CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW 1.1 INTRODUCTION Early man had to rely directly on nature for all his needs because he had no other means to cater for these needs. His food, shelter, clothing, weapons for defence were all provided for by nature. In addition, he got his health and cure needs from nature –this marked the beginning of medicine. Primitive man had used materials from plants, animals and minerals to cater for his health, however, plant materials have been used the most. Although, many of the earliest practices of medicine have been found to be inappropriate (for example, the use of Catharanthus roseus in the treatment of diabetes), many others have been proven to be appropriate and are still used in modern medicine. Examples include the use of Digitalis leaves for the treatment of heart failure, leaves of Ocimum gratissimum for its anti-diarrhoeal property, barks of Cinchona and leaves of Artemisia annua for the treatment of malaria. How primitive man got to know what plants could be used for particular conditions is not sure, but it is not unlikely that he got the knowledge by intuition, trial and error or observing other animals that had consumed these plants (Sofowora, 2008). 1.1.1MEDICINAL PLANTS A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs. A good number of drugs prescribed by orthodox medical practitioners were first isolated from plant 1 and many of these drugs are still obtained from their natural sources. Examples include: dtubocurarine, obtained from Chondrodendron sp.; vinblastine and vincristine from Catharanthus roseus; atropine and hyoscine from Atropa belladonna leaves; ergot alkaloids from Claviceps purpurea; digoxin and digitoxin from Digitalis purpurea L. leaves; reserpine from Rauwolfia vomitoria or R. serpentina root. However, some are now prepared synthetically for commercial purpose. An example is papaverine, formerly obtained from Papaver somniferum poppy (Sofowora, 2008). Due to the enormous number of drug that have been discovered from plant, and the current upsurge in drug resistant diseases, the search for novel bioactive compounds and alternative sources of known medicinally useful compounds continues. 1.1.2 THE NEED FOR STANDARDIZATION AND QUALITY CONTROL Of the major criticism levied against the use of herbal medicines, the most important are standardization and quality control (Sofowora, 2008). It is already known and well accepted that both standardization and quality control begins from collection of the raw materials. Various factors can affect the composition and proportion of active principles in plant materials. These factors include the species, the geographical location, climatic conditions/seasonal variations, age of the plant and time of collection, hence, the need for standardization and quality control. According to regulatory guidelines and pharmacopoeias; macroscopic and microscopic evaluation, chemical profiling as well as selection of chemical markers are important for quality control and standardization of botanicals. 1.1.2.1 CHEMICAL PROFILING Chemical profiling is the first task in the standardization of traditional medicines in China (Liang et al., 2014). This can be adopted worldwide. The existence and variability of the constituents of 2 botanicals makes it difficult to evaluate their safety and efficacy by measuring the amount of a particular constituent(s). A chemical profile or fingerprint of such a product is most reliable. This is because a chemical profile evaluates the product as a whole and indicates the presence of multiple chemical markers, other constituents and impurities that maybe present within a sample (Liang et al., 2014). The most commonly used techniques in profiling of botanicals are chromatographic techniques. Chromatographic techniques often used include TLC, HPTLC, HPLC and GC – this is known as chromatographic fingerprinting. Chromatographic fingerprinting has been demonstrated to be a powerful technique for the quality control of herbal medicines, and according to WHO guidelines, it is one of the parameters required for quality evaluation of these drugs (Rasheed, et al., 2012). 1.1.2.2 CHEMICAL MARKERS Chemical markers are chemically-defined constituents of a herbal drug which are of interest for quality control purposes independent of whether they have any therapeutic activity or not. Ideally, chemical markers should be components that contribute to the therapeutic effect(s) of herbal medicines, as their measurement could also serve as a means of measuring the potency of the drug; other chemical components are used as markers. This is because only small amounts of active principles are usually produced and these amounts are affected by various extrinsic factors such as changes in climatic conditions. However, any compound(s) selected as markers should be neutral to environmental changes (Li, et al., 2008; Kushwaha, et al., 2010; Rasheed, et al., 2012). Therefore, the process of identifying and selecting of chemical markers should be a highly validated process; plant materials should be collected from different geographical location, at different seasons, at different times of the day and at different ages. All these samples should be 3 analyzed separately before a conclusive decision can be made regarding the constituents to be selected as markers. 1.2 LITERATURE REVIEW 1.2.1 THE GENUS: Ixora Ixora is a genus belonging to the family Rubiaceae. Plants of this genus are evergreen, flowering shrubs or trees. The genus holds around 500 species, the most common being Ixora coccinea, commonly known as “Flame of the Woods” or “Burning Love”. The name “Ixora” is derived from an Indian deity. The flowers are reputed to be part of Hindu worship offerings at statues of incarnations of Paramathma and are commonly used as well in Indian folk medicine. The genus is widely distributed in the tropics; from Africa to Southeast Asia and through Oceania and Australia, with a few found in tropical America and is mostly cultivated for ornamental purposes. Ixora also grows commonly in subtropical climates in the United States, such as Florida (Radhakrishnan, 2002; Idowu, et al., 2010; Kharat, et al., 2013). There are numerous species, differing in leaf size, flower colour and size, and/or plant height. There are also numerous named cultivars, most of which can be distinguished by the size of the leaves and flowers as well and the colour of the flowers. The colour of the flowers vary widely. The flower could be red, orange, yellow, pink, or purple in colour, again differing according to species or cultivar (Kharat, et al., 2013; Rockledge Gardens). 1.2.2 DESCRIPTION 4 Plants of this genus are densely branched; leaves are coriaceous, up to 10 cm long, sessile, subsessile, oblong or obtuse. Flowers are numerous; they grow in clusters in a corymb manner. The colour of flowers varies, depending on the species or variety. Fruits are globose, fleshy, 2-seeded and are pea-sized. Several popular cultivars are dwarfs, usually staying under 3feet (1 meter) in height. The plant usually flowers in April - May and fruits in May–June (Kharat, et al., 2013). The following are photographs of some species of Ixora. 5 1.2.3 TAXONOMY (Kharat, et al., 2013) Kingdom: Plantae Division: Angiosperms Class: Eudicots Subclass: Asterids Order: Gentianales Family: Rubiaceae Subfamily: Ixoroideae Tribe: Ixoreae Genus: Ixora Some of the species include (Idowu, et al., 2010; GRIN) Ixora abyssinica (Fresen.) Oliv. Ixora acuminata Roxb. Ixora aggregata Hutch. 6 Ixora albersii K.Schum. Ixora amoena Wall. exG. Don Ixora aneimenodesma K.Schum. Ixora arborea Roxb. ex Sm. Ixora assimilis (Sond.) Kuntze Ixora asteriscus K.Schum. Ixora atrata Stapf Ixora baldwinii Keay Ixora barbata Roxb. Ixora batesii Wernham Ixora bauchiensis Hutch. & Dalziel Ixora brachycalyx (Hiern) Kuntze Ixora brachypoda DC. Ixora carniflora K.Krause Ixora casei Hance Ixora chinensis Lam. Ixora coccinea L. Ixora congesta Stapf Ixora crebrifolia (Hiern) Kuntze Ixora delicatula Keay Ixora edentula (Sond.) Kuntze Ixora fastigiata (R.D.Good) Bremek. Ixora finlaysoniana Wall. ex G.Don 7 Ixora foliosa Hiern Ixora gardeniifolia (A.Rich.) Kuntze Ixora gerrardii (Harv.) Kuntze Ixora guineensis Benth. Ixora hookeri (Oudem.) Bremek. Ixora incana (Klotzsch) Kuntze Ixora javanica (Blume) DC. Ixora kavalliana K.Schum. Ixora laxiflora Sm. Ixora lutea Hutch. Ixora macrothyrsa Ixora mannii (Hiern) Kuntze Ixora nigerica Keay Ixora nitida Schumach. & Thonn. Ixora pavetta Andrews Ixora radiata Hiern Ixora rhodesiaca Bremek. Ixora rosea K.Schum. Ixora siamensis G.Don Ixora stricta Roxb. Ixora thomsonii Hiern Ixora villosa (Vahl) Poir. Ixora viridiflora K.Schum. 8 1.2.4 CHEMICAL CONSTITUENTS Several chemical constituents have been reportedly isolated and identified from different parts of the plant species belonging to the Ixora genus, ranging from flowers, seed oil, fruits, leaves or aerial part and root. This study has reported the names and structures of virtually all chemical compounds that have been isolated from the genus and they are shown both in table and figures below: 9 Table 1. Folkloric uses, pharmacological activities and reported isolated chemical constituents of Ixora species Ixora species Plant Part Folkloric use Reported Used activities/properties I. acuminata Leaves and Fresh leaves (3-4 at a time) are eaten Roxb. roots (raw/ boiled) thrice daily as a remedy for headache and cooling of forehead. The infusion of the leaves is taken orally in the treatment of jaundice (Purkayastha et al., 2005; Laloo et al., 2006; Srivastava & Nyishi 2010). Ixora alba Leaves and It has been reported to used in the stem-bark treatment of whooping cough, anemia, highly coloured urine and general debility ( Talpade & Chachad, 2016). I. Stems amplexicaulis I. arborea Flowers Fruit Root Its flowers are used in pulmonary troubles and whooping cough (Aktar et al., 2009). & Fruits and roots are given to females when urine is highly coloured (Aktar et al., 2009). 10 biological Reported isolated chemical constituents The stems of I. amplexicaulis afforded 6α,16α-dihyroxyent-kaurane(1), (24R)-6βhydroxy-24-ethyl-cholest-4en-3-one(2), 7βhydroxysitosterol(3), maslinic acid(4), 3,3'-bis(3,4-dihydro4-hydroxy-6-methoxy-2H-1benzopyran)(5) and protocatechuic acid(6) (Chen et al., 2016). Leaf stem I. brachiata Leaf Roxb (Rasna) Root The decoction of bark is used for anaemia and general debility and remedy for urinary diseases (Aktar et al., 2009). and The root is medically important in the treatment of skin diseases (SadeghiNejad et al., 2014) Antispasmodic effect on isolated Betulin(7), erythrodiol(8), guinea pig ileum, hypotension in lupeol(9), and stigmasterol cat/dog and CNS depressant effect in (10)(Aktar et al., 2009). mice. Antioxidant (free radical scavenging) activity (Aktar et al., 2009; Usha et al., 2016). Chrysin-5-O-β-Dxylopyranoside (11) (Chauhan et al., 1984; Darwish and Ibraheim, 2003) The ethanolic extracts of the leaf and the root have been reported to possess antifungalactivity against tested dermatophytic species. According to Siddha literature, I. brachaita Roxb have been reported to possess antiinflamatory, aromatic and antipyretic properties (Sadeghi-Nejad and Deokule, 2009). The ethanolic and methanolic root extracts has been reported to have anti-Leishmania effect (SadeghiNejad et al., 2014) 11 Flowers The methanolic extract of the flowers was found to possess significant antiinflammatory activity against cotton pellet induced subacute inflammation in rats (Vimala et al., 1997; Dahanukar et al., 2000). Ixora chinensis Lamk Flowers Infusion of the fresh flowers drunk ad libitum is said to be good as a remedy against incipient tuberculosis, haemorrhage and headache, and for contusion (Batugal et al., 2004; Kharat, et al., 2013). Seeds Leaves Palmitic acid(12), stearic acid(13), oleaic acid(14), linoleic acid(15), crepenynic acid (octadec-cis-9-en- 1 2ynoic acid) (16), cis,cis,cis, trans-8,10,12,14octadecatetraenoic acid (ixoric acid) (17)(Minquan, 1990; Johnson and wyatt, 2004; Kharat, et al., 2013) I. chinensis leaves have beenused to treat headache and stomachache and Ixoroside (18) and ixoside (7,8-dehydroforsythide) (19) 12 as a remedy for incipient tuberculosis (Kharat, et al., 2013). Root Ixora coccinea L. Flowers In Malaysia, the root decoction is given after birth (parturition) and are also use for urinary trouble. In Indonesia, the decoction of roots is used for bronchial disorders (Kharat, et al., 2013) Decoction of the flowers is given for thetreatment of cancer, leucorrhoea, dysentery, diarrhoea, dysentery, catarrhal bronchitis, dysmenorrhoea, haemoptysis, andhypertension, amenorrhea and irregular menstruation(Maniyar, et al., 2010; Kharat, et al., 2013; Rajeevand Rajamanoharan,2014). Decoctionof flowers or bark is used as a lotion for eyetroublesand eruptions in children (Joshi,et al., 2013; Ratnasooriya et al., 2005; Batugal et al., 2004; Ghani, 2003; Annapurna et al., 2003 Saha et al., 2008, Latha et al., 1995) and healing of ulcers (Nayak et al., 1999). The flowers are used externally in the treatment sores,chronic ulcer, scabies, and some type of dermatitis and also human internally for cholera, leucorrhoea, antitumor and gonorrhea (Arunachalam et al., 2009). The fresh juice of flower have along with known geniposidic acid (20) (Takeda et al., 1975) Pharmacological investigations of the flower had revealed: analgesic and anti-inflammatory activities (Nayak et al., 1999; Aher et al., 2013); antioxidant (Saha et al., 2008)antidiarrheal activity (Maniyar, et al., 2010); antileucoderma activity (Joy et al., 1998); cytotoxic and antitumour activities (Annapurna et al., 2003, Latha and Panikkar, 1998; Ratnasooriya et al., 2005); chemoprotective effects of the flowers on cisplatin-induced toxicity in mice (Latha and Panikkar, 2001); modulatory effects of the flowers on cyclophosphamide-induced toxicity in mice (Latha and Panikkar, 1999, Latha et al., 2004) as well as hepatoprotective effects of the flowers against paracetamol overdose-induced hepatotoxicity in rats (Latha et al., 2003). Aqueous ethanolic extracts of I. coccinea dry flowers have also been 13 Cycloartenol 3-palmitate(21), Cycloartenol 3myristate(22),lupeol (23), oleanolic acid (24), sitosterol (25), lupeol 3-palmitate (26), and ursolic acid (27) (Ragasa et al., 2004; Latha and Panikkar, 1999; Latha et al., 2001;Reena et al., 1994). Biochin A, Myricetin(28), Quercetin(29), Rutin(30), Ursolic acid(27), Diadzein(31) and formononetin(32), cyanadin-3-Rutinoside(33) and delphinidin 3-Oglucoside(34). (Sumathy et al., 2011; Joshi et al., 2013; Kharat, et al., 2013) 21,23-Epoxy-tirucall-7-en-3βol (Ixoroid)(35), stigmast-5en-3-O-β-D-glucoside (36), 5O-caffeoylquinic acid (37) and D-mannitol (38) (Versiani protective action against electroconvulsions Flower paste is applied daily once on cuts and wounds till it cures (Policepatel and Manikrao, 2013). reported to possess antimicrobial activity (Latha et al., 1995). The hexane fraction of the flowers has been reported to show significant antigenotoxic properties (Ratnasooriya et al., 2005, Latha et al., 2001). The wound healing activity of the flowers has been reported (Nayak et al., 1999). Cardioprotective effect of methanolic extract of Ixora coccinea flowers on doxorubicin induced cardiac Ixora flower has traditionally been myopathy has been reported (Momin associated with enhanced sexuality and et al., 2011). the re-kindling of passion. Decoction of roots used as sedative in the treatment of nausea, hiccups and loss of appetite and fever. Flowers are used to treat diarrhea, dysentery, leucorrhoea, dysmenorrhea, haemoptysis and catarrhal bronchitis. Leaves are used for treating sores, ulcers, and skin disease. In tropical Asia, infusion of flowers and bark are used for blood-shot eyes, leaves for sores and ulcers. Fruits The fruits, when fully ripe, are used as a dietary source (Naskar et al., 2013; Dontha et al., 2015) Aerial part The decoction and poultice fresh leaves Amptothecin isolated from the leaves 14 et al., 2012) Amptothecin(39) (Saravanan (Leaves and stem) and stems are used in folk medicine for the treatment of sprains, eczema, boils and contusions, wounds / sores, ulcers, eye troubles and diarrhoea(Ghani, 2003, Annapurna et al., 2003, Ratnasooriya et al., 2005, Latha et al., 1995). The leaves are also used to pacify vitiated pitta, skin diseases, colic, flatulence, diarrhea,indigestion, ulcers, wounds, and used as antiseptic (Kharat, et al., 2013). of I. coccinea has been reported to demonstrate strong anticancer activity (Saravanan and Boopalan, 2011). Using in vitro HIV-1 replication assays with a CD4+ T cellline and peripheral blood mononuclear cells, pure ixoratannin A-2 isolated from the leaves of I. coccinea has been confirmed to have antiviral activity and minimal cytotoxicity activity (Tietjen et al., 2015). Peptide I (1) exhibited selective potency against Hep3B liver cancer while compound II (2)and compound 2 did not show notable cytotoxicity toward cancercell lines but peptide 2showed significant antiinflammatory effects on neutrophils (Lee, et al., 2010). The cardioprotectiveeffect of the leaves on doxorubicin induced cardiac myopathy and have proved effective in maintaining the hemodynamic, electrocardiographic, biochemicaland histopathological parameters close to normal, by boosting the endogenous antioxidantstores and blunting the oxidative stress (Dontha et al., 2015) Antiulcer, Anti-inflammatory, antimitotic and antimicrobial activities have been reported for the 15 and Boopalan, 2011). 17β-dammara-12,20-diene3β-ol (Ixorene) (40), βsitosterol (25), lupeol(23)and D-mannitol (38). (Ikram, et al., 2013; Dontha et al., 2015). Ixorapeptide I (41) and Ixorapeptide II (42) (Lee, et al., 2010). Epicatechin(43), procyanidin A-2(44), ixoratannin A-2 (45), cinnamtannin B1(46),kaempferol 7-O-α-Lrhamnoside(47), kaempferol 3-O-α-L-rhamnoside(48), quercetin-3-O-α-Lrhamnopyranoside(49), kaempferol 3, 7-O- α-Ldirhamnoside (kaempferitrin)(50) ( Idowu, et al., 2010). GC-MS analysis of the leaves extract of Ixora coccinea afforded the following: 3, 7, 11, 15- Tetramethyl 2hexadecen-1-ol(51); Ethyl 15-methyl-hexa decanoate(52); Z,Z-6,28Heptatriacontadien-2-one(53); Ethyl 9,cis,11,transoctadecadienoate (54); Ethyl 9,12,15,octadecatrienoate(55); leaves (Arunachalam et al., 2009; Kharat, et al., 2013; Dontha et al., 2015) Hypoglycaemic and the hypolipidemic activity of the aqueous extract of the leaves of Ixora coccinea Linn in alloxan induced diabetic albino rats have also been reported (Neelima et al., 2011; Maniyar and Bhixavatimath, 2011; Kharat, et al., 2013; Dontha et al., 2015). Report have shown the antidiarrhoeal activity of aqueous extract of the leaves and flowers of I. coccinea against a castor oil induced diarrhoea model in rats (Dontha et al., 2015; Prabhu et al., 2010; Maniyar et al., 2010). Anti-inflammatory, antimitotic and antimicrobial activities have been reported for the leaves while CNS depressant, hypothermic and semen coagulant activities for the aerial parts have also been reported (Annapurna et al., 2003; Latha and Panikkar, 1999).The leaves have also been found to be active against a plant pathogenic fungus, Drechslera oryzae(Annapurna et al., 2003) as well as possessing antinociceptive / anti-inflammatory, antihistamine and antioxidant activities (Bose et al., 16 Bi cyclo (5.1.0)octane,8methylene(56); Methyl 6,9,12,15,18 heneicosapentaenoate(57); Squalene(58); Dodecane,1fluoro (59); Trans-2,4Dimethylthiane S,S- Dioxide (60); Z,Z-6,28 Heptatriactontadien (61); Methyl 2-HydroxyPentadicanoate (62); Methyl 17-Methyl-Octadecanoate (63); Ethyl 9,12Hexadecadienoate (64); Ethyl 9,12,15 Octadecatrienoate (65); Bicyclo [5.1.0]octane,8methyl (66); 1,3-Di methyl 3-isobutyldiaziridine(67); Cyclohexane, hexyl (68); 1,2,4,5 –cyclo hexanetetrol (69); Trans-2-Methyl-4-NPentylthiane S,S-dioxide(70); Chloroacetic acid,tetradecyl ester (71); Heptasanoic acid, 25-methyl, methyl ester(72) (Shyam and Suresh, 2013) 2011; Ratnasooriya et al., 2005, Saha et al., 2008). Lupeol, isolated from the petroleum ether fraction of the ethanol extract of the leaves of Ixora coccinea, showed anti-inflammatory activity in the carrageenan-induced paw edema test in rats (Reena et al., 1994, Ratnasooriya et al., 2005). Ixora coccinea extracts have also been reported to possess wound healing properties (Saifuddin et al., 2006) The in vitro antileishmanial activity of the leaves extract has also been reported (Naskar et al., 2013; Dontha et al., 2015). Evaluation of anti-asthmatic activity of the leave extract has also been reported(Missebukpo et al., 2011; Kharat, et al., 2013) Root Diluted tincture and decoction of the roots are used for mouthwash and as gargles for sore throat, nausea, loss of appetite (anorexia), fever, hiccups, diarrhea, dysentery, sores, chronic ulcers and skin diseases, gonorrhea (Batugal et al., 2004; Annapurna et al., 2003, Ratnasooriya et al., 2005; Arunachalam et al., 2009;Joshi,et al., 2013) The anthelmintic activity of the roots in different extracts against Indian earthworm, Pherituma posthuma has been as reported ( Surana et al., 2011; Dontha et al., 2015). Antileucoderma activity for the root has also been reported (Joy et al., 1998) The roots of I. coccinea showed wound healing and antimicrobial 17 Palmitic acid methyl ester, PME (73), stearic acid methyl ester (74), oleic acid methyl ester (75) and linoleic acid methyl ester (76), cis Δ9, trans Δ11-octadecadienoic acid (77) Palmitic acid(12),stearic acid(13), oleic acid(14), linoleic acid (15) (Annapurna et al., 2003; Kharat, et al., Roots ground into pulp, mixed with activity (Kharat, et al., 2013). water and as tincter are used for . diarrhea and dysentery (Maniyar, et al., 2010; Vadivu et al., 2010). The root is alsoused as astringent and antiseptic against scabies and other skin diseases (Kharat, et al., 2013). Ixora congesta Roxb. Ixora cuneifolia (GRIN) Ixora elliptica R.Br et Ridl Ixora ferrea (Jacq.) Benth Ixora finlaysoniana Remedy for fevers, toothache, measles, Anti-inflammatory (Iwu, 1993). and bronchial infections (Iwu, 1993). Root and stem are used to cure nervous disorders and fever (De Block, 1998; Johnson and Wyatt, 2004). Leaf and root, mixed with root of Badaduk or Decaspermum fruticosum or root of Biliusa or Arytera sp. Is boiled and the infusion, about 150ml two times daily is taken, to treat warm body and fatigue. The decoction is also used for bath (Kulip and Majawat, 2000). Useful in the treatment of Stomachic (Johnson and Wyatt, 2004). I. finlaysoniana flowers has been scientificallydocumented to possess estrogenic,abortifacient and antiimplantation properties (Kharat, et al., 2013) Anti-gestagenic activity (oral administration of extract to adult female rats at 250mg/kg dose on days 1-5 or 1-7 post-coitum prevented pregnancy in 100% rats) (Kharat, et al., 2013). 18 2013). 9, 12-Octadecadienoic(78), di-n-octyl phthalate(79), βAmyrin(80), Kaempferol-7O-glucoside(81), Kaempferitrin(50) and Quercitrin(49) (Joshi,et al., 2013; Dontha et al., 2015) Leaves Ixora fulgens Roxb. Ixora Roots grandifolia Leaves Ixora javanica (Blume) DC. Flowers Root I.finlaysoniana leaves showed antigestagenicactivity. Used in treatment of toothache(Johnson and Wyatt, 2004) Thea root is used in delivery and Stomachache (Kharat, et al., 2013; Johnson and Wyatt, 2004) The leaves of I. grandiflora are used as poultice in fresh form for treatment of sprain,eczema, boils and concussions. The decoctionof the leaves is also used in treatment of wounds and skin ulcer (Kharat, et al., 2013). Flower eaten as vegetable. Leaf and I. javanica flower extract have been flower are used in a cure-all infusion reported to demonstrate anti-tumours activity (Nair et al., 1991; Kharat, et al., 2013) Root crush with lime water/poultice for 19 n-nonacosanol(82), αamyrin(83), β-sitosterol(25), 3-hydroxyhexan-5-olide(84), sitosterol -3-O- β –Dglucoside(36), protocatechuic acid(6), and Gallic acid(85), (3R, 5R)-3-(βglucopyranosyloxy)-5hexanolide (parasorboside)(86), D-1-Omethyl-myo-inositol(87) and Galactitol (88) Unny et al., 2003; Singh, 1993, Darwish and Ibraheim, 2003; Kharat, et al., 2013 . Ferulic acid(89), protocatechuic acid(6)and caffeic acid(90). Nair and Panikkar, 1990; Nair et al., 1991; Kharat, et al., 2013 Ixora laxifora Ixora macrophylla Barth. I. macrothyrsa Methanol flower extract Flowers I. nigricans R. Br. ex Wight & Arn Ixora pavetta Flowers Stem-bark Roots and snake bite (Johnson and Wyatt, 2004) Used in the treatment of inflammation Anti-nociceptive and antiand painful conditions such as inflammatory ((Iyadi, et al., 2005)) rheumatic arthritis, sprains, spasmodic colics (Iyadi, et al., 2005) Used to treat headache and puerperium Johnson and Wyatt, 2004. I. macrothyrsa pacifies vitiated kapha, pitta, burning sensations, eczema, ringworm other skin diseases menorrhagia,leucorrhea and general weakness. The flowers of I. macrothyrsa are used toimpart color to herbal preparation (Kharat, et al., 2013). Used to treat dysentery and root crush with lime water/poultice for snake bite (Johnson and Wyatt, 2004). Traditionally the plant is used for the treatment of diarrhoea, dysentery, urinary disorders, leucorrhoea, veneral diseases and sedative(Srinivas, K.; Celestin Baboo, 2011). Anti-bacterial (including Methicillin Wahab, et al., 2012; Kharat, Resistant Staphylococcus aureus et al., 2013 MRSA and Acinetobacter baunmannii) . The flowers of I. pavetta have been extracted by ethanol and evaluated for theantiulcer activity by Aspirin induced and pylorus ligation of rats (Kharat, et al., 2013). Two spoonfuls of stem bark extract is administered daily twice for 9 days in the treatment of jaundice(Bapuji & Ratnam, 2009) Theroots are reported for its use in 20 3-butyn-2-ol(91), 3-butyn-1ol(92), amyl nitrite(93), 2octyn-1-ol(94), 1, 9decadiyne(95) and glyoxylic acid (96) from the flowers of I. pavetta Vahl. (Srinivas, K.; Celestin Baboo, 2011). I. Fruits philippinensis I. parviflora Root Vahl Bark Leaves Flowers Ixora pendula Jack Ixora rhodesiaca Bremek Ixora stricta Roxb I. undulata Leaves urinary diseases (Talpade & Chachad, 2016) The fruits are edible Root used in treatment of dysuria and Antioxidant activity (Wen et al., Menorrhagia ( Kharat, et al., 2013) 2011) The fruits and roots are used as antidote (Bachheti et al., 2011). The decoction of bark is used for anemia and debility (Bachheti et al., 2011). Antioxidant (Bose et al., 2011) β-sitosterol(25), The leaf extract of I. parviflora kaempferol(97), β-sitosterolshowed antiviral, hypotensive and 3-O- β -D-glycoside (36), spasmolytic activity (Kharat, et al., kaempferol-7-O-methyl ether(98) 2013). (Bachheti et al., 2011; Kharat, et al., 2013) The flowers of I. parviflora are pounded in milk and given for relieving whooping cough and in treatment of ulcers (Bachheti et al., 2011; Kharat, et al., 2013). Used to treat rhinosis and sore (Johnson and Wyatt, 2004). Remedy for measles, cold, skin Anti-microbial (Iwu, 1993). diseases (Iwu, 1993). Remedy for nervous disorders and also Whitening activity (Johnson and to treat stomach ache (Johnson and Wyatt, 2004, Kuo-Ching, 2004). Wyatt, 2004, Kuo-Ching, 2004). Rubiothiazepine isolated from the root Rubiothiazepine(99). 21 has been reported to show cytotoxic (Mohammed et al. 2014). activity against EL4 (Murine Leukemia) and also showed cytotoxic and HIV-1 activity against MT-4 and HIV-1IIIB with (Mohammed et al. 2014) 22 1-(R)-phenylethanol βgentiobioside(100), 2-methylphenylmethanol βgentiobioside(101), 3,4dimethylphenol βgentiobioside(102), (5R,6R,Z)-5,6-dihydroxy-5,6dihydro-2H-thiopyran-2oneO-methyloxime β-Dglucopyranoside(103), (5R,6R,Z)-5,6-dihydroxy-5,6dihydro-2H-thiopyran-2-one O-methyl oxime βgentiobioside(104), kaempferol 3-O-α-Lrhamnopyranosyl-(1→6)-(4"trans-p-coumaroyl)-β-Dgalactopyranoside7-O-α-Lrhamnopyranoside(105),corch oionoside C(106), icariside B1(107), 3-methoxy-4hydroxyphenol 1-O-β-Dglucopyranoside(108), kaempferol 3-Orobinobioside(109), quercetin 3-O-robinobioside(110), variabiloside E(111), and acteoside(112)(Sugimoto et al., 2014; Ragasa et al., 2015). D-mannitol(38) (Mohammed et al. 2014) CH3 CH3 H 3C H 3C CH3 H CH3 CH3 CH3 H H OH O HO 6,16-dihyroxy-ent-kaurane (1) OH OH 7-hydroxysitosterol (3) (24R)-6-hydroxy-24-ethyl-cholest-4-en-3-one (2) CH3 H3C COOH OH OH O CH3 CH3 HO H HO H 3C O OH H O O CH3 OH O 3,3'-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran)(5) CH2 H H CH2OH CH3 OH CH3 H H3C CH3 CH3 H3C H3C H3C H C H2 CH3 CH3 CH3 Betulin (7) HO H H3C Erythrodiol (8) CH3 H3 C CH3 CH3 CH3 CH3 HO H3C stigmasterol (10) HO CH3 OH Protocatechuic acid (6) Maslinic acid (4) CH3 H FIG. 1 Chemical constituents from the stem of Ixora amplexicaulis HO CH3 CH3 CH3 H CH3 H3C CH3 OH H3C CH3 Lupeol (9) FIG. 2 Chemical constituents from the leaves of Ixora arborea 23 HO O O HO O O HO OH Chrysin-5-O- -D-xylopyranoside (11) FIG. 3 Chemical constituents from the stem of Ixora arborea O O OH OH Stearic acid (13) Palmitic acid (12) OH OH O Oleic acid (14) O Linoleic acid (15) OH OH O O cis,cis,cis, trans-8,10,12,14- octadecatetraenoic acid (ixoric acid) (17) crepenynic acid (octadec-cis-9-en- 1 2-ynoic acid) (16) FIG. 4 Chemical constituents from the seed oil of Ixora chinensis HO O HO O H H H HO O O OH O OH O H OH O CH3 O OH H HO O HO H HO H OH O O HO O HO Ixoroside (18) HO O HO HO Ixoside (7,8-dehydroforsythide) (19) FIG. 5 Chemical constituents from the leaves of Ixora chinensis 24 HO Geniposidic acid (20) CH2 H3 C H3C H3C H3C H 3C H OH O OH H3C O CH3 O CH3 CH3 O H3 C CH3 H 3C H CH3 CH3 HO H3C H CH3 Cycloartenol 3-palmitate (21) Cycloartenol 3-myristate (22) H 3C CH3 H Lupeol (23) CH2 H3C CH3 H H3C H3C CH3 H3C CH3 CH3 COOH O H3C H 3C H CH3 CH3 O HO H3C HO CH3 H CH3 Sitosterol (25) Oleanolic acid (24) CH3 Lupeol 3-palmitate (26) H3 C CH3 CH3 COOH CH3 HO H3 C CH3 H Ursolic acid (27) FIG. 6 Chemical constituents from the flowers of Ixora coccinea 25 CH3 OH HO O OH OH OH HO HO O O OH OH O Myricetin (28) O OH OH OH OH OH OH OH HO OH O O O O Rutin (30) Quercetin (29) H 3C O HO HO HO O OH OH OH HO O O + OH O O CH3 Formononetin (32) HO OH O OH O H3C OH OH HO O HO + OH O OH O HO Cyanadin-3-rutinoside (33) OH HO OH O O OH Delphinidin 3-O-glucoside (34). FIG. 7 Chemical constituents from the flowers of Ixora coccinea CH3 O CH3 H3C CH3 H3C CH3 H H CH3 H H H3C CH3 O H H O HO HO OH CH3 21,23-Epoxy-tirucall-7-en-3ol (35) (Ixoroid ) CH3 CH3 H OH H H HO OH Stigmast-5-en-3-O--D-glucoside (36) (Sitosterol-3-O--D-glucoside) OH OH OH HO OH OH D-mannitol (38) 26 CH3 FIG. 8 Chemical constituents from the flowers of Ixora coccinea H2C CH3 CH2 CH3 CH3 CH3 H3C H CH3 H3C H3C CH3 CH3 H CH3 H 3C H CH3 17-dammara-12,20-diene-3-ol (40) (Ixorene) HO H3C H CH3 Lupeol (23) H3C H3C OH OH H 3C OH HO OH HO Sitosterol (25) OH D-mannitol (38) CH3 O OH O O O N H O NH O HN N N H H3CO N O Ixorapeptide II (42) Ixorapeptide I (41) FIG. 9 Chemical constituents from the leaves of Ixora coccinea 27 OH OH OH OH HO OH HO O O OH OH OH HO O O OH OH HO OH OH O HO O OH O O HO O HO OH HO OH OH Epicatechin (43) Procyanidin A-2 (44) OH HO OH Ixoratannin A-2 (45) OH OH HO O OH OH OH O H3C HO OH O HO O OH O O HO OH OH O HO OH O OH kaempferol 7-O--L-rhamnoside (47) HO HO OH O HO Kaempferol 3-O--L-rhamnoside (48) OH OH H3C HO HO HO HO O OH OH O O O OH HO OH HO OH Cinnamtannin B-1 (46) O OH O HO CH3 OH O O O OH CH3 O Kaempferol 3, 7-O- -L-dirhamnoside (kaempferitrin) (50) OH O O HO CH3 OH O HO Quercetin-3-O--L-rhamnopyranoside (49) (Quercetrin) FIG. 10 Chemical constituents from the leaves of Ixora coccinea 28 HO O O Ethyl 15-methyl-hexa decanoate (52) O 3, 7, 11, 15- Tetramethyl 2 hexadecen-1-ol (51) O O Ethyl 9,cis,11,trans octadecadienoate (54) Z,Z-6,28-Heptatriacontadien-2-one (53) OR (6Z,28Z)-heptatriaconta-6,28-dien-2-one Bi cyclo (5.1.0) octane,8-methylene (56) O O Ethyl 9,12,15,octadecatrienoate (55) F O Dodecane,1-fluoro (59) O Methyl 6,9,12,15,18 heneicosapentaenoate (57) O S O Trans-2,4-Dimethylthiane S,S- Dioxide (60) Squalene (58) O HO O Methyl 2-Hydroxy-Pentadicanoate (62) Z,Z-6,28 Heptatriactontadien (61) O O O O Ethyl 9,12-Hexadecadienoate (64) Methyl 17-Methyl-Octadecanoate (63) FIG. 11 Chemical constituents through GC-MS from the leaves of Ixora coccinea 29 HO OH HO OH O O Ethyl 9,12,15 Octadecatrienoate (65); 1,2,4,5 -cyclo hexanetetrol (69) O S O Bicyclo [5.1.0]octane,8-methyl (66) Trans-2-Methyl-4-N-Pentylthiane S,S-dioxide (70) N O NH O 1,3-Di methyl -3-isobutyldiaziridine (67) Cl Chloroacetic acid,tetradecyl ester (71) O Cyclohexane, hexyl (68) O Heptasanoic acid, 25-methyl, methyl ester (72) FIG. 12 Chemical constituents through GC-MS from the leaves of Ixora coccinea contd. O O O O Palmitic acid methyl ester, PME (73) Stearic acid methyl ester (74) O Oleic acid methyl ester (75) O Linoleic acid methyl ester (76) O O O COOH OH cis, trans-9, 11-octadecadienoic acid (77) Palmitic acid (12) O OH OH Stearic acid (13) Oleic acid (14) OH Linoleic acid (15) O FIG. 13 Chemical constituents from the root of Ixora coccinea 30 O H3C O CH3 O OH CH3 H3C O CH3 O CH3 9, 12-Octadecadienoic (78) O HO Di-n-octyl phthalate (79) CH3 H3C -amyrin (80) OH HO HO OH O OH OH H 3C HO O O OH HO O O O OH HO O OH OH OH O O OH CH3 O Kaempferol 3, 7-O- -L-dirhamnoside (kaempferitrin) (50) Kaempferol-7-O-glucoside (81) OH OH O HO OH O HO O O CH3 OH HO Quercetin-3-O--L-rhamnopyranoside (49) (Quercetrin) FIG. 14 Chemical constituents from the root of Ixora coccinea contd. 31 CH3 H 3C H 3C H 3C OH CH3 CH3 CH3 nonacosanol (82) CH3 CH3 HO HO CH3 COOH HO H3C Sitosterol (25) -amyrin (83) CH3 H 3C H 3C CH3 CH3 H O OH O OH Protocatechuic acid (6) 3-Hydroxyhexan-5-olide (84) COOH H O HO HO OH H CH3 H OH O CH3 Stigmast-5-en-3-O--D-glucoside (36) (Sitosterol-3-O--D-glucoside) OH HO HO HO O O CH3 OH Parasorboside (86) OH OH OH O OH HO HO HO O O CH3 D-1-O-methyl-myo-inositol (87) Gallic acid (85) HO HO OH HO OH HO Galactitol (88) FIG. 15 Chemical constituents from the leaves of Ixora finlaysoniana COOH HO O OH HO O O OH OH OH Ferulic acid (89) caffeic acid (90) OH (4-hydroxy-3-methoxy cinnamic acid) Protocatechuic acid (6) OR [(E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid] FIG. 16 Chemical constituents from the flowers of Ixora javanica 32 CH3 OH OH O O N amyl nitrite (93) 3-butyn-1-ol (92) 3-butyn-2-ol (91) O O OH OH 1, 9-decadiyne (95) Glyoxylic acid (96) 2-octyn-1-ol (94) FIG. 17 Chemical constituents from the flowers of Ixora pavetta CH3 H3C H 3C CH3 H 3C H O Sitosterol (25) HO OH H3C O O OH OH O Kaempferol (97) H Stigmast-5-en-3-O--D-glucoside (36) (Sitosterol-3-O--D-glucoside) OH OH O CH3 O HO HO HO H CH3 H OH H 3C CH3 OH OH O Kaempferol-3-O-methyl ether (98) FIG. 18 Chemical constituents from the leaves of Ixora parviflora 33 HO OH O HO O HO S OH N O CH3 Rubiothiazepine (99) 7-[(-D-glucopyranosyl)oxy]-6- hydroxy-2-methoxy-4,5-dihydro-1,3-thiazepine Glc Glc 6' GlcO 1-(R)-phenylethanol -gentiobioside (100) 6' GlcO 2-methylphenylmethanol-gentiobioside (101) HO Glc 6' GlcO GlcO S 3,4-dimethylphenol -gentiobioside (102) N HO O Glc 6' GlcO (5R,6R,Z)-5,6-dihydroxy-5,6-dihydro-2H-thiopyran-2one O-methyl oxime 6-O--D-glucopyranoside (103) S OH N O RhaO O (5R,6R,Z)-5,6-dihydroxy-5,6–2H-thiopyran2-one O-methyl oxime-gentiobioside (104) OH O 4'' OGal E-p-coumarate 6'' Rha kaempferol 3-O--L-rhamnopyranosyl-(1 6)-(4"-trans-pcoumaroyl)--D-galactopyranoside 7-O--L-rhamnopyranoside (105) FIG. 19 Chemical constituents from the leaves of Ixora undulata 34 H C OH OH OH OH OH O O HO HO HO O O O O OH OH Corchoionoside C (106) Icariside B1 (107) OH OH HO OH O OH O O HO HO OCH3 OH O OH OH OH O O O 3-methoxy-4-hydroxyphenol-1-O--D-glucopyranoside (108) H 3C O HO OH HO OH kaempferol 3-O-robinobioside (109) O HO OH OH OH OH O OH OH O O HO O O O OH H 3C O OH HO OH quercetin 3-O-robinobioside (110 O variabiloside E (111) OH O HO OH OH OH Rha (6'')--D-Gal (4'-E-coumarate) O O HO H 3C HO Rha (3')-Glu (4'-E-caffeate) O O O O O OH OH O OH H3C OH HO O HO HO OH OH O HO HO OH OH acetoside (112) OH OH D-mannitol (38) NOTE: Robinobioside: [ -D-(6'-O--L-rhamnopyranosyl)galactopyranoside] FIG. 20 Chemical constituents from the leaves of Ixora undulatacontd 35 CHAPTER TWO METHODOLOGY 2.1 COLLECTION The leaves of five different species of Ixora were collected from different locations at Obafemi Awolowo University, Ile-Ife (pictures of the aerial parts of these species are shown in Figure 21 below). Voucher specimens have been deposited at the department of Botany, Obafemi Awolowo University, for proper identification. For the purpose of this study, these plants are known as small red (SR) – having small leaves and red flowers; small yellow (SY) – having small leaves and yellow flowers; medium red (MR) – having medium-sized leaves and red flowers; medium yellow (MY) – having medium-sized leaves and yellow flowers; and big red (BR) – having big leaves and red flowers. 36 aghgh SMALL YELLOW (SY) SMALL RED (SR) MEDIUM YELLOW (MY) MEDIUM RED (MR) BIG RED (BR) 37 2.2 EXTRACTION The leaves from each plant were separately air-dried at ambient temperature and labeled. The dried leaves were then size-reduced and extracted with methanol by percolation. The filtrates form each plant material was concentrated using a rotary evaporator. 2.3 FRACTIONATION About 2g of each crude extract was dissolved using small volumes of methanol, adsorbed unto silica gel with the aid of a mortar and pestle, and then, it was left to dry. The dried silica gel was packed into a column that had been laid with cotton wool, and after packing, the column was overlaid with cotton wool. The adsorbed extract was then eluted with n-hexane, ethyl acetate, acetone and methanol (in order of increasing polarity). The eluate of each solvent were combined, concentrated to dryness and then reconstituted using the same solvent. This yielded the following fractions: n-Hexane fractions, Ethyl acetate fractions, Acetone fractions, Methanol fractions and Crude extracts each of SR, SY, MR, MY, and BR. Partition was not done because the crude extracts were not soluble in water. 2.4 THIN LAYER CHROMATOGRAPHY (TLC) Each fraction (i.e. n-hexane, ethyl acetate, acetone and methanol) and the crude extracts from all the species were analyzed using silica gel pre-coated on alumina plates. All TLC analyses were carried 38 out at ambient temperature, in a 10ml chromatographic tank. Different solvent systems were tried before the reported solvent systems were used. Fractions Solvent system used n-Hexane Ethyl acetate EtOAc (100%) EtOAc:n-Hex (1:1) EtOAc:n-HEx(7:3) Acetone MeOH:EtOAc:AcOH (1:10:0.5) Methanol MeOH:EtOAc:AcOH (3:7:0.5) Crude n-Hex:EtOAc:AcOH (1:10:0.5) 39 CHAPTER THREE RESULT EtOAc fractions Solvent system: EtOAc 100% EtOAc fractions Solvent system: EtOAC:n-Hex 1:1 EtOAc fractions Solvent system: n-Hex:EtOAc 3:7 Acetone fractions Solvent system: MeOH:EtOAc:AcOH 1:10:0.5 40 MeOH fractions Solvent system: MeOH:EtOAc:AcOH 3:7:0.5 Crude extract Solvent system: n-Hex:EtOAc:AcOH 1:10:0.5 41 CHAPTER FOUR DISCUSSION AND CONCLUSION The extensive literature survey revealed that Ixora species have different important medicinal properties with diverse pharmacological spectrum. This study reviews the various chemical constituents, pharmacological, therapeutic applications and traditional knowledge of the genera Ixora. The ethno-pharmacological uses and biological activities attributed to species of Ixora suggest that this genus is a rich source of medicinally useful phytochemicals. This study has revealed that most of the compounds that have been reported from this genus were isolated form Ixora coccinea, followed by Ixora undulata. It is also clear from the study that only few of these compounds exist in other species as well. The chemical profiling of five different species (SR, SY, MR, MY and BR) in our laboratory using TLC revealed that less than 40% of the chemical constituents are the same which further proved that there are significant differences in their chemical constituents as shown in the discussion of results below. ETHYL ACETATE FRACTION (EtOAc fr) Solvent system: EtOAc:Hex 7:3 The constituent at Rf-0.839 is present in both MR and MY, but absent in all others. Likewise, the constituent at Rf-0.805 is present in both SR and SY, but absent in others. The constituent at Rf0.782 is only present in MR, it is absent in others, while the constituent at Rf-0.736 is present in all except BR, but is lightly expressed. The constituent at Rf-0.701 is only present in MR and MY. Two different bands (having different colours) appear at Rf-0.609. One is present in SR and SY, while the other is only present in MR. The constituent at Rf-0.471 is only present in SR and SY. A constituent appears at Rf-0.437, which is only present in MR. The constituent at Rf-0.425 is present in MY, and it seems to be present in BR. The constituent at Rf-0.345 is present in SR and SY, but absent in others. Two different bands appear at Rf-0.287, one is only present in BR, while the other 42 is present in SR, SY and MY. Two different bands appear at 0.253, one is present in MR, while the other in MY. Two different constituents appear at Rf-0.218 and 0.138, both are present only in MR. The constituent at Rf-0.23 is only present in SR and SY. The constituent at Rf-0.195 is only present in BR. Two different constituents appear at Rf-0.184, one is present only in MY, while the other is present only in SR and SY. The constituent at Rf-0.172 is present in all. The constituent at Rf-0.161 is only present in BR.The constituent at Rf-0.115 is present only in SR, SY and MR. The constituent at Rf-0.080 is only present in SR, SY and MY. ACETONE FRACTION Solvent system: MeOH:EtOAc:AcOH 1:10:0.5 The constituent at Rf-0.651 is present in SR, SY and MR, but not conspicuous in others. The constituents at Rf-0.535 is again present in SR, SY and MR, but not obvious in others. The constituent at Rf-0.151 is present in all except MY. METHANOL FRACTION (MeOH fr) Solvent system: MeOH:EtOAc:AcOH 3:7:0.5 The constituents at Rf-0.391 and 0.092 are present in all. CRUDE EXTRACT Solvent system: n-Hex:EtOAc:AcOH 1:10:0.5 The constituent at Rf-0.860 is present in BR and MY, but is absent in MR. The constituent at Rf0.849 is only conspicuous in MR. The constituent at Rf-0.814 is present in BR and MY. The constituents at Rf-0.709, 0.663, 0.360 and 0.058 are present in all extracts. The constituent at Rf0.628 is present in all except BR, but is lightly expressed in MR. The constituent at Rf-0.198 is present in all except in BR. The review also revealed that many of the isolated chemical compounds from the various species possess good anti-oxidant, anti-cancer, anti-inflammatory, anti-HIV or antiviral etc activities. 43 The systematically analyzed batteries of in-vitro assays and various chemical constituents isolated from these genus has in no doubt provided insight into the factors contributing to their bioactivity and hence, their various important medicinal uses. However, despite the fact that different species had been extensively used traditionally, various pharmacological activities of isolated chemical compounds have not extensively tested. 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