phytochemical and biological studies of croton
TRANSCRIPT
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Phytochemical and biological studies of Croton bonplandianum (Euphorbiaceae)
By
Muhammad Naeem Qaisar
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctorate of Philosophy in Pharmaceutical Chemistry
FACULTY OF PHARMACY
BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN
PAKISTAN
2015
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Brief Contents
Serial No. Contents Page No.
1 List of Abbreviations
2 List of Tables
3 List of Figures
4 Introduction 1
5 Literature review 9
6 Materials and Methods 64
7 Results 89
8 Discussions 117
9 References 120
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Acknowledgement
In the name of Allah, who has given me strength and courage to accomplish this work in the
benefit of mankind. I bow my head on thanks and gratitude to Allah for his countless blessings.
It is great pleasure to express my indebted gratitude to my supervisor Professor Dr. Bashir
Ahmad Chaudhary for instilling in me the value of hard work, dedication and thirst for
knowledge. I am greatly thankful to him for his constant care, encouragement and especially for
his kind behavior. I am also thankful to Dr. Khalid Husain Janbaz, Dean Faculty of Pharmacy,
Bahauddin Zakariya University Islamabad for his supportive attitude. I wish to express best
regards to my co supervisor Dr. Muhammad Uzair for providing me the opportunity to work
under his kind guidance and for his supportive attitude. I am also thankful to administrative staff
of Faculty of Pharmacy, Bahauddin Zakariya University. I am blessed by having a friend like
Sajid Nawaz Hussain who always provided support and motivation to me. His encouraging
behaviour and help were always there where things didn’t seem to work. I would also like to
thank my friends and lab fellows Farook Azam, Khurram Afzal, and Sadd Ullaha for providing
me nice company during my research work. I would like to pay my heartiest thanks to my
parents and my grandmother for their prayers, untiring efforts, supporting and encouraging
behavior. Perhaps I would not be able to present this work in present form without co-operation
of Higher Education Commission (HEC) Pakistan for funding me through Indigenous PhD
fellowship programme.
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Abbreviations
13C-NMR Carbon-13 Nuclear Magnetic Resonance
1H-NMR Proton Nuclear Magnetic Resonance
CC Column chromatography
DCM Dichloromethane
DPPH 1, 1-Diphenyl-2-picrylhydrazyl
HR-EI Masspec High Resolution Electron Impact mass spectroscopy
IR Infrared
MeOH Methanol
HRMS High Resolution mass spectrometry
NaOH Sodium Hydroxyde
NMR Nuclear Magnetic Resonance
TLC Thin layer chromatography
UV Ultraviolet
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Abstract
The research work was carried out for the phytochemical and biological studies of Croton
bonplandianum (Euphorbiaceae). Preliminary phytochemical screening revealed the presence of
alkaloids, saponins, flavonoids, tannins and terpenoids while anthraquinone glycosides and
cardiac glycosides were absent. The extraction of dried plant material was affected by
dichloromethane and methanol successively. Both dichloromethane and methanol extracts were
subjected to biological activities such as antibacterial, antifungal, antioxidant, α-chymotrypsin
inhibitory, urease inhibitory, α-glucosidase inhibitory and butyrylcholinesterase inhibitory
activities along with brine-shrimp toxicity, phytotoxicity against Lemna minor. Dichloromethane
extract has shown in vitro α-glucosidase inhibitory activity of 97.89 % with IC50 value of 14.93
µg/ml compared to the standard acarbose, which exhibited 92.23 % inhibition with IC50 value of
38.25 µg/ml. Methanol extract appeared with potent butyrylcholinesterase inhibitory activity of
84.14 % with IC50 found to be 31.01 µg/ml compared to the standard eserine, which exhibited
82.82 % inhibition with IC50 value of 30.01 µg/ml. Methanol extract was found toxic with LD50
value of 115.76 (0.0048 - 13.76) µg/ml against Artemia salina and also showed radical
scavenging activity (%RSA) of 59.62% with IC50 value of 396.20 µg/ml . Based on these results
activity guided isolation of constituents from dichloromethane and methanol extracts were done.
Fractionation of dichloromethane extract by column chromatography on silica gel and Sephadex
LH 20 using different mobile phase systems led to the purification of compounds (A-I). The
structures of these isolated compounds were established by spectroscopic technique such as UV
and IR spectroscopy. Proton Nuclear Magnetic Resonance (1H NMR), 13C NMR and Mass
spectrophotometry (EIMS, HRMS) were used for elucidation of structure. On the basis of
physical and spectral data from literature, these compounds were identified as n-pentacosanyl-
n-nonadeca-7′-en-9′-α-ol-1′-oate (A), n-tridecanyl n-octadec-9,12-dienoate (B), nonacosyl
hexadecanoate (C), heptacosanoic acid (D), 1,3,5-trihydroxy-2-hexadecanoylamino-(6e,9e)-
heptacosdiene (E), coumarin (F), betulin (G), stigmasterol (H), and 3,5-dimethoxy 4-hydroxy
cinnamic acid (I) were isolated. All these compounds were screened for in vitro α-glucosidase
inhibitory activity, compound F, G and I possessed significant α-glucosidase inhibitory activity
in a concentration-dependent manner and explained more potent inhibitory activity with IC50
values ranging from 23.0 to 26.7 μg/ml than that of a positive control acarbose (IC50, 38.2
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µg/ml). Fractionation of methanol extract by column chromatography on silica gel using
different mobile phase system afforded five compounds (J-N). Based on spectral data the
chemical structure has been established as 4-hydroxy-3,5-dimethoxybenzoic acid (J), 5,8-
dihydroxycoumarin (K), stigmasterol 3-O- β -D-glucoside (L), sparsifol (M) and 6-O-β-D-
glucopyranosyl-β-D-(1-O-sinapoyl,6'-O-sinapoyl)-glucopyranose (N) were isolated from
methanol extract of Croton bonplandianum. The compounds J, K, L and N exhibited significant
butyrylcholinesterase inhibitory activity in a concentration-dependent manner and exhibited
potent inhibitory activity with IC50 values ranging from 21.0 to 36.0 μg/ml, than that of a
positive control eserine (IC50, 32.0 µg/ml).
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CONTENTS
1. Introduction 1
1.1 Secondary metabolites 1
1.1.1 Alkaloids 2
1.1.2 Phenolics 2
1.1.3 Terpenoids 3
1.1.4 Tannins 3
1.1.5 Glycosides 4
1.2 Botanical aspects of Euphorbiaceae 4
1.2.1 Classification 4
1.2.2 Botanical aspects of genus Croton 5
1.2.3 Croton bonplandianum 5
1.3 Aims and objective 8
2 Literature review 9
2.1 Ethnomedicinal uses of Croton species 9
2.2 Previous phytochemical reports on genus Croton 20
2.2.1.1 Aporphine 20
2.2.1.2 proaporphine 21
2.2.1.3 Morphinane Dienone 24
2.2.1.4 Protoberberine 25
2.2.1.5 Glutarimide 26
2.2.1.6 Guaiane 26
2.2.1.7 Harman 27
2.2.1.8 Tyramine 27
2.2.1.9 Benzylisoquinoline 27
2.2.1.10 Peptide derivatives 27
2.2.1.11 Miscellaneous alkaloids 27
2.2.2 Flavonoids 29
2.2.3 Terpenoids 31
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2.2.3.1 Monoterpenes and sesquiterpenes 31
2.2.3.2 Diterpenoids 32
2.2.3.2.1 Acyclic diterpenoids 32
2.2.3.2.2 Bicyclic diterpenoids 33
2.2.3.2.3 Clerodane diterpenoids 33
2.2.3.2.4 Halimanes and an indane derivatives 37
2.2.3.2.5 Labdanes 38
2.2.3.3 Tricyclic diterpenoids 40
2.2.3.3.1 Abietanes 40
2.2.3.3.2 Daphnanes 41
2.2.3.3.3 Pimaranes and isopimaranes 41
2.2.3.4 Tetracyclic diterpenoids 42
2.2.3.4.1 Atisanes 42
2.2.3.4.2 Kauranes 42
2.2.3.5 Pentacyclic diterpenoids 47
2.2.3.6 Macrocyclic diterpenoids 48
2.2.3.7 Limonoids 50
2.2.3.8 Triterpenoids 50
2.2.4 Phytosterols 53
2.2.5 Fixed oils 55
2.3. Previous pharmacological reports on Genus Croton 55
2.3.1 Antioxidant activity 55
2.3.2 Antidiarrhial activity 56
2.3.3 Antimicrobial activity 56
2.3.4 Antimalarial activity 57
2.3.5. Antiulcer activity 58
2.3.6. Anticancer activity 58
2.3.7. Antihypertensive activity 60
2.3.8. Antiinflammatory and antinociceptive 60
2.3.9. Antidepresant activity 61
2.3.10 Antihyperlipidemic and antihypercholestrolemic activity 61
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2.3.11 Antiviral activity 62
2.3.12 Vasorelaxant activity 62
2.3.13 Antioestrogenic activity 62
2.3.14 Insecticidal activity 62
2.3.15 Antileishmanial activity 62
2.3.16 Antispasmodic activity 63
2.3.17 Phyt
otoxic activity 63
3. Material and methods 64
3.1 Collection of plant material 64
3.2 Solvents and chemicals 64
3.3 Preparations of reagents 64
3.3.1 Wagner’s reagent 64
3.3.2 Mayer’s reagent 64
3.3.3 Hager’s reagent 64
3.3.4 Dragendroff’s reagent 65
3.3.5 Godine reagent 65
3.4 Preparation of solutions 65
3.4.1 Preparation of dilute HCl 65
3.4.2 Preparation of dilute ammonia solution 65
3.4.3 Preparation of 70% alcohol 65
3.4.4 Preparation of lead subacetate solution 65
3.4.5 10 M NaOH 65
3.4.6 10% Ferric chloride solution 66
3.4.7 3.5% Ferric chloride in glacial acetic acid 66
3.4.8 1% Gelatin solution in 10% Sodium chloride 66
3.4.9 10% Sulphuric acid 66
3.5 Phytochemical methods 66
3.5.1 Preliminary phytochemical screening of plant material 66
3.5.1.1 Detection of alkaloids 66
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3.5.1.2 Detection of anthraquinone glycosides 67
3.5.1.3 Detection of cardioactive glycosides 67
3.5.1.4 Detection of tannins 67
3.5.1.4.1 Ferric chloride test 67
3.5.1.4.2 Gelatin test 68
3.5.1.4.3 Catechin test 68
3.5.1.5 Tests for saponin glycosides 68
3.5.1.6 Detection of flavonoids 68
3.5.1.7 Detection of terpenoids 68
3.6 Extraction 68
3.7 Chromatographic Method 69
3.7.1 Thin Layer Chromatography 69
3.7.1.1 Visualisation of components on TLC plates 69
3.7.2 Column Chromatography 69
3.8 Spectroscopy 71
3.9 Physical and Spectroscopic data of isolated compound(A-I) 72
3.9.1 Compound A 72
3.9.2 Compound B 73
3.9.3 Compound C 73
3.9.4 Compound D 74
3.9.5 Compound E 75
3.9.6 Compound F 76
3.9.7 Compound G 77
3.9.8 Compound H 77
3.9.9 Compound I 78
3.9.10 Compound J 79
3.9.11 Compound K 80
3.9.12 Compound L 81
3.9.13 Compound M 82
3.9.14 Compound N 82
3.10 Biological methods 83
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3.10.1 Antibacterial assay 83
3.10.2 Antifungal assay 84
3.10.3 Antioxidant assay 84
3.10.4 Cytotoxic assay 85
3.10.5 Phytotoxic assay 85
3.10.6 Urease inhibition assay 86
3.10.7 α-Chymotrypsin inhibition assay 86
3.10.8 α-glucosidase inhibition assay 87
3.10.9 Butyrylcholinesterase inhibition assay 87
4. Results 89
4.1 Phytochemical studies 89
4.1.1 Detection of secondary metabolites 89
4.1.2 Extraction 89
4.2 Biological screening of crude extracts 90
4.3 Thin layer Chromatography 95
4.3.1 TLC analysis of dichloromethane extract of Croton bonplandianum 95
4.3.2 TLC analysis of methanol extract of Croton bonplandianum 96
4.4 Isolation of compounds 97
4.4.1 Isolation of compounds from dichloromethane extract 97
4.4.2 Isolation of compound (J-N) from methanol extract (CBM) 99
4.5 Structure elucidation of the isolated compounds 101
4.5.1 Compound A (n-Pentacosanyl-n-nonadeca-7’-en-9’-α-ol-1’-oate) 101
4.5.2 Compound B (n-Tridecanyl n-octadec-9,12-dienoate) 102
4.5.3 Compound C (Nonacosyl hexadecanoate) 103
4.5.4 Compound D (Heptacosanoic acid) 104
4.5.5 Compound E (1,3,5-Trihydroxy-2-hexadecanoylamino-
(6E,9E)-heptacosdiene) 105
4.5.6 Compound F (2H-1-Benzopyran-2-one) 106
4.5.7 Compound G (Betulin) 107
4.5.8 Compound H (Stigmasterol) 108
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4.5.9 Compound I (3,5-Dimethoxy-4-hydroxy cinnamic acid) 109
4.5.10 Compound J (4-Hydroxy-3,5-dimethoxybenzoic acid) 110
4.5.11 Compound K (5,8-Dihydroxycoumarin) 111
4.5.12 Compound L (Stigmasterol 3-O-β-D-glucoside) 112
4.5.13 Compound M (Sparsifol) 113
4.5.14 CompoundN(6-O-β-D-Glucopyranosyl-β-D-(1-O-sinapoyl,6’-O-
sinapoyl)- glucopyranose 114
4.6 Biological activity of isolated compounds 115
5. Discussion 117
6. References 120
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LIST OF FIGURES
1.1 Taxonomical classification of Croton bonplandianum 6
1.2 Croton bonplandianum 7
4.1 Results of TLC analysis of dichloromethane extract of
C.bonplandianum 95
4.2 Results of TLC analysis of methanol extract of
C. bonplandianum 96
4.3 Isolation scheme of compounds (A-1) from dichloromethane
extract of Croton bonplandianum 98
4.2 The schematic representation of isolation of compounds (J-N) from
methanol extract of Croton bonplandianum 100
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LIST OF TABLES
2.1 Ethnomedicinal uses of Croton species 9
3.1 Solvent systems used for the analysis of dichloromethane extracts of
Croton bonplandianum 70
3.2 Solvent systems used for the analysis of methanol extracts of Croton
bonplandianum 71
4.1 Results of phytochemical screening of Croton bonplandianum 89
4.2 Results of extraction of plant material with different solvents 89
4.3 Results of antibacterial bioassay of methanol and dichloromethane
extracts of Croton bonplandianum 90
4.4 Results of antifungal bioassay of methanol and dichloromethane
extracts of Croton bonplandianum 91
4.5 Results of phytotoxic bioassay of methanol and dichloromethane
extracts of Croton bonplandianum 91
4.6 Results of Brine Shrimp Lethality bioassay of methanol and
dichloromethane extracts of Croton bonplandianum 92
4.7 Results of antioxidant activity of methanol and dichloromethane
extracts of Croton bonplandianum 92
4.8 Results of α-chymotrypsin inhibition assay of methanol and
dichloromethane extracts of Croton bonplandianum 92
4.9 Results of urease inhibitory activity of methanol and dichloromethane
extracts of Croton bonplandianum 93
4.10 Results of α-Glucosidase inhibition assay of methanol and
dichloromethane extracts of Croton bonplandianum 93
4.11 Results of butyrylcholinesterase inhibition assay of methanol and
dichloromethane extracts of Croton bonplandianum 94
4.12 Results of α-Glucosidase inhibition assay of compounds (A-1)
isolated from dichloromethane extracts of Croton bonplandianum 115
4.13 Results of butyrylcholinesterase inhibition assay of compounds (J-N)
isolated from methanol extracts of Croton bonplandianum
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1 Introduction
Since time immemorial and in almost all cultures, man has relied on nature for basic needs such
as food, shelter, clothing, fragrances and medicines (Cragg and Newman, 2005). The oldest
records of the use of plants as medicinal agents came from Mesopotamia and from the ancient
period of about 2600 BC. Plants have been used as medicines for various ailments such as
cancer, antitumor, hypolipidemic, cardiovascular diseases, ant platelet and for other purpose
such as immune-stimulating agents (Liu, 2011). The medicines initially used in the form of
crude drugs such as tinctures, teas, poultices, powders and other herbal formulations whose
dosage was developed through experience and experimentation (Balick and Cox, 1997). Due to
the development of separation techniques and pharmacological evaluation, the medicines are
nowadays made of active compounds isolated from the plants, or their synthetic equivalents. The
information regarding specific plants used for particular ailment and the method of application
were originally by oral traditional mode but later became documented in herbal pharmacopoeias
(Balunas and Kinghorn, 2005).
1.1: Secondary metabolites
The plant constituents are classified as primary and secondary metabolites. Primary metabolites
are widely distributed in nature, occurring in one form or another in virtually all organisms. In
higher plants such compounds were often concentrated in seeds and vegetative storage organs
and are needed for physiological development because of their role in basic cell metabolism.
Plants generally produce many secondary metabolites which are biosynthetically derived from
primary metabolites. Secondary metabolites have been directly or indirectly playing an important
role in the human society to combat diseases (Wink et al., 2005). Secondary metabolites have no
apparent function in a plant’s primary metabolism, but often have an ecological role, as
pollinator attractants, represent chemical adaptations to environmental stresses or serve as
chemical defense against micro-organisms, insects and higher predators. Secondary metabolites
are frequently accumulated by plants in smaller quantities than the primary metabolites
(Karuppusamy, 2009; Sathishkumar et al., 2009). In contrast to primary metabolites, they are
synthesized in specialized cell types and at distinct developmental stages, making their extraction
and purification difficult. As a result, secondary metabolites that are used commercially as
biologically active compounds are generally high value-low volume products than the primary
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metabolites, which are used in drug manufacture by the pharmaceutical industries. A simple
classification of secondary metabolites includes alkaloids, phenolics, terpenoids, tannins and
glycosides.
1.1.1: Alkaloids
The alkaloids represent the group of secondary metabolites that include basic nitrogen atoms.
The compounds with neutral and weakly acid properties are also incorporated in the alkaloids.
Along with carbon, hydrogen and nitrogen, the group also holds oxygen, sulfur and rarely other
element such as chlorine, bromine and phosphorus (Nicolaou et al., 2011). Alkaloids are
produced by a large variety of organisms, such as bacteria, fungi, animals, but mostly by plants
as secondary metabolites. Most of them are toxic to other organisms and can be extracted by
acid-base. They have diverse pharmacological effects and have a long history in medications
(Aniszewski, 2007.) The boundary between alkaloids and other nitrogen-containing natural
compounds is not clear-cut (Giweli et al., 2013). Compounds like amino acids, proteins,
peptides, nucleotides, nucleic acid, and amines are not usually called alkaloids. Compared with
most other classes of secondary metabolites, alkaloids are characterized by a great structural
diversity and there is no uniform classification of them (Verpoorte, 1998). First classification
was based on the common source because no information about chemical structure was yet
available. Recent classification is based on similarity of the carbon skeleton (Savithramma et al.,
2011)
1.1.2: Phenolics
Phenolic compounds from plants are one of largest group of secondary plants constituents. They
are characterized by the antioxidant, anti-inflammatory, anticarcinogenic and other biological
properties (Park et al., 2001). Hydroxybenzoic acids and hydroxycinnamic acids represent two
main phenolic compounds found in plants. In tea, coffee, berries and fruits, the total phenolic
comounds could reach up to 103 mg/100 g fresh weigh (Manach et al., 2007). The approach to
classifying plant phenolics are based on: (1) a number of hydroxylic group, phenolic compounds
containing more than one OH-group in aromatic ring are polyphenols; (2) chemical composition:
mono-, di, oligo- and polyphenols; (3) substitutes in carbon skeleton, a number of aromatic rings
and carbon atoms in the side chain. According to the latter principle, phenolic compounds are
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divided into four major groups: phenolics with one aromatic ring, with two aromatic rings,
quinones and polymers.
Phenolic compounds with one aromatic ring are simple phenols (C6), phenol with attached one
(C6-C1), two (C6-C2) and three (C6-C3) carbon atoms. Phenolic compounds with two aromatic
rings: this group includes benzoquinones and xanthones (C6-C1-C6) containing two aromatic
rings which are linked by one carbon atom; stylbenes (C6-C2-C6) which are linked by two
carbon atoms; and flavonoids, containin three carbon atoms (C6-C3-C6). Flavonoids, depending
on the structure of propane unit and an attaching place of side chain, are divided into flavonoids
in strict sense, which are derived from chromane or chromone, isoflavonoids and neoflavonoids.
Polyphenolics are more than 8,000 different compounds identified to date. That is why the
terminology and classification of polyphenols is complex and confusing. Although all
polyphenols have similar chemical structures, there are some distinctive differences. Based on
these differences, polyphenols can be subdivided into two classes, flavonoids and non
flavonoids, like tannins (Somasegaran and Hoben, 1994).
1.1.3: Terpenoids
Terpenoids constitute a large family of phytoconstituents such as steroids, carotenoids, and
gibberelic acid. They are the most important group of active compounds in plants with over than
23,000 known structures. They are polymeric isoprene derivatives and synthesized from acetate
via the mevalonic acid pathway. During their formation, the isoprene units are linked in head and
tail fashion. The number of units incorporated into a particular terpene serves as a basis for their
classification. Many of them have pharmacological activity and are used for diseases treatment
both in humans and animals. Diterpenes tend to be most abundant in Lamiaceae family and have
antimicrobial and antiviral properties (Beaulieu and Baldwin, 2002). Some interesting
compounds are extensively used in the industry sector as flavors, fragrance and spices (Styger et
al., 2011). Several thousand different types of molecules from very different plant groups have
been isolated and characterized.
1.1.4: Tannins
Tannins are the phenolic compounds that precipitate proteins. They can form complex with
proteins, starch, cellulose and minerals. They are synthesized via shikimic acid pathway, also
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known as the phenylpropanoid pathway. The same pathway leads to the formation of other
phenolics such as isoflavones, coumarins, lignins and aromatic amino acids. Tannins are water
soluble compounds with exception of some high molecular weight structures. They are usually
subdivided in two groups, hydrolysable tannins that include gallotannins, elligatannins, complex
tannins, and condensed tannins (Lancini and Lorenzetti, 1993). The tannins also constitute the
active principles of plant-based medicines. According the literature, the tannins containing plants
are used as astringents (Fujiki et al., 2012), diuretic, antitumor (Trouillas et al., 2003).
1.1.5: Glycosides
Polt (1995) notes that Glycosides are characterized by a sugar portion or moiety attached by a
special bond to non-sugar portions. Many plants store chemicals in the form of inactive
glycosides which can be activated by enzyme hydrolysis. So, most glycosides can be classified
as prodrugs since they remain inactive until they are hydrolyzed in the large bowel leading to the
release of the aglycone, the right active constituent. Concerning the therapeutic action in
different studies it has been shown that glycosides have anticancer (Zhou et al., 2007)
expectorant (Fernández et al., 2006), sedative and digestive properties (Galvano et al., 2004).
1.2 Botanical aspects of Euphorbiaceae
1.2.1: Classification
Euphorbiaceae has 300 genera and 5000 species mainly shrubs, trees and non-succulent herbs.
It is widely distributed in the world but with strongest representation in the humid tropics and
subtropics region of the both hemispheres (Nasir and Ali, 1986). According to the most recent
research, this notoriously difficult family is divided into f i ve subfamilies the
Acalyphoideae, the Crotonoideae, the Euphorbioideae, the Phyllandthoideae and the Old
fieldioideae. Out of these, first three are uni-ovulate and the last two are bi-ovulate families.
Now, three uni-ovulate subfamilies have become strict ly Euphorbiaceae. The last two
have been separated from the Euphorbiaceae and now treated as the family
Phyllanthaceae (Wurdack., et al 2005). This family has very characteristic smell and cup-
shaped flowers. The male and female flowers of some species of this family are present in
the single flower and each contributes by single stamen. However, some species have
separate male and female plants and some species may produce a mixture of male, female and
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bisexual flowers. In Pakistan, the Euphorbiaceae is represented by 24 genera of which 11 are not
native (Nasir and Ali, 1986). Taxonomical classification is given in figure 1.1.
1.2.2: Botanical aspects of genus Croton
The genus croton, established by Linnaeus in 1737 is the significant genus of the
Euphorbiaceae family and comprises 1300 species as shrubs, herbs and trees of the tropical and
subtropical areas (Salatino et al. 2007).
The leaves are mostly alternate but may be opposite or whorled they are simple, or
compounds, or sometimes highly reduced. The flowers are unisexual and usually
antinomorphic. The genus Croton contains monoecious or more rarely dioecious trees, shrubs,
herbs or lianas indumentums stellate, lepidote or both (Nasir and Ali, 1986).
1.2.3: Croton bonplandianum
The plant grows in S. Balivia, Paraguay, Soth west Brazil, North Argentina, Bangladesh,
South America, South India and Pakistan (Pande and Tewari, 1962, Satish and Bhakuni,
1972). In Pakistan, this plant is found near Khyber, Attock, Wah, Rawalpindi, Sargodha,
Gujarat, Sialkot, Lahore and Karachi. The botanical characteristics are given as under.
Croton bonplandianum Baill is a monoecious woody shrub, which is 1-5 m in height, but
more usually c. 30-40 cm, with whorled branches. Nasir and Ali, (1986) commented the plant
grows in sandy clay soil along roadside, irrigation canal banks, in plantations and on waste
ground. Whole plant Croton plandianum is depicted in figure 1.2.
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Kingdom Planate
Subkingdom Vascular plants Broyophytes
Subgroup Angiosperms Gymnosperms
Class Dicotyledon Monocotyledon
Subclass Rosidae
Order Euphorbiales
Family Euphorbiaceae
Genus Croton
Species Croton bonplandianum
Figure 1.1: Taxonomical classification of Croton bonplandianum.
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Figure 1.2: Croton bonplandianum
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1.3: Aims and objective
The changing climate and lifestyle have emerged as serious global concerns because of certain
issues like; health disorders i.e. cancer, hepatitis, stress-related disorders, urinary disorders, and
bacterial infections. Plants have been reported to possess good therapeutic action against many of
such diseases. Different classes of secondary metabolites, alkaloids and terpenoids have been
accounted for Croton species. Croton species, such as Croton cajucara, Croton zambesicus,
Croton nepetaefolius and Croton celtidifolius have been depicted as medicinal plants with their
biological activities assessed. Amongst such plants studied to date, several have been discovered
to exhibit multiple biological activities, for example Croton celtidifolius has been accounted to
possess anti-inflammatory, antioxidant, antinociceptive, anticonvulsant and anxiolytic activities.
Along these aforementioned studies, many other works are currently underway to assess the
biological activities of the extracts, fractions and active components from plants of the genus
Croton. The literature survey indicated that alkaloids, crotsparine, N-methyl-crotsparine and 3-
methoxy-4, 6-dihydroxymorphinandien-7-one were secluded from Croton bonplandianum,
phenolics compounds and terpenoids were not reported for Croton bonplandianum.
Antimicrobial, antimalarial and phytotoxic activity has been subjected for Croton
bonplandianum. It is of worth significance that apart from these limited studies, no systematic
work has yet been initiated for biological investigation and isolation of compounds from Croton
bonplandianum.
The proposed research was carried out by the application of modern analytical techniques and
bioassay methods, and the set aims and objectives of the research were to;
Evaluate the biological activities of the crude extracts of the selected plant.
Isolate compounds from the crude extracts of selected plant.
Elucidate the chemical structure of the isolated compounds.
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2 Literature review
2.1: Ethnomedicinal uses of Croton species
Croton plants in folk medicine have been extensively used all over the world. A notable
example is sangre de drago, a sap from a number of American Croton species including C.
lechleri Muell.-Arg which is marketed as an herbal remedy for diarrhea, inflammation,
insect bites, viral infections and wounds (Cai et al., 1993a, b; Chen et al., 1994). Croton
plants are used in the treatment of cancer, constipation, diabetes, digestive problems,
dysentery, external wounds, fever, hypercholesterolemia, hypertension, inflammation,
intestinal worms, malaria, pain, ulcers and weight-loss (Salatino et al., 2007). Specific
ethno-medicinal applications of various species across the globe are given in table 2.1.
Table 2.1: Ethnomedicinal uses of Croton species
Name of species
(Region)
Plant part Condition managed Reference
C. alienus Pax
(Kenya)
Unspecified Body weaknesses Gachathi, 2007
C. antanosiensis
Leandri)
(Madagascar)
Stem bark Leafy
branches
Induce virility during
circumcision ceremonies,
Ordeal poison in ancient
times Fumigate houses in
case of epidemic diseases
Schmelzer and
Gurib-Fakim, 2008
C. antisiphiliticus
(Brazil)
Entire plant Stimulant, Wound
healing, Veneral diseases,
Rheumatic fever
Elisabetsky et al.,
1992
C. arboreous
Millsp. (Cascarillo
Mexico)
Aerial parts Auxiliary anti-
inflammatory in
respiratory ailments
Aguilar-
guadarrama and
Rios, 2004
C. argyratus
(Malaysia)
Dried flowers Purgative Ilham et al., 1995
Schmelzer and
Gurib-Fakim, 2008
24
C. barorum Leandri
(Madagascar)
A decoction of
stem and root
barks Aromatic
leafy branches
Malarial fever, Cough,
Diarrhea, Leukaemia and
Breast Cancer Insect
Repellent (lice) and
Perfumery in soap
Rakotonandrasana
et al., 2010
Schmelzer and
Gurib-Fakim, 2008
C. bonplandianus
Baill (Argentina
although it has
gotten its way into
Kenya where it is
found as a common
weed)
Entire plant Antiseptic Bandoni et al.,
1976
C. cajucara Benth.
(Sacaca, Peru and
Brazil)
Stem bark and
Leaves (in form
of tea or pills)
Diabetes, Diarrhea,
Malaria, High Blood
Cholesterol Levels,
Gastrointestinal
disturbances, Hepatic
disturbances, weight loss
Duke, 1984; Duke,
1994; Campos et
al.,2002.
Grassi-Kassisse et
al., 2003
C. californicus
Mueller Arg.
(California, U.S.A.)
Leaves Rheumatism, Malaria,
Pain reliever
Williams et al.,
2001, Chavez et
al., 1982, Wilson
et al., 1976;
Farnsworth et al.,
1969
C. capitatus Mitchx Unspecified Malaria Farnsworth et al.,
1969
C. caudatus
(Indonesia, India)
Stem bark Stomach disorders,
Malaria
Banerji et al., 1988
C. celtidifolius
Baill. (“Sangue-de-
adave”, Brazil)
Stem bark and
Leaf infusions
Inflammatory diseases,
Leukemia, Ulcers and
Rheumatism
Nardi et al., 2003
25
C.ciliatoglandulifer
(Syn. C. ciliato-
glandulosus,
Mexico)
Entire plant Purgative Farnsworth et al.,
1969.
C. cortesianus
(Mexico)
Aerial parts Veneral diseases and
Wound healing
Dominguez and
Alcorn, 1985
C. corymbulosus
(U.S.A)
Aerial parts Purgative Coon, 1974
C. decaryi Leandri
(Madagascar)
Leafy branches
Decoction from
aerial parts
Mattress filler to Repel
Lice Calm patients
suffering from Paranoid
Psychosis
Schmelzer and
Gurib-Fakim, 2008
C. dichogamus Pax
(Kenya, Uganda,
Tanzania, Rwanda
and Ethiopia)
Leaves, Roots
Whole plant
decoction
Fever, Chest ailments,
Stomach diseases,
Tuberculosis, Impotence
Malaria
Kokwaro, 1993
and 2009
Jeruto et al., 2011
C. draco , bearing
a red sap widely
used in traditional
medicine in
Mexico and
Central America)
Aerial parts Fever, Tumors, Bleeding,
Cough, Flu, Diarrhoea
and Stomach ulcers,
Topically as wound
healing for cuts, open
sores, herpes, Anti-septic
after tooth extraction and
Oral sores
Murillo et al.,
2001
C. draconoides
(Peru)
Latex Cancer, Wounds,
Inflammation
Piacente et al.,
1998
C. eluteria Bennett
(“Cascarilla”, Syn.
C. eluteria (L.)
Wright, West Indies
and Northern South
Stem bark (used
as substitute for
Chinchona and
Cascara,
Dysentry, Dyspepsia,
Malaria, Fever,
Bronchitis, Tonic and
Bitters, Flavoring for
liqueurs and Scenting
Duke, 1984, Vigor
et al., 2001
26
America-Bahama
Island)
tobacco
C. flavens
Curacao, Venezuela
Leaves Rheumatism, Fever,
Menstrual Pains
Flores and
Ricalde, 1996.
C. fragilis
Mexico
Entire plant Stomach-aches, Hepatic
pains
Hecker, 1984
C. geayi
Madagascar
Infusion of its
Leafy twigs
Fevers, Coughs, Asthma
and Constipation in new-
born babies
Schmelzer and
Gurib-Fakim,
2008; Palazzino et
al., 1997.
C.glabellus
Mexico
Leaves Ulcers Flores and
Ricalde, 1996
C.glandulosus
Mexico
Entire plant Stomach-aches Heinrich et al.,
1992
C.goudotii
Madagascar
Leaves Stem bark Chronic blennorrhea,
Cough and an Aphrodisiac
Malaria, Chronic
gonorrhea
Rakotonandrasana
et al., 2010
C. gratissimus Leaves Rheumatism, Perfume,
Dropsy, Fever, Bleeding
gum, Perfume Carthatic,
Eruptive irritant,
Respiratory condition,
Intercostals neuralgia,
Dropsy,
Farnsworth et al.,
1969
C.antunesii
Western and
Southern Regions
of Africa
Stem bark Indigestion, Pleurisy,
Uterus disorder, Fish
poison
Watt and
Breyer-Brandwijk,
1962.
Farnsworth et al.,
1969.
27
C. gubouga S.
Moore South
Africa, Tanzania,
Botswana, Caprivi
strip, Malawi,
Zambia and
Zimbabwe.
Seed and stem
bark
Emesis, Pugartive,
Febrifuge, Fish poison,
Laxative, Malaria
Watt and Breyer-
Brandwijk,1962;
Neuwinger, 1996,
2000 and 2004.
C. guatemalensis
(Guatemala)
Stem bark and
Leaves
Malaria Franssen et al.,
1997
C. haumanianus
Congo
Stem bark,
Leaves
Blennoragy, Gastric
diseases, Hypertension,
Epilepsy
Tchissambou et
al., 1990
C.hovarum
Madagascar
Stem bark –
Aerial parts
Leaves
Fish poison Molluscicidal
Colic and Acute Body
Weakness
Krebs and
Ramiarantosa,
1996
Schmelzer and
Gurib-Fakim, 2008
Krebs and
Ramiarantsoa,
1997
C. humilis
Jamaica
Entire plant Insecticide Asprey and
Thornton, 1955
C.insularis
Caledonia,Australia
Entire plant Abortifacient Rageau, 1973
C.jatrophoides
Tanzania
Roots Colds, Intestinal worms
and Stomachache
Schmelzer and
Gurib-Fakim,
2008;Kokwaro,
2009
C.joufra;
Thailand
Stem bark
Decoction of
Blood purification Anti-
dysentery and Peptic
Mokkhasmit et al.,
1971;
28
Leaves and Stem
bark Decoction
of the flowers
promoter Anthelmintic Sutthivaiyakit et
al., 2001.
C.kongensis;
Thailand; China
Entire plant Sores Pei, 1985
C. lechleri
Ecuador and Peru;
Cai et al., 1993a, b
and 1991)
Latex from stem
bark
Wound healing, Cancer,
Stomach ulcers,
Rheumatism
Duke, 1994
C. lobatus
Senegal, Eritrea
and Ethiopia;
Carribean, South
America and The
Arabian Peninsula)
Leaves, Leaves
combined with
seeds and bark
Malaria, Pregnancy
troubles, Dysentery,
Rheumatic pain
Whooping Cough,
Convulsions, Mouth
infections Eye diseases,
un consciousness Lotion
for female sterility
Purgative Anti-
hypertensive medication
Neuwinger, 1996,
2000and 2004;
Attioua et al.,2007
Schmelzer and
Gurib-Fakim,
2008
Neuwinger, 1996,
2000 and 2004.
C.longiracemosus
Gabon
Roots Antheimintic,,Anti-
inflammatory
Akendengue and
Louis,1994
C.macrostachys
Madagascar,
Somali, Sudan,
Eritrea, East Africa,
Angola Guinea,
Liberia, Malawi,
Zambia and
Zimbabwe
Entire plant and
Seeds Decoctions
Malaria, Dysentry,
Rheumatism, Taenacide,
Venereal diseases,
Conjuctivitis, Purgative,
blood clotting, mumphs,
skin rashes Anthelmintic,
vermifuge, Female
infertility, Constipation,
Stomach pains, Chest
Kew, 2012 and
2013 Schmelzer
and Gurib-Fakim,
2008; Klauss and
Adala, 1994;
Mazzanti et al.,
1987
29
pains, Bloat, wound
healing, Diabetics
C.malabaricus
(India)
Fresh shoots Joint Pains, Rheumatic
Arthritis
Pushpangadan and
Atal, 1984
C. malambo
Venezuela and
Colombia
Stem bark
infusion
Diabetes, Diarrhoea,
Rheumatism, Gastric
Ulcer, Anti-
Inflammatory, Analgesic
Suárez et al., 2003
C. mayumbensis J.
Leonard Gabon,
Cameroon and The
Central African
Republic
Stem bark and
Leaves
Microbial Infections,
Human Parasitic Diseases
such as Amoebiasis
Yamale et al.,
2009
C.mauritianus
Reunion Island
Entire plant Fever Vera et al., 1990
C.megalobotrys
Zimbabwe
Stem bark, Roots,
Seeds
Purgative, Malaria,
Abortion, Tape worms
Nyazema, 1984
C.megalocarpus
Kenya Eastwards
to The Democratic
Republic of Congo
and Southwards to
Mozambique. \
Entire plant Stem
bark Decoction
Root decoction
Sap issuing from
its leaves
Gall bladder problems,
Chest pains, Internal
swellings, Malaria
Anthelmintic, Whooping
Cough Pneumonia
Bleeding Wounds
John et al., 1994,
Kokwaro, 2009,
Kew, 2012 and
2013.
C. membranaceus
Mull Arg.( West
Africa)
Root and Leaf
extracts Essential
oils from the
Stem bark
Aromatize tobacco
(Bahamas), Improve
Digestion (Nigeria),
Benign Prostate
Hyperplasia and Measles
(Ghana) Aromatherapy to
Asare et al., 2011;
Adesogan, 1981
30
treat cough, Fever,
Flatulence, Diarrhoea and
Nausea
C. menyhartii
Eastern Africa,
Somalia
Roots Malaria, Dymenorrhea,
Intestinal obstruction,
Influenza
Kokwaro, 1993
and 2009
C.mongue
Madagarscar
Stems and seeds
Stem
Toxic Match
manufacturing
Ralison et al.,
1986
C. mubango
Congo, Ivory
Coast, Angola
Entire Plant Female sterility, Spiritual
madness, Asthma,
Paralysis, Hepatalgia,
Sleeping Sickness,
Diarrhea, Furgative,
Vermifuge
Watt and Breyer-
Brandwijk, 1962;
Bossard et al.,
1993; Bouquet
and Debray, 1974;
Otshudi et al.,
2000
C.mucronifolius
Brazil
Leaves Syphilis, Rheumatism,
Influenza
Lemos et al., 1992
C.nepetaefolius.
Brazil
Infusions or
decoctions of the
stem bark and
leaves
Antispasmodic properties,
Relieve flatulence,
Increase appetite, Sedative
Santos et al., 2008
C.oblongifolus
Chucka; India,
Thailand and
China.
Entire plant and
seeds
Sores, Ringworm,
Migraine, Leprosy,
Dysentery, Diarrhea,
Purgative, Insecticide,
Blood Purification, Anti-
Pyretic, Gastric Ulcers,
Liver enlargement and
remittent fever, Hepatitis
Pei, 1985., Sommit
et al., 2003;
Ngamrojnavanich
et al., 2003
31
C. onacrostachyus
Kenya
Entire tree Psychotherapeutic effect
on muphs- “ngumbu”
Kokwaro, 2009
C. palanostigma
Peru
Stem bark latex,
Leaves,
Boils and sores, Uterine
ulcers, Wounds, Snake
bites, Gastro- intestinal
cancer
Lahlou et al., 2000
C. penduliflorus
Sierra Leone
Eastwards to
Nigeria , Central
African Republic
and Gabon.
Roots, Seeds,
Stem bark Leaf
infusion Seed
extract
Purgative, Stomach-
aches, Labor pains,
Headaches, Impotence
Menstrual disorders,
Fever Uterine tumors and
Stomach complaints
Anika and Shetty,
1983
Adesogan, 1981
Schmelzer and
Gurib-Fakim, 2008
C.polytrichus
Kenya
Roots Headache and labour
pains
Kokwaro, 2009
C.
pseudopulchellus
Mali, Nigeria,
Somalia, Kenya,
Ethiopia, Angola,
Zimbabwe,
Mozambique and
South Africa
Leaves Roots
Stem
Anthrax, Insecticide
Syphilitic ulcers, Chest
infections, Tuberculosis
Asthma, Colds, Viral and
Tissue infections
Condiment, Burnt and
smoke used to flavor fresh
milk
Hedberg et al.,
1983
Langat et al.,
2012
C.regelianus;
Brazil
Leaf Infusion Rheumatism, Malignant
tumors, Stomach aches
Torres et al., 2010
C. repens
Mexico
Entire plant Dysentery, Diarrhea Heinrich et al.,
1992
32
C. roxburghii
India
Entire plant Antivenin, Clear bowels,
Malaria, Cardiotonic
Selvanayahgam et
al., 1994
C. ruizianus
Peru
Leaves Anti-spasmodic,
Vulnerary
Piacente et al.,
1988
C. sakamaliensis
(Madagascar)
Stem bark
infusion
Diarrhea, Cough, Fever,
Purgative
Radulovic et al.,
2006
C. salutaris
Peru
Leaves Fever Brandao et al.,
1985
C.schefleri
(Tanzania)
Roots Insanity, Remedy for
miscarriage
Watt and
Breyer-Brandwijk
, 1962; Mathias,
1982.
C.soliman
(Mexico)
Latex Skin infections, Warts Zamora-martinez
and Pola, 1992
C. steenkampianus
Tanzania,
Mozambique and
Southern Africa
Fresh leaves
Vapor inhalation
Relieve body pains Schmelzer and
Gurib-Fakim,
2008; Adelekan et
al., 2008
C. sublyratus
South-Eastern
Asian Countries
and Thailand.
Its mixture with
C. oblongifolius
Stem bark
Gastric ulcers and gastric
cancer Anthelmintic and
dermatological problems
Kawai et al., 2005
Vongchareonsathit
and De-Eknamkul,
1998; Ogiso et al.,
1981.
C.sylvaticus;
Distributed from
Ethiopia in the
Northern parts of
Africa to the
Eastern Cape in
South Africa,
Stem bark Roots
Unspecified
Leaves Decoction
Leaves infusion
Abdominal disorders,
Tuberculosis, Chest
pains, Rheumatism, Fish
poison Gall sickness in
cattle , Indigestion,
Pleurisy, Poultices for
swellings/wash for body
Venter and Venter,
1996; Mc Gaw et
al., 2000.
Kokwaro, 2009
Watt and Breyer-
Brandwijk, 1962;
Neuwinger, 1996,
33
more widely found
in Gabon to
Angola.
swellings caused by
kwashiokor , Malaria and
Purgative
2000 and 2004.
Kokwaro, 2009
Beentje, 1994
Venter and
Venter, 1996.
C. tiglium L.
Asia
Fruits, Roots Fish poison, Abortifacient,
Tumors, Laxative, Gout,
Contraceptive, Insecticide,
Cancerous sores,
Purgative
Gimlette, 1929;
Chang et al., 1981.
C. tonkinensis
Gagnep Kho sam
Bac Bo; A
Vietnam)
Leaves Digestive disorders,
Abdominal pains,
Dyspepsia, abscesses,
Impetigo, Gastric and
duodenal ulcers, Malaria,
Urticaria, Leprosy,
Psoriasis, Genital organ
prolapse
Giang et al., 2003;
Minh et al., 2003
C. trinitatis
(Nicaragua)
Entire plant Cough, Bleeding gum,
Influenza
Duke, 1994; Kuo
et al., 2007.
C. urucarana Baill.
(Syn. C. ururucana
Baill.; Brazil and
Argentina)
Red latex of stem
bark
Cancer, Diarrhea,
Respiratory and Urinary
tract infection, Wound
healing, Rheumatism
Perez and Anesini,
1994; Perez et al.,
1997 and 1998.
C. zambesicus
Muell. Arg.
(Syn.C. amabilis
Muell.Arg.;
Originally a
Guineo-Congolese
species but now
Roots/ Leave Menstrual pains
Aperient, Anti-malarial,
Anti-Diabetic Wash for
fevers Dysentery and
Convulsions
Hypertension and
Urinary infections
El-hamidi, 1970;
Mohamed et al.,
2009; Ngadjui et
al., 1999; Baccelli
et al., 2007;
Okokon et al.,
2005 and 2013,
34
Widespread in
Tropical Africa)
(Benin), Anti- microbial,
Fever associated with
malaria Body
strengthening medicine
Watt and Breyer-
Brandwijk, 1962.
C. zehntneri Pax.
et
Hoffm.(Canelade-
cunhã; Brazil)
Leaves and Stem
bark
Seizures, Insomnia,
Anxiety, Sedative,
Appetite stimulating,
Gastro-intestinal
disturbances, Food and
drinks sweetener
Coelho-de-souza
et al.,
1997and1998;
Batatinha et al.,
1995.
2.2 Previous phytochemical reports on genus Croton
The phytochemistry of Croton genus is significantly varied, including the many classes of
natural products mainly, alkaloids, flavonoids, terpenoids and essential oils containing mono and
sesquiterpenoids. Compounds reported from Croton genus are elaborated here.
2.2.1: Alkaloids
Alkaloids are nitrogenous compounds classified according to the nature of the nitrogen
containing carbon skeleton. The alkaloids reported from Croton genus are made up of
Aporphine, proaporphine, peptide derived alkaloids, morphinane, benzylisoquinoline,
protoberberine, harman, tyramine, nicotine, anabasine, and guaiane basic carbon skeletons.
2.2.1.1: Aporphine
Glaucine (1) (Milanowski et al., 2002, Dos Santos et al., 2001), thaliporphine (2) (Milanowski et
al., 2002), norisoboldine (3), (Berry et al., 2005) and isoboldine (4) (Amaral and Barnes, 1997)
have been reported from C. lechleri. Magnoflorine (5) was isolated from C. celtidifolius
(Milanowski et al., 2002). Sparsiflorine (6) and N-methylsparsiflorine (7) were isolated from C.
sparsiflorus (Bhakuni et al., 1970). Wilsonirine (8), hernovine (9), methylhernovine (10) and
dimethylhernovine (11) have been reported from C. wilsonii (Stuart and Chambers, 1967).
Isocorydine (12) was isolated from C. hemiargyeus (Wen-han et al., 2003). Magnoflorine
bromide (13) was isolated from C. turumiquirensis (Casagrande et al., 1975). Hemiargine B (14)
35
and norcorydine (15) have been reported from C. hemiargyeus (Wen-han et al., 2003).
Nornuciferine (16) and nuciferine (17) were reported from C. sparsiflorus (Bhakuni et al., 1979).
2.2.1.2: Proaporphine
Linearisine (18), homolinearisine (19), pronuciferine (20), base E (21) and jacularine (22) were
isolated from C. linearis (Farnsworth et al., 1969; Haynes et al., 1966; Piacente et al., 1998).
Crotsparine (23), N-methylcrotsparine (24) and dimethylcrotsparine (25) were reported from C.
sparsiflorus (Bhakuni et al., 1970; Casagrande et al., 1975; Bhakuni and Dhar, 1968; Chatterjee
and Majumder, 1968). Amuronine (26) was isolated from C. flavens (Charris et al., 2000)
Crotonosine (27) from C. linearis (Farnsworth et al., 1969; Haynes et al., 1966).
Dimethylcrotonosine (28) was reported from C. plumieri (Stuart, 1970). Methylcrotonosine (29),
discolorine (30) and jaculadine (31) have been isolated from C. discolor (Stuart, 1970).
Crotsparinine (32) and methylcrotsparinine (33) were isolated from C. sparsiflorus (Casagrande
et al., 1975; Bhakuni et al., 1979; Bhakuni and Dhar, 1969).
36
37
38
2.2.1.3: Morphinane Dienone
Salutaridine (34) was isolated from C. flavens (Barnes and Soeiro, 1981; Bracher et al., 2004;
Eisenreich et al., 2003; Sanchez and Sandoval, 1982). Norsalutaridine (35) was reported from C.
salutaris (Barnes and Soeiro, 1981). Dihydrosalutaridine (36) and dihydronorsalutaridine (37)
have been isolated from C. linearis (Farnsworth et al., 1969; Sanchez and Sandoval, 1982;
Haynes et al., 1968).
Flavinine (38) was reported from C. flavens (Bhakuni et al., 1979; Stuart et al., 1968 and1969).
O-Methylflavinantine (39) was isolated from C. ruizianus (Farnsworth et al., 1969; Eisenreich et
al., 2003.). Salutarine (40) has been isolated from C. flavens (Eisenreich et al., 2003).
Flavinantine (41) (Piacente et al., 1998; Eisenreich et al., 2003; Stuart et al., 1969; Chambers
39
and Stuart, 1968; Bittner et al., 1997) and Isosalutaridine (42) (Bittner et al., 1997) have been
reported from C. chilensis. Norsinoacutine (43) and sinoacutine (44) were reported from C.
lechleri (Charris et al., 2000; Stuart et al., 1969; Carlin et al., 1995). 4, 5-
dihydroxymorphinandien-7-one (45) has been reported from C. bonplandianum (Tiwari et al.,
1981). Saludimerine A (46) and saludimerine B (47) have been isolated from C. flavens
(Bracher et al., 2004).
2.2.1.4: Protoberberine
Hemiargyrine (48) (Amaral and Barness, 1998), tetrahydropalmatrubine (49) (Wen-han et al.,
2003) and Xylopinine (50) (Wen-han et al., 2003) were isolated from C. hemiargyeus.
Corytenchine (51) and Corytenchirine (52) have been isolated from C. tonkinensis (Pham et al.,
2004). Coreximine (53) and scoulerine (54) were isolated from C. flavens (Eisenreich et al.,
2003). Julocrotine (55) was isolated from C. sylvaticus and C. membranaceus (Mwangi et al,
1998; Aboagye et al., 2000; Bayor et al., 2009).
40
2.2.1.5: Glutarimide
Crotonimide A (56) and Crotonimide B (57) were isolated from C. pullei (Barbosa et al., 2007).
Julocrotone (58) and julocrotol (59) have been reported from C. cuneatus (Suarez et al., 2004).
2.2.1.6: Guaiane
Muscicapine A (60), muscicapine B (61) and muscicapine C (62) were isolated from C.
muscicapa (De Araujo-Junior et al., 2005).
41
2.2.1.7: Harman
2-ethoxycarbonyltetrahydroharman (63) and 6-hydroxy-2-methyltetrahydroharman (64) were
isolated from C. moritibensis (De Araujo-Junior et al., 2004).
2.2.1.8: Tyramine
N-methyltyramine (65) and N-methylhomotyramine (66) were isolated from C. humilis (Stuart
and Byfield, 1971).
2.2.1.9: Benzylisoquinoline
Laudanidine (67) was reported from C.celtidifolius (Amaral and Barnes, 1997). Reticuline (68)
was reported from C.lechleri (Milanowski et al., 2002). Norlaudanosine (69) was reported from
C. hemiargyeus (Wen-han et al., 2003).
2.2.1.10: Peptide derivative
N-benzoylphenylalaninol (70), Aurentiamide acetate (71) and N-benzoylphenylalaninyl-N-
benzoylphenylalaninate (72) have been isolated from C. hieronymi (Catalan et al., 2003).
2.2.1.11: Miscellaneous alkaloids
Taspine (73) was reported from C. lechleri, C. draco and C. campestris (Milanowski et al., 2002;
Risco et al., 2003; Tsacheva et al., 2004; Ribeiro Prata et al., 1993). Hemiargine D (74) and
hemiargine C (75) were reported from C. hemiargyeus (Wen-han et al., 2003). 1, 2, 10-
42
trihydroxycrotosinoline -N-oxide (76) was reported from C. campestris (Ribeiro Prata et al.,
1993). Anabasine (77) was reported from C. muscicapa (De Araujo- Junior et al., 2005). 4-
hydroxyhygrinic acid (78) was reported from C. hovarum (Krebs and Ramiarantosa, 1996 and
1997).
43
2.2.2: Flavonoids
Flavonoids occur naturally, as water-soluble glycosides are phenolic derivatives. Their
classification is based either on their biosynthetic origin or on molecular size. Some flavonoids
are both intermediates in biosynthesis as well as end products which can accumulate in plants.
Ayanin, vitexin, tilirosine, rutin and quercetrin are some of the common flavonoids isolated from
Croton genus. Ayanin (79) was isolated from C. schiedeanus (Puebla et al., 2005). Quercetin-
3,7-dimethyl ether (80) was isolated from C. schiedeanus ( De Garcia et al., 1986) 5-Hydroxy-
7,4 -dimethoxyflavone (81) was isolated from C. betulaster (Barbosa et al., 2003). Kaempferol -
3-O-rutinoside (82) was isolated from C. cajucara (Capasso et al., 1998 and 2000). Kaempferol-
3,4 7- trimethylether (83) was isolated from C. menthodorus (Maciel et al., 2000). Tiliroside (84)
was isolated from C. tonkinensis, C. hovarum and C. zambesicus (Wagner et al., 1970; Capasso
et al., 2000; Phan et al., 2004; Krebs and Ramiarantosa,1996 and 1997; Pham et al., 2004).
Vitexin (85) , Isovitexin (86) and Kaempferol-3,7-dimethylether (87) have been reported from
C. cajucara (Maciel et al., 2000). Rutin (88) was isolated from C. menthodorus (Capasso et al.,
2000). Quercitrin (89) was isolated from C. glabellus (Novoa et al., 1985).Quercetin (90),
Taxmarixetin (91) and eriodictyol (92) were isolated from C. steenkampianus (Schmelzer and
Gurib- Fakim, 2008; Adelekan et al., 2008). Palmeira and co workers isolated artemetin (93)
from leaves and stems of C. brasiliensis (Palmeira et al., 2005).
44
45
2.2.3: Terpenoids
Terpenes are hydrocarbon components of resins and turpentine produced from resins. They
constitute a large and structurally diverse family of natural products derived from C5-isoprene
units. Chemical modifications through oxidation and re-arrangement of their carbon skeletons
produce terpenoids. Mono-, sesqui-, di-, tri-terpenoids and phytosterols have been reported from
Croton genus. Terpenoids are the predominant secondary metabolite constituents in the genus,
chiefly diterpenoids, which may belong to the cembranoid, clerodane, neoclerodane, halimane,
isopimarane, kaurane, secokaurane, labdane, phorbol and trachylobane skeletal types.
Triterpenoids, either pentacyclic or steroidal, have frequently been reported for Croton species.
Volatile oils containing mono and sesquiterpenoids, and sometimes also shikimate-derived
compounds are not rare in the genus.
2.2.3.1: Monoterpenes and sesquiterpenes
Monoterpenes, α-pinene (94), β-pinene (95) and limonene (96) have been reported from dried
aerial parts of C. antanosiensis (Radulovic et al., 2006). Monoterpenes α-pinene (94), β-
pinene (95), linalool coriander oil (97) and β-caryophyllene have been reported from dried
46
stem bark of C. aubrevillei (Menut et al., 1995). Monoterpenes α-phellandrene (98) α–pinene, ρ-
cymene (99) and linalool have been reported from Stem bark of C. stellulifer (Martins et al.,
2000). Leaf oil (sesquiterpenes) stem bark oil (Monoterpenes), both the leaf and stem bark oils
(low amounts of aliphatic compounds of non-terpenic origin) have been reported from leaves
and Stem bark of C. decaryi (Radulovic et al., 2006). Sesquiterpenes, caryophyllene oxide (100),
β-caryophyllene (101), γ-cadinene (102) and α-cadinene and Monoterpenes have been reported
from dried aerial parts of C. geayi (Radulovic et al., 2006). ). Leaf oil (sesquiterpenes) stem bark
oil (Monoterpenes), both the leaf and stem bark oils (low amounts of aliphatic compounds of
non-terpenic origin) have been reported from leaves and Stem bark ofC. Sakamaliensis
(Radulovic et al., 2006). Monoterpenes, Sesquiterpenes and Aliphatic compounds were reported
from C. zambesicus (Boyom et al., 2002).
2.2.3.2: Diterpenoids
Acyclic and cyclic diterpenoids are the most abundant natural products to have been isolated
from Croton genus.
2.2.3.2.1:Acyclic diterpenoids
Phytol (103) is the simplest acyclic diterpenoid that easily gets biosynthetically oxidised to
plaunotol (104) (2, 6, 10, 14-phytatetraene-1, 19-diol), the chief constituent of the leaves of Thai
medicinal plant C. sublyratus, later renamed C. stellatopilosus. This phytochemical is marketed
as “Plau noi” or “Kelnac” that is used as an anti-ulcerative (Wungsintaweekul and De-Eknamkul,
2005). Other acyclic phytanes from Croton genus include:- 3, 12-dihydroxy-1, 10, 14-
phytatriene-5, 13-dione (105) from C. salutaris (Tansakul and De-Eknamkul, 1998); trans-
phytol and isomers of phytol (103) from C. zambesicus (Catalan et al., 2003; Block et al., 2004)
and geranylgeraniol (106), from C. lobatus (Attioua et al.,2007; Chabert et al., 2006).
47
2.2.3.2.2: Bicyclic diterpenoids
Clerodanes, labdanes, halimanes and an indane derivative are some of the bicyclic diterpenoids
reported from croton genus, clerodane and labdane being the major classes.
2.2.3.2.3: Clerodane diterpenoids
Clerodane diterpenoids are the most prevalent compounds reported from Croton genus, trans-
dehydrocrotonin, a nor-ent - clerodane diterpenoid (107) and cis-dehydrocrotonin (108) were
reported from C. Cajucara and C. Schieddeanus (Maciel et al., 1997 and 2000; Babili et al.,
1998; Merritt and Levy, 1992; Rodriguez et al., 2004; Grynberg et al., 1999). Derivatives of
trans-dehydrocrotonin (109) and (110) were isolated from C. Sonderianus (Agner et al., 2001).
5β-hydroxy-cis-dehydrocrotonin (12r)-12-hydroxycascarillone (111) from C. Schieddeanus
(Maciel et al., 2006). Entclerodane crotocorylifuran, (112), (113) and (114) C. Zambesicus
(Ngadjui et al., 1999) and C. Haumanianus (Tchissambou et al., 1990). Corylifuran (115) C.
Corylifolius (Tchissambou et al., 1990 and Burke et al., 1976). Compound (116) and (117) from
Brazilian C. Campestris (Babili et al., 1998). Cascallin, cascarillone, cascarillin a, cascarillin b
(118), cascarillin c (119) and cascarillin d (120). All these cascallin derivatives are reported
from C. Eluteria (Vigor et al., 2001). Sonderianin (121, 122) and 12-epi-methyl-barboscoate
48
(123) from C. Ururucana (Puebla et al., 2003). Clerodane diterpenoid (124) C. Cajucara
(Maciel et al., 1997). Furano-clerodane, crotomembranafuran (125) C. membraneaceus (Bayor et
al., 2009) (126-129) from C. Hovarum (Krebs and Ramiarantosa, 1996 and1997). Isoteucvin
(130) jatropholdin (131) teucvin derivative (132) and teucvin (133) are reported from C.
Jatrophoides (Mbwambo et al., 2009) chiromodine (134), epoxy-chiromodine (135) C.
Megalocarpus (Addae-Mensah et al., 1989; Marko et al., 1999). Crotepoxide, crotomacrine,
floridoline and 12-oxo-hardwickiic acid (136) C. Macrostachys (Addae-Mensah et al., 1989;
Kapingu et al., 2000)
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50
51
2.2.3.2.4: Halimanes and an indane derivative
Biosynthetically, halimane diterpenoids possessing the halimane carbon skeleton lay between the
labdanes and clerodanes in their general structure. Halimane diterpenoids that have been reported
from croton genus include centrafine (137) from C. Membranaceous, penduliflaworosin (138),
from C. Jatrophoides (Mbwambo et al., 2009), C. Penduliflorus hutch (Adesogan, 1981) and C.
Sylvaticus leaves (Schneider et al., 1995). Compound (139) from C. Hovarum (Krebs and
Ramiarantosa, 1996 and 1997) and neoclerodane-5, 10-en-19, 6β, 20,12-diolide (140) from C.
Macrostachys (Addae-Mensah et al., 1989). An indane derivative (141) from C. Steenkampianus
(Adelekan et al., 2008) is another of the bicyclic phytanes reported from Croton species.
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2.2.3.2.5: Labdanes
Hundreds of labdanes and their pharmacological values have been reported from higher plants.
2α,3α–dihydroxylabda- 8(17),12,14-triene (142) and 2α-acetoxy-3α–dihydroxylabda-
8(17),12,14-triene (143) have been reported from C. Ciliatoglanduliferus (nabeta et al., 1995).
Labdane-8α, 15-diol (144) , 5-acetoxylabdan-8α-ol (145) have been reported C. Eluteria (vigor
et al., 2001). Austroinulin, 6-o-acetylaustroinulin (146) has been reported C. Glabellus (morales-
flores et al., 2007). Labda-7,12(e) 14-trien-17-oic acid (147), labda-7,12 (e),14-trien-17-al (148),
, 17-hydroxylabda-7,12,14-triene (149), 17-acetoxylabda-7,12,14-triene (150), labda-7, 13 -dien-
17,1 2-olide (151), 15- hydroxylabda-7, 13 -diene- 17,12- olide (152), 12,17-dihydroxylabda-
7,13-diene (153), ent-3α-hydroxymanoyl oxide labda-7,12 (e),14-triene (154) have been reported
from C. Oblongifolius (sommit et al., 2003; garcia et al., 2006). Crotonadiol (155) has been
reported C. Zambesicus (ngadjui et al., 1999) maruvic acid (156) has been reported C.
Matourensis (chaichantipyuth et al., 2005). 2,3-dihydroxy-labda-8(17),12(13), 14(15)-triene
(157) has been reported C. Joufra (sutthivaiyakit et al., 2001). Gomojoside h (158) has been
reported C. Membraneaceus (bayor et al., 2009, asare et al., 2011). Geayinine (ent-8,13-
epoxylabd-14-enes) (159) isogeayinine (160) have been reported C. Geayi (radulovic et al.,
2006). Crotomachlin (161) has been reported C. Macrostachyus (addae-mensah et al., 1989)
.compound (162) has been reported C. Pseudopulchellus (langat et al., 2012).
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54
2.2.3.3: Tricyclic diterpenoids
Tricycloditerpenoids reported from croton genus include abiatanes, daphnanes, pimaranes, and
isopimaranes.
2.2.3.3.1: Abietanes
Related parent diterpene hydrocarbons include 13, 16-cycloabiatanes (163); 17 (15-16)-abeo-
abietanes (164) in which the methyl group, c-17 has shifted from c-15 to c-16 and totaranes
(165) which arise from abietane when the isopropyl group migrates from c-13 to c-14. African C.
Zambesicus is the only croton species reported to have produced abietane diterpenoids but their
names were not included in the report accessed (aiyar and seshadri, 1970).
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2.2.3.3.2: Daphnanes
Included in this category is rhamnofolanes such as (-)-20-acetoxy-9-hydroxy-1, 6, 14-
ramnofolatriene-3, 13- dione reported from C. Rhamnifolius (breitmaier, 2006). Daphnanes are
similar in structure to rhamnofolanes, differing only in the position of the isopropyl group, c-15
whereby, in daphnanes, it is on c-2 while in rhamnofolane, it is on c-1. However, rhamnofolanes
and other constituents from jatropha species rarely occur in plants. Instead, daphnanes are more
frequently found ((breitmaier, 2006). Two daphnanes, steenkrotin b (166) and its triacetyl
derivative (167) have been reported from C. Steenkampianus (adelekan et al., 2008).
2.2.3.3.3: Pimaranes and isopimaranes
Pimaranes and isopimaranes are 13-14, 8-cyclolabdanes with the perhydrophenanthrene basic
skeleton, differing only in their configuration at c-13. Ent-isopimarane, yucalexin p-4 (168) has
been reported from Argentinian C. Sarcopetalus (mwangi et al., 1998; de heluani et al., 2000).
56
3β-hydroxy-19-acetoxy-ent-isopimara-8, 15-dien-7-one (169), plaunol a and c, swassin and 3β-
hydroxy-19-o-acetyl-pimara-8(9), 15-dien-7-one which has been found to be weakly cytotoxic
are reported from thai C. Joufra (sutthivaiyakit et al., 2001 neuwinger, 2000). From asian C.
Oblongifolius, ent-pimara-7, 15 – dien – 19 – oic acid (170) was isolated (de heluani et al., 2000)
while from african C. Zambesicus, three isopimaranes, isopimara-7, 15- dien-3β-ol (171), (172)
and (173) are reported (block et al., 2004).
2.2.3.4: Tetracyclic diterpenoids
Atisanes, kauranes and tiglianes are the reported tetracyclic diterpenoids from croton genus.
2.2.3.4.1: Atisanes
Atisane is the basic carbon skeleton of various diterpene alkaloids (aconitum-alkaloids) found in
the plant families of rhanunculaceae and garryaceae ((breitmaier, 2006). Two 3, 4-seco-atisane
diterpenoids with cytotoxic potency crotobarin (174) from C. Barorum and crotogaudin (175)
from C. Goudotii have been reported (rakotonandrasana et al., 2010).
2.2.3.4.2: Kauranes
Kauranes are the commonest class of the tetracyclic diterpenoids reported from croton genus,
these includes, twelve kauranes and ent-kauranes (176 -187) isolated from vietnamese C.
Tonkinensis (crude extract significantly cytotoxic (kuo et al., 2007). Fifteen ent-kauranes( 188 -
57
203, 208) from the leaves of C. Tonkinensis (minh et al., 2003; ngadjui et al., 2002; giang et al.,
2005) . Argyrophilic acid (204), a stereoisomer of cunabic acid found to be active against gram
positive bacteria in vitro was reported from C. Argyrophylloides (giang et al., 2004). Ent –15 -
oxokaur – 16– en – 18 – oic acid (205) was reported from C. Argyrophylloides (fernandes et al.,
1974). Ent-16β, 17-dihydroxykaurane (206) japanese C. Sublyratus (monte et al., 1988). Two
ent-kauranes including this one (207) from asian C. Kongensis (kitazawa and ogiso, 1981) . Ent-
kauran-16β, 17-diol and ent-kauran-16β, 17, 19-triol C. Hutchinsonianus (Chen et al., 2007).
Three ent-kauranoids (209-211) C. Lacciferus (li et al., 1990). Geayine (212), 7-oxogeayine
(213) were isolated from C. Geayi (Radulovic et al., 2006). Compound (214) was C. Zambesicus
(aiyar and seshadri, 1970). Compounds (215-220) were reported from C. Pseudopulchellus
(langat et al., 2012).
58
59
60
61
2.2.3.5: Pentacyclic diterpenoids
in this category, only trachylobanes are reported from two african croton species from beninian
C. Zambesicus, ent-18-hydroxy-trachyloban-3-one (221) and its vaso-relaxant properties (jogia
et al., 1989), ent-trachyloban-3-one (222), (223), (224), ent-trachyloban-3β-ol (225) and (226)
were reported (ngadjui et al., 1999; block et al., 2004; aiyar and seshadri, 1970). Cameroonian C.
Zambesicus is reported to have produced compounds (227-228), 7β-acetoxytrachyloban-18-oic
(229) and trachyloban-7β-18-diol (230) (ngadjui et al., 1999). Compounds (231- 232),
trachyloban-18-oic acid (233), trachyloban-19-oic acid (234), 3α, 19- dihydroxytrachylobane
(235), 3α, 18, 19-trihydroxytrachylobane (236), 3β,19 – dihydroxytrachylobane (237) and
3β,18,19 – trihydroxytrachylobane (239) are reported from eastern africa C. Macrostachyus
(addae-mensah et al., 1989; kapingu et al., 2000).
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2.2.3.6: Macrocyclic diterpenoids
Cembranoids are the macrocyclic diterpenoids reported from croton genus, C. Gratissimus
predominantly yielded cembrane diterpenoids (pudhom et al., 2007; mulholland et al., 2010).
Neocrotocembranal (239) (baccelli et al., 2007) , crotocembranoic acid (240) and
neocrotocembranoic acid (241)(roengsumran et al., 1999) from the stem bark of C.
Oblongifolius. Poilaneic acid (242) from the stem bark of C. Poilanei (roengsumran et al.,
2002) . Furano-cembranoids (243-245) lactonized cembranoid (246) from C. Oblongifolius
(roengsumran et al., 1998; sato et al., 1981). (+)-[1r,10r]-cembra-2e,4e,7e,11z-tetraen-20, 10-
olide (247), (+)-[1r,4s,10r]-4-hydroxycembra- 2e, 7e,11z-trien-20,10-olide, (-)-[1r,4r,10r]-4-
hydroxycembra- 2e, 7e, 11z-trien-20, 10-olide, (+)-[1r,2s,7s,8s,12r]-7,8-epoxy-2,12-
cyclocembra-3e,10z-dien-20,10-olide, (+)-[1s, 4s, 7r, 10r]-1,4,7-trihydroxycembra-2e, 8
(19),11z-trien-20, 10-olide (epimers at c-7), (-)-[1s, 4s, 10r]-1, 4-dihydroxycembra-2e, 7e, 11z-
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trien-20, 10-olide, (+)-[10r]-cembra-1e, 3e, 7e,11z,15-penten-20,10-olide and (+)-[1s, 4r, 8s,
10r]-1, 4, 8-trihydroxycembra-2e,6e,11z-trien-20, 10-olide were all isolated from C. Gratissimus
(mulholland et al., 2010).
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2.2.3.7: Limonoids
Only one research group has reported the isolation of limonoids from a croton plant, C.
Jatrophoides (kubo et al., 1990; nihei et al., 2002, 2005 and 2006). The chemical structures of
the limonoids that were reported supposedly from C. Jatrophoides (kubo et al., 1990 and nihei
et al., 2002, 2004, 2005 and 2006, lemos et al., 1992, santos et al., 2008, sommit et al., 2003,
ngamrojnavanich et al., 2003). Their names are dumsin (248); zumsin (249); zumketol (250);
zumsenin ,zumsenol); dumnin dumsenin); musidunin and musiduol .compounds showed potent
anti-feedant activity (pc50 < 2.0 μg/ml) against the larvae of the pink bollworm, pectinophora
gossypiela and fallworm, spodoptera frugiperda (nihei et al., 2004 and 2006)
2.2.3.8: Triterpenoids
Triterpenoids are c30 compounds derived from six isoprene units and are widely distributed in
plant kingdom in a free state or as esters or glycosides. They are further sub-grouped into
tetracyclic and pentacyclic triterpenoids.. Triterpenoids of various carbon skeletons have been
reported from the croton genus. Taraxerane, acetylealeuritolic acid (251) was reported from C.
Cajucara, C.tonkinesis, C. Megalocarpus, C. Hovarium, C. Urucarana (addae-mensah et al.,
1989; maciel et al., 1997; krebs and ramiarantosa, 1996 and 1997; puebla et al., 2003; pham and
pham, 2002). Lupane, lupeol (252) was reported from C. Zambesicus, C. Megalocarpus , C.
65
Gratissimus and C. Haumanianus (ngadjui et al., 1999; addae-mensah et al., 1989; mulholland
et al., 2010; tschissambou, 1990) . 3β-o acetoacetyl lupeol (253) and betulin (254) C.
Megalocarpus (addae-mensah et al., 1989) . Lupenone (255) (barbosa et al., 2003) 20-
hydroxylupan-3-one (256) from C. Betulaster. Friedelane , friedelin (257) C. Hovarum (krebs
and ramiarantosa, 1996 and 1997). Oleanane , β-amyrin (258) , 3-oxo-olean-12-en-28-oic acid
(259), 3-oxo-olean-18-en-28-oic acid (260) from C. Betulaster (barbosa et al., 2003) . Ursane ,
α-amyrin (261) C. Hieronymi (block et al., 2004) α-amyrin acetate (262) C. Hieronymi, C.
Tonkinensis (addae-mensah et al., 1989; pham and pham, 2002) . Taraxastane , 3-oxo-20β-
hydroxytarastane (263) C. Betulaster (barbosa et al., 2003). Hopane 3-oxo-22-hydroxyhopane
(264), hop-22-(29)-en-3β-ol (265) C. Hieronymi (risco et al., 2003) .
66
67
2.2.4: Phytosterols
quite a number of phytosterols have been reported from croton genus. Included is sitosterol
(266) from C. Zambesicus (ngadjui et al., 1999) and C. Membranaceus (bayor et al., 2009);
sitosterol -3-d-glucoside (267) , dl- threitol (268) (bayor et al., 2009) and ethylcholesta 4, 22-
diene-3-one (269) from C. Gratissimus (mulholland et al., 2010); cholestan-5,7-dien-3-ol (270),
3-hydroxycholest-5-en-7-one (271), cholestan-3-one (272) and ergosterol (273) from C.
Pseudopulchellus (langat et al., 2012).
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69
2.2.5: Fixed oils
Perhaps, one of the great values of the croton genus is the discovery of C. Megalocarpus seeds
as a potential source of fixed oils that could be a suitable alternative bio-diesel. Linoleic acid (a
fixed oil common in seeds) was found to be the major fatty acid, constituting 74.3% of all the
fatty acids present in the oil (wu et al., 2013). Earlier reports on the same oil had indicated that it
possessed epstein-barr virus-activating potency (wu et al., 2013). The seeds of C. Macrostachys
were found to contain 48% oils (linoleic acid (80%), palmitic acid (12%), stearic acid (6%) and
myristic acid (2%)). The purgative and inflammatory activities of these oils have been
demonstrated rationalizing the ethno-botanical use of C. Macrostachys as a purgative (mazzanti
et al., 1987). C. Penduliflorus seeds produced essential oils that were found to be
hypocholesterolemic but could predispose anaemia (ojokuku et al., 2011). From C. Stellulifer
[syn. C. Stelluliferus], oils having anti-microbial activities except against aspergillus niger were
isolated (martins et al., 2000).
2.3 Previous pharmacological reports on Genus Croton
2.3.1: Antioxidant activity
C.celtidifolius bark possesed antioxidant activity which results from the direct action of
constituents on specific targets. The extracts of C. celtidifolius indicated antioxidant properties in
vitro, all were able to scavenge superoxide anions at a concentration of 100 μg · ml–1. They were
most effective in the deoxyribose assay, IC50 0.69 (0.44–1.06), 0.20 (0.11–0.39), 0.55 (0.28–
1.08) μg · ml–1 respectively (Nardi et al., 2003). C. urucurana red latex has antioxidant effect
against lipid peroxidation and free radical scavenging activity (Orlandi et al., 2002). C. lechleri
sap possesses significant antioxidant activity against the oxidative damages induced by
apomorphine and hydrogen peroxide in Saccharomyces cerevisiae and maize plantlets (Lopes et
al., 2004). Leaf extracts of C. cajucara were observed to exert antioxidant effects against the free
radical DPPH and in paraquat treated yeast cells (Tieppo et al., 2006). C. lechleri latex has
antioxidant, free radical scavenging (Desmarchelier et al., 1997). Studies were carried out to
investigate the effect of Croton bonplandianum leaves on experimental wounds and in vitro
antioxidant activities like effect on DPPH and Nitric oxide. Ethanol and aqueous extract of shade
dried leaves of Croton bonplandianum extract is formulated as 10% ointment and topically
70
applied to experimental wounds in rats. The plant showed a definite positive effect on wound
healing with significant increase in wound contraction (Divia et al., 2011).
2.3.2: Antidiarrhial activity
The red sap from C. urucurana showed promising potential for the control of pathologies
associated with secretory diarrhea (Gurgel et al., 2001). Proanthocyanidin isolated from C.
lechleri is a potent inhibitor of cholera toxin-induced fluid accumulation and chloride secretions
(Fischer et al., 2004) and hence useful to control fluid loss and diarrhea. SP-303 has been
indicated particularly for patients of AIDS, common victims of diarrhea (Holodniy et al., 1999).
2.3.3: Antimicrobial activity
Red latex of C. lechleri showed antimicrobial properties (Chen et al., 1994). The red latex from
C. urucurana inhibited the growth of the fungi Tricophyton tonsurans, Tricophyton
mentagrophytes, Tricophyton rubrum, Microsporum canis and Epidermophyton floccossum,
showing a potential utility as an alternative treatment for dermatophytosis (Gurgel et al., 2005).
The red latex of C. draco and its ethyl acetate and ethyl ether extracts exhibited high inhibition
on the classical activation pathway of the complement system using hemolytic assay (Tsacheva
et al., 2004). The antimicrobial studies revealed that methanol extract of leaf and fruit of Croton
bonplandianum is more effective against tested microbes than aqueous and acetone extracts. The
methanol extract appeared with maximum activity against gram positive bacteria and acetone
extract of leaves showed maximum activity against gram negative bacteria. None of the extracts
showed activity against Pseudomonas aeruginosa (Manjit et al., 2011). Plaunotol has displayed
activity against twenty methicillin-resistant and fourteen methicillin-sensitive strains of
Staphylococcus aureus (Matsumoto et al., 1998) (Inoue et al., 2004).
The results suggested that plaunotol might be useful in the prevention of infection and skin care
for patients with atopic dermatitis. Catechin and acetyl aleuritolic acids obtained from C.
urucurana, are effective against S. aureus and Salmonella typhimurium, acetyl aleuritolic acid
showed minimum inhibitory concentration ten fold higher than catechin (Peres et al., 1997). The
volatile oil from leaves of C. cajucara, composed mostly by linalool, inhibits the growth of
Candida albicans, Lactobacilus casei, Porphyromonas gengivalis, Staphylococus aureus and
71
Streptococcus mutans, all involved in diseases of the oral cavity (Alviano et al., 2005). Among
these microorganisms, the authors noted that linalool is active almost exclusively against
Candida albicans, and that the volatile oil is not toxic to mammalian cells. The phenylpropyl
benzoates 3'-(4"-hydroxy-3",5"-dimethoxyphenyl)-propyl benzoate, 3'-(4"-hydroxy-phenyl)
propyl benzoate and 3'-(4"-hydroxy-3"-methoxy-phenyl)-propyl benzoate obtained from stems of
C. hutchinsonianus, were shown to exert effect against Candida albicans. The three
phenylpropyl benzoates (1—3) were found to exhibit antifungal activity against Candida
albicans (IC50 5.36— 11.41 mg/ml). Compounds 1—2 (IC50 2.11—4.95 mg/ml) exhibited potent
but non-selective activity against the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2
(COX-2) whereas 3 (IC50 1.88 mg/ml) preferentially inhibited the enzyme COX-2
(Athikomkulchai et al., 2006).
2.3.4: Antimalarial activity
Numbers of Croton species are traditionally used as antimalarials throughout endemic malarial
areas. Antiplasmodial activity was demonstrated in vitro for C. pseudopulchellus Pax a specie
from southern Africa (Prozesky et al., 2001). Methanolic extracts from aerial parts of C. lobatus
L. (a widespread species in tropical America, from Florida to Argentina) were active toward
Plasmodium falciparum 3D7 chloroquine sensitive strains, while root methanolic extracts
inhibited growth of K1 resistant strains (Weniger et al., 2004). It has been found that the 8,9-
Secokauranes from C. kongensis exhibited anti-mycobacterial activity at the minimum
inhibitory concentration Two new 8,9-secokaurane diterpenes, ent-8,9-seco-7α,11β-
diacetoxykaura-8(14),16-dien-9,15-dione (1) and ent-8,9-seco-8,14-epoxy-7α-hydroxy-11β-
acetoxy-16-kauren-9,15-dione (2), together with two known compounds, ent-8,9-seco-7α-
hydroxy-11β-acetoxykaura-8(14),16-dien-9,15-dione (3) and ent-7β-hydroxy-15-oxokaur-16-en-
18-yl acetate, were isolated from Croton kongensis. This is the first report on the presence of 8,9-
secokauranes in the plant genus Croton. Diterpenes 1−3 exhibited antimycobacterial activity with
minimum inhibitory concentrations (MICs) of 25.0, 6.25, and 6.25 μg/mL, respectively, and
possessed antimalarial activity with IC50 ranges of 1.0−2.8 μg/mL (Thongtan et al., 2003).
72
2.3.5: Antiulcer activity
Extracts of the bitter bark of C. eluteria (cascarillai) has been shown to strengthen the histamine-
stimulated gastric acid secretion, giving experimental support to the use of cascarilla in bitter
preparations aimed to improve digestion (Appendino et al., 2003). The “sangre de grado” of C.
lechleri has shown wound-healing activity (Chen et al., 1994) in cutaneous disorders and orally
in a dilute form to facilitate the healing of gastric ulcers, reducing ulcer size and bacterial content
of the ulcer (Jones et al., 2003). The volatile oil from the bark of C. cajucara has been shown to
exert gastric ulcer healing activity as well as protection of the gastric mucosa (Hiruma et al.,
2000). Low molecular weight sesquiterpenes appear to be important active constituents of the
volatile oil (Bighetti et al., 1999). Efficacy of the oil seems to be based on its ability to stimulate
local mucus synthesis and prostaglandin production by the gastric mucosa (Hiruma et al., 2002).
Dehydrocrotonin showed strong antiulcerogenic activity (Souza-Brito et al., 1998, Hiruma-Lima
et al., 1999). The A-ring of both crotonin and dehydrocrotonin is not directly involved in the
antiulcerogenic activity (Bighetti et al., 1999).
Dehydrocrotonin has good antiulcerogenic activity (Rodriguez et al., 2006). A semi-synthetic
crotonin, namely 4SRC, was synthesized and it was shown to have a significant preventive effect
against gastric ulcer induced by different agents (Almeida et al., 2003). Presence of a lactone
moiety or Michael acceptor is probably essential for the anti-ulcerogenic effect, a mechanism of
gastric cytoprotection being mediated by an action on prostaglandin biosynthesis and by a
Michael reaction between the SH-containing compounds of the mucosa on the Michael acceptors
present in antiulcerogenic compounds (Melo et al., 2003). Volatile oil from the bark of Croton
cajucara significantly reduced the gastric injury induced in rats (Hiruma-Lima et al., 1999,
Hiruma-Lima et al., 2000). Plaunotol is an anti-peptic ulcer agent, (Wada et al., 1997)
commercially available under the name Kelnac (Vongchareonsathit et al., 1998). The anti-ulcer
effect of plaunotol is probably linked to its activity against Helicobacter pylori (Takagi et al.,
2000, Koga et al., 2002).
2.3.6: Anticancer activity
Shoots of C. hieronymi have shown strong activity against lung carcinoma cells and mouse
lymphoma and some activity against human colon carcinoma (Catalán et al., 2003). The
73
dichloromethane extract of leaves of C. zambesicus showed in vitro cytotoxicity against human
cervix carcinoma cells (Block et al., 2002). The red latex of C. lechleri has been shown to have
anti-tumor activity (Chen et al., 1994). Tran-dehydrocrotonin exhibits anti-tumor efficacy and
immunomodulatory actions in vivo, which may be related to its chemical structure (Melo et al.,
2004). The dehydrocrotonin and its synthetic derivative dimethylamide-crotonin inhibit cells
growth in vitro partly by apoptosis induction and cell differentiation, but do not cause serious
damage to immune cells (Anazetti et al., 2004). However, dehydrocrotonin is not cytotoxic (and
also not genotoxic) to bone marrow cells of Swiss mice submitted to acute intraperitoneal
treatment in vivo (Agner et al., 1999) and antimutagenic with regard to cyclophosphamide, in
particular if administered by gavage 180 (Agner et al., 2001) (Correa et al., 2005) developed a β-
cyclodextrin complex to improve delivery of dehydrocrotonin.
A lower cytotoxicity of the complex β-cyclodextrin-dehydrocrotonin to V79 fibroblasts and rat
cultured hepatocytes, compared to free dehydrocrotonin, proposes that such complex may be
useful for in vivo dehydrocrotonin administration. The furoclerodane croblongifolin from C.
oblongifolius showed significant cytotoxicity against human breast ductal carcinoma, human
undifferentiated lung carcinoma, human liver hepatoblastoma, gastric carcinoma and colon
adenocarcinoma tumor cell lines (Vilaivan et al., 2002). It is found that the halimane and
cembranoid diterpenes from C. soblongifolius showed antitumoral activity, but 12-
benzoyloxycrotohalimaneic acid was inactive (Roengsumran et al., 2004). Ent-Kauranes of C.
tonkinensis also have been shown to be cytotoxic (Giang et al., 2005). Trachylobane (ent-
trachyloban-3β-ol) a constituent of leaves of C. zambesicus has recently been shown to induce
apoptosis in human promyelocytic leukemia cells in a concentration-dependent manner (Baccelli
et al., 2005). Plaunotol, an acyclic diterpene present in C. sublyratus leaves, has recently been
shown to have anti-cancer activity through inhibition of angiogenesis (Kawai et al., 2005).
Anethole, a phenylpropanoid constituent of C. zehntneri volatile oil, has been shown to have
anti-carcinogenic effect (Chainy et al., 2000). Taspine is active against KB and V-79 cells, a fact
that makes it a likely responsible for the purported anticancer activity of C. lechleri red sap
(Chen et al., 1994). Antitumor properties of twigs extract of Croton bonplandianum Baill were
proven using potato disc and radish seed bioassays (Islam et al., 2010). Dragon's blood is a dark-
red sap produced by species from the genus Croton (Euphorbiaceae), which has been used as a
74
famous traditional medicine since ancient times in many countries, with scarce data about its safe
use in humans. In this research, we studied genotoxicity and clastogenicity of Croton
palanostigma sap using the comet assay and micronucleus test in cells of mice submitted to acute
treatment. HPLC analysis was performed to identify the maincomponents of the sap. The sap
was administered by oral gavage at doses of 300 mg/kg, 1000 mg/kg and 2000 mg/kg.For the
anal., the comet assay was performed on the leukocytes and liver cells collected 24 h after
treatment, and themicronucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by
scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes
(PCE/NCE ratio). The alkaloid taspine was the main compound indentified in the crude sap of
Croton palanostigma. The results of the genotoxicity assessment revealed that all sap doses
tested produced genotoxic effects in leukocytes and liver cells and also produced
clastogenic/aneugenic effects in bone marrowcells of mice at the two higher doses tested. The
PCE/NCE ratio indicated no cytotoxicity. The data obtained suggestcaution in the use of Croton
palanostigma sap by humans considering its risk of carcinogenesis (Maistro et al., 2013).
2.3.7: Antihypertensive activity
The volatile oil of the bark and leaves of C. nepetaefolius, which contains mainly 18-cineole,
methyleugenol and terpineol exerted antispasmodic effect on gastrointestinal tissues and
antihypertensive activity in the cardiovascular system (Magalhães et al., 2003). Intravenous
treatment with the volatile oil decreases mean aortic pressure and heart rate in either
anaesthetized or non-anaesthetized rats (Lahlou et al., 1999). The aqueous and ethanolic extracts
of C. schiedeanus have a decreasing effect on blood pressure probably by means of an
antihypertensive rather than hypotensive effect (Guerrero et al., 2001). The antihypertensive
activity and vasodilator effects of C. schiedeanus are attributed to a synergistic activity among
flavonoids and terpenoids (Guerrero et al., 2002).
2.3.8: Antiinflammatory and antinociceptive
From the aerial parts of C. arboreous four sesquiterpenes are obtained which have anti-
inflammatory activity against ear edema in mice (Guadarrama et al., 2004). The volatile oil of C.
zehntneri was shown to have antinociceptive activity in mice (Oliveira et al., 2001) and the
volatile oil of C. cajucara has anti-inflammatory and antinociceptive effects in rodents (Bighetti
75
et al., 1999). Cajucarinolide, a diterpene from C. cajucara, was shown to possess anti-
inflammatory activity (Ichiara et al., 1992). The red latex of C. lechleri relieves swelling of
insect bites (Jones, 2003). Dehydrocrotonin, the main component of bark extracts of C. cajucara,
has anti-inflammatory and analgesic effects (Maciel et al., 2000, Carvalho et al., 1996). Crude
leaf extracts of C. cajucara exhibited significant antinociceptive effect in rats (Campos et al
2002). Volatile oil of C. nepetaefolius promoted a dose-dependent antinociceptive effect in hot-
plate test (Abdon et al., 2002). The aqueous extract of the aerial parts of C. cuneatus had
significant activity against plantar inflammation induced by bovine serum albumin (Pereira et al.,
1999). The volatile oil of C. sonderianus had antinociceptive effect in tests with oral
administration, but was inactive in hot-plate tests (Santos et al., 2005). The antinociceptive effect
of the volatile oil of C. zehntneri was evidenced, most likely associated with anti-inflammatory
activity (Oliveira et al., 2001). Crude extract of the stem bark of C. celtidifolius showed
antinociceptive effects stimulants (Dalbo et al., 2005).
2.3.9: Antidepressant activity
The volatile oil from the bark and leaves of C. zehntneri produced antidepressive effects in rats
without anxiety alterations (Lazarini et al., 2000, Norte et al., 2005).
2.3.10: Antihyperlipidemic and antihypercholesterolemic activity
Pharmacological studies carried out with the terpenoids, crotonin, and acetyl aleuritolic acid with
plant extracts gave a striking correlation with the traditional therapeutic use of C. cajucara
species in the Amazon region for the control of hyperlipidemy and associated pathologies
(Maciel, 2002). Hypolipidemic effects were observed by in assays with dehydrocrotonin from C.
cajucara (Silva et al., 2001). In addition to hypolipidemic action, dehydrocrotonin exhibited
hypoglycemic effect in alloxan-induced diabetic, but not in normal rats (Farias et al., 1987).
Extracts of C. cajucara leaves showed significant reductions in the serum total cholesterol, low-
density lipoprotein cholesterol and triglyceride levels, as well as a significant elevation in the
high density lipoprotein in treated rats compared with the control group (Farias et al., 1997).
Experiments treating rats with water extracts gave support to the popular use of C. cajucara bark
in loss-weight programs, the sensitivity of the lipolytic responsess to isoprenaline and adrenaline
being significant higher in adipocytes from treated rats (Grassi et al., 2003).
76
2.3.11: Antiviral activity
C. tiglium seeds contain anti-HIV-1 phorbol esters, 12-O-acetylphorbol-13-decanoate and 12-O-
decadienoylphorbol-13-(2-methylbutyrate) that inhibit the cytopathic effect of HIV-1 12-O-
tetradecanoylphorbol-13-acetate (TPA) is even more active than the mentioned phorbol esters
against HIV-1 (El-Mekkawy et al., 2000). Derivatives of phorbol esters have been evaluated as
inhibitors of proliferation of HIV-1. Among them 12-O-acetylphorbol-13-decanoate has been
shown to be the most potent (Nakamura andYakugaku 2004) (Masuda et al., 1993).
2.3.12: Vasorelaxant activity
Dehydrocrotonin was shown to reduce the mean arterial pressure and heart rate in a dose-
dependent manner in normotensive rats and to relax the tonic contraction in isolated rat aortic
rings induced by phenylephrine (Silva et al., 2005). The neo-clerodanes (12R)-12-
hydroxycascarillone, 5β - hydroxy-cis-dehydrocrotonin, cis- and trans-dehydro-crotonin from C.
schiedeanus relaxed aort rings (Guerrero et al., 2004). A vasorelaxant activity of quercetin-3, 7-
dimethyl ether from C. schiedeanus was observed, the activity probably being influenced by
hydroxylation at positions 3’ and 4’ of the B ring (Guerrero et al., 2002).
2.3.13: Antioestrogenic activity
Dehydrocrotonin obtained from C. cajucara was tested for antioestrogenic activity using
immature rats for bioassay of oestrogen and regularly cycling rats of proven fertility for
antiimplantation effect (Maciel et al., 2000).
2.3.14: Insecticidal activity
The diterpene fraction from C. linearis showed lethal effect on insects (Alexander et al., 1991).
A prenylbisabolane diterpene from C. linearis has insecticidal effect (Smitt et al., 2002). The
same comment applies to hardwickiic acid, a diterpene present in C. aromaticus and C.
californicus (Bandara et al., 1987).
2.3.15: Antileishmanial activity
The linalool-rich volatile oil from leaves of C. cajucara was shown to be a potent agent against
Leishmania amazonensis. The inhibitory concentration for L. amazonensis promastigotes growth
77
is extremely low and the oil has no cytotoxic effects against mammalian cells (Rosa et al., 1895).
Secokauranes obtained from C. kongensis were shown to have anti-malarial activity (Thongtan et
al., 2003).
2.3.16: Antispasmodic activity
The volatile oils of some South-American Croton species are antispasmodic. Cineole,
methyleugenol and terpineol constituents of C. nepetaefolius volatile oil, have been reported to
have antispasmodic effects in laboratory animals (Santos et al., 2006). Experimental results
suggest that C. nepetaefolius volatile oil induces relaxation of guinea-pig ileum (Magalhães et
al., 2004). Anethole and estragole, major components of the volatile oil of C. zehntneri, are
effective relaxants of skeletal muscles (Albuquerque et al., 1995). The volatile oil of C. zehntneri
has relaxing effect on smooth muscle, which supports the use of C. zehntneri in traditional
medicine as a gastrointestinal antispasmodic, an activity that may in part be attributed to
estragole (Coelho et al., 1998).
2.3.17: Phytotoxic activity
Metahanolic extract of Croton bonplandianum leaves are detrimental to at least six weedy
associates, viz. Calotropis procera, Chrysopogona ciculatus, Crotalaria saltiana, Cynodon
dactylon, Eupatorium odoratum and Potygonum orientale (Datta and Sinha-Roy, 1975).
78
3 Materials and methods
3.1: Collection of plant material
The plant material was collected from the different areas of District Sargodha, Punjab, Pakistan.
The plant was identified as Croton bonplandianum by Professor Dr. Altaf Ahmad Dasti and
specimen voucher (SWT-446) was deposited at Institute of pure and applied Biology, Bahauddin
Zakariya University, Multan.
3.2: Solvents and chemicals
All the solvents used for extraction and isolation like methanol, dichloromethane, chloroform, n-
hexane, ethyl acetate, ethanol, propanol, n-butanol, Vanillin, silica gel (70-230 mesh) and TLC
aluuminium sheets 20 × 20 cm, Silica gel 60 F254 were imported from Merck KgaA Darmstadt
Germany. Sephadex LH-20 25-100μm Fluka Chemie GmbH (9041-37-6).
3.3: Preparation of reagents
The reagents were prepared according to the specification of Pharmaceutical Codex (11th edition)
and British Pharmacopoeia.
3.3.1: Wagner’s reagent (Solution of iodine in potassium iodide)
4 g of potassium iodide was dissolve in minimum quantity of water (10 ml). 2g of Iodine was
added, iodine dissolved completely by complex formation. Then volume was made 100 ml with
water.
3.3.2: Mayer’s reagent (solution of potassium mercuric iodide)
Solution (A) of Mercuric chloride was prepared by dissolving 1.36 g of Mercuric chloride in 60
ml of H2O. Solution (B) was prepared by dissolving 5 g of Potassium iodide in 20 ml of water.
Then added the solution (A) into solution (B), mixed and made the volume 100 with water.
3.3.3: Hager’s reagent
Picric acid was dissolved in 100 ml of water till the saturation point was achieved the solution
was filtered.
79
3.3.4: Dragendorff’s reagent (solution of Potassium Bismuth Iodide)
25 g of tartaric acid was dissolved in 100 ml of H2O and added 2.1 g of bismuth oxynitrate.
Shacked for 1 hour and added 50 ml of 40 % solution of Potassium iodide Shacked well allowed
to stand for 24 hours and filtered.
3.3.5: Godine reagent (Godine, 1954)
Godine reagent was prepared by adding equal volume of two solutions
1- 1% Vaniline in ethanol
2- 3% Perchloric acid in water
3.4 Preparation of solutions
3.4.1: Preparation of dilute HCl
The dilute HCl was prepared according to the requirements of the procedures by calculating the
volume of the acid required according to its strength.
3.4.2: Preparation of dilute ammonia solution
375 ml of strong ammonia solution was diluted to 1000 ml with H2O.
3.4.3: Preparation of 70 % alcohol
72.7 ml of alcohol mixed with 27.3 ml of purified water.
3.4.4: Preparation of lead subacetate solution
40 g of lead acetate was dissolved in 90 ml of carbon dioxide free water. Adjust the pH 7.5 with
10 M Sodium hydroxide solution. Centrifuged and collected supernatant liquid. It was lead
subacetate solution.
3.4.5: 10 M NaOH
10 M Sodium hydroxide was prepared by dissolving 40 g of Sodium hydroxide in 100 ml of
water.
80
3.4.6: 10 % Ferric chloride solution
10 g of Ferric chloride was dissolved in sufficient amount of purified water and made the final
volume 100 ml.
3.4.7: 3.5 % Ferric chloride in glacial acetic acid
3.5 % Ferric chloride in glacial acetic acid solution was prepared by dissolving 3.5 g of ferric
chloride in 100 ml of glacial acetic acid.
3.4.8: 1 % gelatin solution in 10 % Sodium chloride
1 g gelatin was dissolved in 100 ml of 10 % Sodium chloride solution.
3.4.9: 10 % Sulfuric acid
10 % sulfuric acid was prepared by diluting concentrated sulfuric acid available in ethanol.
3.5 Phytochemical methods
3.5.1: Preliminary phytochemical screening of plant material
The dried and powdered plant material was investigated for the detection of alkaloids,
glycosides, saponins, flavonoids and tannins in plant material. The detail of the tests employed
is given below.
3.5.1.1: Detection of alkaloids
Brain and Turner, (1975) explained the detection of alkaloids. Three gram of the ground plant
material was boiled with 10 ml of acidified water in test tube for 1 min., cool, and allowed the
debris to settle. Filter the liquid in a test tube. 1 ml of this filtrate was taken and 3 drops of
Dragendorff’s reagent was added, there was no precipitate. The remainder of filtrate was made
alkaline by adding dilute ammonia solution. It was transferred to separating funnel and 5 ml of
chloroform solution was added, two layers were observed. The lower chloroform layer was
pipetted out into another test tube. Chloroform layer was extracted with 10 ml of acetic acid and
then discarded the chloroform. Extracts was divided into three portions; to one portion added few
drops of Dragendorff’s reagent and to second few drops of Mayer’s reagent was added. Turbidity
or precipitate was compared with the third untreated control portion.
81
3.5.1.2: Detection of anthraquinone glycosides
One gram of ground plant material was taken and extracted with 10 ml of hot water for five
minutes, allowed it to cool and filtered. Filtrate was extracted with 10 ml of carbon tetrachloride.
Then carbon tetrachloride layer was taken off, washed it with 5 ml water and then 5 ml dilute
ammonia solution was added. No free anthraquinones was revealed as absence of appearance of
pink to cherry red color in the ammonical layer. One gram of second sample of the same plant
material was extracted with 10 ml of ferric chloride solution and 5 ml of hydrochloric acid then it
was heated on water bath for 10 minutes and filtered. Filtrate was cooled and treated as above.
(Brain and Turner, 1975).
3.5.1.3: Detection of cardioactive glycosides
One gram of ground plant material was taken in a test tube and 10 mL of 70% alcohol was
added. It was then boiled for 2 minutes and filtered. Filtrate was diluted twice of its volume with
water and then 1 ml of strong lead subaceatate solution was added. This treatment leads to the
precipitation of chlorophyll and other pigments, which was then filtered off. Filtrate was
extracted with an equal volume of chloroform. Chloroform layer was pipetted out and evaporated
to dryness in a dish over a water bath. Residue was dissolved in 3 mL of 3.5% ferric chloride in
glacial acetic acid and was transferred to test tube after leaving for 1 min. 1.5 ml of sulphuric
acid was then added, which formed a separate layer at the bottom. Cardio active glycosides was
revealed the appearance of brown color at interface (due to deoxy sugar) on standing, and
appearance of pale green color in the upper layer (due to the steroidal nucleus) (Brain and
Turner, 1975).
3.5.1.4: Detection of tannins
Prepared 10% w/v aqueous extract of ground plant material by boiling it with distilled water for
about 10-20 min. Filtered the extract and performed the chemical tests with clear solution.
3.5.1.4.1: Ferric chloride test
Two ml of ferric chloride solution was added to 1-2 ml clear solution of extract. A blue back
precipitate indicated the presence of hydrolysable tannin (Trease and Evans 1983).
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3.5.1.4.2: Gelatin test
Test solution (about 0.5-1%) precipitate 1% solution of gelatin containing 10% sodium chloride
(Trease and Evans, 1983)
3.5.1.4.3: Catechin test
Dipped the match stick in plant extract, dried and then moist it with concentrated hydrochloric
acid. Warmed near flame, a red or pink wood is produced which showed the presence of
catechin (Trease and Evans, 1983).
3.5.1.5: Tests for saponin glycosides
In this test 0.5g of powdered drug was shaken with water. Persistent froth indicated presence of
saponins (Brain et al., 1975).
3.5.1.6: Detection of flavonoids
2 g of the air dried powdered plant material was boiled with 20 ml of distilled water for 10
minutes and filtered. The filtrate was acidified with few drops of dilute HCl. Took 5 ml of
aliquot of the filtrate and made it alkaline (pH 10) with sodium hydroxide (T.S), A yellow colour
was developed indicated the possible presence of flavonoids (El-Olemy et al., 1994).
3.5.1.7: Detection of terpenoids
Plant material was dissolved in 2ml of chloroform and evaporated to dryness. To this, 2ml of
concentrated H2SO4 was added and heated for about 2 minutes. A grayish colour indicated the
presence of terpenoids (Trease and Evans, 1989).
3.6 Extraction
For the purpose of effective extraction, whole plant material was shade dried for 15 days, then
dried plant material was ground in blender and weighed. The extraction of this finely ground
material was affected by simple maceration. The weighed amount of plant material was taken in
extraction bottle and measured volume of dichloromethane was added to it. Filtration was carried
out after 24 hours of addition of solvent. The process was repeated three times with
dichloromethane. The extraction of the marc was done by methanol in the same manner.
83
Dichloromethane and methanol extracts were concentrated separately under reduced pressure by
using rotary evaporator.
3.7 Chromatographic Method
3.7.1: Thin Layer Chromatography
20 mg of each of methanol and dichloromethane extracts were dissolved in 1 ml of methanol and
dichloromethane respectively. The samples were mixed thoroughly by rotating at the Vortex
mixer (Stuart) at 2500 revolutions per minute. After mixing a clear solution was prepared for
spotting on TLC plates. The TLC plates were line marked at 1.5cm for the side which was
intended to be dipped in the mobile phase. 5-10µl of sample was applied to the line mark on the
plate by using micro capillary. Spots of sample applied at the TLC plates were equidistant. Then
the spotted plates were placed in TLC tank and mobile phase was allowed to elute the sample
upto a line that was already marked at a distance of 1cm from corresponding end. Different
solvent systems used to analyze crude DCM, MeOH crude extracts are given in table 3.1 and
table 3.2.
3.7.1.1: Visualization of components on TLC plates
1. Under UV 254 nm
2. Under UV 365 nm
3. Spraying with chemical reagents
With regard to detection, TLC plates were observed with naked eye, in UV light 254 nm, in 365
nm and Godine reagent was sprayed on these plates followed by the spray of 10 percent sulfuric
acid. Plates were kept in oven for 5 minutes at 110 °C. The developed colors were marked.
3.7.2. Column chromatography
Open glass columns were effectively washed, rinsed with methanol and oven-dried. Samples to
be applied onto the column were prepared such that 1g of adsorbent i.e. Silica gel, is loaded with
sample. After loading, silica gel was dried such that it acquired free flowing powder form. Open
glass columns were packed with slurry of Silica gel in suitable solvent and it was allowed to
settle. The amount of silica gel taken to make the slurry was based on a ratio of 1g sample to 30g
silica gel. After settling the length of silica gel column was recorded. When the silica gel column
84
settled completely, the excess amount of solvent present over the level of column was drained
until shiny surface of silica gel appeared. The sizes of the column used were
CR 60/50 (Quickfit-England), CR 40/50 (Quickfit-England) , CR 40/30 (Quickfit-England) and
CR 20/30 (Quickfit-England). Suitable mobile phase were selected with the help of TLC
analysis.
Table 3.1: Solvent systems used for the analysis of dichloromethane extracts of
Croton bonplandianum
Solvent System Ratio
Choloroform:Methanol97.5:2.5
95:5
89:11
80:20
n-hexane:Ethyl acetate 75:25
50:50
30:70
n-hexane: Isopropanol90:10
80:20
n-hexane: Methanol80:20
90:10
Ethyl acetate:Chloroform90:10
80:20
Ethyl acetate:Methanol90:10
80:20
Chloroform:Methanol:Water 80:18:2
Ethyl acetate:Methanol:Water 93:5:3
85
Table 3.2: Solvent systems used for the analysis of methanol extracts of Croton
bonplandianum
Solvent System Ratio
Chloroform:Methanol:Water
85:15:01
80:20:02
70:30:04
Ethyl acetate-Methanol:water
85:15:01
80:20:02
70:30:04
Ethyl acetate-Methanol 80:20:02
70:30:04
Chloroform:Methanol 90:10
80:20
3.8 Spectroscopy
Ultraviolet (UV) spectra were recorded in chloroform on a Shimadzu UV 240 (Shimadzu
Corporation, Kyoto, Japan) and Perkin-Elmer spectrophotometers. Infrared (IR) JASCO A-302
(Japan Spectroscopic Co. Limited) spectrophotometers. Proton magnetic resonance (1H-NMR)
spectra were recorded either in CDCl3, 400 MHz on Bruker AM-300, AM-400 and AMX-500
nuclear magnetic resonance spectrometers, respectively. The 13C-NMR spectra were recorded in
the solvents CDCl3 at 100 MHz, on the same instruments. Mass spectra were recorded on
Finnigan MAT 312 double focusing mass spectrophotometer both connected to PC 386 computer
system or Peak matching, field desorption (FD) measurements performed on the MAT 312 mass
spectrophotometer. High-resolution electron impact mass (HREI MS) spectra were recorded on
JEOL JMS HX 110 mass spectrophotometer. Fraction collector used was Spectra / Chrom CF1,
Oven of Memmert UVIS of DESAGA, weighing balance of SHAIMADZU, Vortex mixer of
Stuart and Melting points were determined in glass capillary tubes using Gallenkamp melting
point apparatus.
86
3.9 Physical and Spectroscopic Data of the isolated Compounds (A-I)
3.9.1: Compound A
State: White amphorous powder
M.P: 158–159 °C
UV λ max MeOHnm (log ε):224 (5.6) nm
IR (KBr) nmax cm-1:3487, 1751, 1618, 752 and 715 cm-1
1H-NMR (CDCl3, 400 MHz) δ;
δ, 5.31 (1 H, dd, J = 18.6, 5.2 Hz), 5.28 (1 H, brs), 4.21 (1 H, J= 11.1 Hz),4.17 (1 H, J= 11.1
Hz)3.87 (1 H, br’m), 2.75 (2 H, brs), 2.32 (1 H, d, J= 6 Hz), 1.65 (2 H, br’s), 1.23 – 1.28 (68 H,
br’s), 0.88,0.91(6 H, brs, 2 CH3).
13C-NMR(100 MHz CDCl3) δ;
δ, 167.7, 130.2, 128.1, 75.8, 68.2, 62.1, 38.6, 32.1, 31.9 – 29.9, 24.7, 23.7, 23.1, 22.7, 14.2 and
14.1
HR-EI-MS;
m/z 662.3329 C44H86O3(calculated. for C44H86O3; 662.3356)
EI-MS;
662 (18.2), 491 (9.3), 465 (11.7), 395 (13.6), 381 (11.6), 367 (22.8), 351 (16.3), 323 (13.6), 295
(16.3), 239 (38.3), 225 (16.1), 211 (16.3), 171 (28.7), 169 (20.3), 155 (21.3), 141 (26.2), 127
(38.3), 113 (37.1).
87
3.9.2: Compound B
State: Amorphous solid
M.P.: 91-92 °C
IR;
1751and 1648 cm-1
1H-NMR(CDCl3, 400 MHz) δ:;
δ 0.83 and 0.90 (t, each, 6H, J = 7.1 Hz), 1.23 – 1.27 (38 H, br’s), 2.42 (1H, d), d 2.23 (1 H, d),
4.39 (2 H, triplet, J=7.5 Hz), δ 4.1 (2 H, triplet, J= 7.0 Hz), d 5.23 (1H, dd, J = 14.9, 7.8 Hz); d
5.21 (1H, dt, J = 14.9, 7.8 Hz); d 5.18 (1H, dt, J = 15.1, 7.1 Hz); 5.05 (1H, dt, J = 15.1, 7.1 Hz)
13C-NMR (100 MHz CDCl3)δ:;
δ, 171.2, 135.6, 130.8, 128.9, 126.9, 68.1, 49.6, 38.7, 33.6, 31.9, 30.3 – 29.3, 28.6, 26.9, 24.1,
23.8, 22.9, 22.6,10.9 and 18.5.
EI-MS;
462 (12), 460 (14), 421 (27), 407 (19), 394 (13), 365 (15), 337 (11), 316 (29), 295 (20), 297
(45), 253 (17), 197 (27), 167 (79), 149 (89), 124 (100), 113 (55), 97 (51), 85 (65), 71 (83), 57
(98) and 43 (80)
HR-EI-MS;
m/z:462.6685 C31H58O2 (calculated. For C31H58O2, 462.6685).
3.9.3: Compound C
State: Colorless amorphous solid
M.P.: 105-106 °C
88
IR;
1652, 1615 and 1538 cm-1
1H-NMR(CDCl3, 400 MHz)δ:;
d 0.87, 0.94 (6H, triplet,J = 6.8 Hz), d1.25 – 1.38 (76H, br’s), 1.69 (quintet),d2.03 (triplet, J=
7.2 Hz), d 4.21 (1H, d, J = 8.7 Hz)
13C-NMR(100 MHz CDCl3)δ:;
d, 169.3, 69.1, 40.1, 33.0, 31.6 – 30.1, 24.9, 24.0, 11.4 and 14.4
EI-MS;
662 (25), 647 (55), 592 (21), 536 (18), 478 (23), 424 (19), 328 (21), 316 (15), 280 (20), 197 (21),
191 (17), 167 (26), 149 (78), 111 (19), 98 (33), 84 (43), 82 (41), 74 (65), 60 (78), 44 (95) and 42
(100)
HR-EI-MSm/z:
m/z 662.3479 (calculated. for C45H90O2; 662.3477)
3.9.4: Compound D
Physical State: Amorphous solid
M.P.: 86-87 °C
IR (KBr) nmax cm-1:
3322, 2688 and 1721
1H-NMR(CDCl3, 400 MHz)δ:;
δ0.91 (3H, triplet, J = 6.5 Hz), 1.59-1.61 (48H, br’s), 1.98 (2 H, quintet), 2.11 (2H, triplet, J= 7.3
Hz)
89
13C-NMR(100 MHz CDCl3)δ:;
δ : 176.1 (C-1), 34.9 (C-2), 32.1 (C-3), 29.6-29.9 (C-4-24), 25.6 (C-25), 22.9 (C-26), 14.2 (C-27)
EI-MS m/z (rel. int.): 410 (21), 367 (23), 341 (35), 320 (19), 306 (13), 273 (17), 253 (18), 239
(17), 205 (19), 192 (22), 169 (32), 149 (44), 137 (54)111 (61), 97 (78), 81 (82), 69 (100), 57 (88)
and 41 (78).
HR-EI-MS
m/z:410.3782 (calculated for C27H54O2, 410.3715)
3.9.5: Compound E
Physical Data;
State; Gummy solid
[a]D25: – 26.2∞ (c 0.10, pyridine)
IR (KBr)
3340, 3220, 1660, 1620 and 1540 cm-1
1H-NMR(CDCl3, 400 MHz) δ:;
d 0.89, 0.94 (6H, triplet, J = 6.8 Hz), 1.28 (18H, br s), 1.32 (18H, br s), 2.03 (2H, t, J = 7.0 Hz,
H-4), 2.15 (2H, t, J = 7.0 Hz, H-2 ), 3.41 (2H, m, H-8), 3.59 (1H, m, H-3),3.65 (1H, dd, J=
11.5, 5.0 Hz, H-1b), 3.94 (1H, m, H-5), 4.22 (1H, dd, J= 11.3, 4.9 Hz, H-1a), 4.87 (1H, dd, J =
15.5, 8.4 Hz); d4.91 (1H, dt, J = 15.5, 8.4 Hz); d 5.05 (1H, dt, J = 16.1, 6.9 Hz); 5.18 (1H, dt, J =
16.1, 6.9 Hz).
13C-NMR(100 MHz CDCl3) δ:;
d: 169.4, 133.6, 132.4, 130.7, 129.8,82.4 (C-3), 74.7 (C-5), 69.1 (C-1), 57.0 (C-2), 33.1 (C-4),
30.4 (C-8), 31.6, 30.8, 30.4, 24.9, 24.0, 23.7, 21.6, 14.4, 11.4
90
EI-MS;
m/z (rel. int. %) 663 (12), 438 (32), 423 (28), 379 (42), 335 (39), 305 (21), 292 (33), 279 (19),
225 (62).
HR-FAB-MS;
m/z 664.6246 (calculated. for C42H82NO4; 664.6243)
3.9.6: Compound F
State: Colourless crystalline solid
M.P.:70 oC
UVlmax MeOHnm (log ε):298 (4.01), 238 (3.31), 225 (2.99)
IR (KBr)
nmax cm-1:1725, 1623, 1589, 1512
1H-NMR (CDCl3, 400 MHz)δ:
δ, 6.41 (1H, d, J= 9.5 Hz, H-3), 7.25 (1H, d, J= 8.7 Hz, H-5), 7.49 (1H, d, J= 8.7 Hz, H-8),7.52
(2H, m, H-6, H-7),7.68 (1H, d, J= 9.5 Hz, H-4)
13C-NMR (100 MHz CDCl3) : 160.1, 152.1 (C-10), 143.5, 130.1 (C-7), 127.1, 125.6 (C-6),
117.91 (C-9), 116.0, 114.9
EI-MS
m/z (rel. int.): 146.0 (29), 118.1 (98), 90.0 (78), 83.0 (100), 63.0 (72), 50.1 (49)
HR-EI-MS
m/z:146.0534 (calcd for C9H6O2,146.0538)
91
3.9.7: Compound G
State: Crystallized from MeOH
M.P.: 251-252°C.
[α]D25: + 20.4° (c 0.42, C5H5N).
IR (KBr) nmax:
3435, 3070, 1635, 880 cm-1.
EI-MS (rel. int. %) m/z:
442 [M]+ (15), 424 (100), 406 (24), 218 (31), 206 (31), 205 (17), 204 (23), 203 (19), 191 (21).
HR-EI-MSm/z:
442.3814 (calculated. for C30H50O2; 442.3810).
1H-NMR (300 MHz; CDCl3) δ;
δ: 4.68 (2H, m, H-29), 3.75 (1H, dd, J = 10.7, 4.2 Hz, H-3), 3.81, 3.42 (1H each, d, J = 11.0 Hz,
H-28), 1.68 (3H, br. s, CH3-30), 1.02 (3H, s, CH3-26), 0.98 (3H, s, CH3-27), 0.92 (3H, s, CH3-
24), 0.89 (3H, s, CH3-23), 0.87 (3H, s, CH3-25).
13C-NMR (125 MHz; CDCl3) δ;
δ: 150.6, 109.6,78.9, 60.4, 55.2, 50.4, 48.7, 47.9, 47.9, 42.8, 40.8, 38.8, 38.6, 37.4, 37.2, 34.2,
33.9, 29.8, 29.1, 28.2, 27.4, 27.1, 25.2, 20.8, 19.1, 18.3, 16.1, 16.0, 15.3, 14.7.
3.9.8:Compound H
Physical Data;
State; Colorless crystalline solid
M.P: 170-171 °C
92
[α]D25; -51.5˚ (c = 0.28, CHCl3)
IR (CHCl3)
nmax cm-1: 3432 (OH), 1648 (C = C)
1H-NMR(CDCl3, 400 MHz) δ;
δ: 5.33 (1H, m, H-6), 5.15 (1H, dd, J = 15.2, 8.4 Hz, H-22), 5.02 (1H, dd, J = 15.2, 8.6 Hz, H-
23), 3.28 (1H, m, H-3), 0.90 (3H, d, J = 6.5 Hz, Me-21), 0.83 (3H, d, J = 6.6 Hz, Me-26), 0.84
(3H, t, J = 7.0 Hz, Me-29), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.80 (3H, s, Me-19), 0.65 (3H, s,
Me-18).
13C-NMR(CDCl3, 100 MHz) δ;
δ: 140.9 , 138.4 , 129.4 , 121.7, 71.9, 57.0, 56.0, 51.3 , 50.3 , 42.5, 42.2, 40.5, 39.7, 37.5, 36.6,
32.2 , 32.0 , 31.9, 31.8, 28.9, 25.4, 24.4, 21.2 , 21.1 , 21.0 , 19.4 , 19.0 , 12.4, 12.0.
EIMSm/z (rel. int. %):
[M]+ 412 (8), 396 (12), 394 (20), 379 (27), 369 (35), 351 (71), 327 (60), 301 (18), 300 (67), 273
(30), 270 (24).
HREIMS;
m/z: 412.3919 (calculated. for C29H48O, 412.3930).
3.9.9: Compound I
Physical Data;
State; yellow solid from acetone
M.p = 89-900C
UVλmax MeOHnm (log ε):
329 (4.01), 239 (3.92), 205 (3.85) nm;
93
IR (KBr)υmax cm-1:
3363, 1704, 1663, 1604, 1449 cm−1
1H-NMR (CDCl3, 400 MHz) δ;
δ, 7.58 (1H, doublet, J=16 Hz), 6.88 ( 2 H, singlet), 6.32 (1 H, doublet, J= 16 Hz) , 4.85 (OH )
and 3.86 (6 H , singlet)13C-NMR (CDCI3, 100 MHz) δ;
δ, 170.87, 149.47, 147.11, 139.51, 126.74, 116.37, 106.85, 147.11, 116.36, 106.84 and 56.85.
EIMS m/z (rel. int) %:
224 (38), 196 (36), 190 (45), 161 (34),149 (45), 131 (12), 119 (24), 107 (15) and 78 (49).
HREIMS
m/z M+224.1233(calculated. forC11H12O5;224.1241)
3.9.10.: Compound J
Physical Data;
State:Crystalline solid from CHCl3
M.P:210 °C
UV lmax MeOHnm (log ε):
216 (4.11), 231 (3.07), 275 (3.87)
IR (KBr)
nmaxcm-1:3510-3320 (O-H), 1708 (C=O), 1626, 1585 (aromatic)
1H-NMR (CD3OD, 400 MHz) δ;
δ: 7.15 (2H, s, H-2, H-6),3.86 (3H, s, MeO-3), 3.83 (3H, s, MeO-5)
13C-NMR (CD3OD, 100 MHz) δ;
94
δ: 168.8 (C-7), 149.1 (C-3, C-5), 140.7 (C-4), 121.4 (C-1),112.4 (C-2, C- 6),56.7 ( MeO-5), δ
52.3 (MeO-3)
EI-MS m/z (rel. int.):
198 (55), 167 (100),155 (4), 139 (20), 124 (12), 83 (15), 53(13)
HR-EI-MS m/z:
198.0526 (calculated for C9H10O5, 198.0528)
3.9.11: Compound K
Physical Data;
State: Colourless crystalline solid
M.P.:229-230 oC
UVλmax MeOHnm (log ε):312 (3.77), 243 (3.82), 218 (4.08)
IR (KBr)
nmax cm-1:3108, 1713, 1607, 1595, 1525, 1503
1H-NMR (CDCl3, 400 MHz) δ;
δ: 7.61 (1H, d, J= 9.5 Hz, H-4), 6.85 (1H, d, J= 8.4 Hz, H-7), 6.75 (1H, d, J= 8.4 Hz, H-6), 6.10
(1H, d, J= 9.5 Hz, H-3)
13C-NMR (CDCl3, 100 MHz) δ;
δ 160.1 (C-1), 150.1 (C-5), 144.5 (C-10), 141.5 (C-8), 137.5 (C-4), 119.1 (C-7), 116.1 (C-6),
114.0 (C-3), 108.9 (C-9)
EI-MS m/z (rel. int.):
178 (100), 150 (84), 122 (14), 94 (28), 66 (43), 51 (14)
95
HR-EI-MSm/z:
178.0261 (calculated for C9H6O4, 178.0267)
3.9.12: Compound L
Physical Data;
State;Colorless amorphous powder
M.P.: 289-290ºC.
[α]D25:-51.5º (c 0.22, C5H5N).
IR (KBr) nmax:
3454 (OH), 3024, 1646 (C=C) cm-1.
EI-MS (rel. int. %) m/z:
412 [M-Glc]+ (72), 397 (15), 394 (22), 379 (28), 369 (35), 351 (71), 327 (55), 301(15), 300 (67),
273 (21), 271 (26)
HR-FAB-MS(+ve)m/z:
575.4229 [M+H]+ (calculated. for C35H59O6; 575.4235).
1H-NMR (400 MHz; CD3OD) δ;
δ: 5.23 (1H, br. d, J = 5.4 Hz, H-6), 5.14 (1H, dd, J = 15.2, 8.4 Hz, H-22), 5.02 (1H, dd, J = 15.2,
8.6 Hz, H-23), 4.78 (1H, d, J = 7.4 Hz, H-1´), 3.84-4.44 (m, Glc-H), 3.83 (1H, m, H-3), 1.01
(3H, s, CH3-19), 0.90 (3H, d, J = 6.2 Hz, CH3-21), 0.83 (3H, d, J = 6.5 Hz, CH3-26), 0.82 (3H, t,
J = 7.0 Hz, CH3-29), 0.80 (3H, d, J = 6.5 Hz, CH3-27), 0.67 (3H, s, CH3-18).
13C-NMR (125 MHz; CD3OD) δ;
δ: 141.5, 138.9, 129.1, 121.1, 102.8, 79.8, 76.9, 76.7 , 74.2 , 70.6 , 62.2 , 57.0 , 56.1 , 52.1 (C-
24), 50.8 , 43.9 , 43.1 , 40.5 , 39.9 , 37.8 , 36.9 (C-10), 32.9 , 32.8 , 31.9 , 31.7 , 28.9 , 25.6 , 24.5
(C-15), 21.9 (C-21), 21.7, 21.5, 19.5, 19.1, 12.6, 12.1
96
3.9.13: Compound M
Physical Data;
State; White crystals (MeOH)
M.P; 186-187 oC
[α]D24: - 76.2 (c = 0.18, H2O)
IR (KBr)
υmax cm-1:3438, 2941 and 1275cm-1
1H NMR (CDCl3, 400 MHz)δ:;
δ, 3.29 (3H,s), 3.30 (1H, br, dd,J = 3.0, 3.0 Hz), 3.56 (1H, br, dd,J = 3.0, 2.4Hz), 3.66 (1H, br,
dd,J= 3.6, 3.0 Hz), 3.82 (1H, br, dd, J = 3.6, 3.0 Hz), 3.91 (1H, br, m), 4.11 (1H, br, m).
13C NMR(100 MHz CDCl3) δ;
δ,82.4,74.6, 73.7, 72.3, 71.3, 69.2, 57.8
EI-MS; (70 e/v) (rel. int %) m/z:
158 [M-2H2O]+ (8), 144 (9), 129 (8), 116 (15), 102 (20), 87 (90), 73 (100), 60 (35), 55 (10)
HR-MS:
m/z 194.1201(calculated. For C7H14O6, 194.1211).
3.9.14: Compound N
State; pale yellowish oil
[α]D24:+1.41 (c 0.92, CH3OH);
UVλmax MeOHnm (log ε): 328 (4.51), 240 (4.44), 202 (4.52) nm;
97
IR;
3363, 2940, 1704, 1633, 1604, 1515, 1456, 1427, 1339, 1285, 1226, 1156, 1115, 910, 828, 766
cm−1
1H-NMR(CDCl3, 400 MHz)δ:;
δ,7.66 (1H, d, J = 16.0 Hz, H-3'''), 7.58 (1H, d, J = 16.0 Hz, H-3''), 6.92 (2H, s, H-5''', 9'''), 6.91
(2H, s, H-5'', 9''), 6.45 (1H, d, J = 16.0 Hz, H-2'''), 6.41 (1H, d, J = 16.0 Hz, H-2''), 5.51 (1H, d, J
= 8.8 Hz, H-1), 5.49 (1H, dd, J = 9.2, 8.8 Hz, H-3), 3.84, 3.87 (12H, s, OCH3 at C-6'', C-8'', C-
6''', C-8'''), 4.68 (1H, d, J = 8.0 Hz, H-1'), 4.49 (1H, brd, J = 10.3 Hz, Ha-6), 3.86 (1H, dd, J =
10.3, 3.9 Hz, Hb-6), 3.25 (1H, d, J = 12.1 Hz, Ha-6'), 4.25 (1H, dd, J= 9.1, 7.5 Hz, H-4), 4.21
(1H, m, H-5), 3.94 (1H, dd, J = 9.1, 7.5 Hz, H-2), 3.80 (1H, dd, J = 12.1, 3.9 Hz, Hb-6'), 3.58
(1H, t, J = 9.1 Hz, H- 3'), 3.63 (2H, m, H-4', H-5'), 3.45 (1H, dd, J = 9.1, 7.5 Hz, H-2')
13C-NMR(100 MHz CDCl3)δ:;
δ169.1, 168.2, 147.4, 147.8, 147.2, 126.6, 126.5, 115.8, 115.4, 107.1, 106.9, 104.8, 92. 6, 84.3,
79.3, 75.1, 74.2, 73.3, 72.5, 72.0, 65.7, 65. 6, 63.8, 56.9, 56.8.
EI-MS m/z (rel. int.):
754 (18), 592 (21), 530 (23), 430 (19), 224 (38), 196 (36), 190 (45), 162 (34),149 (45), 131 (12),
119 (24), 107 (15) and 78 (49).
HREIMS;
m/z 754.5209 (calculated. for C34H42O19;754.5218)
3.10: Biological methods
Biological screening of the selected medicinal plant was done through following bioassays.
3.10.1: Antibacterial assay (Atta-ur-Rehman et al. 2001)
Antibacterial testing is important in those groups of bacteria commonly showing resistance,
primarily staphylococcus species, Niesseria gonorrhea, Streptococcus pneumonia and
Escherchia coli. Antibacterial activity was determined by an agar diffusion method on the
98
already prepared plates of the inoculated media. The required number of holes was bored using a
sterile cork borer ensuring proper distribution in the periphery and one in the centre. The
solutions i.e. the extract, solvent and reference standard (Imepenam) was poured into their
respective hole with the help of sterilized pipette. The plates was left at room temperature for 2
hrs to allow diffusion of the sample and incubated at 37 0C for24-48 hrs. The diameter of the
zones of inhibition was measured to the nearest mm.
3.10.2: Antifungal assay
Atta-ur-Rehman et al., (2001) commented that antifungal testing is important in those groups of
fungi commonly showing resistance. The in vitro antifungal bioassay of the crude
dichloromethane and methanol extracts was performed by agar tube dilution method. The crude
extracts were evaluated against clinical specimens of Candida albicans, Aspergillus flavus,
Microsporum canis, Fusarium solani and Candida glabrata. A control experiment with test
substance (medium supplemented with appropriate amount of DMSO) was carried out for
verification of the fungal growth. The extracts (24 mg) dissolved in sterile DMSO (1 ml), served
as stock solution. Sabouraud Dextrose Agar (SDA) (4 ml), was dispensed into screw cap tubes
which was autoclaved at 121 oC for 15 min and then cooled to 500C. The non-solidified SDA
media was poisoned with stock solution (66.6 µl), giving the final concentration of 400 µg of the
extract/ml of SDA. Each tube was inoculated with a piece (4 mm diameter) of inoculum removed
from a seven day old culture of fungi. For non- mycelial growth, an agar surface streak was
employed. Inhibition of fungal growth was observed after 7 days of incubation at 28±10C.
3.10.3: Antioxidant assay
Mensor et al., (2001) noted that antioxidant assay was assessed by DPPH assay. This assay is
based on the principle that a hydrogen donor is an antioxidant. DPPH radical accepts hydrogen
from an antioxidant. The antioxidant effect is proportional to disappearance of DPPH radical in
the sample. A concentration (0.5µg/ml) of the test extracts was prepared in methanol. To 2.5 ml
solution of each extract concentration was added 1 ml of 0.3 mM of freshly prepared DPPH
solution in methanol and allowed to react in the dark at room temperature for 30 min.
Absorbance of the resulting solution was measured at 518 nm. Methanol (1 ml) added to 2.5 ml
of each sample concentration was used as blank, while 1 ml of 0.3 mM DPPH solution added to
99
2.5 ml of methanol served as a negative control. Gallic acid, prepared in the same concentrations
as the test extracts, was used as reference standards (positive controls) for comparison.
Percentage DPPH scavenging activities of the extracts and reference standards was determined
using the formula.
% scavenging activity 100 - [(As - Ab) /Ac X 100 ]
Where As = Absorbance of sample (extract or reference standard), Ab = Absorbance of blank and
Ac = Absorbance of negative control.
3.10.4: Cytotoxicity assay (Meyer et al. 1982)
The brine shrimp lethality test (BST) was performed. Sample was tested for brine shrimp
lethality. Solutions of the extract was made in DMSO and incubated in duplicate vials with the
brine shrimp larvae. Ten brine shrimp larvae were placed in each of the duplicate vials. Control
brine shrimp larvae were placed in a third vial which contained sea water and DMSO only. After
24 hrs the nauplii was examined against a lighted background, and the average number of
survived larvae in each triplicate was determined. The mean percentage mortality was plotted
against the logarithm of concentrations and the concentration killing fifty percent of the larvae
(LC50) was determined from the graph by taking the antilogarithm of the concentration
corresponding to 50 % mortality rate of the test organisms. Etoposide was used as a standard test
drug.
3.10.5: Phytotoxicity assay
Atta-ur-Rehman et al., (2001) elaborated the method by using Lemna minor assaay. Lemna
minor (Lemnaceae) is a miniature aquatic thaloid monocot consists of a central oral frond with
two attached daughter fronds and a filamentous root. Lemna assay is a quick measure of
phytotoxicity of the material under investigation. An inorganic medium (E. Medium) of pH 5.5-
6.0 was prepared. Vials for testing; 10 vials per dose (500, 50, 5 ppm, control) was prepared as:
15 mg of extract was weighed and dissolved in 15 ml solvent. 1000, 100, and 10 µl solutions was
added to vials for 500, 50, 5 ppm, allowed solvent to evaporate overnight. 2 ml of E. Medium
and then a single plant containing a rosette of three fronds was added to each vial. Vials was
placed in a glass dish filled with about 2 cm water, and container was sealed with stopcock
grease and glass plate. Dish with vials was placed in growth chamber for seven days at 26 0C
100
under fluorescent and incandescent light. Number of fronds per vial was counted and recorded
on day 3 and day 7. Data was analyzed as percent of control with ED50 computer program to
determine FI50 values and 65% confidence interval.
3.10.6: Urease inhibition assay
Lodhi and Abbasi, (2007) are of the view that Urease is an enzyme that catalyzes the hydrolysis
of urea into carbon dioxide and ammonia. The enzyme assay is the modified form of the
commonly known Berthelot assay. A total volume of 85 µl assay mixture contained 10 µl of
phosphate buffer of pH 7.0 in each well in the 96-well plate followed by the addition of 10 µl of
sample solution and 25 µl of enzyme solution (0.1347 units). Contents were pre-incubated at
37ºC for 5 minutes. Then, 40 µl of urea stock solution (20 mM) was added to each well and
incubation continued at 37ºC for further 10 min. After given time, 115 µl phenol hypochlorite
reagents were added in each well (freshly prepared by mixing 45 µl phenol reagents with 70 µl
of alkali reagent). For color development, incubation was done at 37ºC for another 10 min.
Absorbance was measured at 625 nm using the 96-well plate reader Synergy HT. The percentage
enzyme inhibition was calculated by the following formula.
Inhibition (%) = 100 - (Absorbance of test sample / Absorbance of control) × 100.
IC50 values (concentration at which 50% enzyme catalyzed reaction occurs) of compounds was
calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).
3.10.7: α-Chymotrypsin inhibition assay (Canal et al., 1988)
It is involved in the body defense reactions supported by immune system and in most of the
physiological functions of body. It plays an important role in first line defense versus cancer by
clearing away the proteins surrounded the malignant tumors.. The α-chymotrypsin inhibition
activity is performed according to slightly modified method of. A total volume of 100 μl assay
mixture contained 60 μl Tris-HCl buffer (50 mM pH 7.6), 10 μl test compound and 15 μl (0.9
units) purified α-chymotrypsin enzyme (Sigma, USA). The contents was mixed and incubated
for 20 min at 37oC and pre read at 410 nm. The reaction was initiated by the addition of 15 μl
(1.3 mM) substrate (N-succinyl phenyl-alanine-P-nitroanilide). The change in absorbance was
observed after 30 min at 410 nm. Synergy HT (BioTek, USA) 96-well plate reader was used in
all experiments. All reactions were performed in triplicates. The positive and negative controls
101
were included in the assay. Chymostatin (0.5 mM/well) was used as a positive control. The
percentage inhibition was calculated by formula given below.
% Inhibition=100 – (Absorbance of Test/Absorbance of Control) ×100
IC50 values (concentration at which there is 50% in enzyme catalyzed reaction) compounds was
calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).
3.10.8: α- glucosidase inhibition assay (Dong et al., 2012)
The α-glucosidase inhibitory activity was assessed by the standard method with slight
modifications. Briefly, a volume of 60 μl of sample solution and 50 μl of 0.1 M phosphate buffer
(pH 6.8) containing α-glucosidase solution (0.2 U/ml) was incubated in 96 well plates at 37 ºC
for 20 min. After pre-incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside (PNPG)
solution in 0.1 M phosphate buffer (pH 6.8) was added to each well and incubated at 37 ºC for
another 20 min. Then the reaction was stopped by adding 160 μl of 0.2 M NaCO3 into each well,
and absorbance readings (A) was recorded at 405 nm by micro-plate reader and compared to a
control which had 60 μl of buffer solution in place of the extract. For blank incubation (to allow
for absorbance produced by the extract), enzyme solution was replaced by buffer solution and
absorbance recorded. The concentrations of test compounds which inhibited the hydrolysis of
substrates by 50% (IC50) were determined by monitoring the effect of increasing concentrations
of these compounds in the assays on the inhibition values. The IC50 values were then calculated
using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA).
3.10.9: Butyrylcholinesterase inhibition assay (Ellman et al., 1961)
Butyrylcholinesterase inhibiting activity was measured by a slightly modified
spectrophotometric method. Butyrylthiocholine chloride was used as substrates to assay
butyrylcholinesterase. 5, 5′-Dithiobis [2-nitrobenzoic-acid] (DTNB) was used for the
measurement of Butyrylcholinesterase activity. 140 μL of (100 mM) sodium phosphate buffer
(pH=8.0), 10 μL of DTNB, 20 μL of test compound solution and 20 μL of butyrylcholinesterase
solution was mixed and incubated for 15 min (25°C). The reaction was then initiated by the
addition of 10 μL butyrylthiocholine. The hydrolysis of butyrylthiocholine was monitored by the
formation of the yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with
thiocholine, released by the enzymatic hydrolysis of butyrylthiocholine, respectively, at a
102
wavelength of 412 nm (15 min). Test compounds and control was dissolved in EtOH. All the
reactions were performed in triplicate in 96-well micro plates and monitored in a Spectra Max
340 (Molecular Devices, USA) spectrometer. The concentrations of test compounds which
inhibited the hydrolysis of substrates by 50% (IC50) was determined by monitoring the effect of
decreasing concentrations of these extracts in the assays on the inhibition values. The IC50 values
were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc mherst)
103
4 Results
4.1 Phytochemical studies
4.1.1: Detection of secondary metabolites:
Phytochemical studies were carried out for detection of secondary metabolites i.e. alkaloids,
anthraquinone glycosides, cardiac glycosides and saponins, flavonoids, tannins and terpenoids in
plant material. The results of the study are shown in table 4.1.
Table 4.1: Results of phytochemical screening of Croton bonplandianum
Plant namePart
used
Alk
aloi
ds
An
thra
qui
n
one
glyc
osid
es
Car
dia
c
glyc
osid
es
Sap
onin
s
Fla
vono
ids
Tan
nin
s
Ter
peno
ids
Croton
bonplandianum
Whole
plant+ - - + + + +
4.1.2: Extraction
The solvent used for extraction were methanol and dichloromethane. The results are shown in
the table 4.2.
Table 4.2: Results of extraction of plant material with different solvents
Plant NamePart
Used Weight (g)Solvent Used (ml)
Extract
obtained
(g)
Extract codes
Croton
bonplandianum
Whole
plant 1000
Dichloromethane
200020.2 CBD
Methanol
200048.9 CBM
104
4.2 Biological screening of crude extracts
Dried and powdered plant material of Croton bonplandianum was extracted successively at room
temperature with dichloromethane and methanol. Dichloromethane and methanol
extracts screened for antibacterial bioassay, antifungal bioassay, brine-shrimp toxicity,
phytotoxicity against Lemna minor, antioxidant assay, α-chymotrypsin inhibitory activity, and
acetylcholinestrase inhibitory activity. The results of in vitro bioassays performed are presented
below in Tables 4.3-4.
Table 4.3: Results of antibacterial bioassay of methanol and dichloromethane extracts of Croton
bonplandianum .
Name of bacteria
Zone of inhibition of sample
(mm) Zone of inhibition of standard
drug (mm)CBD CBM
Eschericha coli _ _ 25
Bacillus subtilis _ _ 50
Shigella flexinari _ _ 28
Staphylococcus aureus _ _ 48
Pseudomonas aeruginosa _ _ 23
Salmonella typhi _ _ 28
Note: Concentration of extract used, 3 mg/ml and concentration of Standard
drug Imipenum (10µg/ml).
105
Table 4.4: Results of antifungal bioassay of methanol and dichloromethane extracts of Croton
bonplandianum.
Name of fungi
Linear Growth (mm) of
Extracts and control, %Inhibition Standard
MIC
(µg/ml)
CBD CBM CONTROL
Candida albicans 100 100 0 Miconazole 110.8 100
Aspergillus flavus 100 100 0Amphotericin
B20.20 100
Microsporum canis 100 100 0 Miconazole 88.4 100
Fusarium solani 90 100 10 Miconazole 73.25 90
Candida glabrata 100 100 0 Miconazole 110.8 100
Note: Concentration of extract used, 400 µg/ml of DMSO
Table 4.5: Results of phytotoxic bioassay of methanol and dichloromethanr extracts of Croton
bonplandianum
ExtractPlant
Name
Conc. of
Compound
(µg/ml)
No. of Fronds% Growth
Regulation
Conc. of
Standard Drug
(µg/ml)Sample Control
CBM
Lemna
minor
1000 05
20
75
0.015
100 19 05
10 19 05
CBD
1000 05
20
60
100 20 0
10 20 0
106
Table 4.6: Results of Brine Shrimp Lethality bioassay of methanol and dichloromethane extracts
of Croton bonplandianum.
Extract
Code
Dose
µg/ml
No .of
shrimp
No. of
survivors
LD 50
µg/ml STD. Drug
LD 50
µg/ml
CBM
1000 30 04
115.76 Etoposide 7.4625100 30 20
10 30 23
CBD
1000 30 16
1327.85 Etoposide 7.4625100 30 19
10 30 24
Table 4.7: Results of antioxidant activity of methanol and dichloromethane extracts of Croton
bonplandianum
Extract code Conc. mg/ml IC50± SEM.µg/ml % RSA
CBM 0.5 396.205±4.6 59.629
CBD 0.5 inactive 39.37
STD Gallic acid 0.094 4.3±0.43 93.13
Table 4.8: Results of α-chymotrypsin inhibition assay of methanol and dichloromethane extracts
of Croton bonplandianum.
Extract codeConc. µg/ml % inhibition IC50± SEM.µM
CBM 500µg/ml 1.4±2.6% _
CBD 500µg/ml 3.17±2.1% _
STD Chymostatin _ _ 5.97±0.76 µM
107
Table 4.9: Results of urease inhibitory activity of methanol and dichloromethane extracts of
Croton bonplandianum
Extract code Conc. µg/ml % inhibition IC50± SEM. µg/ml
CBM 0.5 i_ _
CBD 0.5 71.27 ±1.21 290.92±2.92
STD Thiourea0.5 98.18±0.13 20.30±0.17
Table 4.10: Results of α-Glucosidase inhibition assay of methanol and dichloromethane extracts
of Croton bonplandianum
Extract code Conc. µg/ml % inhibition IC50± SEM. µg/ml
CBD
500250125
62.0531.2515.6257.3123.656
97.89 ±2.689.58 ±1.679.34 ±1.568.23 ±2.2.60..56 ±2.351.12 ±2.140.67±1.830.34±2.9
14.93±0.37
CBM 0.5 - -
STD Acarbose
500250125
62.0531.2515.6257.3123.656
92.23±0.1481.39±0.2371.09±0.5657.42±0.4448.02±0.2435.99±0.9824.87±1.0113.09±1.12
38.25±0.12
108
Table 4.11: Results of butyrylcholinesterase inhibition assay of methanol and dichloromethane
extracts of Croton bonplandianum.
Extract code Conc. µg/ml % inhibition IC50± SEM. µg/ml
CBM
500
250
125
62.05
31.25
84.14±0.13
83.77±0.13
73.18±0.78
68.73±0.79
50.45±0.2014.93±0.37
CBD 0.5 - -
STD Eserine
500
250
125
62.05
31.25
76.3±0.6
74.2±0.2
70.3±0.1
62.1±1.6
48.9±0.1
0.30±0.01
109
4.3 Thin layer chromatography
4.3.1: TLC analysis of dichloromethane extract of Croton bonplandianum.
The dichloromethane extract of Croton bonplandianum was subjected to TLC based analysis
using precoated silica 60 F254 plates and various combinations of n-hexane and ethyl acetate
based mobile phases systems were used prior to proceed towards fractionation. The ratios were
used in order of increasing polarity (50:50 80:20). The comparative resolutions of
dichloromethane extract into individual components on TLC plates caused by these mobile phase
systems are presented in Figure 1. On comparison it was noted that n-hexane and ethyl acetate
with respective ratio of 60 : 40 affected best resolution of dichloromethane extract into 11
components with Rf values, 0.92, 0.89, 0.84, 0.78, 0.72, 0.66, 0.46, 0.38, 0.32, 0.17, and 0.09.
(Figure1b).
a b c d
Figure 4.1: Results of TLC analysis of dichloromethane extract of C. bonplandianum.
Stationary phase Slica gel 60, F254
Mobile phase a: n-hexane : ethyl acetate ( 50 :50)
b: n-hexane : ethyl acetate ( 60 :40)
c: n-hexane : ethyl acetate ( 70 :30)
d: n-hexane : ethyl acetate ( 80 :20)
Detection
Spot appeared at 254nm
Spot appeared at 366nm
Spot appeared due to Godin reagent ////////
110
4.3.2: TLC analysis of methanol extract of Croton bonplandianum
The dichloromethane extract of Croton bonplandianum was subjected to TLC based analysis
using precoated silica 60 F254 plates and various combinations of chloroform: methanol: water
based mobile phase systems were used prior to precede towards fractionation. The ratios were
used in order of increasing polarity (85: 15: 01 80: 20: 02 70:30:04). The comparative
resolutions of methanol extract into individual components on TLC plates caused by these
mobile phase systems are presented in Figure 1. On comparison it was noted that chloroform:
methanol: water respective ratio 80: 20: 02 of affected best resolution of methanol extract into 09
components with Rf values, 0.90, 0.86, 0.84, 0.78, 0.72, 0.66, 0.46, 0.38, and 0.32, (Figure 2 b).
Figure 4.2: Results of TLC analysis of methanol extract of C. bonplandianum.
Stationary phase Slica gel 60, F254
Mobile phase a: chloromethane : methanol : water (85: 15: 01)
b: chloromethane : methanol : water (80: 20: 02)
c: chloromethane : methanol : water (70 :30:04)
DetectionSpot appeared at 254nm
Spot appeared at 366nm
Spot appeared due to Godin reagent ////////
111
4.4 Isolation of compound
4.4.1: Isolation of compound from dichloromethane extract
Dichloromethane extract (18 g) was subjected to column chromatography on silica gel using
stepwise elution with n-hexane-ethyl acetate ((80:20 →70:30 → 60:40 → 50:50 →ethylacetate)
in increasing order of polarity. six fractions (CBWPD 1- CBWPD 6) were obtained. The fraction
CBWPD 2 (4.07g) subjected to column chromatography on silica gel using n-hexane-
ethylacetate (80:20 →70:30) as eluent resulted two fractions (2a and 2b). The fraction 2a (1400
mg) was subjected to column chromatography on silica gel using n-hexane-ethylacetate (60:40)
as eluent which gave two pure compounds A (11 mg) and B (9 mg). The fraction CBWPD-3
(3.10g) was subjected to column chromatography on silica gel using n-hexane-ethylacetate
(80:20 →70:30) as eluent resulted two fractions (3a and 3b). The fraction 3a (1200 mg) was
subjected to column chromatography on silica gel using n-hexane-ethylacetate (60:40) as eluent
which gave two pure compounds C(12 mg) and D(14 mg).The fraction CBWPD-4 (2.74g)
obtained by n-hexane-ethyl acetate (80:20 →70:30) was subjected to column chromatography on
silica gel using n-hexane – EtOAc (60:40) as eluent resulted two fractions (4a and 4b). The
fraction 4a (850 mg) was subjected to column chromatography on silica gel using n-hexane –
EtOAc (60:40) as eluent which gave two pure compounds E(8 mg) and F(6 mg). The fraction
CBWPD-5 (1.0g) was subjected to column chromatography on silica gel using ethylacetate-
methanol (80:20 →70:30) as eluent resulted two fractions (5a and 5b). The fraction 5a (500 mg)
was subjected to column chromatography on silica gel hexane – EtOAc (60:40) as eluent which
gave two pure compounds G (7 mg) and H (6 mg). Fraction 5b (50 mg) was subjected to column
chromatography on Sephadex LH-20 using methanol as eluent afforded compound I (9 mg).
Isolation scheme of compounds (A-I) from dichloromethane extract of whole plant of croton
bonplandianum (CBWPD) is given in figure 4.3.
112
CBWPD* (18g) CCSilica gel 60 (0.063-0.100 mm)n-hexane – EtOAc (80:20 →70:30 → 60:40 → 50:50 →ethylacetate)
CBWPD-1 CBWPD-2 CBWPD-3 CBWPD-4* CBWPD-5* CBWPD-6 (1.55g) (4.07g) (3.10g) (2.70g) (1.10g) (2.71g)
CC CC CC CC (0.063-0.100 mm) (0.063-0.100 mm) (0.063-0.100 mm) (0.063-0.100 mm) n-hexane – EtOAc n-hexane – EtOAc n-hexane – EtOAc n-hexane – EtOAc (80:20 →70:30 ) (80:20 →70:30) (80:20 →70:30 ) (80:20 →70:30 )
CBWPD2a CBWPD2b CBWPD3a CBWPD3b CBWPD4a* CBWPD4b CBWPD5a* CBWPD5b*
(1400mg) (1200mg) (850mg) (500mg) (50mg)
CC CC CC CC CC(0.04-0.0.063 mm) (0.04-0.0.063 mm ) (0.04-0.0.063 mm ) (0.04-0.0.063 mm) Sephadexn-hexane – EtOAc n-hexane – EtOAc n-hexane – EtOAc n-hexane – EtOAc LH-20 (60:40 ) (60:40 ) (60:40 ) (60:40 ) MeOH
A B C D E F G H I (11mg) (9 mg) (12 mg) (14 mg) (8 mg) (6 mg) (4 mg) (6 mg) (9 mg)
* indicates the fraction having α-glucosidase inhibitory activity
Figure 4.3: Isolation scheme of compounds (A-I) from dichloromethane extract of whole plant of croton bonplandianum (CBWPD).
113
4.4.2: Isolation of compound (J-N) from methanol extract
10 grams of the methanol extract of Croton bonplandianum was dissolved in minimum quantity
of methanol. The solution was filtered and loaded to of silica gel. The open glass column was
packed with 300 grams of silica gel dissolved in the mobile phase. The column was allowed to
be stable with a flow rate 3 ml /min. When the silica was settled properly then after 1 hour with
continuous flow of mobile phase (chloroform: methanol: water 80:20:02), the sample was
applied at the top of the column. The column was eluted continuously with mobile phase
chloroform: methanol: water (80:20:02→70:30:04→65:35:5→60:40:10→methanol) in stepwise
elution development. Each fraction of 400 ml was collected and analyzed by TLC. On the basis
of TLC results, all fractions were combined into 6 fractions namely CBWPM-1, CBWPM-2,
CBWPM-3, and CBWPM-4. Fraction CBWPM-2 on the basis of TLC analysis was
chromatographed using silica gel 60 (0.063-0.100) mm as stationary phase and chloroform:
methanol: water (95:5:0.5→90:12:1→85:15:1→80:20:2) as mobile phase in stepwise elution
development. Each fraction of 10 ml was collected with the help of fraction collector. All these
fractions were analyzed by TLC. It gave 3 fractions finally, fraction CBWPM-2b was
chromatographed using silica gel 60 (0.063-0.100) mm as stationary phase and chloroform:
isopropyl alcohol (98:02) as mobile phase in isocratic manner. This fraction gave compound J (4
mg) and compound K (3.4mg). Similarly, fraction CBWPM-3 on the basis of TLC analysis was
chromatographed using silica gel 60 (0.063-0.100 mm) as stationary phase and chlorofor:
methanol: water (95:5:0.5→90:12:1→85:15:1→80:20:2) as mobile phase in stepwise elution
development. Each fraction of 10 ml was collected with the help of fraction collector. All these
fractions were analyzed by TLC. It gave 2 fractions, fraction CBWPM-3a on the basis of TLC
results was chromatographed using silica gel 60 (0.063-0.100 mm) as stationary phase and
chloroform: isopropyl alcohol (95:05) as mobile phase in isocratic manner. Each fraction of 10
ml was collected with the help of fraction collector. All the fractions were analyzed by TLC and
it yielded 3 compounds L (4.5mg), M (5 mg) and N (3mg). The isolation of these compounds is
schematically represented in Figure 4.4.
114
CBWPM* (10g)
Silica gel 60 (0.063-0.100 mm)Chloroform: Methanol : Water(80:20:2 →70:30:4→65:35:5→60:40:10→methanol)
CBWPM-1 CBWPM-2* CBWPM-3* CBWPM-4 (4.136g) (2.153g) (1.09g) (1.945g)
Silica gel 60 (0.063-0.100) mm CHCl3: MeOH: H2O(95:5:5→90:12:1→85:15:1)
CBWPM -2a CBWPM -2b* CBWPM -2c CBWPM -3a* CBWPM -3b
(0.4g) (0.55g)
Silica gel 60(0.04-0.0.063 mm) CHCl3: IPA (95:05)
Silica gel 60(0.04-0.0.063 mm)CHCl3: IPA(98:02)
J K L M N (4mg) (3.4mg) (4.5mg) (5mg) (3mg)
* indicates the fraction having butyrylcholinesterase inhibitory activity
Figure 4.4: The schematic representation of isolation of compounds (J-N) from methanol extract
of whole plant of Croton bonplandianum (CBWPM).
115
4.5 Structure elucidation of the isolated compounds
4.5.1: Compound A (n-Pentacosanyl-n-nonadeca-7′-en-9′-α-ol-1′-oate)
O
O
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
15'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
OH
22' 18'
17'19'21'23'
Compound A, was attained as colorless amorphous powder. It gives positive tests with
tetranitromethane and bromine water for unsaturation. It gives a M+ peak at m/z 662 consistent
with a molecular formula C44H86O3 (calculated. for C44H86O3; 662.3356) indicated the presence
of two double bond equivalents (i.eolefinic and ester group). Most of the EI mass fragments
were separated by 14 mass units and decreased in abundance with increasing molecular weight
of long straight chain hydrocarbon. Its IR spectra showed the presence of hydroxyl group at
3487 cm-1, ester linkage at 1751 cm-1, unsaturation at 1618 cm-1 and long aliphatic chain
absorption bands at 752, 715 cm-1respectively.
The proton NMR spectra of compound A displayed a six proton peak at 0.86, 0.91(6 H, m)
because of the terminal primary methyl functionalities. Oxygenated methylene proton gives
doublets at 4.21 (J= 11.1 Hz) and 4.17 (J= 11.1 Hz). Another oxygenated methine proton gives
broad multiplet at 3.87.Methylene protons adjacent to ester group gives doublets at 2.75 (br’s)
and 2.32 (m, J= 6 Hz). The remaining methylene protons resonated at 1.65 (2 H) and between
1.23- 1.28.
The Carbon NMR (BB and DEPT) spectrum of A displayed 44 carbon signal consisting of two
methyl, 38 methylene carbon, three methine and one quaternary carbon atoms. The deshielded
carbon peaks at 167.7, 130.2 and 128.1 assigned correspondingly to the ester carbon and vinylic
116
carbon. The oxygenated methine and methylene carbons give signal at 75.8 and 68.2,
respectively. The remaining methylene and methyl carbons appeared in the range of 31.9 –28.9.
On the basis of spectral data analyses and chemical evidences, the structure of the unknown
compound has been elucidated as n-Pentacosanyl-n-nonadeca-7′-en-9′-α-ol-1′-oate (Haq et al.,
2005).
4.5.2: Compound B (n-Tridecanyl n-octadec-9,12-dienoate)
O
O
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
123
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Compound B was obtained in the form of white powder. The EI-MS of the molecule givesM+
peak at m/z 462 corresponding to the molecular formula C31H58O2 (calculated For C31H58O2,
462.6685). It gives positive test for unsaturation with bromine water. Its IR spectrum gives
absorption band of carbonyl moiety at 1751 cm-1and an olefinic group at 1648 cm-1. The position
of ester group was determined from its mass fragmentation pattern.
The Proton-NMR spectrum gives two triplet of methyl group sat δ 0.83 and 0.90 (triplet, each,
6H, J = 7.1 Hz) because of terminal methyl groups. Nineteen methylene protons were observed
at d 1.23 – 1.27 (38 H, br s). The further signals at δ 4.39 (triplet, J=7.5 Hz) corresponding to
methylene of ester group, another peak for two protons due to methylene linked were observed at
δ 4.1 (triplet, J= 7.0 Hz).It further showed two trans-olefinic bonds at d 5.23 (1H, dd, J = 14.9,
7.8 Hz); d 5.21 (1H, dt, J = 14.9, 7.8 Hz); d 5.18 (1H, dt, J = 15.1, 7.1 Hz); 5.05 (1H, dt, J =
15.1, 7.1 Hz). The methylene protons adjacent to olefinic group showed a doublet peak at δ 2.42
and d2.23.The 13C-NMR spectrum corroborated the presence of 31 carbon signals because of
two methyl carbons, 24 methylene carbons, 4 methine carbons and one quaternary carbon. The
117
two terminal methyl groups were observed at 10.9 and 18.5 ppm, respectively. All the values
were in complete agreement to those reported in literature for compound B (Chung et al., 2014).
4.5.3: Compound C (Nonacosyl hexadecanoate)
O
O
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13' 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
14'16'
15'
21
20
19
24 22
29 27 25 23
28 26
The molecular formula of compound C was assigned as C45H90O2 by EIMS, showing a M+ ion
peak at m/z662.3479 (calculated. for C45H90O2; 662.3477) implying saturated fatty ester which
was further confirmed by EIMS fragmentation pattern. Diagnostic fragments with the difference
of 28 or 14 amu were observed in the EI-MS of the compound. The IR spectrum of 3 showed the
absorption bands at 1652, 1615 and 1538 cm-1.
The Proton-NMR spectrum of C showed the presence of two terminal methyls resonating at d
0.87, 0.94 (6H, t, J = 6.8 Hz), 38methylenes at d 1.25 – 1.38 (76H, br s), another methylene
protons appeared at 1.69 (quintet). Its oxymethylene signal were appeared at d4.21 (1H, d, J =
8.7 Hz), and carbonyl methylene protons were resonated at d2.03(triplet, J= 7.2 Hz).The
methylene protons of the long chain hydrocarbon showed a broad signal at δ 1.24. The Carbon
NMR (BB and DEPT) spectrum of C corroborated the presence of two methyl carbon, 42
methylene carbons, and one quaternary carbon atom. The signal of oxymethylene protons were
at δ 69.0 and the other methylenes of hydrocarbon chain resonated at δ 31.6 – 30.1 while the
terminal methyl showed the signal at δ 11.4 and 14.4. All the physical and spectral data were
similar to the reported data; the compound was identified as nonacosyl hexadecanoate (C)
(James Devillers and Minh-Ha Pham-Delegue 2003).
118
4.5.4: Compound D (Heptacosanoic acid)
HO
O
1
3 5 7 9 11 13
14
15
1618
25
20
27
2226 24
12
Compound (D) was isolated as colourless crystalline solid. The molecular formula was deduced
from EI-MS which gave M+ ion peak at m/z 410 (calculated for C27H54O2, 410.3715). In EIMS
the loss of 14between a numbers of fragment ion peaks in its MS showed the presence of a long
aliphatic chain. The IR spectrum displayed the bands at 3322, 2688 and 1721cm-1 in the
molecule.
The 1H-NMR displayed signals for terminal methyl at δ 0.91 (3H, triplet, J = 6.5 Hz,Me-
27)while the rest of the twenty three methylenes appeared at δ 1.59-1.61 as a broad singlet. One
methylene group appeared at δ 1.98 as quintet (H-3). The methylene protons adjacent the
carboxylic moiety appeared as a triplet at δ 2.11 (2H, triplet, J= 7.3 Hz).The 13C-NMR displayed
signal for carbonyl of carboxylic moiety at δ 176.1. The terminal methyl appeared at δ 14.2
while the rest of the methylenes appeared at δ 29.6-29.9 as an envelope and the methylene
adjacent to the carbonyl appeared at δ 34.9. On the basis of these evidences and comparison with
literature, the compound was identified as Heptacosanoic acid (D) (Saini et al., 2009).
119
4.5.5: Compound E (1, 3, 5-Trihydroxy-2-hexadecanoylamino-(6E, 9E)-heptacosdiene)
OH
NH
O
OH
OH
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
15'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Compound E was obtained as gummy solid. The EIMS gives M+ ion peak at 663. It showed the
molecular formula C42H82NO4 by HR-MS, showing a [M+H]+ ion peak at m/z 664.6255
(calculated. for C42H82NO4;664.6251) indicating three degrees of unsaturation. The IR
absorption bands of compound revealed the presence of hydroxyl groups at 3340 and 3220, an
amide group at 1620 and 1540 cm-1 and an olefinic group at 1660 cm-1.
The 1H-NMR spectrum of E showed the presence of two terminal methyls at d 0.89 and 0.94
(6H, triplet, J = 6.8 Hz), nine methylenes at d 1.28 (18H, br s) and another nine methylenes at d
1.32 (18H, br s), and an amide proton signal at d 8.54 (1H, doublet, J = 8.9 Hz). The oxygenated
protons were observed at 3.94 (1H, m, H-5), 4.22 (1H, dd, J= 11.3, 4.9 Hz, H-1a), 3.65 (1H, dd,
J= 11.5, 5.0 Hz, H-1b), 3.59 (1H, m, H-3). The characteristic methylene protons were observed
at 3.41 (2H, m, H-8), 2.15 (2H, t, J = 7.0 Hz, H-2 ), 2.03 (2H, t, J = 7.0 Hz, H-4). It further
showed two trans-olefinic bonds at d4.87 (1H, dd, J = 15.5, 8.4 Hz); d4.91 (1H, dt, J = 15.5, 8.4
Hz); d 5.05 (1H, dt, J = 16.1, 6.9 Hz); 5.18 (1H, dt, J = 16.1, 6.9 Hz).
The Carbon-NMR spectrum (BB and DEPT) of compound E gives 42 peaks, corroborated the
presence of two methyl, thirty-two methylene, seven methine and one quaternary carbons. A
tertiary carbon at d 57.8 and quaternary carbon at d 169.4 supported the presence of a carbon
attached to the nitrogen and an amide carbonyl, respectively. Four methines carbons observed at
d 133.6, 132.4, 129.8 and 130.7 suggested the presence of two double bonds. All of the above
spectral information revealed that E was a 1, 3, 5-Trihydroxy-2-hexadecanoylamino-(6E, 9E)-
heptacosdiene (Mukhtar et al, 2002).
120
4.5.6: Compound F (2H-1-Benzopyran-2-one)
O O1
2
3
45
6
7
8
9
10
It was obtained as colourless crystalline solid. The IR spectrum absorption bands (1625 and 1722
cm-1) indicated the aromatic and lactone moiety in the molecule. Its EIMS gives M+ ion peak at
146. The HR-EI-MS of compound gave the M+ ion peak at m/z146.0541corresponding to the
molecular formula C9H6O2 (calculated for C9H6O2, 146.0539).
Its proton NMR spectrum gave characteristic signal in the aromatic region of O disubstituted
benzene ring. These peaks are at δ 7.68 (1H, d, J= 9.5Hz, H-4) and δ 6.41 (1H, d, J= 9.5Hz, H-
3). While the peaks at δ 7.52 (2H, m, H-6, H-7), δ 7.49 (1H, d, J= 8.7 Hz, H-8), δ 7.25 (1H, d, J=
8.7 Hz, H-5) indicated the coumarin skeleton of the molecule.
Its Carbon-NMR spectrum gives nine carbon signals out of which six methine signals were
observed at δ 143.5, 130.1, 127.1, 125.6, 116.0 and 114.9 while the quaternary signals were
observed at δ 160.1, 152.6 and 117.9 are of typical coumarin skeleton. On the basis of these data
and the compound was identified as 2H-1-benzopyran-2-one (F). This was further confirmed by
the comparison with the published data (Aldrich, 1992).
121
4.5.7: Compound G (Betulin)
HO
CH2OH1
2
34
56
7
8
9
10
11
12
13
14
1516
17
18
19 21
22
2324
26
27
28
25
29
30
20
H
H
H
H
Betulin (G) was isolated as colorless crystals. The EI-MS gives the M+ ion peak at m/z 442
corresponding to the M. F. C30H50O2 (calculated for C30H50O2; 442.3810). The daughter
fragments peaks in the EI-MS of 7 was characteristic of lupene type triterpene and exhibited
important peaks at m/z 442 [M] +, 424 [M-H2O] +, 234, 220 and 207 which are diagnostic for
pentacyclictriterpenes with an isopropenyl group (Budzikiewicz, et al 1963). The IR spectrum
showed absorption bands at 880, 1635, 3070 and 3435.
The proton NMR spectrum gives five tertiary methyl groups at δ 0.87, 0.89, 0.92, 0.98 and 1.02
(3H each, singlet), signals for an isopropylene function at δ 4.68 (2H, multiplet) and 1.68 (3H,
singlet). The carbinolic proton signal were observed at δ 3.75 (dd, J = 10.7, 4.2 Hz) indicating
the β and equatorial configuration of hydroxyl group at C-3. The Carbon-NMR (BB and DEPT)
spectra revealed the presence of six methyl carbon, twelve methylene carbon, six methane
carbon and six quaternary carbon atoms. The physical and spectral data of compound G were in
complete agreement to those published for betulin (Siddiqui et al, 1988)
122
4.5.8: Compound H (Stigmasterol)
HO
1
35
7
9
1113
15
17
18
19
20
21 22
2425
26
27
28
29
23
2
4
10
12
148
H
H
H
6
16
It was obtained as colorless needles from the chloroform soluble fraction. The EIMS gives M+
ion peak at m/z 412 (calculated. for C29H48O, 412.3919). Further daughter fragments were typical
of steroidal skeleton. The nature of oxygen in H was shown to be hydroxyl as indicated by IR
spectrum (3432 cm-1). The mass spectrum showed characteristic fragmentation pattern of Δ5, 22
sterol (Bernard and Tokes, 1977).
The proton NMR spectrum of compound corresponded to the data for stigmasterol. It displayed
signals for two tertiary methyl groups (3 H each, singlet, 0.84, 0.65), two multiplets for three
olefinic protons at δ 5.33 (1H) and 5.15 (2H) and a further peak for the carbinylic proton at δ
3.28 (1 H, m). The 13Carbon-NMR (BB and DEPT) spectra of compound H gives 29 peaks
consisting for six methyl carbon, nine methylene carbon, eleven methane carbon and three
quaternary carbon atoms. The above data was compared with the literature and showed complete
agreement to those of stigmasterol (Holland et al, 1978).
123
4.5.9: Compound I (3, 5-Dimethoxy 4-hydroxy cinnamic acid)
OH
H3CO OCH3
1
2
3
4
5
6
7
HO O
9
8
Compound I was obtained as colorless crystalline solid. The EI-MS of 7 exhibited M+ ion peak
at m/z 224, corresponding to the molecular formula C11H12O5 (calculated. for
C11H12O5;224.1241). The UV maxima in MeOH solvent were observed at 315, 235 and 201 nm.
Its IR spectrum showed hydroxyl and carbonyl bands. The carbon NMR spectrum (BB and
DEPT) showed the presence of eleven carbon signals, containing two methyl carbons, four
methine carbons and five quaternary carbon atoms. In the proton NMR spectrum, signals
corresponding to a 1, 3, 4, 5 tetrasubstituted benzene ring were present. In the 1H NMR spectrum
the H-2 and H-6 of the sinapoyl moiety, were observed at δ 6.77 as a singlet. Furthermore, the
spectrum showed the H-7 and H-8 Trans olefinic protons at δ 7.58 and 6.32 (1 H each, d, J = 16
Hz), 6.88 (2 H, singlet), and two methoxyl groups at δ 3.86 (6 H, singlet).The physical and
spectral data of 7 agreed to those previously reported in literature (Tesaki et al, 1998).
124
4.5.10: Compound J (4-Hydroxy-3, 5-dimethoxybenzoic acid)
COOH
OH
H3CO OCH3
1
2
3
4
5
6
7
Compound J was obtained as colourless crystalline solid. Its EI-MS gave the M+ ion peak at m/z
198corresponding to the molecular formula C9H10O5 (calculated for C9H10O5,198.0528).Its IR
spectra gives absorption bands at 3525 (O-H), 1711 (C=O) and 1615cm-1 (aromatic).
The 1H-NMR spectrum of J displayed a singlet of two protons in aromatic region at δ7.15 (2H,
singlet, H-2, H-6) and further singlet signals due to methoxyl groups atd3.86, 3.83(6H, singlet,
methoxyl-3, 5).The 13C-NMR (BB and DEPT) spectrum of J showed 9 signals out of which 2 for
methyl carbon, two for methine carbon and five for quaternary carbons. The downfield signals at
δ168, 149.1, 146.3, 140.7 and 121.4 were assigned to acid carbonyl and aromatic oxygenated
quaternary carbon atoms, whereas other signal in the aromatic region at δ112.4 and 56.7, 52.3
were assigned to aromatic methine and methoxy carbon atoms. On the basis of above evidences
and by comparison with the literature values (Aldrich, 1992), the compound was identified as 4-
hydroxy-3, 5-dimethoxybenzoic acid (J).
125
4.5.11: Compound K (5, 8-Dihydroxycoumarin)
O O
OH
12
3
45
6
7
8
9
10
OH
Compound K was obtained as colourless crystalline solid. The EI-MS showed the M+ ion peak at
m/z 178corresponding to the molecular formula C9H6O4 (calculated for C9H6O4, 178.0267).Its IR
spectrum showed the absorption bands at 3125, 1721, 1611, 1518 and 809cm-1 which indicated
that K is a coumarin type compound.
The 1H-NMR gave all peaks in the aromatic region, peaks at δ 6.75 (1H, d, J= 8.4 Hz, H-6),
δ6.85 (1H, d, J= 8.4 Hz, H-7), δ 7.61 (1H, d, J= 9.5 Hz, H-4), δ 6.10 (1H, d, J= 9.5 Hz, H-3),
confirming a coumarin type skeleton.
The 13C-NMR spectrum (BB and DEPT) gives 9 carbon peaks corroborated the presence of four
methane carbon and five quaternary carbons. Two downfield carbon peaks are because of
attachment of hydroxyl group. On the basis of these data and comparison with literature values,
compound K was identified as 5, 8-dihydroxycoumarin (Joseph-Nathan et al., 1984).
126
4.5.12: Compound L (Stigmasterol 3-O-β-D-glucoside)
OO
OH
OH
OH
OH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
2122
23
2425
26
27
29
28
1'
2'3'
4'
5'
6'H H
H
H
It was obtained as colorless amorphous solid. The EIMS gives [M-Glc] + ion peak at atm/z 412.
The EIMS fragments peaks showed characteristic pattern of Δ5, Δ22 sterols. Its M. F. was
established as C35H58O6 by HR-EI-MS that showed molecular ion peak at m/z 574.4231
(calculated. for C35H58O6; 574.4233). The IR spectrum gives absorption bands because of the
presence of hydroxyl groups at 3432 cm-1.
The 1H-NMR of compound L completely corresponded to the data for compound H except
additional resonances at δ 5.23 (1H, d, J = 5.4 Hz) confirming its β configurations and signals at
δ 3.84-4.44 corresponding to the sugar moiety. The 13Carbon-NMR spectrum gives 35 carbon
signals having same as those for compound H except additional peaks for sugar moieties. All
values were also in totally agreement with the stigmasterol except additional peaks for sugar
moiety. On the basis of above evidence and mixed m.p. with an authentic sample, the structure of
compound L was established as stigmasterol 3-O-β-D-glucoside (Holland et al, 1978)
127
4.5.13. Compound M (Sparsifol)
2
1
34
56
O
CH3
OHOH
HO
OH
OHH
H
H
H
H
H
Compound M was obtained as white crystals, melting at 186-187 oC. Its IR spectrum showed the
absorption bands hydroxyl group3438, (H-C) 2941 and (O-C) 1275 cm-1. The EI-MS showed the
M+ peak at m/z194which is consistent with a molecular formula C7H14O6 (calculated. for
C7H14O6; 194.1211).
The broad band 13C NMR and DEPT spectra of M showed seven peaks consisting of six methine
carbon and one methyl carbon atoms. The entire carbon atoms signal observed downfield shifts
due to their attachment to oxygen atom. The 1H NMR spectrum showed methoxyl protons as
singlet at δ 3.29 (3H, s) and six oxymethine protons in the range of δ 3.48 to 4.09. Since the
molecular formula showed the presence of 1 double bond equivalent therefore compound M
must be mono cyclic. On the basis of these evidences and comparison with literature, the
compound was identified as Sparsifol M (Mehmood and Malik, 2011).
128
4.5.14: Compound N
(6-O-β-D-Glucopyranosyl-β-D-(1-O-sinapoyl,6'-O-sinapoyl)-glucopyranose)
OH
H3CO OCH3
OO1'
2'3'
4'5'
6' OO
HOHO
HO
H
1''2''
3''
4''5''
6''
OO
HOHOHO
H
O
HO
OCH3
H3CO
1'''
2'''
9'''
8''' 7'''
6'''
5'''
4'''
3'''
9''
8''7''
4
3
5
6
2
1
Compound N was obtained as yellowish oil. EI-MS gives M+ ion peak at 754. Its molecular
formula of C34H42O19 was derived from HR-EI-MS (calculated. for C34H42O19; 754.5218). UV
maxima in MeOH were observed at 328 (4.51), 240 (4.44) and 202 (4.52) nm. The infrared (IR)
spectrum of compound showed the presence of hydroxyl group, ester, double bond, and aromatic
ring. The proton NMR spectrum of N showed the signals for four aromatic protons at δ 6.92 and
6.91 (two each, singlet), two sets of trans-olefinic protons at δ 7.71 and 6.54 (both H, doublet, J
= 16.0 Hz); 7.66 and 6.44 (both H, doublet, J = 16.0 Hz), and one singlet for four methoxy
groups at δ 3.88. These data indicate compound N contains two sinapoyl moieties. The
remaining parts of 1 H NMR showed two anomeric protons (δ 5.75 and 4.36, each 1H, doublet, J
= 8.8, 8.0 Hz, respectively), one proton at δ 5.22, and overlapped eleven protons in the range of δ
3.23-4.21.
The carbon NMR (BB and DEPT) showed 34 carbon signals having 12 oxygenated carbon
signals (showing two sugar moieties). The 13C-NMR signals of sugar moiety corresponded to -
D-glucopyranoside. The sugar unit was assigned as -D-glucose by comparing the NMR
129
chemical shift values with the reported data. The β-configuration of glucose moiety was assigned
on the basis of larger coupling constant of the anomeric proton (J = 7.2 Hz). After hydrolysis
provides the glucose and was further confirmed by the co-TLC with the authentic sample. Thus,
it was suggested that the compound was an ester of trans-sinapic acid with two glucose units.
The structure of compound N was determined to be 6-O-β-D-glucopyranosyl- β-D-(1-O-
sinapoyl, 6'-O-sinapoyl) glucopyranose (Rahman and Moon, 2007).
4.6. Biological activity of isolated compounds
Compounds isolated from dichloromethane and methanol extracts of were tested for α-
Glucosidase inhibition assay and butyrylcholinesterase inhibition assay respectively. The results
of in vitro bioassays performed are being presented below in Tables 4.12 and 4.13.
Table 4.12: Results of α-Glucosidase inhibition assay of compounds (A-I) isolated from
dichloromethane extracts of Croton bonplandianum.
a = standard
CompoundInhibition %
IC50 ( μg/ml)250 (μg/ml) 100 (μg/ml) 50 (μg/ml) 25 (μg/ml) 10 (μg/ml)
A – – – – – >250
B – – – – – >250
C 59.8 ± 1.2 21.2 ± 2.2 9.8 ± 1.4 3.2 ± 1.7 – 214.5
D 81.7 ± 2.4 50.5 ± 1.6 31.0 ± 1.1 6.1 ± 1.2 – 94.7
E – – – – – >250
F 92.5 ± 2.6 87.8 ± 1.4 70.4 ± 2.0 53.2 ± 1.8 28.4± 1.2 25.9
G 95.2 ± 4.2 89.9 ± 3.2 72.9 ± 1.4 51.0 ± 1.4 27.3 ± 1.4 23.0
H 96.4 ± 2.5 65.2 ± 1.9 38.2 ± 1.4 4.5 ± 1.2 – – 72.8
I 92.1 ± 5.6 77.5 ± 1.7 65.4 ± 1.2 46.5 ± 1.3 29.7 ± 2.0 26.7
Acarbose a 92.23±0.14 81.39±0.23 71.09±0.56 57.42±0.44 48.02±0.24 38.25
130
Table 4.13: Results of butyrylcholinesterase inhibition assay of compounds (J-N) isolated from
methanol extracts of Croton bonplandianum.
b = Eserene
CompoundInhibition %
IC50 ( μM)250 ( μM) 100 ( μM) 50 ( μM) 25 ( μM) 10 ( μM)
J 80.7 ± 2.4 60.5 ± 1.6 43.0 ± 1.1 20.1 ± 1.2 – 36.0
K 89.5 ± 2.6 78.8 ± 1.4 60.4 ± 2.0 51.2 ± 1.8 39.4± 1.2 25.0
L 95.2 ± 4.2 89.9 ± 3.2 78.9 ± 1.4 61.0 ± 1.4 49.3 ± 1.4 27.0
M 85.2 ± 4.2 79.9 ± 3.2 62.9 ± 1.4 41.0 ± 1.4 29.3 ± 1.4 82.0
N 96.4 ± 2.5 85.2 ± 1.9 78.2 ± 1.4 68.5 ± 1.2 59.7± 1.3 21.0
Eserene b 93.1 ± 5.6 82.39±0.23 73.09±0.56 59.42±0.44 50.02±0.24 32.0
131
5 Discussion
The research was focused on the phytochemical and biological evaluation of Croton
bonplandianum (Euphorbiaceae). Preliminary phytochemical screening revealed the presence of
alkaloids, saponins, flavonoids, tannins and terpenoids. Dichloromethane and methanol extracts
of whole plant were examined for biological activities such as antibacterial, antifungal,
cytotoxicity, phytotoxicity, antioxidant, α-Chymotrypsin inhibition, urease inhibition, α-
Glucosidase inhibition and butyrylcholinesterase inhibition.
α-glucosidase inhibition activity of the plant extracts was performed in vitro. Dichloromethane
extract exhibited promising activity of 97.89 % with IC50 of 14.93 µg/ml, compared to the
standard acarbose which revealed 92.23 % inhibition with IC50 of 38.25 µg/ml. Diabetes is one
of the world's greatest health problem, affecting about 171 million people and most of these will
be dominated by those suffering from type II diabetes (Gershell, 2005). This increasing trend in
type II diabetes mellitus has become a serious medical concern worldwide, which accounts for 9
% of deaths that prompts every effort in exploring for new therapeutic agents to stem its
progress. Although the drug treatment for type II diabetes mellitus has been improved to some
extent during the last decade drug resistance is still a big concern that needs to be dealt with
effective approaches. One of the strategies to monitor blood glucose for type II diabetes mellitus
is to either inhibit or reduce the production of glucose from the small intestine. Diet rich in
carbohydrate causes sharp rise in the blood glucose level as the complex carbohydrates in the
food is rapidly absorbed in the intestine aided by the α-glucosidase enzyme which breaks
disaccharides into absorbable monosaccharides. α-glucosidase inhibitor prevents the disaccharide
digestion and impedes the postprandial glucose excursion to enable overall smooth glucose
metabolism (Casirola and Ferraris, 2006). Searching of new α-glucosidase inhibitors, thereby
motivating to explore new therapeutic agent for the treatment of type II diabetes.
Considering these valuable facts about the therapeutic potential of croton bonplandianum the
isolation of different constituents from dichloromethane extract was carried out which afforded
nine compounds. Among the isolated compounds, compounds coumarin (F), betulin (G), and
3,5-dimethoxy 4-hydroxy cinnamic acid (I) possessed significant α-glucosidase inhibition
activity in a concentration dependent manner and showed potent inhibitory activity with IC50
132
values ranging from 23.0 to 26.7 µg/ml, than that of a positive control acarbose (IC50 38.2
µg/ml).
Butyrylcholinesterase inhibition activity of methanol extract of croton bonplandianum was
carried out and it exhibited inhibitory activity of 84.14 % with IC50 found to be 31.01 µg/ml,
compared to the standard eserine which exhibited 82.82 % inhibition with IC50 found to be 30.01
µg/ml. Medicinal plants having therapeutic potential for the treatment of neurodegenerative
diseases like alzhemer disease, Epilepsy and Parkinsonism have been extensively explored, still
there is a continuous search for new drugs like galanthamine (Heinrich and Teoh, 2004;
Ngkaninan et al., 2003). Recent studies showed that the main cause of the loss of cognitive
functions in AD patients was a continuous decline of the cholinergic neurotransmission in
cortical and other regions of the human brain (Schuster et al., 2010). Acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) are hydrolytic enzymes that act on acetylcholine
(ACh) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline
and acetate. Both enzymes are present in the brain and detected in neurofibrillary tangles and
neuritic plaques. Acetylcholinesterase predominates in the healthy brain, with
butyrylcholinesterase considered to play a minor role in regulating brain ACh levels. However,
BChE activity progressively increases in patients with Alzheimer’s disease, while AChE activity
remains unchanged or declines. Both enzymes therefore represent legitimate therapeutic targets
for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive,
behavioral, and global functioning characteristics of Alzheimer’s disease (Greig et al., 2002). In
our efforts to find phytochemical agents that could be effective in the prevention and
management of neurodegenerative conditions, activity guided isolation of compounds from
methanol extracts of Croton bonplandianum was done. Among the isolated compounds,
compounds 4-hydroxy-3,5-dimethoxybenzoic acid (J), 5,8-dihydroxycoumarin (K), stigmasterol
3-O- β -D-glucoside (L) and 6-O-β-D-Glucopyranosyl-β-D-(1-O-sinapoyl,6'-O-sinapoyl)-
glucopyranose (N) possessed significant butyrylcholinesterase inhibitory activity in a
concentration dependent manner, and showed potent inhibition activity with IC50 values ranging
from 21.0 to 36.0 µg/ml than that of positive control eserine (IC50, 32.0µg/ml).
The methanol extract of Croton bonplandianum was found toxic with LD50 value of 115.76
(0.0048 - 13.76) µg/ml against Artemia salina when tested in vitro, pointed to a possibility that
133
the extract may contain a toxic compounds. Bioactive compounds were often toxic to shrimp
larvae therefore lethality to shrimp larvae can be used as a rapid and simple preliminary monitor
for plant extract lethality which in most cases correlates reasonably well with cytotoxicity and
antitumour properties (McLaughlin, 1991). Methanol extracts of the whole plant Croton
bonplandianum showed considerable antioxidant activity when analyzed by DPPH free radical
scavenging assay and had radical scavenging activity (RSA) of 59.62% with IC50 value of
396.205 µg/ml. Antioxidants are responsible for various mechanisms including prevention of
chain initiation, decomposition of peroxides, radical scavenging and reducing capacity (Cook
and Samman, 1996). These free radicals may oxidize nucleic acids, proteins, lipids and can
initiate degenerative diseases. The presence of flavonoids and tannins in all the plants is likely to
be responsible for the free radical scavenging effects observed. Flavonoids and tannins are
phenolic compounds and plant phenolics are a major group of compounds that act as primary
antioxidants or free radical scavengers (Potterat, 1997). It has been displayed that compounds A,
B, C, D, K and N were isolated for the first time in the family (Euphorbiaceae) and compounds
E, F, G, H, I, J and L were isolated for the first time from Croton bonplandianum.
The results revealed the presence of medicinally important constituents in the Croton
bonplandianum. Biological studies confirmed the presence of these phytochemicals contribute
medicinal as well as physiological properties to the Croton bonplandianum. Therefore, extracts
and isolated compounds from Croton bonplandianum could be seen as a good source for useful
drugs. The traditional medicine practice is recommended strongly for Croton bonplandianum. It
is hoped that the strong knowledge of natural products coupled with combinatorial sciences and
high-throughput screening techniques will improve the ease with which natural products and
formulations can be used in drug discovery campaigns and development process, thereby
providing new functional leads for various diseases.
134
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