CHEMISTRY AND INSECTICIDAL POTENTIAL OF PARTHENIN AND ITS TRANSFORMATION REACTION

PRODUCTS AGAINST Tribolium castaneum (Herbst).

Thesis

Submitted to the Punjab Agricultural University in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE in

CHEMISTRY (Minor Subject: Biochemistry)

By

Ramandeep Kaur (L-2010-BS-196-M)

Department of Chemistry College of Basic Sciences and Humanities

© PUNJAB AGRICULTURAL UNIVERSITY LUDHIANA – 141 004

2012

CERTIFICATE I

This is to certify that the thesis entitled Chemistry and insecticidal potential of

parthenin and its transformation reaction products against Tribolium castaneum

(Herbst) submitted for the degree of M.Sc., in the subject of Chemistry (Minor subject:

Biochemistry) of the Punjab Agricultural University, Ludhiana, is a bonafide research work

carried out by Ramandeep Kaur (L-2010-BS-196-M) under my supervision and that no part

of this thesis has been submitted for any other degree.

The assistance and help received during the course of investigation have been fully

acknowledged.

_________________________ MajorAdvisor Dr. (Mrs.) K. K. Chahal Professor-cum-Head Department of Chemistry Punjab Agricultural University Ludhiana - 141004

CERTIFICATE II

This is to certify that the thesis entitled, Chemistry and insecticidal potential of

parthenin and its transformation reaction products against Tribolium castaneum

(Herbst) submitted by Ramandeep Kaur (Admn. No. L-2010-BS-196-M) to the Punjab

Agricultural University, Ludhiana, in partial fulfillment of the requirements for the degree of

M.Sc. in the subject of Chemistry (Minor subject: Biochemistry) has been approved by the

Student’s Advisory Committee along with Head of the Department after an oral examination

on the same.

_____________________ ______________________ {Dr. (Mrs.) K. K. Chahal} {Dr. (Mrs.) K. K. Chahal} Head of the Department Major Advisor ______________________ (Dr. Gursharan Singh) Dean Postgraduate Studies

Acknowledgements Firstly, I bow my head with utmost reverence before the Almighty whose eternal blessing has enabled me to accomplish this noble effort.

It gives me immense pleasure to record my thanks and sense of profound gratitude to my Major Advisor Dr. (Mrs.) K. K. Chahal, Professor-cum-Head, Department of Chemistry, Punjab Agricultural University, Ludhiana, for her expert guidance, encouragement, inspiration and advice throughout my research work. It was my privilege to be guided by a person of calibre, whose blessings bring best in every one of my endeavours.

Special thanks are accorded to Dr. B. R. Chhabra, Professor Adjunct for his excellent technical guidance and support.

I owe my unpayable debt to the other esteemed members of my advisory committee. Dr. (Mrs.) Manpreet Kaur, Assistant Professor, Department of Chemistry, Dr. (Mrs.) Bavita Asthir Senior Biochemist, Department of Biochemistry, Dr. (Mrs.) B.K. Kang, Associate Professor, Department of Entomology, Dr. (Mrs.) S.K. Uppal ,Senior Biochemist-cum-Incharge of Sugarcane Section, Department of Plant Breeding and Genetics, for their able guidance , constructive suggestions and continuous support.

I am indebted to my respected family for their constant words of encouragement, deep affection and heartful blessings that enabled me to this stage of career.

Friends are always a moral support which is extremely important when one is feeling low. I take great pleasure in thanking my friends Ajay, Dalvir, Amanpal and Amit for giving me moral support, sharing the burden of my work and making things smooth.

My sincerest thanks to Mr. Banjit Singh, Mr. Mukesh Kumar and Mr. Raj Singh for their invaluable and generous help in the laboratory. I feel proud to be a part of PAU, Ludhiana where I learnt a lot and spent some unforgettable moments of my life.

I thankful to Punjab Agricultural University for providing merit fellowship during final year of my M.Sc.

Last but not least, I duly acknowledge my sincere thanks to all who love and care for me.

_____________ (Ramandeep Kaur)

Title of the Thesis : Chemistry and insecticidal potential of parthenin and its transformation reaction products against Tribolium castaneum (Herbst)

Name of the Student : Ramandeep Kaur and Admission No. L-2010-BS-196-M Major Subject : Chemistry Minor Subject : Biochemistry Name and Designation : Dr. (Mrs.) K. K. Chahal of Major Advisor Professor- cum-Head

Degree to be Awarded : M.Sc. Year of Award of Degree : 2012 Total Pages in Thesis : 73 + VITA Name of University : Punjab Agricultural University, Ludhiana-141 004

ABSTRACT

The present investigation deals with Chemistry and insecticidal potential of parthenin and its transformation reaction products against Tribolium castaneum (Herbst).The shade dried and powdered leaves of Parthenium hysterophorous were extracted in chloroform using Soxhlet extraction method. Parthenin was isolated by column chromatography using chloroform:acetone (5%) solution as the eluent. Parthenin was subjected to reaction with diazoester which resulted into the formation of two compounds- pyrolysis product and diazoester adduct. Parthenin on reactions with dry hydrochloric acid gas and formic acid gets converted into anhydroparthenin. Parthenin on irradiation with microwave gets converted into anhydroparthenin. Parthenin and its derivatives were characterised on the basis of melting point, TLC, FT-IR and 1H NMR. Parthenin and its derivatives were tested for their bioefficacy against adults of Tribolium castaneum (Herbst) by releasing them in wheat grains spiked with various concentrations of test compounds viz. 1,000, 2,000, 4,000, 5,000, 10,000 and 20,000 μg g-1 of wheat respectively. The observations of mortality were noted every 24 hours till complete or constant mortality was obtained. The corrected per cent mortality was calculated using Abbott’s formula. All the compounds exhibited complete mortality at the spiking level of 10,000 and 20,000μg g-1. Parthenin was found to be most potent followed by anhydroparthenin, pyrolysis product and diazoester adduct. Key words: Parthenium hyseterophorous, Bioefficacy, Tribolium castaneum, Soxhlet

extraction. ______________________ ____________________ Signature of Major Advisor Signature of the Student

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CONTENTS

CHAPTER TOPIC PAGE

I. INTRODUCTION 1-4

II. REVIEW OF LITERATURE 5-24

III. MATERIAL AND METHODS 25-32

IV. RESULTS AND DISCUSSION 33-57

V. SUMMARY 58-60

REFERENCES 61-73

VITA

CHAPTER – I

INTRODUCTION

Wheat (Triticum aestivum L.) of Family Gramineae is an important staple foodstuff

in North India. Post-harvest losses of wheat are due to biotic (insects, molds, rodents and

birds) as well as abiotic (temperature, relative humidity and moisture content of the grains)

factors. Among the biotic processes, insect pests are the major agent, which cause

considerable losses in terms of quality and quantity of food grains. The damage caused by

insect pests to wheat grain has been estimated at 10 to 20 percent (Ramzan et al 1991, Khan

et al 2010). Tribolium castaneum has been found to be one of major insect pest of wheat

according to surveys conducted (Mahmood et al 1996, Ghizdavu and Deac 1994, Khalil and

Irshad 1994, Desimpelaere 1996, Bandyopadhyay and Ghosh 1999).

Red flour beetle, T. castaneum (Herbst) is one of the major insect pests of stored

grains with cosmopolitan distribution (Ghizdavu and Deac 1994, Desimpelaere 1996, Abro

1996, Wong et al 1996 and Hulasare et al 2003). Although, T.castaneum is considered a pest

of flour and other milled cereal products and is also considered as a secondary pest in stored

wheat (Le Cato 1975, Hamed and Khattak1985, Irshad and Talpur 1993). A single larva can

attack 88 grains during its life time which leads to a considerable loss of quality and viability

of grain (Atanasov 1978). Apart from loss of weight and quality of food grains, insects of

genus Tribolium secrete a variety of toxic quinones which are known to be carcinogenic.

Presence of Tribolium species in the food grains give pungent smell and infested flour

becomes dirty yellow in colour (El-Mofty et al 1989) which affect baking quality of flour

(Flogliazza and Pagani 2003).

To prevent the loss during storage, farmers usually rely on synthetic chemical

insecticides. Methyl bromide was used as fumigant in past. Its use has been restricted due to

ozone layer depletion (Zhang and Van Epenhuijsen 2004).These problems lead to

increasingly stringent environment regulation of pesticides (Isman 2006, Pavela 2007). At

present there is an urgent need to develop safer, more environmentally friendly and efficient

alternatives that have the potential to replace synthetic pesticides.

Among the various alternatives, use of natural plant products called allelochemicals

offer a new approach for the management of noxious weeds and pests in sustainable manner

(Macias et al 2001).Their toxicities as well as repellent effects on the pests were of special

interest during the last decade. Plant essential oils are alternative to synthetic pesticides

possess insecticidal, ovicidal, repellent and ovipositional activities against various stored

product insects (Chiasson et al 2004, Tripathi and Kumar 2007,Tripathi et al 2009, Aboua et

al 2010).

2

Secondary metabolites are known to exhibit a broad spectrum of biological activities.

Among them, sesquiterpene lactones are most widely distributed in the members of family

Compositae. Sesquiterpenes containing α-methylene-γ-lactone moiety have attracted a great

deal of interest to explore their role as cytotoxic agents (Liu et al 2008), anticancer agents

(Zhang et al 2005), anti-inflammatory agents (Hall et al 1979), antioxidants (Jung et al 2004)

and plant growth regulators (Chhabra et al 1998). Due to their ability to undergo a Michael

reaction with biological nucleophiles, α-methylene-γ-lactone has been reported to possess

biological activity (Macias et al 1996). Due to their biologically active nature, these

compounds have been investigated the most for their chemistry, mechanistic pathways,

chemical transformations and synthesis (Rodriguez et al 1976). The possibility that they can

provide a lead in the search for new plant growth regulators have helped in isolation and

partial synthesis of some of the most potent compounds in which structural features other

than α-methylene-γ-lactone moiety are significant which keep the field open for further

chemical studies.

Parthenium hysterophorous L. is rich source of α-methylene-γ-lactone containing

sesquiterpenoids. This annual or biennial herbaceous plant originated from tropical America

but has spread throughout the world’s tropical areas. It can grow and reproduce itself any

time of the year. During a favorable growing season, four or five successive generations of

seedlings can emerge at the same site. Low temperature considerably reduces plant growth,

mainly flowering and seed production by reducing leaf area index, relative growth rate, net

assimilation rate, and leaf area duration (Pandey et al 2003). The weed grows fast and

comfortably on alkaline to neutral clay soils. However, its growth is slow and less prolific on

a wide range of other soil types (Rezene et al 2005). It has become a serious problem in

many parts of the world due to its threat to agricultural activities, biodiversity and human

health and has also been labelled as a useless weed. The weed is particularly problematic in

India and Australia where it was first noticed in the 1950s and has continued to spread at an

alarming rate (Navie et al 1996). In South Africa, the weed first invaded the warmer and

wetter eastern parts of the country in the 1880s, and is currently spreading to several

prominent game reserves, including Hluhluwe-I Mfolozi and the Kruger National Park

(KNP) (Strathie et al 2005). P. hysterophorus is a known invader of disturbed areas such as

roadsides, agricultural fields and wastelands (Navie et al 1996). Characteristics that make the

weed such an effective invader include tolerance of a wide range of ecological and climatic

conditions, a fast growth rate, high fecundity and efficient utilization of resources (Hedge

and Patil 1982). The plant, however, is a folk remedy (Towers et al 1977) against various

afflictions:

3

SOURCE USES

Barbados Flowers and leaves used for inflammation eczema, skin rashes.

Cuba Common name Escobar Amarga, medicinal plant, febrifuge, bitter and

corroborant (Roig 1953).

Gaudeloupe Febrifuge, used for herpes and rheumatic pains.

Guadeloupe and

Martinique

Common names Absinthe batard, herbe, a pian, metricaire allude to

cure of female ailments (Duss 1972).

Guyana Used for skin eruptions.

Jamaica Supposed to be used in resolutive baths and infusions and for treatment

of wounds. Country people use it to prepare a decoction for colds and to

make a bath for fleas on dogs. The plant is said to contain bitter

glucosides. The plant is still used for ‘bush baths’ in the Kingston area

and perhaps, elsewhere as well.

Trinidad Used, along with other herbs, in the preparation of bush baths for

cleansing the skin.

Mexico Analgesic properties, particularly in muscular rheumatism. Used by

aztecs as remedy against headache and ulcerated sores (Herz et al

1962).

U. S. Virgin Islands

Used for muscular strains, analgesic, vermifuge and heart trouble.

United states In Montgomery, Ala, it was purposed to be efficacious as a skin tonic

by older people.

The aqueous extracts of this allelopathic weed are important as potential agents to be

manipulated for biological control of pathogenic fungi, only when these extracts are used at

lower concentrations; whereas, at higher concentrations, a potent increase in biomass

production may prove to be beneficial for mass production of mycoherbicides to control the

weeds of economically important crops.

Parthenin has been reported to be located in various plant parts with especially high

concentrations occurring in trichomes on the leaves (Kanchan 1975, McFadyen 1995,

Reinhardt et al 2004). On the molecular level, sesquiterpene lactone biosynthesis is regulated

at the transcriptional level, and these compounds generally originate from the mevalonic acid

pathway. It has been suggested that all terpenes originate from the common precursor,

isopentenyldiphosphate. Reinhardt et al (2004) determined that one trichome type in

4

particular, the capitate-sessile trichome, contained virtually 100 percent parthenin. Reinhardt

et al (2004) further quantified the amount of parthenin present in one capitate-sessile gland

at 0.3 μg parthenin per gland and suggested that these trichomes are the main source of

parthenin that is released from the plant. P. hysterophorous contains diverse allergenic

sesquiterpene lactones, as shown by chemical, phytochemical and biological analysis.

Picman et al (1982) classified the plants collected in several continents into seven types,

according to the lactones present in them:

Type I: Parthenin, coronopilin and tetraneurin A

Type II: Parthenin, coronopilin

Type III: Coronopilin

Type IV: Hymenin, coronopilin and dihydrohymenin

Type V: Hymenin, coronopilin and hysterin

Type VI: Hymenin and hysterin

Type VII: Hymenin

Parthenin, having α-methylene-γ-lactone moiety along with other functionalities and

five chiral centres, is interesting for its structural pattern and biological activity including

antimalarial, antiameobic, allelopathic, antitumour, antifungal, nematicidal and antibacterial

etc. It has been associated with dermatitis and related skin disorders. Derivatisation of

parthenin is known to affect the biological activity and some of the derivatives have been

found to show the activity comparable with Azadirachtin (Datta and Saxena 2001).

The objectives of present study were:

1. Isolation of parthenin.

2. Transformation reactions of parthenin.

3. Bioefficacy studies of parthenin and its derivatives against Tribolium castaneum.

The thesis runs into several chapters, namely, review of literature, materials and

methods, results and discussion, which is followed by summary.

Since almost the entire investigation incorporated in this thesis is on isolation and

transformation reactions of parthenin and its bioefficacy against stored products insect pest

of wheat i.e. T. castaneum (Herbst), a review of literature on the parthenin, its transformation

reaction products and their bioefficacy against stored product insect pests was thought to be

appropriate. This review is given in chapter II of the thesis. In chapter III a brief outline of

various methods and techniques employed in the investigation are described. Chapter IV is

devoted to results and discussion. Chapter V gives the summary of the research work carried

out. The references cited in the text are alphabetically arranged at the end of the thesis.

CHAPTER – II

REVIEW OF LITERATURE

Insect pests are the major problem in stored grains because they affect their quantity

and their quality (Madrid et al 1990). The susceptibility of stored grains to insect infestation

depends on some factors such as harvest, environmental conditions, bulk purity, storage

facilities and pest control methods (Lee et al 2001). Insect damage accounts for 10-40

percent of loss in stored grains, worldwide. It is obvious that the red flour beetle Tribolium

castaneum is a cosmopolitan and polyphagous stored product pest. It is among the major

pests of stored grains and stored products throughout the world (Small 2007). In Tunisia and

North Africa, Jarraya (2003) reported that this insect is the most important and destructive

pests in mills.

Fumigation is the most economical tool for managing these stored pests (Azelmat et

al 2006). There is a great need, for the development of alternative control methods that

would be both effective and environment friendly. Several alternatives have been tested as

replacement for methyl bromide for the control of stored pests. These include fumigants such

as phosphine, sulfuryl fluoride and carbonyl sulphide (Fields and White 2002), as well as

ethyl formate (Desmarchelier et al 1998) and compounds like alkylphosphines (Chaudhry et

al 2000), cyanogens (O’Brien et al 1999) and isothiocyanates (Shaya et al 2003). Moreover,

carbon dioxide has been used for disinfesting storage commodities. Annis (1987) reported

the toxic effects of carbon dioxide-rich atmospheres on several insect pests of stored

products. Furthermore, Gamma radiation using Cobalt 60 (synthetic radioactive  isotope of

cobalt) could be employed to disinfest stored product insects (Ramos et al 2007). In addition,

insect growth regulators (IGR) may be used to manage stored insect pests (Mohandass et al

2006). Furthermore, potential use of semiochemicals was reported to protect stored products

from insect infestation (Cox 2004). Although, the effectiveness of these methods seems

good, these are of global concerns due to their negative effects (Kostyukovsky et al 2002,

Negahban et al 2006 and Ogendo et al 2003). Therefore, among Integrated Pest Management

tactics, plants played a significant role because they constitute an important source of

insecticides (Golob and Webley 1980). In recent years, use of biopesticides is preferred in

comparison to synthetic pesticides as they are ecofriendly and biodegradable (Kumar et al

2008).

Plant extracts contain many secondary metabolites. These metabolites feature several

properties against insects, like insecticidal, antifeedant and growth regulatory activity.

Secondary metabolites considered as that substance or mixture of substances that exert

biocide action due to their chemical nature (Celis et al 2008). However, most of the plants

6

used against insects have an insectistatic effect, rather than insecticidal. This refers to the

inhibition of the insect’s development and behaviour, and it is divided into: repellence,

antifeeding activity, growth regulation, feed deterrents (Koul 2004) and oviposition

deterrents.

Repellent activity is presented in plants that have compounds with fouling smell or

irritating effects, which cause insects to get away from them (Peterson and Coats 2001).

Antifeeding activity is exerted by compounds that once ingested by the insect, prevent

feeding and eventually leading to death due to starvation (Isman 2006). Growth regulating

compounds inhibit metamorphosis or provoke precocious moulting. They alter the growth

regulating hormones and cause malformations, sterility or death in insects (Celis et al 2008).

Sesquiterpene lactones with α-methylene-γ-lactone moiety fused on various

skeletons are a rapidly expanding group of natural products comprising over 200 skeletal

types and 1350 individual types. These are known to be associated with a wide spectrum of

biological activities (Rodriguez et al 1976). Some of them have been shown to have

considerable biological activities such as insecticidal (Munekata et al 1973, Singh 2010),

fungicidal (Sabanero et al 1995 and Tan et al 1998), antimicrobial (Purohit et al 1997),

cytotoxic (Robles et al 1997), phytotoxic (Pandey 1996), anticancer (Douglas 2000), anti-

inflammatory (Cho and Baik 2000; Schinella et al 1998), antitumor (Cho and Park 1998),

ischemic (Singh et al 1993), antifeedant (Hough and Hahan 1992), antihistosomal (Ando et

al 1987), nematicidal (Mahajan et al 1986), antimalarial (Tani et al 1985), allelopathic

(Batish et al 2002) and plant growth regulators (Kalsi et al 1977; Chhabra et al 1998). It has

been reported that the biological activity is attributed to the presence of α-methylene-γ-

lactone moiety (Shibaoka et al 1967).

Pseudoguanolides, a class of sesquiterpene lactones are C15 compounds lactonised at

C6 and C8 positions and have various oxygen functions at different positions. These

compounds with abnormal carbon skeletons (Herz et al 1962) are bitter, colourless, and

relatively stable and have lyophilic behavior. A careful observation of different

pseudoguaianolides indicates that these compounds contain α-β-unsaturated-γ-lactone moiety

as an essential feature. There is a wide range indicating as this structure is associated with

remedy against ulcered sores, skin diseases, facial neuralgia, fever and anaemia (Kohli and

Rani 1994). They also exhibit certain allergic diseases like dermatities and also act as

antibacterial, antifeedent (Srivastava et al 1990) cytotoxic (Ruangrungsi et al 1987),

cytoprotective and weed germination stimulatory agents, but for plants, these can play

adverse effects. Even due to their toxic nature pseudoguaianolides have an important place in

7

research field due to diverse structural features, facile chemical transformations they undergo

and their easy availability.

α-methylene-γ-lactone, an α,β-unsaturated cyclopentenone or a conjugated ester,

chemically α, β-unsaturated carbonyl structures, are type of sesquiterpene lactones,

described as active compounds of various medicinal plants used in traditional medicine and

are known to possess a wide variety of biological and pharmacological activities. Schmidt

(1997) reported the reaction of these moieties with nucleophiles, especially cysteine

sulfhydryl groups, by a Michael-type addition. Therefore, exposed thiol groups, such as

cysteine residues on proteins, appear to be the primary targets of sesquiterpene lactones,

thereby inhibiting a variety of cellular functions which directs the cells into apoptosis.

Various biological activities described for sesquiterpene lactones include anticancer (Zhang

et al 2005), antidiarrheal (Wendel et al 2008), anti-inflammatory (Talhouk et al 2008),

fungicidal (Wedge et al 2000), antileukemic (Nasim and Crooks 2008), antimycobacterial

(Cantrell et al 1998), cytotoxic (Jung et al 1998 and Scotti et al 2007), nematicidal (Mahajan

et al 1986), trypanocidal, leishmanicidal (Sulsen et al 2008) and plant growth regulatory

activity (Chhabra et al 1998).

Heilmann et al (2001) reported that the difference in activity among individual

sesquiterpene lactones may be explained by different numbers of alkylating structural

elements. However other factors such as lipophilicity, molecular geometry, and the chemical

environment or the target sulfhydryl may also influence the activity of these compounds

(Crammer et al 1988). Due to the diverse bioactivities of sesquiterpene lactones along with

their structural complexity, these compounds are important targets for synthetic purposes. A

number of sesquiterpene lactones isolated from plant sources have been chemically

transformed with the aim of relating variable biological properties with the variation in

functional moieties associated with the molecule.

2.1 CONGRASS GRASS- Parthenium hysterophorous

Parthenium hysterophorous is an aggressive ubiquitous annual herbaceous weed,

which has invaded all parts of India (Tower et al 1977, Singh et al 2008), has been declared a

national hazard and is commonly known as white top Gajarghas, Carrot weed, Star weed;

Fever few, White top, Chatak Chandani, Bitter weed and Ramphool etc. the plant was first

reported in Pune in 1956. This alien weed is believed to have been introduced into India as

contaminants in PL-480 wheat imported from USA in 1950’s. The rate of infestation has

become very severe.Approximatelytwo million hectares of land in India have been infested

with their herbaceous menace (Dwivedi et al 2009) and it is rapidly invading in the North-

8

western Indian Himalayas (Dogra et al 2011).It has been branded as cosmopolitan weed

(Rodriguez et al 1976) in addition to national culprit (Mani and Gautam 1976).

P. hysterophorous is an erect, ephemeral herb reaching about 1 m in height. Its stem

is longitudinally grooved and bears green leaves. The leaves are pale green in color,

irregularly dissected and pubescent. The flower heads are usually 0.5mm in diameter. The

fruits are broadly obvoid in shape and have dark brown color (Anonymous 2001). It is

considered to be a native of North –East Mexico and is endemic in America and was

introduced in Africa, Asia and Oceania in cereal and grass seed shipments from USA in

about 1950s. It has achieved the status of Worst Weed in Australia and India (Bajwa et al

2004 and Shelke 1984).

Chemical analysis of P. hysterophorous has indicated that all its parts including

trichomes and pollen contain toxins called sesquiterpenes lactones. Maishi et al (1998)

reported that P. hysterophorus contains a bitter glycoside parthenin (1), a major

sesquiterpene lactone. Other phytotoxic compounds or allelochemicals are hysterin,

ambrosin, flavonoids such as quercelagetin 3, 7-dimethylether (2), 6-hydroxyl kaempferol 3-

0 arabinoglucoside and fumaric acid (3). All these compounds along with parthenin are

responsible for various biological activities. Parthenin has a cyclopentenone ring and an α-

methylene-γ-lactone moiety. It has been found to be of interest due to its anticancer

(Rodriguez et al 1976), antibacterial (Kupchan et al 1971, Mew et al 1982), antimalarial

(Picman and Towers 1983) and allelopathic properties (Hopper et al 1990). It is also reported

to be toxic and cause allergic contact dermatitis in humans and animals (Kanchan 1975, Patil

and Hedge 1988) and extensive eczematous eruption of exogenous type in human beings

(Khan et al 2011).

           

 

9

       

The major compounds present in P. hysterophorous are parthenin (1), ambrosin (4),

coronopilin (5), hymenin (6), dihydroparthenin (7) and dihydroisoparthenin (8) but the

composition of constituents in P. hysterophorous varies with geographical location. Plant

from Southern Texas was found to be rich of hymenin (Towers et al 1977) while plant from

Mexico was found to berich in hysterin (Vivar et al 1966) while in India it is rich in

parthenin and coronopilin (Chhabra et al 1999). Two sesquiterpene lactones hysterin and

dihydroisoparthenin have been isolated from plants growing in Argentina and Jamaica

(Picman et al 1982). Histamine (0.6 percent) is present in the aerial parts of the plants

(Kamal and Mathur 1991).

 

10

Syringaresinol has also been isolated from this weed (Das et al 1999). Three

ambrosanolides; 8α- epoxymethylacrylyloxyparthenin, its11α, 13-dihydro derivative and 8α-

epoxymethylacrylyloxyambrosin have been isolated from chloroform extract of the aerial

parts (Chhabra et al 1999). A novel sesquiterpenoid, charminarone (the first seco-

pseudoguaianolide) has been isolated from the whole plant (Venkataiah et al 2003). Ramesh

et al (2003a) have reported isolation of four new pseudoguaianolides parthenin, coronopilin,

2β-hydroxycoronopilin and tetraneurin-A from the flowers. Four new acetylated

pseudoguainolides along with several known constituents have also been isolated from the

flowers of P. hysterophorous (Das et al 2007).

Parthenin, a sesquiterpene lactone was isolated in 1959 from P. hysterophorus and it

was suggested to have guaianolide skeleton (Herz and Watanabe 1959), but later on NMR

spectra and different chemical transformations confirmed it to have pseudoguaianolide

skeleton (Herz et al 1962). The synthesis of this pseudoguaianolide was reported by

Heatchcock et al (1982). It is believed to play a major role in the allelopathy of P.

hysterophorus and it may be important in the displacement of naturally occurring vegetation

for the weed to become established in an area. On the molecular level, sesquiterpene lactone

biosynthesis is regulated at the transcriptional level and these compounds generally originate

from the mevalonic acid pathway and have been suggested to originate from common

precursor iso-pentyldiphosphate. Parthenin has been reported to be located in various plant

parts with especially high concentration in trichomes on leaves. Parthenium leaves contain

about 5percent parthenin (Anonymous 2003). Reinhardt et al (2004) determined that one

capitate sessile trichome contained virtually 100 percent parthenin and further quantified that

the amount of parthenin present in one capitate-sessile gland at 0.3 microgram parthenin per

gland and suggested that these trichomes are main source of parthenin that is released from

the plant.

Parthenin (1), hexacosanol, myricyl alcohol, β-sitosterol (9), campesterol,

stigmasterol, betulin, ursolic acid, β-D-glucoside of β-sitosterol, saponin and five flavonoids

(10-14) have been isolated (Shen et al 1976) from leaves of P. hysterophorus.

 

11

 

The saponin on hydrolysis yields oleanolic acid and glucose. The aqueous extract

contains free amino acids, glucose, galactose and potassium chloride (Gupta et al

1977).Methoxy pseudoguaianolides viz. 13-methoxydihydroambrosin, 13-

methoxydihydroparthenin and 2β, 13α-dimethoxydihydroparthenin have been isolated from

leaves (Bhullar et al 1997). The leaves also contain parthenin, caffeic (15), chlorogenic (16),

p-hydroxybenzoic (17), p-anisic (18), vanilic (19), salicylic, gentisic, neo-chlorogenic and

proto-catechuic acids (Anonymous 2003). Methanolic extract of flowers contains several

constituents such as 8β-hydroxycoronopilin (20), 2β-hydroxycoronopilin (21), 11-H, 13-

hydroxyparthenin (22), parthenin (1) and coronopilin (5) (Sethi et al 1987).

 

 

12

`

Parthenin (up to 8 percent) is present in capitulum (Anonymous 2003). Das et al

2005 isolated a highly oxygenated pseudoguaianolide (8-β-acetoxyhysterone C) along

withparthenin (1), coronopilin (5) and hysterone C from the flowers. Another

pseudoguaianolide 8-β-acetoxyparthenin has been isolated from the aerial parts of P.

hysterophorous (Das and Das 1997).

Histamine (0.35 percent) is present in the roots of plant (Kamal and Mathur 1991).

The roots also contain parthenin, caffeic, chlorogenic, p-hydroxybenzoic, p-anisic, vanilic,

salicylic, gentisic, neo-chlorogenic and proto-catechuic acids (Anonymous 2003).

2.2 REACTIONS OF PARTHENIN

Parthenin is a sesquiterpenoid having a pseudoguainolide structure. Parthenin

contains an α-methylene-γ-butyrolactonemoiety along with other functionalities and five

chiral centers. The compound is interesting for its structural pattern (Herz et al 1962), as well

as for its bioactivity. It has been transformed chemically and photochemically into various

derivatives. The derivatives of parthenin can be prepared by various methods.

2.2.1 Reduction reactions

Various derivatives of parthenin by regioselective and stereoselective chemical

modifications followed by reduction with reducing agents like Polymethylhydrosiloxane

(PMHS) / palladium on carbon (Pd-C), HCOONH4/ Pd-C and NaBH4/ CoCl2. 6H2O have

13

been reported. More derivatives formed by reaction of parthenin with reagents like

Trimethlorthoformate (TMOF), NaHSO4.SiO2 and NaN3/ CAN have been reported (Ramesh

et al 2003b).

Hymenin (6) on dehydration with known reagents afforded anhydroparthenin (30)

(Toribio and Geissman 1968). Attempts to dehydrate cornopilin (5) resulted in its failure.

Treatment of coronopilin (5) with acetic acid and sulfuric acid resulted in rearranged

carboxylic acid derivative (23) (Geissman and Matsueda 1964).

Herz and Watanabe (1959) reported reduction of parthenin (1) with lithium-

aluminium hydride followed by dehydrogenation with Pd-C to yield atremazulene (24).

Formation of norparthenone (25) after ozonolysis of parthenin (1) in methanol at -78°C

wasalso reported (Herz and Watanabe 1959).

(1) (24)

(5)

(23)

14

  Herz and Hogenauer (1961) reported the reduction of coronopilin (5) to yield

norpathenone (25) under similar conditions.

Hydrogenation of parthenin (1) in ethanol on 5 percent Palladium on carbon at room

temperature afforded dihydroisoparthenin (8) and tetrahydroparthenin (26) as minor product

and hydrogenation of coronopilin (5) under similar reaction conditions also yielded the same

product (Herz et al 1962).

   

Ambrosin (4) on hydrogenation with platinum oxide in presence of acetic acid

containing perchloric acid gave a mixture of (27) and (28) (Vivar et al 1966).

(1) (5)

(25)

(1) (8) (26)

15

Hymenin (6) on hydrogenation in presence of platinum oxide of room temperature

afforded dihydroisohymenin (29) (Toribio et al 1968)

Bhat and Nagasampagi (1989) reported the conversion of parthenin (1) into

epiallodamsin (31) when anhydroparthenin (30) on reaction with dimethylamine gave a

lactone product which was further hydrogenated and relactonised to give the desired product.

(30)

(31)

OH

OO

O

PtO2 / H2

OH

OO

O(6) (29)

16

  Ramesh et al (2003b) reported the formation of different products on reduction if the

reagents are different. Parthenin (1) gives dihydrocoronopilin (32) with ammonium formate

and Pd/C is reported and dihydroisoparthenin (8) with polymethylhydrosiloxane (PMSH) and

Pd/C in tetrahydrofuran.

2.2.2 Adduct formation

Diazomethane (Smith and Pings 1937) is a useful reagent for carbon insertion inα,β-

unsaturated esters to give pyrazoline derivatives. The addition occurs via 1, 3-dipolar

cycloaddition where nitrogen gets attached to α- carbon atom. Thermal decomposition of the

pyrazoline adduct leads to olefinic and cyclopropane derivatives.

(1)

(32) (8)

17

Dehydrocostus lactone (33) when treated with diazomethane (Kalsi et al 1979)

yielded a crystalline pyrazoline derivative (34) which on pyrolysis, gave two compounds i.e.

13-methyl dehydrocostus lactone (35) and 11-spirocyclopropyl derivative (36).

 

Parthenin (1) was transformed into its pyrazoline adduct (37) which on pyrolysis

afforded two products (38 and 39). Of these, the cyclopropyl derivative (38) was found to

show significant bioregulatory properties (Saxena et al 1991).

(1) (37)

(38) (39)

18

  γ-Hydroxy ester (40) derived from cyclocostunolide on reaction with diazomethane

yielded two isomeric pyrazolines (41 and 42) viatransesterification of the hydroxy ester

while another γ-hydroxy ester (43) on a similar reaction afforded a mixture of two

pyrazolines (44) epimeric at C-11 (Singh and Kalsi 1992).

Zdero et al (1990) reported the formation of a single crystalline adduct (46) of 7α-

hydroxyl isoalantolactone (47) on addition of diazomethane.

(40)

(41) (42)

(43) (44)

(45) (46)

19

2.3 BIOLOGICAL ACTIVITY

Plants produce a diverse range of bioactive molecules, making them rich source of

different types of medicines. Most of the drugs today are obtained from natural sources or

semi synthetic derivatives of natural products and used in the traditional systems of medicine

(Sukanya et al 2009). P. hysterophorous has been known to show a wide range of biological

activity. Parthenin the major compound, possess α-methylene-γ-butyrolactone. Several

studies on the relationship between biological activity and structure have shown that α-

methylene-γ-lactone moieties must be considered as alkylating agents of biological systems

which undergo a Michael reaction (Lee et al 1977) with biological nucleophiles such as L-

cysteine or thiol containing enzymes (Enz-SH).

Later on, the idea that the biological activity was due to α-methylene-γ-lactone

moiety (Shibaoka et al 1967), was modified as the bioactivity could not be associated solely

to the above said moiety and hence other structural features must, therefore, be significant

(Larson and Craig 1992).

2.3.1 Insecticidal activity

Datta and Saxena (2001) studied the eleven derivatives from P. hysterophorus and

recorded that it has the ability to act as antifeedent which has minimized the damage caused

by different insect pests. P. hysterophorus plant extracts have the ability to minimize the

population below critical threshold level of Red Pumpkin Beetle in Bitter gourd. So, the

extract can be used as an alternative to synthetic pesticides or can be supplemented to avoid

excessive use of chemicals for the safe and friendly environment (Ali et al 2011). Diethyl

ether extracts of P. hysterophorous proved to be the most effective oviposition deterrent and

ovicidal agent while the least effective as irritant extract against Aedes aegypti (Kumar et al

2011).

Parthenin and its derivatives showed insecticidal activity against first instar larvae of

Trogoderma granarium, third instar of Spodoptera litura and second juviline stage of

Meloidogyne incognita. Pyroparthenin was found to be most effective as insecticide as

percent weight loss in wheat grains was found to be 0.64 and number of progeny adults was

lowest (4.0) compared to 39.5 and 78.6, respectively with parthenin at 300 μg

g-1concentration (Shakil et al 2005).Petroleum ether extracts of leaves, stem and

20

inflorescence of P. hysterophorus at different concentrations was tested against mustard

aphid, Lipaphis erysimi (Kalt.).Out of three exracts, the leaf extract showed the most

significant effect (Sohal et al 2002). Singh (2010) reported insecticidal activity of parthenin

and its derivatives against Tribolium castaneum. The reduction products are found to be

more active as insecticide followed by parthenin, diethanolamine adduct, methanol adduct

and diazomethane adduct.

2.3.2 Antibacterial activity

Antibacterial activity of methanolic exract of P. hysterophorous against Escherichia

coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Enterococcus spp.,

and Staphylococcus aureus was tested. The activity was highest for S. aureus while it was

negative for K. pneumonia (Fazal et al 2011). Madan et al (2011) have showed antimicrobial

activity of petroleum ether extract of P. hysterophorous against Staphylococcus aureus,

Pseudomonas aeruginosa and Escherichia coli. Sukanya et al (2009) reported antibacterial

activity against Escherichia coli and Ralstonia solanacearum.

Ramesh et al (2003b) reported the antibacterial activity of parthenin and its

derivatives against B. subtilis, B. spharicus, Staphylococcus aureus, Kleibsiellaa erogenes

and Chromobacterium violaceum. α-methylene-γ-butyrolactone moiety was indispensible for

the activity of compounds as those lacking this moiety. Crude ethanolic extract (50 percent)

of P. hysteroporus flowers exhibited trypanocidal activity against Trypanosoma evansi both

in vitro and in vivo (Talakal et al 1995).

2.3.3 Anticancer and cytotoxic

Extensive research work has been carried out to characterize the anticancer activity,

the molecular mechanisms and the potential chemopreventive and chemotherapeutic

application of sesquiterpene lactones (Zhang et al 2005). A recent survey showed that out of

the 87 approved anticancer drugs over the past ten years, 62 per cent are of natural origin or

are modelled on natural product parents.

Mew et al (1982) have demonstrated that sublethal doses of parthenin exhibit

antitumour activity in mice. An association was found with cytotoxicity, since concomitant

nuclear alternations such as pycnosis, micronuclei and karyorrhexis (Ramos et al 2002).

Parthenin has also been reported to show remarkable cytotoxicity to carbohydrates

(Abramowski and Towers 1985) and bovine kidney cells and inhibition to DNA, RNA and

protein synthesis as well as enzymes like succinate dehydrogenase and oxidative

phosphorylation (Narsimahan et al 1985).

Antitumor effects of methanolic flower extract of P. hysterophorus have been

studied in mice bearing transplantable lymphocytic leukemia. Markers such as glutathione,

21

cytochrome P-450, glutathione transferase and UDP-glucuronyltransferase in liver tissues

showed significant changes leading to slow development of tumors. The extract also results

in increased survival of the leukemic mice (Mukherjee and Chatterjee 1993).

Jha et al (2011a) concluded that the methanolic extract of P. hysterophorous exhibit

CNS depressantactivity in tested animal models. It showed significant reduction in blood

glucose level in the diabetic (P<0.01) rats. The extract showed less hypoglycemic effect in

fasted normal rats, (P<0.05).The study also revealed that the active fraction of flower extract

is very promising for developing standardized phytomedicine for diabetes mellitus (Patel et

al 2008). Yadav et al (2010) revealed that the wistar albino rats become anemic after

treatment with methanolic extracts of P. hysterophorous. There is overall significant

reduction in WBC count which signified that rat immune system becomes weak after oral

treatment of P. hysterophorous extract. Skeleton muscle relaxant activity was seen with

methanolic extracts of P. hysterophorous in swiss albino mice. The muscle relaxation may be

produced due to depolarizing blockage of neuromuscular junction (Jha et al 2011b).

Haq et al (2011) reported in vitro cytotoxicity of P. hysterophorus extracts against

human cancerous cell lines. The extracts exhibited cytotoxic effect on wide range of human

cancer cell lines and to select a better extract is a matter of screening. Methanol extract

showed highest percentage growth inhibition and activity and other three extracts did not

follow a regular pattern because of different cell specificity and cascade mechanisms

followed by different cells. Further, these extracts have a role in apoptosis when analyzed in

human leukemia HL-60 cells. In future there is a bright hope to advance this lead into a

capable anticancer therapeutics.

A spiro-isoxazolidine derivative of parthenin namely SLPAR13 (47) induced cell

death in three human cancer cell lines namely HL-60 (acute promyelocytic leukaemia), SiHa

and HeLa (cervical carcinoma) with various inhibitory concentrations (Saxena et al 2012).

   

            (47) 

22

Analogues of parthenin have displayed significant cytotoxicity inhuman cervical

carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compoundsalso

induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction (Shah et al

2009).

2.3.4 Health hazards to humans and livestock

This weed is known to cause many health hazards which have now reached epidemic

proportions. Agriculturists are concerned about P. hysterophorus affecting food and fodder

crops, since the pollen and dust of this weed elicit allergic contact dermatitis in humans

(Gunaseelan1987; Morin et al 2009). Dermatitis is a T cell-mediated immune injury and the

disease manifests as itchy erythematous papules and papulovesicular lesions on exposed

areas of the body (Akhtar et al 2010). These effects have been related to cytotoxicity of the

sesquiterpene lactone parthenin (Narasimban et al 1984). Persons exposed to this plant for

prolonged period manifest the symptoms of skin inflammation, eczema, asthma, allergic

rhinitis, hay fever, black spots, burning and blisters around eyes.

P. hysterophorus also causes diarrhoea, severe papular erythematous eruptions,

breathlessness and choking (Maishi et al1998). Exposure to P. hysterophorus pollens causes

allergic bronchitis (Towers and Subba Rao1992). Ramos et al (2001) assessed the mutagenic

potential of a crude extract of P. hysterophorus in the Salmonella/microsome (Ames) assay

and the mouse bone marrow micronucleus test. However, it did not show genotoxic potential.

Sharma et al (2005) observed that the clinical pattern of Parthenium dermatitis progresses

from airborne contact dermatitis to mixed pattern or chronic actinic dermatitis pattern.

Eczema herpeticum is reported to complicate parthenium dermatitis. Sriramarao et al (1993)

worked on the use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the

diagnosis of P. hysterophorus hypersensitivity. Parthenium-sensitive patients with rhinitis

who had positive results on skin prick tests to P. hysterophorus pollen extracts responded

with a positive skin reaction to mAb-2. Akhtar et al (2010) studied the involvement of TH

type cytokines in Parthenium dermatitis.

Exposure to P. hysterophorus also causes systemic toxicity in livestock

(Gunaseelan1987). Alopecia, loss of skin pigmentation, dermatitis and diarrhoea has been

reported in animals feeding on P. hysterophorus. Degenerative changes in both the liver and

kidneys and inhibition of liver dehydrogenases have been reported in buffalo and sheep

(Rajkumar et al1988). The milk and meat quality of cattle, buffalo and sheep deteriorate on

consumption of this weed (Lakshmi and Srinivas 2007). Significant reduction in rat WBC

count after oral treatment of Parthenium extract signifies its immune system weakening

ability (Yadav et al 2010).

23

2.3.5 Reducing agricultural and pasture productivity

Singh et al (2003) explored the allelopathic properties of unburnt (UR) and burnt

(BR) residues of P. hysterophorus on the growth of winter crops, radish and chickpeas. The

extract prepared from both UR and BR was toxic to the seedling length and dry weight of the

test crops. BR extract was more toxic due to its highly alkaline nature. Growth studies

conducted in soil amended with UR and BR extracts revealed phytotoxic effects towards test

crops, UR being more active than BR unlike crude extracts. These effects were attributed to

the presence of phenolics. Parthenin leaching as root exudate plays a pivotal role in

allelopathic interference with surrounding plants (Belz et al 2007).

Parthenin has also been reported as a germination and radicle growth inhibitor in a

variety of dicot and monocot plants and it enters the soil through the decomposing leaf litter

(Gunaseelan 1998). Burning of P. hysterophorus in fields reduced germination, biomass

growth, plumule and radicle length of Phaseolus mungo (Kumar and Kumar 2010). Poor

fruiting of leguminous crops and reduction in chlorophyll content of crop plants were

observed in P. hysterophorous infested fields (Lakshmi and Srinivas 2007). P.

hysterophorous played important role as alternate host for crop pests functioning as an

inoculum source. This weed has been reported to serve as a reservoir plant of scarab beetle, a

pest of sunflower. Its invasion causes changes in above-ground vegetation and below-ground

soil nutrient contents, disturbing the entire grassland ecosystem in Nepal as reported by

Timsina et al (2010).

P. hysterophorus is a serious invasive weed of pasture systems, reducing pasture

productivity by 90 percent (Evans 1997). It has become a major weed of grazing lands in

central Queensland and New South Wales in Australia. It squeezes grasslands and pastures,

reducing the fodder supply. Dhileepan (2007) observed dwindling effect of P. hysterophorus

on grass biomass of grazing fields in Queensland, Australia.

2.3.6 Health benefits of P. hysterophorus

The decoction of P. hysterophorus has been used in traditional medicine to treat

fever, diarrhoea, neurologic disorders, urinary tract infections, dysentery, malaria and as

emmenagogue (Surib-Fakim et al 1996). Ethnobotanically, it is used by some tribes as

remedy for inflammation, eczema, skin rashes, herpes, rheumatic pain, cold, heart trouble

and gynaecological ailments. P. hysterophorus has been found to be pharmacologically

active as analgesic in muscular rheumatism, therapeutic for neuralgia and as vermifuge

(Maishi et al 1998). This weed is also reported as promising remedy against hepatic

amoebiasis. Parthenin, the major constituent of the plant, exhibits significant medicinal

attributes including anticancer property (Venkataiah et al 2003).

24

The methanol extract of the flowers showed significant antitumour activity and

parthenin exhibited cytotoxic properties against T cell leukaemia, HL-60 and Hela cancer

cell lines (Das et al 2007). Previously, Ramos et al (2002) had established the antitumour

potential of P. hysterophorus extracts in vitro and in vivo with positive results in terms of

tumour size reduction and overall survival of cell lines. Aqueous extract of P. hysterophorus

has hypoglycaemic activity against alloxan-induced diabetic rats (Patel et al 2008). So,

flower extract of this weed can be used for developing drug for diabetes mellitus.

Parashar et al (2009) reported the synthesis of silver nanoparticles by reducing silver

ions present in the aqueous solution of silver nitrate complex using the extract of P.

hysterophorus. This discovery can promote this noxious plant into a valuable weed for

nanotechnology-based industries in future. Applications of such eco-friendly nanoparticles in

bactericidal, wound healing and other medical and electronic applications makes this method

potentially exciting for the large-scale synthesis of other nanomaterials.

CHAPTER – III

MATERIAL AND METHODS

This chapter includes the information about the experimental procedures employed

during the course of investigation. The various chemicals used, methods employed for

extraction of parthenin, column chromatography for isolation of parthenin, preparation of

derivatives of parthenin, thin layer and column chromatography for purification of parthenin

and reaction products formed, rearing of Tribolium castaneum and testing of bioefficacy of

parthenin and different products against T. castaneum, are included in this chapter. All the

melting points were determined in open capillaries, on a Büchi B-545 melting point

apparatus. IR spectra were measured in chloroform solution on Perkin Elmer, Model RX-1

FT-IR spectrophotometer. 1H NMR spectra were recorded with Bruker AC (400 MHz) as

solutions (in CDCl3) using tetramethylsilane (TMS) as internal reference. The 1H NMR

spectroscopic analysis was obtained from Central Instrumentation Laboratories (CIL), Panjab

University, Chandigarh. The chemical shifts are expressed in δ (ppm) values and the

abbreviations 's', 'brs', 'd', 't' and 'm' stand for singlet, broad singlet, doublet, triplet and

multiplet respectively.

3.1 MATERIALS

3.1.1 Plant materials

Leaves of Parthenium hysterophorous were collected from PAU campus and around

roadsides.

The following adsorbents were used for chromatographic separation:

3.1.2 Silica Gel

i. Silica gel for column chromatography : Qualigens fine chemicals,

Mumbai.

Pore size : 60-120 mesh

pH (10 per cent aqueous suspension) : 7

Activity according to Brockman and Schodder : 2-3

Chloride max : 0.02 per cent

Iron max : 0.03 per cent

ii. Silica gel for thin layer chromatography : Qualigens fine chemicals,

Mumbai.

3.1.3 Various Reagents Used

i. Acetone – Loba Chemie Pvt. Ltd, Mumbai.

ii. Chloroform - Thermo Electron Pvt. Ltd, Mumbai.

26

iii. Dichloromethane – S.D. Fine Chemicals Ltd, Mumbai.

iv. Methanol - Samir Tech-Chem Pvt. Ltd, Vadodara.

v. Petroleum ether - Thermo Electron Pvt. Ltd, Mumbai.

vi. Sulfuric acid - S.D. Fine Chemicals Ltd, Mumbai.

vii. Vanillin - S.D. Fine Chemicals Ltd, Mumbai.

viii. Diethyl ether - S.D. Fine Chemicals Ltd, Mumbai.

3.1.4 Apparatus

Apart from the common laboratories glassware and apparatus, the following specific

equipments were used:

1. Glass columns (1.5 cm id x 40 cm long)

2. Thin layer chromatographic equipment

i. TLC plates 20 x 20 cm glass plates

ii. Slurry applicator (Perfit, Ambala, India)

iii. Development tank (Kontes, USA)

3. Soxhlet apparatus

4. Rotary vacuum pump

5. Electrical grinder

6. Electrical shaker

7. Rearing jars

3.2 ANALYTICAL TECHNIQUES

3.2.1 Chromatographic Techniques

Since chromatography is the commonly used method for the isolation and

purification of compounds of interests from a mixture so the brief description of various

chromatographic techniques used during the work is given below:

3.2.1.1 Column Chromatography (CC)

Column chromatography involves the separation of compounds from a mixture by

eluting the column with solvents of increasing polarity in a step wise manner and the

collection of fractions according to the sequence regarding the eluted products being

monitored by TLC. Column was packed with silica gel for column chromatography with 60-

120 mesh size activated at 1100 C for 1 hr. The material to be chromatographed was adsorbed

on silica gel for 5 min.The extraction was carried out by eluting the column with solvent of

27

increasing polarity and the various fractions were collected. For the recovery of the material

the solvent was distilled using rotary vacuum pump.

3.2.1.2 Thin Layer Chromatography (TLC)

Chromatography denotes a procedure in which a solution of substance to be

separated is passed in a direction determined by the arrangement of the apparatus (bottom to

top in case of TLC) over more or less finely divided insoluble organic or inorganic solid

resulting in the retention of the individual components to different extent. The underlying

mechanism is the partitioning of the moving compounds between the liquid phase and also

their being reversibly bound to the surface of the adsorbent. The amount transferred to the

solvent will be a function of the distribution of the compound, least strongly adsorbed will be

in the highest concentration.

Out of variety of adsorbents available (silica gel G, aluminium oxide and charcoal),

the most commonly used is silica gel G (containing gypsum as binder). The wide

applicability is due to the fact that their adsorbing power towards various classes of

compounds can be altered by pre-treatment. The rate of migration of compound on a given

adsorbent depends upon the solvent used. The solvent in order of their increasing polarity

(increasing eluting powers) are:

Petroleum ether < cyclohexane < carbon tetrachloride < toluene < benzene <

dichloromethane < chloroform < ether < acetone < alcohol.

3.2.1.3 Preparation of Thin Layer Chromatographic Plates

The silica gel G (10 g) was dissolved in water (100 mL) to prepare slurry.

Chromatoplates 20 x 20 cm were coated with slurry with the aid of an applicator, giving 0.25

mm thickness. The chromatoplates were air dried at room temperature and finally activated

in oven at 1100 C for 45 min. The chromatoplates were used after cooling for 10-15 min. The

spotting of plates was done with the help of micropipettes. The spot was applied 1 cm

upward from the lower end of chromatoplate. After the initial spotting on the stationary

phase, the chromatoplate was placed inside the developing chamber and mobile phase

(chloroform: acetone mixture::6: 1) was allowed to rise up the plate. When the chromatoplate

was developed i.e. mobile phase had reached the upper end of the chromatoplate (15 cm).

The chromatoplate was removed from the developing chamber and air dried. When the

chromatoplate was completely dried, the spots were visualized by spraying with vanillin

spray reagent. The plates after spraying were placed in the oven maintained at 1100 C for

5min in order to visualize the spots. The advantage of TLC is its ability to detect a wide

range of compounds using spray reagents.

28

3.2.1.4 Spray Reagents

The TLC plates were developed in suitable solvent chloroform: acetone in ratio 6:1

and visualization of spots was done by spraying the plates with vanillin or methanol: sulfuric

acid (19:1) as the spray reagents.

a) Vanillin spray reagent: To 10 mL of methanol added 3 drops of glacial acetic acid. Then

0.5mL of concentrated sulfuric acid was added drop wise. After about 5 to 10 min when the

solution temperature dropped down to room temperature, vanillin (0.5 g) was added to the

solution. Stirred the solution to dissolve vanillin and spray reagent was ready for use.

b) Methanol: Sulfuric acid spray reagent: To 95 mL of methanol 5 mL of concentrated

sulfuric acid was added dropwise. Cooled the solution to room temperature and spray reagent

was ready for use.

3.3 PREPARATION OF REAGENTS

3.3.1 Preparation of diazoester

A solution of glycine ethyl ester hydrochloride (14.0g) and sodium acetate (0.3g) in

water (15mL) was added to the flask and cooled to 2°C using ice-salt bath. A cold solution of

sodium nitrite (0.8g) in water (10mL) was added to it and the mixture was stirred until the

temperature reached 0ºC. To the ice-cold mixture cold 10 percent sulfuric acid (3mL) was

added. The reaction mixture was added to cold, alcohol-free ethyl ether (80mL) in a

separatory funnel. The ether layer was removed and immediately washed with 50mL cold 10

percent sodium carbonate solution. The ether solution was finally dried over sodium sulfate

and then distilled off. The yellow oil was obtained which was identified as pure diazoester

(TLC).

3.4 ISOLATION OF PARTHENIN

The leaves of Parthenium hysterophorous collected from PAU campus were dried in

shade, powdered and extracted using Soxhlet extraction method. 250 g of powdered plant

material was extracted with 1.0 L of chloroform for 24 hrs. The chloroform extract so

obtained was distilled to yield thick syrup (2.3 g). Extract of four batches (9.5 g) was

collected and to this was added methanol (150 mL), water (150 mL), lead acetate (5.0 g) and

glacial acetic acid (5mL) and kept overnight. The clear solution, yellow in color, was filtered

and the filtrate was concentrated to a minimum volume by distilling off methanol. The

residue was diluted with water in 1:1 ratio and thoroughly extracted with chloroform (3 x 50

mL). The organic layer was dried over anhydrous sodium sulfate and chloroform was

distilled to yield crude extract (7.5 g). The extract so obtained was dissolved in minimum

amount of chloroform and chromatographed over silica gel (450 g). The details of

chromatography are given in Table 1.

29

Table 1: Chromatography of extract of Parthenium hysterophorous

S. No. Eluent (mL) Weight (g) TLC based remarks

1. Chloroform (5 x 100) - -

2. Chloroform (7 x 100) 1.2 Mixture

3. Chloroform:Acetone 5percent (4 x 100) 0.8 Mixture

4. Chloroform:Acetone 5percent (10 x 100) 3.8 Pure compound mp162°C, may be parthenin

5. Chloroform:Acetone 10percent (6 x 100) - -

6. Chloroform:Acetone 10percent (6 x 100) 1.1 Highly polar liquid Fraction 4 (Table 1) was identified as Parthenin (1) mp 162°C lit.165°C (Herz et al 1962).

IR: 3600, 3418, 3070, 1760, 1720, 1645 and 880 cm-1 1H NMR signals (CDCl3, 300 MHz) δ at : 1.11 (d, 3H, J= 7.56 Hz, C14-Hs), 1.27 (s, 3H,

C15-Hs), 3.54 (m, 1 H, exchangeable), 5.03 (d, 1H, J = 7.83 Hz, C6-H), 5.62 and 6.26 (d, 1H

each, J = 2.70 Hz, C13-Hs), 6.15 (d, 1H, J = 6.00 Hz, C3-H), 7.62 (d, 1H, J = 5.99 Hz, C2-H) 13C NMR signals (CDCl3, 75.45 MHz) δ at : 17.43 (C15-q), 18.35 (C14-q), 28.4 (C9-t),

29.81 (C8-t), 40.47 (C7-d)a, 44.19 (C10-d)b, 59.05 (C5-s), 79.06 (C6-d), 84.28 (C1-s), 121.87

(C13-t), 131.43 (C3-d), 140.47 (C11-s), 163.77 (C2-d), 171.19 (C12-s), 211.12 (C4-s), (a) and

(b) are interchangeable.

3.5 REACTIONS OF PARTHENIN

3.5.1 Reaction of parthenin (1) with Diazoester

Parthenin (1, 2.0 g) in ether was treated with an excess of diazoester. Reaction

mixture was kept for four days. The completion of reaction was confirmed by thin layer

chromatography. The reaction mixture was subjected to chromatography over silica gel

(100g). The details of chromatography are shown below:

Table 2: Chromatography of reaction mixture of parthenin with diazoester

S. No. Eluent (mL) Weight (g)

TLC based remarks

1. Hexane( 10 x 100) 0.2 Yellow coloured liquid

2. Dichloromethane ( 10 x 100) 1.6 Pure compound

Fraction (2) contained pure compound whose IR and NMR data is as under:

IR: 3600, 3418, 1760, 1750 and 1720 cm-1

1H NMR signals (CDCl3, 300 MHz) δ at: 1.10(d,3H, J=7.10 Hz, C14-Hs), 1.20 (s, 3H, C15-

Hs), 3.2 (m,1H,exchangeable), 5.20 (d, 1H, J=7.2 Hz, C6-H), 6.25 (d, 1H,J=6.5 Hz,C3-H),

30

7.62 (d,1H, J=6.5Hz, C2-H), 1.15 (t, 3H, J=7.00Hz, -COOCH2CH3), 4.18 (q, 2H, J=7.00,

COOCH2CH3)

The second batch for the above reaction mixture was again subjected to column

chromatography by changing solvent system. The details of chromatography of reaction

mixture are given in Table 3:

Table 3: Chromatography of reaction mixture of parthenin with diazoester

S. No. Eluent (mL) Weight(g) TLC based remarks

1. Chloroform ( 10 x 100) - Yellow coloured liquid

2. Chloroform:Acetone 5 % ( 6 x 100) 0.4 Mixture

3. Chloroform: Acetone 10 % (6 x 100) 1.4 Pure compound

Fraction (3) was pure compound and its IR and NMR data is given below:

IR: 3600, 3418, 1765, 1754, 1718 and 1500 cm-1

1H NMR signals (CDCl3, 300 MHz) δ at: 1.15(d, 3H, J=7.00 Hz, C14-Hs), 1.22 (s, 3H, C15-

Hs), 3.6 (m, 1H, exchangeable), 5.00 (d, 1H, J=7.2 Hz, C6-H), 6.20 (d, 1H, J=6.20 Hz,C3-H),

7.60 (d, 1H, J=6.20Hz, C2-H),1.10 (t, 3H, J=7.00Hz, -COOCH2CH3), 4.20(q, 2H, J=7.00,

-COOCH2CH3)

3.5.2 Reaction of parthenin (1) with dry hydrochloric acid gas using different solvents

3.5.2.1 A slow stream of dry hydrochloric acid gas was bubbled through a solution of

parthenin (1.0 g) in tetrahydrofuran (20mL) at 0ºC till the solution gets saturated. The

reaction mixture was diluted with water and thoroughly extracted with chloroform (3x 50

mL). The mixture was finally dried over sodium sulfate.

3.5.2.2 A slow stream of dry hydrochloric acid gas was bubbled through a solution of

parthenin (1.3 g) using methanol (20mL) as the solvent at 0ºC till the solution gets saturated.

The reaction mixture was diluted with water and thoroughly extracted with chloroform (3x

50 mL). The mixture was finally dried over sodium sulfate.Evaporation of the solvent

yielded pure anhydroparthenin.

3.5.3 Treatment of parthenin (1) with microwave radiation

Parthenin (1.0 g) was dissolved in small quantity of dichloromethane. To this was

added silica gel (30 g) and mixed well. Excess of solvent was evaporated off to obtain silica

gel which was then exposed to microwave irradiation at power level 12 (800 W) for 5 min.

The product mixture was then eluted with dichloromethane. The solvent was evaporated and

a product (0.8 g) was obtained which was identified as anhydroparthenin (30) from the

spectral data.

31

3.5.4 Reaction of parthenin (1) with formic acid

A solution of parthenin (2.0 g) in formic acid (25mL) was refluxed for 20 hrs. The

yellowish brown solution was diluted with water and thoroughly extracted with chloroform

(4x 20 mL). The organic layer was neutralized by washing with water (2x20mL) followed by

washing with saturated solution of sodium bicarbonate and dried over sodium sulfate.

Evaporation of solvent yielded yellow oil (1.8g) which on dilution with ether solidified into a

yellow mass. It was dissolved in hot chloroform (5mL) and diluted with ether when a fine

yellow crystalline compound (1.5g) separated out with melting point 125ºC. It was identified

as anhydroparthenin from the spectral data. The IR and 1HNMR of the compound are as

under:

IR: 3070, 1760, 1700, 1650, 1550, 1370 and 880 cm-1 1H NMR signals (CDCl3, 300 MHz) δ at: 1.33(s, 3H, C15-Hs), 2.00 (s, 3H,C14-Hs), 4.47( d,

1H , J = 8H, C6-H), 5.56 ( d,1H, J = 3 Hz), 6.20 (1H, J = 3Hz, C13-Hb), 6.00 (d, 1H, J= 6Hz,

C3-H) , 8.06 ( d, 1H, J= 6Hz, C2-H)

3.6 BIOEFFICACY STUDIES

3.6.1 Test Insects

Rust red flour beetle – Tribolium castaneum (Herbst)

3.6.2 Experimental grains

The wheat grains (Triticum aestivum variety PBW 343, moisture content 11.0 per

cent) used throughout these studies were obtained from Department of Plant Breeding and

Genetics, PAU, Ludhiana.

3.6.3 Rearing and handling of test insect

Adults were collected from the local grain market and released in glass jar (10 × 15

cm) containing wheat flour mixed with 5 per cent yeast powder. Before culturing, the flour

was kept at 60±10C in oven for 2 hrs to eliminate contamination with other organisms. The

culture jars were placed in incubators maintained at 30±10C and 70±1 per cent relative

humidity. After seven days of oviposition period, the adults were removed and the eggs were

allowed to develop to the pupae stage. The pupae were sifted from the flour with a 50 mesh

sieve and put in small glass jars (5 × 10 cm) containing wheat flour plus yeast. From, these

jars, the adults of known age (1-2 weeks) i.e. F1 generation were obtained for the

experimental purposes. The average weight of 100 freshly emerged adults was 190 mg.

3.6.4 Preparation of standard of parthenin and its derivatives

Standard A (2, 00,000 µgg-1): 2 g of the test compound was dissolved in acetone and

volume was made to 10 mL.

32

Standard B (1, 00,000 µgg-1): 2.5g of the test compound was dissolved in acetone and

volume was made to 25 mL.

3.6.5 Testing the bioefficacy of Parthenin and its derivatives

The bioefficacy study of parthenin and its derivatives against T. castaneum adults

was carried out using F1 progeny. For the experiment, wheat (20g) was taken in a bottle.

Wheat was spiked with different concentrations i.e. 20,000, 10,000, 5,000, 4,000, 2,000 and

1,000 µg g-1 of parthenin using standard of 1,00,000µg g-1 and 2,00,000 µg g-1 (Table 4).

There were three replications for each treatment and for control treatment, only wheat and

acetone was used. The bottles were put in electric shaker for 5 minutes to enable thorough

mixing of parthenin with wheat grains. Twenty adults of same age were released into each

bottle and mouth of bottle was covered with muslin. The observation of mortality of T.

castaneum was taken after every 24 hrs till complete or constant mortality was obtained. The

corrected percent mortality was calculated using Abbott’s formula (Abbott 1925):

Corrected per cent mortality = Per cent mortality in treated–Per cent mortality in control

x 100 100 – Per cent mortality in control

Similar treatments were carried out with all the synthesized compounds from parthenin.

Table 4: Spiking of wheat at different concentrations using parthenin and its derivatives using standards (1, 00,000 and 2, 00,000 µgg-1) of the respective test compound

Sr. No.

Spiking level (µg g-1)

Weight of wheat grains taken (g)

Volume of standard used

(mL)

Volume of acetone used (mL)

1 20,000 20 2 (A) -

2 10,000 20 2 (B) -

3 5,000 20 1(B) 1

4 4,000 20 0.8(B) 1.2

5 2,000 20 0.4(B) 1.6

6 1,000 20 0.2(B) 1.8

7 control 20 - 2

3.6.6 Statistical Analysis

The statistical analysis of the parent compound and its various reaction products was

carried out and CD (5%) was calculated.

CHAPTER – IV

RESULTS AND DISCUSSION

4.1 EXTRACTION AND CHARACTERIZATION OF PARTHENIN

The extract of Parthenium hysterophorus was obtained from the shade dried and

powdered leaves by Soxhlet extraction method using chloroform as the solvent. Pure

parthenin (1) was isolated from the extract by column chromatography. It showed mp 162º C

and the purity of the compound was checked by TLC. IR spectrum of the compound showed

bands at 3600 and 3418 cm-1 (free and bonded –OH group), 1760 cm-1 (γ-lactone), 1720 cm-1

(α, β– unsaturated cyclopentenone moiety and 3070, 1645 and 880 cm-1 (due to

exomethylene double bond). This data was supported by 1H NMR signals (CDCl3, 300 MHz)

δ at : 1.11 (d, 3H, J= 7.56 Hz, C14-Hs), 1.27 (s, 3H, C15-Hs), 3.54 (m, 1 H, exchangeable),

5.03 (d, 1H, J = 7.83 Hz, C6-H), 5.62 and 6.26 (d, 1H each, J = 2.70 Hz, C13-Hs), 6.15 (d, 1H,

J = 6.00 Hz, C3-H), 7.62 (d, 1H, J = 5.99 Hz, C2-H) coupled with 13C NMR signals (CDCl3,

75.45 MHz) δ at : 17.43 (C15-q), 18.35 (C14-q), 28.4 (C9-t), 29.81 (C8-t), 40.47 (C7-d)a, 44.19

(C10-d)b, 59.05 (C5-s), 79.06 (C6-d), 84.28 (C1-s), 121.87 (C13-t), 131.43 (C3-d), 140.47 (C11-

s), 163.77 (C2-d), 171.19 (C12-s), 211.12 (C4-s), (a) and (b) are interchangeable. All this data

proved this compound to be parthenin (1).

4.2 REACTIONS OF PARTHENIN

4.2.1 Reaction with diazoester

Sesquiterpene lactones having α-methylene-γ-lactone moiety are known to undergo

1, 3-dipolar addition to diazomethane activated by electron attracting group of which α-

methylene-γ-lactone is a promising site. Dehydrocostus lactone has been reported to undergo

1, 3-dipolar addition with an excess of ethereal solution of diazomethane to yield solid

compound mp 92 º C, identified as pyrazoline (48). Thermal decomposition of pyrazoline

which has been known to give olefin (49) and cyclopropane (50) (Kalsi et al 1979), has been

34

of interest from both synthetic and mechanistic point of view (Wulfman et al 1978, Machezie

1975).

Diazoester has successfully been used to add to a double bond in the presence of Cu

(I) salts to give corresponding cyclopropane derivatives (51) (Vig et al 1979). The reaction

may involve the intermediate formation of a pyrazoline which is decomposed to

carboxyethyl cyclopropane. 

 

With this view in mind parthenin (1) was allowed to react with an excess of

diazoester by avoiding the use of Cu (I) salt so as to get the corresponding pyrazoline. But

the usual work up yielded a solid crystalline compound (52) which was further purified by

recrystallisation. It showed IR bands at 3600 and 3418 cm-1 (free and bonded –OH group),

1760 cm-1 (γ-lactone), 1720 cm-1 (α, β– unsaturated cyclopentenone moiety and an additional

band at 1730 (due to the presence of an ester grouping) suggested by the structure 52 in this

35

compound. This structure was supported by1H NMR signals (CDCl3, 300 MHz) δ at: 1.10(d,

3H, J=7.10 Hz, C14-Hs), 1.20 (s, 3H, C15-Hs), 3.2 (m, 1H, exchangeable), 5.20 (d, 1H, J=7.2

Hz, C6-H), 6.25 (d, 1H, J=6.5 Hz,C3-H), 7.62 (d, 1H, J=6.5 Hz,C2-H),1.15 (t, 3H, J=7.00Hz,-

COOCH2CH3), 4.20(q,2H, J=7.00,-COOCH2CH3).

 

The formation of compound and its structure was confirmed by absence of IR bands

3070, 1645 and 880 cm-1 (due to exomethylene double bond) and 1H NMR signal at 5.62 and

6.26 (d, 1H each, J = 2.70 Hz, C13-Hs). The appearance of 1H NMR signal at 1.10 (t, 3H,

J=7.00Hz,-COOCH2CH3), 4.20(q, 2H, J=7.00,-COOCH2CH3) supported the formation of

compound (52).The formation of this compound may be attributed to the decomposition of

pyrazoline (if all it was formed as the intermediate) on silica gel or during work up. 

The formation of cyclopropane derivatives might have not been surprising since this

type of reaction has often been used from the synthesis of cyclopropane (Vig et al 1979)

without concern for the presumed intermediate pyrazoline. However, the surprising feature

of this observation was the fact that spontaneous nitrogen evolution even when the reaction

was carried out in the absence of Cu (I) salt. This is in contrast to the fact that in most cases

where the reaction of a diazoalkane with an α, β- unsaturated addend is used to effect the

synthesis of a cyclopropane, the pyrazoline is first formed and then it should be heated to

varying temperatures to give the desired products. A more careful perusal of literature

showed that diphenyl diazomethane with some addends also result in spontaneous

cyclopropane formation (Walborsky and Hornyak 1955). The spontaneous nitrogen evolution

from the reactions of diazoalkanes with α, β- unsaturated addends has been recognized as

being anomalous and this anomaly has been attributed to reaction conditions with no solid

explanation and is still a topic of further research.   

Further it was observed that when column was run in chloroform, diazoester

pyrazoline adduct (53) was recovered whose structure was confirmed by spectroscopic

36

analysis. The 1H NMR signals (CDCl3, 300 MHz) δ at: 1.15(d, 3H, J=7.00 Hz, C14-Hs), 1.22

(s, 3H, C15-Hs), 3.6 (m, 1H, exchangeable), 5.00 (d, 1H, J=7.2 Hz, C6-H), 6.20 (d, 1H,

J=6.20 Hz,C3-H), 7.6 (d, 1H, J=6.20 Hz,C2-H),1.10 (t, 3H, J=7.00Hz,-COOCH2CH3),

4.20(q,2H, J=7.00,-COOCH2CH3). The appearance of IR bands near 1500 cm-1gives

indication about the presence of -N=N-. The formation of adduct may be due to less contact

time between silica gel and pyrazoline derivative.

 

4.2.2 Reaction of parthenin with dry HCl gas

Parthenin (1) contains an α -methylene-γ -lactone moiety which plays a vital role for

its bioactivity. It was thought that with extending conjugation the biological activity of the

compound might increase. Parthenin on irradiation with microwave (M.W.) for 8 min

resulted in the formation of anhydroparthenin (Das and Venkataiah 1999).

    Anhydroparthenin (30) has been prepared by refluxing parthenin with formic acid

(Herz et al 1962). Bhat and Nagasampagi (1989) have reported that the reaction of parthenin

(1) with formic acid not only yielded expected anhydroparthenin (30) but also rearranged

products (54 and 55)

37

To improve the yield of anhydroparthenin the reaction of parthenin with dry HCl

gas was thought to be a better choice, to achieve this parthenin was dissolved in

tetrahydrofuran and was allowed to react with dry HCl gas. The reaction resulted in a total

mess. The failure of reaction was attributed to the solvent, in order to improve the reaction

conditions methanol was used as solvent and the reaction with dry HCl gas gave

anhydroparthenin (30) as a major product. It was confirmed by absence of peak near 3500

cm-1 in IR spectrum. It was further confirmed by 1H NMR spectroscopy. The 1H NMR

spectrum of compound (30) showed a typical singlet at δ 1.33 corresponding to the methyl

group at C-5.Another singlet at δ 2.0 was obtained due to C-14 Hs. A doublet at δ 4.47(J=8

Hz) was seen due to presence of H-atom at C-6 position. Two doublets at δ 5.56 and δ 6.20

with J=3Hz were obtained showing the presence of the two H-atoms at C-13. Another pair of

doublets at δ 6.0 (J= 6 Hz) and δ 8.06 (J= 6Hz) were obtained suggesting the unsaturation at

C-2 and C-3 position. The data is quite similar to parthenin itself but for the absence of –OH

group and presence of an additional double bond. This compound might have been produced

by elimination of –OH group under acidic conditions. This data was found to be similar to

that obtained for the anhydroparthenin (30). Thus the structure of compound is:

 

38

 

The parthenin (1) was irradiated with microwave and anhydroparthenin (30, 0.8 g)

was obtained as the product. The yield was comparable to that of the already employed

methods.

4.4 Bioefficacy studies of parthenin and its derivatives

The bioefficacy studies were conducted using Parthenin (1), anhydroparthenin (30),

pyrolysis product of parthenin (52) and diazoester adduct of parthenin (53) against Tribolium

castaneum (Herbst). The compounds were tested at six different concentrations and there

were three replications and control. The mortality in control was also noted. The

observations were taken till complete or constant mortality was obtained. The corrected

percent moratlity was calculated using Abbot’s formula (1925).

4.1.1 Bioefficacy of parthenin against Tribolium castaneum The bioefficacy studies of parthenin on Tribolium castaneum were carried out at six

different concentrations ranging from 1000μg g-1 to 20,000μg g-1 respectively. Corrected per

cent mortality of parthenin (1) against Tribolium castaneum is shown in Table 6 and Fig 4.1.

No mortality was observed on the first day upto 10,000μg g-1 concentration however

corrected per cent mortality of 8.33 was observed at 20,000μg g-1 concentration. There was

no mortality till the third day at the application rate of 1,000-5,000μg g-1 concentration

whereas at 10,000 and 20,000μg g-1, the corrected percent mortality of 5.00 and 48.33 was

observed. On fifth day of treatment, corrected per cent mortality of 15.00 and 68.33 was

observed at 10,000 and 20,000μgg-1 concentrations respectively whereas corrected per cent

mortality of 1.50 was observed at concentration of 4,000 and 5,000μg g-1. No mortality was

observed at 1,000 and 2,000μg g-1 concentrations respectively on the fifth day of exposure.

On the eighth day of treatment, corrected per cent mortality of 3.35, 13.35, 26.50, 68.35 and

98.33 was observed at 2,000, 4,000, 5,000, 10,000 and 20,000 μg g-1concentrations

respectively. Complete corrected per cent mortality was observed on tenth day of treatment

at 20,000μg g-1 concentration. On tenth day of exposure, the corrected per cent mortality at

39

other concentrations increased to 5.00, 8.35, 21.50, 40.00 and 85.00at 1,000, 2,000, 4,000,

5,000 and 10,000 μg g-1concentrations respectively. Complete corrected per cent mortality

was achieved at 10,000 μg g-1on fifteenth day of treatment whereas 1,000, 2,000, 4,000 and

5,000 μg g-1 concentrations showed corrected per cent mortality of 10.00, 13.35, 40.00, and

70.00 respectively. On eighteenth day of treatment the corrected percent mortality was found

to be 10.00, 25.00, 51.50 and 73.35 at 1,000, 2,000, 4,000 and 5,000μg g-1concentrations

respectively .On twentieth day of experiment, corrected per cent mortality of 10.00, 30.00,

55.00 and 75.00 was reported whereas corrected per cent mortality of 11.50, 35.00, 58.35

and 76.65 was observed on twenty-fifth day of application at 1,000, 2,000, 4,000 and

5,000μgg-1concentrations respectively. There was slow increase in corrected percent

mortality from twentieth to twenty fifth day of exposure. The corrected per cent mortality

was found to be15.00, 38.35, 58.35 and 78.35 per cent at 1,000, 2,000, 4,000 and 5,000μg g-1

concentrations respectively on thirtieth day of treatment. The corrected per cent mortality

was recorded as 15.00, 40.00, 73.35 and 91.60 at 1,000, 2,000, 4,000 and 5,000 μg g-1

concentrations respectively on fortieth day of exposure which did not change till forty five

days of treatment. Complete corrected per cent mortality was achieved on fifty and fifty fifth

day of application at 5,000 and 4,000μgg-1concentrations respectively. The corrected percent

mortality on fiftieth day was 15.00, 46.50 and 85.00 at 1,000, 2,000, 4,000μgg-1

concentrations respectively whereas it was 35.30 and 78.43 at 1,000 and 2,000μgg-1

concentrations respectively on fifty-fifth day of exposure. Complete corrected percent

mortality was observed after sixty- two days of exposure at 2,000μg g-1 concentration. The

complete corrected percent mortality was observed on tenth, fifteenth, fiftieth, fifty fifth and

sixty-two days at 20,000, 10,000, 5,000, 4,000 and 2,000μgg-1 concentrations respectively.

Complete mortality was not shown at low concentration of 1,000 μgg-1 even after sixty two

day of exposure. The mortality became constant after sixty two days and did not show any

change after that. This showed that parthenin at low concentration of 1000 μg g-1was not

exerting its toxic influence on insects. The data showed that the corrected per cent mortality

increases with increase in concentration of parthenin and time of application.

40

Table 6: Corrected percentage mortality of T. castaneum with parthenin

Days of application

Concentrations (μg g-1)

20,000 10,000 5,000 4,000 2,000 1,000

1 8.33 0 0 0 0 0

3 48.33 5.00 0 0 0 0

5 68.33 15.00 1.50 1.50 0 0

8 98.33 68.35 26.50 13.35 3.35 0

10 100.00 85.00 40.00 21.50 8.35 5.00

15 - 100.00 70.00 40.00 13.35 10.00

18 - - 73.35 51.50 25.00 10.00

20 - - 75.00 55.00 30.00 10.00

25 - - 76.65 58.35 35.00 11.50

30 - - 78.35 58.35 38.35 15.00

40 - - 91.60 73.35 40.00 15.00

45 - - 91.60 73.35 40.00 15.00

50 - - 100.00 85.00 46.50 15.00

55 - - - 100.00 78.43 35.30

60 - - - - 96.10 37.25

62 - - - - 100.00 39.21

65 - - - - - 39.21

41

 

 

Fig. 4.1 Corrected percent mortality of Tribolium castaneum using parthenin at indicated time interval

0

10

20

30

40

50

60

70

80

90

100

20,000 10,000 5,000 4,000 2,000 1,000

Cor

rect

ed p

er c

ent m

orta

lity

Conc. (µg g-1 of grains)

Day 1 Day 3 Day 5 Day 8 Day 10 Day 15 Day 18 Day 20 Day 25Day 30 Day 40 Day 45 Day 50 Day 55 Day 60 Day 62 Day 65

42

4.1.2 Bioefficacy of anhydroparthenin against T. castaneum

The bioefficacy studies of anhydroparthenin on Tribolium castaneum were carried

out using six different concentrations ranging from 1000μg g-1 to 20,000μg g-1 respectively

Corrected per cent mortality of anhydroparthenin (30) against Tribolium castaneum adults is

shown in Table 7 and Fig 4.2. No mortality was observed after 48 hours of treatment at all

the concentrations tested except at 20,000μgg-1 where low corrected per cent mortality of

1.67 was observed on first day. On third day of treatment, corrected per cent mortality of

5.00and 11.67 was observed at 10,000 and 20,000μg g-1 concentrations respectively whereas

all other lower concentrations showed no mortality. Corrected per cent mortality of 1.67,

8.33 and 20.00was observed at 5,000, 10,000 and 20,000μg g-1on fifth day of treatment and

5.00, 6.67, 18.33 and 35.00 was observed at 4,000, 5,000, 10,000 and 20,000μg g-

1concentrations respectively on eighth day of treatment. There was no mortality at the

concentrations of 1000-4000 μg g-1 on fifth day and at 1000- 2000μg g-1 on eighth day. On

tenth day of exposure, the corrected per cent mortality of 1.67, 5.00, 13.33,25.00 and 41.67

was achieved at 2,000, 4,000, 5,000 and 10,000 and 20,000μgg1concentration respectively

whereas at 1,000μg g-1 concentration no mortality was observed. On fifteenth day of

exposure corrected percent mortality of 1.67, 6.67, 11.67, 23.33, 28.33 and 75.00 was

observed at 1,000, 2,000, 4,000, 5,000, 10,000 and 20,000 μg g-1concentrations respectively.

Corrected percent mortality of 6.67 was observed at 1,000 and 2,000μg g-1concentrations and

13.33, 25.00 and 33.33 was observed at 4,000, 5,000 and 10,000μg g-1concentrations

respectively on eighteenth day of exposure. Complete corrected percent mortality was

observed at 20,000μg g-1concentration on eighteenth day of treatment. On twentieth day the

corrected percent mortality of 6.67, 18.33, 13.33, 25.00 and 36.67 was observed at 1,000,

2,000, 4,000, 5,000 and 10,000μg g-1concentrations respectively. The corrected per cent

mortality of 10.00, 13.33, 23.33, 33.33and 46.67 was observed at1,000, 2,000, 4,000,

5,000and 10,000μg g-1 concentrations respectively on twenty-fifth day of exposure. There

was very little increase in corrected percent mortality in five days. Corrected per cent

mortality of18.33, 23.33, 33.33, 45.00 and 58.33 was observed at 1,000, 2,000, 4,000, 5,000

and 10,000μg g-1 concentrations respectively on thirtieth day of application. On fortieth day

of exposure the corrected percent morality of 75.00was achieved at 10,000 μg g-1

concentration and 53.33 at 5,000 and 4,000 μg g-1 concentrations respectively whereas the

corrected percent mortality of 31.67 and 35.00 was observed at 1,000 and 2,000μg g-1

concentrations respectively. On forty-fifth day the corrected percent mortality of35.00,

50.00, 60.00, 66.67 and 86.67 was observed at 1,000, 2,000, 4,000, 5,000 and 10,000 μg g-1

43

concentrations respectively. Complete corrected percent mortality was observed on fiftieth

day at 10,000μg g-1concentration.Corrected per cent mortality of41.67, 51.67, 63.33 and

78.33 was at 1,000, 2,000, 4,000 and 5,000μg g-1concentration respectively was observed on

fiftieth day whereas corrected per cent mortality of 55.00, 56.67, 73.33 and 80.00 was

observed at 1,000, 2,000, 4,000 and 5,000μg g-1concentrations respectively on fifty fifth day

of exposure. The corrected percent mortality of 55.00, 70.00, 86.67 and 91.67 was observed

on sixty fifth day of exposure at 1,000, 2,000, 4,000 and 5,000μg g-1concentrations

respectively and it became constant. This data showed that compound is active at high

concentrations only but at low concentrations mortality rate was very low in the beginning

which increased to more than fifty percent with time. Complete mortality was obtained on

eighteenth and fiftieth day of exposure at concentrations of 20,000 and 10,000 μg g-

1concentrations respectively. The corrected percent mortality was found to increase with

increase in concentration and time.

Table 7: Corrected percentage mortality of T. castaneum with anhydroparthenin

Days of application

Concentrations (μg g-1)

20,000 10,000 5,000 4,000 2,000 1,000

1 1.67 0 0 0 0 0

3 11.67 5.00 0 0 0 0

5 20.00 8.33 1.67 0 0 0

8 35.00 18.33 6.67 5.00 0 0

10 41.67 25.00 13.33 5.00 1.67 0

15 75.00 28.33 23.33 11.67 6.67 1.67

18 100.00 33.33 25.00 13.33 6.67 6.67

20 - 36.67 25.00 13.33 18.33 6.67

25 - 46.67 33.33 23.33 13.33 10.00

30 - 58.33 45.00 33.33 23.33 18.33

40 - 75.00 53.33 53.33 35.00 31.67

45 - 86.67 66.67 60.00 50.00 35.00

50 - 100.00 78.33 63.33 51.67 41.67

55 - - 80.00 73.33 56.67 55.00

65 - - 91.67 86.67 70.00 55.00

70 - - 91.67 86.67 70.00 55.00

44

Fig. 4.2: Corrected percent mortality of Tribolium castaneum using anhydroparthenin at indicated time interval

0

10

20

30

40

50

60

70

80

90

100

20,000 10,000 5,000 4,000 2,000 1,000

Cor

rect

ed p

er c

ent m

orta

lity

Conc. (µg g-1 of grains)

Day 1 Day 3 Day 5 Day 8 Day 10 Day 15 Day 18 Day 20 Day 25 Day 30 Day 40 Day 45 Day 50 Day 55

45

4.1.3 Bioefficacy of pyrolysis product of Parthenin against T. castaneum

The bioefficacy studies of pyrolysis product of parthenin on Tribolium castaneum

were carried out at six different concentrations ranging from 1000μg g-1 to 20,000μg g-1

respectively Corrected per cent mortality of pyrolysis product (52) of parthenin against

Tribolium castaneum adults is shown in Table 8 and Fig 4.3.No mortality was observed on

first day of treatment at all the concentrations tested. On third day, 10,000 and 20,000 μg g-

1concentratios showed low corrected percent mortality of 1.67 and 8.33 respectively. On fifth

day corrected percent mortality of 1.67 was observed at 4,000 and 5,000 μg g-1

concentrations respectively whereas corrected per cent mortality 6.67 and 13.33 at 10,000

and 20,000μg g-1 concentrations respectively. On eighth day the compound showed corrected

per cent mortality of 1.67at lower concentrations of 1,000 and 2,000 μg g-1and at higher

concentrations of 4,000, 5,000 and 10,000 and 20,000 μg g-1respectively, the corrected per

cent mortality 6.67, 8.33, 10.0 and 23.33 was observed. On tenth day there was not much

increase in corrected percent mortality at all the concentrations tested and the corrected

percent mortality of 8.33, 10.00, 15.00, 18.33, 23.30 and 56.67 was observed at 1,000, 2,000,

4,000, 5,000, 10,000 and 20,000μg g-1 concentrations respectively on fifteenth day. The

corrected percent mortality was 21.67, 28.33 and 78.33 at 5,000, 10,000 and 20,000 μgg-1

concentrations respectively and it reached a value of 10.00, 15.00 and 18.33 at 1,000, 2,000

and 4,000μg g-1 concentrations respectively on eighteenth day of exposure. On twentieth day

the corrected percent mortality of13.33, 18.33, 20.00, 25.00 and 31.67was observed at 1,000,

2,000, 4,000, 5,000 and 10,000μg g-1 concentrations respectively. Complete corrected percent

mortality was observed on twentieth day of treatment only at 20,000μg g-1concentration.

There was slow increase in corrected percent mortality on twenty-fifth day. The corrected

percent 15.00, 20.00, 23.30, 26.67 and 33.33was observed at 1,000, 2,000, 4,000, 5,000 and

10,000μg g-1 concentration on twenty-fifth day of treatment. On thirtieth day, the corrected

percent mortality was 18.33, 25.00, 26.67, 31.67 and 36.67 was observed at 1,000, 2,000,

4,000, 5,000and 10,000μg g-1 concentrations respectively. The corrected percent mortality

of25.00, 31.67, 36.67, 41.67 and 53.33 was observed at 1,000, 2,000, 4,000, 5,000 and

10,000μg g-1 concentrations respectively on fortieth day. On forty-fifth day of exposure the

corrected percent mortality reached a value of 45.00, 48.33 and 63.33at 4,000, 5,000 and

10,000μg g-1 concentrations respectively and 36.67 and 38.33for rest of the concentrations

respectively. Complete corrected percent mortality was achieved on fifty first day at 10,000

μg g-1 concentration and corrected percent mortality was 90.00 at 5,000μg g-1 concentration

46

after fifty five days. The corrected percent was 58.33, 78.33 and 81.67 at 1,000, 2,000 and

4,000μg g-1 concentrations respectively on fifty fifth day of exposure. These data showed

that the corrected per cent mortality increased with increase in concentration of the

compound. More than fifty percent was obtained even at low concentrations whereas it was

nearly 80.00 percent at 2,000 and 4,000μg g-1 respectively. Ninety percent mortality was

obtained at 5,000 μg g-1 concentrations. The mortality became constant after fifty five days.

This showed that compound at these concentrations was exerting less effect on insects.

Table 8: Corrected percentage mortality of T. castaneum with pyrolysis product of

parthenin

Days of application

Concentrations (μg g-1)

20,000 10,000 5,000 4,000 2,000 1,000

1 0 0 0 0 0 0

3 8.33 1.67 0 0 0 0

5 13.33 6.67 1.67 1.67 0 0

8 23.33 10.00 8.33 6.67 1.67 1.67

10 35.00 15.00 8.33 10.00 3.33 1.66

15 56.67 23.30 18.33 15.00 10.00 8.33

18 78.33 28.33 21.67 18.33 15.00 10.00

20 100.00 31.67 25.00 20.00 18.33 13.33

25 - 33.33 26.67 23.3 0 20. 00 15.00

30 - 36.67 31.67 26.67 25.00 18.33

40 - 53.33 41.67 36.67 31.67 25.00

45 - 63.33 48.33 45.00 38.33 36.67

50 - 93.33 73.33 65.00 53.33 46.66

51 - 100.00 73.33 71.67 60.00 48.33

55 - - 90.00 81.67 78.33 58.33

65 - - 90.00 81.67 78.33 58.33

47

Fig. 4.3 Corrected percent mortality of Tribolium castaneum using pyrolysis product of parthenin at indicated time interval

0

10

20

30

40

50

60

70

80

90

100

20,000 10,000 5,000 4,000 2,000 1,000

Cor

rect

ed p

er c

ent m

orta

lity

Conc. (µg g-1 of grains)

Day 1 Day 3 Day 5 Day 8 Day 10 Day 15 Day 18 Day 20Day 25 Day 30 Day 40 Day 45 Day 50 Day 51 Day 55 Day 70

48

4.1.4 Bioefficacy of diazoester adduct of Parthenin against T. castaneum

The bioefficacy studies of diazoester adduct of parthenin on Tribolium castaneum

were carried out at six different concentrations ranging from 1000μg g-1 to 20,000μg g-1

respectively Corrected per cent mortality of diazoester adduct (53) of Parthenin against

Tribolium castaneum adults is shown in Table 8 and Fig 4.4. No mortality was observed on

first day of treatment. On the third day of exposure no effect was observed at concentrations

of 1,000,2,000,4,000,5,000 and 10,000 μg g-1 and low corrected percent mortality of 6.67

was observed at 20,000μg g-1 concentration. On fifth day of treatment there was no insect

mortality at concentrations of 1,000, 2,000, 4,000 and 5,000μg g-1 but at 10,000 and 20,000

μg g-1concentrations the corrected percent mortality of 10.00 and 13.33 was observed.

At2,000, 4,000, 5,000, 10,000 and 20,000 μg g-1 concentration corrected percent mortality of

1.67, 6.67, 8.33, 13.33 and 21.67 was observed on the eighth day of treatment. On tenth day

the corrected percent mortality was 3.33, 6.67, 10.00, 16.67 and 28.33 at 2,000,

4,000,5,000,10,000 and 20,000 μg g-1 concentrations respectively. No mortality was

observed till ten days at 1,000 μg g-1. The corrected percent mortality of 1.67, 6.67, 13.33,

15.00, 18.33 and 41.67 was observed at 1,000, 2,000, 4,000, 5,000, 10,000 and 20,000 μg g-1

concentrations respectively on fifteenth day of treatment. On eighteenth day corrected

percent mortality of 3.33, 8.33, 13.33 and 20.00 was observed at 1,000, 2,000, 4,000 and

5,000 μg g-1 concentrations respectively, whereas it was 20.00 and 65.00 at 10,000 and

20,000 μg g-1 concentrations respectively. The corrected percent mortality of 21.67 was

observed at 5,000 and 10,000 μg g-1 concentrations respectively and that of 6.67, 8.33, 13.33

was observed at 1,000, 2,000, 4,000μg g-1 concentrations respectively on twentieth day.

Complete corrected percent mortality was observed at 20,000 μg g-1 concentration on

twenty-fifth day of exposure. The corrected percent mortality of 13.33, 15.00, 20.00, 21.67

and 26.67 was observed at 1,000, 2,000, 4,000, 5,000 and 10,000μg g-1 concentrations

respectively on twenty-fifth day of exposure. The corrected percent of 21.60, 25.00 and

33.33 was observed at 4,000, 5,000 and 10,000μg g-1 concentrations respectively on thirtieth

day whereas mortality remains constant at 2,000 and 1,000 μg g-1 concentrations till thirtieth

day. The corrected percent mortality of 20.00, 23.33, 28.33, 31.67 and 41.67 was observed

on fortieth day at all the concentrations tested. The corrected percent mortality of 23.33,

26.67, 35.00, 38.33 and 48.33 was observed at 1,000, 2,000, 4,000, 5,000 and 10,000μg g-1

concentrations respectively on forty fifth day of exposure. The corrected percent mortality of

48.33, 61.67, 70.00, 70.00 and 76.66 was observed at 1,000, 2,000, 4,000, 5,000 and

49

10,000μg g-1 on fifty fifth day of exposure. Complete corrected percent mortality was

observed on twenty fifth and sixtieth day of exposure at 20,000 and 10,000 μg g-1

concentrations respectively. The concentration of 20,000 μg g-1 was most effective whereas

1,000μg g-1 was least effective where low mortality of 55.00 was observed even on sixtieth

day of application. The data showed that diazoester was more active against Tribolium

castaneum at higher concentrations whereas at lower concentrations effectiveness increased

with increase in number of days for a particular concentration. The corrected percent

mortality remained constant after sixty days.

Table 9: Corrected percentage mortality of T. castaneum with diazoester adduct of

parthenin

Days of application

Concentrations (μg g-1)

20,000 10,000 5,000 4,000 2,000 1,000

1 0 0 0 0 0 0

3 6.67 0 0 0 0 0

5 13.33 10 0 0 0 0

8 21.67 13.33 8.33 6.67 1.67 0

10 28.33 16.67 10.00 6.67 3.33 0

15 41.67 18.33 15.00 13.33 6.67 1.67

18 65.00 20.00 20.00 13.33 8.33 3.33

20 76.67 21.67 21.67 13.33 8.33 6.67

25 100.00 26.67 21.67 20.00 15.00 13.33

30 - 33.33 25.00 21.60 15.00 13.33

40 - 41.67 31.67 28.33 23.33 20.00

45 - 48.33 38.33 35.00 26.67 23.33

55 - 76.66 70.00 70.00 61.67 48.33

60 - 100.00 83.33 83.33 76.66 55.00

65 - - 83.33 83.33 76.66 55.00

50

Fig. 4.4 Corrected percent mortality of Tribolium castaneum using diazoester adduct of parthenin at indicated time interval

0

10

20

30

40

50

60

70

80

90

100

20,000 10,000 5,000 4,000 2,000 1,000

Cor

rect

ed p

er c

ent m

orta

lity

Conc. (µg g-1 of grains)

Day 1 Day 3 Day 5 Day 8 Day 10 Day 15 Day 18 Day 20 Day 25 Day 30 Day 40 Day 45 Day 50 Day 60 Day 70

51

4.1.5 Comparison of bioefficacy of parthenin and reaction products formed against

T. castaneum at 20,000 μg g-1

The comparison of the corrected percent mortality observed as a result of the

treatment of parthenin and its reaction products (anhydroparthenin, diazoester adduct and

pyrolysis product) against Tribolium castaneum at 20,000 μg g-1is shown in Table 10 and Fig

4.5. The corrected percent mortality of 8.33 and 1.67 was observed in case of parthenin and

anhydroparthenin respectively whereas no corrected per cent mortality was observed in case

of diazoester adduct and pyrolysis product on first dayof exposure. On third day of exposure

corrected percent mortality of 48.33 was observed in case of parthenin whereas 11.67, 8.33

and 6.67 was observed for anhydroparthenin, pyrolysis product and diazoester adduct

respectively. On fifth day of experiment, corrected per cent mortality of 68.33 and 20.00 was

observed using parthenin and anhydroparthenin, whereas diazoester adduct and pyrolysis

product showed corrected percent of 13.33 respectively. The corrected percent mortality

increased to 98.33, 35.00, 23.33 and 21.67 per cent respectively on eighth day of exposure.

Parthenin showed complete corrected per cent mortality on tenth day of exposure. Corrected

percent mortality of 41.67, 35.00 and 28.33 was observed using anhydroparthenin, pyrolysis

product and diazoester adduct respectively on tenth day of exposure. The corrected percent

mortality of 75.00, 56.67 and 41.65 was observed for anhydroparthenin, pyrolysis product

and diazoester adduct on fifteenth day of exposure. Anhydroparthenin showed complete

mortality on eighteenth day of exposure. On eighteenth day the corrected percent mortality of

78.33 and 65.00 was observed using pyrolysis product and diazoester adduct. Complete

mortality was achieved in case of pyrolysis product on twentieth day and diazoester adduct

after twenty five days of exposure. From the above discussion it was found that parthenin

was most active against T. castaneum adults where corrected percent mortality was obtained

on tenth day where as the diazoester adduct was least active at 20,000 μg g-1 concentration

where corrected percent mortality was obtained on twenty-fifth day. The complete percent

mortality was obtained on tenth, eighteenth, twentieth and twenty-fifth day in case of

parthenin, anhydroparthenin, pyrolysis product and diazoester adduct. Hence we can

conclude that parthenin is most active followed by anhydroparthenin, pyrolysis product and

diazoester adduct. It was also concluded that diazoester adduct and pyrolysis product had

almost similar insecticidal activity against T. castaneum which was lower than

anhydroparthenin. The decreasing order of activity of different compounds against T.

castaneum adults at 20,000 μg g-1 concentration is as follows:

Parthenin >Anhydroparthenin> Pyrolysis product >Diazoester Adduct

52

Table 10: Comparison of corrected percentage mortality of T. castaneum with parthenin and its reaction products at indicated time interval after treatment at 20,000 μg g-1

Days of

application Concentrations

Parthenin

Anhydroparthenin

Pyrolysis product

Diazoester adduct

1 8.33 1.67 0 0

3 48.33 11.67 8.33 6.67

5 68.33 20.00 13.33 13.33

8 98.33 35.00 23.33 21.67

10 100.00 41.67 35.00 28.33

15 - 75.00 56.67 41.67

18 - 100.00 78.33 65.00

20 - - 100.00 76.67

25 - - - 100.00

4.1.6 Structure activity relationship

The biological activity shown by the parthenin may be due to the presence of:

1) Hydroxyl group at position C-1

2) α-methylene-γ-lactone moiety

Removal of hydroxyl group during dehydration reaction of parthenin with dry

hydrochloric acid gas, leads to decrease in insecticidal activity as in case of

anhydroparthenin. Conversion of exomethylene double of α-methylene-γ-lactone moiety to

cyclopropyl ester decreases the insecticidal activity of pyrolysis product and decrease in

activity of diazoester adduct may be due to formation of adduct at the exomethylene double

bond of α-methylene-γ-lactone moiety. The insecticidal activity of pyrolysis product is more

than that of diazoester adduct which may be due to more ring strain in cyclopropyl ester than

that of diazoester adduct. Anhydroparthenin showed more insecticidal activity as compared

to pyrolysis product and diazoester adduct may be due to extended conjugation.Hence

parthenin was the most active compound followed by anhydroparthenin which in turn was

followed by pyrolysis product and diazoester adduct.

53

Fig. 4.5: Comparison of corrected percentage mortality of T. castaneum with parthenin and its reaction products (30, 52 and 53) at indicated time interval after treatment at 20,000 μg g-1

0

10

20

30

40

50

60

70

80

90

100

Parthenin Anhydroparthenin Pyrolysis product Diazoester adduct

Cor

rect

ed p

er c

ent m

orta

lity

Different compounds

Day 1 Day 3 Day 5 Day 8 Day 10 Day 15 Day 18 Day 20 Day 25

54

Table 11: Effect of Parthenin and anhydroparthenin and their different concentrations on T. castaneum at different time intervals.

Day Parthenin Anhydroparthenin

C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6

0 1.3±0.47 0 0 0 0 0 0.3±0.58 0 0 0 0 0

5 13.67 ±12.5 3 ±1 0.3±0.58 0 0 0 3.3 ±1.5 1.7±0.58 0.3 ±0.58 0 0 0

10 20±0 17±2 8 ±2.6 4.3 ±1.25 1.7 ±1.2 1.1±0.99 8±2 5 ±1 2.7 ±1.5 1±0 0.3 ±0.58 0

15 20±0 20±0 14 ±1.7 8 ±1.7 2.7 ±1.2 1.7±0.58 15±1 5.7 ±0.58 4.7 ±1.2 2.3 ±0.58 1.3 ±0.58 0.3 ±0.58

20 20±0 20±0 15 ±1 11±3 6 ±1 2±1 20±0 7.3 ±1.5 5±1 2.7± 0.58 1.67±0.58 1.3 ±0.58

25 20±0 20±0 15.3±0.58 11.7±2.1 7 ±1 2.3±0.58 20±0 9.3 ±1.2 6.7 ±1.2 4.7 ±1.2 2.7 ±0.58 2 ±1

30 20±0 20±0 15.7 ±1.2 11.7±2.1 7.7 ±1.5 2.7 ±1.2 20±0 11.7±0.58 9±1 6.7 ±0.58 4.7 ±0.58 3.7 ±0.58

35 20±0 20±0 18±1 14.7 ±1.2 8±2 3±1 20±0 13 ±1 10 ±1 8±1 6 ±1 4.3 ±0.58

40 20±0 20±0 18.3 ±1.2 14.7 ±1.2 8±2 3±1 20±0 15 ±1 10.7±0.58 10.7 ±1.2 7 ±1 6.3 ±0.58

45 20±0 20±0 19.3 ±1.2 17±1 10.3 ±1.5 4.7 ±1.5 20±0 17.3±0.58 13.3 ±1.2 12±2 10 ±1 7±1

50 20±0 20±0 20±0 18.7 ±1.5 13.7 ±1.5 7.3 ±1.5 20±0 20±0 15.7±0.58 12.7 ±1.5 10.3 ±1.5 8.7 ±0.58

55 20±0 20±0 20±0 20±0 16.3 ±1.5 9±1 20±0 20±0 16±1 14.7±0.58 11.3±2.3 11 ±1.73

60 20±0 20±0 20±0 20±0 19±1 9±1 20±0 20±0 18 ±1 17.3 ±1.2 13.4 ±1.5 11±1.7

65 20±0 20±0 20±0 20±0 20±0 9.7 ±1.5 20±0 20±0 16.3 ±0.58 17.3 ±1.2 14 ±1.7 11±1.7

Values are Mean ± S.E. Where, C1=20,000 μg g-1, C2=10,000 μg g-1, C3=5000μg g-1, C4=4000μg g-1, C5=2000μg g-1, C6=1000μg g-1 C.D (5%) - Compounds (0.17), Concentrations (0.20) , Days (0.30) , Compound × Concentration (0.40) , Compound × Days (0.60), Concentration × Days (0.74)

55

Table 12: Effect of Pyrolysis product and diazoester adduct and their different concentrations on T. castaneum at different time intervals.

Day Pyrolysis product Diazoester adduct

C1 C2 C3 C4 C5 C6 C1 C2 C3 C4 C5 C6

0 0 0 0 0 0 0 0 0 0 0 0 0

5 2.3±0.58 1.3±0.58 0.3±0.58 0.3 ±0.58 0 0 2.7±0.58 2±0 0 0 0 0

10 7±1 3±1 1.7±0.58 2±0 0.7±0.58 0.3±0.58 5.7±0.58 3.3±1.2 2±0 1.3±0.58 0.7±0.58 0

15 11.3±1.2 4.6 ±1.5 3.7±0.58 3±1 2±1 1.7±1.2 8.3±0.58 3.7±0.58 3±0 2.3±0.58 13.4±0.58 0.34±0.58

20 20±0 6.3 ±.058 5 ±1 4±1 3.7±1.2 2.7±1.2 16±1 4.3±1.2 4.3±0.58 2.7±0.58 2±1 1±1

25 20± 0 6.7±0.58 5.3 ±1.2 4.7±0.58 4±1 3±1 20±0 5.3±1.2 4.3±0.58 3.3±0.58 2.3±0.58 2 ±1

30 20± 0 7.3±0.58 6.3 ±1.2 5.3±0.58 5±1 3.7±0.58 20±0 6.7 ±1.6 5±1 4.3±0.58 3±1 2.7±0.58

35 20±0 7.7±0.58 7.3 ±1.5 6±1 5.3±0.58 4.3±0.58 20±0 7±1 5.3±0.58 4.7±0.58 3±1 2.7±0.58

40 20±0 10.7 ±1.2 8.3 ±0.58 7.3±0.58 6.4±0.58 5±1 20±0 8.3±0.58 6.3±1.5 5.7±0.58 4.7±0.58 4±1

45 20±0 12.7 ±1.5 9.7 ±1.5 9±1 7.7±1.2 7.3± 1.5 20± 0 9.7±0.58 7.7±1.6 7±1 5.3±0.58 4.7 ±1.2

50 20±0 18.7±0.58 14.7 ±1.2 13±1 10.7±0.57 9.3±1.5 20±0 10.7±0.58 11.7±0.58 7.7±0.58 8.3 ±1.5 6.7 ±1.5

55 20±0 20±0 18±1 16.3±0.58 5.7 ±1.5 11.7±1.5 20±0 15.3 ±1.5 14±1 14±1 12.3±0.58 9.7 ±0.58

60 20±0 20±0 18±1 16.3±0.58 5.7±1.5 11.7±1.5 20±0 20 ±0 17.3±0.58 16.7±1.2 15.7±1.2 11.3 ±1.2

65 20±0 20±0 18 ±1 16.3±0.58 5.7±1.5 11.7±1.5 20±0 20±0 17.3±0.58 16.7±1.2 15.7±1.2 11.3 ±1.2

Values are Mean ± S.E. Where, C1=20,000 μg g-1, C2=10,000 μg g-1, C3=5000μg g-1, C4=4000μg g-1, C5=2000μg g-1, C6=1000μg g-1 C.D (5%) - Compounds (0.17), Concentrations (0.20) , Days (0.30) , Compound × Concentration (0.40) , Compound × Days (0.60), Concentration × Days (0.74)

56

4.1.7 Statistical Analysis

Table 13: Analysis of factorial experiment in CRD for CD (5%)

For comparison CD

Compounds (A) 0.16

Concentrations (B) 0.20

Days (C) 0.30

Compounds × Concentrations (A ×B) 0.40

Compounds × Days (A × C) 0.60

Concentrations × Days (B × C) 0.74

A= Compounds, B= Concentrations, C= Days, AB= Interaction between compound and concentration, AC= Interaction between Compound and days, BC= Interaction between Concentrations and days, C.D. = Critical difference.

The statistical analysis of the parent and all the compounds prepared was carried out

and the results are given in Tables 11, 12 and 13. Crticial difference was calculated for all the

compounds. The crticial difference values for compounds, concentration and days were 0.16,

0.20 and 0.30, respectively. The results showed that all the compounds, concentrations and

days behaved significantly different. The interaction of the compound was statistically

analyzed with respect to concentration and number of days. The crticial difference values of

0.40, 0.60 and 0.74 were obtained indicating that the interaction between the compound and

concentration, compound and days and concentration and days is also significantly different.

4.1.8 Conclusions

From the bioefficacy studies of parthenin and its reaction products obtained by

carrying out different reactions it was found that at all concentrations parthenin showed more

biological activity as compared to its different compounds prepared by carrying out different

recations.

Parthenin (1) was found to be more active than anhydroparthenin (30) against

Tribolium castaneum. The activity in parthenin may be due to the presence of hydroxyl

group at C-1 and α-methylene-γ-lactone moiety. The decrease of activity in

anhydroparthenin may be attributed to the loss of hydroxyl group during the reaction. Hence

the activity of parthenin may be due to the presence of hydroxyl group at the C-1. Parthenin

showed more activity as compared to pyrolysis product (52). The decrease in activity is

thought to be due to the loss of double bond and instability of newly formed cyclopropyl

ester. In case of diazoester adduct (53) the activity is less than the parent compound, due to

57

formation of adduct at α-methylene-γ-lactone moiety. The result revealed that the decrease in

activity may be due to loss of exomethylene double bond.

Dehydration product anhydroparthenin (30) showed more insecticidal potential as

compared to pyrolysis product (52) and diazoester adduct (53) which may be due to extended

conjugation after removal of hydroxyl group due to dehydration during the reaction. All the

compounds showed high activity at higher concentrations whereas at lower concentrations

the insecticidal activity was low. The insecticidal activity of diazoester adduct and pyrolysis

product was almost comparable in the beginning but with the passage of time the pyrolysis

product was found to be more active than diazoester adduct. It may be due to lowering of

activity due to formation of adduct whereas cyclopropyl ring may be more active as

compared to adduct.

Hence we can conclude that the insecticidal activity of parthenin against Tribolium

castaneum may be due to the presence of hydroxyl group and α-methylene-γ-lactone moiety.

The order of insecticidal potential of compounds is:

Parthenin > Anhydroparthenin > Pyrolysis product > Diazoester Adduct

CHAPTER – V

SUMMARY

Stored grain insect pests have been damaging our economy by infesting agricultural

stored products. The continuous increasing pressure of expanding human population has

created a critical problem of food scarcity. Thus protection of stored grains and other

agricultural products from insect infestation is essential to feed the increasing population.

Various synthetic insecticides have been used to minimize the loss caused by insect pests but

pests developed resistance against most of these synthetic pesticides. The uncontrolled use of

these synthetic insecticides also causes great hazards for environment and consumers due to

residual property Therefore, it is an urgent need to develop bioinsecticides which should be

ecologically safe, biodegradable and cause no toxicity in non-target organisms.So the present

work on chemistry and insecticidal potential potential of parthenin and its derivatives was

undertaken.

The work incorporated in the present thesis reports the isolation and chemical

transformations of parthenin (1), a sesquiterpene lactone obtained from the leaves of P.

hysterophorus, followed by testing the bioefficacy studies of parthenin and its derivatives

against Tribolium castaneum (Herbst).

The thesis has been divided into five chapters including summary. The first chapter

incorporates introduction regarding the research problem. The second chapter contains an

exhaustive review of literature followed by fine details of experimental section (Chapter III).

Chapter IV deals with results and discussion. The leaves of P. hysterophorous were plucked,

shade-dried, powdered and extracted in chloroform using Soxhlet extraction method. Extract

of four batches (9.5 g) was collected and to this was added methanol (150 mL), water (150

mL), lead acetate (5.0 g) and glacial acetic acid (5mL) and kept overnight. The clear

solution, yellow in color, was filtered and the filtrate was concentrated to a minimum volume

59

by distilling off methanol. The residue was diluted with water (1:1) and then thoroughly

extracted with chloroform (3 x 50 mL). The organic layer was dried over anhydrous sodium

sulfate and chloroform was distilled to yield crude extract (7.5 g). The extract so obtained

was dissolved in minimum amount of chloroform and chromatographed over silica gel (450

g). Pure parthenin was recovered using column chromatography when five percent

chloroform: acetone was used as eluent. Characterization of parthenin was done by

determining melting point (1620C); checking purity by thin layer chromatography and

confirmation of structures was done by spectral studies.

Different reactions carried out during the work include:

• Reaction with diazoester

• Reaction of parthenin with dry HCl gas

• Reaction of parthenin with formic acid

• Irradiation of parthenin with microwave radiation

Parthenin was subjected to reaction with diazoester pyrolysis product (52) and

diazoester adduct (53) were obtained as the product.

               

  Parthenin was subjected to reaction with dry hydrochloric acid gas and formic acid

which afforded anhydroparthenin (30) as the major product. On irradiation of parthenin with

microwave anhydroparthenin was found to be the major product. Parthenin was also

subjected to reaction under microwave conditions. Anhydroparthenin was obtained as the

product.

60

All the synthesized compounds anhydroparthenin (30), pyrolysis product (52) and

diazoester adduct (53) were characterized on the basis of thin layer chromatography, infrared

and nuclear magnetic resonance spectroscopy.

Parthenin (1) and its derivatives anhydroparthenin (30), pyrolysis product (52) and

diazoester adduct (53) were evaluated for their bio-efficacy against T. castaneum. The insects

were reared and F1 generation adults were selected for studying the bio-efficacy at different

spiking levels from 1,000 to 20,000 μg/g. The mortality was noted every 24 hrs till complete

or constant mortality was observed. All the compounds showed complete mortality at 10,000

and 20,000 μg/g whereas the compounds at low concentrations showed less mortality. All the

compounds synthesized are less active as compared to the parent i.e. parthenin. It was seen

that dehydration product anhydroparthenin showed high activity as compared to diazoester

adduct and pyrolysis product of parthenin. Mortality rate was very low at lower

concentration (1,000 μg/g) for all the compounds tested. The concentration of 20,000 μg g-1

was most effective whereas 1,000μg g-1 was least effective. Results indicate that mortality

increased with increase in concentration of the compound applied and also with increase in

time of application. The decreasing order of biological activity shown by parthenin and its

reaction products is given below:

Parthenin > Anhydroparthenin > Pyrolysis product > Diazoester Adduct

Statistical analysis of the parthenin and its various reaction products showed that

they behaved significantly different from each other. Different days and concentrations

employed also behaved significantly different in terms of their insecticidal activity against

Tribolium castaneum.

Hence, it was concluded that parthenin was more active as insecticide against stored

grain pest of wheat i.e. Tribolium castaneum as compared to all its derivatives prepared.

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VITA

Name : Ramandeep Kaur

Father's name : S. Lakhwinder Singh

Mother's name : Mrs. Hardip Kaur

Nationality : Indian

Date of birth : 09.12.1989

Permanent Home Address : 58, Gurdyal Enclave, P.O. Box-Jamalpur Awana, Ludhiana

EDUCATIONAL QUALIFICATION

Bachelor's degree : B.Sc. (Medical)

University and year of award : Panjab University, Chandigarh (2010)

%age of marks : 82.2

Master's degree : M.Sc. (Chemistry)

University and year of award : Punjab Agricultural University, Ludhiana (2012)

OCPA : 8.44/10.00

Title of Master's Thesis : Chemistry and insecticidal potential of Parthenin and its transformation reaction products against Tribolium castaneum (Herbst) Awards/Distinctions/Scholarships/ : University Merit Scholarship holder Fellowships during Master degree programme (third and fourth semester).