian plant Costus speciosus Sm (Bauer, 1988).
This compound inhibited the growth of Aspergillus niger, C ladosporium cladosporioides,
Colletotrichum gloeosporioides, Curvularia spp ,and Penicillium app (Bauer, 198 8). Plants
of the Curcuma genus also yielded antifungal agents. The ethyl acetate extract of the
rhizomes of Curcuma amada showed antifungal activity against Candida albicans,
Epidermophyton floccosum, T richophyton rubrum, Aspergillus niger, Aspergillus flavus,
Penicillium notatum, Helminthosporium oryzae, Bonytis alli, Paecilomyces spp., F usarium
spp., Saccharomyces cerevisae, Pythium spp., Phytophthora parasitica, Rhizopus spp.,
Mucor spp., Curvularia spp., and Sclerotium spp (Ghosh, 1980). The essential oil of
rhizomes of Curcuma caesia displayed activity against Aspergillus niger, Aspergillusflavus,
Penicillium lilacinum, Pencillium javanicum, T richoderma viride, Curvularia oryzae,
Helminthosporium oryzae, Pestalotia lapagericola, Microsporum gypseum, and
T richophyton mentagrophytes (Banerjee, 1976).

Among pathogenic yeast types, Candida species are the most common and stubborn

51

of infectious agents in humans (Montes, 1985). Although a number of treatments existed for
yeast infections, many of them involved oral administration, and their bad taste made them
unattractive to patients. There is always a need for new treatments to combat yeast infection
since diagnostic problems can lead to systematic infection and death. Considering the
discovery of effective antifungal agents fi'om Zingiberaceae family, particularly the Curcuma
germs, our laboratory assayed extracts from C. longa L. against some important agricultural
and human pathogens. In this chapter, we report the isolation of an antibiotic diterpene,
labda-8(17),lZ-diene-15,16-dial, from the leaves of C. Ionga. This is the first report of the
isolation and characterization of labda-8(17),12-diene-15,16-dial from C. longa and its

antibiotic and mosquitocidal activities.
Experimental

Plant Materials - Turmeric plants were grown in large plastic pots, using a mixture
of 50 % loamy field soil and 50% bacto soil. The C. longa rhizomes were obtained from a
grocer in Detroit, Mchigan. Several rhizomes were planted just below the surface of the soil
in each pot. The plants were watered every two to three days, and fertilized with 20-20-20
Peters brand fertilizer. The turmeric plants were harvested for rhizomes in nine-month
cycles. Leaves were collected every two weeks for a three-month period, lyophilized and

milled, and kept in a -20 °C freezer until extraction.

52

Chemicals and cell culture media- YMG medium (yeast extract 4 g-L", maltose 10
g-L", glucose 4 g-L'l and agar 12 g-L"), PDA (potato dextrose agar; potato infirsion 200
g0L", dextrose 20 goL", agar 15 g-L"), Emmons medium (neopeptone 10 g-L", glucose 20
g-L" and agar 15 g-L"), NG medium (NaCl 3.0 g-L“, bacto peptone 2.5 g-L", cholesterol
1 rnl-L'l of 5 mgmrl‘l stock solution, CaCl2 1 ml-L'1 of l M stock solution, MgSO,, 1 ml-L"
of 1 M stock solution and potassium phosphate buffer 25 ml-L'l of stock solution of KHzPO,
11.97 gorootttrl and KzHPO, 2.0 34 00m1l ) and YPDA medium (yeast extract 20 g‘L ,
peptone 10 g-L", dextrose 20 got1 and adenine sulphate 2ml-I:l of 0.5% stock solution)
were prepared were prepared as published (Nair et al., 1989) in our lab. The ingredients were
purchased from Difco Lab (Detroit, Michigan, USA). Sterile saline was prepared by

dissolving NaCl (8.5 g0L“) in an appropriate volume of deionized water.

Extraction and isolation of the antifimgal compound 5 - The initial extraction method
for compound Sis summarized in Scheme 3.1. Lyophilized and milled turmeric leaves (100
g) were extracted successively with hexane, ethyl acetate and methanol in an extraction
column Each solvent was used in 3X500 ml aliquots over a 24 h period. Each extract was
evaporated to dryness, yielding 6.0 g of hexane extract (GR-19-224A), 2.0 g of ethyl acetate
extract (GR-19-224B), and 15.0 g of methanol extract (GR-19-224C). The hexane extract
exhibited activity against C. albicans. The TLC of the hexane extract exhibited a UV-
fluorescing major component in the extract. The hexane extract GR-l9-224A (2.5 g) was

dissolved in hexane (3 00 ml) and partitioned with methanol (3 X 100 ml) in a separatory

53

Scheme 1 Extraction of Leaf

 

Lyophilized turmeric leaves
(100 9)

 

 

 

Extracted with hexane
(500mLX3). 24h

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

f
GR-19-224A ,
Brown on (6.09) Res'due
Extracted with EtOAc
(500mLX3). 24h
GR-19-224B
Dark Green Solid (2.09) Residue
Extracted with MeOH
(500mLXl. 24 h3
l
GR-19-224C

Green Gum(159) Resrdue discarded

 

 

54

funnel. The methanol extracts then were combined and extracted further with hexane (3 X
100 ml). The hexane and methanol fi'actions were evaporated to dryness separately, yielding
a light yellow oil and a dark green solid, respectively. TLC analysis showed that most of the
fluorescing compound was in the methanol-partitioned fraction (1.8 g). A small amount of
this compound was still present in the hexane-partitioned fraction and then was extracted
further with MeOH (10X5 ml). The MeOH fraction was evaporated to dryness, and
combined with the original methanol fraction, yielding a total of 2.1 g of GR-19-273B. The
yield of the hexane fi'action, GR-19-273A, was 440 mg. Methanolzwater (7:3) was used to
dissolve GR-19-273B (1.8 g) (14 ml X 3), in order to precipitate the chlorophyll. The
suspension was centrifuged, and supernatant was collected, and removal of solvent in vacuo
yielded 1.38 g of a brown oil (GR-19-274A). A portion of this oily residue (560 mg) was
purified by VLC using 85:15 methanolzwater as the mobile phase, and fractions were
collected in aliquots of 50[I], 50[II], 25[III], 30[IV], 35[V], 40[VI], and 40[VII] ml,
respectively. The final elution was with chloroform in 60ml[VIII] and 200 ml[IX] aliquots,
altogether to yield fractions I-IX. Bioassays revealed that fractions III-VII were antifungal,
and were combined to yield an oily residue (128 mg).. Repeated purification of this oily
residue by reverse phase preparatory TLC, using a 7:1 methanolzwater mobile phase, gave
a UV-flourescing band, GR/19/277A . Elution of this band afforded an oily compound 5

(21.1 mg). (Appendices VII, VIII, IX, X, XI, XII, XIII for spectra of compound 5).

Spectral - NMR spectra were recorded at the Max T. Rogers NMR facility at

55

Michigan State University on a Varian VXR 500 MHZ spectrometer (Varian, California,
USA) at ambient temperature. Mass spectra were acquired at the Michigan State University
Mass Spectrometry Facility on a JEOL HX-l 10 double focusing mass spectrometer (JEOL,
Tokyo, Japan). Reverse phase preparative TLC plates were purchased from Whatman Inc.

(Clifton New Jersey).

Compound 5. lI-INMR (CDCl,):6 0.71, 0.80, and 0.87 (each 3H, s, 18, 19, 20 CH,),
1.04 (1H, m,J= 4,13 Hz, 1-H), 1.11 (1H, dd, J= 3, 13 Hz, H—S), 1.17 (1H, m, H-3), 1.33
(1H, m, J= 4, 13 Hz, H—6), 1.40 (1H, m, H—3), 1.49 (1H, m, J=3.3, 14 Hz, H—2), 1.57 (1H,
m, H-2), 1.67 (1H, m, H-l), 1.73 (1H, m, H-6), 1.88 (1H, dd, J= 1.5, 11 Hz, H-9), 2.00
(1H, m, H-14), 2.31 (1H, m, H-1 1), 2.40 (II-I, m, H-14), 2.48 (II-I, m, H-1 1), 3.35 (1H, d,
J= 17 Hz, H-14), 3.42, (1H, d, J= 17 Hz, H-l4), 4.34 (1H, d, J= 1 Hz, H-17), 4.84 (1H,
d, J= 1 Hz, H—17), 6.74 (1H, t, J=6.5 Hz, H-12), 9.38 (1H, s, H-16), and 9.61 (1H, t, J=1.5,
H-15). 13c1~11er(c1)c1,): 6 14.4 (020), 19.3 (02), 21.7 (019), 24.1 (C-6), 24.7 (on),
33.6 (C-18 , 04), 37.8 (014), 39.2 (C-1), 39.3 (07), 39.6 (010), 42.0 (03), 55.4 (C3),
56.5 (09), 107.8 (cm, 134.8 (013), 148.0 (C-8), 159.9 (012), 193.5 (C-16), and 197.3
(015). EIMS: m/z (rel. Int); 302 (114* 39) 287 (7), 284 (5), 273 (9), 137 (100), 69 (56), 55

(36).

Circular Dichroism (CD) of compound 5
Circular Dichroim Analyses. - The CD analysis of compound 5 was carried out using

a JASCO J-710 71CD-ORD spectropolarimeter (Jasco, Incorporated, Japan). The spectra

56

were plotted on a DeskJet 855C Hewlett Packard plotter (Hewlett Packard Corporation,
Palo, Alto, California). Nitrogen (99.99%) was generated by a nitrogen generator model NG-
150 (Peak Scientific, Chicago, Illinois) at a rate of 15 L-m". Pure labda 8(17), 12- diene-
15,16-dial was dissolved in EtOH (1.4 mg/ 1 ml), and the CD was determined under the
following conditions: scan mode (wavelength), bandwidth (0.5 nm), sensitivity (50 mdeg),
response (1 s), wavelength range (200-400 nm), step resolution (1 nm), scan speed (200
nm/min), and accumulation (1) (Appendix XIII).

Antimicrobial assay - Antifirngal and antibacterial assays of compound 5 were
conducted according to the reported procedure (Nair et al., 1989). Cultures ofFusarium
orysporum (MS-SM-l322), Fusarium monilrforme (MS-SM-1323), Gleosporum spp. and
Rhizoctonia spp. were raised on potato dextrose agar (PDA) medium in petri dishes.
Cultures of Candida albicans, Candida louseii, Candida parapsilosis and Aspergillusflavus
(MS strains) were raised on YMG medium in petri dishes. Cultures of Staphylococcus
epidermidis (ATCC 25923 ), Streptococcus aureus (MS strain), and Escherichia coli (ATCC

25922) cultures were grown on Emmons medium in petri dishes.

Anticancer (topoisomerase inhibition) assay - Topoisomerase assay of compound
5 was conducted according to the reported procedure (Chang et al., 1995). Mutant
Saccharomyces cerevisae cell cultures JN 3 94, JN3 94 t-1 and JN 394 t2_, were provided by
Dr. John Nitiss at St. Jude Children’s Research Hospital (Nitiss et al., 1993). JN394 is
hypersensitive to topoisomerase I and II poisons because of deletions that irradicated the

RAD52 repair pathway. JN394 t-, is isogeneic to JN394, except that the topl gene is

57

deleted. The deletion of this gene causes a lack of response to topoisomerase I poisons. JN
394 t“, that contains the top2-5 gene, is resistant to topoisomerase II poisons, but it responds
to topoisomerase I poisons. The mutant yeasts were cultured on YPDA medium in petri

dishes containing 20 ml of media.

The cell suspension for bacteria and mutant yeast and spore suspension for fungi were
prepared by suspending the bacteria or yeast or spores fi'om a fully grown petri-dish culture
in 10 ml of sterile saline solution, and transferring the suspension to a sterile culture test tube.
The cell or spore suspension concentration was adjusted to 10° colony forming units per
milliliter (CFU/ml) by conducting a serial dilution of the stock culture and by plate count, to
determine the CFU/ml of the test organism. Bioassays were conducted by lawning the
desired cell or spore suspension (100 pl) on petri dishes of the corresponding medium. The
test compound in DMSO then was spotted carefully on the bioassay plates at desired
concentrations along with 25 pl of DMSO which served as a control. The plates were
allowed to dry in a laminar flow hood, and then incubated at 27° C for 72 h. The zone of

inhibition was measured in mm.

Mosquitocidal assay - Aedes aegwtii mosquito larvae were used to test for
mosquitocidal activity of crude leaf extracts and pure compounds according to the established
procedure of Nair et al. (1989). A. aegwtii eggs were provided by Dr. Raikhel at Michigan
State University, Dept. of Entomology. Approximately 200 mosquito eggs were placed in

500 ml of degassed, deionized water prepared by sonication. About 5 mg of bovine liver

58

powder was added as a food source for the larvae. After four days, 4th-instar mosquito
larvae were transferred in 980 pl of deionized water in a test tube. 20pl of compound 5 at
the desired concentrations in DMSO was added to each tube and left at room temperature.
20pl of DMSO alone served as control. The mortality of the larvae was recorded at 2-, 4-,

6-, 24-, and 48-h intervals. The assay was conducted in triplicate.

Nematicidal assays - Panagrellus redivivus and Caenorhabditis elegans were used
to test for nematicidal activity of crude leaf extracts and compound 5 according to the
established procedure of Nair, et al. (1993). A stock culture of nematodes in media was
maintained at 14 day intervals to provide uniform young nematodes. 10-20 nematodes in
48 pl growth media were transferred to wells in a 96-well tissue-culture plate. 2pl of 50%
aqueous DMSO solution of test material was added to this. 2 pl of 50% aqueous DMSO
alone was used as control. The experiment was conducted in triplicate.

Gypsy moth, forest tent caterpillar, corn earworm and tobacco homworm assays -
These assays were conducted using artificial diets. Dry diet ingredients for the gypsy moth
consisted of wheat germ (36g), casein (7 .5g), Wesson’s salt mix (2.4g), sorbic acid (0.6 g),
methylparaben (0.3 g), and Hoffman-LaRoche #26862 vitamin mix (3 g). 0.845g of dry diet
mix was used for each treatment. The corn earworm diet was obtained from North Carolina
State, and 0.94 g was used for each treatment. The tobacco homwonn diet also was obtained
from North Carolina State, and 0.95 g was used for each treatment. Dry diet was weighed
out in a scintillatiOn vial, one for each test compound. The test compound was dissolved to

a desired concentration in DMSO, so that 20 pl of the solution was added to the diet. 20pl

59

of DMSO alone served as the control. Agar solution was added to bring the total weight to
5.0 g, and the mix was homogenized with a spatula. The mixture was poured immediately
(before jelling) and equally into 15 micro beakers. The beakers were randomized and allowed
to cool for 3 h. One insect larvae of the type being tested was transferred to each of the 15
micro beakers with a small paint brush. The beakers were capped, and transferred to a tray
in an incubation chamber at 28 °C for six days. At the end of this period, each larvae was
weighed, and the average (in mg) for each sample was compared against weight of the control
to determine bioactivity. Data were analyzed using Dunnet’s test, where means of the

treatments are compared with the control.

Results and Discussion

Leaves of C. longa were extracted sequentially with hexane, ethyl acetate and
methanol. Preliminary bioassays were performed on these extracts to test for antimicrobial,
anticancer, mosquitocidal, nematicidal, and Gypsy moth, forest tent caterpillar, tobacco
homwonn and corn earworm growth inhibition.

Hexane extract was found to inhibit C. albicans at 250 ppm concentration. In
addition, it exhibited activity against A aegwtii with LDloo at 250 ppm in 48 h. The hexane
extract showed only minor activity against Rhizoctonia spp. Preliminary anticancer assays
of the hexane extract exhibited marginal activity on all three mutant yeast strains, rendering
it impossible to distinguish if the extract was inhibiting topoisomerase, or exhibiting antifirngal
activity, or both. Hence the topoisomerase inlubition assay was not studied further using this

extract. The hexane extract did not exhibit activity against any of the other microbial

60

pathogens tested, including F usarium oxysporum (MS-SM-1322), F usarium moniliforme
(MS-SM-1323 ), Gleosporum spp,. Rhizoctonia spp. Aspergillus flavus (MS strains)
Staphylococcus epidermidis (ATCC 25923), Streptococcus aureus (MS strain), and
Escherichia coli (ATCC 25922). The methanol and ethyl acetate extracts did‘ not show any
antimicrobial, anticancer or mosquitocidal activities. In addition, none. of the three solvent
extracts exhibited activity against nematodes or caused growth inhibition in the gypsy moth,
forest tent caterpillar, tobacco homwonn or corn earworm assays.

. Purification of the hexane extract afforded compound 5 as the active compound. It
was evaluated for antifungal activity on C. albicans, C. louseii, and C. parapsilosis at 100,
50, 25, 10, and 1 ppm concentrations. Activity was observed at 1 ppm for C. albicans, and
at 25 ppm for C. lauseii, and C. parapsilosis. Mosquitocidal activity was evaluated for
compound 5 at 100, 50, 25, 12.5, 10, 6.25, and 1 ppm concentrations. Compound 5 had
ID“," at 100 ppm on A. aegyptii in 12 h. In 48 11, compound 5 exhibited LDloo on A. aegyptii
at 10 ppm. The mechanism of this activity has not been determined. However, upon
application of the test material to the mosquitoes, they began to behave in a fi'enzied manner.
Within 20 min, the mosquitoes gathered at the water- air interface, until death occurred, and

sank to the bottom of the assay tube.

The 1I-INMR lE'CNMIL and DEPT spectra of compound 5 showed that there were
30 protons and 20 carbons, respectively. In the lI-INMR spectrum, a singlet appeared at 9.38
ppm, integrated for one proton, was characteristic of an aldehydic group adjacent to an

olefinic moiety. Also, a proton triplet at 9.61 ppm, with a coupling constant of 1.5 Hz was

61

typical of an aldehydic group adjacent to a methylene group. It was assigned to C-15 in
compound 5. The C-14 methylene protons appeared as two separate doublets, at 3.37 and
3.43 ppm, respectively. These protons were deshielded by the adjacent aldehyde group. Two
doublets in the olefinic region at 4.35 and 4.84 ppm integrated for one proton each and were
assigned to C-17. A single proton triplet in the olefinic region at 6.74 ppm was assigned to
C-12. Also, the lHNIvm spectrum showed three singlets at 0.71, 0.80, and 0.87 ppm,
respectively, for C-18, 19 and 20 methyl groups.

l3C NMR supported the presence of two aldehydic carbons at C-15 and 16, two
aliphatic methylene carbons at C-11 and 14, one aliphatic methine carbon at C-12, one
exocyclic methylene carbon at C-17, and three aliphatic methyl carbons at C-18, 19 and 20,
respectively, in compound 5. The DEPT spectrum of 5 showed 4 quaternary carbon signals
at 134.8, 148.0, 39.6 and 33.6 ppm, respectively. The signal at 134.8 ppm was in agreement
with the reported value for olefinic carbons vicinal to an aldehydic substituent (Silverstein,
1,991), and was assigned to C-13. The signal at 148 ppm was assigned to 08. The two
remaining quaternary carbons at 33.6 and 39.6 ppm, were assigned to C-4, and 010,
respectively.

The HMQC spectra proved very helpful in elucidating the structure of compound 5.
It confirmed the assignments of protons and carbons at residence at C-1, C-2, C-3, C-6 and
C-7. The COSY spectrum further proved these assignments and showed the proton couplings
between C-1 and C-2, and C-2 and C-3. Similar HMQC analysis provided assignments of C-
6 and C-7 at 24.1 and 39.3 ppm, respectively. The COSY data showed the connectivities

ofC-S to C-6, and O9 to C-ll (Figure 3.2 ). From the NMR data, the molecular formula

62'

Figure 3.1 Structure of
Labda 8(17)-12-diene, 15,16 dial

 

63

Figure 3.2 COSY Correlations
for Labda 8(17)-12-diene, 15,16 dial

 

64

Figure 3.3-Possible EIMS Fragmentation Patterns For
Labda 8917l-diene - 15,16-dial

mlz=302
LI. = 39% Qgfl/M

mlz = 284 \ / mlz = 287

r.i. = 5% r.i. = 7%

gt/ ©9290

mlz=273
r.i. = 9%

mlz=69 mlz=55
r.i. = 56% r.i. = 36%

65

of the compound was calculated as €311,002 The EIMS of compound 5 gave the molecular
ion at mlz 302. Also, the FABMS spectra confirmed the molecular weight as 302, as
indicated by the W peak at m/z 303.

The EIMS gave a peak at m/z 287, indicative of a sigma bond dissociation (a-
cleavage) of a methyl group, (M’-15). The peaks at m/z 284 and 273 were resulting from
the loss of water (M’-18), and an aldehydic group, respectively. The peak at m/z 137 was
100% intense. The peak at m/z 69 resulted from an tat-cleavage at the C-14 carbonyl to give
a stable acylium ion, and the peak at m/z 55 signified two sigma ring bond dissociations with
concomitant double bond formation (Figure 3.3). Based on the spectral data, compound 5
was confirmed to be the diterpene labda 8(17),12-diene-15,16- dial (Figure 3.1).

The molar elipticity of compound 5 was at the extrenum of -17 .23 mdeg at 320 nm
(Appendix XIII). Compound 5 with a negative optical rotation was isolated previously
from the seeds ofAlpinia galanga (Morita, 1987). The positive form of 8 (17), 12 -labd-
diene-15,l6-dial was isolated previously fi'om rhizomes of A lpinia speciosa (Itokawa, 1980).
Also, Morita (1987) determined the absolute configuration of 8 (17), 12 -labd-diene-15,16-
dial fi'om A. galanga by the ozonolysis of the diterpene 8 (17), lZ-labd-diene-15,16- dial,
yielding compound 6 (Figure 3.4). The CD spectrum of 6 gave a molar ellipticity of -2.79 at
289 nm (Morita, 1987). The sign value of the ellipticity would be the same for compound 6
as in the case of the natural product from A. galanga, since no chiral centers were lost or
introduced during the ozonolysis. The negative value of the ellipticity of compound 5
indicated that both compound 5 and 8 (17), 12 -labd-diene-15,16-dial from A. galanga are

identical in their configuration. Also, due to the double bond and aldehydic group in

66

Figure 3.4

67

conjugation, a higher ellipticity value was obtained for 5 than for 6. This is the first report of
the CD for labda 8( 17) 12-diene-15, 16-dial.

. . Compound 5 exhibited activity against C. albicans, C. kruseii and C. parapsilosis
at 1, 25, and 25 ppm concentrations, respectively. Candida is the causative agent in vaginal
yeast infections. As such, there are relatively few antibiotics available to combat this
pathogen It is possible that this natural product or its analogues may be useful in the firture
as a topical treatment to combat yeast infections. Compound 5 exhibited an LDloo at 10 ppm
on A. aegyptii larvae The mosquitocidal activity may be exploited in the future to combat
growing populations of A. aegjptii and manage them as vectors of human disease. These
activities are novel. The mechanisms of these bioactivities are unknown, and warrant further

research.

CHAPTER IV

Summary and Conclusions

Fresh rhizomes collected fiom the greenhouse-grown Curcuma longa Linn plants
were lyophilized. The volatile oil was extracted from the ice accumulated on the condenser
coil of the lyophilizer. Lyophilized rhizomes of C. longa L. were extracted sequentially with
hexane, ethyl acetate, and methanol, separately. A similar process was conducted for the
lyophilized leaves. Preliminary bioassays were carried out on all these extracts at 250 ppm
concentrations to determine the presence of antibacterial, antifungal, anticancer,
mosquitocidal, and nematicidal activities. All crude extracts were tested for anticancer
activity in the form of topoisomerase inhibition, using mutant Saccharomyces cerevisae
cultures. These crude extracts also were evaluated for growth inhibition of gypsy moth
larvae, (Lymantria dispar), forest tent caterpillar larvae (Malacosoma dystria), tobacco
homwonn larvae (Manduca sexta), and corn earworm larvae flielicoverpa zea).

The ethyl acetate extract from the rhizome displayed good anticancer activity. The

essential oil fiom the rhizome and the ethyl acetate extract exhibited activity on anticancer and

68

69

mosquitocidal assays, respectively. The crude methanol extract from the rhizome displayed
no antimicrobial, insecticidal, anticancer, or growth inhibition activity when tested on
Aspergillus spp., Bonytis spp., F usarium moniliforme, F usarium oxysporum, Gloesporum
spp., Rhizoctonia spp., Candida albicans, Streptococcus spp., Staphylococcus spp.,
Escherichia coli, Aedes aegwtii mosquitoes, Panagrellus redivivus Goody nematodes,
Caenorhabditis elegans nematodes, Saccharomyces cerevisiae mutant cultures IN 3 94, IN
394t-1, JN3 94t2-5, Lymantria dispar, Malacosoma aystria, Manduca sexta, or Helicoverpa
zea.

Bioassay-directed fiactionation was carried out to isolate the active compounds from
rhizomes of C. longa. Rhizomes yielded three anticancer compounds, curcumin I (1),
curcumin II (2), curcumin III (3). Structures of compounds 1-3 were confirmed using
lHNMR experiments, and these compounds were evaluated for anticancer activity
(topoisomerase inhibition) (Chapter II). Curcumin III gave the most potent anticancer
activity, represented by the inhibition of topoisomerase I and II enzymes at 25 ppm
concentration. However, curcumin I and curcumin H inhibited topoisomerase I and II at 50
ppm concentration.

The volatile oil of the rhizome yielded a mosquitocidal sesquiterpene ketone, ar -
turmerone (4). The structure 'of compound 4 was confirmed using lHNMIL li’CNMR, and
EIMS experiments (Chapter 11). Compound 4 was evaluated for mosquitocidal activity
(Chapter II). It displayed an LDloo at 50 ppm on A. aegmtii.

The hexane extract fiom the leaves exhibited good antifungal and mosquitocidal

activities. The crude leaf methanol and ethyl acetate extracts displayed no antimicrobial,

70

insecticidal, anticancer, or growth inhibition activity when tested on Aspergillus spp.,
Boaytis mp" F usarium moniliforme, F usarium oxysporum, Gloesporum spp., Rhizoctonia
spp., Candida albicans, Streptococcus spp., Staphylococcus spp., Escherichia coli, A.
aeg'ptii mosquitoes, Panagrellus redivivus Goody nematodes, Caenorhabditis elegans
nematodes, Saccharomyces cerevisiae mutant cultures JN3 94, IN 394t-1, JN394t2-5,
Lymcma'ia dquar, Malacosoma dystria, Manduca sexta, or Helicoverpa zea (Chapter III).

Bioassay-directed fractionation was carried out to isolate the active compound from
the leaf extracts of C. longa. The hexane extract yielded an antifirngal diterpene labda 8(17)
diene-lS, 16,-dial, Compound 5. The structure of compound 5 was confirmed using lHNMR,
”CNMR, DEPT, HQMC, COSY, EIMS and FABMS experiments (Chapter III). Compound
5 was evaluated for antifimgal activity on C. albicans, C. kruseii, and C. parapsilosis.
Compound 5, labda 8(17) 12-diene-15,16- dial, was antifungal against C. albicans at 1 ppm.
Also, this diterpene inhibited the growth of C. kruseii and C. parapsilosis at 25 ppm. It also
displayed an LDloo at 10 ppm in mosquitocidal assays against A. aegyptii.

The most efi‘ective mosquitocidal component was the labdane type diterpene, labda
8(17) 12-diene -15,16-dial, compound 5, which displayed an LDm at 10 ppm in
mosquitocidal assays, compared to the activity of ar-turmerone, compound 4, which
displayed an LDloo at 50 ppm on A. aegyptii. Interestingly, ar-turmerone caused mortality
in 18 h at 50 ppm, while labda 8(17) l2-diene -15, 16-dial took 48 h at 10 ppm concentration
to cause mortality.

The mechanism for the inhibition of these compounds has not been determined.

However, some observations regarding inhibition displayed by the curcuminoids can be made

71

based on their structural differences. Curcumin 111 displayed greater anticancer activity than
either curcumin II, or curcumin I, and curcumin II displayed more activity than Curcumin I.
A literature search revealed that this trend is not unusual for most of the activities reported
for curcuminoids. The lack of a methoxy substituent in a curcuminoid increased its activity.
Also, removal of both methoxy substituents further enhanced activity as displayed by
curcumin III. It is possible that the elimination of the methoxy group(s) increased the
phenolic nature of the curcuminoid, and hence it can act as an effective antioxidant or a free
radical scavenger. Additional research is required to confirm this hypothesis.

The work reported in this thesis has provided additional insight into known
compounds with novel activities. Also, this is the first report of the isolation of compound

5, (-) labda 8(17)-12- diene - 15,16-dial from C. longa.

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84

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