UNDER PEER REVIEW

1 Biological, Pharmacological and Therapeutic Activities of africana

2 (Lam.) Benth

3 Sunday Ene-Ojo Atawodi * and Olufunsho Dayo Olowoniyi

4 Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria

5 Abstract

6 Occurring widely in and beyond is Kigelia africana Benth, a medicinal with several

7 attributes and considerable potentials. Various parts of the plant are used locally to treat cancer,

8 ulcer, gynecological disorders, genital infections, skin diseases, diabetes, epilepsy, bacterial and

9 fungal infections as well as being used as cosmetics to enhance beauty. The antioxidant and anti-

10 inflammatory properties of some parts of the plant have also been reported. Phytochemical

11 analyses revealed the presence of naphthaquinones such as lapachol, kigelinol and isopinnatal in

12 the root and iridoids such as specioside, verminoside and minecoside in the stem bark, while the

13 are reported to be rich in hydrocarbons like n-hentriacontane, 1-tricosene and esters like

14 pentafluoroheptadecyl ester. The are reported to contain iridoids such as jiofuran,

15 jioglutolide and ajugol as well as flavonoids and steroids. Toxicological evaluation showed that

16 the LD50 for the ethanolic extracts of the and stem bark are 1.3g/kg and 4g/kg respectively,

17 while the methanolic extract of the fruit had an LD50 of 3.98g/kg. A 50% methanol-water extract

18 of the gave an LD50 greater than 3g/kg. In this review we provide an up-to-date information

19 on the established biological, pharmacological and toxicological properties as well as

20 phytochemicals responsible for these activities in Kigelia africana.

21 Keywords: Kigelia africana; sausage ; Bioactive compounds; Biological activity; 22 Toxicological effects 23 24

25 1.0 Introduction

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26 Kigelia africana (syn. Kigelia pinnata, Kigelia aethiopica) is commonly called sausage or

27 cucumber tree because of its huge fruits. It belongs to the family. Due to its wide

28 occurrence, it has vernacular names in many African languages: Rawuya (Hausa); Uturubein

29 (Igbo); Pandoro, Iyan (Yoruba); Bechi (Nupe); Mwegea (Swahili); Umfongothi (Zulu) [1, 2] and

30 Ebie in Igala. In Hindi it is known as Balmkheera [3]. Kigelia africana occurs throughout

31 tropical Africa. It grows particularly well in wetter areas, spreading across the wet savannah and

32 riverine areas [3].

33

34 The tree can grow up to 20m tall. It is evergreen where rainfall occurs throughout the year, but

35 where there is a long dry season. The leaves are opposite or in whorls of three, 30-

36 50cm long, pinnate, with six to ten oval leaflets up to 20cm long and 6cm broad; the terminal

37 leaflet can be present or absent. The (and later the fruit) hang down from branches

38 on long flexible stems. Flowers are produced in panicles; they are bell-shaped, orange to reddish

39 or purplish green and about 10cm wide. Individual flowers do not hang down but are oriented

40 horizontally, some birds are attracted to these flowers and the strong stems of each make

41 ideal footholds. Their scent is most notable at night indicating their reliance on pollination by

42 , which visit them for pollen and nectar [4].

43

44 Kigelia africana is characterized by its huge fruits which can weigh between 5 to 10kg [5]. The

45 fruit is a woody from 30 – 100cm long and up to 18cm broad, hanging down on rope-like

46 peduncles [4]. The fruit is indehiscent, with woody wall and heavily marked with lenticels at the

47 surface. It is grey-brown and many seeded when matured. are ovoid, ca. 10mm x 7mm

48 with leathery testa, embedded in a fibrous pulp. The fruit is eaten by several species of

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49 , including , bush pigs, savannah elephants, hippopotami, monkeys and

50 porcupines. The seeds are dispersed in their dung. The seeds are also eaten by parrots and brown-

51 headed parrots, and the foliage by elephants and [4].

52

53 This review on Kigelia africana is patterned into biological activities such as antiprotozoal,

54 antibacterial, antifungal activities; pharmacological properties such as anti-inflammatory and

55 analgesic, anticancer, antidiarrhoeal, anti-ulcer effects as well as toxicological effects with the

56 responsible phytochemicals.

57

58 2.0 Uses of Kigelia africana in traditional medicine and their pharmacological evaluation

59 2.1 Anti-protozoal activity

60 One of the several uses of Kigelia africana is for treating malaria. In-vitro studies revealed the

61 efficacy of hexane, dichloromethane, ethyl acetate and ethanol extracts of the root bark against

62 Plasmodium falciparum [6] and Trypanosoma brucei brucei and T.b. rhodesiense [3], the

63 causative organisms for malaria and sleeping sickness respectively. The growth of Entamoeba

64 histolytica was also inhibited by the stem bark butanol extract [7]. Four compounds that

65 exhibited significant anti-plasmodial activity were isolated from the ethyl acetate extract of

66 Kigelia Africana. Three of the four compounds showed good activity against all the different

67 parasite strains, the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum,

68 with IC50<5µM. Specicoside exhibited the highest activity on W-2 (IC50=1.5µM) followed by

69 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid (IC50=1.60µM) and atranorin (IC50=4.41µM) while

70 p-hydroxycinnamic acid was the least active (IC50=53.84µM) [6]. Lapachol in the methanol

71 extract of the root and another compound (aquinone) obtained from the wood show anti-malarial

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72 activity [3]. In a study of the anti-amoebic activity of the butanol extract of the stem bark, three

73 known iridoids, specioside, verminoside and minecoside were isolated, purified and identified to

74 be responsible. Verminoside exhibited two fold anti-amoebic activities as compared to the

75 standard drug while specioside showed comparable activity with metronidazole [7]. The anti-

76 trypanosome activity of the stem bark and root bark extracts are attributed to 2-(1-hydroxyethyl)-

77 naphtho-[2,3-b]-furan-4,9-quinone and three naphthoquinoids: isopinnatal, kigelinol and

78 isokigelinol [3].

79

80 2.2 Antibacterial and antifungal activities

81 Various parts of Kigelia africana are employed to treat bacterial and fungal infections. In a study

82 to verify these properties, crude extracts of stem bark and fruits were prepared with distilled

83 water, ethanol or ethyl acetate. In the microtitre plate bioassay, the stem bark and fruit extracts

84 showed similar antibacterial effects against Gram-negative and Gram-positive bacteria. A

85 mixture of three fatty acids exhibiting antibacterial effects was isolated from the ethyl acetate

86 extract of the fruits using bioassay-guided fractionation. Palmitic acid was the major antibacterial

87 compound in this mixture thus supporting the traditional use of the plant in therapy of bacterial

88 infections [3]. A biologically monitored fractionation of the methanolic extracts of the root and

89 fruits led to the isolation of the naphthoquinones, kigelinone, iso-pinnatal, dehydro-α-lapachone

90 and lapachol and the phenylpropanoids, ρ-coumaric acid and ferulic acid as the compounds

91 contributing to the observed antibacterial and antifungal activities [4].

92

93 In another antibacterial and antifungal study using the agar diffusion technique, Owolabi and co-

94 workers reported that like amoxillin standard antibiotics, crude ethanolic extract exhibited

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95 antibacterial and antifungal activities against Staphylococcus aureus and Candida albicans with

96 zones of inhibition measuring 15.0±0.95 and 20.75±4.6mm respectively but the aqueous extract

97 exhibited no antibacterial or antifungal activity. The minimum inhibitory concentration for the

98 ethanol extract was found to be 6.25±1.07mg/ml for S. aureus and 7.92±1.52mg/ml for C.

99 albicans. In another report, it was also established that the stem bark extract inhibited a number

100 of harmful micro-organisms, including Escherichia coli (responsible for abscesses),

101 Pseudomonas aeruginosa(which causes skin sepsis and infections), Staphylococcus aureus

102 (which causes impetigo and skin abscesses) and Candida albicans, a fungal organism that causes

103 thrush [4]. Similarly, evaluation of the antibacterial activities of ethanolic and aqueous extracts

104 of Kigelia africana fruit against multi drug resistant Pseudomonas aeruginosa, both ethanolic

105 and aqueous extracts showed antibacterial activity, with the ethanolic extract showing more

106 potency [8-10].

107

108 2.3 Anti-inflammatory and analgesic activity

109 The use of the bark, stem, twigs, leaves and fruits of Kigelia africana to relieve rheumatism,

110 tooth and headache has been documented [3, 11]. Picerno and co-workers [12] reported that the

111 anti-inflammatory property of Kigelia africana fruit polar extract was due to the constituent

112 verminoside which is known to cause significant anti-inflammatory effects inhibiting both iNOS

113 expression and NO release in the LPS-induced J774.A1 macrophage cell line [12].

114

115 The ethanolic extract of the stem bark has been evaluated for analgesic property using acetic acid

116 induced mouse writhing and hot plate reaction time; and anti-inflammatory property using the

117 carrageenan-induced paw oedema. The extract showed a dose dependent significant reduction of

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118 the number of writhes (p<0.001) with 500mg/kg body weight dose giving the highest reduction.

119 The extract showed an insignificant elongation of the hot plate reaction time (p>0.05). In the

120 carrageenan-induced paw oedema, a dose dependent significant inhibition was observed

121 (p<0.001) between the second and the fifth hour, confirming that the ethanolic stem bark extract

122 has significant analgesic and anti-inflammatory properties. Inhibition of the synthesis of

123 prostaglandins and other inflammatory mediators has been suggested to be responsible for the

124 analgesic and anti-inflammatory properties [13].

125

126 2.4 Anti-diarrhea activity

127 One important local use of Kigelia africana is the use of the leaf for treating diarrhea. Scientific

128 evaluation showed that administration at a dose of 100 or 200mg/kg of aqueous leaf extract to

129 experimental animals caused anti-diarrhea activity, as evidenced by the reduced fecal output in

130 castor oil – induced diarrhea in animals and remarkably decreased the propulsive movement of

131 the gastro-intestinal contents [14]. On the isolated guinea pig ileum, the extract did not

132 appreciably affect acetylcholine and histamine induced contractions. In an antidiarrheal activity

133 studied in vivo using castor oil to induce diarrhea in rats and in vitro using isolated jejunum, 500

134 and 1000mg/kg ethanol root extract significantly reduced the frequency of diarrheal stool and the

135 spontaneous propulsive movement of isolated jejunum. Kigelia africana root extract also

136 produced reversible inhibition of acetylcholine induced mobility of isolated rabbit jejunum. The

137 observed spasmolytic effects of the extract may explain its continual use in the management of

138 chronic abdominal pains associated with diarrhea [2] The intra-peritoneal LD50 of the extract in

139 mice was estimated to be 785.65±24 mg/kg [15].

140

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141 2.5 Anti-diabetic and antioxidant activities

142 Like many other African food [16, 17] and medicinal [18 – 20], the use of Kigelia

143 africana to manage diabetes is traditionally practiced and reported [2,3]. A decoction of the dried

144 fruit is made by boiling in water for an hour. The anti-diabetic activity and the antioxidant effect

145 were studied [21]. Also, in a polyherbal preparation, ADD-199, Kigelia africana is in

146 combination with three other plants: Maytenus senegalenses, Annona senegalenses and Lannea

147 welwitchii. The anti-diabetic and antioxidant effects were investigated in streptozotocin-induced

148 diabetic C3H mice and results compared with two allopathic hypoglycaemic drugs,

149 glibenclamide and metformin. Plasma glucose, insulin and lipids as well as liver glycogen and

150 lipid peroxidation were measured following treatment for eight weeks. The results indicated that

151 plasma insulin levels in normal controls at termination were about 76µmol/L compared to trace

152 levels in untreated diabetic mice. Like glibenclamide, ADD-199 increased insulin levels in

153 diabetic mice up to 70% of levels in untreated non-diabetic mice whilst metformin had no effect.

154 Also, basal plasma glucose levels in diabetic controls (18.8 mM) were reduced to 14.0 mM by

155 100 mg/kg ADD-199 in < 2 weeks compared to 4 to 6 weeks for glinbenclamide and metformin,

156 respectively. This hypoglycaemic effect of ADD-199 was associated with the alkaloidal content

157 of the extract. Treatment with ADD-199 or the hypoglycaemic agents reversed the observed

158 elevation in plasma lipids but increased hepatic glycogen, triacylglycerol and cholesterol levels.

159 Treatment also increased hepatic glucose uptake by isolated diaphragms and attenuated hepatic

160 lipid peroxidation. These antihyperglycaemic and antioxidant actions of ADD-199 was

161 comparable to those of the maximum daily therapeutic doses of glibenclamide (0.25 mg/kg) and

162 metformin at 50 mg/kg [21].

163

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164 Olaleye and Rocha, [22] carried out an in-vitro assessment of the antioxidant property of Kigelia

165 africana extracts in rat liver homogenate. Increased formation of thiobarbituric acd reactive

166 substances (TBARS) was stimulated by different pro-oxidants: 10µM iron (II) sulphate, (FeSO4),

167 5µM sodium nitroprusside (SNP), and 2mM 3-nitropropionic acid administered, which indicates

168 lipid peroxidation in the liver. Administration of Kigelia africana statistically (p<0.05) reduced

169 the production of TBARS in a concentration-dependent manner in all the pro-oxidant-induced

170 oxidative stress, suggesting that the use of the plant in the treatment of various diseases,

171 especially liver diseases could be due to its ability to act as an antioxidant [22]. Saini and co-

172 workers [3] attributed the antioxidant potential of Kigelia africana to caffeic acid derivatives and

173 other compounds unique to the plant.

174

175 2.6 Anti-ulcer effect of Kigelia africana

176 The use of Kigelia africana fruit, bark and root to treat ulcer has been reported [3]. Owolabi and

177 Nworgu [23] investigated the anti-ulcer activity of the ethanol extract of Kigelia africana stem

178 bark in Wistar albino rats. In both preventive and curative models of ulcer respectively induced

179 by absolute ethanol and indometacin, the extract caused marked inhibition of ulceration,

180 suggesting a dose-dependent gastro-protective effect by the plant in the two models of ulcer [23].

181

182

183

184 2.7 Toxicity of Kigelia africana parts

185 2.7.1 Acute toxicity

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186 Acute toxicity tests of the fruits indicate they are non-toxic [2]. In a study on the diuretic activity

187 of aqueous extract of the bark in experimental rats, Sharma and colleagues [24] reported that it

188 was safe up to 5g/kg. A determination of acute toxicity of the methanol fruit extract using male

189 Sprague Dawley rats showed that the extract was well tolerated by the animals as there were no

190 observable signs of acute toxicity effects like restiveness, seizure or dizziness after the

191 administration of 400mg/kg. However at 6400mg/kg, the animals showed signs of toxicity like

192 jerks and writhes with 60% death. At 12,800mg/kg there was 80% death of the animals. The

193 LD50 was estimated from a log-dose curve to be 3,981.07mg/kg [25-27]

194

195 In another study using paracetamol (acetaminophen)-induced liver toxicity model, administration

196 of 100mg/kg aqueous extract counteracted the effects of paracetamol on levels of aspartate

197 transaminase (AST), alanine transaminase (ALT), superoxide dismutase (SOD), catalase (CAT),

198 gluthathione peroxidase (GPx) and δ-aminolevulinate dehydrogenase (δ-ALA-D), suggesting

199 that it can act as hapato-protective agent against toxicity possibly through its antioxidant action

200 [22].

201

202 2.7.2 Sub-chronic and chronic toxicity

203 The sub-chronic toxicity of the aqueous anti-diabetic polyherbal extract ADD-199 containing

204 Kigelia africana and three other plants which was administered at a daily dose of 100 or 500

205 mg/kg body weight over 30 days, to male Wistar albino rats appeared to show no effect on many

206 haematological, urinary and plasma biochemical parameters as well as on some modulators of

207 some hepatic cytochrome P450 (CYP) isozymes normally measured as indices of organ specific

208 toxicity or potential for drug interactions. Specifically ADD-199 containing Kigelia africana did

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209 not affect plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline

210 phosphatase (ALP) and albumin or creatinine kinase (CK) levels. It also did not affect plasma

211 creatinine and urea levels. Furthermore, ADD-199 neither affected packed cell volume (PCV),

212 nor red blood cells (RBC), reticulocytes, platelets, lymphocytes and granulocyte levels. It

213 however, caused significant dose-dependent reductions in white blood cell counts at day 15 with

214 varying degrees of recovery by day 30. Also, it reduced the rate of body weight increases after

215 week 3. However, no changes were observed in organ weight at termination. The ADD-199 did

216 not significantly affect zoxazolamine-induced paralysis and pentobarbital-induced sleeping times

217 as well as certain CYP isozyme activities in rats, suggesting that ADD-199 had no overt organ

218 specific toxicity and did not demonstrate a potential for drug interactions via CYP-mediated

219 metabolism in rats following sub-chronic administration [26].

220

221 The protective effect of methanol extract of Kigelia africana fruit extract against cisplatin-

222 induced renal toxicity in male rats has been studied [27]. Whereas over 28 days, cisplatin-treated

223 rats suffered loss in body weight, elevation in blood urea nitrogen and serum creatinine levels as

224 well as tubular necrosis, pre-treatment with Kigelia africana fruit methanol extract as a

225 prophylaxis significantly prevented these changes. Though post-treatment of animals with the

226 extract after cisplatin treatment did not completely restore serum catalase activity, it caused some

227 alleviating effects, suggesting that Kigelia africana fruit extract may protect against cisplatin-

228 induced renal toxicity, and hence might serve as a novel agent to limit renal injury [27].

229

230 2.7.3 Cytotoxic activity

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231 The cytotoxicity of hexane, chloroform, ethyl acetate, ethanol and methanol extracts of different

232 parts of Kigelia africana has been studied on Artemia salina using the brine shrimp lethality test

233 (BSLT). Some workers [28] have reported moderate toxicity of the ethanol extract of the root

234 and fruit at a dosage of 593 and 124µg/ml respectively while the ethyl acetate extract of the fruit

235 was also moderately toxic at 495µg/ml. Other workers [11, 28, 29] reported a moderate

236 cytotoxicity of ethanol extract of the fruit to Artemia salina at a dosage of 1000µg/ml.

237

238 2.8 Molluscidal and piscicidal effects

239 The molluscidal and piscidal effects of the aqueous extract of Kigelia africana bark has been

240 reported [25]. In a study to evaluate the piscicidal effect of Kigelia africana aqueous bark extract

241 against Clarias gariepinus fingerlings, graded concentrations of the extract, 40,80,120ppm were

242 prepared into which twenty fingerlings were added in replicates. The toxicity test lasted 24

243 hours during observations were made at 1, 2, 4, 8, 12, 16, 20 and 24 hours. Varying degrees of

244 mortality was recorded, with 100% death after 4 hours in the tank of 120ppm concentration and

245 the causative agent identified as coumarins.[30] Table 1 summarizes the toxic effects of Kigelia

246 africana extracts on different species of animals.

247

248

249 2.9 Anticancer activity of Kigelia africana parts

250 There are many reports in literature suggesting the use of Kigelia africana to either prevent or to

251 treat cancer [4, 30, 31]. In a study to determine the effect of Kigelia africana oil on cell

252 proliferation in culture, human colon adenocarcinoma (Caco-2) and human embryonic kidney

253 (HEK-293) cells were maintained and treated with various concentrations (0, 20, 40, 80, 100 and

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254 120mg/l) of Kigelia africana seed oil. The trypan blue dye exclusion method was used to

255 determine cell growth 48 hours after oil treatment. The seed oil suppressed both Caco-2 and

256 HEK-293 cell growth in a dose dependent manner. The seed oil did not cause increase cell death

257 as the number dead cells remained unchanged under control and oil-treated conditions. The oil

258 significantly suppressed Caco-2 cell growth compared to HEK-293 cell growth at all oil

259 concentrations. The suppression of Caco-2 and HEK-293 cell proliferation by Kigelia africana

260 seed oil suggest a potential antiproliferative effect of the oil on the two cell lines [11]

261 Methanolic extract of the root of Kigelia africana contains the constituent lapachol [30] which is

262 reported to be effective in the treatment of solar keratosis, skin cancer and Kaposi sarcoma, an

263 HIV-related skin ailment [31]. Serial dilutions of standardized aqueous, ethanol and

264 dichloromethane extracts of the stem bark and fruits of Kigelia africana were tested for their

265 growth inhibitory effects against four melanoma cell lines and a renal cell carcinoma line (Caki-

266 2) using two different assays (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,

267 MTT and Sulforhodamine B, SRB assays). Lapachol, a possible constituent of these extracts,

268 together with known therapeutic anti-neoplastic agents evaluated this way, showed significant

269 inhibitory activity of the dichloromethane extract of the stem bark and lapachol in a dose-

270 dependent and time-dependent manner. Chemosensitivity of the melanoma cell lines to the stem

271 bark was greater than that seen for the renal adenocarcinoma line, but in marked contrast

272 sensitivity to lapachol was similar amongst the five cell lines, suggesting that lapachol is the

273 active ingredient that exhibit anti-cancer property.

274

275

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276 2.10 Effect on the Central Nervous System

277 Among the variety of uses of Kigelia africana is in the treatment of epilepsy, CNS stimulating

278 activity and as antidotes against snake poisons. Snake bite antidotes are made with an infusion of

279 the fruits, stem, leaves, twig or bark taken orally or rubbed onto the bite [2]. The CNS stimulant

280 activity of the ethanolic stem bark extract has been verified [9]. The barbiturate induced sleeping

281 time and the Rota rod bar were used to study the effect of the extract on muscle coordination in

282 mice. The results showed that the extract at all doses tested reduced the duration of sleeping time

283 when compared to the control group that received distilled water. This difference in sleeping

284 time was significant (p<0.0001 at all doses tested) and was found to be dose dependent. Its effect

285 was also compared with caffeine (a known stimulant) and the extract gave a shorter duration of

286 sleeping time compared to caffeine (p<0.05 at 400 mg/kg dose) indicating better stimulant

287 properties. In comparison with diazepam, the extract at all doses tested also gave a statistically

288 significant shorter duration of sleep (p<0.0001). On the Rota rod, the extract had no sedative

289 effect as the animals maintained their balance on the rod through the entire period of the

290 experiment [25].

291

292 2.11 Effects on animal reproductive organs and reproductive system

293 In traditional medicine, sexual complaints such as infertility, poor libido, sexual asthenia and

294 impotence are treated with herbal prescriptions containing the fruit, roots or leaves of Kigelia

295 africana. A small amount of unripe fruit is chewed or an aqueous preparation of the fruit is taken

296 orally as a sexual stimulant, and the intoxicating traditional beer to which they are added is drunk

297 as an aphrodisiac [5]. The fruits are also applied on the breast to improve flow of milk in

298 lactating women [3]. Kigelia africana fruit aqueous extract has been successfully used as fertility

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299 enhancing agent in rats [32]. The steroidal components are thought to enhance reproductive

300 ability since steroids as androgen and estrogen have shown to contain fertility properties

301 necessary for the improvement and production of reproductive organs [33]. A study to

302 investigate the effects of varying dietary supplementation of Kigelia africana on the sperm

303 quality and fertility in African catfish, Clarias gariepinus showed that dietary inclusion of the

304 plant positively affected some parameters of sperm quality in the fish, with increases in sperm

305 counts, percentage motility, milt volume and motility duration [33].

306

307 2.12 Use as cosmetics

308 Traditionally, Kigelia africana is used as cosmetic to enhance beauty [5]. Some preparations

309 contain extract of one or more parts of the plant, mainly the fruit, stem bark or the pendulum

310 (where the fruit hangs from, or a product thereof). Typically, the preparation contains 50%

311 extract mixed with carrier, excipients and colorants (SumobrainTM). Aqueous or alcohol extracts

312 are ideal for water based cosmetic products such as gels, lotion, water or oil emulsions and

313 creams. The products are used to make anti ageing and regenerating skin care products, skin

314 tightening cosmetics such as bust firming products. Anti-inflammatory, antioxidant and

315 antibacterial agents are other products that are commercially made from Kigelia Africa

316 (PhytoTrade Africa).

317

318 2.13 Diuretic activity

319 The diuretic activity of Kigelia Africana aqueous bark extract was investigated by the

320 determination of urine volume, electrolyte concentration and diuretic potency in male albino rats.

321 Different concentrations of the extract, 250 and 500mg/kg were orally administered to hydrated

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322 rats and their urine output was immediately measured after 5 hours of treatment. Fusemide

323 (10mg/kg) was used as reference drug while normal saline (0.9%) solution was used as control.

324 The result showed that the bark extract exhibited dose dependent diuretic property. The onset of

325 diuretic action was within 1 hour and lasted up to 5 hours, with 500mg/kg displaying a potency

326 of 0.8 and 250mg/kg gave 0.32. The extract also caused a marked increase in Na+, K+ and Cl-

327 labels. The result suggests that the aqueous extract possess significant diuretic activity, justifying

328 its use in folk medicine.

329

330 3.0 Nutritional value of Kigelia africana

331 Kigelia africana provides a nutritious source of food during times of famine when the seeds are

332 roasted to eat. The fruit and bark are used in the brewing process to aid fermentation and enhance

333 the flavor of traditional beer. The fruit pulp is not edible as it may cause blistering of the tongue

334 and skin. However, fallen fruits along with leaves and flowers are browsed or foraged by

335 livestock and game [5]. In a study of the effect of Kigelia africana fruit meal (KAFM) on sperm

336 quality of African catfish, Clarias gariepinus, the KAFM supplement may have enhanced

337 nutrient utilization which is reflected by improvement in weight gain by testes [33].

338

339 The pro-fertility effect of dried fruit meal (KAFM) was investigated on reproductive

340 performance of female Clarias gariepinus fed with increasing levels for 90 days in relation to

341 egg production and quality (number, shape, structure, fecundity) and hatchability (%

342 fertilization, % hatching, % survival). The decrease in % deformity in hatchlings of Clarias

343 gariepinus fed dietary KAFM compared with the control diet suggest that KAFM improves the

344 quality of larvae. The highest % survival of hatchlings was recorded in the fish fed with dietary

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345 KAFM (100g KAFM/kg diet). Egg sizes for fish fed the control diet and dietary levels revealed

346 no significant difference in egg size. The result showed that dried KAFM had greater fertility on

347 male than on female Clarias garipinus [34].

348

349 4.0 Chemical Constituents of Kigelia africana

350 The large occurrence of secondary metabolites in different parts of Kigelia africana is

351 responsible for its several medicinal applications. These compounds include naphthaquinones,

352 iridoids, sterols, coumarins, flavonoids and alkaloids among others [3, 13, 35]. Structures of

353 isolated compounds have been characterized and identified using gas chromatography,

354 ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance and other

355 spectroscopic techniques like, mass spectrometry, or gas chromatography-mass spectrometry.

356 The use of Kigelia africana in traditional African medicines has been verified by corresponding

357 pharmacological properties.

358

359 4.1 Chemical constituents of Kigelia africana fruit

360 Gouda and colleagues [36] reported that a new furanone derivative formulated as 3-(2'-

361 hydroxyethyl)-5-(2''-hydroxypropyl) dihydrofuran-2-(3H)one and four new iridoids named: 7-

362 hydroxyviteoid II, 7-hydroxyeucommic acid, 7-hydroxy-10-deoxyeucommiol and 10-

363 deoxyeucommiol have been isolated from the fruits in addition to seven known iridoids namely,

364 jiofuran, jioglutolide,1-dehydroxy-3,4-dihydroaucubigenin, des-p-hydroxybenzoyl kisasagenol

365 B, ajugol, verminoside and 6-transcaffeoyl ajugol. Further phytochemical investigation of the

366 fruits of Kigelia africana yielded a new phenylpropanoid derivative identified as 6-p-

367 coumaroylsucrose together with ten known phenylpropanoid and phenylethanoid derivatives and

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368 a flavonoid glycoside [37]. A biologically monitored fractionation of the fruit led to the isolation

369 and identification of the naphthoquinones, kigelinone, isopinnatal, dehydro-alpha lapachol and

370 the phenylpropanoids p-coumaric acid and ferulic acid.

371

372 4.2 Chemical constituents of Kigelia africana stem

373 A study of the antimicrobial properties of the aqueous stem bark extract of Kigelia africana

374 revealed the presence of two naphthoquinones kigelinone and isopinnatal [35,3].Three known

375 iridoids: specioside, verminoside and minecoside have also been isolated from the stem bark

376 [40]. The dichloromethane extract of the stem bark contain naphthoquinones which possess anti

377 trypanosomal properties [3,4] while kigelin, β-sitosterol, 1,3-dimethylkigelin and ferulic acid

378 have been isolated from the bark, while the isolation of kigeliol from the wood and balaphonin

379 from the stem bark have been reported [38, 39].

380

381 4.3 Chemical constituents of Kigelia africana root

382 Earlier workers [40] reported the isolation and identification of the naphthoquinones, kigelinone,

383 isopinnatal, dehydro-alpha-lapachol and the phenylpropanoids p-coumaric acid and ferulic acid

384 from the root of Kigelia africana. Steroids, iridoids and coumarins have been isolated from the

385 root bark [38] as well as three isocoumarins: 6-methoxymellein, kigelin and 6-demethylkigelin

386 [41]. The isolation of kiglin and 6-methoxymellein together with two known compounds,

387 stigmasterol and lapachol from the root has also been reported [43]. Naphthoquinones that

388 possess anti-trypanosomal properties have been reported in the dichloromethane extract of the

389 root [3,4] while two non-quinonoid aldehydes, norviburtinal and pinnatal have been obtained

390 from the root bark [4, 43].

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391

392 4.4 Chemical constituents of Kigelia africana leaf

393 The hexane extract of the leaf of Kigelia africana has been reported to be rich in hydrocarbons

394 and some volatile compounds. In a study that qualitatively and quantitatively analyzed the

395 hexane extract for various chemical compositions, it was revealed to contain twelve compounds

396 with the major ones identified as n-hentriacontane, 1-tricosene, 11-(2,2-

397 dimethylpropylheneicosane, 2,6,10-trimethyldodecane, pentafluoroheptadecyl ester, 2-

398 ethylhexyloctadecyl sulfurous acid ester, heneicosane and hexyloctylsulfurous acid ester. Others

399 are 4,4-dimethylundecane, methyl-12-methyltetradecanoate, 1-iodohexadecane and 1-

400 iododecane. Hentriacontane have been reported to have a possible anti-tumour activity while

401 methyl-12-methyltetradecanoate has also been reported for its inhibition capacity on the

402 development of coneal angiogenesis, which is responsible for blindness and other infections [35].

403 Flavonoids and iridoids [36] and a 7-O-glucoside [42-44] have also been found in the leaves

404 (Table 2). The structural formulae of some of the chemical constituents isolated from Kigelia

405 africana are presented in Figure 1.

406

407 5.0 Conclusion

408 As a medicinal plant with a large array of secondary metabolites, Kigelia africana is applied in

409 traditional medicine to treat a wide range of diseases. There is the need to scientifically verify the

410 various healing attributes ascribed to it, following which establishment of the exact identity of

411 the chemical constituent responsible for any confirmed biological activity would enrich the

412 available chemical lead for new drugs.

413

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414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435 References

436

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437 [1] Mann A, Gbate M. and Umar AN.Medicinal and Economic Plants of Nupeland, Jube- 438 Evans Books & Publications, Bida. 1st Edition. .2003; 277 pp 439 440 441 [2] Otimenyin SO. Uzochukwu DC. Spasmolytic and Anti-diarrhea effects of the bark of 442 Erythrina senegalensis and root of Kigelia africana, Asian J Pharm Clin Res 2012; 3 (4): 443 11-14 444 445 [3] Saini S, Kaur H,Verma,B.,Ripudaman, Singh S. Kigelia africana (Lam.) Benth. An 446 overview. Nat Prod Rad 2009; 8 (2): 190-97. 447 448 [4] Olatunji AG. Atolani O. Comprehensive scientific demystification of Kigelia africana:a 449 review. Afr J Pure Appl Chem 2009; 3(9): 158-64. 450 451 452 [5] Oyelami OA. Yusuf KO. Oyelami AO. The use of Kigelia africana in the management of 453 polycystic ovary syndrome (PCOS). Chin Med 2012; 3, 1-3. 454 455 (6) Oyeku A. Oyelami, Kafayat O. Yusuf, Atinuke O. Oyelami. The Use of Kigelia 456 africana in the Management of Polycystic Ovary Syndrome (PCOS) Chin Med 2012; 457 3:1-3 458 459 (7) Bharti N, Singh S. Fermida N, Amir A. Isolation and in vitro antiamoebic activity of 460 iridoids isolated from Kigelia pinnata, ARKIVOC 2006: 69-76 461 462 (8) Binutu OA. Adesogan KE. Okogun,JI. Antibacterial and antifungal compounds from 463 Kigelia pinnata, Planta Med 1996; 62 (4): 352-353. 464 465 (9) Owolabi OJ,Omogbai EKI. Obasuyi, O. Antifungal and antibacterial activities of the 466 ethanolic and aqueous extracts of Kigelia africana (Bignoniaceae) stem bark. Afri J 467 Biotechnol 2007; 6 (15):1677-80. 468 469 470 (10) Tyagi A, Singh V. Bharadwaj M, Kumar A, Thakur K. Isolation and antibacterial 471 susceptibility testing of multi drug resistant Pseudomonas aeruginosa causing urinary 472 tract infection. J Chem Pharm Res 2011; 3(4): 342-347. 473 474 475 (11) Chivandi, E, Cave, E, Davidson, BC, Eriwanger, KH, Mayo, D and Madziva, MT. 476 Suppression of Caco-2 and HEK-293 cell proliferation by Kigelia africana, Mimusops 477 zeyheri and Ximenia caffra seed oils. In vivo 2012; Jan-Feb, 26 (1): 99-105. PubMed. 478 479 (12) Picerno P, Autore,G.,Marzocco,S.,Meloni,M..Sanogo R Aquino RP. Anti- 480 inflammatory activity of verminoside from Kigelia africana and evaluation of 481 cutaneous irritation in cell cultures and reconstituted human epidermis. J Nat Prod 482 2005; 68:1610-16.

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483 484 (13) Owolabi OJ. Omogbai EKI. Analgesic and anti-inflammatory activities of the 485 ethanolic stem bark extract of Kigelia africana (Bignoniaceae), Afri J Biotechnol 486 2007; 6 (5): 582-85. 487 488 (14) Hemamalini. K Suvidha. S, Anurag Bhargav2 And Uma Vasireddy 489 Evaluation Of Anti-Ulcer Activity Of Methanolic Extracts Of Kigelia Africana, 490 Sophora Interrupta And Holoptelea Integrifolia Leaves In Experimental Rats. Int J 491 Curr Pharm Res, Vol 4, Issue 4, 61-66 492 493 [15]. K. Hemamalini , Anurag Bhargav, Uma Vasireddy, S. Suvidha, V. Ramya 494 Krishna G. L. Soujanya. Phytochemical And Anti-Cholinesterase Of Leaves Of 495 Different Herbs On Frog’s Experimental Models International Journal Of Biological 496 & Pharmaceutical Research. 2012; 3(3): 373-376. 497 498 [16]. Atawodi SE. Evaluation of the hypoglycemic, hypolipidemic and antioxidant effects of 499 methanolic extract of “Ata-ofa” polyherbal tea (A-Polyherbal) in alloxan-induced diabetic rats. 500 Drug Inv Today 2011; 3(11): 270-76 501 502 [17] Atawodi SE, Atawodi JC. Azadirachta indica (neem): A plant of multiple biological and 503 pharmacological Activities. Phytochem Rev 2009; 8(3): 601–20 504 505 506 [18] Gupta, R, Mathur, M, Bajai, VK. Katarachiya, P. Yadav, S. Kamal, R. Gupta, RS. 507 Evaluation of antidiabetic and antioxidant activity of Molinga oleifera in experimental 508 diabetes. J Diabetes, 2012; 4 (2): 164-171 509 510 [19] Gidado A, Ameh D.A. Atawodi, S.E and Ibrahim S. Antidiabetic Effect of Nauclea 511 latifolia Leaf Ethanolic Extract in Streptozotocin-induced Diabetic Rats. Pharmacogn Res 512 2009;1(6): 392-95 513 514 [20] Okine LKN. Nyarko AK, Osei-Kwabena N, Oppong IV,Barnes,F. Ofosuhene,M. The 515 antidiabetic activity of the herbal preparation ADD-199 in mice: a comparative study with 516 two oral hypoglycaemic drugs. J Ethnopharmacol 2004; 97 (1): 31-8. 517 518 [21] Nyarko AK. Okine LKN.Wedzi RK. Addo PA, Ofosuhene M. Sub-chronic toxicity 519 studies of the antidiabetic herbal preparation ADD-199 in the rat: absence of organ 520 toxicity and modulation of cytochrome p450. J Ethnopharmacol 2005; 97(2): 319-25 521 522 [22] Olaleye MT. Rocha JB. Commonly used medicinal plants exhibit distinct in-vitro 523 antioxidant activities against hepatotoxins in rat liver. Expl Toxicol Path 2007; 58 (6): 524 433-8. 525 526 527 . 528

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529 [23] Anvesh H, Vimal KV, Shailaja R . Antiulcer Effect of The Methanolic Extract of Kigelia 530 Africana.Lam, Benth (Bignoniaceae). Pharmacologyonline 2010; 1: 344-51 531 532 [24] Sharma US, Singh A. Agarwal V. Diuretic activity of Kigelia pinnata bark extract. J 533 Pharmacol Res 2010; 1 (2): 17-20 534 535 [25] Ashraf, M. Ayub, M. Sajjad, T. Elahi, N. Ali, I. Ahmed, Z. Replacement of rotenone by 536 locally grown herbal extracts. Int J Agr Biol 2010; 12:77-80 537 538 [26] Azu OO, Duru FIO, Osinubi AA, Noronha CC, Elesha SO, Okanlawon AO. Protective 539 agent, Kigelia africana fruit extract, against cisplatin-induced kidney injury in Sprague- 540 Dawley rats. Asian J Pharm Clin Res 2010; 3(2): 84-8. 541

542 [27] Zofou D, Kengne AB, Tene M, Ngemenya MN, Tane P, Titanji VP. In vitro anti- 543 plasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark 544 of Kigelia africana (Lam.) Benth (Bignoniaceae). Parasitol Res. 2011;108(6):1383-90

545 546 [28] Adoum, OA, Determination of toxicity effects of some savannah plants using brine 547 shrimptest (BST) Int Jor P App. Scs 2008; 2(3): 1-5 548 549 550 [29] Fafioye, OO. Plants with piscicidal activities in Southwestern Nigeria. Turk. J Fish Aquat 551 sci. 2005; 5, 91-97. 552 553 [30] Hussain,H.,Krohn,K.,Ahmad,U.V.,Miana,G.A. and Green, I.R. Lapachol: an overview, 554 ARKIVOC 2007; 2:145-171. 555 556 [31] Jackson,S.J.,Houghton,P.J.,Retsas,S.,and Photiou, A Cytotoxicity of Norviburtinal and 557 Isopinnatal from Kigelia pinnata against cancer cell lines, Planta Med 2000; 66: 758-761. 558 559 [32] Abioye AIR, Duru,FIO, Noronha CC. Okanlawon AO. Aqueous extract of the bark of 560 Kigelia africana reverses early testicular damage induced by methanol extract of Carica 561 papaya. Nig J Hlth Biomed Sci 2003; 2(2): 81-7 562 563 [33] Adeparusi EO, Dada,A.A. Alale OV. Effects of Medicinal Plant (Kigelia africana on 564 Sperm Quality of African Catfish Clarias gariepinus (Burchell, 1822) Broodstock. J Agric 565 Sci 2010; 2(1): 193-99 566 567 [34] Dada AA, Adeparusi EO. Alale OV. Dietary dried Kigelia africana as fertility enhancer 568 in female Clarias gariepinus (Burchell 1822). Agric Biol J North Am 2010; 1(5):791-95 569 570 [35] Atolani O, Olatunji AG. Epicuticular Wax and Volatiles of Kigelia pinnata Leaf Extract. 571 Ethnobotan Leafl 2010; 14: 797-06. 572

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573 [36] GoudaYG, Abdel-baky AM., Darwish FM, Mohammed KM, Kasai R Yamasaki, K. 574 Iridoids from Kigelia pinnata DC. Fruits, Phytochem 2003; 63 (8) 887-92. 575 576 [37] GoudaYG, Abdel-baky AM, Mohammed KM, Darwish FM, Kasai R Yamasaki, K.. 577 Phenylpropanoid and phenylethanoid derivatives from Kigelia pinnata DC. Fruits, Nat 578 Prod Res 2006; 20(10): 935-939. 579 580 [38] Akunyili DN. Houghton PJ. Monoterpenoids and naphthoquinones from Kigelia pinnata 581 bark, Phytochem 1993; 1015-1018. 582 583 [39] Akunyili DN, Houghton PJ. Raman A. Antimicrobial activities of the stem bark of 584 Kigelia pinnata J Ethnopharmacol 1991; 35(2), 173-177. 585 586 [40] Neelam B, Shailendra S, Fermida N, Amir A. Isolation and in vitro anti amoebic activity 587 of iridoids isolated from Kigelia pinnata. General papers. ARKIVOC(x) 2006: 69-76 588 589 [41] Folashade O. Oyedeji, Olufunsho Samuel Bankole-Ojo 590 Quantitative evaluation of the antipsoriatic activity of sausage tree (Kigelia africana). 591 African Journal of Pure and Applied Chemistry Vol. 6(13), pp. 214-218, November 2012 592 593 [42] Moideen SVK, Houghton PJ, Rock P, Croft SL, Aboagye-Nyame F. Activity of extracts 594 and naphthoquinones from Kigelia pinnata against Trypanosoma brucei brucei and 595 Trypanosoma brucei rhodesiense. Planta Med 1999; 65(6): 536-540. 596 597 [43] Houghton PJ, Photiou A, Uddin S, Shah P, Browning M, JacksonSJ. Retsas, S. Activity 598 of extracts of Kigelia pinnata against melanoma and renal carcinoma cell lines. Plant Med 599 1994; 60 (5): 430-433. 600 601 602 603 604 605 606 607 608 .

609

610

611

612

613

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614 615 616 617 O O

O CH3

OH O 618 Kigelinone 619 620 621 622 623 COOH

HO

624 Coumaric acid 625 626 OH

HO O OH

OH O 627 Luteolin

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OH

HO O OH

OH O 628 Luteolin 629 630 631

OH O CH3 OH R

R' CH O 3 R=OH, R'=H Pinnatal 632 R=H, R'=OH Isopinnatal 633 634 O

O CH3

OH

O 635 2-acetylnaphthao[2,3-b]furan-4,9-quinone 636 637

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H3C CH3 H C 3 H CH3

H3C H

H Stigmasterol HO 638 639 640

CH3

H3C CH3 H C 3 H CH3

H3C H

H

HO 641 Sitosterol 642 643 O H3C CHO R

O

R CH O 3 Kigelinol 644 Isokigelinol 645 646 647

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648

OCH3

OH

Caffeic acid COOH

OH

OH

Ferulic acid COOH

O

O OH H

CH3

O 2-(1-Hydroxyethyl)-2-acetylnaphtho-[2,3-b]-furan-4,9-dione

O

O

649 1,4 Benzoquinone 650 651 652 653 654

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O

OH

CH3

O CH3

655 Lapachol 656 657

CH3O CH3

O H3C

OH O 658 Kigelin 659 660 661

OHC

O 662 Norviburtinal 663 664 665 666 667 668

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H COOH C C H

OCH3 669 OH 670 671 Ferulic acid 672 673 OH OH

HO O

C OH

674 OH O 675 676 Quercetin 677 678 679 Figure1: Structural formulae of some compounds found in Kigelia africana 680

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Table 1 Toxic effects of Kigelia africana extracts on different species of animals S/N Animal Species Observed effect Plant part Extract Dose Route Reference: 1 Artemia salina Moderate toxicity Root Ethanol 593µg/ml Whole body Adoum,2008 Fruit Ethanol 124µg/ml Fruit Ethyl acetate 495µg/ml 2 Artemia salina Moderate cytotoxicity Fruit Ethanol 7500µg/ml Whole body Kolodziej,1997 3 Artemia salina Low toxicity Leaves Methanol 250µgml Whole body Ayuko et al, 2009 4 Fish Increased opercular Bark Aqueous Oral/whole Ufodike and ventilation and tail fin body Omoregie, beat leading to 1994 eventual fatique and eventual death 5 Sprague-Dawley Protective effect Fruit Methanol 500mg/kg Oral Azu et al,2010 rats against cisplatin- 100mg/kg induced kidney oxidant injury 6 Wistar albino No overt organ Fruit Aqueous 100-500mg/kg Oral Nyarko et al, rats specific toxicity and 2005 did not demonstrate a potential for drug interaction via cytochrome P450- mediated metabolism 7 Mice Reversed the effects Leaves Aqueous 100mg/kg Oral Olaleye and of severe hepatic Rocha, 2008 necrosis induced by a large dose of paracetamol

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Table 2: Chemical Constituents of Kigelia Africana

S/No Compound Plant Part Bioactivity Reference

Kigelinone Stem bark Antibacterial, Akunyili and Houghton,1993; Antifungal Akunyili et al,1991;Binutu et al, 1997 Isopinnatal Stem bark Antibacterial, Akunyili et al,1991; Weiss et antifungal, anti-malaria al, 2000; Jackson et al, 2000, Binutu et al,1997 Kigelinol Stem bark Anti-malaria Weiss et al, 2000 Isokigelinol Stem bark Anti-malaria Moideen et al,1999; Jackson et al, 2000 Lapachol Anti-cancer Binutu et al, 1997;Hussain et al, 2007 Dehydro-alpha-lapachone Binutu et al,1997 2-(1-hydroxyethyl)naphtha[2,3-b]furan-4,9- Stem bark Anti-trypanosoma Moideen et al, 1999 quinine Kigeliol Wood Houghton,2007 Balaphonin Stem bark Houghton,2007 Β-Sitosterol Bark Cytotoxicity Desai et al,1971;Khan,1998 Caffeic acid Bark Grace et al, 2002

Specioside Stem bark Antibacterial, Neelam et al, 2006 Antifungal Verminoside Stem bark Neelam et al, 2006; Gouda et al, 2003; Picerno et al, 2005 Minecoside Stem bark Neelam et al, 2006 Carbohydrates, alkaloids, tannins, saponins Bark Owolabi and Omogbai, 2007 and glycosides 7-Hydroxyviteoid II Fruit Gouda et al, 2003

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7-Hydroxyeucommic acid Fruit Gouda et al, 2003 7-Hydroxy-10-deoxyeucomiol Fruit 10-Deoxyeucommiol Fruit Gouda et al, 2003 Jiofuran Fruit Gouda et al, 2003 Jioglutolide Fruit Gouda et al, 2003 1-Dehydroxy-3,4-dihydroxy-3,4- Fruit Gouda et al, 2003 dihydroaucubigenin des-p-hydroxybenzoyl kisasagenol B Fruit Gouda et al, 2003 Ajugol Fruit Gouda et al, 2003 6-trans caffeoyl ajugol Fruit Gouda et al, 2003 6-p-Coumaroyl sucrose Fruit Gouda et al,2006 β-Sitosterol Fruit Desai et al,1971;Khan,1998 Quercetin Fruit Grace and Davis,2002 Luteolin Fruit Grace and Davis,2002 3-(2'-hydroxyethyl)-5-(2''-hydroxypropyl) Fruit Gouda et al,2003 Dihydrofuran-2-(3H)one

Isocoumarins 6-Methoxymellein Root Govindachari et al,1971 Kigelin Root 6-Demethylkigelin Root 3-Dimethylkigelin Root

Phenylpropanoids p-Coumaric acid Root bark Antibacteria, Binutu et al, 1997; Grace et al, Antifungal 2002 Ferulic acid Root bark Binutu et al, 1997; Grace et al,2002

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Stigmasterol Root Govindachari et al, 1971

Non-quinonoid aldehydes Norviburtinal Root bark Joshi et al, 1982 Pinnatal Root bark Joshi et al, 1982

Hydrocarbons n-Hentriacontane Leaf Antitumour Atolani and Olatunji, 2010 1-Tricosene Leaf ,, 11-(2,2-Dimethylpropyl)heneicosane Leaf ,, 2,6,10-Trimethyldodecane Leaf ,, Heneicosane Leaf ,, 4,4-Dimethylundecane Leaf ,, ,,

Pentafluoroheptadecyl ester Leaf ,, 2-ethylhexyloctadecyl sulphurous acid ester Leaf ,, Hexyloctyl sulphurous acid ester Leaf ,, Methyl-12-methyltetradecanoate Leaf Inhibition of corneal Atolani and Olatunji, 2010; angiogenesis Grace et al, 2002

1-Iododecane Leaf Atolani and Olatunji, 2010 11-Iodohexadecane Leaf ,,

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