Phytochemical and biological studies of bonplandianum ()

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

1 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

2 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.

3 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

4 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 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

5 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).

6 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 Croton 5 1.2.3 Croton bonplandianum 5 1.3 Aims and objective 8

2 Literature review 9 2.1 Ethnomedicinal uses of Croton 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

7 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

8 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

9 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

10 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

11 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

12 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

13 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

14 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 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

15 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

16 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 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

17 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, 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 fi 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 strictly 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 . However, some species have separate male and female plants and some species may produce a mixture of male, female and

18 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 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.

19 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.

20 Figure 1.2: Croton bonplandianum

21 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.

22 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 Plant part Condition managed Reference (Region) C. alienus Pax Unspecified Body weaknesses Gachathi, 2007 (Kenya) C. antanosiensis Stem bark Leafy Induce virility during Schmelzer and Leandri) branches circumcision ceremonies, Gurib-Fakim, 2008 (Madagascar) Ordeal poison in ancient times Fumigate houses in case of epidemic diseases C. antisiphiliticus Entire plant Stimulant, Wound Elisabetsky et al., (Brazil) healing, Veneral diseases, 1992 Rheumatic fever C. arboreous Aerial parts Auxiliary anti- Aguilar- Millsp. (Cascarillo inflammatory in guadarrama and Mexico) respiratory ailments Rios, 2004 C. argyratus Dried flowers Purgative Ilham et al., 1995 (Malaysia) Schmelzer and Gurib-Fakim, 2008

23 C. barorum Leandri A decoction of Malarial fever, Cough, Rakotonandrasana (Madagascar) stem and root Diarrhea, Leukaemia and et al., 2010 barks Aromatic Breast Cancer Insect Schmelzer and leafy branches Repellent (lice) and Gurib-Fakim, 2008 Perfumery in soap C. bonplandianus Entire plant Antiseptic Bandoni et al., Baill (Argentina 1976 although it has gotten its way into Kenya where it is found as a common weed) C. cajucara Benth. Stem bark and Diabetes, Diarrhea, Duke, 1984; Duke, (Sacaca, Peru and Leaves (in form Malaria, High Blood 1994; Campos et Brazil) of tea or pills) Cholesterol Levels, al.,2002. Gastrointestinal Grassi-Kassisse et disturbances, Hepatic al., 2003 disturbances, weight loss C. californicus Leaves Rheumatism, Malaria, Williams et al., Mueller Arg. Pain reliever 2001, Chavez et (California, U.S.A.) al., 1982, Wilson et al., 1976; Farnsworth et al., 1969 C. capitatus Mitchx Unspecified Malaria Farnsworth et al., 1969 C. caudatus Stem bark Stomach disorders, Banerji et al., 1988 (Indonesia, India) Malaria C. celtidifolius Stem bark and Inflammatory diseases, Nardi et al., 2003 Baill. (“Sangue-de- infusions Leukemia, Ulcers and adave”, Brazil) Rheumatism

24 C.ciliatoglandulifer Entire plant Purgative Farnsworth et al., (Syn. C. ciliato- 1969. glandulosus, Mexico) C. cortesianus Aerial parts Veneral diseases and Dominguez and (Mexico) Wound healing Alcorn, 1985 C. corymbulosus Aerial parts Purgative Coon, 1974 (U.S.A) C. decaryi Leandri Leafy branches Mattress filler to Repel Schmelzer and (Madagascar) Decoction from Lice Calm patients Gurib-Fakim, 2008 aerial parts suffering from Paranoid Psychosis C. dichogamus Pax Leaves, Roots Fever, Chest ailments, Kokwaro, 1993 (Kenya, Uganda, Whole plant Stomach diseases, and 2009 Tanzania, Rwanda decoction Tuberculosis, Impotence Jeruto et al., 2011 and Ethiopia) Malaria C. draco , bearing Aerial parts Fever, Tumors, Bleeding, Murillo et al., a red sap widely Cough, Flu, Diarrhoea 2001 used in traditional and Stomach ulcers, medicine in Topically as wound Mexico and healing for cuts, open Central America) sores, herpes, Anti-septic after tooth extraction and Oral sores C. draconoides Latex Cancer, Wounds, Piacente et al., (Peru) Inflammation 1998 C. eluteria Bennett Stem bark (used Dysentry, Dyspepsia, Duke, 1984, Vigor (“Cascarilla”, Syn. as substitute for Malaria, Fever, et al., 2001 C. eluteria (L.) Chinchona and Bronchitis, Tonic and Wright, West Indies Cascara, Bitters, Flavoring for and Northern South liqueurs and Scenting

25 America-Bahama tobacco Island) C. flavens Leaves Rheumatism, Fever, Flores and Curacao, Venezuela Menstrual Pains Ricalde, 1996. C. fragilis Entire plant Stomach-aches, Hepatic Hecker, 1984 Mexico pains C. geayi Infusion of its Fevers, Coughs, Asthma Schmelzer and Madagascar Leafy twigs and Constipation in new- Gurib-Fakim, born babies 2008; Palazzino et al., 1997. C.glabellus Leaves Ulcers Flores and Mexico Ricalde, 1996 C.glandulosus Entire plant Stomach-aches Heinrich et al., Mexico 1992 C.goudotii Leaves Stem bark Chronic blennorrhea, Rakotonandrasana Madagascar Cough and an Aphrodisiac et al., 2010 Malaria, Chronic gonorrhea C. gratissimus Leaves Rheumatism, , Farnsworth et al., Dropsy, Fever, Bleeding 1969 gum, Perfume Carthatic, Eruptive irritant, Respiratory condition, Intercostals neuralgia, Dropsy, C.antunesii Stem bark Indigestion, Pleurisy, Watt and Western and Uterus disorder, Fish Breyer-Brandwijk, Southern Regions poison 1962. of Africa Farnsworth et al., 1969.

26 C. gubouga S. Seed and stem Emesis, Pugartive, Watt and Breyer- Moore South bark Febrifuge, Fish poison, Brandwijk,1962; Africa, Tanzania, Laxative, Malaria Neuwinger, 1996, Botswana, Caprivi 2000 and 2004. strip, Malawi, Zambia and Zimbabwe. C. guatemalensis Stem bark and Malaria Franssen et al., (Guatemala) Leaves 1997 C. haumanianus Stem bark, Blennoragy, Gastric Tchissambou et Congo Leaves diseases, Hypertension, al., 1990 Epilepsy C.hovarum Stem bark – Fish poison Molluscicidal Krebs and Madagascar Aerial parts Colic and Acute Body Ramiarantosa, Leaves Weakness 1996 Schmelzer and Gurib-Fakim, 2008 Krebs and Ramiarantsoa, 1997

C. humilis Entire plant Insecticide Asprey and Jamaica Thornton, 1955 C.insularis Entire plant Abortifacient Rageau, 1973 Caledonia,Australia C.jatrophoides Roots Colds, Intestinal worms Schmelzer and Tanzania and Stomachache Gurib-Fakim, 2008;Kokwaro, 2009 C.joufra; Stem bark Blood purification Anti- Mokkhasmit et al., Thailand Decoction of dysentery and Peptic 1971;

27 Leaves and Stem promoter Anthelmintic Sutthivaiyakit et bark Decoction al., 2001. of the flowers C.kongensis; Entire plant Sores Pei, 1985 Thailand; China C. lechleri Latex from stem Wound healing, Cancer, Duke, 1994 Ecuador and Peru; bark Stomach ulcers, Cai et al., 1993a, b Rheumatism and 1991) C. lobatus Leaves, Leaves Malaria, Pregnancy Neuwinger, 1996, Senegal, Eritrea combined with troubles, Dysentery, 2000and 2004; and Ethiopia; seeds and bark Rheumatic pain Attioua et al.,2007 Carribean, South Whooping Cough, Schmelzer and America and The Convulsions, Mouth Gurib-Fakim, Arabian Peninsula) infections Eye diseases, 2008 un consciousness Lotion Neuwinger, 1996, for female sterility 2000 and 2004. Purgative Anti- hypertensive medication

C.longiracemosus Roots Antheimintic,,Anti- Akendengue and Gabon inflammatory Louis,1994 C.macrostachys Entire plant and Malaria, Dysentry, Kew, 2012 and Madagascar, Seeds Decoctions Rheumatism, Taenacide, 2013 Schmelzer Somali, Sudan, Venereal diseases, and Gurib-Fakim, Eritrea, East Africa, Conjuctivitis, Purgative, 2008; Klauss and Angola Guinea, blood clotting, mumphs, Adala, 1994; Liberia, Malawi, skin rashes Anthelmintic, Mazzanti et al., Zambia and vermifuge, Female 1987 Zimbabwe infertility, Constipation, Stomach pains, Chest

28 pains, Bloat, wound healing, Diabetics C.malabaricus Fresh shoots Joint Pains, Rheumatic Pushpangadan and (India) Arthritis Atal, 1984 C. malambo Stem bark Diabetes, Diarrhoea, Suárez et al., 2003 Venezuela and infusion Rheumatism, Gastric Colombia Ulcer, Anti- Inflammatory, Analgesic C. mayumbensis J. Stem bark and Microbial Infections, Yamale et al., Leonard Gabon, Leaves Human Parasitic Diseases 2009 Cameroon and The such as Amoebiasis Central African Republic C.mauritianus Entire plant Fever Vera et al., 1990 Reunion Island

C.megalobotrys Stem bark, Roots, Purgative, Malaria, Nyazema, 1984 Zimbabwe Seeds Abortion, Tape worms C.megalocarpus Entire plant Stem Gall bladder problems, John et al., 1994, Kenya Eastwards bark Decoction Chest pains, Internal Kokwaro, 2009, to The Democratic Root decoction swellings, Malaria Kew, 2012 and Republic of Congo Sap issuing from Anthelmintic, Whooping 2013. and Southwards to its leaves Cough Pneumonia Mozambique. \ Bleeding Wounds C. membranaceus Root and Leaf Aromatize tobacco Asare et al., 2011; Mull Arg.( West extracts Essential (Bahamas), Improve Adesogan, 1981 Africa) oils from the Digestion (Nigeria), Stem bark Benign Prostate Hyperplasia and Measles (Ghana) Aromatherapy to

29 treat cough, Fever, Flatulence, Diarrhoea and Nausea C. menyhartii Roots Malaria, Dymenorrhea, Kokwaro, 1993 Eastern Africa, Intestinal obstruction, and 2009 Somalia Influenza C.mongue Stems and seeds Toxic Match Ralison et al., Madagarscar Stem manufacturing 1986

C. mubango Entire Plant Female sterility, Spiritual Watt and Breyer- Congo, Ivory madness, Asthma, Brandwijk, 1962; Coast, Angola Paralysis, Hepatalgia, Bossard et al., Sleeping Sickness, 1993; Bouquet Diarrhea, Furgative, and Debray, 1974; Vermifuge Otshudi et al., 2000 C.mucronifolius Leaves Syphilis, Rheumatism, Lemos et al., 1992 Brazil Influenza C.nepetaefolius. Infusions or Antispasmodic properties, Santos et al., 2008 Brazil decoctions of the Relieve flatulence, stem bark and Increase appetite, Sedative leaves C.oblongifolus Entire plant and Sores, Ringworm, Pei, 1985., Sommit Chucka; India, seeds Migraine, Leprosy, et al., 2003; Thailand and Dysentery, Diarrhea, Ngamrojnavanich China. Purgative, Insecticide, et al., 2003 Blood Purification, Anti- Pyretic, Gastric Ulcers, Liver enlargement and remittent fever, Hepatitis

30 C. onacrostachyus Entire Psychotherapeutic effect Kokwaro, 2009 Kenya on muphs- “ngumbu” C. palanostigma Stem bark latex, Boils and sores, Uterine Lahlou et al., 2000 Peru Leaves, ulcers, Wounds, Snake bites, Gastro- intestinal cancer

C. penduliflorus Roots, Seeds, Purgative, Stomach- Anika and Shetty, Sierra Leone Stem bark Leaf aches, Labor pains, 1983 Eastwards to infusion Seed Headaches, Impotence Nigeria , Central extract Menstrual disorders, Adesogan, 1981 African Republic Fever Uterine tumors and and Gabon. Stomach complaints Schmelzer and Gurib-Fakim, 2008

C.polytrichus Roots Headache and labour Kokwaro, 2009 Kenya pains C. Leaves Roots Anthrax, Insecticide Hedberg et al., pseudopulchellus Stem Syphilitic ulcers, Chest 1983 Mali, Nigeria, infections, Tuberculosis Langat et al., Somalia, Kenya, Asthma, Colds, Viral and 2012 Ethiopia, Angola, Tissue infections Zimbabwe, Condiment, Burnt and Mozambique and smoke used to flavor fresh South Africa milk C.regelianus; Leaf Infusion Rheumatism, Malignant Torres et al., 2010 Brazil tumors, Stomach aches C. repens Entire plant Dysentery, Diarrhea Heinrich et al., Mexico 1992

31 C. roxburghii Entire plant Antivenin, Clear bowels, Selvanayahgam et India Malaria, Cardiotonic al., 1994 C. ruizianus Leaves Anti-spasmodic, Piacente et al., Peru Vulnerary 1988

C. sakamaliensis Stem bark Diarrhea, Cough, Fever, Radulovic et al., (Madagascar) infusion Purgative 2006 C. salutaris Leaves Fever Brandao et al., Peru 1985 C.schefleri Roots Insanity, Remedy for Watt and (Tanzania) miscarriage Breyer-Brandwijk , 1962; Mathias, 1982. C.soliman Latex Skin infections, Warts Zamora-martinez (Mexico) and Pola, 1992 C. steenkampianus Fresh leaves Relieve body pains Schmelzer and Tanzania, Vapor inhalation Gurib-Fakim, Mozambique and 2008; Adelekan et Southern Africa al., 2008 C. sublyratus Its mixture with Gastric ulcers and gastric Kawai et al., 2005 South-Eastern C. oblongifolius cancer Anthelmintic and Vongchareonsathit Asian Countries Stem bark dermatological problems and De-Eknamkul, and Thailand. 1998; Ogiso et al., 1981. C.sylvaticus; Stem bark Roots Abdominal disorders, Venter and Venter, Distributed from Unspecified Tuberculosis, Chest 1996; Mc Gaw et Ethiopia in the Leaves Decoction pains, Rheumatism, Fish al., 2000. Northern parts of Leaves infusion poison Gall sickness in Kokwaro, 2009 Africa to the cattle , Indigestion, Watt and Breyer- Eastern Cape in Pleurisy, Poultices for Brandwijk, 1962; South Africa, swellings/wash for body Neuwinger, 1996,

32 more widely found swellings caused by 2000 and 2004. in Gabon to kwashiokor , Malaria and Kokwaro, 2009 Angola. Purgative Beentje, 1994 Venter and Venter, 1996. C. tiglium L. Fruits, Roots Fish poison, Abortifacient, Gimlette, 1929; Asia Tumors, Laxative, Gout, Chang et al., 1981. Contraceptive, Insecticide, Cancerous sores, Purgative C. tonkinensis Leaves Digestive disorders, Giang et al., 2003; Gagnep Kho sam Abdominal pains, Minh et al., 2003 Bac Bo; A Dyspepsia, abscesses, Vietnam) Impetigo, Gastric and duodenal ulcers, Malaria, Urticaria, Leprosy, Psoriasis, Genital organ prolapse C. trinitatis Entire plant Cough, Bleeding gum, Duke, 1994; Kuo (Nicaragua) Influenza et al., 2007. C. urucarana Baill. Red latex of stem Cancer, Diarrhea, Perez and Anesini, (Syn. C. ururucana bark Respiratory and Urinary 1994; Perez et al., Baill.; Brazil and tract infection, Wound 1997 and 1998. Argentina) healing, Rheumatism C. zambesicus Roots/ Leave Menstrual pains El-hamidi, 1970; Muell. Arg. Aperient, Anti-malarial, Mohamed et al., (Syn.C. amabilis Anti-Diabetic Wash for 2009; Ngadjui et Muell.Arg.; fevers Dysentery and al., 1999; Baccelli Originally a Convulsions et al., 2007; Guineo-Congolese Hypertension and Okokon et al., species but now Urinary infections 2005 and 2013,

33 Widespread in (Benin), Anti- microbial, Watt and Breyer- Tropical Africa) Fever associated with Brandwijk, 1962. malaria Body strengthening medicine C. zehntneri Pax. Leaves and Stem Seizures, Insomnia, Coelho-de-souza et bark Anxiety, Sedative, et al., Hoffm.(Canelade- Appetite stimulating, 1997and1998; cunhã; Brazil) Gastro-intestinal Batatinha et al., disturbances, Food and 1995. drinks sweetener

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)

34 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).

35 36 37 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

38 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).

39 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).

40 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-

41 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).

42 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).

43 44 2.2.3: Terpenoids 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

45 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).

46 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

47 (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)

48 49 50 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.

51 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).

52 53 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).

54 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).

55 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 -

56 203, 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).

57 58 59 60 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).

61 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-

62 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).

63 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.

64 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) .

65 66 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).

67 68 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

69 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

70 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).

71 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

72 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

73 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

74 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).

75 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

76 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).

77 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.

78 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.

79 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.

80 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).

81 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.

82 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

83 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

97.5:2.5 Choloroform:Methanol 95:5 89:11

80:20 n-hexane:Ethyl acetate 75:25 50:50 30:70

90:10 n-hexane: Isopropanol 80:20

80:20 n-hexane: Methanol 90:10

90:10 Ethyl acetate:Chloroform 80:20

90:10 Ethyl acetate:Methanol 80:20

Chloroform:Methanol:Water 80:18:2

Ethyl acetate:Methanol:Water 93:5:3

84 Table 3.2: Solvent systems used for the analysis of methanol extracts of Croton bonplandianum

Solvent System Ratio 85:15:01

Chloroform:Methanol:Water 80:20:02

70:30:04 85:15:01

Ethyl acetate-Methanol:water 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.

85 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

-1 -1 IR (KBr) nmax cm :3487, 1751, 1618, 752 and 715 cm

1 H-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).

13 C-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).

86 3.9.2: Compound B State: Amorphous solid

M.P.: 91-92 °C

IR; 1751and 1648 cm-1

1 H-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)

13 C-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

87 IR; 1652, 1615 and 1538 cm-1

1 H-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)

13 C-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

-1 IR (KBr) nmax cm : 3322, 2688 and 1721

1 H-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)

88 13 C-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

25 [a]D : – 26.2∞ (c 0.10, pyridine)

IR (KBr) 3340, 3220, 1660, 1620 and 1540 cm-1

1 H-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).

13 C-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

89 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) -1 nmax cm :1725, 1623, 1589, 1512

1 H-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)

13 C-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)

90 3.9.7: Compound G State: Crystallized from MeOH

M.P.: 251-252°C.

25 [α]D : + 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).

1 H-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).

13 C-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

91 25 [α]D ; -51.5˚ (c = 0.28, CHCl3)

IR (CHCl3) -1 nmax cm : 3432 (OH), 1648 (C = C)

1 H-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).

13 C-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;

92 1 IR (KBr)υmax cm- : 3363, 1704, 1663, 1604, 1449 cm−1

1 H-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) 13 C-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) -1 nmaxcm :3510-3320 (O-H), 1708 (C=O), 1626, 1585 (aromatic)

1 H-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)

13 C-NMR (CD3OD, 100 MHz) δ;

93 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) -1 nmax cm :3108, 1713, 1607, 1595, 1525, 1503

1 H-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)

13 C-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)

94 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.

25 [α]D :-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).

1 H-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).

13 C-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

95 3.9.13: Compound M Physical Data; State; White crystals (MeOH)

M.P; 186-187 oC

24 [α]D : - 76.2 (c = 0.18, H2O)

IR (KBr) -1 -1 υmax cm :3438, 2941 and 1275cm

1 H 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).

13 C 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

24 [α]D :+1.41 (c 0.92, CH3OH);

UVλmax MeOHnm (log ε): 328 (4.51), 240 (4.44), 202 (4.52) nm;

96 IR; 3363, 2940, 1704, 1633, 1604, 1515, 1456, 1427, 1339, 1285, 1226, 1156, 1115, 910, 828, 766 cm−1

1 H-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')

13 C-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

97 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

98 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

99 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

100 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

101 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)

102 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 s n s s s i s s d d s e e c i u i d n i n a d d

i o

Part o q i i i i o e n n s s a n l n d

Plant name n e o r o o o a r n o p c c p h v a k

used a t l r y y a a l l C l T e n S A g g F T A Croton Whole + - - + + + + bonplandianum 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 Extract Part Plant Name Solvent Used (ml) obtained Extract codes Used Weight (g) (g) Dichloromethane 20.2 CBD Croton Whole 2000 bonplandianum plant 1000 Methanol 48.9 CBM 2000

103 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 . Zone of inhibition of sample Name of bacteria (mm) Zone of inhibition of standard CBD CBM drug (mm) 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).

104 Table 4.4: Results of antifungal bioassay of methanol and dichloromethane extracts of Croton bonplandianum. Linear Growth (mm) of MIC Name of fungi Extracts and control, %Inhibition Standard (µg/ml) CBD CBM CONTROL Candida albicans 100 100 0 Miconazole 110.8 100 Amphotericin Aspergillus flavus 100 100 0 20.20 100 B 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 Conc. of No. of Fronds Conc. of Plant % Growth Extract Compound Standard Drug Name Sample Control Regulation (µg/ml) (µg/ml) 1000 05 75 CBM 100 19 20 05 Lemna 10 19 05 0.015 minor 1000 05 60 CBD 100 20 20 0 10 20 0

105 Table 4.6: Results of Brine Shrimp Lethality bioassay of methanol and dichloromethane extracts of Croton bonplandianum. LD 50 LD 50 Extract Dose No .of No. of µg/ml STD. Drug µg/ml Code µg/ml shrimp survivors

1000 30 04

100 30 20 115.76 Etoposide 7.4625 CBM 10 30 23

1000 30 16

100 30 19 1327.85 Etoposide 7.4625 CBD 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.

Conc. µg/ml % inhibition IC50± SEM.µM Extract code CBM 500µg/ml 1.4±2.6% _ CBD 500µg/ml 3.17±2.1% _ STD Chymostatin _ _ 5.97±0.76 µM

106 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 Thiourea 0.5 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

500 97.89 ±2.6 250 89.58 ±1.6 125 79.34 ±1.5 62.05 68.23 ±2.2. CBD 14.93±0.37 31.25 60..56 ±2.3 15.625 51.12 ±2.1 7.312 40.67±1.8 3.656 30.34±2.9

CBM 0.5 - -

500 92.23±0.14 250 81.39±0.23 125 71.09±0.56 62.05 57.42±0.44 STD Acarbose 38.25±0.12 31.25 48.02±0.24 15.625 35.99±0.98 7.312 24.87±1.01 3.656 13.09±1.12

107 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

500 84.14±0.13 250 83.77±0.13 CBM 125 73.18±0.78 62.05 68.73±0.79 14.93±0.37 31.25 50.45±0.20

CBD 0.5 - -

500 76.3±0.6 250 74.2±0.2 STD Eserine 125 70.3±0.1 0.30±0.01 62.05 62.1±1.6 31.25 48.9±0.1

108 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 ////////

109 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)

Detection Spot appeared at 254nm Spot appeared at 366nm Spot appeared due to Godin reagent ////////

110 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.

111 CBWPD* (18g) CC Silica 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) Sephadex n-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).

112 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.

113 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).

114 4.5 Structure elucidation of the isolated compounds 4.5.1: Compound A (n-Pentacosanyl-n-nonadeca-7′-en-9′-α-ol-1′-oate)

OH O

23' 21' 19' 17' 15' 13' 11' 7' 5' 3' 1' 9' O 22' 18' 14' 12' 10' 8' 6' 4' 2'

17 9 5 15 13 11 7 3 1 19

10 18 16 14 12 8 6 4 2

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

115 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

13' 11' 9' 7' 5' 3' 1'

O 12' 10' 8' 6' 4' 2'

17 15 13 11 9 7 5 1 2 3 18 16 14 12 10 8 6 4

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

116 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

15' 13' 11' 9' 7' 5' 3' 1 3 5 1'

O 6 16' 14' 12' 10' 8' 6' 4' 2' 2 4

28 26 24 22 20 18 16 14 12 10 8

7 19 15 13 11 9 29 27 25 23 21 17

+ 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).

117 4.5.4: Compound D (Heptacosanoic acid)

22 26 24 20 18 16

15 27 25 12 1 HO 14

3 5 7 9 11 13 O

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).

118 4.5.5: Compound E (1, 3, 5-Trihydroxy-2-hexadecanoylamino-(6E, 9E)-heptacosdiene)

O

15' 13' 11' 9' 7' 5' 3' 1'

14' 12' 10' 8' 6' 4' 2' NH

26 24 22 20 18 16 14 12 10 8 6 OH 4 2 OH

27 25 23 21 19 17 15 13 11 9 7 5 3 1

OH

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).

119 4.5.6: Compound F (2H-1-Benzopyran-2-one)

5 4 9 3 6

2 7 1 10 O 8 O

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).

120 4.5.7: Compound G (Betulin)

29

20

30 19 21 H

12 18 22 11 25 26 13 17 H 1 9 14 16 CH2OH 15 28 2 10 8 H 27 3 5 7 4 6 HO H

24 23

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)

121 4.5.8: Compound H (Stigmasterol)

29

28

21 22 26 24 18 20 23 25 12 27 11 17 16 19 13 H 1 9 15 14 2 10 8 H H 3 7 5 HO 4 6

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).

122 4.5.9: Compound I (3, 5-Dimethoxy 4-hydroxy cinnamic acid)

HO O 9

8

7 1 6 2

5 3 H3CO OCH 4 3 OH

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).

123 4.5.10: Compound J (4-Hydroxy-3, 5-dimethoxybenzoic acid)

7 COOH

1

6 2

5 3 H CO 3 OCH3 4

OH

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).

124 4.5.11: Compound K (5, 8-Dihydroxycoumarin)

OH 5 4 9 6 3

2 7 1 10 O 8 O 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).

125 4.5.12: Compound L (Stigmasterol 3-O-β-D-glucoside)

29 28

22 21 26 24 18 20 23 25 12 H 27 11 19 H 13 17 16 1 9 14

2 10 8 15 H H 6' OH 3 5 7

O 4 6 5' O 4' OH 1' OH 3' 2' OH

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)

126 4.5.13. Compound M (Sparsifol)

OH OH OH 4 2 3 OH HO H H H O 6 CH 5 1 3 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).

127 4.5.14: Compound N (6-O-β-D-Glucopyranosyl-β-D-(1-O-sinapoyl,6'-O-sinapoyl)-glucopyranose)

6 6' O 5 O O 4 9''' O ' 1 O O O 5 1' 4' HO 3 2 9'' ' HO HO 3' 2 HO ''' HO H H 8 7''' HO 8'' 7'' 1''' 1'' '' 2'' 6' 2''' 6''

''' 3'' H CO 3 3 5''' H3CO 5'' OCH '' 3 4''' OCH3 4 OH HO

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

128 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.

Inhibition % Compound 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

a = standard

129 Table 4.13: Results of butyrylcholinesterase inhibition assay of compounds (J-N) isolated from methanol extracts of Croton bonplandianum.

Inhibition % Compound 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 b = Eserene

130 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

131 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

132 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.

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