Full Length Research Paper
ABSTRACT
Berlinia grandiflora is used for managing numerous ailments including tumor in South-west, Nigeria. The phytochemical contents, antioxidant and antiproliferative activities of B. grandiflora leaf were determined in this study. The absolute ethanol extract was evaluated by high performance liquid chromatography-diode array detection (HPLC-DAD). The antioxidant activity was carried out by standard analytical methods. The antiproliferative activity was done using growth inhibition activity of Sorghum bicolor seed radicle with methotrexate as the reference drug. The presence of some phytochemicals known to have antioxidant properties and exert anticancer effects is revealed in this study. The HPLC analysis revealed the presence of betulinc acid, ferulic acid, rutin and caffeic acid. However, ferulic acid, rutin, and caffeic acid were identified for the first time in B. grandiflora. Free radical scavenging activity, reducing power capacity and nitric oxide inhibitory activity were exhibited by the extract, with IC50 values of 88.3, 7.4 and 99 µg/mL, respectively. While ascorbic acid, the reference compound, used in this study has IC50 values of 0.7, 3.4 and 7.4 µg/mL, respectively. The total phenol content expressed in gallic acid equivalent (GAE) was 75 mg/g. A dose-dependent antiproliferative activity was observed with IC50 values of 0.50, 0.59 and 0.29 µg/mL after 48, 72 and 96 h of treatment, respectively. This result justifies the folklore use of the plant for managing cancer in Nigeria and the plant’s potential as a new phyto-chemo-therapy.
Key words: Antiproliferative, antioxidant, phytochemicals, cancer, Berlinia grandiflora, betulinic acid.
INTRODUCTION
Berlinia grandiflora (Vahl) Hutch. & Dalziel is a medicinal plant which is widely distributed across African countries; it belongs to the family Fabaceae. The genus Berlinia has about 20 species which are found in eastern, western and southern Africa, especially in countries like Guinea, Mali, Nigeria, Central African Republic and DR Congo (Hutchison and Dalziel, 1963). Locally in Nigeria, B. grandiflora is called “Apado” by the Yoruba tribe and “Ububa” by the Igbo ethnic groups (Asuzu et al., 1993). The medicinal value of B. grandiflora is well known to herbalists and traditional medicine practitioners in South west of Nigeria. The plant is used for the treatment of several disease conditions in ethnomedicine. The bark sap is used for dressing wound and sores, while the bark decoction is used for managing haemorrhoids and liver problems. The bark extract is also used to ease labor pain during childbirth and for treating gastrointestinal disorder (Josephs et al., 2012). Leafy twigs decoction is used to ameliorate fever conditions and to combat vomiting and nausea. The leaf decoction is drunk as a tonic and used to improve blood circulation (Ode et al., 2013). The leaf extract is used to manage cancer in south-western Nigeria, as a vermifuge, and for treating diarrhea and diabetes (Soladoye et al., 2010; Ode et al., 2013). Previous researchers have reported the phytochemical analysis, analgesic activity (Asuzu et al., 1993), antihelminthic activity (Enwerem et al., 2001), antioxidant activity, antimicrobial activity (Josephs et al., 2012; Ode et al., 2013) and the anti-diabetic activity (Duru et al., 2014) of B. grandiflora. Also, the cytotoxicity of chemical compounds in the seed of the plant has been reported by Duru et al. (2014).
The leaves and stem barks of B. grandiflora have been reported to possess antimicrobial activity against a wide range of bacteria such as Staphylococcus aureus, Escherichia coli, Alcaligenes faecalis, Serratia marcescens, Enterobacter bacteraerogenes, Kliebsiella pneumonia, Pseudomonas aeruginosa and Proteus vulgaris (Josephs et al., 2012). The antimicrobial property of B. grandiflora is attributed to betulinic acid (Enwerem et al., 2001). The major compounds in the aqueous extract of B. grandiflora seeds are: α-methyl-mannofuranocide (21%), Pentadecanoic acid (15%), 9-octadecanoic acid (31%), Octadecanoic acid (17%) and 9-octadecinamide (16%). These compounds may be responsible for the acute toxicity of the seed of B. grandiflora in animals (Duru et al., 2014). The methanol extract of the leaf also shows acute toxicity in mice (Josephs et al., 2012). Methanol extract of the leafs and stem barks of B. grandiflora contain tannins, alkaloids, flavonoids, starch and glycosides (Asuzu et al., 1993; Ode et al., 2013). Similarly, the aqueous extract of the seeds was reported to contain alkaloids, saponins, tannins, phenol, flavonoids and starch, while anthraquinones, phenols, steroids and cyanogenic glycoside were absent (Josephs et al., 2012).
Although B. gradiflora is used in ethnomedicine as an anti-tumor agent, no scientific report of the antiproliferative activity of the leaf of the plant exists in literature. Therefore, the aim of this study is to scientifically evaluate the antiproliferative activity and antioxidant properties of the ethanol extract of the plant leaf with a view to providing scientific information to justify its use for managing cancer and tumors.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Phytochemical constituents
The extraction yield of the dried leaves of B. grandiflora was 7.28%. Phytochemical screening of the extract reveals the presence of alkaloids, saponins, terpenes, steroids, flavonoid, phenols and tannins. However, anthraquinones were not detected in the test sample. Previous studies have shown that B. grandiflora leaf contains tannins, alkaloids, flavonoids, glycosides, saponins, phenols while anthraquinones and carbohydrates were absent (Ode et al., 2013). The result of the phytochemical analysis in the present study is also consistent with the report of Asuzu et al. (1993) and Duru et al. (2014). The therapeutic property of plants is widely due to the presence of phytochemicals in them. Previous studies have shown that phytochemicals possess several pharmacological activities such as; anti-inflammatory activity, anti-atherosclerotic activity, anti-tumor activity, anti-mutagenic activity, anti-cancer activity, anti-bacterial activity, anti-viral activity to mention a few (Adamu et al., 2017). Specifically, phenolics which comprise flavonoids, tannins and phenols represent phytochemicals linked to antioxidant properties of plant. Phenolic compound has been proven to exhibit preventive and therapeutic effects against diseases where oxidative stress has been implicated, including cardiovascular diseases, cancer, neurodegenerative disorders and aging (Adamu et al., 2016).
The reverse phase HPLC-DAD analysis of the BG extract shows the presence of some phenolic compounds. A total of nine peaks appeared with retention times (Rt) of 3.32, 3.92, 5.53, 5.98, 6.49, 6.97, 7.77, 9.03 and 10.53 min, respectively (Figure 1). The most prominent peak Rt = 3.32 min corresponds to betulinic acid (Figure 2), a well-known anticancer agent which is derived from plant (Biswas et al., 2015). This is in agreement with the report of Enwerem et al. (2001), who isolated betulinic acid from the leaf of B. grandiflora. Three other peaks corresponded with that of known antioxidant phenolic compounds, caffeic acid (Rt = 6.49 min), rutin (Rt = 6.97 min) and ferulic acid (Rt = 7.76 min) (Figures 3 and 4). Caffeic acid, rutin and ferulic acid have been reported to exhibit antioxidant activity (Rice-Evans et al., 1997).
Antioxidant activity
From Figure 5, 125 and 250 μg/mL of the B. grandiflora extract exhibit antioxidant activity of 59 and 60%, respectively. While the control drug, ascorbic acid, exhibits an antioxidant activity of 97.6 and 97.8%, respectively.
In this assay, the ethanol extract of B. grandiflora leaves shows some degree of free radical scavenging capacity. At 250 and 125 µg/mL, the free radical scavenging capacity of the extract is above 50%. However, when compared with the reference compound ascorbic acid (IC50 = 0.7 µg/mL), B. grandiflora (IC50 88.3 µg/mL) exhibited a weaker free radical scavenging capacity. The result of this study is similar to the report of Ode et al. (2013).
Nitric oxide is a potent inhibitor of physiological processes such as smooth muscle relaxation, neuronal signaling, and inhibition of platelet aggregation and regulation of cell- mediated toxicity; it is also implicated in inflammation, cancer and other pathological conditions (Esievo et al., 2018). From Table 1, B. grandiflora showed very weak nitric oxide (NO) inhibition (IC50 99 µg/mL) when compared with the NO inhibition of ascorbic acid (IC50 = 3.4 µg/mL).
Several studies have confirmed the correlation between antioxidant activity and reducing power in plant extracts. Reducing power may be used as a pointer to potential antioxidants. The occurrence of antioxidants (reducing species) in extracts causes reduction of Fe (III) to Fe (II) complexes which can be determined by the formation of prussain blue color. This reduction may be due to the donation of a hydrogen atom from phenolic compounds (Shimada et al., 1992) which also correlates to the presence of a reducing agent. The reducing power of B. grandiflora (IC50 = 15 µg/mL) is low compared to that of ascorbic acid (IC50 = 7.4 µg/mL).
The total phenol content in BG extract expressed in gallic acid equivalent (GAE) was 75.1 mg/g (Table 1). Plant phenols are a major group of the compounds acting as free radical scavengers and as such, it is important to estimate the content of phenol in the extract (Brum et al., 2013). Phenols contain hydroxyls that are responsible for the antioxidant properties which may be due to their redox properties (Rice-Evans et al., 1997). According to this study, the phenol content may be responsible for the antioxidant activity of the extract.
Growth inhibitory effects of B. grandiflora (leaves) on S. bicolor (Guinea corn) seeds
The result of antiproliferative activity of BG showed that there is a significant reduction in the length of radicles of S. bicolor seeds treated with the various concentrations of the extract when compared with the length of radicles of S. bicolor seeds treated with only distilled water. After 48 h of incubation, the percentage inhibition of 0.5, 1.0, 2.0 and 4.0 mg/mL of ethanol extract of BG was 44.5, 64.3, 88.0, and 93.1%, respectively. However, methotrexate has a percentage inhibition of 90.7% after 48 h of incubation. It was observed that the percentage inhibition increased with corresponding increase in the concentration of the extract (Table 2). This suggests that the extract exhibits a concentration-dependent inhibition. The extract also showed a time-dependent inhibition; the percentage inhibition of each concentration increases with increase in incubation time; the percentage inhibition after 48, 72 and 96 h for 0.5 mg/mL extract test solution was 44.5, 45.5 and 66.9%, respectively; that of 1 mg/mL was 64.3, 54 and 70.6%, respectively. 2 mg/mL exhibited 88.0, 83.2, and 85.8%, respectively and 4 mg/mL exhibited 93.1, 95.6 and 98.6%, respectively. Furthermore, the percentage inhibition for 2 and 4 mg/mL of the extract solution was similar to the percentage inhibition of the positive control (0.05 mg/mL methotrexate) which is a well known anticancer drug. The IC50 values of the antiproliferative activity of the ethanol extract of BG were 0.50, 0.59 and 0.29 µg/mL after 48, 72 and 96 h of treatment, respectively. Therefore it can be implied that the ethanol extract of BG can mitigate the fast proliferation of cancer cells. The fact that 0.05 mg/mL of methotrexate showed a significant percentage inhibition justifies the use of this method.
The results of this study have shown that the ethanol extract of B. grandiflora contains phytochemicals which possess antioxidant properties and are reported to exert anticancer effects too. It also supports the report of the ethnomedicinal use of B. grandiflora for the management of tumors in South-west Nigeria (Soladoye et al., 2010). However, this is the first work reporting the antiproliferative activity of the plant. Furthermore, this is the first study to report the presence of some plant polyphenols specifically; rutin, ferulic acid and caffeic acid in B. grandiflora. Rutin is widely found in plants as glycoside, combining the flavonol quercetin and the disaccharide rutinose. The ability of flavonoids to act as potent antioxidants depends on their molecular structures, the position of the hydroxyl group and other features in their chemical structure (Iqbal et al., 2015). Ferulic acid and caffeic acid are also phenolic compounds. High content of phenolic compound has been linked to anticancer activities (Iqbal et al., 2015). Polyphenolic compounds have an aromatic benzene ring with substituted hydroxyl groups and other functional derivatives. These functional groups can bind to free radicals and also metal ions that could catalyze formation of reactive oxygen species (ROS) which promotes lipid peroxidation.
The antiproliferative activity of the plant could be due to the presence of betulinic acid which is the major constituent of B. grandiflora from HPLC analysis. Previously, it has been reported that betulinic acid from Zizyphus species namely; Zizyphus rugosa, Zizyphus oenoplia and Zizyphus mauritian showed excellent anticancer properties (Fulda, 2008). Furthermore, betulinic acid was reported to be a highly selective growth inhibitor of human melanoma, neuroectodermal and malignant tumor cells and to also induce apoptosis in these cells (Yogeeswari and Sriram, 2005). Betulinic acid has been identified as a potent anticancer agent and is currently undergoing clinical trials as an anticancer drug (Biswas et al., 2015).
CONCLUSION
The ethanol extracts of B. grandiflora leaves exhibited significant growth inhibitory effects on fast proliferating cells (S. bicolor seed radicle). This suggests that it can inhibit cancerous cells. This study provided preliminary evidence that supports the ethnomedicinal use of BG in the treatment of tumor. Furthermore, the presence of betulinic acid, rutin, caffeic acid and ferulic acid may be working in synergy with all other phytochemicals to exert the antiproliferative activity of the plant. The authors intend to conduct subsequent studies on cancer cell line.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
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