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Phytochemical, anti-inflammatory and anti-trypanosomal properties of Anthocleista vogelii Planch (Loganiaceae) stem bark
Journal of Ethnopharmacology 238 (2019) 111851
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Phytochemical, anti-inflammatory and anti-trypanosomal properties of Anthocleista vogelii Planch (Loganiaceae) stem bark
T
Fabian Ifeanyi Ezea,b,∗, Xavier Siwe Noundoub,c, Patience O. Osadebea,∗∗, Rui W.M. Krauseb,c a
Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria Nsukka, 410001, Nigeria Medicinal Organic Chemistry Laboratory, Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa c Marine Natural Products Chemistry Laboratory, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anthocleista vogelii Anti-inflammatory Anti-trypanosomal Cytotoxicity Phytochemical analysis
Ethnopharmacological relevance: Anthocleista vogelii Planch (Loganiaceae) is used in African Traditional Medicine for the treatment of pain and inflammatory disorders as well as sleeping sickness. Aim of the study: To determine the in vivo anti-inflammatory and in vitro anti-trypanosomal activities of the extracts of A. vogelii stem bark and identify the phytochemical classes of the fractions responsible for the activities. Materials and methods: The in vivo anti-inflammatory activity of the extracts was evaluated using the egg albumin-induced rat paw oedema model while the in vitro anti-trypanosomal activity was assessed on Trypanosoma brucei brucei. The in vitro cytotoxicity was assessed on HeLa (human cervix adenocarcinoma) cell line. Results: The methanolic extract of A. vogelii stem bark, with 11.2% yield, gave LD50 > 5000 mg/kg. The nhexane fraction of the extract contains steroids, terpenes and fatty acids and yielded non-cytotoxic terpenoidal column fraction with anti-trypanosomal IC50 of 3.0 μg/mL. The ethylacetate fraction at 100 mg/kg dose significantly (p < 0.05) provoked 37.8, 62.5 and 69.7% inhibition of oedema induced by egg-albumin at the second, fourth and sixth hours respectively. Conclusion: The study demonstrated that the anti-inflammatory and anti-trypanosomal activities of A. vogelii are probably due to non-cytotoxic terpenoids and validated the traditional use of A. vogelii in the treatment of inflammation and sleeping sickness.
1. Introduction Anthocleista vogelii Planch (Loganiaceae) is a medicinal plant used extensively in Southern Nigeria for treating various diseases. It usually grows around the riverine or marshy areas of the tropical humid forest of West Africa (Irvine, 1961) and is commonly used in the management of pain (Adjanohoun et al., 1996), inflammatory disorders (Dalziel, 1955) and sleeping sickness (Nwodo et al., 2015). Combinations of the stem or root bark and the leaves are used as anti-inflammatory and antidiabetic agents (Sunday et al., 2016) and also in the treatment of wounds (Dalziel, 1955; Leeuwenberg, 1972). Combined extracts of Ficus exasperate and A. vogelii at different doses have been reported to
exhibit up to 91.7% chemosuppresion of Plasmodium berghei berghei (Okon et al., 2014). The petroleum ether leaf extract has been reported to demonstrate good anti-plasmodial activity, which may be due to the constituent decussatin, stigmasterol, swertiaperennin and hexadecanoic acid (Alaribe et al., 2012). Antimicrobial compounds have been isolated from the stem bark of A. vogelii (Tene et al., 2008). Both the root and stem barks possess hypoglycemic activity in both normal and alloxaninduced diabetic animals (Abuh et al., 1990; Osadebe et al., 2014), supporting the traditional use of the plant in diabetes management (Sunday et al., 2016). The aqueous extract has been reported to exhibit good anti-nociceptive activity and very low toxicological profile (Mbiantcha et al., 2013), and most plants with anti-nociceptive activity
Abbreviations: AV, Anthocleista vogelii crude extract; AVEF, Anthocleista vogelii ethylacetate fraction; AVHF, Anthocleista vogelii n-hexane fraction; AVMF, Anthocleista vogelii methanol fraction; AVMK, Anthocleista vogelii water marc (insoluble residue); AVWF, Anthocleista vogelii water fraction; DMEM, Dulbecco's Modified Eagle's Medium; DCM, Dichloromethane; DMSO, Dimethyl sulphoxide; EtOAc, Ethyl acetate; Em590, Emission at 590 nm; Exc560, Excitation at 560 nm; HeLa, human cervix adenocarcinoma cell line; IC50, 50% Inhibitory concentration; InterCEDD, International Center for Ethnomedicine and Drug Development; LD50, Lethal dose at 50% population; NSAIDs, Non-steroidal anti-inflammatory drugs; SD, standard deviation; TLC, Tin layer chromatography ∗ Corresponding author. Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria Nsukka, 410001, Nigeria. ∗∗ Corresponding author. E-mail addresses: [email protected] (F.I. Eze), [email protected] (P.O. Osadebe). https://doi.org/10.1016/j.jep.2019.111851 Received 19 June 2018; Received in revised form 23 March 2019; Accepted 31 March 2019 Available online 09 April 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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2.4. Phytochemical analysis
also exhibit anti-inflammatory activity due to involvement of the same mediators (Swathy et al., 2010). Most analgesic and anti-inflammatory drugs in use today are associated with severe adverse effects, including mucosal damage to the stomach and the small intestine (Al-Saeed, 2011; Harirforoosh et al., 2013; Nikose et al., 2015); hence, the need for continuous search for safer alternatives. A. vogelii has been reported to show potent anti-ulcer activity (Ateufack et al., 2008, 2014). No extract, fraction or compound from A. vogelii has been scientifically investigated for anti-inflammatory or anti-trypanosomal activity or cytotoxicity on HeLa cells. Though the phytochemical composition of the aqueous extract of the plant has been reported (Jegede et al., 2011), the compositions of the different solvent fractions have not been reported. We herein report the phytochemical, anti-inflammatory, anti-trypanosomal and cytotoxic properties of the extract and fractions of the stem bark of A. vogelii.
Qualitative phytochemical study was carried out on the extracts and fractions using standard protocols (Harborne, 1998; Ram and Sinha, 2015; Sahira and Cathrine, 2015; Santhi and Sengottuvel, 2016). 2.5. Acute toxicity study Acute toxicity study was carried out on the extract using the Lorke's method (Lorke, 1983). No adverse effects or mortalities were detected in the mice up to 5000 mg/kg, p.o. during 24 h observation period. 2.6. Anti-inflammatory activity studies Egg-albumin-induced rat paw oedema model, as reported by Akah and Nwabie (1994), Okoli et al. (2007) and Adebayo et al. (2004), was used. The different solvent fractions: AVHF, AVEF, AVMF and AVWF, at 200 mg/kg, were first screened for anti-inflammatory activity. The best result was obtained for AVEF. Then, AVEF test samples at 50, 100 and 400 mg/kg respectively were administered orally to animal groups (n = 4). Control animals received equivalent volume of vehicle (3% v/v Tween 80) or 10 mg/kg piroxicam. Thirty minutes after administration, inflammation was induced by sub-plantar injection of 0.1 ml of fresh undiluted egg albumin into the left hind paw of the animals. Oedema volumes were determined by measuring the volume of water displaced in a plethysmometer, before and after 1, 2, 3, 4, 6 and 12 h after induction of inflammation. The percentage inhibition of oedema was calculated using the relation: (Tanko et al., 2012).
2. Materials and methods 2.1. Plant collection and preparation A. vogelii stem bark was harvested wild in June from Calabar, Nigeria and authenticated by Mr. A. Ozioko of the International Center for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Nigeria where voucher specimen (with voucher number: InterCEDD/ 30) was deposited. The plant was air-dried under shade and pulverized to tiny chips before solvent extraction. The study was carried out according to the international, national and institutional rules concerning biodiversity rights.
Inhibition (%) Mean paw diameter (control ) − Mean paw diameter (treated ) = Mean paw diameter (control )
2.2. Experimental animals Adult Swiss albino mice (21–34 g weight) and Wistar rats (132–250 g) of both sexes, aged 8–10 weeks, were used for the studies. Animals were housed and maintained in standard conditions. To keep the hydration rate constant, food and water were stopped 12 h before the experiments. The experimental protocol was in accordance with the guidelines of the ethics committee of the University of Nigeria as registered by the National Health Research Ethics Committee of Nigeria (ref: NHREC/05/01/2008B). The research was conducted in accordance with the internationally accepted principle for laboratory animal use and care as found in European Community Guidelines (EEC Directive of 1986; 86/609/EEC). The ethics for use of experimental animals were followed carefully.
× 100 The data are expressed as the mean ± SEM analyzed by one-way analysis of variance (ANOVA) and Dunnett's t-test was used as the test of significance. P value < 0.05 was considered as the minimum level of significance. All statistical tests were carried out using SPSS statistical software. 2.7. Anti-trypanosomal assay To assess the anti-trypanosomal activity, test samples were added to in vitro cultures of Trypanosoma brucei brucei (s427) in 96-well plates at a fixed concentration of 20 μM (0.06 μg/mL) for pentamidine (used as drug standard) and 25 μg/mL for test extracts and fractions. The parasites were cultured in HMI11 medium, maintained at 37 °C with 5% CO2 and subcultured by 1: 100 dilutions every two days. After an incubation period of 48 h at 37 °C in a carbon dioxide incubator (5% CO2), the number of parasites surviving drug exposure was determined by adding a resazurin-based reagent. The reagent contains resazurin, which is reduced to resorufin by living cells. Resorufin is a fluorophore (Exc560/Em590) and can, thus, be quantified in a multi-well fluorescence plate reader (Merghoub et al., 2009). Results were expressed as % parasite viability – the resorufin fluorescence in compound-treated wells relative to untreated controls. Compounds were tested in triplicate wells and standard deviation (SD) obtained. Non-cytotoxic samples that reduced parasite viability to < 20% were considered for further testing (dose-response assays). Serial dilutions of the test samples were added to the in vitro cultures and again incubated for 48 h. For each sample, percentage viability was plotted against Log concentration and the IC50 obtained from the resulting dose-response curve by non-linear regression. Pentamidine (an existing drug treatment for trypanosomiasis) was used as a drug standard and yields IC50 values in the range 0.001–0.05 μM.
2.3. Solvent extraction and fractionation The air-dried, pulverized plant materials (1 kg) were exhaustively extracted with 90% aqueous methanol by cold maceration at ambient temperature within 72 h and the extracts concentrated in vacuo at 40 °C using a rotary evaporator to yield the crude extract, AV (11.2%). The crude extract was fractionated by re-dissolving it in 90% aqueous methanol and partitioning the solution successively with n-hexane and ethylacetate to yield n-hexane (AVHF, 3.5%) and ethylacetate (AVEF, 13.9%) fractions. The ethylacetate marc was successively extracted with absolute methanol and water to yield methanol (AVMF, 57.7%) and aqueous (AVWF, 7.3%) fractions respectively as well as insoluble residue (AVMK, 17.3%). The AVHF (1 g) was subjected to gradient elution column chromatography on silca gel 60 (60Ǻ, 70–230 mesh) with sample to silica ratio of 1:75, and eluted successively with dichloromethane (DCM, 100%), DCM/EtOAc (10:1; 1:1) and, lastly, EtOAc (100%). Based on the thin layer chromatography (TLC) profiles, eight sub-fractions i.e. AVHF B1e B8, were obtained. B3 and B4 have the same TLC profiles and were, therefore, combined to give B4. 2
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2.8. Cytotoxicity assay
Table 2 Column sub-fractions of n-hexane fraction of A. vogelii.
The overt cytotoxicities of the extracts and fractions were evaluated according to the method previously described by Merghoub et al., (2009) and Chithambo et al., (2017). The extracts and fractions were incubated for 48 h at 37 °C in a carbon dioxide incubator (5% CO2), at a fixed concentration of 50 μg/ml in 96-well plates containing HeLa cells in a culture medium of Dulbecco's Modified Eagle's Medium (DMEM). The number of cells surviving drug exposure was determined by using the resazurin-based reagent and reading resorufin fluorescence in a multi-well plate reader. Results were expressed as % viability–the resorufin fluorescence in compound-treated wells relative to untreated controls. Compounds were tested in triplicate wells and standard deviation (SD) obtained. Emetine (which induces cell apoptosis) was used as a positive control drug standard.
The percentage yields of the crude extract and fractions are given in section 2.3. The yield of the crude extract is given as a percentage of weight of plant material and those of the fractions as percentage of the crude extract. The stem bark has very poor yield of the non-polar fractions, AVHF and AVEF, which are the repository of the secondary metabolites largely responsible for the anti-trypanosomal and anti-inflammatory activities. Therefore, it will be more profitable to isolate the pure compounds, obtain their chemical structures and develop new potent drugs from them than use the plant in the crude form.
3.4. Column fractions of AVHF The column sub-fractions of AVHF and their descriptions are given in Table 2. The terpenoidal sub-fractions gave single spot each on analytical silica gel TLC plates suggesting that they might be pure compounds. Structural elucidation of the possibly new compounds is, therefore, necessary. 3.5. Anti-inflammatory activity The dose-dependent inhibition of egg-albumin-induced rat paw Table 1 Phytochemical constituents of the different extracts.
AVHF AVEF AVMF AVWF AVMK
++ + -
++ + -
+++ + -
+++ +++ +++
+ ++ + +++
-
Remaining, immovable components.
The cytotoxicity profile of the extracts and fractions on HeLa cells at a fixed concentration of 50 μg is given in Fig. 2. The percentage viability of the cells after drug exposure was very high, > 50%, for all the tested extracts, except AVHF-B8 which gave ∼ 44%. Extracts which reduced the cells percentage viability to < 25% are considered cytotoxic (Merghoub et al., 2009; Chithambo et al., 2017). The stem bark extract of A. vogelii showed LD50 greater than 5000 mg/kg, indicating high margin of safety as opposed to the NSAIDs, which elicit life threatening adverse effects (Harirforoosh et al., 2013). This is further supported by the cytotoxicity profile (Fig. 2). Almost all the tested samples gave very high percentage viabilities (> 50%) at 50 μg/ml, indicating that even at such high concentration of the extracts, more than 50% of the HeLa cells were still active. This implies very high selectivity index and makes the extracts more advantageous as antiinflammatory drug over the currently used NSAIDs which are used as rat poisons at high concentration due to their inherent toxicity. The secondary metabolites present in A. vogelii stem bark are mainly saponins, flavonoids, terpenoids, steroids and alkaloids. The ethylacetate fraction of the stem bark of A. vogelii contains mainly alkaloids and flavonoids while the n-hexane fraction contains mainly terpenoids and steroids and these are largely responsible for the anti-trypanosomal and anti-inflammatory activities of the plant. The polar fractions: AVMF, AVWF and AVMK did not show any significant anti-trypanosomal activity (Table 3) despite their higher solubility in the DMSO assay medium. They also gave no significant anti-inflammatory activity; hence, excluding polar phytochemicals, such as polyphenolics, in the activities. From the results of anti-inflammatory studies (Fig. 1), the optimal dose of AVEF at 100 mg/kg, though crude fraction with very complex composition, beats pure Piroxicam (10 mg/kg) in activity except at the fourth hour. This suggests that further purification might lead to pure
The crude extract, AV, showed LD50 > 5000 mg/kg. Within the first 30 min of administration the animals that received 1000 mg/kg and above were restless, hyperactive and gradually faded up. Animals that received < 1000 mg/kg showed no external sign of toxicity.
Tannins
Terpene Terpenoid Terpenoid Fatty acid Terpenoid Terpenoid Steroid, alkaloid
3.7. Cytotoxicity assay
3.3. Acute toxicity profile
Flavonoids
Greenish oily liquid Yellow solid Greenish semi solid White powder Light green semi-solid Green oily solid Brown solid
The IC50 values obtained for the individual samples are given in Table 3. Non-cytotoxic samples with trypanosomal residual percentage viability < 25% were considered active; hence, put forward for IC50 assay. The polar fractions (AVMF, AVWF and AVMK) showed no significant activity at 50 μg/mL, as indicated by high percentage viability values, and, hence, have IC50 values > 50 μg/mL. All the non polar fractions showed trypanosoidal activity. The terpenoidal fraction, AVHF-B8 gave very low percentage parasite viability.
The phytochemical composition of the different extracts is shown in Table 1. The polar fractions are composed mainly of saponins, flavonoids and no tannins, while the non-polar fractions contain mainly terpenes, alkaloids and steroids.
Saponins
AVHF-B1 AVHF-B2 AVHF-B4 AVHF-B5 AVHF-B6 AVHF-B8 AVHF-Bna
3.6. Trypanosomal assay
3.2. Qualitative phytochemical analysis of the different extracts
Terpenes
Phytochemical class
oedema, as a function of anti-inflammatory activities of the ethylacetate fraction, AVEF, of A. vogelii, is given in Fig. 1. The best activity was obtained at 100 mg/kg dose which significantly (p < 0.05–0.01) provoked 38.5%, 37.8%, 55.6%, 62.5%, 69.7%, 81.0% and 85.7% inhibition at the first, second, third, fourth, sixth, eight and twelfth hours respectively. The Piroxicam standard at 10 mg/kg provoked 2.6%, 18.9%, 44.4%, 67.5%, 60.6%, 76.2% and 78.5% respectively at those hours.
3.1. Extraction yield
Steroids
Description
a
3. Results and discussion
Alkaloids
Fraction
+ trace, ++ moderate, +++ very abundant, - not detected. 3
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Fig. 1. Percentage inhibitions of rat paw edema.
our preliminary screening of the solvent fractions for anti-inflammatory activities, we noted that the most active fraction was AVEF followed by AVHF. This implies that alkaloids, flavonoids, terpenes, steroids and or fatty acids are responsible and may be working in synergy (Chithambo et al., 2017). This is consistent with numerous previous reports on the anti-inflammatory activities of many other plants where the activity is attributed to the presence of terpenoids, sterols or flavonoids (Parmar and Ghosh, 1978; Ahmad et al., 1983; Calixto et al., 2000; Sabu and Kuttan, 2002; Ghannadi et al., 2005; Silva et al., 2005). Reduction in activity at higher dose may be attributed to increased toxicity, which may result in further release of the mediators of inflammation. Oedema induced by egg albumin results from the release of histamine and serotonin (Pearce, 1986). According to the report by Akindele et al. (2012), egg albumin produces mast cell degranulation with subsequent release of inflammatory mediators such as histamine, which has been associated with increased vasodilatation and increased permeability of blood vessels leading to the exudation of plasma proteins and fluids into the tissues. A. vogelii appreciably inhibited oedema in this model (Fig. 1), suggesting the possible inhibition of histamine and serotonin in its anti-inflammatory property. Further works on the measurement of these mediators may confirm their involvement. Human African trypanosomiasis, a neglected tropical disease caused by Trypanosoma species, remains one of the serious health problems confronting many African countries. The current anti-trypanosomal agents such as suramin and pentamidine are associated with serious adverse effects (Wang, 1995) and require lengthy parenteral administration. They are also expensive in addition to drug resistance issue (Mpia and Pepin, 2002; Horn, 2014; Kofi and Nguyen, 2016). In view of these concerns, continued search for more potent, cheaper and safer alternatives to the existing anti-trypanosomal drugs is warranted. Natural products from plants have been used to treat human ailments since the origin of man and remain an important source of drug leads (Kingston, 1996). We investigated the anti-trypanosomal properties of A. vogelii based on the folkloric use of the plant to remedy sleeping sickness. Bioactivity-guided purification of n-hexane fraction yielded potent terpenoids (AVHF-B2 and AVHF-B4) with anti-trypanosomal IC50 of 5.8 and 3.0 respectively. This result agrees with previous reports on the involvement of triterpenoids in the anti-trypanosomal activity of medicinal plants (Abe et al., 2002; Abiodun et al., 2012; Kofi and
Table 3 Anti-trypanosomal IC50 values for the different extracts and fractions. Sample at 50 μg/mL
Percentage viability (%) ± SD
IC50 (μg/ml for samples, μM for Pentamidine)
AVHF AVHF-B2 AVHF-B4 AVHF-B5 AVHF-B8* AVHF-Bn AVEF AVMF AVWF AVMK Pentamidine
2.14 ± 0.99 0.54 ± 0.89 3.05 ± 2.15 7.70 ± 6.05 0.71 ± 0.06 7.47 ± 6.60 3.06 ± 0.42 105.65 ± 0.15 103.35 ± 0.79 102.37 ± 0.95
20.9 5.72 ∼ 3.011 31.79 ND 24.67 ∼ 36.33 > 50 > 50 > 50 0.004363
ND: Not determined. * cytotoxic; hence, IC50 not determined.
Fig. 2. Cytotoxicity profile of the different extracts and fractions.
compounds with high efficacy. Piroxicam is a known prostaglandins inhibitor. Its higher activity at the fourth hour is attributed to greater inhibition of prostaglandins than the extract at the tested dose. From 4
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Nguyen, 2016), but is a success story in terms of the low cytotoxicity profile (Fig. 2) and the corresponding high selectivity index.
of Anthocleista vogelii Planch in Rats. Hindawi Publishing Corporation, Ulcers 2014, Article ID 172096, 6 pages. Calixto, J.B., Beirith, A., Ferreira, J., Santos, A.R., Cechinel, F.V., Yunes, R.A., 2000. Naturally occurring antinociceptive substances from plants. Phytother Res. 14, 401–418. Chithambo, B., Siwe Noundou, X., Krause, R.W.M., 2017. Anti-malarial synergy of secondary metabolites from Morinda lucida Benth. J. Ethnopharmacol. 199, 91–96. Dalziel, J.M., 1955. The Useful Plant of West Tropical Africa. Crown Agents for the Colonies, London, pp. 318–330. Ghannadi, A., Hajhashemi, V., Jafarabadi, H., 2005. An investigation of the analgesic and anti-inflammatory effects of Nigella sativa seed polyphenols. J. Med. Food 8, 488–493. Harborne, J.B., 1998. Phytochemical Methods: a Guide to Modern Techniques of Plant Analysis, second ed. Chapmann and Hall Publishers, London, pp. 21–72. Harirforoosh, S., Asghar, W., Jamali, F., 2013. Adverse effects of nonsteroidal antiinflammatory drugs: an update of gastrointestinal, cardiovascular and renal complications. J. Pharm. Sci. 16, 821–847. Horn, D., 2014. High-throughput decoding of drug targets and drug resistance mechanisms in African trypanosomes. Parasitology 141, 77–82. Irvine, F.R., 1961. Woody Plants of Ghana. Oxford Univ. Press, London, pp. 878. Jegede, I.A., Ibrahim, J.A., Kunle, O.F., 2011. Phytochemical and pharmacognostic studies of the leaf and stem-bark of Anthocleista vogelii (Planch). J. Med. Plants Res. 5 (26), 2011. Kingston, D.G., 1996. Natural products as pharmaceuticals and sources of lead structures. In: Wermuth, C.G. (Ed.), The Practice of Medicinal Chemistry. Acad. Press, London, pp. 101–116. Kofi, D.K., Nguyen, H.T., 2016. Antitrypanosomal activities and mechanisms of action of novel tetracyclic iridoids from Morinda lucida Benth. Antimicrob. Agents Chemother. 60 (6), 3283–3290. Leeuwenberg, A.J.M., 1972. Flore du Cameroun 12, 5–22. Lorke, D., 1983. A new approach to practical acute toxicity testing. Arch. Toxicol. 53, 275–289. Mbiantcha, M., Nguessom, K., Ateufack, G., Oumar, M., Kamanyi, A., 2013. Analgesic properties and toxicological profile of aqueous extract of the stem bark of Anthocleista vogelii planch (Loganiaceae). Int. J. Pharm. Chem. Biol. Sci. 3 (1), 1–12. Merghoub, N., Amzazi, S., Morjani, H., 2009. Cytotoxic effect of some Moroccan medicinal plant extracts on human cervical cell lines. J. Med. Plants Res. 3 (12), 1045–1050. Mpia, B., Pepin, J., 2002. Combination of eflornithine and melarsoprol for melarsoprolresistant Gambian trypanosomiasis. Trop. Med. Int. Health 7, 775–779. Nikose, S., Arora, M., Singh, P., Nikose, D., Gadge, S.V., 2015. Gastrointestinal adverse effects due to use of non-steroidal anti-Inflammatory drugs (NSAIDs) in non-traumatic painful musculoskeletal disorders. J. Gastrointest. Dig. Syst. 5, 348. Nwodo, N.J., Ibezim, A., Ntie-Kang, F., Adikwu, M.U., Mbah, C.J., 2015. Anti-trypanosomal activity of Nigerian plants and their constituents. Molecules 20, 7750–7771. Okoli, C.O., Akah, P.A., Nwafor, S.V., Anisiobi, A.I., Ibegbunam, I.N., Erojiekwe, O., 2007. Anti-inflammatory activity of hexane leaf extract of Aspila africana C.D. Adams. J. Ethnopharmacol. 109, 219–225. Okon, E.O., Gboeloh, L.B., Udoh, E.S., 2014. Antimalarial effect of combined extracts of the leaf of Ficus exasperate and stem bark of Anthocleista vogelii on mice experimentally infected with Plasmodium berghei berghei (Nk 65). Res. J. Med. Plant 8 (3), 99–111. Osadebe, P.O., Uzor, P.F., Omeje, E.O., Agbo, M.O., Obonga, W.O., 2014. Hypoglycemic activity of the extract and fractions of Anthocleista vogelii Planch stem bark. Trop. J. Pharmaceut. Res. 13 (9), 1437–1443. Parmar, N.S., Ghosh, M.M.N., 1978. Current trends in flavonoid research. Indian J. Pharm. 12, 213–228. Pearce, F.L., 1986. On the heterogeneity of mast cells. Pharmacology 32 (2), 61–71. Ram, S., Sinha, V.S., 2015. Qualitative phytochemical analysis of some plants used to cure malaria in Kolhan region of Jharkhand, India. J. Med. Plants Stud. 3 (6), 60–62. Sabu, M.C., Kuttan, S., 2002. Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J. Ethnopharmacol. 81, 155–160. Sahira, B.K., Cathrine, L., 2015. General techniques involved in phytochemical analysis. Int. J. Adv. Res. Comput. Sci. 2 (4), 25–32. Santhi, K., Sengottuvel, R., 2016. Qualitative and quantitative phytochemical analysis of Moringa concanensis Nimmo. Int. J. Curr. Microbiol. App. Sci. 5 (1), 633–640. Silva, G.N., Martins, F.R., Matheus, M.E., 2005. Investigation of anti-inflammatory and antinociceptive activities of Lantana trifolia. J. Ethnopharmacol. 100, 254–259. Sunday, R.M., Ilesanmi, O.R., Obuotor, E.M., 2016. Anti-diabetic effect of Anthocleista vogelii ethanolic root extract in alloxan-induced diabetic rats. Res. J. Med. Plant 10, 79–88. Swathy, B., Mohana, S., Saravana, A., 2010. Review on herbal drugs for analgesic and anti-inflammatory activities. IJBPR 1 (1), 7–12. Tanko, Y., Mohammed, A., Saleh, M.I., Mohammed, K.A., Etta, E., Bako, I.G., Muhammad, A., Yerima, M., 2012. Antinociceptive and anti-inflammatory activities of ethanol extract of Bryophyllum Pinnatum in laboratory animals. JDMS 3 (1), 46–49. Tene, M., Tane, P., Kuiate, J., Tamokou, J., Connolly, J.D., 2008. Anthocleistenolide, a new rearranged nor-secoiridoid derivative from the stem bark of Anthocleista vogelii Planch. Letter, Planta Med. 74, 80–83. Wang, C.C., 1995. Molecular mechanisms and therapeutic approaches to the treatment of African trypanosomiasis. Annu. Rev. Pharmacol. Toxicol. 35, 93–127.
4. Conclusion Results showed that A. vogelii holds promise as a potential source of new anti-inflammatory and anti-trypanosomal drugs. Further purification and spectral characterization of the alkaloids and terpenoids from the stem bark will result in potent anti-inflammatory and anti-trypanosomal compounds which might be used as safer alternatives to the existing ones. Also, the cytotoxicity and acute toxicity profile indicated a wide therapeutic margin. Furthermore, A. vogelii extracts and compounds have been shown to possess anti-ulcer activities. This is advantageous over the conventional NSAIDs which provoke erosion of gastric mucosa. Based on these observations and results, there is a clear justification for the ethnomedicinal and folkoric use of A. vogelii for treating sleeping sickness, pain and inflammatory disorders such as rheumatoid arthritis in Nigeria and other parts of Africa. Conflicts of interest The authors declare that there is no conflict of interests. Acknowledgements This work was sponsored by the Nigeria’s Tertiary Education Trust Fund (TETFund) under the TETfund Institution-Based Research (IBR) Intervention and supported by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package, and Rhodes University SandisaImbewu. The authors are grateful to Michelle Isaac for assistance in the bioassays. X. Siwe Noundou is grateful for a Rhodes University Postdoctoral Research Fellowship. References Abe, F., Yamauchi, T., Nagao, T., Kinjo, J., Okabe, H., Higo, H., Akahane, H., 2002. Ursolic acid as a trypanocidal constituent in Rosemary. Biol. Pharm. Bull. 25 (11), 1485–1487. Abiodun, O.O., Gbotosho, G.O., Ajayiyeoba, E.O., Brun, R., Oduola, A.M., 2012. Antitrypanosomal activity of some medicinal plants from Nigerian ethnomedicine. Parasitil. Res. 110 (2), 521–526. Abuh, F.Y., Wambebe, C., Rai, P.P., Sokomba, E.N., 1990. Hypoglycaemic activity of Anthocleista vogelii (Planch) aqueous extract in rodents. Phytother Res. 4 (1), 20–24. Adebayo, H.A., John-Africa, L.B., Agbafor, A.G., Omotosho, O.E., Mosaku, T.O., 2004. Anti-nociceptive and anti-inflammatory activities of extract of Anchomanes difformis in rats. Pak. J. Pharm. Sci. 27 (2), 265–270. Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbilé, Z.O., Kamanyi, A., KamsuKom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabié, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Contribution to Ethnobotanical and Floristic Studies in Cameroon. CSTR/OUA, Cameroon, pp. 273. Ahmad, M.M., Quresh, S., Shah, A., Qazi, N.S., Rao, R.M., Albakiri, M., 1983. Anti-inflammatory activity of Caralluma tuberculata alcoholic extract. Fitoterapia 46, 357–360. Akah, P., Nwabie, A.I., 1994. Evaluation of Nigerian traditional medicines: plants used for rheumatic disorder. J. Ethnopharmacol. 42, 179–182. Akindele, A.J., Ibe, I.F., Adeyemi, O.O., 2012. Analgesic and antipyretic activities of Drymaria cordata (Linn.) willd (Caryophyllaceae) extract. Afr. J. Tradit., Complementary Altern. Med. 9 (1), 25–35. Alaribe, C.S.A., Coker, H.A.B., Shode, F.O., Ayoola, G., Adesegun, S.A., Bamiro, J., Anyim, E.I., Anyakora, C., 2012. Antiplasmodial and phytochemical investigations of leaf extract of Anthocleista vogelii Planch. J. Nat. Prod. 5, 60–67. Al-Saeed, A., 2011. Gastrointestinal and cardiovascular risk of nonsteroidal anti-inflammatory drugs. Oman Med. J. 26, 385–391. Ateufack, G., Nguelefack, T.B., Wabo, H.K., Watcho, P., Tane, P., Kamanyi, A., 2008. Antiulcer effects of the aqueous and organic extracts of the stem bark of Anthocleista vogelii in rats. Pharm. Biol. 44 (3), 166–171. Ateufack, G., Nguelefack, T.B., Wabo, H.K., Tane, P., Kamanyi, A., 2014. Antiulcerogenic Activity of 1-Hydroxy-3,7,8-Trimethoxyxanthone Isolated from the Methanol Extract
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Phytochemical, anti-inflammatory and anti-trypanosomal properties of Anthocleista vogelii Planch (Loganiaceae) stem bark
T
Fabian Ifeanyi Ezea,b,∗, Xavier Siwe Noundoub,c, Patience O. Osadebea,∗∗, Rui W.M. Krauseb,c a
Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria Nsukka, 410001, Nigeria Medicinal Organic Chemistry Laboratory, Department of Chemistry, Rhodes University, Grahamstown, 6140, South Africa c Marine Natural Products Chemistry Laboratory, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anthocleista vogelii Anti-inflammatory Anti-trypanosomal Cytotoxicity Phytochemical analysis
Ethnopharmacological relevance: Anthocleista vogelii Planch (Loganiaceae) is used in African Traditional Medicine for the treatment of pain and inflammatory disorders as well as sleeping sickness. Aim of the study: To determine the in vivo anti-inflammatory and in vitro anti-trypanosomal activities of the extracts of A. vogelii stem bark and identify the phytochemical classes of the fractions responsible for the activities. Materials and methods: The in vivo anti-inflammatory activity of the extracts was evaluated using the egg albumin-induced rat paw oedema model while the in vitro anti-trypanosomal activity was assessed on Trypanosoma brucei brucei. The in vitro cytotoxicity was assessed on HeLa (human cervix adenocarcinoma) cell line. Results: The methanolic extract of A. vogelii stem bark, with 11.2% yield, gave LD50 > 5000 mg/kg. The nhexane fraction of the extract contains steroids, terpenes and fatty acids and yielded non-cytotoxic terpenoidal column fraction with anti-trypanosomal IC50 of 3.0 μg/mL. The ethylacetate fraction at 100 mg/kg dose significantly (p < 0.05) provoked 37.8, 62.5 and 69.7% inhibition of oedema induced by egg-albumin at the second, fourth and sixth hours respectively. Conclusion: The study demonstrated that the anti-inflammatory and anti-trypanosomal activities of A. vogelii are probably due to non-cytotoxic terpenoids and validated the traditional use of A. vogelii in the treatment of inflammation and sleeping sickness.
1. Introduction Anthocleista vogelii Planch (Loganiaceae) is a medicinal plant used extensively in Southern Nigeria for treating various diseases. It usually grows around the riverine or marshy areas of the tropical humid forest of West Africa (Irvine, 1961) and is commonly used in the management of pain (Adjanohoun et al., 1996), inflammatory disorders (Dalziel, 1955) and sleeping sickness (Nwodo et al., 2015). Combinations of the stem or root bark and the leaves are used as anti-inflammatory and antidiabetic agents (Sunday et al., 2016) and also in the treatment of wounds (Dalziel, 1955; Leeuwenberg, 1972). Combined extracts of Ficus exasperate and A. vogelii at different doses have been reported to
exhibit up to 91.7% chemosuppresion of Plasmodium berghei berghei (Okon et al., 2014). The petroleum ether leaf extract has been reported to demonstrate good anti-plasmodial activity, which may be due to the constituent decussatin, stigmasterol, swertiaperennin and hexadecanoic acid (Alaribe et al., 2012). Antimicrobial compounds have been isolated from the stem bark of A. vogelii (Tene et al., 2008). Both the root and stem barks possess hypoglycemic activity in both normal and alloxaninduced diabetic animals (Abuh et al., 1990; Osadebe et al., 2014), supporting the traditional use of the plant in diabetes management (Sunday et al., 2016). The aqueous extract has been reported to exhibit good anti-nociceptive activity and very low toxicological profile (Mbiantcha et al., 2013), and most plants with anti-nociceptive activity
Abbreviations: AV, Anthocleista vogelii crude extract; AVEF, Anthocleista vogelii ethylacetate fraction; AVHF, Anthocleista vogelii n-hexane fraction; AVMF, Anthocleista vogelii methanol fraction; AVMK, Anthocleista vogelii water marc (insoluble residue); AVWF, Anthocleista vogelii water fraction; DMEM, Dulbecco's Modified Eagle's Medium; DCM, Dichloromethane; DMSO, Dimethyl sulphoxide; EtOAc, Ethyl acetate; Em590, Emission at 590 nm; Exc560, Excitation at 560 nm; HeLa, human cervix adenocarcinoma cell line; IC50, 50% Inhibitory concentration; InterCEDD, International Center for Ethnomedicine and Drug Development; LD50, Lethal dose at 50% population; NSAIDs, Non-steroidal anti-inflammatory drugs; SD, standard deviation; TLC, Tin layer chromatography ∗ Corresponding author. Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria Nsukka, 410001, Nigeria. ∗∗ Corresponding author. E-mail addresses: [email protected] (F.I. Eze), [email protected] (P.O. Osadebe). https://doi.org/10.1016/j.jep.2019.111851 Received 19 June 2018; Received in revised form 23 March 2019; Accepted 31 March 2019 Available online 09 April 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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2.4. Phytochemical analysis
also exhibit anti-inflammatory activity due to involvement of the same mediators (Swathy et al., 2010). Most analgesic and anti-inflammatory drugs in use today are associated with severe adverse effects, including mucosal damage to the stomach and the small intestine (Al-Saeed, 2011; Harirforoosh et al., 2013; Nikose et al., 2015); hence, the need for continuous search for safer alternatives. A. vogelii has been reported to show potent anti-ulcer activity (Ateufack et al., 2008, 2014). No extract, fraction or compound from A. vogelii has been scientifically investigated for anti-inflammatory or anti-trypanosomal activity or cytotoxicity on HeLa cells. Though the phytochemical composition of the aqueous extract of the plant has been reported (Jegede et al., 2011), the compositions of the different solvent fractions have not been reported. We herein report the phytochemical, anti-inflammatory, anti-trypanosomal and cytotoxic properties of the extract and fractions of the stem bark of A. vogelii.
Qualitative phytochemical study was carried out on the extracts and fractions using standard protocols (Harborne, 1998; Ram and Sinha, 2015; Sahira and Cathrine, 2015; Santhi and Sengottuvel, 2016). 2.5. Acute toxicity study Acute toxicity study was carried out on the extract using the Lorke's method (Lorke, 1983). No adverse effects or mortalities were detected in the mice up to 5000 mg/kg, p.o. during 24 h observation period. 2.6. Anti-inflammatory activity studies Egg-albumin-induced rat paw oedema model, as reported by Akah and Nwabie (1994), Okoli et al. (2007) and Adebayo et al. (2004), was used. The different solvent fractions: AVHF, AVEF, AVMF and AVWF, at 200 mg/kg, were first screened for anti-inflammatory activity. The best result was obtained for AVEF. Then, AVEF test samples at 50, 100 and 400 mg/kg respectively were administered orally to animal groups (n = 4). Control animals received equivalent volume of vehicle (3% v/v Tween 80) or 10 mg/kg piroxicam. Thirty minutes after administration, inflammation was induced by sub-plantar injection of 0.1 ml of fresh undiluted egg albumin into the left hind paw of the animals. Oedema volumes were determined by measuring the volume of water displaced in a plethysmometer, before and after 1, 2, 3, 4, 6 and 12 h after induction of inflammation. The percentage inhibition of oedema was calculated using the relation: (Tanko et al., 2012).
2. Materials and methods 2.1. Plant collection and preparation A. vogelii stem bark was harvested wild in June from Calabar, Nigeria and authenticated by Mr. A. Ozioko of the International Center for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Nigeria where voucher specimen (with voucher number: InterCEDD/ 30) was deposited. The plant was air-dried under shade and pulverized to tiny chips before solvent extraction. The study was carried out according to the international, national and institutional rules concerning biodiversity rights.
Inhibition (%) Mean paw diameter (control ) − Mean paw diameter (treated ) = Mean paw diameter (control )
2.2. Experimental animals Adult Swiss albino mice (21–34 g weight) and Wistar rats (132–250 g) of both sexes, aged 8–10 weeks, were used for the studies. Animals were housed and maintained in standard conditions. To keep the hydration rate constant, food and water were stopped 12 h before the experiments. The experimental protocol was in accordance with the guidelines of the ethics committee of the University of Nigeria as registered by the National Health Research Ethics Committee of Nigeria (ref: NHREC/05/01/2008B). The research was conducted in accordance with the internationally accepted principle for laboratory animal use and care as found in European Community Guidelines (EEC Directive of 1986; 86/609/EEC). The ethics for use of experimental animals were followed carefully.
× 100 The data are expressed as the mean ± SEM analyzed by one-way analysis of variance (ANOVA) and Dunnett's t-test was used as the test of significance. P value < 0.05 was considered as the minimum level of significance. All statistical tests were carried out using SPSS statistical software. 2.7. Anti-trypanosomal assay To assess the anti-trypanosomal activity, test samples were added to in vitro cultures of Trypanosoma brucei brucei (s427) in 96-well plates at a fixed concentration of 20 μM (0.06 μg/mL) for pentamidine (used as drug standard) and 25 μg/mL for test extracts and fractions. The parasites were cultured in HMI11 medium, maintained at 37 °C with 5% CO2 and subcultured by 1: 100 dilutions every two days. After an incubation period of 48 h at 37 °C in a carbon dioxide incubator (5% CO2), the number of parasites surviving drug exposure was determined by adding a resazurin-based reagent. The reagent contains resazurin, which is reduced to resorufin by living cells. Resorufin is a fluorophore (Exc560/Em590) and can, thus, be quantified in a multi-well fluorescence plate reader (Merghoub et al., 2009). Results were expressed as % parasite viability – the resorufin fluorescence in compound-treated wells relative to untreated controls. Compounds were tested in triplicate wells and standard deviation (SD) obtained. Non-cytotoxic samples that reduced parasite viability to < 20% were considered for further testing (dose-response assays). Serial dilutions of the test samples were added to the in vitro cultures and again incubated for 48 h. For each sample, percentage viability was plotted against Log concentration and the IC50 obtained from the resulting dose-response curve by non-linear regression. Pentamidine (an existing drug treatment for trypanosomiasis) was used as a drug standard and yields IC50 values in the range 0.001–0.05 μM.
2.3. Solvent extraction and fractionation The air-dried, pulverized plant materials (1 kg) were exhaustively extracted with 90% aqueous methanol by cold maceration at ambient temperature within 72 h and the extracts concentrated in vacuo at 40 °C using a rotary evaporator to yield the crude extract, AV (11.2%). The crude extract was fractionated by re-dissolving it in 90% aqueous methanol and partitioning the solution successively with n-hexane and ethylacetate to yield n-hexane (AVHF, 3.5%) and ethylacetate (AVEF, 13.9%) fractions. The ethylacetate marc was successively extracted with absolute methanol and water to yield methanol (AVMF, 57.7%) and aqueous (AVWF, 7.3%) fractions respectively as well as insoluble residue (AVMK, 17.3%). The AVHF (1 g) was subjected to gradient elution column chromatography on silca gel 60 (60Ǻ, 70–230 mesh) with sample to silica ratio of 1:75, and eluted successively with dichloromethane (DCM, 100%), DCM/EtOAc (10:1; 1:1) and, lastly, EtOAc (100%). Based on the thin layer chromatography (TLC) profiles, eight sub-fractions i.e. AVHF B1e B8, were obtained. B3 and B4 have the same TLC profiles and were, therefore, combined to give B4. 2
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2.8. Cytotoxicity assay
Table 2 Column sub-fractions of n-hexane fraction of A. vogelii.
The overt cytotoxicities of the extracts and fractions were evaluated according to the method previously described by Merghoub et al., (2009) and Chithambo et al., (2017). The extracts and fractions were incubated for 48 h at 37 °C in a carbon dioxide incubator (5% CO2), at a fixed concentration of 50 μg/ml in 96-well plates containing HeLa cells in a culture medium of Dulbecco's Modified Eagle's Medium (DMEM). The number of cells surviving drug exposure was determined by using the resazurin-based reagent and reading resorufin fluorescence in a multi-well plate reader. Results were expressed as % viability–the resorufin fluorescence in compound-treated wells relative to untreated controls. Compounds were tested in triplicate wells and standard deviation (SD) obtained. Emetine (which induces cell apoptosis) was used as a positive control drug standard.
The percentage yields of the crude extract and fractions are given in section 2.3. The yield of the crude extract is given as a percentage of weight of plant material and those of the fractions as percentage of the crude extract. The stem bark has very poor yield of the non-polar fractions, AVHF and AVEF, which are the repository of the secondary metabolites largely responsible for the anti-trypanosomal and anti-inflammatory activities. Therefore, it will be more profitable to isolate the pure compounds, obtain their chemical structures and develop new potent drugs from them than use the plant in the crude form.
3.4. Column fractions of AVHF The column sub-fractions of AVHF and their descriptions are given in Table 2. The terpenoidal sub-fractions gave single spot each on analytical silica gel TLC plates suggesting that they might be pure compounds. Structural elucidation of the possibly new compounds is, therefore, necessary. 3.5. Anti-inflammatory activity The dose-dependent inhibition of egg-albumin-induced rat paw Table 1 Phytochemical constituents of the different extracts.
AVHF AVEF AVMF AVWF AVMK
++ + -
++ + -
+++ + -
+++ +++ +++
+ ++ + +++
-
Remaining, immovable components.
The cytotoxicity profile of the extracts and fractions on HeLa cells at a fixed concentration of 50 μg is given in Fig. 2. The percentage viability of the cells after drug exposure was very high, > 50%, for all the tested extracts, except AVHF-B8 which gave ∼ 44%. Extracts which reduced the cells percentage viability to < 25% are considered cytotoxic (Merghoub et al., 2009; Chithambo et al., 2017). The stem bark extract of A. vogelii showed LD50 greater than 5000 mg/kg, indicating high margin of safety as opposed to the NSAIDs, which elicit life threatening adverse effects (Harirforoosh et al., 2013). This is further supported by the cytotoxicity profile (Fig. 2). Almost all the tested samples gave very high percentage viabilities (> 50%) at 50 μg/ml, indicating that even at such high concentration of the extracts, more than 50% of the HeLa cells were still active. This implies very high selectivity index and makes the extracts more advantageous as antiinflammatory drug over the currently used NSAIDs which are used as rat poisons at high concentration due to their inherent toxicity. The secondary metabolites present in A. vogelii stem bark are mainly saponins, flavonoids, terpenoids, steroids and alkaloids. The ethylacetate fraction of the stem bark of A. vogelii contains mainly alkaloids and flavonoids while the n-hexane fraction contains mainly terpenoids and steroids and these are largely responsible for the anti-trypanosomal and anti-inflammatory activities of the plant. The polar fractions: AVMF, AVWF and AVMK did not show any significant anti-trypanosomal activity (Table 3) despite their higher solubility in the DMSO assay medium. They also gave no significant anti-inflammatory activity; hence, excluding polar phytochemicals, such as polyphenolics, in the activities. From the results of anti-inflammatory studies (Fig. 1), the optimal dose of AVEF at 100 mg/kg, though crude fraction with very complex composition, beats pure Piroxicam (10 mg/kg) in activity except at the fourth hour. This suggests that further purification might lead to pure
The crude extract, AV, showed LD50 > 5000 mg/kg. Within the first 30 min of administration the animals that received 1000 mg/kg and above were restless, hyperactive and gradually faded up. Animals that received < 1000 mg/kg showed no external sign of toxicity.
Tannins
Terpene Terpenoid Terpenoid Fatty acid Terpenoid Terpenoid Steroid, alkaloid
3.7. Cytotoxicity assay
3.3. Acute toxicity profile
Flavonoids
Greenish oily liquid Yellow solid Greenish semi solid White powder Light green semi-solid Green oily solid Brown solid
The IC50 values obtained for the individual samples are given in Table 3. Non-cytotoxic samples with trypanosomal residual percentage viability < 25% were considered active; hence, put forward for IC50 assay. The polar fractions (AVMF, AVWF and AVMK) showed no significant activity at 50 μg/mL, as indicated by high percentage viability values, and, hence, have IC50 values > 50 μg/mL. All the non polar fractions showed trypanosoidal activity. The terpenoidal fraction, AVHF-B8 gave very low percentage parasite viability.
The phytochemical composition of the different extracts is shown in Table 1. The polar fractions are composed mainly of saponins, flavonoids and no tannins, while the non-polar fractions contain mainly terpenes, alkaloids and steroids.
Saponins
AVHF-B1 AVHF-B2 AVHF-B4 AVHF-B5 AVHF-B6 AVHF-B8 AVHF-Bna
3.6. Trypanosomal assay
3.2. Qualitative phytochemical analysis of the different extracts
Terpenes
Phytochemical class
oedema, as a function of anti-inflammatory activities of the ethylacetate fraction, AVEF, of A. vogelii, is given in Fig. 1. The best activity was obtained at 100 mg/kg dose which significantly (p < 0.05–0.01) provoked 38.5%, 37.8%, 55.6%, 62.5%, 69.7%, 81.0% and 85.7% inhibition at the first, second, third, fourth, sixth, eight and twelfth hours respectively. The Piroxicam standard at 10 mg/kg provoked 2.6%, 18.9%, 44.4%, 67.5%, 60.6%, 76.2% and 78.5% respectively at those hours.
3.1. Extraction yield
Steroids
Description
a
3. Results and discussion
Alkaloids
Fraction
+ trace, ++ moderate, +++ very abundant, - not detected. 3
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Fig. 1. Percentage inhibitions of rat paw edema.
our preliminary screening of the solvent fractions for anti-inflammatory activities, we noted that the most active fraction was AVEF followed by AVHF. This implies that alkaloids, flavonoids, terpenes, steroids and or fatty acids are responsible and may be working in synergy (Chithambo et al., 2017). This is consistent with numerous previous reports on the anti-inflammatory activities of many other plants where the activity is attributed to the presence of terpenoids, sterols or flavonoids (Parmar and Ghosh, 1978; Ahmad et al., 1983; Calixto et al., 2000; Sabu and Kuttan, 2002; Ghannadi et al., 2005; Silva et al., 2005). Reduction in activity at higher dose may be attributed to increased toxicity, which may result in further release of the mediators of inflammation. Oedema induced by egg albumin results from the release of histamine and serotonin (Pearce, 1986). According to the report by Akindele et al. (2012), egg albumin produces mast cell degranulation with subsequent release of inflammatory mediators such as histamine, which has been associated with increased vasodilatation and increased permeability of blood vessels leading to the exudation of plasma proteins and fluids into the tissues. A. vogelii appreciably inhibited oedema in this model (Fig. 1), suggesting the possible inhibition of histamine and serotonin in its anti-inflammatory property. Further works on the measurement of these mediators may confirm their involvement. Human African trypanosomiasis, a neglected tropical disease caused by Trypanosoma species, remains one of the serious health problems confronting many African countries. The current anti-trypanosomal agents such as suramin and pentamidine are associated with serious adverse effects (Wang, 1995) and require lengthy parenteral administration. They are also expensive in addition to drug resistance issue (Mpia and Pepin, 2002; Horn, 2014; Kofi and Nguyen, 2016). In view of these concerns, continued search for more potent, cheaper and safer alternatives to the existing anti-trypanosomal drugs is warranted. Natural products from plants have been used to treat human ailments since the origin of man and remain an important source of drug leads (Kingston, 1996). We investigated the anti-trypanosomal properties of A. vogelii based on the folkloric use of the plant to remedy sleeping sickness. Bioactivity-guided purification of n-hexane fraction yielded potent terpenoids (AVHF-B2 and AVHF-B4) with anti-trypanosomal IC50 of 5.8 and 3.0 respectively. This result agrees with previous reports on the involvement of triterpenoids in the anti-trypanosomal activity of medicinal plants (Abe et al., 2002; Abiodun et al., 2012; Kofi and
Table 3 Anti-trypanosomal IC50 values for the different extracts and fractions. Sample at 50 μg/mL
Percentage viability (%) ± SD
IC50 (μg/ml for samples, μM for Pentamidine)
AVHF AVHF-B2 AVHF-B4 AVHF-B5 AVHF-B8* AVHF-Bn AVEF AVMF AVWF AVMK Pentamidine
2.14 ± 0.99 0.54 ± 0.89 3.05 ± 2.15 7.70 ± 6.05 0.71 ± 0.06 7.47 ± 6.60 3.06 ± 0.42 105.65 ± 0.15 103.35 ± 0.79 102.37 ± 0.95
20.9 5.72 ∼ 3.011 31.79 ND 24.67 ∼ 36.33 > 50 > 50 > 50 0.004363
ND: Not determined. * cytotoxic; hence, IC50 not determined.
Fig. 2. Cytotoxicity profile of the different extracts and fractions.
compounds with high efficacy. Piroxicam is a known prostaglandins inhibitor. Its higher activity at the fourth hour is attributed to greater inhibition of prostaglandins than the extract at the tested dose. From 4
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Nguyen, 2016), but is a success story in terms of the low cytotoxicity profile (Fig. 2) and the corresponding high selectivity index.
of Anthocleista vogelii Planch in Rats. Hindawi Publishing Corporation, Ulcers 2014, Article ID 172096, 6 pages. Calixto, J.B., Beirith, A., Ferreira, J., Santos, A.R., Cechinel, F.V., Yunes, R.A., 2000. Naturally occurring antinociceptive substances from plants. Phytother Res. 14, 401–418. Chithambo, B., Siwe Noundou, X., Krause, R.W.M., 2017. Anti-malarial synergy of secondary metabolites from Morinda lucida Benth. J. Ethnopharmacol. 199, 91–96. Dalziel, J.M., 1955. The Useful Plant of West Tropical Africa. Crown Agents for the Colonies, London, pp. 318–330. Ghannadi, A., Hajhashemi, V., Jafarabadi, H., 2005. An investigation of the analgesic and anti-inflammatory effects of Nigella sativa seed polyphenols. J. Med. Food 8, 488–493. Harborne, J.B., 1998. Phytochemical Methods: a Guide to Modern Techniques of Plant Analysis, second ed. Chapmann and Hall Publishers, London, pp. 21–72. Harirforoosh, S., Asghar, W., Jamali, F., 2013. Adverse effects of nonsteroidal antiinflammatory drugs: an update of gastrointestinal, cardiovascular and renal complications. J. Pharm. Sci. 16, 821–847. Horn, D., 2014. High-throughput decoding of drug targets and drug resistance mechanisms in African trypanosomes. Parasitology 141, 77–82. Irvine, F.R., 1961. Woody Plants of Ghana. Oxford Univ. Press, London, pp. 878. Jegede, I.A., Ibrahim, J.A., Kunle, O.F., 2011. Phytochemical and pharmacognostic studies of the leaf and stem-bark of Anthocleista vogelii (Planch). J. Med. Plants Res. 5 (26), 2011. Kingston, D.G., 1996. Natural products as pharmaceuticals and sources of lead structures. In: Wermuth, C.G. (Ed.), The Practice of Medicinal Chemistry. Acad. Press, London, pp. 101–116. Kofi, D.K., Nguyen, H.T., 2016. Antitrypanosomal activities and mechanisms of action of novel tetracyclic iridoids from Morinda lucida Benth. Antimicrob. Agents Chemother. 60 (6), 3283–3290. Leeuwenberg, A.J.M., 1972. Flore du Cameroun 12, 5–22. Lorke, D., 1983. A new approach to practical acute toxicity testing. Arch. Toxicol. 53, 275–289. Mbiantcha, M., Nguessom, K., Ateufack, G., Oumar, M., Kamanyi, A., 2013. Analgesic properties and toxicological profile of aqueous extract of the stem bark of Anthocleista vogelii planch (Loganiaceae). Int. J. Pharm. Chem. Biol. Sci. 3 (1), 1–12. Merghoub, N., Amzazi, S., Morjani, H., 2009. Cytotoxic effect of some Moroccan medicinal plant extracts on human cervical cell lines. J. Med. Plants Res. 3 (12), 1045–1050. Mpia, B., Pepin, J., 2002. Combination of eflornithine and melarsoprol for melarsoprolresistant Gambian trypanosomiasis. Trop. Med. Int. Health 7, 775–779. Nikose, S., Arora, M., Singh, P., Nikose, D., Gadge, S.V., 2015. Gastrointestinal adverse effects due to use of non-steroidal anti-Inflammatory drugs (NSAIDs) in non-traumatic painful musculoskeletal disorders. J. Gastrointest. Dig. Syst. 5, 348. Nwodo, N.J., Ibezim, A., Ntie-Kang, F., Adikwu, M.U., Mbah, C.J., 2015. Anti-trypanosomal activity of Nigerian plants and their constituents. Molecules 20, 7750–7771. Okoli, C.O., Akah, P.A., Nwafor, S.V., Anisiobi, A.I., Ibegbunam, I.N., Erojiekwe, O., 2007. Anti-inflammatory activity of hexane leaf extract of Aspila africana C.D. Adams. J. Ethnopharmacol. 109, 219–225. Okon, E.O., Gboeloh, L.B., Udoh, E.S., 2014. Antimalarial effect of combined extracts of the leaf of Ficus exasperate and stem bark of Anthocleista vogelii on mice experimentally infected with Plasmodium berghei berghei (Nk 65). Res. J. Med. Plant 8 (3), 99–111. Osadebe, P.O., Uzor, P.F., Omeje, E.O., Agbo, M.O., Obonga, W.O., 2014. Hypoglycemic activity of the extract and fractions of Anthocleista vogelii Planch stem bark. Trop. J. Pharmaceut. Res. 13 (9), 1437–1443. Parmar, N.S., Ghosh, M.M.N., 1978. Current trends in flavonoid research. Indian J. Pharm. 12, 213–228. Pearce, F.L., 1986. On the heterogeneity of mast cells. Pharmacology 32 (2), 61–71. Ram, S., Sinha, V.S., 2015. Qualitative phytochemical analysis of some plants used to cure malaria in Kolhan region of Jharkhand, India. J. Med. Plants Stud. 3 (6), 60–62. Sabu, M.C., Kuttan, S., 2002. Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J. Ethnopharmacol. 81, 155–160. Sahira, B.K., Cathrine, L., 2015. General techniques involved in phytochemical analysis. Int. J. Adv. Res. Comput. Sci. 2 (4), 25–32. Santhi, K., Sengottuvel, R., 2016. Qualitative and quantitative phytochemical analysis of Moringa concanensis Nimmo. Int. J. Curr. Microbiol. App. Sci. 5 (1), 633–640. Silva, G.N., Martins, F.R., Matheus, M.E., 2005. Investigation of anti-inflammatory and antinociceptive activities of Lantana trifolia. J. Ethnopharmacol. 100, 254–259. Sunday, R.M., Ilesanmi, O.R., Obuotor, E.M., 2016. Anti-diabetic effect of Anthocleista vogelii ethanolic root extract in alloxan-induced diabetic rats. Res. J. Med. Plant 10, 79–88. Swathy, B., Mohana, S., Saravana, A., 2010. Review on herbal drugs for analgesic and anti-inflammatory activities. IJBPR 1 (1), 7–12. Tanko, Y., Mohammed, A., Saleh, M.I., Mohammed, K.A., Etta, E., Bako, I.G., Muhammad, A., Yerima, M., 2012. Antinociceptive and anti-inflammatory activities of ethanol extract of Bryophyllum Pinnatum in laboratory animals. JDMS 3 (1), 46–49. Tene, M., Tane, P., Kuiate, J., Tamokou, J., Connolly, J.D., 2008. Anthocleistenolide, a new rearranged nor-secoiridoid derivative from the stem bark of Anthocleista vogelii Planch. Letter, Planta Med. 74, 80–83. Wang, C.C., 1995. Molecular mechanisms and therapeutic approaches to the treatment of African trypanosomiasis. Annu. Rev. Pharmacol. Toxicol. 35, 93–127.
4. Conclusion Results showed that A. vogelii holds promise as a potential source of new anti-inflammatory and anti-trypanosomal drugs. Further purification and spectral characterization of the alkaloids and terpenoids from the stem bark will result in potent anti-inflammatory and anti-trypanosomal compounds which might be used as safer alternatives to the existing ones. Also, the cytotoxicity and acute toxicity profile indicated a wide therapeutic margin. Furthermore, A. vogelii extracts and compounds have been shown to possess anti-ulcer activities. This is advantageous over the conventional NSAIDs which provoke erosion of gastric mucosa. Based on these observations and results, there is a clear justification for the ethnomedicinal and folkoric use of A. vogelii for treating sleeping sickness, pain and inflammatory disorders such as rheumatoid arthritis in Nigeria and other parts of Africa. Conflicts of interest The authors declare that there is no conflict of interests. Acknowledgements This work was sponsored by the Nigeria’s Tertiary Education Trust Fund (TETFund) under the TETfund Institution-Based Research (IBR) Intervention and supported by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package, and Rhodes University SandisaImbewu. The authors are grateful to Michelle Isaac for assistance in the bioassays. X. Siwe Noundou is grateful for a Rhodes University Postdoctoral Research Fellowship. References Abe, F., Yamauchi, T., Nagao, T., Kinjo, J., Okabe, H., Higo, H., Akahane, H., 2002. Ursolic acid as a trypanocidal constituent in Rosemary. Biol. Pharm. Bull. 25 (11), 1485–1487. Abiodun, O.O., Gbotosho, G.O., Ajayiyeoba, E.O., Brun, R., Oduola, A.M., 2012. Antitrypanosomal activity of some medicinal plants from Nigerian ethnomedicine. Parasitil. Res. 110 (2), 521–526. Abuh, F.Y., Wambebe, C., Rai, P.P., Sokomba, E.N., 1990. Hypoglycaemic activity of Anthocleista vogelii (Planch) aqueous extract in rodents. Phytother Res. 4 (1), 20–24. Adebayo, H.A., John-Africa, L.B., Agbafor, A.G., Omotosho, O.E., Mosaku, T.O., 2004. Anti-nociceptive and anti-inflammatory activities of extract of Anchomanes difformis in rats. Pak. J. Pharm. Sci. 27 (2), 265–270. Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enow-Orock, E.G., Focho, D., Gbilé, Z.O., Kamanyi, A., KamsuKom, J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.L., Mbome, I.L., Mubiru, N.K., Nancy, W.L., Nkongmeneck, B., Satabié, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Contribution to Ethnobotanical and Floristic Studies in Cameroon. CSTR/OUA, Cameroon, pp. 273. Ahmad, M.M., Quresh, S., Shah, A., Qazi, N.S., Rao, R.M., Albakiri, M., 1983. Anti-inflammatory activity of Caralluma tuberculata alcoholic extract. Fitoterapia 46, 357–360. Akah, P., Nwabie, A.I., 1994. Evaluation of Nigerian traditional medicines: plants used for rheumatic disorder. J. Ethnopharmacol. 42, 179–182. Akindele, A.J., Ibe, I.F., Adeyemi, O.O., 2012. Analgesic and antipyretic activities of Drymaria cordata (Linn.) willd (Caryophyllaceae) extract. Afr. J. Tradit., Complementary Altern. Med. 9 (1), 25–35. Alaribe, C.S.A., Coker, H.A.B., Shode, F.O., Ayoola, G., Adesegun, S.A., Bamiro, J., Anyim, E.I., Anyakora, C., 2012. Antiplasmodial and phytochemical investigations of leaf extract of Anthocleista vogelii Planch. J. Nat. Prod. 5, 60–67. Al-Saeed, A., 2011. Gastrointestinal and cardiovascular risk of nonsteroidal anti-inflammatory drugs. Oman Med. J. 26, 385–391. Ateufack, G., Nguelefack, T.B., Wabo, H.K., Watcho, P., Tane, P., Kamanyi, A., 2008. Antiulcer effects of the aqueous and organic extracts of the stem bark of Anthocleista vogelii in rats. Pharm. Biol. 44 (3), 166–171. Ateufack, G., Nguelefack, T.B., Wabo, H.K., Tane, P., Kamanyi, A., 2014. Antiulcerogenic Activity of 1-Hydroxy-3,7,8-Trimethoxyxanthone Isolated from the Methanol Extract
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