CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 251 REVIEW Phytochemical and Biological Studies of Ochna Species by Anil Kumar Reddy Bandi a ), Dong-Ung Lee b ), Raphaël Ghogomu Tih c ), Duvvuru Gunasekar* d ), and Bernard Bodo e ) a ) Institute of Bioconvergence Technology, Dongguk University, Gyeongju 780714, Republic of Korea b ) Division of Bioscience, Dongguk University, Gyeongju 780714, Republic of Korea c ) Department of Organic Chemistry, Faculty of Science, University of Yaoundé, P.O. Box 812, Yaoundé, Cameroon d ) Natural Products Division, Department of Chemistry, Sri Venkateswara University, Tirupati 517502, India (phone: þ 91-877-2249035; e-mail: duvvurusekarg@rediffmail.com) e ) Laboratoire de Chimie et Biochimie des Substances Naturelles, USM 0502 MNHN – UMR 5154 CNRS, 63 rue Buffon, F-75005 Paris The genus Ochna L. (Gr, Ochne; wild pear), belonging to the Ochnaceae family, includes ca. 85 species of evergreen trees, shrubs, and shrublets, distributed in tropical Asia, Africa, and America. Several members of this genus have long been used in folk medicine for treatment of various ailments, such as asthma, dysentery, epilepsy, gastric disorders, menstrual complaints, lumbago, ulcers, as an abortifacient, and as antidote against snake bites. Up to now, ca. 111 constituents, viz. flavonoids (including bi-, tri-, and pentaflavonoids), anthranoids, triterpenes, steroids, fatty acids, and a few others have been identified in the genus. Crude extracts and isolated compounds have been found to exhibit analgesic, anti-HIV-1, anti-inflammatory, antimalarial, antimicrobial, and cytotoxic activities, lending support to the rationale behind several of its traditional uses. The present review compiles the informations concerning the traditional uses, phytochemistry, and biological activities of Ochna. Contents 1. Introduction 2. Traditional Uses 3. Chemical Constituents 3.1. Flavonoids 3.1.1. Monoflavonoids 3.1.2. Biflavonoids 3.1.3. Triflavonoids 3.1.4. Pentaflavonoids 3.2. Anthranoids 3.3. Triterpenes and Steroids 3.4. Fatty Acids 3.5. Others 4. Biological Activities 4.1. Antimicrobial Activity 4.2. Cytotoxic Activity  2012 Verlag Helvetica Chimica Acta AG, Zrich 252 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 4.3. NF-kB Activity 4.4. Analgesic and Anti-Inflammatory Activity 4.5. Antimalarial Activity 4.6. Anti-HIV-1 Activity 4.7. b-Lactamase Inhibitory Activity 5. Conclusions 1. Introduction. – Use of plant-derived medicines in both traditional and modern medicine is widespread and has shown a significant increase. According to the World Health Organization (WHO), more than 80% of the worlds population in developing countries depends primarily on plant based medicines for basic healthcare [1]. Furthermore, a growing world-wide interest in the use of phytopharmaceuticals as complementary or alternative medicine, either for prevention or amelioration of many diseases, has been noted in recent years. Among the plants often used in traditional medicine, Ochna species, which belong to the family Ochnaceae, play a vital role. The genus Ochna includes ca. 85 species of evergreen trees, shrubs, and shrublets, and is distributed widely in tropical Asia, Africa, and America [2], of which eleven species occur in India [3]. They are generally referred to as Ochnas or Mickey-Mouse plants, due to their drupaceous fruit shape. Several members of this genus are cultivated as decorative plants due to their colorful flowers and unusual fruits, e.g., Ochna kirkii, O. mossambicensis, O. schweinfurthiana, O. serrulata, and O. thomasiana (Fig.). Previous phytochemical studies have revealed that the genus is a prolific source of complex flavonoids and related phenolic compounds. Some of these compounds and their crude extracts exhibit an array of interesting biological activities, including analgesic, anti-inflammatory, anti-HIV-1, antimalarial, antimicrobial, and cytotoxic properties, etc. In the past few decades, several Ochna species have been investigated from a phytochemical and pharmacological point of view. To gain a comprehensive and systematic understanding of this genus, a compilation of the constituents isolated from various members of Ochna, covering the literature up to December 2010, including their traditional uses and biological activities, is presented in this review. 2. Traditional Uses. – A number of Ochna species have a long history of use as herbal remedies in Asia and Africa. For example, Ochna squarrosa L. (known as erra juvvi), a small shrub, has been used in indigenous systems of medicine for treating various ailments, i.e., the bark as a digestive tonic and the roots for its curative effect against asthma [3]. The stem bark of O. lanceolata Spreng. (syn. O. heyneanaWight et Arn. ), a semi-evergreen tree found widely in Central and Peninsular India, is used by the Palliyar tribes as an abortifacient, and for treatment of gastric complaints and menstrual disorders [4]. Similarly, O. pumila (called champa baha) roots are used as an antidote to snake bites. In addition, its use by Mundas for treatment of epilepsy has been reported, and the leaves have been used as a poultice for treatment of lumbago and ulcers. In Thai folk medicine, the bark of O. integerrima (Lour. ) Merr. has been used as a digestive tonic, and the roots as an anthelmintic; in Indonesia, an infusion of its roots and leaves is reputed for its antidysenteric and antipyretic properties [5]. O. afzelii R.Br. ex Oliv. and O. calodendron Gilg. et Mildbr. have been used in CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 253 Figure. Some Ochna species. Photo credits: O. kirkii, Richard Foo TH, Flickr; O. pulchra, Rotational, Wikipedia; O. serrulata, South African National Biodiversity Institute, South Africa; O. thomasiana, Forest & Kim Starr, Hawaiian Ecosystems at Risk Project. 254 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) traditional Cameroonian medicine as a remedy for jaundice, toothache, female infertility, liver infections, and dysentery [6], while, in Tanzania, Washambaas used O. macrocalyx Oliv. bark for treatment of dysmenorrhoea, diarrhea, hemorrhoids, and stomach pain [7]. O. schweinfurthiana F.Hoffm. is a shrub found in African woodlands, where its powdered bark is used as an antimalarial and antihelmintic, and the decoction of the roots or leaves is used in wound dressing. In addition, it is used in Northern Nigeria for treatment of measles, typhoid fever, and fungal skin infections. 3. Chemical Constituents. – Reported chemical constituents from the genus Ochna, so far in total ca. 111, include flavonoids, anthranoids, triterpenes, steroids, fatty acids, and a few other compounds [8 – 50]. Their structures are shown below, i.e., compounds 1 – 111, and their names and the corresponding plant sources are compiled in Tables 1 – 5. Of these, biflavonoids are the predominant constituents within the genus Ochna. 3.1. Flavonoids. 3.1.1. Monoflavonoids. Forty-three compounds, viz., eight flavones, 1 – 8, eight flavonols; 9 – 16, five flavanonols, 17 – 21, 19 isoflavones, 22 – 40, and three flavan-3-ols, 41 – 43, were reported from various Ochna species [8 – 28] (Table 1). Of four C-glycosylflavones, 1 – 4, Nair et al. isolated vitexin (1), orientin (3), and isoorientin (4) from the acetone-insoluble fraction of the EtOH extract of O. jabotapita leaves by preparative PC [8], while isovitexin (2) was obtained from the leaves of O. squarrosa together with 1 and 3 [9]. The isolation of 5-methoxyfurano[3’’,2’’: 7,8]flavone (5) from the stems of O. squarrosa by Reddy et al. [11] represent its first occurrence in a natural source, while from the MeOH extract of the root bark of O. squarrosa, furanoflavones 6 – 8 were obtained as potent analgesic and anti-inflammatory compounds. In addition, the structures of newly isolated compounds 6 and 7 were further confirmed by their synthesis, as outlined in Scheme 1 [12]. Kaempferol (9), its 3-O-glycosides, 10 and 11, quercetin 3-O-glucoside (12) and quercitrin (13), have been isolated from O. beddomei, O. lanceolata, O. obtusata, and O. calodendron [13 – 18]. In 2007, Reutrakul et al. isolated five new anti-HIV-1 flavonoid glucosides, 14 – 18, together with known 6-(g,g-dimethylallyl)taxifolin 7-O-b-d-glucoside (19) from the active AcOEt fraction of the leaves and twigs of O. integerrima by bioassay-guided fractionation [10]. Taxifolin 3-O-rhamnoside (20) was reported from the leaves of O. beddomei [13], and calodendroside A (21), a new di-O-glucoside, was obtained from the stem bark of O. calodendron [20]. It is noteworthy that the majority of flavonoid glycosides are based on flavone/flavanone, found almost exclusively in the leaves, rarely in the root and stems barks, i.e., 13 and 21. In 1989, Rao and Gunasekar isolated a new isoflavone, named squarrosin (30), from the heartwood of O. squarrosa [26]. Isolation of 30 constitutes the first report on the occurrence of isoflavones in the genus Ochna. Since then, eighteen more isoflavones, 22 – 29, and 31 – 40, have been reported from several Ochna species [10] [12 – 14] [21 – 28]. Among them, compounds 27 – 38 possess an OCH2O moiety linking C(6) with C(7) of ring A, except for 5,7,8trimethoxy-3’,4’-(methylenedioxy)isoflavone (39), isolated from O. afzelii [24] and O. squarrosa [27] [28], having an OCH2O moiety between C(3’) and C(4’) of ring B. Lanceolone (40), the only example of an isoflavone with a pyran moiety, was obtained from the leaves of O. afzelii [24], and three flavan-3-ols, i.e., ()-epicatechin (41), (þ)catechin (42), and (þ)-epicatechin (43), were reported from several Ochna species [13] [14] [16] [17] [27]. CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 255 256 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) Table 1. Monoflavonoids from the Genus Ochna No. Compound class and name Source Part Ref. O. O. O. O. O. O. O. O. O. O. O. jabotapita squarrosa integerrima squarrosa jabotapita squarrosa jabotapita squarrosa squarrosa squarrosa squarrosa Leaves Leaves Leaves, twigs Leaves Leaves Leaves Leaves Stem Root bark Root bark Root bark [8] [9] [10] [9] [8] [9] [8] [11] [12] [12] [12] O. O. O. O. O. O. O. O. O. O. O. O. O. beddomei lanceolata beddomei lanceolata obtusata beddomei lanceolata obtusata calodendron calodendron integerrima integerrima integerrima Leaves Stem bark Leaves Stem bark Leaves Leaves Leaves Leaves Stem bark Root bark Leaves, twigs Leaves, twigs Leaves, twigs [13] [14] [13] [14] [15] [13] [16] [15] [17] [18] [10] [10] [10] Flavanonols 17 6-(g,g-Dimethylallyl)dihydrokaempferol 7-O-b-d-glucoside O. integerrima Leaves, twigs 18 6-(3-Hydroxy-3-methylbutyl)taxifolin 7-O-b-d-glucoside O. integerrima Leaves, twigs 19 6-(g,g-Dimethylallyl)taxifolin 7-O-b-d-glucoside O. integerrima Leaves, twigs O. integerrima Leaves 20 Taxifolin 3-O-rhamnoside O. beddomei Leaves 21 Calodendroside A O. calodendron Stem bark [10] [10] [10] [19] [13] [20] 1 Flavones Vitexin 2 3 Isovitexin Orientin 4 5 6 7 8 Isoorientin 5-Methoxyfurano[3’’,2’’:7,8]flavone 3’,4’-Dihydroxyfurano[3’’,2’’: 6,7]flavone 4’-Hydroxy-3’-methoxyfurano[3’’,2’’: 6,7]flavone 5-Methoxyfurano[3’’,2’’: 6,7]flavone 9 Flavonols Kaempferol 10 Kaempferol 3-O-b-d-glucoside 11 Kaempferol 3-O-a-l-rhamnoside 12 Quercetin 3-O-glucoside 13 Quercitrin 14 6-(g,g-Dimethylallyl)kaempferol 7-O-b-d-glucoside 15 6-(g,g-Dimethylallyl)quercetin 7-O-b-d-glucoside 16 6-(3-Hydroxy-3-methylbutyl)quercetin 7-O-b-d-glucoside Isoflavones 22 Afrormosin 23 Prunetin 24 Isoprunetin 25 Gerontoisoflavone A 26 5,7,4’-Trimethoxyisoflavone 27 Irilone 28 3’-O-Methylirilone 29 4’-O-Methylirilone O. O. O. O. O. O. O. O. O. O. O. O. O. O. beddomei lanceolata calodendron integerrima integerrima integerrima afzelii afzelii calodendron calodendron calodendron calodendron integerrima calodendron Leaves Stem bark Leaves Stem wood Root bark Stem wood Stem bark Leaves Leaves Leaves Stem heartwood Leaves Stem wood Stem heartwood [13] [14] [21] [22] [22] [22] [23] [24] [21] [21] [25] [21] [22] [25] CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 257 Table 1 (cont.) No. Compound class and name Source 30 Squarrosin O. integerrima O. squarrosa 31 5-O-Methylsquarrosin O. integerrima O. squarrosa O. integerrima O. integerrima O. integerrima O. squarrosa 32 Methylirisolin O. integerrima O. integerrima O. integerrima O. integerrima O. afzelii O. afzelii 33 5,4’-Dihydroxy-3’-methoxy-6,7-(methylenedioxy)isoflavone O. calodendron 34 3’-Hydroxy-5,5’-dimethoxy-6,7-(methylenedioxy)isoflavone O. afzelii 35 5,3’-Dihydroxy-4’,5’-dimethoxy-6,7-(methylenedioxy)isoflav- O. afzelii one 36 4’-Hydroxy-5-methoxy-6,7-(methylenedioxy)isoflavone O. afzelii 37 5,4’-Dimethoxy-3’-hydroxy-6,7-(methylenedioxy)isoflavone O. afzelii 38 4’-Hydroxy-5,3’-dimethoxy-6,7-m(ethylenedioxy)isoflavone O. afzelii 39 5,7,8-Trimethoxy-3’,4’-(methylenedioxy)isoflavone O. afzelii O. afzelii O. squarrosa 40 Lanceolone O. afzelii Flavan-3-ols 41 ()-Epicatechin 42 (þ)-Catechin 43 (þ)-Epicatechin O. beddomei O. lanceolata O. lanceolata O. calodendron O. afzelii Scheme 1. Synthesis of Furanoflavones 6 and 7 Part Ref. Stem wood Heartwood Leaves, twigs Root bark Stem wood Root bark Root wood Heartwood Leaves, twigs Stem wood Root bark Root wood Stem bark Leaves Stem heartwood Stem bark Stem bark [22] [26] [10] [12] [22] [22] [22] [26] [10] [22] [22] [22] [23] [24] [25] [27] [27] Stem bark Stem bark Stem bark Leaves Stem bark Root bark Leaves [27] [27] [27] [24] [27] [28] [24] Leaves Stem bark Leaves Stem bark Stem bark [13] [14] [16] [17] [27] 258 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 3.1.2. Biflavonoids. The genus Ochna is known to produce biflavonoids. Structurally, these are small polyphenolic molecules comprising two identical or non-identical flavonoid units joined in a symmetrical or unsymmetrical manner through an alkyl(CC) or an alkoxy-based (COC) linker of varying length. These possibilities allow significant structural variation in biflavonoids, which is further amplified by the positions of the functional groups, e.g., OH, MeO, C¼O groups, or C¼C bonds, and stereogenic centers on the flavonoid scaffold. To date, 43 biflavonoids, 44 – 86, have been isolated from Ochna (Table 2). Ochnaflavone (44), the taxonomic marker of the genus, and its mono- and dimethyl ethers, 45 and 46, respectively, were reported from the leaves of O. squarrosa by Kawano and co-workers in 1973 [29]. Compounds 44 – 46, reported for the first time, represent a new series of biflavone ethers linked via two phenyl rings. Later, nine more ochnaflavone derivatives, 47 – 55, were obtained from several Ochna species and are classified as follows: a) flavoneflavone, 47; b) flavanoneflavone, 48 – 51; c) flavoneflavanone, 52 and 53, and d) flavanoneflavanone, 54 and 55 [10] [13 – 15] [19] [30] [31] [33]. Among them, 7’’-O-methylochnaflavone (48) and 2’’,3’’-dihydroochnaflavone 7’’-O-methyl ether (53), isolated from O. integerrima, have shown anti-HIV-1 activity, based on the results of HIV-1 RT and cell based assays [10], while tetrahydroochnaflavone (54), a new biflavanone reported by Gunasekar et al. from the leaves of O. beddomei, has been found to exert significant cytotoxicity against human nasopharynx carcinoma (KB) cells [33]. Tetrahydroamentoflavone (56) and its 7’’-O-methyl ether, 57, obtained from the leaves of O. pumila, were the first CC linked biflavanones reported from the Ochna genus [34]. Recently isolated biflavanones 58 – 62 with C(3)C(3’’) linkage were chamaejasmine (58) from the root bark of O. calodendron [18], 7,4’,7’’,4’’’-tetramethylisochamaejasmine (59) from O. lanceolata stem bark [14], ent-ruixianglangdusu B (60), an antipode of ruixianglangdusu B from O. lanceolata leaves [16], and biflavanones I and II (61 and 62, resp.) from the outer bark of O. integerrima [35]. Compounds 61 and 62, known only as biotransformation products from chalcones with peroxidases of cultured plant cells, have been isolated for the first time from natural plant extracts. Both 61 and 62 displayed significant antimalarial activities in vitro against Plasmodium falciparum strains, K1 and FCR-3, respectively. Biisoflavanones, hexaspermone C (63) and its dehydro derivative 64, together with a tetrahydrofuran derivative, ochnone (85), and a furobenzopyran derivative, cordigol (86), obtained from the bark of O. macrocalyx, possess antibacterial and cytotoxic activities [36]. Compounds 65 – 73, isolated from O. squarrosa, O. afzelii, O. integerrima, and O. calodendron [12] [22] [24] [27] [37 – 40] belong to the lophirone series, of which 5’’-hydroxylophirone B 7’’-O-b-d-glucoside (67) is the only biflavonoid O-glucoside known so far from the genus Ochna [37]. In 2003, Pegnyemb et al. investigated the stem bark of O. afzelii and reported afzelones A – D (74 – 77, resp.) [23] [27], of which afzelone A (74) is the first biflavonoid bearing a tetrahydrofuran and six-membered rings on the same aromatic ring, and the rearranged biflavonoid, afzelone D (77), is a derivative of lophirone A (65). Calodenone (78), calodenins A – C (79, 80, and 82, resp.), and dihydrocalodenin B (81) were isolated from O. squarrosa, O. calodendron, O. afzelii, O. integerrima, and O. macrocalyx [12] [18] [22 – 24] [27] [36 – 38] [40 – 42]. Among these, calodenin C (82), obtained from the stem bark of O. calodendron, is the first reported proanthocyanidin dimer from the genus Ochna [42]. Chalcone dimers lophirones C and K (68 and 72, resp.), and CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 259 260 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) calodenins A and B (79 and 80, resp.), possessing structures with either a benzofuran or a dihydrobenzofuran ring system, might have been derived biosynthetically from CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 261 condensation of an isoliquiritigenin unit involving the a-C-atom, and either the C(3) (ring A) of a second identical unit (i.e., 68 and 72, resp.) or the C(3’) (ring B) of a chalconaringenin unit (i.e., 79 and 80, resp.), as shown in Scheme 2. Compounds 83 and 84 were obtained from O. integerrima [22] [37]. 262 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) Table 2. Biflavonoids from the Genus Ochna No. Compound name Source Part Ref. 44 Ochnaflavone 45 46 47 Ochnaflavone 4’-O-methyl ether Ochnaflavone 7,4’-di-O-methyl ether 7’’-O-Methylochnaflavone 48 2,3-Dihydroochnaflavone 49 2,3-Dihydroochnaflavone 7-O-methyl ether 50 51 52 53 2,3-Dihydroochnaflavone 7’’-O-methyl ether 2,3-Dihydroochnaflavone 7,4’,7’’-tri-O-methyl ether 2’’,3’’-Dihydroochnaflavone 2’’,3’’-Dihydroochnaflavone 7’’-O-methyl ether 54 55 56 57 58 59 60 61 62 63 64 65 Tetrahydroochnaflavone 7-O-Methyltetrahydroochnaflavone Tetrahydroamentoflavone 7’’-O-Methyltetrahydroamentoflavone Chamaejasmin 7,4’,7’’,4’’’-Tetramethylisochamaejasmin ent-Ruixianglangdusu B Biflavanone I Biflavanone II Hexaspermone C Dehydroxyhexaspermone C Lophirone A 66 5’’-Hydroxylophirone B 67 68 5’’-Hydroxylophirone B 7’’-O-b-d-glucoside Lophirone C O. integerrima O. beddomei O. lanceolata O. obtusata O. lanceolata O. squarrosa O. beddomei O. pumila O. artopurpurea O. squarrosa O. squarrosa O. integerrima O. pumila O. beddomei O. lanceolata O. obtusata O. beddomei O. beddomei O. obtusata O. lanceolata O. beddomei O. integerrima O. integerrima O. integerrima O. beddomei O. beddomei O. pumila O. pumila O. calodendron O. lanceolata O. lanceolata O. integerrima O. integerrima O. macrocalyx O. macrocalyx O. squarrosa O. integerrima O. integerrima O. afzelii O. afzelii O. integerrima O. calodendron O. integerrima O. integerrima O. integerrima O. integerrima O. integerrima O. integerrima O. integerrima O. afzelii O. calodendron Leaves, twigs Leaves Stem bark Leaves Leaves Leaves Stem bark Leaves Leaves Leaves Leaves Leaves, twigs Leaves Leaves Stem bark Leaves Stem bark Leaves Leaves Stem bark Stem bark Leaves Leaves, twigs Leaves Leaves Leaves Leaves Leaves Root bark Stem bark Leaves Outer bark Outer bark Bark Bark Root bark Root bark Root wood Leaves Stem bark Stem bark Stem bark Root bark Root wood Stem bark Stem bark Root bark Root wood Stem bark Stem bark Stem bark [10] [13] [14] [15] [16] [29] [30] [31] [32] [29] [29] [10] [31] [13] [14] [15] [30] [13] [15] [14] [30] [19] [10] [19] [33] [13] [34] [34] [18] [14] [16] [35] [35] [36] [36] [12] [22] [22] [24] [27] [37] [38] [22] [22] [37] [37] [22] [22] [37] [39] [40] CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 263 Table 2 (cont.) No. Compound name Source Part Ref. 69 70 71 72 73 74 75 76 77 78 Isolophirone C Dihydrolophirone C Lophirone H Lophirone K Lophirone L Afzelone A Afzelone B Afzelone C Afzelone D Calodenone 79 Calodenin A 80 Calodenin B 81 Dihydrocalodenin B 82 83 Calodenin C 1-[3-(2,4-Dihydroxybenzoyl)-4,6-dihydroxy-2(4-hydroxyphenyl)-1-benzofuran-7-yl]-3(4-hydroxyphenyl)prop-2-en-1-one 1-[3-(2,4-Dihydroxybenzoyl)-2,3-dihydro-4,6dihydroxy-2-(4-hydroxyphenyl)-1-benzofuran7-yl]-3-(4-hydroxyphenyl)prop-2-en-1-one Ochnone Cordigol O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. Stem bark Stem bark Root bark Stem bark Root bark Stem bark Stem bark Stem bark Stem bark Root bark Root bark Root bark Root wood Stem bark Stem bark Stem bark Stem bark Stem bark Leaves Stem bark Bark Stem bark Bark Stem bark Stem bark Root bark Root wood Stem bark Root wood Stem bark [39] [39] [12] [40] [12] [23] [23] [23] [27] [12] [18] [22] [22] [27] [37] [38] [40] [23] [41] [24] [23] [41] [36] [40] [36] [41] [42] [22] [22] [37] [22] [37] Bark Bark [36] [36] 84 85 86 afzelii afzelii squarrosa calodendron squarrosa afzelii afzelii afzelii afzelii squarrosa calodendron integerrima integerrima afzelii integerrima calodendron calodendron afzelii afzelii afzelii macrocalyx calodendron macrocalyx afzelii calodendron integerrima integerrima integerrima integerrima integerrima O. macrocalyx O. macrocalyx 3.1.3. Triflavonoids. Two new triflavonoid pigments, caloflavans A and B (87 and 88, resp.), were isolated from the CH2Cl2 extract of the leaves of O. calodendron [21]. From a biogenetic point of view, both 87 and 88 may arise from condensation of isombamichalcone with afzelechin, either at C(6) or C(8) of ring A, respectively. 3.1.4. Pentaflavonoids. In 2001, Messanga and co-workers isolated a novel pentaflavonoid, ochnachalcone (89), from the more polar portion of the AcOEt fraction of the stem bark of O. calodendron [17]. It is built up from two bichalcone units connected via the flavan-3-ol (afzelechin), and it has been reported for the first time from an Ochnaceae species. 3.2. Anthranoids. Eight anthranoids, i.e., two anthraquinones, 90 and 91, five anthrones, 92 – 96, and a trans-bianthrone, 97, have been isolated from O. squarrosa, O. obtusata, and O. pulchra [12] [43 – 45] (Table 3). Among them, przewalskinone B (91), 264 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) Scheme 2. Proposed Biosynthesis of Lophirone C (68) Table 3. Anthranoids from the Genus Ochna No. Compound name Source Part Ref. 90 91 92 93 94 95 96 97 Chrysophanol Przewalskinone B Vismione D Acetylvismione D Vismione L Vismione M 3-O-Geranylemodinanthrone ()-Ochnabianthrone O. O. O. O. O. O. O. O. Root bark Stem bark Root bark Root bark Root bark Root bark Root bark Root bark [12] [43] [44] [44] [44] [44] [44] [44] [45] squarrosa obtusata pulchra pulchra pulchra pulchra pulchra pulchra vismiones L and M (94 and 95, resp.), and ()-ochnabianthrone (97) are new compounds. 3.3. Triterpenes and Steroids. An oleanane triterpene, oleanolic acid (98), from the heartwood of O. squarrosa [25], and three steroids, b-sitosterol (99), b-sitosterol b-dglucoside (100), and campesterol (101), from O. squarrosa, O. afzelii, and O. calodendron, were isolated [21] [25] [27] [38] [46] [47] (Table 4). 3.4. Fatty Acids. A total of eight fatty acids, 102 – 109, have been reported from various Ochna species (Table 5). Among them, calodendrosides B and C (102 and 103, resp.) are two novel fatty acid glycosides, isolated by Messanga et al. [18] from the root bark of O. calodendron; while from the whole plant of O. squarrosa, octacosan-1-ol CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 265 (104) was reported [47]. In another study, Osman and co-workers reported on O. squarrosa and O. artopurpurea seed oil as rich sources of palmitic acid (105; 73.5%), 266 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) tripalmitin (106; 50%), linoleic acid (107; 10.9%), oleic acid (108; 14.1%), and palmitoleic acid (109; 25.6%) [48 – 50]. 3.5. Others. A glucoside derivative, lanceoloside A (110), and a cyanoglucoside, menisdaurin (111), were reported from the stem bark and leaves of O. afzelii and O. calodendron, respectively [23] [21]. 4. Biological Activities. – 4.1. Antimicrobial Activity. In 2003, Tang et al. [36] reported on evaluation of the antibacterial activity of biflavonoids, 63, 64, 80, 81, and CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 267 Table 4. Triterpenes and Steroids from the Genus Ochna No. 98 99 Compound class and name Triterpene Oleanolic acid Steroids b-Sitosterol 100 b-Sitosterol b-d-glucoside 101 Campesterol Source Part Ref. O. squarrosa Heartwood [26] O. calodendron O. squarrosa O. squarrosa O. calodendron O. afzelii O. calodendron O. squarrosa O. squarrosa Leaves Heartwood Leaves Leaves Stem bark Stem bark Whole plant Whole plant [21] [26] [46] [21] [27] [38] [47] [47] Table 5. Fatty Acids from the Genus Ochna No. Compound name Source Part Ref. 102 103 104 105 106 107 108 109 Calodendroside B Calodendroside C Octacosan-1-ol Palmitic acid Tripalmitin Linoleic acid Oleic acid Palmitoleic acid O. calodendron O. calodendron O. squarrosa O. squarrosa O. squarrosa O. squarrosa O. squarrosa O. artopurpurea Root bark Root bark Whole plant Seed oil Seed oil Seed oil Seed oil Seed oil [18] [18] [47] [48] [49] [48] [48] [48] [50] 85, isolated from the EtOH extract of O. macrocalyx by the broth dilution assay method [51]. Among them, dihydrocalodenin B (81) showed good activity against the three strains of multidrug-resistant (MDR) Staphylococcus aureus (RN4220, XU212, and SA-1199-B), with a minimum inhibitory concentration (MIC) value of 8 mg/ml (15 mm), while calodenin B (80) was found to be active against the XU212 strain only. Compounds 63, 64, and 85 were inactive at a concentration of 64 mg/ml. These results indicated that the strong antibacterial activity of 81 against MDR S. aureus may render it a good candidate for further investigation. The crude bark has traditionally been used in treatment of diarrhea; when tested against Escherichia coli, which is known to cause gastrointestinal problems, especially diarrhea, the crude bark extract showed no activity at 512 mg/ml. More recently, the antimicrobial activities of crude acetone and MeOH extracts of O. schweinfurthiana leaves were examined by Abdullahi et al. by using disc-diffusion and nutrient broth-dilution techniques [52]. Both extracts (600 mg/disc) have shown a broad spectra of activities on susceptibility tests, with a mean zone of inhibition ranging from 15 to 21 mm against Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (NCTC 6750), Salmonella typhi (ATCC 19430), and Staphylococcus aureus (ATCC 021001); however, no activity was observed against methicillin-resistant Staphylococcus aureus, Neisseria gonorrhea, Corynebacterium ulcerans, Bacillus subtilis (NCTC 8236), Escherichia Coli (NCTC 10418), and Candida albicans (ATCC 10231). 268 CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) Sparfloxacin (100 mg/disc), a standard antibiotic, inhibited the growth of all of the organisms tested, with the exception of C. albicans. Except for Streptococcus pyogenes, the acetone extract showed higher activity against the tested organisms, as indicated by MIC and minimum bactericidal concentration (MBC) test results, with a mean zone of inhibition of 10 – 20 and 20 – 40 mg/ml, respectively, which provides validation for the ethnomedicinal use of O. schweinfurthiana leaves in wound dressing and treatment of forms of bacterial infections. 4.2. Cytotoxic Activity. Compounds 63, 64, 80, 81, and 85 were assayed for cytotoxic activity against MCF-7 breast cancer cells on 96-well plates by the MTT (3-(4,5dimethylthiazol-2-yl-)-2,5-diphenyl-2H-tetrazolium bromide) reduction method, using doxorubicin as a reference compound [36]. Moderate-to-good cytotoxicities were observed for compounds 80, 81, and 85, with IC50 values of 56  7, 35  7, and 7  0.5 mm, respectively, whereas compounds 63 and 64 did not show any toxicity below 100 mm. The crude extract exhibited cytotoxicity with an IC50 value of 52  10 mg/ml. 4.3. NF-kB Activity. The crude EtOH bark extract and column subfractions of O. macrocalyx were found to have nuclear factor kappa B (NF-kB) inhibitory activity at 200 mg/ml in the electrophoretic mobility shift assay (EMSA); by the IL-6 luciferase reporter gene assay method, crude and AcOEt extracts showed inhibitions of 42  8% and 30  9%, respectively, at 100 mg/ml [36]. For both assays, parthenolide was used as a positive control [53]. Interestingly, none of the isolated compounds, 63, 64, 80, 81, 85, and 86, exhibited any activity. 4.4. Analgesic and Anti-Inflammatory Activity. The crude AcOEt fraction and the new constituents 6, 7, and 73, isolated from the root bark of O. squarrosa, were examined for analgesic (tail-flick method in Swiss mice) and anti-inflammatory (carrageenan-induced paw edema method in albino rats) activities by Anuradha et al. in 2006 [12]. According to the results, all of the test compounds exhibited significant analgesic activity at 25 mg/kg, compared with control (1% Tween 80). Moreover, the crude extract and compound 7 showed more promising protection at early reaction time and potency than at standard reaction time. The crude extract showed promising anti-inflammatory activity at 25 mg/kg, comparable to standard (diclofenac sodium; 20 mg/kg), while compounds, 6, 7, and 73 were active at higher doses than standard. The potent analgesic and anti-inflammatory activities of the crude extract may be due to the synergistic effect of the mixture of compounds occurring in natural proportion. 4.5. Antimalarial Activity. Bioassay-guided purification of the 80% EtOH extract of the outer bark of O. integerrima, which preliminarily showed significant in vitro antimalarial activity against the MDR strain (K1) of Plasmodium falciparum and sensitive strain FCR-3 (IC50 of 6.5 and 4.5 mg/kg, resp.), furnished biflavanones I and II (61 and 62, resp.) as new antimalarial principles [35]. Test results indicated that the activity of the major active compound 61 (IC50 80 ng/ml) against the K1 strain was three times higher than against FCR-3 strain, with a selectivity of 375, while its stereoisomer 62 was 65 and 17 times weaker for both strains. It appears that 61 could be a promising compound to be used in investigations of antimalarial activity in vivo in animal models. 4.6. Anti-HIV-1 Activity. In 2007, Reutrakul et al. examined the anti-HIV-1 activity of crude MeOH extract, AcOEt fraction, and the pure isolated compounds, 1, 15 – 19, 31, 47, and 53, of the leaves and twigs of O. integerrima [10] by using syncytium ( DTat/revMC99 virus and the 1A2 cell line) [54] and reverse transcriptase (RT) assay CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 269 methods [55]. Flavonoids, 15 – 19, with an isoprenyl and sugar groups on ring A, showed significant anti-HIV-1 activities in the syncytium assay, with EC50 values ranging from 14.0 to 102.4 mg/ml. Biflavonoids 7’’-O-methylochnaflavone (43) and 2’’,3’’-dihydroochnaflavone 7’’-O-methyl ether (53) were found to be very active in the syncytium assay (EC50 2.0 and 0.9 mg/ml, resp.), and demonstrated potent inhibition of HIV-1 RT (IC50 2.0 and 2.4 mg/ml, resp.), comparable with a non-nucleoside RT inhibitor, nevirapine. Vitexin (1), with only a sugar moiety on ring A, exhibited weak activity in the HIV-1 RT assay. The lack of anti-HIV-1 activity of 31 in both assays might be due to the absence of an isoprenyl moiety in its structure. Therefore, it appears that both isoprenyl and sugar moieties on ring A were crucial for anti-HIV-1 activity. 4.7. b-Lactamase Inhibitory Activity. In 2007, Gangoué-Piéboji et al., while searching for anti-b-lactamase inhibitors, found that the crude MeOH bark extracts of O. afzelii exhibited strong inhibition activity in vitro against OXA-10 and P99 b-lactamases (IC50 98% and 94%, resp.) [56]. 5. Conclusions. – Plants of the genus Ochna have a wide distribution, and have been used in folk medicine for treatment of various ailments. So far, 111 chemical constituents, viz., flavonoids, anthranoids, triterpenes, steroids, fatty acids, and a few others, have been identified in the genus, and many of these have been evaluated for their biological activity. 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