INTRODUCTION
Section I: Bioactive Molecules from
Mimusops elengi & Artemisia pallens
Section II: Characterization of Molecules by
Single Crystal X- Ray Diffraction Section I Bioactive Molecules from Mimusops elengi & Artemisia pailens
1.1 Ayurveda - The Art of Life Man has sought out plants with medicinal properties since ancient times. Evidence of this is the thousand years old traditions and records of proper healing. Traditional medicine (also known as indigenous or folk medicine) comprises medical knowledge systems that developed over centuries within various societies before the era of modern medicine. Traditional medicines include Unani system in Greece, Ayurvedic system in India, Amachi in Tibet, traditional Chinese medicine in China, Siddha or more recently Homoeopathy in Germany. Researchers are investigating not only the classical plants but also the related species that may contain similar active constituents as well as hitherto unknown plants which have no previous history of medicinal use\ Ayurveda is a holistic system of medicine from India that uses a constitutional model. Its aim is to provide guidance regarding food and lifestyle so that healthy people can stay healthy and folks with health challenges can improve their health. Ayurveda is an intricate system of healing that originated in India thousands of years ago. Vast ethno botanical knowledge exists in India from ancient time. Written records of the use of plants for curing human or animal diseases in India can be traced back to the earliest (4500-1600 BC) scripture of the Hindus, the Rigveda. Ayurveda, the Indian indigenous system of medicine dating back to Vedic ages (1500-800 BC), has been the integral part of Indian culture. Several plants were described in Atharvaveda. This was followed by monumental ancient treatise on the subject like Charak Samhita (1000-800 BC), Sushrut Samhita (800-700 BC) and Vagbhata's Ashtang Hridaya ^. As a science of life, Ayurveda is applicable to every living thing. The knowledge of Ayurveda is an integral part of the universal reality and manifests with each manifestation of the universe. In Ayurveda a person is viewed as a unique individual made up of five primary elements. The elements are ether (space), air, fire, water and earth. Just as in nature, we too have these five elements in us. Ether and air combine to form what is known in Ayurveda as the Vata dosha. Vata governs the principle of movement and therefore can be seen as the force which directs nerve impulses, circulation, respiration and elimination. Fire and water are the elements that combine to form the Pitta dosha. The Pitta dosha is the process of transformation or metabolism. The transformation of foods into nutrients that our bodies can assimilate is an example of a pitta function. Pitta is also responsible for metabolism in the organ and tissue systems as well as cellular metabolism. Finally, it is predominantly the water and earth elements which combine to form the Kapha dosha. Kapha is what is responsible for growth, adding structure unit by unit. Another function of the Kapha dosha is to offer protection. Cerebral- spinal fluid protects the brain with spinal column and is a type of Kapha found in the body. Also, the mucosal lining of the stomach is another example of the Kapha dosha protecting the tissues. We are all made up of unique proportions of Vata, Pitta and Kapha. These ratios of the doshas vary in each individual; and because of this, Ayurveda obsen/es each person as a special mixture that accounts for our diversity ^. The use of drugs and dietary supplements, derived from plants, has accelerated in recent years. Advances in science, notably during the last two centuries, better understanding of human body, and its physiology led to the isolation of many of the active ingredients of these herbs and plants in pure form and formulated synthetic compounds with or without herbal extracts, obtaining the drugs mostly used in the control of disease. The medicinal plants continue to receive attention of scientists from chemical, pharmacological and clinical angles in India and abroad. The studies on folk medicines through ethno botanical surveys are gaining importance'*.
1.2 Active Ingredients of Plants used as Drugs All plants produce chemical compounds as part of their normal metabolic activities. These include primary metabolites, such as sugars and fats, found in all plants, and secondary metabolites acquire in vahous proportions of active ingredients and only in a particular genus or species. The functions of secondary metabolites are varied, for example, some secondary metabolites are toxins used to deter predation, and others are pheromones used to attract insects for pollination. Phytoalexins protect against bacterial and fungal attacks. Allelochemicals inhibit rival plants that compete for soil and light. Organic chemicals from crude drugs provide a model which can be copied or modified by the organic chemist to produce a more potent drug or a better drug with fewer side effects. Many active principles have been isolated from plant sources such as digoxin (i) and acetyl digoxin (ii) from the Digitalis lanata leaf ^'®. Acetyl digoxin possesses cardio tonic and cardio toxic properties^. Plant-derived alkaloids have traditionally been of interest due to their pronounced physiological activities. Lopes F.C. et al isolated guanidine alkaloid pterogynidine (iii) from the Brazilian plant Alchornea glandulosa. It can be used potentially against pathological situations where angiogenesis is stimulated as tumor development^. Mentha longifolia L. (Lamiaceae) leaves have been traditionally implemented in the treatment of minor sore throat and minor mouth or throat irritation by the indigenous people of Iraq. Al-Bavati FA. reported antimicrobial activity of menthol (iv) and other isolates which validates the use of this plant in the treatment of minor sore throat and minor mouth or throat irritation^. The fruits and seeds of Semecarpus anacardium are used widely for the treatment of human cancers and other diseases in the Ayurvedic and Sidda systems of medicine in India. 3-(8'(Z),11'(Z)-pentadecadienyl) catechol (v) isolated from this plant is cytotoxic to tumor cell lines with IC(50) values lower than doxorubicin (vi) ^°, Gaultheria yunnanensis possesses analgesic and anti-inflammatory activities. The active principle of this is found to be gaultherin''^ (vii). Flavonoids, quercetin (viii) and kaempferol (ix) isolated from Danae racemosa are the active principles responsible for the antinociceptive action against several pain models^^, Scaphyglottis livida and Maxillaria densa are used in folk medicine for treatment of painful complaints. Activity of the isolated compounds in mice and rats support the popular use of this species ^^. Papaver somniferum, yields a sap of narcotic opium, from which the potent pain killer morphine (x) is made. Activator protein 1 is a transcription factor involved in the regulation of proinflammatory mediators'"*. For centuries, malaria was treated with the bark of Cinchona calisaya and Cinchona succirubra plants named "quinas" in Brazil. Quinine (xi) is the active principle isolated from chinchona plants. Other plant species like Deianira erubescens (roots and leaves), Strychnos pseudoquina (bark) and Remijia ferruginea (bark) are also used to treat fever and malana ^^. The history of aspihn (xii) can be traced back to ancient Egypt where extract of willow bark was used to treat inflammation. The active component of the extract was identified as the glucoside of salicylic alcohol. The severe gastric side effects associated with the use of sodium salicylate prompted the synthesis of the o-acetyl-derivative as a possible pro-drug. Acetylsalicylic acid was synthesized one hundred years ago and was mass-produced under the commercial name of 'Aspirin' ^^. The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a plant species known for its production of terpenoid indole alkaloids (TIAs), many of which are pharmaceutically important. Ajmalicine (xiii) and serpentine (ivx) are preschbed for the treatment of hypertension, whereas the bisindoles vinblastine (xv), vincristine (xvi) and 3',4'-anhydrovinblastine (xvii) are used for their antineoplastic activity in the treatment of many cancers". Rauwolfia serpentine is reported to be the first herbal antipsychotic plant^^. It is used extensively in India for sleeplessness, anxiety and high blood pressure^^. Reserpine (xviii) and deserpidine (xix) alkaloids are the active principals from Rauwolfia species which are responsible for their antihypertensive action^°. A plant flavonide quercetin -3-0-glucoside (Q3G) isolated by preparative HPLC from aerial parts of Prangos ferulaceae is cytotoxic, phytotoxic, antimicrobial and antioxidant ^^ Medicinal plants are also reported to be useful for the treatment of diseases like eye infections. IHippobromus pauciflorus was the most commonly used plant species ^^. Seeds of Murraya l HN HN HN "°:drxlHO°' HO H (i) (iii) ":.dr3cta "^o H°O (ii) (iv) O OH .^^OH .0 O OH 0/„ V^ OH OH NH, (V) (vi) Of! 0 OH o HO O o te OH OH OH OH OH OM C (vii) (viii) OH O (ix) (X) (xi) 0.\ /OH O O (Xii) (xiv) OH OH •N '^ // HN°Y- A HN ^. .^., N-X H >:-^ 0-----Ao^ A-\'' / t * o (xvi) /^N •\< OCH3 0 /' OCH. H3COOC OCH3 (xvii) (xviii) OCH3 y^^ocH3 OCH. H3COOC OCH3 (xix) (XX) OH (xxi) (xxii) (xxiii) O— Rhamnose-Glucose (xxiv) (xxv) Ergot Alkaloids HOOCA. /\ / N H \ •N H (xxvi) (xxvii) (xxviii) (xxix) (xxx) (xxxi) Cfi6^H5 H (xxxii) (xxxiii) 1.3 Family Sapotaceae The Sapotaceae family consists of large evergreen trees and less commonly shrubs^^. The plants are distributed throughout the tropic of Asia, Africa and America. The family consists of about forty genera and six hundred species. The members of this family are widely distributed throughout the tropics^^'^^. The important ones are Argania, Butyrospermum, Colacarpum, Chrysophyllum, Mimusops, Payenne, Sarcosperm etc.^'*. Sapotaceae seeds are mostly nuts and their kernel fat ranges from 30% - 50%^^. Economically the members of Sapotaceae are of use in several ways. The seeds of Butyrospermum parkii are used as 'shea butter'. Those valued for delicious and edible fruits include Colacarpum sapota, Chrysophyllum cainite, Lucuma mammosa, Manilkara achras etc. The seeds of Madhuca butyracca produce the vegetable butter, called 'phulwa', used as a cold cream, lip salve and luminent. The latex of Manilkara achras yields 'chickle', used for making chewing gum. Mimusops globosa is the source of 'balata', commertially important latex. Chrysophyllum olivaeforme furnishes cabinet wood^^. The Mimusops hexandra bark is reported to be febrifuge and general tonic. It retards fermentation. The Achras sapota bark (aqueous extract) contains tuberculostatic principle^^. Proteins from the seeds of Pouteria torta with lectin like properties were found to possess insecticidal and antifungal activity^^. Pouteria campechiana, Pouteria sapota and Pouteria viridls are tropical plants in the Sapotaceae family that bear edible fruits, among these P. sapota had the highest antioxidant activity^®. Orally administered saponins from Madhuka longifolia had both antiulcerogenic and anti-inflammatory activities in rats. The anti-inflammatory activity was 1/5 that of phenyl butazone"^^. Argan oil from Argania spinosa is found to be beneficial in the treatment of the hyperlipidemia and hypercholesterolemia'*". Argania spinosa is a tree that has played an essential function in the South-western Moroccan micro-economy. By providing food for human beings and animals as well as fuel, it has played a key role for the native population of these regions for centuries. The main traditional use of argan oil is for nutritional purposes. Natives either directly eat the oil on toasts, generally for breakfast, or use it for frying. As cosmetic, the oil is traditionally indicated to cure all kind of pimples on the skin and more particularly juvenile acne and chicken pox pustules. It is also recommended to reduce dry skin problems and slow down the appearance of wrinkles. It is also used in rheumatology. For these indications, the oil is used as a skin lotion and applied on the area to be cured. In addition and as olive oil, argan oil is also used by mouth and is traditionally prescribed as hepatoprotective agent, or in case of hypercholesterolemia or atherosclerosis'^V 1.3.1 Genus Mimusops Mimusops is a genus of trees distributed in the tropics of the Old World. It comprises of thirty species. One of the species, commonly found in India, is Mimusops elengi. The other common plants are Mimusops hexandera and Mimusops manill 1.4 Family Compositeae Asteraceae or Compositeae is one of the largest families comprising nine hundred genera and thirteen thousand species, which are cosmopolitan in distribution. The members of this family are well represented in the semi-arid 12 parts of the tropical regions of the world, and are poorly represented in the tropical rain forests^^. Asteraceae are most usually herbs, but some shrubs, trees and climbers do exist. They are generally easy to distinguish, mainly because of their characteristic inflorescence and many shared apomorphies. The family is represented in India by one hundred and thirty eight genera and seven hundred and twenty three species with high concentrations in both the Himalayan range as well as peninsular India. 71 species and 2 varieties i.e. approximately 10% are endemic to the peninsular India. The Compositeae members of Peninsular India are closely related to the African species. The temperate genera also have a fairly good degree of representation in peninsular India ''. Commercially important plants in the Asteraceae include the food crops Lactuca sativa (lettuce), Cichorium{ch\cory), Cynara scolymus (globe artichoke) Helianthus annuus (sunflower), Smallanthus sonchifoliusiyacon), Carthamus tinctorius (safflower) and Helianthus tuberosus (Jerusalem artichoke). 1.4.1 Genus Artemisia Artemisia is a fairly large genus within the family Asteraceae with two hundred individual species known, which are usually found in dry areas. They are invariably found as small fragment shrubs or herbs and most yield essential oils. Some of these oils have found uses in perfumery and medicine (for example, vermifuges, stimulants etc.) whereas the leaves of some species are used as culinary herbs. The plants themselves as are popular among gardeners as cultivated ornamentals. 1.4.1.1. Bioactivity of Genus Artemisia As summarized by different groups ^°"^^, many species have been used since ancient times as folk remedies for some treatment purposes, relieving cough, invigorating blood circulation, stopping pain, inducing sweat, diuresis, antihypertension, anthelmintic antitoxic and anti-allergy. Essential oils from Artemisia (wormwood) are of botanical and pharmaceutical interest. They are used in traditional remedies in many parts of the world. In pure form the oil is a narcotic poison. Nearly all species are intensively bitter and strongly aromatic. Extracted substances from the plant have an antibacterial action and some of these substances have potential use in mosquito control. Other properties include toxicity to nematodes and inhibition of seedling growth®^. A. dracunculus L. (Asteraceae) has been used orally as an antiepileptic remedy in Iranian folkloric medicine. The anticonvulsant potential and composition of the essential oil obtained from the aerial parts of the plant were assessed by Sayyah et al^'*. A. absinthium L. is a commonly used medicinal plant for parasitic diseases all over the world^^. The antimicrobial activities against the common human pathogens of the isolated oils from A. absinthium and A. vulgaris were reported by Blagojevi et al. The oils showed a broad spectrum of antimicrobial activity. Therefore, these oils can be used as flavor and fragrance ingredients^^. The antibacterial ^^, antifungal ^^ and antioxidant ^^ activities of the essential oils isolated from A. absinthium, A. santonicum and A. spicigera were evaluated by different groups. The oils exhibited potent antifungal activity at a wide spectrum on the growth of agricultural pathogenic fungi. A. santonicum and A. spicigera oils showed antibacterial activities over a very wide spectrum. Essential oil of A. molinieri has shown a strong inhibition of the growth of Candida albicans and Saccharomyces cerevisiae var. chevalieri. The oils have shown interesting antioxidant activities on the basis of a-tocopherol as reference compound^^. The essential oil of A. lavandulaefolia exhibited considerable inhibitory effects against all obligate anaerobic bacteria^°. Antileshmanial activity of aqueous extract and essential oil of A. herba-alba Asso was reported by Hatimi et al^V Chemical composition and antibacterial activity of the essential oil from A. feddei was reported by Cha JD et al. It showed a considerable inhibitory effect on the obligate anaerobic bacteria^^. 1.4.1.2 Compounds Isolated From Genus Artemisia Members of the Artemisia genus are important medicinal plants found throughout the world. Arteminolides have been isolated from the aerial parts of Artemisia, which have an inhibitory activity on farnesyl-protein transferase (FPTase) in in-vitro assay. The arteminolides inhibit tumor cell growth in a dose- dependent manner. Furthermore, one of the arteminolides blocked in-vivo growth of human colon and lung tumor xenograft without the loss of body weight in nude mice^^''^'*. Guaianolide sesquiterpene lactones, sesquiterpenes along with arteminolides were isolated by bioassay-guided fractionation from the methanol extract of the aerial parts o^ Artemisia sy/vaf/ca using the NF-KB mediated reporter gene assay. All isolated compounds displayed inhibitory activity on the LPS-induced NF-KB activation, NO production, and TNF-a production with IC50 values of 0.49- 7.17, 1.46-6.16, and 3.19-27.76 pM, respectively, in RAW264.7 cells. It was also established that one of the isolated arteminolide suppressed the expression of NF-KB target genes such as iNOS and COX-2. NF-KB inhibitory activities of these compounds support the pharmacological use of Artemisia sylvatica, which has been employed as an herbal medicine for the treatment of inflammation^^. The effect of Dehydroleucodine (DhL) isolated from Artemisia douglasiana on gastric acid secretion in rats and its anti-inflammatory effect was studied by Guardia T et. aP^. DhL and aqueous extract of the plant exhibit significant antioxidant capacity^^. The isolation and antifungal activities of Methyleugenol, 5- phenyl-1,3-pentadiyne and capillarin from the steam-distilled essential oil fraction of A. dracunculus were described by Meepagala et al. The isolates were tested against Colletrotictium fragariae, Colletroticlium gloeosporioides, and Colletrotictium acutatum^^. The oils of A. absinttiium of French origin contain (Z)- epoxyocimene and chrysanthenyl acetate as major components. Antimicrobial screening performed on these samples showed that A. absinthium oil inhibited the growth of yeasts {Candida albicans and Saccliaromyces cerevisiae var. cIrievaiierP. Piperitone and trans-ethyl cinnamate isolated from the essential oil of Artemisia judaica showed pronounced insecticidal and antifeedant activity against the third instar larvae of Spodoptera littoralis (Boisd). When tested for antifungal activity against plant pathogenic fungi, the isolated compounds exhibited a moderate to high activity ^°. Ethanol extract from aerial parts of Artemisia annua L. exhibited a strong depressant activity on the CNS ^\ A. annua L. is an annual herb native of Asia, it has been used for many centuries for the treatment of fever and malaria. The active principle of A. annua, artemisinin, is currently being developed to a registered antimalarial drug. Artemisinic acid, a possible biogenetic precursor of artemisinin, was isolated from the hexane extract of/A. annua ^^. A number of monoterpenes, diterpenes and sesquiterpenes natural product also have been isolated from the seeds of >4. annua by Brown GD. et. ai^^. The antifungal activity of A. herba alba was found to be associated with two major volatile compounds canzone and piperitone, isolated from the fresh leaves of the plant ^^ The essential oil of A. iwayomogi exhibited a wide range of antibacterial activity. The major components reported from this are camphor, borneol, camphene and beta-caryophyiiene^^. Terpenoids like a-amyrin, (3-amyrin, p-sitosterol and coumarins like 5,6,7- trimethoxycoumarin and 6-methoxy-7,8-methylenedioxycoumarin were isolated from the non-polar fraction of/A. apiacea by Lee S. et. al ^^. The isolation of 6,7-dimethoxycoumarin, 6-methoxy-7,8-methylene dioxycoumarin, 5,6-dimethoxy-7,8-methylenedioxycoumarin, 6-hydroxy-7,8- methylenedioxycoumarin and 5-hydroxy-6,8-dimethoxycoumarin from A. apiacea is reported by Kim KS et. al^^. Wong HF and Brown GD reported the isolation of dimehc guaianolides and a fulvenoguaianolide from A. myriantha^^. They have also reported the presence and isolation of germicranolides along with their confirmation^^. Flavones namely cirsilineol, cirsimaritin, arcapillin and cirsiliol were isolated by Zhang QW et al from flower buds of >A. scoparia^°. As summarized, many different species of Mimusops and Artemisia have been used since ancient times as folk medicine. Thus chemical screening of chemo taxonomically related species have been undertaken for its phytochemical investigation and evaluation or broadening the source of bioactive compounds particularly when their synthesis is very costly. The present Research Work describes the chemical investigation and biological activity of a potent medicinal plant Mimusops elengi, an aromatic medicinal shrub Artemisia pailens and the Single Crystal X-Ray diffraction study. 16 Section 11 Characterization of Molecules by Single Crystal X- Ray Diffraction X-Ray Diffraction X-rays were discovered in 1912 by Rontgen. An electron hits at an anode and it creates radiations of a continuous distribution of wavelengths. It causes sharp atomic transitions, resulting in X-rays (Fig 1) with definite wavelengths. Electromagnetic radiations of very short wavelength (0.01 - 100 nm) are produced when an electron hits a piece of metal in an evacuated tube. Any radiation can be diffracted by appropriate scatterers, but as a rule, the most important information arises when the wavelength of the radiation is similar to, or smaller than, the size of the spacing between the objects being studied. i -30 rn.A 4^-^ i 50 kV imom -^y. X-rays Fig1 Generation of X-Rays Bragg's law of X-ray diffraction Diffraction from a sample is governed by Bragg's law (Fig 2). Sir William Lawrence Bragg, Australian-born British physicist and X-ray crystallographer, discovered the law, named as Bragg's law, of X-ray diffraction in 1912, which is basic for the determination of crystal structure. He was joint winner with his father. Sir William Bragg of the Nobel Prize for Physics in 1915. Diffraction from crystals is described by the scientist Bragg^^ 17 Fig 2 Bragg's law Mathematical form of Bragg's law is as: n X.= 2 d sin 9 where n is an integer (the order of scattering), X is the wavelength of the radiation, d is the spacing between the scattering entities (e.g. planes of atoms in the crystal) and 0 is the angle of scattering. Diffraction pattern displayed the important two facts: • The crystal structure of an unknown material; and the average spacing between layers or rows of atoms • Orientation of a single crystal or grain. Diffraction from powder sample^^ A powdered sample consists of thousands of crystalline particles oriented in different directions. Diffraction from a powdered sample forms a continuous cone (Fig 3) liL^TTVS H :ffi *9 w Fig 3 Fig 4 Formation of cones Strip of pliotographic film 18 These cones intersect a strip of photographic film (Fig 4) located in the cylindrical camera to produce a characteristic set of arcs on the film. It shows the diffraction lines and the holes for the incident and transmitted beams. Back reflections produced when 26 > 90°, distance from the beam source S2, can be measured. The distance S1 corresponds to a diffraction angle of 20. The angle between the diffracted and the transmitted beam is always 29. The data (distances S1 and S2) obtained from above figure, Bragg's Law, n A,= 2 d sin 9 and hkl planes, unit cell parameters can be determined. In such cases diffracted intensities are very low so it is more difficult and less reliable to find the correct structure. Powder diffraction can be used to examine the phase changes as a function of temperature, pressure etc. Diffraction from Single Crystals Single crystal X-ray diffraction is a powerful method which gives the three dimensional structure and confirms the stereochemistry, thus there will not be any ambiguity in the confirmation of the molecules. X-ray studies revealed the precise bond distances and angular relations among the atoms in the molecule. This has helped to design required molecular architecture. The ohentations of the functional groups contribute some of the physical properties as well as biological activities. The single crystal X-ray diffraction study establishes precised stereo chemical structures of synthetic organic molecules, biologically important molecules, natural products, inorganic metal clusters, organometallics, high-energy materials, catalytically active coordination compounds etc^^. A crystal consists of atoms and molecules arranged in a regular pattern in space. The orientation of atoms whether above the plane (P) or below the plane (a) can be distinguished. The steric crowding in the molecule modifies bond angles which establish stereochemistry. This regularity is responsible for diffraction of beams. Diffraction from a single crystal (Fig 5a and 5b) is shown as sphere of reflections. Each spot corresponds to diffraction from an hkl plane. The intensity of the spot depends on type of atoms that are responsible for diffraction from the particular plane and location of the atoms in space. An experimental set up (Fig 6) for single crystal X-ray diffraction is shown below. Figure 5a Figure 5b Diffraction pattern of a Single Crystal Single-crystal X-ray analysis has three basic steps. • To obtain an adequate crystal of the material under study. It should be sufficiently large and regular in structure, with no significant internal imperfections such as cracks or twinning. • An intense beam of monochromatic X-rays, produces regular pattern of reflections from the crystal. The diffracted intensity get recorded at every orientation of the crystal. Multiple data sets are received, to acquire complete information of diffracted intensities. • Data processing and combining computationally with complementary chemical information. The refined model of the atomic arrangement gives unambiguous crystal structure. The calculations of H-bonding and geometrical parameters are stored in a public database. Ai'Ga DGtfictor(! 50keV Electrons PrlmaLry X- ray Beam Anode 4-Circle OonDuiiieter ( Eulerian or Kappa Geonrietry) Figure 6 An experimental set up 20 Single crystal mounted on a goniometer head is placed in the X-ray beam. The diffracted intensities are recorded on film / detector / CCD camera. The Unit cell parameters can be determined using few reflections, depending on cell parameters - cell length and cell angle. They are classified as Monoclinic, Triclinic, Trigonal, Tetragonal, Hexagonal, Orthorhombic or Cubic. These seven crystal systems when combined with face centered and body centered lattice gives fourteen Bravais lattices (Fig 7). With the knowledge of all cell parameters, volume of the unit cell and the density can be calculated using the following formula: (Density of Crystals) p = (Molecular Weight X Z) / (Volume X 6.023 X 10"' g/cc •i>c'-~~.~ .-.:i^-' P Tetragonal | Trigonal/Hexagonal P Trigonal R h- 17 -i Cubic Fig 7 Bravais lattices 21 Characterization of the various crystal systems 94 Crystal Point Groups Bravis Unit Cell system types Properties Triclinic 1,-1 P a, b, c, a, p, y Monoclinic 2, m, 2/m P, C a, b, c, 90,p, 90 Orthorhombic 222, mm2, mmm P, 1, F a, b, c, 90,90, 90 Tetragonal 4, -4, 4/m, 422, P, 1 a, a, c, 90,90, 90 4mm, 42m, 4/mmm Trigonal 3, -3, 32, 3m, -3m P, R a, a, c, 90,90, 120 Hexagonal 6, -6, 6/m, 622, P a, a, c, 90,90, 120 62m, 6/mmm, Cubic 23, m-3, 432, -43m, P, F, 1 a, a, a, 90,90, 90 m3m Symmetry Symmetry plays an important role in crystallography^^ • Inversion • Mirror plane • Glide • Translation Centers of symmetry, mirror planes, two-, three-, four- and six fold and the corresponding inversion axes of symmetry are encountered. An inversion axis involves rotation along with inversion through a point. One-fold inversion axis is equivalent to a center of symmetry and a two fold inversion axis is equivalent to a mirror plane. A glide line or plane is a composite symmetry element, which is made up of a reflection and a translation. The translation must be in the direction of the plane of the mirror. A screv; axis is a translational symmetry element and consists of a rotation followed by a translation. It exists for each rotation axis. An 11 infinite crystal or repeating three dimensional structures possesses translational symmetry because after a translation it can be made to coincide with itself. Translational symmetry elements include glide lines and planes, and screw axes. These symmetry elements operate on infinite crystals or structures and express the way in which symmetry elements of reflection and rotation can combine with the lattice to create extra symmetry types. These symmetries when operated on crystal systems give 32-point groups. Symmetry operations, screws and glides, which include translational components and Bravais translations, yield systematic absences in the diffraction pattern regardless of the cell content. Thumb Rule of Symmetry Operation for a point x, y, z Mirror 1 'b' axis x, -y, z Two fold axis parallel to 'b' -x, y, -z 2i Screw axis parallel to 'b' -x, 1/2+y, -z Rotation by 360 / 2 degrees + 1/2 Translation along 'b' (Rotation by 360 / n degrees + 1/2 Translation along 'b') 'a' glide ± to 'b' means Mirror plane 1 to 'b' + 1/2 Translation along 'a' 1/2 + x, -y, z Symmetry present in the compound can be identified by specific information in the intensities of the diffraction pattern. In particular, centre of symmetry, screw axes and glide plane operations as they result in the systematic absences. Thus from systematic absences one can find the symmetry present and the space group to which the crystal belongs^^. Structure Determination: Intensities of each reflection are measured by recording the diffraction pattern using a detector. A diffraction pattern is obtained according to Bragg's law, 2d sin 6 = nX. The scattering from different planes depend on type of atom and where it is present in a three dimensional space. ^^ Considering X-Rays as wave function, the intensity I of a reflection is 2 proportional to the square of the complex structure factor F, I a F • It is obtained by the summation of all atoms of unit cell in the crystal. The calculations reflect the atomic scattering factor of each atom and the position of the atom in the structure. To reconstruct the electron density obtained by X-ray diffraction and 2"i to find out the size of the atoms present in a molecule Fourier Transform technique is used. Here two components are required for calculations as shown below^^: n i=1 Electronic property Structural property of atom (Position) The absence of phase information poses a serious problem to the otherwise straightforward reconstruction of the electron density p (xyz) from the experimental structure factor amplitudes \Fhkl\. The electron density p can be calculated using the following relation: p (xyz) = (1A/) Ihki Fhki exp [ -2TC i(hx +ky+lz)] Contours for electron density map (Fig 8) are displayed. Fig 8 Contours for electron density map 24 Once the structure is solved and the atomic coordinates x,y,z are obtained, the intensities are calculated. These intensities and the observed intensities are Least square refined. The positional and thermal parameters are refined to get an accurate structure. From the x, y, z co-ordinates of each atom, geometrical parameters like bond lengths, bond angles, torsion angles along with intra and inter molecular hydrogen bonding can be established to a great accuracy. Presence of water molecule and its coordination can be recognized. Single Crystal X-Ray Diffraction ^K*^ i«I^H ^^^^^L'^ '>--.^^^^B - "HBH1 1 ifA^B ^^^^^•EZL_- i^^^pi^pl ^^^H ''^'tBaMfcj-..*- Fig 9 Fig 10 Crystals viewed through microscope Leica polarizing microscope Crystallization The crystallization process consists of two major events, nucleation and crystal growth. Nucleation is the step where the solute molecules dispersed in the solvent start to gather into clusters on the nanometer scale that become stable under the current operating conditions. These stable clusters constitute the nuclei. The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical cluster size. Nucleation and growth continue to occur simultaneously while super saturation exists. Commonly used techniques include solvent evaporation; slow cooling of the solution, vapour diffusion, sublimation and many variations incorporate in such methods^^. 25 Preliminary Characterization of Single Crystalline Sample A good quality crystal, as mentioned above (Fig 9) is selected for the single crystal x-ray analysis by watching carefully using polarizing microscope (Fig 10). This single crystal is mounted on the tip of thin glass fiber which is fitted on goniometer (Fig 11) for the X-ray studies. The crystal is centered and set right by the instrument. A video microscope photograph (Fig 12) of this crystal which is mounted on the glass fiber is shown below. It is used for the data collection. The rotation photograph taken for 60 seconds is shown (Fig 13). ^ tmn^asmmammaaRe Edt Grab v«w Toott Tvoeti Mndov . Help LJi|a;|H| a| z.|z.|z,| 8>m [T ~| _J#4-|BH-| A|T| Fig 11 Fig 12 Goniometer Crystal mounted on glass fiber «• E* 'jyita UoMt *r.»i» lien™ IJi«iliF Li>4l F«to ^^^^^^^^B^^'' * 1 K>0 3i5 Fig 13 Rotation photograph 26 Fig 14 SMART APEX Single Crystal X-Ray Diffractometer Unit Cell Determination Unit cell determination is carried out on BRUKER's SMART APEX Single Crystal X-ray CCD Diffractometer (Fig 14). The diffracted frames collected are processed to get the proper orientation matrix and unit cell parameters. X-ray intensity measurements (Data Collection) are carried out at room temperature (296K) on a Bruker SMART APEX CCD Diffractometer with graphite-monochromatized (MoK(,= 0.71073A) radiations. Crystal to detector distance is 6.05 cm, 512 x 512 pixels / frame, maximum detector swing angle is 27 -30.0°, beam center is (260.2, 252.5), in plane spot width is 1.24, using SAINT integration. The X-ray data collection is monitored by Bruker's SMART program. Using various crystallographic systems and systematic absences, different data sets are collected and the exposure time per frame is chosen depending on the best diffraction of the mounted crystal. The structures are solved by direct methods using SHELXTL program. The data are corrected for Lorentzian, polarisation and absorption effects. SHELX-97 (ShelxTL)^°° is used for structure solution and full matrix least squares refinement on F^. The refinements are carried out using SHELXTL-97. A typical diffracted frame (Fig 15) is displayed. Fig 15 Diffracted Frame 28 References 1. Vinod D. Rangari, Pharmacognosy & Phytochemistry, Part I First Edition, Career Publications, pp. 10 and 18, 2002, 2. J.K. Maheshwari, Ethnobotany and Medicinal Plants of Indian Subcontinent, pp.(6) Scientific Publisher (India), Jodhpur, India, 2000. 3. Robert E. Svoboda, Ayurveda Life Health and Longevity, Penguin Books India, Ltd, p-7, 1992. 4. J.K. 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Bassi (eds.), Computing in Crystallography , pp. 93-107, Delft, University Press 1978. 99. http:/v\AAAA/.iucr.org 100. G. M. Sheldhck, SHELX-97 program for crystal structure solution and refinement. University of Gottingen, Germany, 1997 34 Chapter 1 Chemical Investigation of Mimusops elengi Section I Isolation and Characterization of 3, 4 bis (1, 3-benzodioxol-5-yl methyl) tetrahydrofuran-2-ol - Compound 1 - Cubebin Section II Isolation, characterization and hydrolysis study of an aromatic ester by LC-MS Section III Quantification of metabolites by HPTLC PUBLICATIONS A. Metals, amino acids and carbohydrate contents of medicinally important Plant - Mimusops elengi Oriental Journal of Chemistry 25 (4), 1109-1112, 2009 B. GC-MS Study Of A Steam Volatile Matter From Mimusops elengi; International Journal of ChemTech Research, 1 (2), 158-161, 2009 C. Dibutyl Phthalate, A Secondary Metabolite from Mimusops elengi Chemistry of Natural Compounds, 213.09, No. 6, 2010. 36 1.1 Introduction - Mimusops elengi Mimusops elengi is a potent medicinal plant having historic importance. It is cultivated in North and Peninsular India and Andaman Islands. All parts (Panchang) have been reported for their medicinal uses in Ayurvedic systems of medicine. From the ancient times the bark material has been used as a preventive medicine for oral gum diseases and also used as chewing sticks. The tree is lopped for fodder. The wood is valuable and an oil is expressed from the seeds. 1.1.1 Botanical Characterization Mimusops elengi belongs to the family Sapotaceae. It is an evergreen tree up to 15 m high with dark grey fissured bark and densely spreading crown. The trunk is short and erect. Leaves are oblong, glabrous and leathery with wavy margins. Flowers are white, fragrant, axillary, solitary or fascicled. Fruits are ovoid or ellipsoid berries. Seeds are ovoid, compressed, greyish brown and shinyV 1.1.2 Distribution Mimusops is a genus of trees distributed in the tropics of the Old World. One of the species is found in India. The tree attains large dimensions in the moist evergreen forests of Western Ghats. In the estern ghats it is found in dry areas, often on laterite and is comparatively small in size. In Andamans it attains a height up to 35 meter. It is grown as an avenue or shade tree in many parts of India. It is frequently cultivated in gardens as an ornament. It is also grown as an avenue or shade tree through out the greater part of India^'^. 1.1.3 Important biological uses and actions of the plant parts Literature survey revealed that all parts of the plant are used for the treatement of various diseases as it contains bioactive molecules. The flowers, fruits and bark are acrid, astringent, cooling and anthilmentic^. In Ayurveda, the important preparation of Mimusops is "Bakuladya Taila", applied on gum and teeth for strengthening them. 37 1.1.3.1 Leaves Leaves are used as an antidote for snakebite '*. The nutritive value of leaves of M. eleengi growing in west Bengal has been evaluated along with other 36 species ^. 1.1.3.2 Bark The bark is used as a gargle for odontopathy, inflammation and bleeding of gums. Tender stems are used as tooth brushes ^ The bark is used as snuff for high fever accompanied by pains in various parts of the body. It is used as tonic and febrifuge. It is also useful in urethrorrhoea, cystorrhoea, diarrhoea and dysentery^. The bark, flowers and fruits are acrid, astringent, cooling and anthelmintic ^. Anthelmintic activity of bark was studied by Mali R.G. et al^. 1.1.3.3 Flowers Garlands made of its flowers are ever in good demand due to its long lasting scent. Flowers are used for preparing a lotion for wounds and ulcers. Powder of dried flowers is a brain tonic and is useful as a snuff to relieve cephalgia"'. The flowers are considered expectorant and smoked in asthma. 1.1.3.4 Fruits A lotion prepared from unhpe fruits and flowers is used for smearing on sores and wounds\ Unripe fruit is used as a masticatory and helps to fix loose teeth. Pulp of ripe fruit is antidysentehc^. 1.1.3.5 Seeds Seeds are used for preparing suppositories in cases of constipation especially in children. Saponins from seeds are spermicidal and spasmolyticV The bark and seed coat are used for strengthening the gum and enter into the composition of various herbal tooth powders, under the name of "VajradanW, one of the famous Indian tooth powder, where they may be used along with tannin-containing substances like catechu {Acacia catechu), pomegranate {Punica granatum) bark, etc. Nutritional evaluation of the refined seed oil was earned out in rats. It Vv/as found to be satisfactory and quite comparable to that of peanut oiT. 38 1.1.4 Supplimentary Uses Tree is also lopped for medium quality fodder. Wood is used for building purpose, piles, bridges, boats, furniture, panels, cabinet work, musical instruments, walking sticks etc.^' ^. In Thiland an infusion of the flowers is used as a cosmetic after bath. Steam volatile matter of flowers is used for cosmetic purposes. The ripe fruit is edible. It is some times used for making preservatives and pickles. The seed kernel oil is used for edible and lighting purposes ^. The bark is used in some parts of India for dyeing and tanning purposes due to presence of tannins^. Bhuyan et. al. extracted and identified the colour components from the bark material. The dyeing behavior of these colour components on wool suggests that these dyes might be an alternative to synthetic dyes®. The plant is found to be potential biomass source for hydrocarbons and other phytochemicals^°. 1.1.5 Shioka Mimusops elengi having high medicinal value is described in Ayun/eda as, W^geR^^Rt^: ^?^^^TRR# ^: I (•Hlc|Mc|,|^|) Bitter juice of Bakul is good for soothing effect. It helps in various conditions like cough, pitta, poisoning, worms, leprasy and dental diseases. This sweet and astrigent juice also prevent loose motions. Fruit of Bakul reduces sensitivity of teeth and strengthens the gum tissues. 39 1.1.6 Classification Kingdom: Plantae Division: Magnoliophyta Class : Magnoliopsida Order : Ericales Family : Sapotaceae Genus : Mimusops Species : elengi 1.1.7 Common names ^ Bengali: Bakul Hindi: Bakul, Maulsiri Gujarathi: Barsoli, Bolsari Telugu: Pogada, Tamil: Vagulam, Magadam Assam: Gokul Oriya: Bokulo, Baula English: Bulletwood tree 1.1.8 Previous work The investigations of different parts of the plant carried out in the past showed that the plant contains free triterpenoids^^ The fruit and seed were found to contain quercitol (I), ursolic acid (II), triterpene alcohol, glucose (III), dihydroquercitin (IV), p-D-glucoside of p-sitosterol (V)^^. The leaves, heartwood and root were found to contain hentriacontane (VI), P-carotene (VII), lupeol (VIM), a-spinasterol (IX), hederagenin (X) , triterpene acids, quercitol, glucose etc^^. Isolation of a sterol, an epimer of chondrillasterol, was reported by Jahan N. et. al^"*. Steroidal glycosides 3-0-(3-D-glucopyranoside and the 3-0-p-D-galactopyranoside of the sterol - (24R)-stigmasta-7,22(E)-dien- SP-ol (XI) have been isolated by Jahan Nusrat et. a^^ They have also isolated a lupene-type triterpene, 3p-hydroxy-lup-20(29)-ene-23,28-dioic acid (XII), along with the known triterpenes, (3-amyrin (XIII), lupeol, a-taraxerol (XIV) and ursolic acid""^. Triterpenes, 3p, 19a, 23-trihydroxy-urs-12-ene and 3p-(p-hydroxy-cis- cinnamoyloxy) urs-12-en-28-oic acid had been isolated from the methanolic extract along with 3p-(p-hydroxy-trans-cinnamoyloxy) urs-12-en-28-oic acid and ursolic acid, respectively^^. Pentacyclic triterpenes, 3[3,6p,19a,23-tetrahydroxy- urs-12-ene (XV) having moderate inhibiting activity against p-glucuronidase enzyme and ip-hydroxy-3p-hexanoyllup-20(29)-ene-23,28-dioic acid (XVI), were reported 18 ^ ^13^91 40 Mimusops elengi 41 Critical evaluation of the therapeutic potential of bassic acid (XVII), an unsaturated triterpenic acid isolated from M.elengi, was earned out for its antileishmanial properties by Lala Sanchaita et. aP^. A steroidal saponin, 5a-stigmast-9(11) en-3-O-p-D-glucopyranosyl (1 -^ 5)-0-pD-xylofuranoside was isolated from the roots of M. eleng'i^^. The volatile components of the flowers were reported by Wong, K. C. A total of seventy four compounds were identified^\ D-mannitol (XVIII) was isolated from the acetone extract of M. elengi flowers. The ether soluble fractions from an ethanol extract of fresh flowers yielded [3-sitosterol. An ethyl acetate fraction gave (3 -sitosterol-D-glucoside^^. The protein content of the leaves was reported to be a condensation polymer of L-cystine, L-lysine, L-arginine, DL-serine, DL-threonine, L-glutamic acid, L-proline, tryptophan, L-tyrosine, DL-methionine, DL-valine, isoleucine, and L-leucine^^. Pentacyclic triterpenic acids, mimusopic acid (XIX) and mimusopsic acid (XX), possessing the novel migrated oleanane skeleton, mimusopane were isolated from the seeds by Sen et. al^"*. Mimusopic acid exhibited anti-HIV reverse transcriptase activity and modification of this novel compound may lead to more potent bioactive substances. Moreover, the saponins present also demonstrated to be antifungal against some human pathogens ^^. Seed kernels yielded 22.4% oil composed of palmitic (XXIII), stearic (XXIV), behenic (XXV), oleic (XXVI) and linoleic acid (XXVII) ^^. The oil was also found to contain 9-keto-octadec-15-enoic acid (XXI) in addition to usual myristic (XXII), palmitic, stearic, and oleic acids ^^. Presence of erucic acid (XXVIII) in small amount from the seed oil was reported by Shweta Sharma et. al^^. Lipid composition of the seed oil was reported by Javed et. al. The oil was found to contain hydrocarbons , wax esters , triacyl glycerols , free fatty acids , 1,3-diacylglycerols , 1,2-diacylglycerols , alcohols , sterols , 2-monoacylglycerols , and 1-monoacylglycerols ^^. Unsaponifiable lipid constituents of seed oils were studied by Saeed M. et. al^". Triterpenoid saponins from the seeds had been isolated by number of scientists'^ Triterpenoid saponins mimusopside A and B were isolated by Sahu, N.P. along with taxifolin (XXIX), a-spinasterol glucoside and Mi-glycoside'^. Triterpenoid saponins, mimusopin (3-0-p-D-glucopyranosyl-2p, 3(3, 6(3, 23-tetrahydroxyolean- 12-en-28-oic acid 28-0-a-l-rhamnopyranosyl-(1 -^ 3)- f3-D-xylopyranosyl - 42 (1-> 4)[a-L-rhamnopyranosyl-(1 -> 3)]-a-L -rhamnopyranosyl-(1 -^ 2)-a-L- arabinopyranoside) and mimusopsin 3-0-[(3-D-glucopyranosyl-(1^3)(3-D-gluco- pyranosyl]-2p, 3p, 6p, 23-tetrahydroxyolean-12-en-28-oic acid28-0-a-L- rhamnopyranosyl-(1 ^3)-|3-D-xylopyranosyl-(1 ^4)-a-L-rhamno pyranosyl-(1 -»'2)- a-L-arabinopyranoside were isolated by Sahu et. al ^^. Isolation of a triterpene, mimusic acid, 2p,3p,16a,23-tetrahydroxyoleana- 5,13(18)-dien-28-oic acid, from the seed was also reported^"*. Pentacyclic triterpenes, mimusopgenone (XXX) and mimugenone (XXXI), were isolated from the seeds by Sen et. al^^. A minor triterpenoid saponin, mimusin, {3-0-[p-D- glucopyranosyl-(1 ->6)- p-D-glucopyranosyl]-2p,3p,6[3,23-tetrahydroxyolean-12- en-28-oic acid 28-0-a-L-rhamnopyranosyl-(1^3)- f3-D-xylopyranosyl-(1->4)-a - L-rhamnopyranosyl-(1^2)- a-L-arabinopyranoside} was isolated from the seeds of the plant ^^ Another minor triterpenoid saponin, elengin {3-0-[|3-D-glucopyranosyl-(1^6)- (3- D-glucopyranosyl]-2p,3p,6p,16a,23-pentahydroxyolean-12-en-28-oic acid 28-0- a-L-rhamnopyranosyl-(1 ^3)- (3-D-xylopyranosyl-(1 -^4)-a-L- arabino-pyranoside} was also isolated from the seeds''^.Spasmolytic activity of saponins was studied on guinea pig ileum by Banerji et. al. The results showed that saponins having a triterpenoid moiety were more active than those with a steroid moiety ^^. The seeds were found to contain a mixture of triterpenoid glycosides^^. Pentahydroxy flavones 2,3-dihydro-3,3'4'5,7-penta hydroxyflavone and 3,3',4',5,7-pentahydroxyflavone isolated from the seeds have strong antibacterial activity against gram positive and gram negative bacteria'*". The bark was found to contain a-spinasterol (XXXII), taraxerone (XXXIII), taraxerol (XXXIV), sodium salt of ursolic acid (XXXV) and bitulinic acid, fatty acid ester of a-spinasterol (XXXVI), (3-D-glucoside of p-sitosterol, quercitol'*\ A small amount of alkaloid isolated from the bark consists largely of a tiglate ester of a base having a mass spectrum identical with that of laburnine and isoretronecanol'*^. Compounds isolated previously are displayed (Chart 1). Literature survey reveals that very few individual constituents have so far been isolated and characterized from the bark material. 43 The present work is undertaken to identify the molecules of therapeutic potential from the bark Chart 1 Previous worl< OH HO OH HO '• / \ OH o=Ay^V OH HO OH (I) (II) (III) Cr OH HO-^.<^\^0\.-^V>\, OH OH OH O Glu —0' 27 (IV) (V) (VI) (VII) (VIII) (IX) (X) (XI) 44 COOH GOGH (XII) (XIII) (XIV) OH' COOH CH3-[GH2}4-GO—G / OH HO CG%H (XV) (XVI) COOH OH OH (XVII) (XVIM) (XIX) COOH 0 HO^\[CH2]i2-CH3 (XX) (XXI) (XXII) 45 O O o HO^\[CH2]i4-CH3 HO \[CH2]i6-CH3 HO^ ^ [CH2]20-CH3 (XXIII) (XXIV) (XXV) CH3-[CH2]6-CH=CH-[CH2]7-COOH CH3-[CH2]7-CH=CH-[CH2]7-COOH (XXVI) (XXVII) O [CH2]i/ ^OH O OH (XXVIII) (XXIX) (XXX) o (XXXI) (XXXII) (XXXIII) ONa R= C16 or C18 fatty acid (XXXIV) (XXXV) (XXXVI) 46 1.1.9 Preliminary screening With regards to the medicinal importance of the bark, as mentioned earlier, preliminary studies for the detection of metal content, the presence of amino acids and carbohydrates was carried out for the first time. Metal analysis showed the presence of potassium, calcium, magnesium, zinc, copper and iron along with nitrogen and phosphorous. Interestingly high percentage of calcium was observed (Table 1)^. Acid soluble and insoluble ash was brought about 6 % and 0.9 % respectively. The crude powdered material was analyzed for fat, proteins and carbohydrates as per Indian Standard methods of test for animal feeds and feeding stuffs, Official methods of analysis and Indian Standards. It was found to be 0.38, 8.74 and 74.75 g/100g. Energy value was calculated and was furnished 337.38 Kcal/100g. It had significant energy content. The amino acids are being basic units of proteins, their presence was detected. Qualitative estimation of amino acids by paper chromatography showed the presence of glycine, tryptophan, proline, lysine, alanine and methionine (Table 2)^. The qualitative estimation of carbohydrates showed presence of different sugars like maltose, xylose, fructose, arabinose and dextrose. The detection of carbohydrates was carried out by using conventional method of paper chromatography. Steam Distillation Volatile organic materials, the products, secondary metabolites of the plants, are generally consisting of complex mixtures of mono-, di-, sesqui-, th-terpene hydrocarbons and oxygenated materials, biogenically derived from them. One of the ways by which essential oils or the volatile organic matter extracted from plant material is by performing steam distillation. Steam distillation of bark material was carried out to yield 0.18% of volatile organic matter. The volatile organic matter from the bark of this plant was analyzed for the first time using GC-MS. This is one of the best techniques to identify the constituents of volatile matter like long chain, branched chain hydrocarbons, alcohols, acids, esters etc. Use of GC-MS enabled identification of chemical constituents present in it which may be of therapeutic potential. The major constitutents were a-cadinol, x-muurolol, hexadecanoic acid, di-isobutyl phthalate, penta-decanoic acid etc. Their structures were confirmed by genesis.^ 47 Table 1 Percentage of minerals MINERAL PERCENTAGE METHOD Nitrogen 0.3300 Atomic absorption spectrometer Phosphorus 0.3300 Atomic absorption spectrometer Potassium 1.2500 Atomic absorption spectrometer Calcium 0.3900 Atomic absorption spectrometer Magnesium 0.0600 Atomic absorption spectrometer Zinc 0.0029 Atomic absorption spectrometer Copper 0.0014 Atomic absorption spectrometer Iron 0.0409 Atomic absorption spectrometer Aluminium 0.0074 Atomic absorption spectrometer Manganese 0.0052 Atomic absorption spectrometer Table 2 Amino acids Name of Amino Acid Rf for Standard Rf for Sample Amino Acid Tryptophan 0.63 0.64 Lysine 0.73 0.75 Methionine 0.74 0.75 Proline 0.75 0.75 Glysine 0.63 0.64 Alanine 0.76 0.75 Solvent system : Isopropyl alcohol: Ethyl acetate: Water (4: 4: 3) 48 1.2 Section I Isolation and Characterization of 3, 4 bis (1, 3-benzodioxol-5-yl methyl) tetrahydrofuran-2-ol - Compound 1 - Cubebin 1.2.1 Lignans Compound 1, cubebin, isolated from the bark belongs to ligans. Lignans are phyto-nutrients, a class of plant compounds beneficial to human health but not classified as vitamins. Lignans are a normal part of a healthy diet and widely distributed in foods and plants in small amounts. The most common dietary sources of lignans are unrefined grain products, seeds such as sesame and flaxseed and berries, fruits and vegetables. The fhendly bacteria in our intestine convert plant lignans into the "human" lignans, primarily enterolactone, that have a weak estrogen like activity. Estrogens are small molecules responsible for controlling of many reactions in the body. When there are low estrogen levels in the body these weak lignan 'estrogens' make up some of the insufficiency; when natural estrogen is abundant, the lignan 'estrogens' appear to reduce the activity of natural estrogen hormones by occupying the cellular estrogen-binding sites. As a result, recent research has shown that plant lignans may influence the development of tumors such as breast, prostate and colon cancers that depend on hormones to start and progress"*^ ' '*^. Lignans may also support good cardiovascular health'*^ and help to moderate other estrogen dependant conditions such as menopausal symptoms'*^ and osteoporosis''^ 1.2.2 Review of Literature A bioactive lignan 3,4 bis(1,3-benzodioxol-5-yl methyl)tetrahydrofuran-2-ol, (cubebin), was isolated previously from Aristolochia odoratissima'^^' ^°. The secondary metabolite in the leaves of Piper cernuum ^\the roots and stems of Aristolochia triangularis^^, Aristolochia elegans^^, the leaves of Justicia hyssopifolia ^'*, roots of Aristolochia cymbifera ^^, Drymis winterii ^^ , mature seeds and seedlings (roots) of Virola venosa ^^ , the seeds of Virola surinamensis ^^, stems and roots o^ Aristolochia pubescens ^^, leaves, bark and wood of Zanthoxylum monophyllum ^°, Virola multinervia:;^^Sspf^s ^""'^^ , seeds 49 of Piper cubeba ®^, Tarenna madagascariensis ^'*, Aristolochia constricta, 65 roots and leaves o\ Aristolochia malmeana ^^ produce the lignan cubebin. 1.2.3 Present Work Plant Material The plant material is collected from the local market of Pune, Maharashtra state, India. It is authenticated at Agharkar Research Institute, Pune, India. Its authentication number is AHMA S/B - 065. Air shade dried powdered bark material is extracted with soxhiet extractor using different solvents like hexane, chloroform, ethanol and methanol for 18 hours. During the removal of hexane under reduced pressure, white solid is separated out. Purification of this solid is carried out using mixed solvent system of chloroform-hexane. Repeated crystallization gives fine needles of 3, 4 bis -(1,3- benzodioxol-5-yl methyl) tetrahydrofuran-2-ol (Compound 1), cubebin, which has been isolated for the first time from this plant material. Compound 1 1.2.4 Results and Discussion Compound 1 is isolated as white crystalline needles, obtained by repeated crystallization using mixed solvent system of chloroform - hexane. It exhibits a sharp melting nature at 130 °C. The LC-MS on negative mode has exhibited a molecular ion peak at 355 amu. While EIMS (Fig 1) at m/z [338 + 18]'' due to removal of water molecule from the said compound. This indicates that the molecular formula is to be C20H20O6. Fragmentation pattern (FP 1) is in agreement with the structure. 50 IR spectrum (Fig 2) is displayed a characteristic broad hydroxy band at 3336 cm"\ aromatic stretching at 1609 cm'^ and 1485 cvn'\ The absorption at 1240 cm'^ and 1038 cm'^ is noticed for C-0 stretching. ^H NMR spectrum (Fig 3, Table 3) has indicated multiplets at 5 2.32 (1 H) for C-4 proton, 5 2.41 (1 H) for C-3 proton, 6 2.61 (4 H) for C-1' and C-1" protons. Triplets at 5 3.56 (1 H, J= 6 Hz) and 5 4.10 (1 H, J= 6 Hz) are observed for C-5 methylene protons. A broad singlet at 5 5.22 (1 H) is noticed for C-2 anomahc proton. A sharp singlet at 5 5.92 (4 H) is specified for the methylene dioxy protons at C-4' and C-4". A multiplet at 5 6.56 (2 H) is observed for C-3' and C-3" aromatic protons. A multiplet at 5 6.62 (2 H) is indicated for C-6' and C-6" aromatic protons. A doublet at 5 6.72 (2 H, J= 8 Hz) is observed for C-5' and C-5" aromatic protons. ^^C NMR spectrum (Fig 4, Table 4) has displayed 13 signals accounting for 20 carbon atoms. The signals at 6 147.57, 5 145.93 and 5133.31 are assigned for tetra substituted carbon atoms (3'a & 3"a), (5'a & 5"a) attached to methylene dioxy ring and C (2' & 2") of aromatic ring respectively. The downfield doublets at 6 121.39, 5 108.93 and 5 108.10 are noticed for the C (6' & 6"), C (5'&5") and C (3' & 3") aromatic carbons respectively. The doublet at 5 103.40 is observed for C-2 methine carbon atom flanged ny two oxygen atoms. The up field doublets at 5 53.15 and 5 45.91 are observed for C-3 and C-4 carbon atoms. The most downfield triplet flanged by two oxygen atoms is observed at 5 100.82. Another downfield triplet at § 72.29 is noticed for C - 5 carbon atom. The upfield triplets attached to oxygen at 5 39.25 and 5 33.64 are assigned for C-1' and C-1" methylene carbon atoms respectively The nature of carbon signals are determined by DEPT (Fig 5) experiment. It has assigned the presence of nine methine, five methylene and (by difference) six quaternary carbon atoms. Table 3 ^H NMR of the Compound 1 (CDCI3 at 400 MHz) Protons Chemical shift in ppm- 5 H4 2.32 (m, 1 H) H3 2.41 ( m, 1 H) H r&HI" 2.61 (m, 4H) H5 3.56 and 4.10 (t, 1 H, J= 6 Hz) H2 5.22 ( br. S, 1 H) H 4' & H 4" 5.92 ( s, 4 H) H 3' & H 3" 6.56 (m ,2 H) H6'&H 6" 6.62 ( dd, 2 H, J=6 & J=4 Hz) H5'&H 5" 6.72(d,2H, J = 8Hz) Table 4 '13X NMR of the Compound 1 (CDCI3 at 100 MHz) Atom No. Chemical shift in ppm- 5 C 1" 33.64 (t) c r 39.25 (t) C 4 45.91 (d) C 3 53.15 (d) C 5 72.29 (t) C 4' & C4" 100.82 (t) C 2 103.40 (d) C 3' & C 3" 108.10 (d) C 5' & C 5" 108.93 (d) C 6' & C6" 121.39 (d) C 2' & C 2" 133.31 (s) C 5'a & C 5"a 145.93 (s) C 3'a & C 3"a 147.57 (s) 52 X-Ray Analysis Single crystals of the compound 1 were grown by slow evaporation of the solvent hexane. Colourless needles of the compound with approximate size 0.26 x 0.05 X 0.03 mm were used for data collection. Exposure / frame = 20.0 sec / frame, Total scans = 3, total frames = 1598 , 9 range = 2.14 to 23.49 °, completeness to e of 23.49 ° is 99.8 %. SADABS correction applied, C20H20O6, M = 356. Crystals are belong to Monoclinic, space group P2i, a=11.629(2), b= 5.6076(10), c=13.587(3) A,V= 873.7(3) A^ Z = 2, Dc = 1.355 g /cc /v (Mo-Kp) = 0.094 mm"\ 4927, reflections measured, 2573 unique [l>2a(l)], R value 0.0665, wR2 = 0.1507. Largest difference peak and hole 0.167 and -0.149 e. A "^ ORTEP diagram of the molecule (Fig 6) is drawn at 40 % probability. Five-member ring adopts envelop conformation. Substituents at C 9 and C 12 on five-membered ring are trans to each other. X-ray analysis revealed that there are 2 molecules in the unit cell (Fig 7). Data collection and refinement parameters (Table 5) are listed. Fig 6 ORTEP diagram of Compound 1 Fig 7 Molecules In the unit cell 53 Table 5 Crystal data & structure refinement parameters for Compound 1 Empirical formula C20H20O6 Formula weight 356.36 Temperature 296(2) K Wavelength 0.71073 A Crystal system Monoclinic Space group P2i Unit cell dimensions a = 11.629(2) A a = 90° b = 5.6076(10) A [3 = 99.573(3)° c = 13.587(3) A Y=90° Volume 873.7(3) A^ Z 2 Calculated density 1.355 g/cc Absorption coefficient 0.100 mm"^ F(000) 376 Crystal size 0.26x0.05x0.03 mm 9 range for data collection 2.14 to 23.49° Limiting indices -11<=h<=13,-6<=k<=6,- 15<=l<=15 Reflections collected / unique 4927/2573 [R(int) = 0.0536] Completeness to 6 = 23.49 99.8 % Max. and min. transmission 0.9968 and 0.9748 Refinement method Full-matrix least-squares on F^ Data / restraints / parameters 2573/1 /236 Goodness-of-fit on F^ 1.025 Final R indices [l>2sigma(l)] R1 =0.0665, wR2 = 0.1217 R indices (all data) R1 =0.1342, wR2 = 0.1507 Absolute structure parameter 3(3) Largest difference peak and hole 0.167 and-0.149 e.A"^ 54 1.2.5 Conclusion Considering the medicinal importance of the plant, chemical investigation carried out from the bark material, produce a compound of therapatic potential - cubebin. It has been isolated for the first time from M. elengi. The compound is found to be active against 4'*^ instar larvae of Aedes aegypti (L) . Such entomological activity has been observed for the first time for this plant. The entomological activity study of compound 1 and various extracts is discussed in detail separately in Chapter 3. 1.2.6 Experimental Air shade dried powdered bark material (300 g) was extracted with soxhiet extractor. The extraction was carried out using different solvents like hexane, chloroform, ethanol and methanol successcesively for 18 hours. The solvents were recovered under reduced pressure to get their respective extracts. The yield of hexane, chloroform, ethanol and methanol extract was found to be 1.36, 1.33, 8.59 and 1.43 % respectively. During the removal of hexane under reduced pressure, white solid was separated out. Purification of this solid was carried out using mixed solvent system of chloroform-hexane coupled with preparative thin layer chromatography. The compound was further purified by preparative thin layer chromatography. Repeated crystallization of the compound was carried out using n-hexane. It gave colourless fine needles of 3, 4 bis (1, 3-benzodioxol-5-yl methyl) tetrahydrofuran-2-ol. Compound 1 White crystalline solid, Molecular Formula C20 H20 Oe, M.P. 130 °C, IR: (KBr) cm"^: 3336, 1609 , 1485 ,1240 , 1038 LC-MS : m/z 355 [M-^]\ 55 1.2.7 Spectral Data of Compound 1 100-, Ji, ° iiiii "• L, i r '?" ''•' "" in :« :3i :" ^"O'"' -" ?« ^i- «>• , '•' >-•" ^«" •"' '" •" iiir.|iili|.,i|i„i|.ii^.i,|il|i|Ji,i.ii|li«i..i|.ii.i.ii|iii.!iii|ii(i|.iiif.niiiiifiii|,ij|im|liiifiii|iii.fiii,i.!|i.iirf^i.i|...fi.l.ilii, j|.i,i,iiifi,.iif...riivin...i|iiiiin.|i..i.."|iitTT» Fig 1 Mass Spectrum Fig 2 IR Spectrum s i i i i i IV I 5" 1- 1' 3' o3- i I _. h _ A '••,,/\,A^/ 1 ( H "' ~ i) " ' Fig 3 ^H NMR Spectrum 56 \mmiimimMfimiUJ i¥*^m lH Fig 4 ^^C NMR Spectrum Fig 5 DEPT Spectrum • 2 e' transfer / Ij /?;/r-338 / single e" tran^Ter <°Xf0 i p 0 % = Fragmentation Pattern (FP 1) 57 1.3 Section II Isolation, characterization and hydrolysis study of an aromatic ester by LC- MS 1.3.1 Phthalates Phthalates are widely used as the additives in many plastics and consumer products ^^' ^^. Some of them are bis-(2-ethylhexyl) phthalate, diethyl phthalate, diisobutyl phthalate, dibutyl phthalate etc. Dibutyl phthalate is used for cellulose plastics, food wraps, adhesives, perfumes and also in cosmetics ^°. R.N.Roy et. al. reported the strong bioactive nature of it against bacteria as well as unicellular and filamentous fungi ^\ It possesses a new pharmacological activity leading to selective deterioration of leukemic cells with less harmfull effect on the growth of normal hemopoietic progenitors ^^. About the toxicity of phthalates to humans, re-evaluations of the risks of phthalates were conducted by U.S. state governments and European authorities. The rules in the form of laws have been implemented for public health. Analysis of all of the available data leads to the conclusion that the risks are low, even lower than originally thought, and that there is no convincing evidence of adverse effects on humans. Since the scientific evidence strongly suggests that risks to humans are low, phthalate regulations that have been enacted are unlikely to lead to any marked improvement in public health ". 1.3.2 Review of Literature Various phthalates are present in nature. Its presence is reported from soil bacteria, marine algae, fungi and plants. Dibutyl phthalate has been isolated previously from mahne algae^'*'^^, bacteria^^' ^^ and fungi^^. It has also been produced as a secondary metabolite of the plant. Roots of some plants like Achyrathes bidentata ^^ and Rheum glabricaule ^° contain dibutyl phthalate. The leaves of "Mironovskaya 808" wheat ^\ Alstonia scholaris ^^ and the Torreya grandis ^^ produce dibutyl phthalate. The neutral essential oil from the pericarp of Trichosanthes rosthornii \Nas also found to contain dibutyl phthalate ^'*. 58 1.3.3 Present work Air shade dried powdered bark material is extracted with soxhiet extractor using different solvents like hexane, chloroform, ethanol and methanol for 18 hours. The solvent is removed under reduced pressure to get their respective extracts. Chromatographic separation of the components from chloroform extract of the M. elengi bark has been carried out. The presence and isolation of dibutyl phthalate has been reported for the first time*^. The structure of the isolated compound is elucidated by spectral analysis (MS, ^H-NMR, ^^C-NMR, DEPT etc.). Saponification of the isolate and examination of the resultant products led to the conclusion that the compound is dibutyl phthalate. Compound 2 1.3.4 Results and Discussion The compound isolated is a colourless transparent liquid. The mass spectrum (Fig 8) of the compound exhibits a molecular ion peak at m/z 279 [m+l]"" , which suggestes the molecular formula C16H22O4. Fragmentation pattern (FP2) is in agreement with the structure. IR spectrum (Fig 9) displays a characteristic absorption frequency at 1728 cm"^ for ester carbonyl. The absorption at 1600 cm"^ and 1579 cm'^ is noticed for aromatic stretching. The absorption at 1124 cm"^ and 1074 cm'^ is noticed for C-0 stretching. ^H NMR spectrum (Fig 10, Table 6) has displayed an upfield triplet at 8 0.98 (6 H, J=5 Hz) for C 5' and C 5" methyl protons. The multiplets at 5 1.47 (4 H) and § 1.74 (4 H) are observed for C (4' & 4") and C (3' & 3") methylene protons respectively. A down field triplet at 5 4.33 (4 H, J = 5 Hz) is noticed for C 2' and C 2" methylene protons. The doublet of doublets at 5 7.56 (2 H, J = 10 and 5 Hz) 59 and 5 7.73 (2 H, J = 10 and 5 Hz) are indicated for C (3 &4) and C (2&5) aromatic protons respectively. The ^^C NMR spectrum (Fig 11, Table 7) displays eight signals accounting for sixteen carbon atoms. A quartet at 6 13.7 is assigned for C - 5' and C - 5" methyl carbon atoms. The triplets at 6 19.18, 5 30.59 and 5 65.55 are noticed for C (4' & 4"), C (3' & 3") and C (2' & 2") methylene carbons respectively. The downfield doublets at 5 128.84 and 5 130.89 are observed for C (2 & 5) and C (3 & 4) aromatic carbon atoms. A singlet at 5 132.35 is indicated for C-1 and C-6 tetrasubstituted aromatic carbon atoms. The most downfield singlet at 6 167.69 is assigned for C-1' and C-1" carbonyl carbon atoms. The DEPT pulse sequence (Fig 12) indicates six signals accounting for 12 carbon atoms. Thus it reveales the presence of four methine, six methylene, two methyl and four quaternary carbon atoms. Saponification of compound 2 using HPLC - LC-MS method indicates various mass fragments which are presented in Scheme I. These fragmients of the compound 2 establish the structure of the molecule, an aromatic diester, dibutyl phthalate. Conventional fragmentation pattern (FP2) also makes an apperaence for the presence of dibutyl phthalate. The information obtained from chromatographic profiles is displayed (Fig 13). It clearly indicates emergence and increase of new peaks for hydrolytic products and decrease of the peak for the compound. The LC-MS chromatograms for the hydrolytic products are indicated (Fig 14). LC - MS of the hydrolytic degradation products are reported (Table 8). 1.3.5 Conclusion A bioactive molecule, dibutyl phthalate has been isolated for the first time from this plant. The strong bioactive nature of the molecule is against gram positive and gram negative bacteria as well as unicellular and filamentous fungi. It is also found to be an a-glucosidase inhibitor. It has many other applications like in cosmetics, cellulose plastics, food wraps, adhesives, perfumes etc. 60 7} 2 e transfer 0 C15H2204 C13H16O4 m/z=278 m/z=236 O o C15H2204 m/z^ 278 1^ 1^ H C10H10O4 C16H22O4 m/2=194 m/z= 278 C12H14O4 C9H8O4 y m/z= 222 m/z= 180 Scheme I 1.3.6 Experimental Extraction and Isolation Air shade dried powdered bark material (300 g) was extracted with soxhiet extractor using different solvents like hexane, chloroform, ethanol and methanol for 18 hours. The solvents were removed under reduced pressure to get the respective extracts. Chloroform extract (1.33 %, 4.018 g) was further purified. Broad fractionation of the crude chloroform extract (4.0 g) was carried out using gradient polarity solvents on silica gel (60- 120, 160 g) to get ten fractions. Fractions were monitored by thin layer chromatography. The details are given (Table 9). Fractions 3 and 4 were mixed together (1.2 g) for re-column chromatography using gradient polarity solvents on silica gel (60- 120, 60 g) and total nine fractions (A to I) were collected. Fractions were monitored by thin layer chromatography. The details are given (Table 10). Fraction B (180 mg) was fractioned using hexane: ethyl acetate with increasing percentage of ethyl acetate over silica gel (60- 120, 50 g) to obtain four major fractions (B-l to B-IV) to yield an impure compound 1. Details are given (Table 11). The compound is further purified by preparative TLC. The yield of pure compound obtained was 0.95 % as a liquid Saponification by HPLC- LC - MS Saponification was performed using HPLC -LC-MS instrument. The Instrumental conditions are as under: HPLC Column YMC ODS - A ( 5 cm X 4.5 cm X Sp) A = 0.1 % TFA in H2O B = 0.1 % TFA in acetonithle Gradient = 5 % B to 90 % B in 8 min hold for 2min Injection volume = 3 pL ; Column oven temperature = 30 °C ; Flow rate = 1.0 ml/min LC-MS Conditions : Scan mode = Positive ; CDC temperature = 250 °C ; Heater block = 200 X ; Detector voltage = 1.5 kV ; Scan speed = 1000 amu / sec ; Nebulizing gas flow = 1.5 L / min ; Ionization mode = ESI (Atmospheric Pressure Ionization ) Approximately 100 pg of compound was dissolved in 100 pL of methanol and diluted to 1000 pL with 0.1 N NaOH. The sample was injected in LC-MS system at 0, 1, 2, 4, 6 and 8 hours time intervals. The degradation products were analyzed by mass spectra. Compound 2 Colourless transparent liquid , Molecular Fo rmula C16H22O4 b.p. > 280 °C, IR : 1728 , 1600 , 1579 , 1464, 1385, 1285, 1124 , 1074 cm"^ LC-MS -.m/z 279 [m+1]^ 62 Table 6 ^H NMR of the Compound 2 (CDCI3 at 500 MHz) Protons Chemical shift in ppm- 5 H 5' & H 5" 0.98 (f, J=5 Hz, 6 H) H 4' & H 4" 1.47 (m, 4 H) H 3' & H 3" 1.74(A77, 4H) H 2' & H 2" 4.33 {t, J=5 Hz, 4 H) H3&H4 7.56(dd, J=10, 2Hz, 2 H) H2&H5 7.74(dc/, J=10, 2Hz, 2H) Table 7 'X NMR of the Compound 2 (CDCI3 at 125 MHz) Atom No. Chemical shift in ppm- 5 C 5' & C 5" 13.70 (Q) C 4' & C 4" 19.18(f) C 3' & C 3" 30.59 it) C 2' & 0 2" 65.55 (0 C2&C5 128.84(d) C3&C4 130.59(d) 01 &C6 132.35 (s) C1'&C1" 167.69 (s) Table 8 LC - MS of the Hydrolytic Degradation Products Degradation Products Retention Time ( minutes ) miz Values of Fragments [M+lf 1 9.5 279 II 8.2 237 III 6.7 223 IV 6.4 195 V 4.8 181 Table 9 Chromatographic separation of chloroform extract Fraction Eluent Volume Weight of Approximate No. Collected the fraction composition (ml) (g) 1 Hexane 250X2 0.025 Mixture of unidentified compounds. 2 Hexane: Toluene 250X2 0.035 Mixture of unidentified (50:50) compounds. 3 Toluene 250X6 0.029 Mixture of unidentified compounds + compound 2 4 Toluene: Ethyl acetate 250X2 1.189 Mixture of unidentified (75:25) compounds + compound 2 5 Toluene: Ethyl acetate 250X8 0.073 Mixture of unidentified (75:25) compounds. 6 Toluene: Ethyl acetate 250X6 0.367 Mixture of unidentified (50:50) compounds. 7 Toluene: Ethyl acetate 250X2 0.042 Mixture of unidentified (25:75) compounds. 8 Ethyl acetate 250X2 0.383 Mixture of unidentified compounds. 9 Ethyl acetate: Ethanol 250X2 0.012 Mixture of unidentified (50:50) compounds. 10 Ethanol 250X2 0.153 Mixture of unidentified compounds. 64 Table 10 Rechromatography of fraction (3+4) Fraction Eluent Volume Weight of Approximate No. Collected the fraction composition (ml) (miligram) A Toluene 100X19 141 Mixture of unidentified compounds. B Toluene: Ethyl acetate 100X2 180 Mixture of unidentified (90: 10) compounds + compound 2 C Toluene: Ethyl acetate 100X13 317 Mixture of unidentified (90: 10) compounds. D Toluene: Ethyl acetate 100X8 125 Mixture of unidentified (80: 20) compounds. E Toluene: Ethyl acetate 100X6 154 Mixture of unidentified (70: 30) compounds. F Toluene: Ethyl acetate 100X6 70 Mixture of unidentified (50: 50) compounds. G Toluene: Ethanol 100X6 10 Mixture of unidentified (80: 20) compounds. H Toluene: Ethanol 100X4 10 Mixture of unidentified (50: 50) compounds. 1 Ethanol 100X2 7 Mixture of unidentified compounds. 65 Table 11 Rechromatography of fraction B Fraction Eluent Volume Weight of Approximate No. Collected the fraction composition (ml) (miligram) B-l Hexane 100X8 8 Mixture of unidentified compounds B-ll Hexane : Ethyl acetate 100X10 70 Mixture of unidentified ( 95 : 5 ) compounds + compound 2 B-lll Hexane : Ethyl acetate 100X10 37 Mixture of unidentified (90 : 10) compounds B-IV Hexane : Ethyl acetate 100X6 27 Mixture of unidentified (85:15) compounds 66 1.3.7 Spectral Data of Compound 2 Fig 8 Mass Spectrum Fig 9 IR Spectrum 06 -^ \^ _1UIL^ ! ' ' ' ' 1 ' ' ' M ' I ' ' 1 ' ' ' ' 1 ' Fig 10 ^H NMR 67 Fig 12 DEPT [M + 1] 279 I [M + 231 301 m/z = 278 (25%) Co y V-' III ^ single e" y 1 + O—CH2-CH2-CH2-CH3 transfer O O o O m/z = 205 O (60%) +^0-^^^H2^C-CH2-CH3 2 e' tran^r + 0-H m/z = 149 (100%) Fragmentation Pattern (FP 2) 68 rnAU -,... - —_ ^.._.—_ — —— 4000 3000- 1 1 2000- 1 1000 1 i^ _ -_._ .__.i I J._„J 0- JL I IPDA Multi 1 0.0 2.5 5.0 7.5 10.0 min mAU 40OO '5 'i f 3000 1 1 . 2000 i 1 j 1 i ; 1 it 1 lOOO i 11 { 1 1 1 i : i 11 I _ . '•- - - - j O ' ., —" IPDA Multi 1 0.0 2.5 5.0 7.5 10.0 "^"4000 - I ^ 1 ,1 3000 5 i 1 1 3 2000 1 1 j 1 J 1 i 1000 i 1 1 1 .•J i y 3 i ^ 0 1 .1 .Ji! i... »,- . - 1; ; IPDA Multi 1 "•"T" """" I 0.0 2.5 5.0 7.5 10.0 min Fig 13 Chromatographic profile of saponified ions of Compound 2 69 100 l«0-95 0 l.?3.05 ^^i=^'^^N^"\ ^ _,,.,.„ 190.05 0 100 200 300 400 500 600 700 800 900 in / z 100 194.95 162.95 c4c: 299.00 11 344.15 . .. J - 1-. ^. 100 200 300 400 500 600 700 800 900 m/z 100 223.00 oo nn 148.90 0 °-'"" • . 264.00 100 200 300 400 500 600 700 800 900 m/z 100 237.00 162.95 74.05 194.95 304.25 .a J,.-...... ii r ,J.- 100 200 300 400 500 600 700 800 900 m/z 100 27^.05 399.10 148.90, 324.05 I 467.35 600.35 ^^'^•^^ .... _...J.,.i..d,_i .- 100 200 300 400 500 600 700 800 900 m/z Fig 14 IVIass profile of saponified ions 70 1.4 Section III Quantification of cubebin and dibutyl phthalate by HPTLC 1.4.1 Introduction The plant based medicines are being used globally as home remedies and offering a broad spectrum of activity since ancient times. The drug activity depends upon the several active compounds and components present in it. Quality standards of the herbal drugs can be achieved through systematic evaluation of plant material using modern analytical techniques. HPTLC is very important, essential and viable tool for qualitative and quantitative analysis of herbal products. As per proverb "Nothing works - TLC works", this universally applicable visual chromatography is based on validated methods and globally accepted ®^. 1.4.1 Review of literature Cubebin Cubebin possess various biological activities. Anti-inflammatory, Analgesic and antipyretic activity of cubebin ^^' ^^ and its derivatives ^^"^° are reported by different group of scientists. The trypanocidal activities of lignan compounds obtained by partial synthesis from cubebin, which was isolated from the seeds of Piper cubeba, were evaluated against free amastigote forms of Trypanosoma cruzF\ A lignan profile of Piper cubeba containing cubebin was determined using GC, GC-MS and HPLC^^. Cubebin is also found to possess histamine release inhibitory activity ^^. The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated by Juliana Saraiva et. al ^'*. Literature survey revealed that there are no reports available for quantification of cubebin using any analytical tools. Dibutyl phthalate (DBP) A gas chromatographic method for the identification and quantification of dibutyl phthalate from sediments and biota from estuarine environments was reported^^. A sensitive and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an 71 antibody-coated plate format. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water and leachate from plastic drinking water bottles ^^. Identification and quantification of phthalates from the underground waters, stream waters, spring water and tap water from the Zagreb area were performed by the method of gas chromatography (GC-ECD) ^^. Detection and quantification of phthalates in commercial whole milk were performed by gas chromatography coupled to mass spectrometric (GC-MS) detection using an appropriate surrogate (4-n-nonylphenol) and internal standard [deuterated bis (2-ethylhexyl) phthalate] ^^. Literature survey also revealed no reports on quantification of dibutyl phthalate using HPTLC. Keeping the above facts in view and considering the wide applications of cubebin and dibutyl phthalate attempts have been made to develop HPTLC method, which is simple and reliable for their quantification in crude extracts. 1.4.2 Present Work A simple High Performance Thin Layer Chromatographic (HPTLC) method has been developed for the analysis and quantification of cubebin and dibutyl phthalate present in different extracts and their fractions of M.eleng bark. A suitable solvent system is acquired by attempting various mobile phases on pre-coated aluminium plates (Silica gel Merck 60 F254) for quantification of analytes. An appropriate mobile phase is found as toluene: ethyl acetate (9.5:0.5) for both the compounds. The densitometric determination is carried out after derivatization with anisaldehyde sulphuric acid reagent for cubebin and vanilline sulphuric acid reagent for dibutyl phthalate. The plates are scanned at 527 nm by absorption / reflection mode. The amount of cubebin and dibutyl phthalate in the extracts has been estimated by comparing the peak area with the standards. The proposed High Performance Thin Layer Chromatographic method is found to be simple, faster and reliable for quantification of analytes. 72 1.4.3 Results and Discussion Different mobile phases were tested and the desired resolution was achieved by toluene: ethyl acetate 9.5:0.5 v/v. The spots are visualized after spraying the appropriate reagents for the both. The maximum absorption of the standard peaks are compared with the spots of the extracts. Calibration cun/es of the standards, cubebin (Fig 15) and dibutyl phthalate (Fig 16) are obtained by plotting peak areas against concentration applied. HPTLC scans for standard cubebin (Fig 17) and dibutyl phthalate (Fig 18) are displayed. HPTLC quantification of cubebin (Fig 19) and dibutyl phthalate (Fig 20) in test samples are indicated. Equation of calibration curve for cubebin is Y= 5167.5x - 2260.3. The correlation coefficient is found to be 0.9669 and thus it exhibits good linearity between concentrations and area. The amount of cubebin in the extract HSE is found to be 52.23 mg/g while in ACE and EA it is 46.15 and 42.65 mg/g of extract respectively. Cubebin content of fractions Fri, Fr2 and Fr3 are found to be 58.15, 54.25 and 67.13 mg/g of extract respectively while Fr4 does not indicate presence of cubebin. Equation of calibration curve for dibutyl phthalate is Y= 323.43x + 1445.4. The correlation coefficient is found to be 0.9806 and thus it exhibits good linearity between concentration and area. The amount of dibutyl phthalate in the extract HSE is found to be 13 mg/g of extract. Amount of dibutyl phthalate in Fr6 is found to be 24.5 mg/g of extract. Dibutyl phthalate is isolated from this fraction. 1.4.4 Conclusion The proposed HPTLC method is found to be simple, rapid, accurate and reproducible for the estimation of cubebin and dibutyl phthalate in M. eleng bark. 1.4.5 Experimental Preparation of Standard A stock solution of standards (1 mg / ml) was prepared by dissolving 10 mg of accurately weighed sample in methanol and making up the volume up to 10 ml. The stock solution was further diluted with methanol for working standard solution of appropriate concentration (0.2 mg/ml). 73 Chromatographic conditions Stationary Phase: Pre-coated silica gel plates Merck 60 F254 (10x 10, 0.2 mm thickness) Experimental conditions: Temperature 25± 2° C Relative humidity: 40% Mobile Phase: Toluene: Ethyl acetate (9.5:0.5 v/v) Spotting device: Linomat III Automatic sample spotter, CAMAG Development Mode: CAMAG twin trough chamber, CAMAG Densitometer: TLC Scanner III, CATS software, CAMAG Calibration curve of the standard Standard solution of cubebin and dibutyl phthalate (1pg to 5 pg per spot) was applied separately on precoated silica gel 60 F254 HPTLC plates (E. Merck), of uniform thickness, 0.2mm. The plates were developed in a solvent system of toluene: ethyl acetate (9.5:0.5 v/v) in CAMAG twin trough chamber up to a distance of 8 cm. After development the plates were dried in air and sprayed by using anisaldehyde sulphuric acid reagent for cubebin and vaniline sulphuric acid reagent for dibutyl phthalate. The plates were subsequently activated at 100°C for derivetization. These plates were scanned at 527 nm absorbance / reflection mode using reflectance mode by CAMAG Scanner III and CATS software was used to analyze the plates. The peak areas were recorded. Respective calibration curves were prepared by plotting peak area against concentration of cubebin and dibutyl phthalate. Sample preparation Air shade dried powdered bark material (300 g) was extracted with soxhiet extractor. The extraction was carried out using solvents hexane (HSE, 1.36 %) and chloroform (CSE, 1.33%) successively for 18 hours. The plant material (100 g) was also refluxed with acetone (ACE, 8.83 %) and ethyl acetate (EA, 11.6 %) separately for 18 hours. Solvent was removed under reduced pressure to obtain their respective crude extracts. The crude ethyl acetate extract (EA) was broad fractioned on silica gel (60-120, 10 g) using n-hexane (Fr1, 26.6%), Hexane: ethyl acetate (Fr2, 9:1, 26.78%), Hexane: ethyl acetate (Fr3, 8:2, 9.88%) and the residual methanol fraction (Fr4, 16.23 %). 74 The solvents were removed under reduced pressure to get their respective extracts. The crude chloroform extract (CSE) was further fractioned using various solvents like hexane (Fr5, 6.72%), toluene (Fr6, 56.30%) and the residual matter (Fr7, 25.2%). HPTLC quantification of the extracts: The extracts were accurately weighed and stock solutions of 10 mg/ ml were prepared. Theses stock solutions were further diluted with methanol to get solutions of 1 mg/ml. 20 pi per spot of these solutions were applied on to a precoated silica gel (60 F254 ) HPTLC plates in triplicates. The plates were developed by ascending mode to a distance of 8 cm and scanned as per above mentioned conditions. The cubebin (Compound 1) and dibutyl phthalate (Compound 2) content of various extracts and their fractions were determined by comparing the peak area of chromatogram with the calibration curve of working standards. The Rf value of standards were compared with the Rf value of the extracts. The average content of the cubebin and dibutyl phthalate in different bark extracts were expressed in mg per g of the extract. 3300 16000 = 323.43x •H45.4 y = 5167.5x-2260.3 3100 R'=0,9806 14000 f? = 0.9669 12000 2900 10000 2700 8000 2500 6000 2300 2100 4000 1900 2000 1700 0 0.5 1.5 2.5 3.5 1500 Fig 15 Fig 16 Calibration curve of Cubebin Calibration curve of DBF 75 Fig 17 Fig 18 HPTLC Scan (Standard Cubebin) HPTLC Scan (Standard DBP) Figure 19 Figure 20 Quantification in test samples Quantification in test samples (cubebin) (DBP) Fig 19: [1] HSE [2] EA [3] Fr1 [4] Std [5] Fr2 [6] Fr3 [7] Fr4 [8] ACE Fig 20: [1] Std [2] HSE [3] Fr5 [4] Fr6 [5] Fr7 76 References 1. Medicinal Plants, P. P. Joy, J. 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