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This is to certify that the

dissertation entitled

BIOACTIVE COMPOUNDS FROM
OCIMUM SANCTUM LINN. LAMIACEAE

presented by
MARK A. KELM

has been accepted towards fulﬁllment
of the requirements for

Ph.D. Horticulture

 

 

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Major professor

Date May 12, 1999

 

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use muss-nu

BIOACTIVE COMPOUNDS FROM OCIMUM SANCTUM LINN. LAMIACEAE

By

Mark Allen Kelm

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Horticulture

1999

ABSTRACT
BIOACTIVE COMPOUNDS FROM OCIMUM SANCTUM LINN. LAMIACEAE
By

Mark Allen Kelm

A phytochemical investigation was conducted on various solvent extracts
of the herb Ocimum sanctum Linn. Lamiaceae resulted in the isolation and
characterization of mosquitocidal, anti-oxidant, and anti-inﬂammatory
compounds. Mosquito-bioassay-guided fractionation led to the isolation of two
mosquitocidal compounds, eugenol and (E)-6-hydroxy-4,6-dimethyl-3-heptene-2-
one with minimum inhibitory concentrations at 200 and 6.25 ug-ml", respectively.
A novel triglyceride, 1,3-dilinoleneoyl-Z-palmitin also was identiﬁed. In two other
separate studies, anti-oxidant and anti-inﬂammatory assays yielded eugenol,
cirsilineol, cirsimaritin, isothymonin, isothymonin, apigenin, and rosmarinic acid,
which were isolated from the acetone extract of fresh leaves and stems of O.
sanctum. Anti-oxidant activities of many of these compounds namely cirsilineol,
isothymonin, and rosmarinic acid were comparable to synthetic anti-oxidants
when assayed at 10 uM concentrations. At 1000 uM concentration nearly all of
these compounds demonstrated very good anti-inﬂammatory activity as
demonstrated by the inhibition of cyclo-oxygenase-1. In another study, corn
eanNorm-bioassay-guided fractionation led to the characterization of a bioactive

porphyrin that exhibited toxicity/anti-feedant activity at 100 ppm concentration.

Structural identiﬁcation and characterization was facilitated by the use of various
spectroscopic experiments (NMR, MS, IR, and UV).

Due to the plant’s anti-oxidant activity, it could potentially be used as a
cancer preventative agent or even to slow the growth of existing malignant
tissues. The anti-inﬂammatory activity 0. sanctum, in particular its ability to
inhibit cyclo-oxygenase-2 could provide an alternative to currently available
drugs. Incorporation of O. sanctum either as a ground powder or crude extracts
into diet supplements or phytoceuticals could aid in the treatment of the
aforementioned maladies. The use of O. sanctum has potential in the control of
insect pests. Furthermore, because 0. sanctum is a plant, the acceptance and
marketability of it would be enhanced considering the current lean towards

natural remedies for human and agriculture pest management.

”GM/6m, @Md/Iw/ng

ACKNOWLEDGMENTS

I wish to thank my best friend and future wife, Sarah Breitkreutz for all the
help, encouragement, understanding, and love that she has given through the
course of my doctorate program. She has made a difﬁcult task a lot less

burdensome.

I also wish to thank my family for the support they have given me during
the last several years. The encouragement and support that they gave were

deeply appreciated

The advice and discussions academic and othenNise, provided by my lab
mates; Dr. Amitabh Chandra, Dr. James Nitao, Dr. Russel Ramsewak, Dr. S.
Balasubramanian (a.k.a. “Bala”), Andy Erickson, Dr. Haibo Wang, Dr. Yu-Chen
Chang, and Dr. Holly Herner were also greatly valued and appreciated. A
special thanks also goes to Di Zhang, Laura Clifford, Di Wu, and Raﬁkali Momin.

Thanks to the guys at the NMR facility, Drs. Kermit Johnson and Long Lee.

Finally I want like to thank the members of my graduate committee, Drs.
Jack Kelly, Doug Gage, Bob Schutzki, Jim Miller, and my major advisor Dr.

Muralee Nair for the guidance an advice they were able to offer.

TABLE OF CONTENTS

LIST OF FIGURES .............................................................................. vii
LIST OF ABBREVIATIONS .................................................................. viii
Introduction ........................................................................................... 1
CHAPTER I — Literature Review ................................................................ 5

CHAPTER II - Mosquitocidal compounds and a triglyceride,

1, 3-dilinoleneoyl-Z-palmitin from Ocimum sanctum Linn ............................... 28
CHAPTER III — Anti-oxidants from Ocimum sanctum Linn ............................. 39
CHAPTER IV - Anti-inﬂammatory compounds from Ocimum sanctum Linn ...... 60

CHAPTER V — A porphyrin compound from Ocimum sanctum Linn. with corn

earworm anti-feedant/toxicity activity ........................................................ 68
CHAPTER VI - Summary ...................................................................... 76
REFERENCES .................................................................................... 80

vi

LIST OF FIGURES

Figure 1. Anti-oxidant activity of synthetic and isolated compounds assayed at

10 uM ................................................................................................ 59

Figure 2. Anti-oxidant activity at 21 minutes for synthetic and isolated

compounds at 10 uM ............................................................................ 59
Figure 3. In vitro anti-inﬂammatory assay of eugenol, compounds 1-6 and
synthetics. Eugenol, compounds 1-6, and aspirin tested at 1000 uM and

ibuprofen and naproxen tested at 10 pM ................................................... 64

Figure 4. In vitro anti-inﬂammatory activity of eugenol, compounds 1,2,5,6

and aspirin tested at 1000 uM and ibuprofen tested at 10 uM ........................ 66

Figure 5. Corn eanNonn anti-feedant/toxic activity of two fractions containing

isolated porphyrin. Signiﬁcant at P < 0.005 ................................................ 73

vii

BHA

BHT
CHCb
cox
13CNMR
DMSO
DQFCOSY
d

dd
DPA-PA

DEPT
E

EIMS
Et20
EtOH
EtOAc
FABMS
GC
1HNMR
HPLC
LC

m
MeOH
MIC
MOPS
MPLC
MS

m/z
NMR
PG
PGHS-1
PGSH-2
PTLC

s

TBA
TBHQ
TLC
UV

6

J

VLC

LIST OF ABBREVIATIONS

Butylated hydroxyanisole

Butylated hydroxytoluene

Chloroform

Cyclooxygenase

Carbon nuclear magnetic resonance
Dimethyl sulfoxide

Double quantum ﬁltered correlated spectroscopy
Doublet

Doublet of doublet

3-(p-(6-phenyl)-1 ,3,5-hexatrienyl)phenylpropionic
acid

Distortionless enhancement polarization transfer
Entgegen

Electron impact ionization mass spectroscopy
Diethyl ether

Ethanol

Ethyl acetate

Fast atom bombardment mass spectroscopy
Gas chromatography

Proton nuclear magnetic resonance

High pressure lipid chromatography

Lethal concentration

Mulitplet

Methanol

Minimum inhibitory concentration
3-[N-morpholino] propanesulfonic acid
Medium pressure lipid chromatography
Mass spectroscopy

Mass-to-charge ratio

Nuclear magnetic resonance

Prostaglandin

Prostaglandin endoperoxide H synthase-1
Prostaglandin endoperoxide H synthase-2
Preparative thin layer chromatography
Singlet

2-thiobarbituric acid

tert-butylhydroquinone

Thin layer chromatography

Ultraviolet

Chemical shifts

Coupling constant

Vacuum liquid chromatography

viii

INTRODUCTION

Ocimum sanctum Linn. Lamiaceae has been used for generations in
Southeast Asian traditional medicine and cuisine. O. sanctum (synonym O.
tenuiﬂorum) more commonly known by its numerous vernacular names; Holy
Basil, Sacred Basil, Tulsi (Unani), Tulasi, Tulssi, Surasa, Krishnamul, Vishnu-
priya (Sanskrit), Kala-tulasi (Hindi), Krishna-tulasi (Bengali), and Thulasi (Tamil),
has been used to treat a wide variety of ailments. This plant, as many of the
common names imply, holds religious signiﬁcance as well. Sacred Basil is
thought to be the transformed nymph, Tulsi, the beloved of Lord Krishna, and
thus has been regarded as sacred and as a consequence, it can be found in and
around many Hindu temples and homes (Mallick et al., 1989). There are even
some Christian sects that consider the herb as holy, since it can be found
growing near the tomb of Christ. According to Ayurveda, the traditional Indian
system of medicine, teas made from the leaves are prescribed for colds, stomach
disorders, and as a source of nourishment for the old and weak (Thakur, 1989).
In addition, the roots are alleged to posses aphrodisiac qualities. A decoction
made from the roots has been used to treat malarial fever. The leaf juices have
been applied to the skin for treatment of ringworms and other cutaneous
afflictions (Butani, 1982). According to African folk medicine, 0. sanctum was
used to repel mosquitoes and have a subterfuge and poultice-like effects (Batta

and Santhakumari, 1970).

II

Besides its use in plantings around Hindu temples and homes, 0.
sanctum has little horticultural importance. However, the plant has been
considered as an alternative and cheaper source of the highly valued phenyl
propanoid, eugenol (Asthana and Gupta, 1984). Eugenol can comprise 30-70%
(Asthana and Gupta, 1984) of the essential oil and is used mainly in
pharmaceutical formulations, perfumes, toiletries, and as a ﬂavoring agent in
foods. Currently eugenol is obtained from the costly spices clove, Eugenia

caryophyllata L. and cinnamon, Cinnamon zeylanicum L.

Much of the impetus for research on O. sanctum has been due to its role
in traditional folk medicine. Most of these studies on O. sanctum were focused
primarily on the bioactivity of crude extracts. These studies not only examined
pharmacological activities, but also activities pertaining to agriculturally related
pest problems such as insects, nematodes, and fungal diseases. Interestingly, a
limited number of papers published on O. sanctum dealt with the isolation and
identiﬁcation of bioactive compounds and the determination of bioactivity of

compounds previously identiﬁed in O. sanctum.

Many studies pertaining to various biological activities have been
completed on crude extracts from O. sanctum, as discussed in Chapter II. Also,
a number of articles have focused more speciﬁcally on the bioactivities of
compounds identiﬁed from O. sanctum, are discussed in Chapter II. It has been

my hypothesis that given the body of pre-existing research, as well as preliminary

studies carried out in the Bioactive Natural Products Laboratories (BNPL), O.
sanctum has the potential to generate bioactive compounds for human and
agricultural pest control. Therefore, the primary purpose of the current research
was to isolate and identify the presence of biologically active compounds not
previously examined. In addition, it was hoped that the present work will add to
the body of phytochemcial knowledge that already exists for O. sanctum. O.
sanctum plants were obtained initially from the Hare Krishna of Detroit and grown
in the BNPL greenhouses, Department of Horticulture and National Food Safety
and Toxicology Research Center, Michigan State University. As previously
stated, primarily the crude extracts of O. sanctum have been examined for
pharmacological as well as agricultural pest related studies. The potential health
and pesticidal beneﬁts from O. sanctum are therefore apparent given this body of
literature and its long use as a traditional medicine. Therefore, I hypothesized
that O. sanctum contains compound(s) capable of demonstrating antioxidant and
anti-inﬂammatory activities. In addition, 0. sanctum contains compounds that

possess insecticidal and/or antifeedant activity.

This study, extracts and compounds were examined for biological
activities. Insect bioassays utilizing mosquito larvae (Aedes aegyptii Linn.) and
corn ean~orm (Heliocoverpa zea Hubner) were carried out to determine toxicity
and antifeedant activities, respectively. Antioxidant and anti-inﬂammatory assays

were conducted on isolated compounds.

The body of this dissertation is comprised of several chapters. Each
chapter, excluding the literature review is organized in a scientiﬁc journal format,
which includes an abstract, introduction, materials and methods, and a results

and discussion section.

Chapter 1

LITERATURE REVIEW

This chapter reviews the breadth of literature on the bioactivities of crude
extracts and pure compounds from O. sanctum. Among the pharmacological
activities reported for O. sanctum crude extracts were anti-carcinogenic,
chemopreventative, anti-oxidant, radioprotective, anti-mutagenic, analgesic, anti-
pyretic, anti-inﬂammatory, anti-spermatogenic, anti-fertility, hypoglycemic, anti-
ulcerogenic, immunostimulatory, anti-stress, CNS effects, anti-bacterial, and
effect on blood lipid proﬁles. Bioactivities associated with agricultural pest and
other pest problems were; anti-fungal, anti-bacterial, insecticidal, pulicidal,
nematicidal, and herbicidal activities. Papers about bioactivities of pure
compounds from O. sanctum were far fewer, these consisted of antioxidant,
hepatoprotective, anti-inﬂammatory, anti-hyperlipidemia, anti-tumor promotion,
and anti-stress. Other miscellaneous articles pertaining to phytochemical studies

on O. sanctum are presented.

Kelm, M. A. and Nair, M. G. (submitted 1999) Bioactivity and Phytochemical

review of Ocimum sanctum Linn. J. Ethnopannacology

Pharmacologically related studies

An investigation of the anticarcinogenic effects of the commonly
consumed Indian herb, Tulsi revealed that it signiﬁcantly decreased the
occurrence of benzo[a]pyrene-induced neoplasia and 3'methyl-4-
dimethylaminoazo benzene induced hepatomas in Swiss mice and Vlﬁnstar rats,
respectively (Aruna and Sivaramakrishnan, 1992). Also, 0. sanctum exhibited
chemopreventative activity against chemical dimethylbenz[a]anthracene (DMBA)
induced carcinogenesis. In one study, a topical application of alcoholic O.
sanctum leaf extract followed by application of DMBA resulted in a signiﬁcant
reduction in skin papillomagenesis of male Swiss albino mice (Prashar et al.,
1994). The authors also noted a 25% increase in glutathione-S-transferase
activity and a decrease in glutathione content in mouse skin following the topical
application of O. sanctum leaf extract. In a later study, treatment of mice with the
ethanolic extract introduced via gastric intubation and orally were found to be
more effective than topical applications in the prevention of DMBA-induced tumor
formation (Prashar and Kumar, 1995). Similar results were obtained using
Syrian golden hamsters (Karthikeyan et al., 1999). Researchers determined the
inﬂuence of alcoholic extracts on the activity of certain detoxifying enzymes
(Banerjee et al., 1996). Oral doses of O. sanctum extract at 400 and 800
mg-kg'1 body weight for 15 days signiﬁcantly elevated the activities of
cytochrome p-450, cytochrome b5, aryl hydrocarbon hydroxylase, and

glutathione-S-transferase (Banerjee et al., 1996). These enzymes are

considered to be very important in the detoxiﬁcation process of carcinogens,

mutagens and other poisons.

The anti-thyrodic and anti-oxidant activity of oral doses of aqueous
extracts of O. sanctum was demonstrated by observing a rise in serum thyroxine
concentrations and an increase in hepatic lipid peroxidation, glucose-6-
phosphate, and endogenous anti-oxidant enzymes super oxide dismutase and
catalase (Panda and Kar, 1998). In another study, it was determined that O.
sanctum extract possesses free radical scavenging activity in vitro (Maulik et al.,
1999). It is well known that free radicals, in particular oxygen free radicals, play
a major role in the pathology of numerous diseases which include heart disease,
arrhythmia, stroke, brain damage, respiratory syndrome, liver damage, cancer,
and inﬂuenza (Mukhopadhyay et al., 1994 and Das and Maulik 1994). Ursolic
acid, a triterpene, isolated from O. sanctum demonstrated protective effect
against lipid peroxidation (Balanehru and Nagarajan, 1991). In a follow up
article, Balanehru and Nagarajan (1992) found that ursolic acid prevented the
cardiotoxicity of adriamycin by acting as an antioxidant. Furthermore, a
pharmacological review article on ursolic acid discussed its hepatoprotective,

anti-inﬂammatory, anti-tumor, and anti-hyperlipidemia activity (Liu, 1995).

lntraperitoneal injections of 50 mg-kg‘1 followed by 10 mg-kg‘1 per day of
O. sanctum extract for ﬁve consecutive days gave the best protection (70%

survival) against whole body exposure to 60Co gamma radiation in albino mice

 

(Devi et al., 1995). In a following report it was found that O. sanctum extracts
gave in vivo protection against 60Co-gamma-radiation induced cytogenic damage
(Ganasoundari et al., 1997). The authors speculated that the likely mechanism
of the observed radioprotection involved free radical scavenging activity by the O.
sanctum extracts administered interperitoneally. In a related article, researchers
found that the same extracts were beneﬁcial in the protection of bone marrow
stem cells in adult Swiss mice against the deleterious effects of 6°Co gamma

radiation (Ganasoundari et al., 1997). This report also demonstrated the lack of a

 

toxic effect on the part of O. sanctum water extracts as compared to the well
established radioprotector, WR-2721 (amifostine), an organic thiophosphate.
Given this, 0. sanctum extracts may have an advantage over toxic WR-2721 in
clinical uses as a natural and potentially benign substance. In a follow-up report,
the same authors had shown the radioprotective effect of bone marrow by the
administration of O. sanctum water extracts, in addition, the toxicity of WR-2721
was reduced by the presence of O. sanctum extracts which also resulted in an
increase in the beneﬁcial radioprotective effects synergistically (Ganasoundari et
al., 1998). The authors also demonstrated a signiﬁcant rise in chromosome
protection by the use of O. sanctum extracts when administered intraperitoneally
to Swiss mice. Again, it was suggested that the radioprotection was caused by

the free radical scavenging qualities of O. sanctum extracts (Ganasoundari et al.,

1998).

The anti-mutagenic effect of O. sanctum juice were demonstrated in mice
that were administered the bone marrow cell mutagens; methylmetnaesulfonate,
mitomycin C, and dimethylnitroamine (Lim-Sylianco et al., 1988). O sanctum
extracts were given orally by gavage, whereas the mutagens were given

intraperitoneally.

The analgesic activity of the oil from the seeds of O. sanctum was
determined in rats and mice (Singh and Majumdar, 1995). Using the tail ﬂick, tail
clip, and tail immersion methods, researchers concluded that O. sanctum was
not effective in raising the pain threshold, indicating that the O. sanctum is not
centrally active. However, using the acetic-acid-induced writhing method, the oil
showed a signiﬁcantly elevated responses in a dose-dependent manner, which is
indicative of peripherally mediated pain. A dose of 3 ml-kg‘1 of O. sanctum oil
gave similar results as produced by a 100 mg-kg‘1 of aspirin. These treatments
were given intraperitoneally. In an earlier study, it was observed that rises in
hypouricemic and uricosuric effect followed by administration of Tulsi leaves or
seeds in rabbits may result in a reduction of uric acid levels (Sarkar et al., 1990).
High levels of uric acid are associated with gouty arthritis. These results may,
therefore, substantiate one of the traditional uses of Tulsi as a treatment for

gouty arthritis or other joint inﬂammations.

Methanolic and water suspensions of O. sanctum both showed analgesic

activity in the mouse hot plate method, anti-inﬂammatory activity in rats, and anti-

 

pyretic activity in rats (Godwani et al., 1987). 500 mg-kg‘1 doses of the
methanolic extract and water suspensions were found to achieve the same
response as seen with 300 mg-kg’1 aspirin. However, at the same dose levels,
the methanolic extract and the crude water suspension displayed weaker and

shorter duration of analgesic activity than that of aspirin.

The ﬁxed oil of O. sanctum was 100% effective in the treatment of bovine
mastitis after ﬁve days alone and after three days when combined with cloxacillin
(Singh et al., 1995). Bovine mastitis effects the udder and is characterized by
inﬂammation and bacterial infections largely due to Staphylococcus aureus. In
another study, the anti-asthmatic and anti-inﬂammatory of ethanol extracts of
both dry and fresh leaves of O. sanctum were analyzed. Guinea pigs were used
for the study. Interestingly, only the fresh extracts along with the volatile and
ﬁxed oils had shown protection against histamine-induced acetyl choline pre-
convulsive dyspnea (Singh and Agrawal, 1991). Results from anti-inﬂammatory
studies involving carrageenan-induced hind paw edema indicated inhibition by
fresh-leaf volatile and ﬁxed oils. Furthermore, in a later study, anti-inﬂammatory
activity was demonstrated by the oil from O. sanctum, since it inhibited
arachidonic acid and leukotriene-induced paw edema. Furthermore, the authors
postulated that O. sanctum may be used as a potential anti-inﬂammatory agent
that blocks both cyclooxygenase and lipoxygenase pathways in arachidonic acid

metabolism (Singh et al., 1996).

10

Non-polar (benzene, petroleum ether, and ether) and polar extracts
(acetone and EtOH) of O.sanctum had shown 80-60% and less than 50% anti-
fertility, respectively. Anti-fertility activity of O. sanctum extracts was determined
by the examination of fertile female rats for the presence of spermatozoa in
vaginal smears, followed by laparomety to determine the number of implantation

sites (Batta and Santhakumari, 1970).

High amounts of O.sanctum leaves in the diets of male albino mice
decreased in seminal plasma pH, which corresponded to an increase in reducing
substances such as alkaline phosphatase, acid phosphatase, mucoproteins, and
electrolytes (Kasinathan et al., 1972). Histologically, there was a slight
impairment of spermatogenesis. Taken together, the observed sterility was in
large part due to these changes. A more recent report compliments these
ﬁndings (Seth et al., 1981) in which a benzene leaf extract of O. sanctum
reduced the count and motility of sperm in adult male rats. A reduction in the
weight of testes was also observed. Furthermore, over the course of three
months of diets including Tulsi leaves, the mating behavior of both male and
female rats was severely inhibited (Khanna et al., 1986). This study also showed
a decreased sperm count and sperm motility and weight reduction in male sex
organs of rats over long-term exposure to Tulsi diets. Contrary to these studies
by Khanna et al. (1986) and Kasinathan et al. (1972), researchers found an

increase in testes size vs. controls and an increase in cone weight vs. control in

11

White leghorn male birds (Arneja et al., 1987). These studies, therefore,

suggested that antifertility activity is species dependent.

Incorporation of seeds and leaves of O. sanctum into artiﬁcial diets
displayed a progressive and signiﬁcant hypoglycemic effect during the ﬁrst week
in adult albino rabbits. Both seed- and leaf-containing diets reduced blood sugar
levels when compared with the control. However, on a dry weight basis, the
leaves were more effective at reducing blood sugar (Sarkar and Pant, 1989).
Doses of 100, 150, 200, and 400 mg-kg'1 of Tulsi extract were given along with a
normal diet to adult male Wistar rats. Signiﬁcant decreases in sexual behavior
were observed by those rats receiving 200 mg-kg'1 and 400 mg-kg'1 (Kantak and
Gogate, 1992). Sexual behavior was observed and scored followed by
presenting male rats with a primed ovariectomise female in which 2 pg of
estrogen were administered the previous three days followed by progesterone on
the fourth day. Reghunandanan et al., (1995) reported a reduction in testicular
sperm count and glutamyl transpeptidase activity in Wistar rats after 24 h

following intraperitoneal injections of benzene extract of O. sanctum leaves (300

make").
A deﬁnite lowering of blood sugar level in normal, glucose-fed

hypoglycemic and streptozotocin-induced diabetic rats was observed following

oral administration of alcoholic extracts of O.sanctum. Enhancement of the

12

action of exogenous insulin in normal rats also was observed (Chattopadhyay,

1993)

Researchers concluded that O.sanctum extracts have anti-ulcerogenic
properties against experimental ulcers, since it can cause a decrease in acid
secretion and increased mucous secretion in VWstar albino rats (Mandel et al.,
1993). O.sanctum-pretreated rat stomach mucosa was able to resist increases
in gastric lesions caused by HCl-ethanol treatments. Pretreated rats also
displayed a decrease in acidity and an increase in mucosal defense. Both
studies (Mandel et al., 1993 and Janardhanan, et al., 1999) seem to justify the

use of O.sanctum as an anti-ulcerogenic compound.

Steam distillates from fresh 0. sanctum leaves were used in a study to
determine its effects on humoral immune responses in vitro. It was found that
O.sanctum could keep in check or maintain the humoral immune responses by
acting on different aspects involved with the immune system such as antibody
production, the release of mediators of hypersensitivity reactions, and tissue
responses to mediators in target organs (Mediratta et al., 1988). A more recent
study reported on the humoral immune response in albino rats by the
quantiﬁcation of agglutinating antibodies and E-rosette formation. Results
showned an increase in antibody titre, E-rosette formation and lymphocytosis
which are indicative of immunostimulation of humoral immunological responses

(Godhwani et al., 1988). Diets containing dry powdered leaf of O.sanctum were

13

 

fed to birds at 500 mg-day'1 orally. lmmunomodulating and immunopotentiating
activities by dried leaf of O.sanctum were shown in naturally IBD (infectious
bursal disease) virus-infected poultry (Sadekar et al., 1998). IBD virus is one of

the poultry industry's toughest problems.

Connected in part with the immuno-stimulatory properties, researchers
also discovered that O. sanctum possess anti-stress or adaptogenic properties
(Wagner, et al., 1994, Sen et al., 1982, Bahrgava and Singh, 1981,) . O.sanctum
was found to enhance physical endurance of swimming mice, inhibit stress-
induced ulcers in rats, protect from carbon tetrachloride-induced hepatotoxicity in
mice and rats, and inhibit milk-induced leucocytosis (Bahrgava and Singh, 1981).
Sen and coworkers (1992) analyzed the effects of O. sanctum extract and
eugenol (major component in O.sanctum) on various biochemical stress-induced
changes. Both O.sanctum and eugenol reduced cholesterol levels caused by
restraint stress in male Wistar rats (Sen et al., 1992). Also, O.sanctum and
eugenol effectively lowered lactate dehydrogenase and alkaline phosphatase
levels typically caused by stress (Sen et al., 1992). Red blood cell membrane
dynamics also changed due to induction by restraint stress. Increased
membrane protein clusterization, increased membrane ﬂuidity and reduced
membrane thickness occurred in response to stress; these are lessened or

reversed in the presence of either O.sanctum or eugenol (Sen et al., 1992).

14

Action of O. sanctum on the central nervous system (CNS) was studied in
mouse models (Ahumada et al., 1991 and Sakina et al., 1990). In these studies,
it was found that O. sanctum extracts acted in synergy with the drug
pentobarbital in depressing CNS responses. Sakina et al. (1990) reported a
decrease in recovery time and severity due to electroshock and
pentylenetetrazole-induced convulsions, in decreased apomorphine-induced
ﬁghting time and ability to walk in rodents. This study also indicated a possible
interaction of O. sanctum extracts with dopaminergic neurons and a synergistic
action when combined with bromocryptine, a potent Dz-receptor antagonist. In
addition, longer narcosis times were observed in male mice compared to female

mice (Ahumada et al., 1991).

Anti-bacterial studies showed ethanolic extracts of O. sanctum to be
effective against Staphylococcus aureus (100 rig-disc“), S. albus (100 jig-disc“),
S. citreus (250 jig-disc“) and E. coli NCTC strain (500 jig-disc") (Phadke and
Kulkarni, 1989). Water extracts of O. sanctum leaves exhibited anti-biotic activity
against several strains of tuberculosis bacteria which included: Mycobacterium
tuberculosis H37 Rv TMC102, M. tuberculosis SmR10(N)-1, and M. tuberculosis
1NHR Rv6 (Reddi et al., 1986). Also, 0. sanctum juice (30 ill-disc") exhibited
inhibitory activity in assays utilizing Sarcina lutea. However, activity was not
observed on Bacillus cereus, B. meaten'um, B. subtillis, S. epidermis, Proteus

sp., Salmonella typhosa, and Vibrio cholerae (Ferdous et al., 1990).

15

The blood lipid proﬁle of albino rats was signiﬁcantly lower following the
administration of fresh 0. sanctum leaves (Sarkar et al., 1994). Total serum
cholesterol, triglycerides, phospholipids, and LDL cholesterol levels all were
reduced signiﬁcantly. In conjunction, blood HDL cholesterol and total fecal sterol
contents were signiﬁcantly greater. Together, these results suggest that O.
sanctum has the potential to prevent or treat heart disease as well as other

disorders associated with high fat intake.

Agricultural Pest and Insect-Related Studies

In addition to the pharmacologically related studies, agricultural insect
control and related studies utilizing O. sanctum have been conducted. Much of
the driving force behind this research was due to the widespread use of O.

sanctum as a pest-managing agent. Only crude extracts of O. sanctum were

studied for pest-managing activity.

Methanol extracts of O. sanctum leaves exhibited some fungicidal and
bacteriacidal activities on Tn'chophyton mertagrophytes and Bacillus subtilis
(Ehrenburg) Cohn at concentrations greater than 104 ppm but were not active on
Collectotn'chum Iagenan'um (Passerini) Ellis et Halstead, Pyn'culan'a oryzae
Cavara, and Cochliobolus miyabeanus (Ito et Kuribayashi) Drechsler ex Dastur
(Sukari and Takahashi, 1988). However, contradicting results were obtained

with ethanolic leaf extracts of O. sanctum. These extracts inhibited the growth of

16

three fungal rice pathogens: rice blast; P. oryzae, brown spot; C. miyabeanus,
and sheath blight; Rhizoctonia solani Kiihn when applied as a spray (2.5 g-l") on
infected rice plants and in agar plate media at 5.0, 10.0, and 10.0 g-I",
respectively (Tewari and Nayak, 1991). Ethanolic extracts and essential oil of O.
sanctum showed fungicidal activity against another species of rice blast, P.
gn’sea Sac. in both conidial germination and mycelial growth (Tewari, 1995).
Ethanolic extracts also showed good performance on par with some synthetic
fungicides (ediphenphos and carbendazim) in ﬁeld studies (Tewar, 1995).
Bananas, Musa spientum var. Malbhog pretreated with aqueous leaf extracts of
O. sanctum then infected with the fungi; Botryodiplodia theobroma, Fusan'um
theobromae, F. oxysporum, Helminthospon'um spiciferum, Culvularia lunata,
Aspergillus ﬂavus, and Tn'chothecium roseum resulted in signiﬁcant retardation of
fungal pathogen growth (Singh et al., 1993). Aqueous leaf extracts of O.
sanctum effectively prevented conidial germination and slowed the growth of pre-
and post-infected by H. spiceferum and F. scirpi in sponge gourd, Luffa
cyclindn'ca L. (Ahmad and Prasad, 1995). Spore germination, spore growth, and
cell wall degrading enzymes of the fruit rot pathogens, Botryodiplodia
theobromae Pat. and Rhizopus anhizus Fisher were inhibited by O. sanctum leaf
extracts (Patil, et al., 1992). A reduction of total protein content of the fungi also

were observed following treatment with O. sanctum leaf extracts.

Soxhletted ethanol extracts of both white and red strains of O. sanctum

did not show any activity on the ﬁsh and shrimp bacterial pathogens, Acromonas

17

hydrophila, Streptococcus sp., and ten strains of Vibrio (Direkbusarakom et al.,

1998).

Contact toxicity bioassays on cockroach (Blatella gennanica L.) and red
bean weevil (Callosbrunchus chinesis L.) gave 100% mortality due to application
of 10 mg of O. sanctum essential oil (Sukari and Takahashi, 1988). Methanol
leaf extracts of O. sanctum proved to be effective as antifeedants and repellents
but were, not insecticidal against Anomis sabulifera Guen., jute semilooper
larvae (Mallick and Banerji, 1989). Ethanolic and methanolic extracts of O.
sanctum were bioassayed against the following aphid species: Myzus persicae
(Sulzer), Metopolophium dirhodum (Walker), Aphis fabae (Scopoli), Sitobion
avenae (Fabricius), and Acyrthosiphon pisum (Harris). Mortality of all species
was observed following application of the test extract (Stein et al., 1988). The
authors concluded that the observed aphidicidal activity cannot be due to
eugenol, rather different compounds are more likely causing the observed effect.
0. sanctum extracts applied at a rate of 10 kg-ha‘1 on rice TKM9 seedlings
resulted in only 5% survival of green leafnopper, Nephotettix virescens Dist.
(Narasimhan and Mariappan, 1988). Only 33% of transmission rate for rice
tungro virus was observed for plants treated with O. sanctum compared to the
75% observed in the control. When O. sanctum seed are placed in water, they
exuded a mucilaginous substance, a polysaccharide of unknown structure. This
substance has been found to effectively trap mosquito larvae (Culex fatigens)

resulting in their death (Hasan and Dec, 1994). Hexane and acetone leaf

18

extracts also showed excellent to moderate toxicity on C. fatigans larvae,
respectively (Deshmukh et al., 1982). Arrekul et al., (1988) found that crude
extracts of O. sanctum a demonstrated repellent effect against the oriental fruit
ﬂy, Daucus doralis. Paradoxically, Roomi and coworkers (1993) found that O.
sanctum leaf extracts were acting as attractants in ﬁeld studies. Methyl eugenol,
present in the essential oil, was used as a positive control which attracted

Daucus spp.

The oviposition deterrent effect of O. sanctum leaf extracts on spotted
bollworm (En‘as Vite/Ia Fabr.) was demonstrated in a study by Sojitra and Patel
(1992). A 5% leaf extract suspension of O. sanctum applied to okra plants
(Hibiscus esculentus L.) was effective at reducing the number of eggs laid. In a
later study, researchers (Adiroubane and Letchoumane, 1988) found that 2.5 and
5% water suspensions of O. sanctum ground leaf alone and in combination with
synthetic pesticides (carbaryl 0.05% and endosulfan 0.035%) were signiﬁcantly
effective in controlling the population of leafhoppers, Amrasca bigulatta in the
ﬁeld. Using similar treatments, infestation of spotted bollworm, Ean'as spp. was

signiﬁcantly less than in the nontreated controls.

Hexane and acetone leaf extracts of O. sanctum demonstrated pulicidal

properties with LC50 at 0.05 and 1.45, respectively (Renapurkar and Deshmukh,

1984). Activities were comparable to dichloro-diphenyl-trichloroethane (DDT),

19

 

dieldrin, malathion, and fenthion with L050 values at 0.31, 0.39, 0.44, and 0.34,

respectively.

Chatterjee et al., (1982) reported nematicidal activity of eugenol and
linalool against the nematode, Meliodogyne incognita. In another article,
eugenol, linalool, cineole, and geraniol (see structures below) were found to be
toxic against nematodes; Anguina tritici, Tylenchulus semipenetrans, M. javanica,
and Heterodera cajani (Sangwan et al., 1990). Okra plants inoculated with M.
incognita followed by treatments with water decoctions of O. sanctum leaves
resulted in signiﬁcant reductions in root protein levels and in the number of root

galls (Santi et al., 1984).

Also, 0. sanctum extracts were found to inhibit several enzymes
completely, namely mitochondrial malate dehydrogenase and malic enzyme in

the metabolic pathways of the ﬁlarial worm, Setan’a digitata (Banu et al., 1992).

Phytochemical Studies

The earliest phytochemical investigations of O. sanctum leaves were
conducted by Nair et al., (1982). Among the compounds identiﬁed were as
follows: ursolic acid, apigenin, luteolin, apigenin-7-O-glucuronide, luteolin-7-O-
glucoronide, orientin, and molludistin. Oleanic acid also was reported from O.

sanctum (Wagner, et al., 1994).

20

OH

COOH R0 0
HO OH 0
ursolic acid R = H; apigenin
R = glucose; apigenin-7-O-glucoside
OH
OH
R0 0
OH O
R =H; luteolin
R = glucose; luteolin-7-O-glucoside
OH
OH HO
OH
Glc O OH
HO
HO O
CH3O O
OH O
OH O
orientin
molludistin

21

Later the chemical composition of O. sanctum seed oil was determined
(Malik et al., 1987). Seed oil represented 18.2% by weight of the seed. GC
indicated the presence of lauric (2.84%), myristic (1.90%), palmitic (5.24%),

stearic (3.12%), oleic (6.0%), linoleic (59.1%), and linolenic (21.7%) acids.

lauric acid palmitic acid myristic acid

0 0
HOW Ito/“1%

oleic acid steric acid
HO 6 — 2 HO 6 —- 3
linoleic acid linolenic acid

A reinvestigation of O. sanctum leaves led researchers to discover the
presence of vicenin-2 (apigenin-6, 8-C-diglucoside), rosmarinic acid, galuteolin

(luteolin-5-O-glucoside), cirsilineol (5, 4'-dihydrxy-6, 7, 3’-trimethoxyﬂavone), and

OH
0
HO \ 0
OH
COOH
HO

rosmarinic acid

 

OH O

vicenin-Z

22

Glc

OR
R = H; 4-allyl- l-O-B-Dglucopyranosyl-
2-hydroxybenzene

R = CH3; 4-allyl-l-O-B-D-glucopyranosyl
2-methoxybenzene

 

cirsilineol

galuteolin

two phenyl propane glucosides; 4-allyl-1-O-j3—D-glucopyranosyl-2-
hydroxybenzene and -allyl-1-O-B—D-glucopyranosyl-2-methoxybenzene (Nbrr
and Wagner, 1992).

Also, gallic acid, gallic acid methyl ester, gallic acid ethyl ester,
protocatechuic acid, vanillic acid, 4-hydroxybenzoic acid, vanillin,
hydroxybenzaldehyde, caffeic acid, and chlorogenic acid were identiﬁed by UV

spectroscopy and HPLC (Nbrr and Wagner, 1992).

23

l i 2..

OH

HO OH HO\¢rOH HO : ,OH

COOH coocn, COOCHZCH3
gallic acid gallic acid methyl ester gallic acid ethyl ester
OH OH OH
OH : ,OMC ©
COOH COOH COOH
protocatechuic 301d vanillic acid 4-hydroxybenzoic acid
OH OH
OMc :
OH
CHO CHO
.. OH
vamllm 4-hydroxybenzaldehyde
OH /
OH
O O
HO
H
OH
//
HOOC H
coon 0”
caffeic acid chlorogenic acid

24

Sukari and coworkers (1995) isolated and identiﬁed stigmasterol,

B—sitosterol and triacontanol ferulate from the dried bark of O. sanctum.

 

stigmasterol

OH
CH O

09' I W“
l
HO O

B—sitosterol
triacontanol ferulate

GC-MS and FTIR experiments of the essential oils of O. sanctum led to
the identiﬁcation of 20 compounds in one study (Laakso et al., 1990) and 12
compounds in another study (Vasudevan et al., 1997). In the former study
eugenol, B—bisabolenes, methyl eugenol, cineole, ocimene, humulene, and 8-
caryophyllene were the major components derived from the steam and water-
distilled oils. Linalool and geraniol were also identiﬁed. In the latter study,
eugenol, and B-caryophyllene, methyl eugenol, and a number of unidentiﬁed

compounds the major compounds found in the subcritical ﬂuid extract.

25

OH

OMe
OMe : ,OMe

 

 

 

 

eugenol methyl eugenol B—bisabolenes cineole
/ \ /
/
/
l I“
B—ocimene a—humulene B—caryophyllene

OH

OH

linalool geraniol

26

Given the above studies and the lack of studies pertaining to bioactive
compounds, this work has shed more light on this deﬁcient area of phytochemical
research on O. sanctum. Through the process of bioassay-guided fractionation,
compounds possessing insecticidal, anti-oxidant, and anti-inﬂammatory activity

were discovered.

27

Chapter 2

MOSQUITOCIDAL COMPOUNDS AND A TRILGLYCERIDE, 1,3-

DILlNOLENEOYL-Z-PALMITIN FROM OCIMUM SANCTUM LINN.*

Abstract - The hexane extract of Ocimum sanctum was investigated using
mosquito bioassay-guided fractionation and yielded compounds 1 and 2. The
isolation of the triglyceride, 1, 3-dilinoleneoyI-2-palmitin (3) from O. sanctum
leaves and stems is novel. The structures of these compounds were established
using 1H- and 13CNMR spectral data. Compounds eugenol (1) and E-6-hydroxy-
4, 6-dimethyl-3-heptene-2-one (2) exhibited mosquitocidal activity at 200 and

6.25 ug-ml'1 in 24 h, respectively, on fourth instar Aedes aegyptii larvae.

* M. A. Kelm and M. G. Nair (1998) Mosquitocidal compounds and a triglyceride,
1, 3-dilinoleneoyl-Z-palmitin, from Ocimum sanctum. J. Ag. Food Chem. 46,

3092-3094.

28

Introduction

Ocimum sanctum L. (Lamiaceae) has been used for generations in
Southeast Asian medicine and cuisine. O. sanctum or “Sacred Basil”, as the
Ayurvedic herbal drug, has been used to treat a variety of human ailments
(Thakur et al., 1989; Butani et al., 1982). Malarial fevers, ringworms, and other
cutaneous afflictions also have been treated with this plant (Butani et al., 1982).
According to African folk medicine, 0. sanctum was reported to repel
mosquitoes, and have subterfuge and poultice effects (Batta and Santhakumari,
1970). Also, the crude extracts from O. sanctum have demonstrated biological
activities against certain insects (Risvi, 1981). For example, crude alcoholic
extracts exhibited aphidcidal properties (Stein et al., 1988), antifeedant activities
on Jute semilooper, Anomis sabulifera (Malik and Raﬁque, 1989), and mosquito
repellent and toxic properties (Batta and Santhakumari, 1970; Deshmukh et al.,

1982).

Previous phytochemical studies of O. sanctum have led to the isolation of
ﬂavones and ﬂavone glycosides, including; apigenin (Nair et al., 1982), apigenin-
7-O-glucoside (Nair et al., 1982), cirsilineol (Nbrr and Wagner, 1992), galuteolin
(Nbrr and Wagner, 1992), luteolin (Nair et al., 1982), luteolin-7-O-glucoside (Nair
et al., 1982), molludistin (Nair et al., 1982), orientin (Nair et al., 1982), and
vicenin-2 (Nbrr and Wagner, 1992). Other compounds identiﬁed in O. sanctum
were eugenol (Laakso et al., 1990), rosmarinic acid (Nbrr and Wagner, 1992), 8-

sitosterol (Sukari et al., 1995), stigmasterol (Sukari et al., 1995), triacontanol

29

ferulate (Sukari et al., 1995), and ursolic acid (Nair et al., 1982). Subsequently,
the phenyl propane glycosides 4-allyl—1-O-8-D-glucopyranosyI-2-hydroxybenzene
and 4-allyl-1-O-I3-D-glucopyranosyl-2-methoxybenzene were isolated (Nbrr and
Wagner, 1992). GC-MS and GC-FTIR analyses of the essential oil of O.
sanctum revealed the presence of 20 compounds (Laakso et al., 1990). From
these studies eugenol, methyl chavicol, and B-bisbolenes were found to be the

major components in the essential oil.

Past research on O. sanctum focused on the biological activity of crude
extract. Typically, phytochemical investigations carried out on O. sanctum
extracts did not lend to the isolation and structural identiﬁcation of biologically
active compounds. The work in our laboratory, in part, involves the preliminary
screening of many plant and microbial extracts in order to determine the
presence of any biologically active compound. In this paper, we report, for the
ﬁrst time, the isolation and structure determination of a novel triglyceride (3) and
two mosquitocidal compounds (1, 2) from the leaf and stem hexane extract of O.

sanctum.

Materials and Methods

General Experimental. 1HNMR, DQFCOSY, NOESY, and HMQC spectra

were recorded at 300 and 500MHz. 13CNMR and DEPT spectra were recorded

at 126 MHz. Chemical shifts were recorded in CDCI3, and the values are in 6

30

(ppm) based on residual of CHCI3 7.24 and CDCI3 77.0. Coupling constants, J,
are in Hz. EIMS were recorded at 70 eV. Particle size of silica gel used in VLC
and MPLC was 35-70 pm. All PLTC puriﬁcations were carried out on 250- and or
500-um silica gel plates. Spots and bands were visualized under UV light (366

and 254 nm).

Gas chromatographic analyses were performed utilizing a ﬂame ionization
detector which was set at 260°C. The capillary column used for the analysis was
a DB-5 (30 m x 0.25 mm ID). The temperature for the analysis was programmed
from 150° (4 min) to 250°C (5 min) at 4°C-min‘1 with a helium carrier gas at a

linear velocity of 34 cm-sec’1 and with split injections.

Plant Material. A voucher plant specimen (MSC 360851) was ﬁled with
the Beal-Darlington herbarium, Department of Botany and Plant Pathology,
Michigan State University. Leaves and stems of O. sanctum were harvested
from plants maintained in the Pesticide Research Center greenhouses at
Michigan State University. Plant materials were then freeze-dried, milled, and

stored at -20°C until extraction.

Mosquitocidal Bioassay. Fourth instar mosquito larvae, Aedes aegyptii L.
were reared in our laboratory from eggs (courtesy of Drs. Alexander Raikal and
Alan Hays, Department of Entomology. Michigan State University). Eggs were

hatched in 500 mL of distilled, degassed water prepared by sonication (30 min).

31

Approximately 5 mg of bovine liver powder was added to the water to provide a
food source. After four days, the fourth instar mosquito larvae were ready for
bioassay. At least 10 larvae were placed in 980 uL of degassed, distilled H20.
To this, 20 pL of DMSO containing the appropriate concentration of test extract
or puriﬁed compounds was added and left at room temperature. Extracts were
tested at 250 ppm. Pure compounds were tested initially at 100-250 ppm then
were diluted serially and subsequently bioassayed to determine LC1oo. 4 mL test
tubes were used for the bioassay. There were three replicates per treatment.
The number of dead larvae were recorded at 2-, 4- and 24-h intervals. The
control tube containing at least 10 larvae received 20 uL of DMSO alone, and
mortality was recorded as in the case of test compounds. These bioassays were
conducted according to previously published works (Roth et al., 1998; Nitao et

al., 1991; Nair et al., 1989).

Saponiﬁcation and Methylation of Compound 3. With stirring, 6.6 mg of 3
was treated with 5% NaOH in MeOH (1 mL) for 5 min. Methanolic 6N HCI then
was added to acidify this solution. This material was dried under a stream of
nitrogen. Diazomethane was prepared by reacting N-nitotroso-N-methyl urea
with concentrated KOH solution under ether. As the diazomethane product
formed, it dissolved into the organic ether phase. This yellow-colored ether
solution containing the diazomethane product then was collected and used to
methylate the free fatty acids obtained in the previous step. The methylated

product was ﬁltered to remove any solids prior to G0 analyses.

32

Extraction and Isolation of Compounds 1, 2, and 3. The freeze-dried O.
sanctum leaves and stems (440 g) were extracted sequentially with hexane,
EtOAc, and MeOH. 750 mL of each solvent was used for the ﬁrst 12 hours.
Thereafter, a second 750 mL of fresh solvent was used to extract the plant
material for an additional 12 hours. The hexane extraction afforded 13.1 g
residue upon removal of solvent. A portion of this residue (10.4 g) was
fractionated on a silica gel VLC (200 9) using: 4:1 pentane-EtZO as the mobile
phase. A 600 mL sintered funnel was used for the VLC. Dimensions of the MP
column were 250 mm x 25 mm. Fraction collection was based on the color of
bands observed through the VLC sintered funnel as well as the MP column.
Flow rates for MPLC experiments were approximately 1-2 mL per min. Fractions
collected in the VLC experiment were 1 (375 mL), 2 (200 mL), and 3 (300 mL).
Next, elution with 2:1 pentane-EtZO yielded fractions 4 (650 mL) and 5 (250 mL).
This was followed by elution with 1:1 pentane-EtZO, which gave fractions 6 (250
mL), 7 (500 mL), and 8 (300 mL), and then elution with 320 which gave fractions
9 (325 mL) and 10 (200 mL). Finally, elution with CHCI3 and MeOH afforded
fractions 11 (500 mL) and 12 (875 mL), respectively. All fractions were
bioassayed, and only fraction 2 was found to be mosquitocidal. Fraction 2 was
puriﬁed further with silica gel MPLC using hexane-MeZCO solvent systems to
yield 7 fractions. The seven solvent systems used and the volume of each
fraction collected were as follows: 1 (hexane; 110 ml), 2 (40:1; hexanezacetone;
90 mL), 3 (25:1; hexanezacetone; 90 mL), 4 (10:1; hexanezacetone; 90 mL), 5

(4:1; hexanezacetone; 190 mL), 6 (acetone; 95 mL), and 7 (acetone; 250 mL).

33

The seventh fraction was found to be mosquitocidal. The MeOH-soluble portion
of this fraction was ﬁnally puriﬁed by repeated preparative TLC to give
compounds 1 (31.9 mg), 2 (10.1 mg) and 3 (8.8 mg). Initially, compound 1 was
puriﬁed using 100% EtOAc as the mobile phase. 8:1 hexane-EtOAc followed by
10:1 hexane-acetone mobile phase ultimately led to the isolation of compound 1.
Like compound 1‘, compounds 2 and 3 were puriﬁed initially with 100% EtOAc as
the mobile phase. Isolation of compounds 2 and 3 was accomplished using 2:1

and 1:1 EtOAc-hexane mobile phases, respectively.

Compound 1, a pale brown oil: 1HNMR (CDCI3) 6: 3.30 (d, 2H, J=6.6 Hz,
H-7), 3.86 (s, 3H, -OCH3), 5.04 (dd, 1H, J=9.0 Hz, J=1.5 Hz, H-9 cis), 5.05 (dd,
1H, J=18.0 Hz, J=2.4 Hz, H-9 trans), 5.47 (s, 1H, -OH), 5.93 (m, 1H, H-8), 6.67
(d, 1H, J=7.8 Hz, H-6), 6.67 (s, 1H, H-3), 6.83 (dd, 1H, J=8.7 Hz, J=1.5 Hz, H-5);
13CNMR (CDCI3) 6: 39.9 (C-7), 55.8 (-OCH3), 111.0 (C-9), 114.1 (C-8), 115.5 (C-
6), 121.1 (C-5), 131.9 (C-3), 137.8 (C-4), 143.7 (C-2), 146.3 (C-1). The spectral
data of this compound were identical to an authentic sample purchased from

Aldrich.

OMe
HO

\
1

Compound 2, a colorless oil: 1HNMR (CDCI3) 6: 1.23 (s, 6H, H-7, 9), 1.88
(s, 3H, H-8), 2.14 (s, 3H, H-1), 2.56 (s, 2H, H-5), 4.25 (s, 1H, -OH), 6.01 (s, 1H,

H-3); 13CNMR (00013) a: 21.0 (08), 27.8 (01), 29.3 (07, 9), 53.8 (05), 69.8

34

(C-6). 124.5 (C-3), 157.4 (C-4), 202.3 (C-2). Therefore, it is identiﬁed as 56-
Hydroxy-4, 6-dimethyl-3-heptene—2-one. 1HNMR data were found to be in

agreement with previously published data (Kimura et al., 1982).

O
6
/
4
123 5 OH

2 7

Compound 3, a pale yellow oil: El-MS m/z (rel. int.): 611 (M"—C16H31O]"
(9), 262 [C18H290+H, 85]+ (85), 261 [C13H2901" (42), 239 (15), 230 (15), 108 (69),
95 (100), 81 (96), 53 (93); 1HNMR (CDCI3) 6: 0.86 (bt, 3H, H-16"), 0.95 (t, 6H,
J=7.5 Hz, H-18'), 1.25 [m, 40H, H-(4'-7')><2 and H-(4"-15")], 1.58 [m, 6H, H-(3'XZ),
3"], 2.01 [m, 8H, H-(8', 17')XZ], 2.29 (t, 4H, J=7.5 Hz, H-2'XZ), 2.28 (t, 2H, J=7.5
Hz, H-2"), 2.76 (m, 8H, H-11', 14'XZ), 4.12 (dd, 2H, J=18.0, 6.0 Hz, H-1a, 33),
4.36 (dd, 2H, J=16.2, 4.2 Hz, H-1b, 3b), 5.24 (m, 1H, H-2), 5.34 (m, 12H, H-9',
10' 12', 13', 15', 16'XZ); 13'CNMR (CDCI3) 6: 14.1 (C-16"), 14.3 (C-18'XZ), 27.2
(C-8', 17'XZ), 22.6, 22.7, 24.8, 24.9, 29.0-29.7 (C-4'-7'xz, 4"-15"), 25.5, 25.6 (C-
11', 14'XZ), 31.5 (C-3'XZ), 31.9 (C-3"), 34.0 (C-2'x2), 34.2 (C-2"), 62.1 (C-1, 3),
68.9 (C-2), 127.1-131.9 (C-9', 10', 12', 13', 15', 16'XZ), 172.3 (C-1'XZ), 173.3 (C-
1"). The spectral data conﬁrmed that this compound is 1, 3-dilinoleneoyl-2-

palmitin.

35

0 6 —3
O
O
14
O
O
6 —3
3

Results and Discussion

Three compounds, 1-3, were isolated from the leaf and stem hexane
extract of O. sanctum by successive silica gel VLC, MPLC, and preparative TLC.
The 1HNMR spectrum of compound 2 contained only ﬁve singlets. The peak
furthest upﬁeld at 6 1.23, integrated for 6 protons, indicated the presence of two
deshielded magnetically equivalent methyl groups, C-7 and C-8. Deshielding of
these methyl groups occurred as a result of a hydroxy group at C-6. Methyl
protons at C-1 and C-9 at 6 2.14 and 1.88, respectively, were indicative of
methyls attached to a carbonyl carbon and an oleﬁnic carbon. From the 13CNMR
spectrum, it was concluded that compound 2 contained an a, 8 unsaturated
ketone moiety (C-3, C-4, and C-2, respectively) with an additional oxygenated
carbon (C-6). This oxygenated carbon was determined to be a tertiary alcohol as
indicated by the disappearance of a singlet at 62.56 following a 020 shake in the
1HNMR spectrum. The DEPT spectrum supported the 1HNMR and 13CNMR data
as well as indicating the presence of three non-protonated carbons at 202.3,
157.4, and 69.8 ppm for one carbonyl, one oleﬁnic, and one oxygenated carbon,

respectively. Finally, the stereochemistry of 2 was determined to be E by

36

NOESY since no correlations were observed for the oleﬁnic proton on C-3 and
the methyl protons on C4. Therefore, compound 2, was conﬁrmed to be E-6-
hydroxy-4, 6-dimethyl-3-heptene—2-one. The 1HNMR spectrum of 2 was identical
to previously published ﬁndings (Kimura et al., 1982). The structure of
compound 3 was determined using 1H- and 13'CNMR, DEPT, and DQFCOSY
spectral data. Also, two-dimensional HMQC proton-carbon correlations
facilitated the assigning of saturated carbons in the fatty acid side chains and
provided further evidence to 1D-NMR experiments. The 1HNMR signals at 6
4.12, 4.36 and 5.24 and 13CNMR signals at 6 62.09 and 68.87 along with MS
fragments at m/z 611 [M*-C15H31O]+, 262 [C13H290+H]", and 261 [C13H2901”
conﬁrmed that compound 3 is a triglyceride with C16 and C18 fatty acid esters.
The overlapping multiplets at 6 5.34 for twelve protons correlated with
unsaturated carbons at 6 127.09 to 131.94 in the HMQC spectrum indicated the
presence of six double bonds in this molecule. Support for the chemical nature
of the side chains came from the GC analyses of the methyl esters of fatty acids
obtained from the hydrolyzed compound 3. The GC proﬁle conﬁrmed the
presence of the methyl esters of linolenic and palmitic acids with a ratio of 2:1,
respectively. Also, retention times for both methyl esters were identical to those
of authentic samples of linolenic and palmitic acid methyl esters analyzed under

the same conditions.

The novel triglyceride, compound 3, was not mosquitocidal. LD100 were

200 and 6.25 ug-mL'1 in 24 h for compounds 1 and 2, respectively, when tested

37

against fourth instar Aedes aegyptii larvae. There was no mortality for control

larvae.

In a previous report the phenylpropanoid, eugenol was found to act as an
attractant to the beetle, Maladera matn'da (Ben-Yakir et al., 1995). Eugenol, in
previous studies, was found to comprise 30-70% of the essential oil in O.
sanctum (Asthana et al., 1984). Compound 2 originally was isolated from green
and red bell peppers, Capsicum annum L. (Kimura et al., 1982) and later
identiﬁed in the culture broth CHCI3 extract of Streptomyces olivaceus (Grote et
al., 1990). The synthesis of 2 has been reported by Duperrier et al. (1975). To
the best of our knowledge, compound 3 has not been reported previously as a

natural product.

The isolation and identiﬁcation of mosquitocidal compounds in O. sanctum
supports earlier ﬁndings that had shown insecticidal activity in crude extracts
(Batta and Santhakumari, 1970; Risvi, 1981; Stein et al., 1988; Malik and
Raﬁque, 1989; Deshmukh et al., 1982). Additional insecticidal compounds will
be isolated from extractions of O. sanctum leaves and stems using more polar
solvents such as acetone. Chapters 3, 4 and 5 describe other biologically active

compounds in the acetone extracts of O. sanctum.

38

Chapter 3

ANTIOXIDANT COMPOUNDS FROM OCIMUM SANCTUM LINN.*

Abstract —Antioxidant bioassay-directed extraction of the fresh leaves and stems
of Ocimum sanctum and puriﬁcation of the extract yielded compounds, cirsilineol
(1), cirsimaritin (2), isothymusin (3), isothymonin (4), apigenin (5) and rosmarinic
acid (6). Appreciable quantities of eugenol (70-80%) also were isolated along
with these compounds. The structures of compounds 1-6 were established by
spectroscopic methods. Compounds 1, 5, and 6 were isolated previously from

O. sanctum, whereas compounds 2, 3, and 4 are identiﬁed for the ﬁrst time in O.
sanctum. Eugenol and compounds 1, 3, 4, and 6 exhibited good antioxidant

activity at 10 uM concentration.

* M. A. Kelm, G. M. Strasburg, and M. G. Nair Antioxidant compounds from

Ocimum sanctum Linn. Phytomedicine.

39

Introduction

Flavonoids represent one of the most ubiquitous classes of polyphenolic
secondary compounds found in higher plants. Many common fruits, vegetables,
herbs, and plant products such as wine, juices, dried fruits are rich sources of
ﬂavonoids. More importantly, many of these compounds have demonstrated
rather potent anti-oxidant activity by being able to block and or scavenge free
radicals (Saija et al., 1995). The formation of oxygen free radicals is a normal
phenomenon carried out in aerobic cells. The interaction of these free radicals
with lipids produces hydroperoxides and peroxides, which in turn may act
adversely with biological systems, resulting in cancer. By way of the free radical
scavenging mechanism, ﬂavonoids effectively negate the deleterious effects of
hydroperoxides and peroxides. Free radical formation also can result from the
presence of transition metal ions such as Fe”, which can act as free radical
initiators. Alternatively, ﬂavonoids can prevent the formation of free radicals by

chelation or complexation with the transition metal free radical initiator.

Concerning structural activity relationships (SAR) of ﬂavanoids, hydroxyl
groups at the C5 and C7 and the C2 - 03 double bond were shown to be
neceSsary for high inhibitory activity on xanthine oxidase (Cos et al., 1998).
Xanthine oxidase forms superoxide radicals and hydrogen peroxide and is
involved in the oxidation of hypoxanthine to xanthine to uric acid. Researchers

also have found that a hydroxyl group at C3' and at 03 were essential for high

40

superoxide scavenging activity (Cos et al., 1998). In addition, substitution
patterns on the B ring were important to antioxidant activity of ﬂavonoids, with
hydroxyl groups increasing activity by preventing lipid peroxidation (Arora et al.,

1998 and Arora et al., 1997).

Many of the previous studies on Ocimum sanctum Linn. Lamiaceae have
focused largely on the biological activity of crude extracts. A triterpene, ursolic
acid, isolated from O. sanctum was shown to be effective in protecting against
lipid peroxidation (Balenehru and Nagarajan, 1991). We have reported
mosquitocidal compounds eugenol and (E)-6-hydroxy-4, 6-dimethyl-3-heptene-2-
one from O. sanctum (Kelm and Nair, 1998). In the present study, we report for
the ﬁrst time the extraction, puriﬁcation, and structural identiﬁcation of anti-
oxidant compounds from the fresh leaves and stems of O. sanctum. Also, the
importance of the A ring substitution relative to anti-oxidant/free-radical

scavenging activity are discussed.

Materials and Methods

General Experimental. 1HNMR spectra were recorded at 300 and
500MHz. 13CNMR and DEPT spectra were recorded at 126 MHz. Chemical
shifts were recorded in DMSO-d5, CD300, and CDCI3. The values are in 6 (ppm)
based on residual of DMSO 2.29; DMSO-d5 39.7 and CHCI3 7.24; CDCI3 77.0.

Coupling constants, J, are in Hz. EIMS were recorded at 70 eV. UV

41

experiments were carried out on a Shimadzu UV-260 spectrophotometer. Shift
reagents were prepared and used according to Markham (1982). UV samples
were prepared at 125-50 ppm concentrations. Particle size of silica gel used in
VLC was 35-70 um. PLTC puriﬁcations were carried out on 250, 500, 1000, and
2000 um silica gel plates and 200 pm KC18 silica gel plates (60 A). Spots and

bands were visualized under UV light (366 and 254 nm).

Anti-oxidant assays. Anti-oxidant assays were conducted on crude
extracts and pure compounds by analysis of model liposome oxidation by
ﬂuorescence spectroscopy peroxidation (Arora et al., 1998 and Arora et al.,
1997). The procedure for the anti-oxidant assay is as follows. A mixture
containing 5 pmol of 1-steroyl-2-Iinoleneoyl-sn-glycerol-3-phosphocholine and 5
umol of ﬂuorescence probe 3-(p-6-phenyl)-1, 3, 5-hexatrienyl) phenyl propionic
acid was dried in a foil-covered round-bottom ﬂask (to prevent degradation of
probe) on a rotary evaporator. The resulting lipid ﬁlm was suspended in 500 uL
of MBSE buffer. The MBSE buffer contained 0.15 M NaCl, 0.1 mM EDTA
(ethylenediamine tetra acetic acid di-sodium salt), 10 mM MOPS [3-(N-
morpholine propane sulfonic acid), adjusted to pH 7.0] buffer and then treated
with Chelex 100 chelating resin to remove trace-metal ions. The lipid-buffer
mixture was subjected to ten-freeze thaw cycles using a dry ice/ethanol bath.
Following the last thaw, the lipid-buffer suspension was extruded 29 times
through a Liposofast extruder containing a polycarbonate membrane with a 100

nm pore size to produce unilamellar liposome. A 20 uL aliquot of this liposome

42

suspension was diluted to 2 mL with 200 uL of HEPES buffer (adjusted to pH
7.0), 100 uL of 1M NaCl (treated with Chelex 100), and 1.68 mL nanopure H20
then incubated for 5 min at room temperature, followed by incubation at 23°C in a

spectrophotometric cuvette. Peroxidation then was initiated by the addition of 20

uL of 0.5 mM FeClz stock solution to achieve a ﬁnal concentration of 5 um of Fe+2

in the presence of test compounds or crude extracts (dissolved in DMSO). Two
controls were used in which one contained test solvent (DMSO), liposome, and
buffer, whereas the second one contained Fe‘z. Fluorescence intensity of these
liposome solutions were measured at an excitation wavelength of 384 nm for
every 3 min over a period of 21 min. The decrease in relative ﬂuorescence
intensity with time indicated the rate of peroxidation. TBHQ, BHT, and Vitamin E

were used as positive controls for this study.

Plant Material. A voucher plant specimen (MSC 360851) of O. sanctum
was ﬁled with the Beal-Darlington herbarium, Department of Botany and Plant
Pathology, Michigan State University. Leaves and stems of O. sanctum were
harvested from plants maintained in the Center for Integrated Plant Systems
greenhouses at Michigan State University. 0. sanctum plants were grown in 11-
inch plastic pots, using a mixture of 50% loam and 50% Bacto mix. Plants were
watered on a daily basis and fertilized once a week with 20-20-20 Peters brand
fertilizer. Leaves and stems were harvested periodically from 4 to 6 month-old

plants.

43

Generalized Extraction of Plant Material. Three separate extractions (l-lll)
were carried out for the isolation of eugenol and compounds 1-6. The following
is a representative protocol used for the extraction of the plant material. 2.58 kg
of fresh 0. sanctum leaves and stems were chopped, blended with acetone (6.5
L), and allowed to set for 24 h. The acetone was removed in vacuo to yield an
aqueous green suspension. Solids were collected in a Bﬁchner funnel lined with
a No. 4 Whatman ﬁlter paper to give extract A (18.33 g). The aqueous ﬁltrate
was sequentially extracted with CHCI3 (5 x 250 mL) followed by EtOAc (5 x 250
mL) to provide extracts B (4.58 g) and C (12.12 g), respectively. The remaining

aqueous phase was dried in vacuo to yield extract D (56.40 g).

Extraction l. 763.9 g of fresh leaves and stems were chopped into pieces
approximately 10-15 mm long. These pieces were blended with acetone (2 L)
the allowed to soak for 24 h. Thereafter, the mixture was ﬁltered through a
Bﬁchner funnel lined with a No. 4 Whatman ﬁlter paper. The plant material was
washed with several aliquots of acetone (3x50 mL). The ﬁltrate was distilled to
remove acetone, resulting in the production of a green precipitate suspended in
an aqueous solution. The solids (Extract A; 10.5 g) were recovered by
centrifuging at 10,000 rpm for 10 min at 4°C. The remaining water was extracted
with CHCI3 (3 x 50 mL) to give upon drying in vacuo, Extract B (0.786 9). Extract

C (17.9 g) was obtained simply by drying the water-soluble phase in vacuo.

44

Extraction II. The second extraction carried out was essentially the same
as the previous with the exception of several modiﬁcations as described below.
450 g of fresh 0. sanctum was chopped, blended with acetone (1.2 L), and
allowed to set for 24 h. The acetone was removed in vacuo to yield a green
precipitate suspended in an aqueous solution. Solids were collected in a
Biichner funnel lined with a No. 4 Whatman ﬁlter paper to give Extract A (4.53 g).
The aqueous ﬁltrate was sequentially extracted with CHCI3 (3 x 50 mL) followed
by EtOAc (3 x 50 mL) to provide Extract B (0.49 g) and Extract C (1.16 g),
respectively. The remaining aqueous phase was dried in vacuo to yield Extract

D (7.1 g).

Extraction III. The third extraction utilized 1.37 kg of plant material. The
same procedure was followed as for the second extraction; however, larger
volumes of solvents were used. Acetone (3.3 L), CHCI3 (5 x 200 mL), and EtOAc
(5 x 200 mL) were used to obtain Extracts A, B, and C, respectively. Weights of

Extracts A, B, C, and D were 3.3, 3.3, 9.1 , and 33.3 g, respectively.

Isolation of eugenol and compound 1. Preparative TLC (silica gel; 3 x
2000 um) of Extract B (477.3 mg) using CHClgzMeOH (15:1) as the mobile
phase, afforded fractions, i-v. Fraction i (342 mg) and ii (14.4 mg) were
determined to be pure by TLC. Fraction iii was puriﬁed further (silica gel; 500
pm) using CHClgzMeOH (25:1) which gave fractions 1-4. Here, fraction 3 (9.7

mg) was determined by TLC to be identical to fraction ii. A second preparative

45

TLC (silica gel; 4 x 2000 pm) of Extract B (290 mg) using CHCI3zMeOH (15:1)
resulted in fractions i-v. In this puriﬁcation fractions i (195 mg) and ii (6.6 mg)
determined to be pure, by TLC, were identical to fractions obtained from the ﬁrst
puriﬁcation of Extract B. Fractions i from the ﬁrst and ﬁnal puriﬁcation were
combined and subsequently determined by NMR to be eugenol.

Eugenol, a pale brown oil: 1HNMR (C003) 6: 3.30 (d, 2H, J=6.6 Hz, H-7), 3.86
(s, 3H, -OCH3), 5.04 (dd, 1H, J=9.0 Hz, J=1.5 Hz, H-9 cis), 5.05 (dd, 1H, J=18.0
Hz, J=2.4 Hz, H-9 trans), 5.47 (s, 1H, -OH), 5.93 (m, 1H, H-8), 6.67 (d, 1H, J=7.8
Hz, H-6), 6.67 (s, 1H, H-3), 6.83 (dd, 1H, J=8.7 Hz, J=1.5 Hz, H-5); 13CNMR
(CDCI3) 6: 39.9 (C-7), 55.8 (-OCH3), 111.0 (C-9), 114.1 (C-8), 115.5 (C-6), 121.1
(C-5), 131.9 (C-3), 137.8 (C-4), 143.7 (C-2), 146.3 (C-1). The spectral data of
this compound was identical to an authentic sample purchased from Aldrich

Chemical Company, Inc. Milwaukee, WI 53233.

OMe
HO
\
eugenol
Fractions ii, iii, and ii from the ﬁrst, second, and third puriﬁcations,

respectively were combined and subsequently determined to be compound 1 by
NMR. Cirsilineol; 5, 4'-dihydroxy-6, 7, 3’-trimethoxyﬂavone (1). UV kmax
(MeOH) 341 nm, 275 nm; (5% AlCl3/MeOH) 378 nm, 284 nm, 262 nm;
(AICl3/HCI) 366 nm, 287 nm, 260 nm. 1HNMR (DMSO-d5) 6: 12.98 (bs, 1H, 5-

OH), 7.67 (d, 1H, J=9 Hz, H-6’), 7.54 (s, 1H, H-2’), 6.92 (s, 1H, H-3), 6.90 (d, 1H,

46

J=8.0 Hz, H-5'), 6.89 (s, 1H, H-8), 3.91 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), 3.72
(s, 3H, -OCH3). 13CNMR (DMSO—d5) 6: 56.56 (-OCH3), 57.09 (-OCH3),

60.70 (—OCH3), 92.28 (C-8), 103.25 (03), 105.65 (010), 115.57 (05'), 120.95
(C-6'), 121.35 (01'), 132.44 (C-6), 148.92 (0.5), 152.45 (09), 152.82 (0.3),

153.31 (cw), 159.13 (07'), 184.78 (0.2), 182.68 (04).

 

Isolation of compound 2. Extract B from Extraction I was fractionated
twice as previously mentioned. The third fraction (iii) from each of the
puriﬁcations, 4.2 and 22.5 mg, respectively were found to be identical by TLC.
Extract B (490.4 mg) from Extraction II was loaded onto preparative silica gel

TLC plates (3x2000 um) and developed with CHCI3: MeOH (15:1) as the mobile
phase. Bands i-v were isolated. Band i (318 mg) was determined to be
compound 1 by comparing its TLC with a previously identiﬁed sample obtained.
Band ii (10 mg) was found to be identical to both fractions iii above and these
subsequently were combined. The combined fractions were subjected to further
puriﬁcation by preparative TLC (2x250 pm) with CHClgzMeOH (20:1) as the
mobile phase. Bands i-iv were obtained. Fraction iii (3.5 mg) was found to be
suitable for NMR and aftenivards was determined to be compound 2.

Cirsimaritin; 5, 4’-dihydroxy-6, 7-dimethoxyﬂavone (2). UV kmax (MeOH) 329

47

nm, 278 nm; (5% AlClg/MeOH) 382 nm, 284 nm; (AlClg/HCI) 358 nm, 288 nm.
‘HNMR (DMSO-d5) 6: 7.94 (d, 2H, J=9.0 Hz, H-2’, 6'), 8.92 (s, 1H, 1+3), 8.90 (d,
2H, J= 9Hz, H-3’, 5'), 8.82 (s, 1H, H-8), 3.91 (s, 3H, -OCH3-6), 3.72 (s, 3H, -
OCH3-7). ”CNMR (DMSO-d5) 6: 56.51 (-oc113-7), 80.10 (-OCH3-6), 91.55 (C-8),
102.30 (03), 105.04 (010), 118.20 (03', 5'), 120.30 (or), 128.48 (02', 6’),
131.83 (C-6), 152.09 (0.5), 152.81 (0.9), 158.55 (07), 182.35 (C-4'), 184.23 (c-
2), 182.18 (C-4).

 

Isolation of compounds 3 and 4. Fraction iii from Extract B of Extraction l
and fraction iv from Extract B of Extraction II were combined (17.6 mg).
Preparative TLC silica gel (2x250 um) using CHClgzMeOH (10:1) as the mobile
phase was carried out on the mixture. Bands 1-4 were obtained, and fraction 4
was determined to be pure by TLC and subsequently was determined to be
compound 3 (6.7 mg) by NMR studies. lsothymusin; 5, 8, 4’-trihydroxy-6, 7-
dimethoxyﬂavone (3). UV kmax (MeOH) 328 nm, 306 nm; (5% AlClg/MeOH) 360
nm, 322 nm, 289 nm, 234 nm; (AICI3/HCI) 352 nm, 322 nm, 288 nm. 1HNMR
(00300) 6: 7.93 (d, 2H, J= 9 Hz, H-2', 6'), 6.92 (d, 2H, J=9.3 Hz, H-3’, 5’), 6.63

(s, 1H, H-3), 4.02 (s, 3H, -oc113), 3.91 (s, 3H, -oc1-13). 13CNMR (coaoo)

48

6: 61.35 (—OCH3), 61.99 (—OCH3), 104.46 (C-3), 107.94 (C-10), 116.99 (C-3’, 5’),
123.30 (C-2’, 6’), 132.25 (C-8), 137.81 (C-6), 142.86 (C-7), 146.42 (C-5), 149.20
(C-9), 162.92 (C-4’), 166.74 (C-2), 184.84 (C-4).

 

Band 4 from both puriﬁcations of Extract B, Extraction l were combined
(5.2 mg) and puriﬁed using silica gel preparative TLC (1x250 um) and
CHClazMeOH (15:1) as the mobile phase. Bands 1-3 were isolated. The NMR
analysis of fraction l resulted in the identiﬁcation of compound 4 (1 mg).
Isothymonin; 5, 8, 4’-trihydroxy-6, 7, 3’-trimethoxyﬂavone (4). UV kmax
(MeOH) 340 nm, 288 nm; (5% AlClg/MeOH) 450 nm, 371 nm, 327 nm, 292 nm,
263 nm; (AICI3/HCI) 450 nm, 364 nm, 331 nm, 293 nm, 259 nm. 1HNMR
I (CD3OD) 6: 7.61 (dd, J= 2.1, 9.0 Hz, 1H, H-6’), 7.60 (d, J=1.8 Hz, 1H, H-2’), 6.93
(d, J=8.7 Hz, 1H, H-5'), 4.03 (s, 3H, -OCH3-6), 3.96 (s, 3H, -OCH3-7), 3.91 (s,

3H, -OCH3-3’).

 

49

Isolation of compound 5. Fractionation of Extract B from Extraction III was
carried out using VLC. Extract B (3 g) was loaded onto a bed of silica gel (75 g)
preconditioned in hexane. Fractions were collected in aliquots of 125 (1), 150
(2), 100(3), 120(4), 160(5), 160(6), 150 (7), 210 (8), and 1,200 (9) mL using
hexane, hexanezacetone (1 :1), hexanezacetone (1 :1), hexanezacetone (1 :1),
acetone, CHCI3, CHClgzMeOH (1:1), CHClazMeOH (1:1), and MeOH,
respectively. The acetone-soluble portion of fraction 3 (190 mg) was dissolved in
MeOH and concentrated. This was allowed to stand overnight at room
temperature, resulting in the precipitation of solids. The mother liquor was
recovered and dried in vacuo to yield a brown gum (160 mg). A portion of this
was subjected to repeated silica gel PTLCs using CHClgzMeOH (30:1, 10:1), to
afford compound 5, apigenin (1.6 mg). Apigenin; 5, 7, 4’-trihydroxyﬂavone (5).
UV kmax (MeOH); (5% AlCl3/MeOH); (AICl3/HCI). 1HNMR (DMSO-d5) 6: 12.86 (bs,
1H, 5-OH), 7.73 (d, 1H, J=8.7 Hz, H-6’), 7.70 (d, 1H, J=8.7 Hz, H-2’), 6.79 (cl, 1H,
J=8.7 Hz, H-5’), 6.76 (d, 1H, J=8.7 Hz, H-3’), 6.34 (s, 1H, H-3), 5.80 (s, 1H, H-6),

5.57 (s, 1H, H-8), 3.44 (bm, 2H, 7, 4’-OH).

 

50

Isolation of Compound 6. Prior to fractionation, the EtOAc extract C (1.16
g) was ﬁrst dissolved in MeOH (1 mL), stirred with CHCI3 (16 mL) and
refrigerated overnight. The precipitate (630 mg) was recovered and dried in
vacuo. This precipitate (630 mg) was puriﬁed by medium pressure silica gel
column chromatography. Fractions were collected in aliquots of 750 (1), 1000
(2), 1000(3), 1000(4), 1500 (5), 200(6), 900 (7), and 850 (8) mL using
CHCI3zMeOH (3:1), MeOHzCHCla (2:1, 4:1, 4:1), 100% MeOH, 2 x 1% HCOOH
(in MeOH), 100% MeOH, respectively. The active fraction 2 (233 mg) was
dissolved in MeOH (5 mL) and precipitated with CHCI3. The pale yellow solids
were recovered and dried in vacuo to yield compound 6 (62.4 mg). Rosmarinic
acid (6). UV Amax (MeOH) 349 nm, 338 nm, 223nm. IR vmax cm'1 3350,
3250,1689, 1600, 1524. 1H-NMR (DMSO-d6) 6: 7.37 (d, 1H, J=15.9 Hz, H-7),
7.04 (d, 1H, J=1.8 Hz, H-2), 6.92 (dd, 1H, J=9.0, 2.1, 2.1 Hz, H-6), 6.75 (d, 1H,
J=2.1 Hz, H-5), 6.67 (d, 1H, J=2.1 Hz, H-2'), 6.60 (d, 1H, J=7.8 Hz, H-5'), 6.49
(dd, 1H, J=9.0, 2.1, 2.1 Hz, H-6'), 6.18 (d, 1H, J=16.2 Hz, H-8), 4.89 (1H, m H-
10), 3.04 (1H,, m, H-11), 2.77 (1H,, m, H-11). 13c-NMR (DMSO-d5) 8: 173.23
(C-12), 166.46 (C-9), 148.38 (C-4), 145.73 (C-3), 144.82 (C-1), 144.50 (C-4'),
143.49 (C-3'), 130.01 (C-1'), 125.76 (C-8), 121.24 (C-7), 119.93 (C-2'), 116.70
(C-2), 116.03 (C-5'), 115.47 (C-5), 114.99 (C-6'), 114.87 (C-6), 75.98 (C-10),
30.76 (C-11).

51

 

Results and Discussion.

Seven compounds, eugenol and 1-6, were isolated from the fresh leaf and
stem extracts of O. sanctum. Structure determination was facilitated by 1H and
13CNMR experiments. Final position assignments of ﬂavone hydroxyl groups
were achieved by the use of different shift reagents in UV ﬂuorescence
experiments. (Markham, 1982). According to Markham’s Interpretation of UV
spectra, compound 1 in MeOH indicated the presence of a ﬂavone with strong
absorption at 341 and 275 nm. In fact, the UV spectra of compounds 2-5
indicated the presence of ﬂavone. Following the addition of AICI3 stock solution,
a +37 nm shift in Band I in the spectrum of compound 1 was observed. This shift
was indicative of a hydroxyl substitution at the C-5 position. The presence of the
5-OH hydrogen bonded to the carbonyl oxygen at C-4 was conﬁrmed by the
chemical shift of the —OH signal at 6 12.98. As deduced from the AlCl3/HCI
spectrum, the presence of A or B ring o—diOH substitutions was not observed.

Two doublets at 6 7.67 (J = 9.0 Hz) and at 6.90 (J = 8.0 Hz) corresponded to

protons H-6’ and H-5’, respectively. Singlets at 6 6.92 and 6 6.89 represented

52

protons at H-3 and H-8, respectively. Intense, strong three proton singlets at 6
3.91, 3.87, and 3.72 were assigned to methoxy protons. These proton data were

found to be in agreement with those previously published (Martinez, et al., 1987).

Compound 2, cirsimaritin was isolated previously from the methanolic
extract of aerial parts of Baccharis trimera (Asteraceae) Less. (Grayer and
Veitch, 1998), the diethyl ether extract of freeze-dried leaves of Becium
grandiﬂorum (Lamiaceae) Pic. Serrn. (Yousseff and Frahm, 1995), the ethanolic
extract of the aerial parts of Centaurea scoparia (Asteraceae) Sieb. (Zhu et al.,
1996), the ethanolic (70%) extract of the air-dried root bark of Clerodendrum
mandarinomm (Verbenaceae) Diels. (Cuvelier et al., 1996), and was identiﬁed by
HPLC in two genera of the Lamiaceae family, Salvia ofﬁcinalis L. and

Rosmarinus ofﬁcinalis L. (Barberan et al., 1996).

The 5-OH and the 4’-OH for compound 2 was observed at 6 12.93 and
10.34, respectively. Two three-proton singlets at 6 3.91 and 3.72 were assigned
to methoxy groups at C-7 and C-6, respectively. Two proton doublets 6 7.94
(J=9.0 Hz) and 6.90 (J=9.0 Hz) indicated the presence of a para substituted
aromatic ring. These data were found to be in agreement with previously
published data (Zhu et al., 1996). A +25 nrn shift in the UV spectrum following
the addition of AlCl3/HCI further indicated the presence of the 5-OH substitution.
With AICI3 alone, shifts in band I were not large enough to indicate the presence

of any o-diOH substitution, in particular the A ring of the molecule.

53

lsothymusin, compound 3, was isolated from a natural source, B.
grandiﬂorum for the ﬁrst time (Yousseff and Frahm, 1995). lsothymusin (6, 7-
dimethoxy-5, 8, 4’-trihydroxyflavone) was previously known as a conversion
product via a Wessely-Moser rearrangement of the natural product, isothymusin
(7, 8-dimethoxy-5, 6, 4’-trihydroxyﬂavone). A protocol for this conversion was

reported (Horie et al., 1995).

In the present study, the identiﬁcation of compound 3 relied largely on UV
experiments, since 1HNMR data for thymusin and isothymusin are
interchangeable. A comparison of AICI3 and AlCl3/HCI spectra of compound 3
revealed only a small change, 8 nm in Band I, which indicated the lack of o—diOH
substitution pattern on the A ring. Our results are in agreement with those
previously published by Barberan et al. (1985). Two 2H proton doublets at 6 7.93
(J = 9.0 Hz) and 6.92 (J = 9.3 Hz) were indicative of a para disubstituted aromatic
ring. The above chemical shifts were assigned to H-2', 6’ and H-3’, 5',
respectively. Two intense three-proton singlets at 6 4.02 and 3.91 were assigned
to methoxy groups on the B ring at C-6 and C-7, respectively. 1HNMR data is
consistent with previously published work (Horie et al., 1995). lsothymusin can
be formed from its isomer by a Wessely-Moser rearrangement (Ferreres et al.,

1985).

Compound 4, isothymonin, is reported herein for the ﬁrst time as a natural

product. Isothymonin, like isothymusin is obtained by means of acidic treatment

54

and Wessely-Moser rearrangement. The UV spectral interpretation for
compound 4 was much like that carried out for compound 3. 1HNMR spectral
data were very similar to data obtained for isothymusin with the addition of a
three-proton singlet at 6 3.91 which was assigned to a methoxy group. The UV
and 1HNMR data for compound 4 were found to match closely previously

published data (Barberan et al., 1985 and Horie et al., 1995).

Apigenin is probably one of the most common of the ﬂavones. Apigenin,
compound 5, was previously identiﬁed in O. sanctum leaves (N6rr and Wagner,
1992), O. basilicum (Grayer et al., 1996), B. trimera (Youseff and Frahm, 1995),
and in the extracts of S. ofﬂcinalis and R. ofﬁncinalis (Cuvelier et al., 1996). The
antioxidant activity of apigenin was reported previously (Cholbi et al., 1991).
However, in another study (Chen et al., 1996), researchers found that apigenin

was unable to prevent the oxidation of lipids in canola oil.

The isolation and identiﬁcation of compound 6, rosmarinic acid was
reported from O. sanctum (Nbrr and Wagner, 1992) and S. ofﬁcinalis (Wang et
al., 1998). Portions of the 1HNMR spectrum, namely chemical shifts at 6 7.37
(J=15.9 Hz), 7.04 (J=1.8 Hz), 6.92 (J = 9.0, 2.1, 1.8 Hz), 6.75 (J = 8.4 Hz), and
6.18 (J=16.2 Hz) matched closely with those of caffeic acid. Furthermore, the
acidic hydrolysis of 6 yielded caffeic acid, as indicated by comparative TLC. The
chemical shifts at 6 7.37 (J = 15.9 Hz) and 6.18 (J = 16.2 Hz) were for doublets

that integrated for one proton each. Coupling constants for both of these oleﬁnic

55

protons (H-7, 8) indicated that they existed in a trans geometry. Protons at
positions 2, 5, and 6 gave identical splitting patterns with 2’, 5', and 6’,
respectively. Chemical shifts for H-2 and 2' were 6 7.04 (J = 1.8 Hz) and 6.67 (J
= 2.1 Hz), respectively. Their J values were indicative of meta coupling protons.
Ortho coupling was observed for H-5 and 5’ doublets. A 1H doublet of doublet
was assigned to H-6 and 6’ at 6 6.92 and 6.49, respectively. Both ortho and
meta coupling was observed for H-6 and 6’ where J values were 9.0, 2.1, 1.8 Hz
and 9.0, 2.1, 2.1 Hz, respectively. 13CNMR experiments indicated the presence
of two carbons at 6 173.23 and 166.46 which corresponded to the carboxylic acid
and ester functional groups, respectively. Both 1HNMR and 13CNMR data

agreed with published data (Wang et al., 1998).

Anti-oxidant assays were conducted according to previously published
procedures (Arora et al, 1998 and Arora et al., 1997). Results for anti-oxidant
bioassay are presented in Figure 1 and 2. All ﬂavones with the exception of
compound 2 demonstrated excellent anti-oxidant activity. Eugenol and
rosmarinic acid (6) also displayed good anti-oxidant activity. The most notable
compound in regards to anti-oxidant activity was compound 3, isothymusin.
These results suggest that hydroxy substitution on the A ring at C-8 enhances
anti-oxidant activity, whereas the addition of a methoxy group at C-3’ position
inhibits the activity, as in the case of compounds 3 and 4. However, in the case
of compounds 1 and 2, when the hydroxy at C-8 is lacking, the more active

compound contains a methoxy at the C-3' position. lsothymusin, compound 3,

56

was the best performer in regards to its anti-oxidant activity. Compound 3 was
about 50% more active than the synthetic antioxidants TBHQ and BHT at the
same 10 0M concentration. Compound 4 performed as well as compound 1 and
better than BHT and TBHQ at 10 uM concentration. Compound 1 performed

equally well if not better than TBHQ and BHT in our anti-oxidant assays (Figure
1, 2). Surprisingly, due to structural similarities, compound 2 displayed poor anti-

oxidant activity in our anti-oxidant assays (Figure 1, 2).

The presence of eugenol in O. sanctum is well known. In fact, eugenol
can comprise 38% of the water distilled essential oil of O. sanctum (Laakso et al.,
1990) and up to 86% in the steam distillate (Sukari et al., 1988). In the same
study, the essential oil of O. sanctum showed some inhibitory activity on
Tn'chophyton mertagrophytes (Robin) Blanchard and Bacillus subtilis (Ehrenberg)
Cohn at 10‘ ppm and 100% mortality on BIatteI/a gennanica L. and
Callosobrunchus chinensis L. in the presence of 10 mg of the neat oil. In a later
study, it was found that eugenol could demonstrate anti-stress activity by
effecting stress-induced changes brought about by various biochemical
parameters and cell membrane dynamics utilizing red blood cells (Sen et al.,
1992). Eugenol’s anti-oxidant activity prior to this paper was reported by
Priyadarsini et al., 1998. In their study, inhibition of lipid peroxidation of rat brain
homogenates induced by ferric ion, ferrous ion, and cumene hydroperoxide was

demonstrated by eugenol at leo 74.2, 11.3, 136.8 umol-dm", respectively.

Eugenol’s anti-oxidant activity was comparable to compound 6 (Figure 1, 2). The

57

presence of eugenol’s 4-hydroxy group could be involved in the observed activity

by formation of a less reactive phenoxyl radical.

Compound 1, cirsilineol was previously identiﬁed in O. sanctum (Nbrr and
Wagner, 1992), O. basilicum (Grayer et al., 1996), and Artemisia assoana VVIIIK.
(Martinez et al., 1987). Cirsilineol was found to have potent 3’, 5’-cyclic
adenosine monophosphate phosphodiesterase (cAMP-PDE) inhibitory activity
(Nagasugi et al., 1998). In this study the authors were able to correlate high

cAMP-PDE-inhibitory activity with averaged 13CNMR chemical shifts.

Cirsimaritin showed a 77% antimutagenic activity at 25 0M per plate in a
modiﬁed Ames test. Apigenin was 85.2% effective at the same concentration in
the same study (Grayer and Veitch, 1998). S. ofﬁcinalis and R. ofﬁcinalis
extracts that contained cirsimaritin and apigenin demonstrated anti-oxidant
activity as measured by accelerated auto-oxidation of methyl linoleneate

(Barberén et al., 1996).

Compound 6 compared to other compounds in the study demonstrated
high anti-oxidant activity. Compound 6 certainly was more active than Vitamin E,
and compounds 2 and 5 (Figure 1, 2). The anti-oxidant activity of rosmarinic acid
was reported previously from sage, Salvia ofﬁcinalis where it was found to
scavenge DPPH (2, 2-diphenyl picryhydrazyl) free radical-induced oxidation
(Wang et al., 1998).

58

Relative Fluorescence

Relative Fluorescence

 

Figure 1. Anti-oxidant activity of synthetic and isolated
oorrpounds assayed at 10uM.

Figure 2. Anti-oxidant activity at 21 minutes for synthetic and
isolated compounds at 10 0M.

 

59

Chapter 4

ANTI-INFLAMMATORY COMPOUNDS FROM OCIMUM SANCTUM LINN.

Abstract — Eugenol and compounds 1-6 described in the previous chapter were
subjected to anti-inﬂammatory assays. Eugenol demonstrated 97% COX-1
inhibitory activity when assayed at 1000 uM concentrations. Compounds 1, 2,
and 4-6 displayed 37, 50, 37, 65, and 58 % COX-1 inhibitory activity,
respectively, when assayed at 1000 uM. Eugenol and compounds 1, 2, 5, and 6
demonstrated COX-2 inhibitory activity at slightly higher levels when assayed at
1000 uM. The activities of compounds 1-6 were comparable to ibuprofen,

naproxen, and aspirin tested at 10, 10, and 100 uM, respectively.

60

Introduction

The formation of prostaglandins from arachidonic acid by prostaglandin
synthase is a well studied process. The formation and subsequent effect of
prostaglandins can result in the stimulation of inﬂammation and associated pain
(Stryer, 1988). Prostaglandin synthase contains both cyclo-oxygenase and
hydroperoxidase components; however, it is well understood that two distinct
isoforms of cyclo-oxygenase (COX) exist, namely COX-1 and COX-2 which are
both involved in the conversion of arachidonic acid to prostaglandins (Lipsky et
al., 1998). COX-1 is found throughout the body, particularly in the
gastrointestinal tract, kidneys and platelets, whereas COX-2 is found
predominantly in inﬂamed tissues. Both COX-1 and COX-2 are inhibited by
traditional non-steroidal anti-inﬂammatory drugs (NSAIDS) such as aspirin,
ibuprofen, naproxen, sulindac, diclofenac, etc. Many of these drugs are taken to
releive inﬂammation pain such as musculoskeletal pain including arthritis and
tendonitis, as well as other general aches and pains. Since traditional NSAIDS
are not speciﬁc in the inhibition of both COX forms, the inhibition of COX-1
seems to be associated with gastrointestal damage, renal dysfunction, and
platelet abnormalities (Simon et al., 1998). Therefore, much of the research in
the 1990s has been directed toward the discovery of NSAIDS with speciﬁc

inhibition towards the enzyme COX-2.

As stated in the previous chapter, ﬂavonoids are widespread components

in the plant portion of the human diet. And, many of these have low toxicity in

61

mammals. Their action as anti-inﬂammatory agents and their low toxicity make
them prime candidates in plant drug research. The anti-inﬂammatory activity of
ﬂavonoids is believed to be controlled, at least in part, by the addition of cyclo-
oxygenase (Kim et al., 1998). Flavonoids such as 3-hydroxyﬂavone, galangin,
quercetin, and kaempferol demonstrated good cyclo-oxygenase inhibitory activity
in assays utilizing rat mixed peritoneal leukocytes (Hoult et al., 1994). Other
researchers have reported similar ﬁndings (Kim et al., 1998, Tordera et al., 1994,
Middleton and Kandaswani, 1992). In a study looking at structural activity
relationships of ﬂavonoids, it was determined that ﬂavonoids lacking 3’, 4’-
dihydroxy substitution as well as fewer overall hydroxy groups had greater cyclo-
oxygenase inhibitory activity (Moroney et al., 1988). More speciﬁcally, it was
demonstrated that B-ring hydroxyl substitution decreased a ﬂavonoid’s ability to
inhibit cyclo-oxygenase. Baumann et al., (1979) indicated that ortho dihydroxy-
substituted phenols can act as initiators of cyclo-oxgenase by acting as cofactors

in prostaglandin generation, speciﬁcally in cell-free assays.

The compounds isolated from O. sanctum are described in Chapter 3
were eugenol, cirsilineol (1), cirsimaritin (2), isothymusin (3), isothymonin (4),
apigenin (5) and rosmarinic acid (6). These compounds were subjected to anti-
inﬂammatory assays in order to determine COX-1 and COX—2 inhibitory
activities. Of the compounds tested, eugenol (Saeed et al., 1995), apigenin (5)
(Todera et al., 1994 and Kim et al., 1998), and rosmarinic acid (6) have
demonstrated anti-inﬂammatory activity. The COX-1 inhibitory activity of

ﬂavones, 1, 2, and 4 are described here for the ﬁrst time. Eugenol, compounds

62

1, 2, 5, and 6 demonstrated better COX-2 inhibitory activity compared to COX-1

inhibitory activity. This would indicate speciﬁc enzyme inhibition towards COX-2.

Materials and Methods

Refer to Chapter 3, Materials and Methods section for General

Experimental, Plant Materials, Extraction, and Fractionation procedures.

Anti-inﬂammatory Assay. Human prostaglandin H synthase isozymes
(hPGHS-1) were expressed in cos-1 cells as described previously (Laneuville et
al., 1994; Meade et al., 1993). Cyclo-oxygenase activity (COX) was measured
by utilizing microsomal membranes (ca. 5 mg protein/ml in 0.1 M TrisHCL, pH
7.4) from sham-transfected cos-1 cells or from cos-1 cells transfected with the
plasmid pOSML - PGHS - 1. Cycle-oxygenase assays were performed at 37 °C
by monitoring the initial rate of 02 uptake using an 02 electrode (Instech
Laboratories, Inc., 5209 Militia Hill Road, Plymouth Meeting PA 19462-1216).
Each assay mixture contained 3 mL of 0.1 M TrisHCl, pH 8.0, 1 mmol phenol, 85
pg hemoglobin and 100 umol arachidonic acid. Reactions were initiated by

adding 5 to 25 pg of microsomal protein in a volume of 10-20 uL.

Instantaneous inhibition was determined by measuring the
cyclooxygenase activity initiated by adding aliquots of microsomal suspensions of
hPGHS—1 (10 pmol Ozlmin cyclooxygenase activity/aliquot) to assay mixtures

containing 10 umol arachidonate and 1000 (N concentrations of the test

63

hPGHS-1 (10 pmol Ozlmin cyclooxygenase activity/aliquot) to assay mixtures
containing 10 umol arachidonate and 1000 (N concentrations of the test
compounds (Figure 3, 4). Ibuprofen and naproxen were assayed at 10 pM and

aspirin at 1000 (M.

Results and Discussion

The in vitro anti-inﬂammatory activity of eugenol, compounds 1, 2 and 4—6

all demonstrated inhibitory activity against COX-1 as expressed in Figure 3.

Figure 3. In vitro anti-inﬂammatory assay of eugenol, compounds
1-6 and synthetics. Eugenol, compounds 1-6, and aspirin tested
at 1000 HM and ibuprofen and naproxen tested at 10 (M.

120

 

% COX-1 inhibitory activity

 

 

 

64

rosmarinic acid demonstrated 58% COX-1 inhibitory activity in comparison to the
other compounds. The activities of eugenol, apigenin (5), and rosmarinic acid (6)
were reported by Saeed et al., 1995, Todera et al., 1994, and Kim et al., 1998.
The COX-1 inhibitory activities of compounds 1, 2, and 4 are reported here for
the ﬁrst time. At 1000 uM, compounds 1-6 demonstrated COX-1 inhibitory
activity comparable to ibuprofen, naproxen, and aspirin at 10, 10, and 100 uM
concentrations, respectively. Ibuprofen, naproxen, and aspirin demonstrated 33,
58, and 46% COX-1 inhibitory activity, respectively. COX-2 inhibitory activities
were demonstrated by eugenol, compounds 1, 2, 5, and 6 when assayed at 1000
(M (Figure 4). Compound 4 was unavailable for testing COX-2 inhibitory activity.
Compound 3 did not inhibit COX-1 or COX-2 activity. This present study
supports earlier ﬁndings and suggests possible phytochemicals relative to
research on the anti-inﬂammatory activity of O. sanctum: Early studies had
shown that methanolic and aqueous suspensions of O. sanctum showed anti-
inﬂammatory activity in rats (Godwani, 1987); however, anti-inﬂammatory activity
was found to be less active than aspirin. Singh et al., 1996, found that the
volatile oil of O. sanctum could inhibit arachidonic and leukotriene-induced

inﬂammation. They concluded that inhibition of cyclo-oxygenase and lipo-

65

Figure 4. In vitro anti-inﬂammatory activity of eugenol,
compounds 1, 2, 5, 6, and aspirin tested at 1000 M and
ibuprofen tested at 10 M.

 

 

 

 

 

.4?

4?; 120

O

“l 100 - _ , .
E 80 a- 73:17: _ ___
g 60 g, 7 1.3155; __ ‘
.E 40 . .
N 1‘ .

5 20

o 0 — *

8\° f

 

oxygenase pathways in arachidonic acid metabolism might be occurring. The
present ﬁndings tend to support these earlier ﬁndings; however, inhibition of
enzymatic pathways might be only part of the overall activity. Tulsi has been
shown to decrease levels of uric acid in rabbits (Sarkar et al., 1990). Elevated

levels of uric acid are associated with gouty arthritis and other joint inﬂammation.

Structural activity relationships (SAR) of ﬂavonoids 1-5, relative to COX-1
inhibitory activity seemed dependent on number and position of hydroxy and
methoxy groups on their A and B rings. Similar conclusions regarding SAR can
be said of compounds 1, 2, 5, and 6 regarding COX-2 inhibitory activity. Due to

the small number of compounds being compared, unequivocal deductions

66

regarding SAR for these compounds cannot be made. Regardless, we can
discuss to some extent, the results obtained. The 5, 7, 4’-trihydroxyﬂavone,
apigenin (6) had the greatest COX-1 inhibitory relative to the other ﬂavones.
Methoxy groups at 6, 7, and 3’ positions resulted in a decrease in activity.
Compound 2, slightly more active that 1, had an additional methoxy group at the
3' position. One notable exception to this is the activity of compounds 3 and 4.
Compound 4 showed modest COX-1 enzyme-inhibitory activity, whereas
compound 3 totally lacked COX-1 enzyme inhibitory activity. The only difference

between these two compounds was a methoxy group at the 3’ position.

These ﬁndings present some compounds that lend support to earlier
observed anti-inﬂammatory activities of crude extracts from O. sanctum.
Furthermore, these results support the traditional use of Tulsi as a remedy for

inﬂammation and pain.

67

Chapter 5

A PORPHYRIN COMPOUND FROM OCIMUM SANCTUM LINN. WITH CORN
EARWORM ANTI-FEEDANT/TOXICITY ACTIVITY

Abstract- A porphyrin compound characterized as phylloerythrin like
compound was isolated from the crude acetone extract of O. sanctum by
bioassay-guided fractionation. This compound was anti-feedant to corn eanivorm,

Helicoverpa zea at 100 ppm.

68

Introduction

Insect toxicity (Narasimhan and Marriappan; 1988, Stein et al., 1988;
Sukari and Takahashi, 1988), anti-feedant, repellant (Mallick and Banerji, 1989,
Arrekul, et al., 1988) and oviposition deterrent activities (Sojitra and Patel, 1992)
were reported for extracts of O. sanctum. In their investigations, only crude
extracts were analyzed for bioactivity against insects. Biologically active

components of these crude extracts, remain unknown.

Preliminary bioassays indicated that the crude acetone extract from O.
sanctum contained a compound or compounds that were demonstrating anti-
feedant activity against Helicoverpa zea, corn earworm. This study undertook
the bioassay-guided fractionation approach to determine the presence of insect
anti-feedants or toxins utilizing H. zea, corn earworm bioassays. My initial
hypothesis of ﬁnding compounds responsible for corn earworm anti-feedant
activity evolved into a search for a porphyrin type compound as the fractionation
progressed. A red ﬂuorescing compound always seemed to be associated with

the active fractions.

Materials and Methods

Refer to Chapter 3, Materials and Methods section for General

Experimental, Plant Materials, and Extraction procedures.

69

Fractionation of crude acetone extract. A quantity of crude acetone
extract (12.5 g) was suspended in acetone (125 mL) and then kept in a freezer at
-20°C overnight to cause precipitation. After 24 h, the suspension was ﬁltered
through a bed of celite. The bioactive ﬁltrate (3.3 g) subsequently was
fractionated using reverse phase (Supelco bonded phase silica C-18) medium
pressure column chromatography (MPLC). Solvents used and corresponding
fractions collected were as follows: A (70:30; MeOHszo), B (90:10; MeOHzH20),
C-l (100% MeOH). Two fractions, H and l were found to be active and identical
according to TLC and subsequently were combined. The combined fractions
(173.2 mg) were subjected to preparative TLC (6x200 um, KCF18 silica gel
plates, 60 A) using 80:20 MeOHzHZO as the mobile phase which resulted in ﬁve
fractions (A-E). The bioactive fraction, fraction D (6 mg) was subjected to a ﬁnal
preparative TLC experiment (1 x 200 um, KCF 18 silica gel plates, 60 A) that
resulted in two bands, A (1.6 mg) and B (1.9 mg). Band A was pure enough to
conduct UV experiments. The UV absorption spectrum obtained was found to be
identical to the published data for the porphyrin, phylloerythrin (Perrin, 1958);
however, this band may contain other related porphyrin compounds as impurities
with similar UV absorption spectra proﬁles. These other porphyrins may have
similar biological activities. Similarities between 1H-NMR data of porphyrins
provided by Kenner et al. (1973) and the isolated compound were found. The
tentative structure for the active compound was elucidated using UV and NMR
studies. Band A; Porphyrin: UV Amax (MeOH) 659 nm, 584 nm, 557 nm, 512

nm, 420 nm. 1H-NMR(DMSO-d6) 8: 12.84 (1H, s, N-H), 13.99 (1H, s, N-H),

70

12.84 (1H, 8, COOH), 9.10, 8.52, 8.36 (3H, s, oleﬁnic), 3.47 (2H, s, -CH2CO-),
3.25 (4H, s, -CH3), 3.37 (4H, m, CH2CH3), 1.09 (6H, t, CH20H3).

3.25
1.09

9.10

 

Com earwonn Anti-feedant Assay. Helicoverpa zea Boddie Noctuidae
eggs were hatched in an incubator at 27°C. Dry diet (940 mg) obtained from
North Carolina lnsectory was placed into glass vials. Crude extracts were
dissolved in DMSO to give a concentration of 1250 mg-20pL". Subsequent
fractions were assayed at progressively lower concentrations. DMSO test
solutions (20 uL) were mixed thoroughly with the dry diet (940 mg). DMSO
(20uL) plus dry diet (940 mg) was used as the control. Agar solution/suspension
(1.4%) was autoclaved 5 min at 15 psi at 125°C to encourage melting of the
agar. The temperature of the agar was dropped to 45°C by the use of a water
bath set. The agar solution then was pipetted into individual vials containing the
test mixtures until a weight of 5 g was obtained. The vials were mixed thoroughly

and poured into 3.5 mL polystyrene vials. One neonate larvae was place in each

71

vial. Vials were capped and placed in a growth chamber with a photoperiod of
16-h days and 8-h nights. Day and night temperatures averaged 27°C. There
were 15 replications per treatment. Larvae were weighed after six days.
Bioassay protocols were adapted from previously published work (Bell, R. A.,
and Joachin, F. G., 1976 and Joyner, K. and Gould, F., 1985). An analysis of
variance (ANOVA) was conducted on the data using a completely randomized

design (CRD).
Results and Discussion

The porphyrin-containing fraction, responsible for the corn earworm anti-
feedant activity, has a UV absorption spectrum identical to previously published
data (Perrin, 1958); however, several other compounds including phylloerythrin
methyl ester, have similar UV absorption spectra proﬁles (Wolf and Scheer,
1973). 1HNMR data indicated a mixture ofcompounds similar to previously
published data (Kenner et al., 1973) namely, peaks for the ethyl, methyl, and

unsaturated protons.

The results of the corn earworm bioassay indicated that the treatments
assayed at 100 ppm had a highly signiﬁcant (p < 0.005) effect on the caterpillar

weight (Figure 5). It is important to point out that the fractions assayed (C and D)

contained the isolated compound in addition to smaller amounts of other

72

porphyrin-type compounds. Therefore, in actuality, biological activity might be

due to other porphyrin type compounds working in concert or independently.

Figure 5. Corn earworm antifeedant/toxic activity of two
fractions containing isolated porphyrin. Significant at P <

 

 

 

0.005.

A 37

0’ 1

gsti-.2 e s f 4,-2 ,.z____z

:5, 2 l-_ _ i ,v , a i i

m ,

3 15 L 4 9 v i i i i , L 7

8, 1: J“? * +2 2 A _

L“

g0.5,-f _ s -5 E. 2 z

< 01—2 , ,. .- - i
c D

Porphyrin-based compounds, such as phylloerythrin, result from the
normal rumen microbial breakdown of chlorophyll from ingested forage plants in
livestock animals. The phylloerythrin is absorbed and transported to the liver.
Healthy livers transfer the phylloerythrin to the bile for excretion. Livers damaged
by the fungal toxin, sporidesmin, cannot properly metabolize phylloerythrin, which
then accumulates in peripheral blood. Therefore, circulating phylloerythrin in the
blood causes the photosensitization reaction in nonpigmented skin (Cheeke,
1997). Photosensitization manifests itself often as facial eczema. The observed

eczema is an example of "secondary photosensitization," in which the skin

73

lesions are really the secondary result of liver damage, rather than the direct
result of a plant toxin (Hansen et al., 1994). Extrapolation of the above
information to our corn earworm bioassays and the activity that was observed,
may not be necessarily relative to photoxicity to H. zea caterpillars. However, it
is possible that phototoxicity resulting from abnormal chlorophyll metabolism
might be occurring in the H. zea gut and resulting in mortality and weight loss.
As of 1995, phototoxin-mediated effects and phototoxicity in insects resulting
from abnormal chlorophyll metabolism is not know to exist (Berenbaum, 1995);
however, akin to this phenomenon, involved the administering of synthetic
compounds which interfere with heme biosynthesis from protoporphyrin IX
(Rebeiz et al., 1990 and 1988). Lethal accumulation of protoporphyrin lX in
Trichoplusia ni Hubner and H. zea larvae was found to result following the
administration of 2, 2’-dipyridyl alone or in combination with 6-aminolevulenic
acid (Rebeiz et al., 1988). Lethal accumulation of protoporphyrin IX in the insect
was associated with regurgitation, convulsions, and loss of body ﬂuids. Larval
death occurred in the dark and light, however, the treatment appeared to be
photodynamic in nature. Also, exogenous applications of protoporphyrin and Mg-
protoporphyrin also exhibited photodynamic damage. The compound, 1, 10-

phenanthroline exhibited similar effects in T. ni (Rebeiz et al., 1990).

The preliminary evidence in this study indicates that a porphyrin like

compound is responsible for the observed mortality of corn earworm larvae.

Whether or not a phototoxic or related effect is occurring, remains unknown.

74

Whatever the mode of action, this work may provide a basis for further research

on the insecticidal activity of porphyrins.

75

Chapter 6

SUMMARY

The body of information about 0. sanctum prior to this work was
presented in Chapter 1 and also formed much of the impetus for the current
work. Much of the literature summarized in Chapter 1 focused primarily on the
biological activity of crude extracts of O. sanctum. This early work stimulated the
formulation of a multidimensional hypothesis namely that, O. sanctum contains
compounds capable of exhibiting anti-oxidant and anti-inﬂammatory as well as
insecticidal and anti-feedant activities. Compounds possessing these activities
were isolated and identiﬁed as discussed in Chapters 2-5. Identiﬁcation of

isolated compounds was determined by the use of various spectral techniques.

Chapters 2 and 5 focused on mosquitocidal and corn earworm anti-
feedant activities, respectively. Isolated as oils, eugenol and E—6—hydr0xy-4, 6-
dimethyl-3-heptene-2-one demonstrated mosquitocidal activity at 200 and 6.25
pg-mL’1 in 24 h, respectively on fourth instar A. aegyptii larvae. Both compounds
with the addition of a novel triglyceride, 1, 3-dilinoleneoyI-2-palmitin were isolated
from the leaf and stem hexane extract of O. sanctum. Bioassay directed
fractionation experiments conducted in Chapter 5 resulted in the identiﬁcation of
a porphyrin compound that displayed toxic activity against corn eanNorm larvae.
Porphyrins are known for their photochemical activity. Therefore, the porphyrin

isolated may possess a phototoxic or photosensitizing effect that results in the

76

death and lower weigh of the test insect by either interfering with heme
biosynthesis or interaction with other chemical aspects of the insect. Given these
preliminary data, further research into the insect toxicology of porphyrins is

warranted.

Preliminary anti-oxidant assays on crude extracts from O. sanctum had
shown excellent anti-oxidant activity. With antioxidant activity in mind, the
isolation and identiﬁcation of eugenol, cirsilineol, cirsimaritin, isothymusin,
isothymonin, apigenin, and rosmarinic acid from acetone extracts of fresh 0.
sanctum leaves and stems was carried out in Chapter 3. Eugenol, cirsilineol,
isothymusin and rosmarinic acid demonstrated good antioxidant activity when
assayed at 10 uM. Cirsimaritin, isothymusin, and isothymonin were identiﬁed for
the ﬁrst time in O. sanctum. lsothymonin was also reported as a natural product

for the ﬁrst time.

The antioxidant compounds isolated and identiﬁed in Chapter 3 were
subjected to COX-1 inhibitory assays (Chapter 4). Eugenol was by far the most
active, showing 97% COX-1 inhibitory activity when assayed at 1000 (M. The
activity of eugenol exceeded the activity of aspirin by 51% at the same molar
concentration. Apigenin and rosmarinic acid also performed well, demonstrating
65 and 58% COX-1 inhibitory activity, respectively. The other compounds

demonstrated comparable activity with ibuprofen and aspirin at 10 pM and 1000

77

pM, respectively. Some of the same compounds demonstrated slightly higher

levels of COX-2 inhibition at the 1000 uM thereby suggesting speciﬁcity.

Natural mosquito control could conceivably be accomplished by utilizing
conventional aerosol sprays or novel chemical dispersion technologies in the
deployment of essential oils from O. sanctum. Human health concerns as well
as an impact on the environment would be alleviated by the use of O. sanctum

as a natural form of insect pest control.

The anti-feedant and/or toxic nature of the porphyrin compound against
corn earworm is new and therefore warrants further investigation. Determination
of mode of action, phototoxicity, and its possible applications would be

worthwhile endeavors for additional research.

These research results could provide support for the use of O. sanctum in
food supplements or nutraceuticals in the prevention of cancer and in the
reduction of inﬂammation. The acceptance of O. sanctum as an alternative to
conventional treatments would be well received by the public considering the

current trend towards natural alternatives to drug treatments.

This research has added to the body of literature dealing with O. sanctum.

More speciﬁcally, the current work has led to the ﬁrst identiﬁcation of biologically

active compounds from O. sanctum. Additional studies should be undertaken to

78

determine the presence of other biologically active compounds contained in O.
sanctum. These compounds may hold potential for pharmaceutical and or

agricultural applications in addition to being a worthwhile research endeavors.

79

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