Abstract
Context: Terminalia macroptera Guill. & Perr. (Combretaceae), a tree that grows in West Africa, has been used in traditional medicine against a variety of diseases such as hepatitis, gonorrhea, skin diseases, and diabetes.
Objective: To investigate enzyme inhibitory activity against α-glucosidase and 15-lipoxygenase (15-LO) and toxicity against brine shrimp of extracts and compounds from T. macroptera leaves.
Materials and methods: Methanol extract, ethyl acetate, and butanol extracts obtained from the methanol extract, six isolated polyphenols (chebulagic acid, chebulic acid trimethyl ester, corilagin, methyl gallate, narcissin, and rutin), and shikimic acid were evaluated for enzyme inhibition and toxicity.
Results: In enzyme inhibition assays, all extracts showed high or very high activity. Chebulagic acid showed an IC50 value of 0.05 µM towards α-glucosidase and 24.9 ± 0.4 µM towards 15-LO, in contrast to positive controls (acarbose: IC50 201 ± 28 µM towards α-glucosidase, quercetin: 93 ± 3 µM towards 15-LO). Corilagin and narcissin were good 15-LO and α-glucosidase inhibitors, as well, while shikimic acid, methyl gallate, and chebulic acid trimethyl ester were less active or inactive. Rutin was a good α-glucosidase inhibitor (IC50 ca. 3 µM), but less active towards 15-LO. None of the extracts or the isolated compounds seemed to be very toxic in the brine shrimp assay compared with the positive control podophyllotoxin.
Conclusion: Inhibition of α-glucosidase in the gastrointestinal tract may be a rationale for the medicinal use of T. macroptera leaves against diabetes in traditional medicine in Mali. The plant extracts and its constituents show strong inhibition of the peroxidative enzyme 15-LO.
Introduction
Terminalia macroptera Guill. & Perr. (Combretaceae), a tree that grows in West Africa, has been used in traditional medicine against a variety of diseases such as hepatitis, gonorrhea, ringworm, skin diseases, and sexually transmitted diseases as well as gastritis, colic, high blood pressure, fever and tuberculosis, as documented by our previous investigations (Pham et al., Citation2011a,b). Even though used for so many different indications, the toxicity of this tree has, to our knowledge, not been investigated previously.
We have recently carried out a survey of the use of T. macroptera by traditional healers in Mali, and diabetes was one of the indications mentioned. Diabetes is a common metabolic disease characterized by abnormally high plasma glucose levels. This disease is regarded as epidemic in many African countries including Mali, and most of the treatment of this disease is carried out by traditional healers (Azevedo & Alla, Citation2008). It is therefore highly important to identify medicinal plants that may have a potential in diabetes treatment. The key enzyme which catalyses the final step in the digestive process of carbohydrates in mammals is α-glucosidase (Anam et al., Citation2009). Hence, α-glucosidase inhibitors can retard the liberation of d-glucose of oligosaccharides and disaccharides from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma glucose levels and suppressed postprandial hyperglycemia (Gao et al., Citation2008).
15-Lipoxygenase (15-LO) is an enzyme present in multiple systems and organs of the body that reacts with unsaturated fatty acids producing active lipid metabolites that are involved in a number of disease states such as cancer, psoriasis, atherosclerosis, and diabetes (both type 1 and 2) (Dobrian et al., Citation2011; Schneider & Bucar, Citation2005). For this reason, 15-LO has emerged as an attractive target for therapeutic intervention (Sadeghian et al., Citation2009). In light of this, targeting inhibitors of 15-LO is a promising pharmacological strategy for the treatment of diabetes, as stated in a recent review (Dobrian et al., Citation2011). Therefore, in order to investigate the rationale for the use of T. macroptera against diabetes, this study was conducted.
Materials and methods
Plant material and test compounds
T. macroptera leaves were collected from Blendio, Mali, in December 2007. The plant was identified by Drissa Diallo, Department of Traditional Medicine (DMT), Bamako, Mali, and a voucher specimen (No. 2468 DMT) was deposited at DMT. The leaves were extracted in a Soxhlet apparatus as previously described (Pham et al., Citation2011b), furnishing dichloromethane (DCM, 5.3% of the plant material) and methanol (MeOH, 35.6% of the plant material) extracts. The latter was further partitioned into ethyl acetate (EtOAc), butanol (BuOH), and aqueous phases. EtOAc and BuOH extracts were chromatographed repeatedly over Sephadex LH20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), reverse phase (C18) silica gel, and MCI CHP20P gel. In some instances, preparative HPLC (reverse phase C18) was used as a final purification step. Substances were identified by NMR spectroscopy (1H, 13C, COSY, APT, HETCOR, HSQC, and HMBC) (Pham et al., Citation2011b). Shikimic acid (1) and the polyphenols methyl gallate (2), corilagin (3), chebulagic acid (4), chebulic acid trimethyl ester (5), rutin (6), and narcissin (7) (structures are shown in ) were isolated from the MeOH crude extract and employed in the present investigation.
Figure 1. Chemical structures of compounds 1–7.
Inhibition of α-glucosidase
Test substances were dissolved in DMSO, and the assay was carried out according to a published procedure, with slight modifications (Matsui et al., Citation1996). α-Glucosidase from Saccharomyces cerevisiae and 4-nitrophenyl α-d-glucopyranoside (PNP-G) were purchased from Sigma-Aldrich, St. Louis, MO. An enzyme solution containing α-glucosidase (0.8 units/mL) in 50 mM phosphate buffer (pH 7.0) containing 100 mM NaCl was made immediately before use and kept on ice during the experiment. Substrate, PNP-G (0.7 mM) in phosphate buffer, was made immediately before use. For each assay, 20 µL test solution and 80 µL enzyme solution were preincubated at 37 °C for 5 min. The reaction was started by adding 1.9 mL of substrate solution, and the solution was then incubated at 37 °C for 15 min. The reaction was stopped by adding 2.0 mL 0.5 M aqueous Tris solution, and the absorbance of PNP released from PNP-G was measured at 400 nm. In blanks, 20 µL DMSO was added instead of test solution. The α-glucosidase inhibitory activity was expressed as the percentage inhibition of α-glucosidase, calculated as 100 × (AB − AS)/AB, where AB and AS represent the absorbance of the blank and sample, respectively. Acarbose (Sigma-Aldrich) was used as a positive control. All samples were analyzed in triplicate, and results are given as averages ± SD.
Inhibition of 15-lipoxygenase (15-LO)
Test substances were dissolved in DMSO, and the assay was carried out as reported previously (Wangensteen et al., Citation2004). Briefly, a solution of linoleic acid (final concentration 134 µM) was oxidized with 15-LO (10 000 U/mL) for 60 s and the increase in absorbance at 234 nm in the absence and presence of test solution was measured. Linoleic acid and 15-LO from soybeans were purchased from Sigma-Aldrich. Quercetin (Sigma-Aldrich), a well known 15-LO inhibitor, was used as a positive control (Wangensteen et al., Citation2004). All samples were analyzed in triplicate and results are given as averages ± SD.
Brine shrimp toxicity assay
Test substances were dissolved in DMSO, and the assay was performed as previously described with minor modifications (Solis et al., Citation1993). Brine shrimp eggs were purchased from Ocean Nutrition, San Diego, CA. After hatching the eggs in 3.3% artificial seawater, 198 µL of this solution containing 10–15 shrimp larvae were transferred to wells of 96-well microplates. Test solution, 2 µL, was then added to the wells. The plate was covered and incubated at room temperature for 24 h. Podophyllotoxin (50 µg/mL) was used as a positive control and DMSO as a negative control. All samples were analyzed in triplicate.
Results and discussion
The results from the α-glucosidase and 15-LO inhibition assays are summarized in and . The inhibitory activity of the extracts toward α-glucosidase was much stronger than the positive control acarbose. The extracts from T. macroptera leaves demonstrated high activity toward 15-LO, as well, and the effects are comparable with or better than the positive control quercetin. The EtOAc extract showed strongest inhibition of both α-glucosidase (0.40 ± 0.02 µg/mL) and 15-LO (IC50: 23.2 ± 0.5 µg/mL). The DCM extract was not tested due to low solubility. As far as we know, T. macroptera extracts have not been tested for inhibition of α-glucosidase or 15-LO previously. However, crude extracts from other Terminalia species have previously been reported as α-glucosidase inhibitors (Anam et al., Citation2009), but most of these appear less active than T. macroptera extracts. Of the substances tested in our assay, chebulagic acid is the most efficient inhibitor of α-glucosidase and 15-LO. In our experiments, chebulagic acid is considerably more active than previously reported (Gao et al., Citation2007). In the previously published work, no positive control was included, so a direct comparison is difficult. The discrepancy may be due to differences in the experimental setup or to different enzyme sources. Corilagin is less active, but it is present in much higher amounts (more than 25-times the amount of chebulagic acid, calculated from yield of substances) and may therefore be the most important contributor to the inhibitory activity of the extract. However, to our knowledge, neither chebulagic acid nor corilagin has been tested for 15-LO inhibitory activity. The flavonoid glycosides rutin and narcissin were both fairly good inhibitors of 15-LO. This is in accordance with the previous results (Bouriche et al., Citation2007) for rutin. Narcissin, however, was reported to be inactive (Robak et al., Citation1988). Of the flavonoids, only rutin showed high activity as α-glucosidase inhibitor, in accordance with previous investigations (De Souza Schmidt Goncalves et al., Citation2010; Shibano et al., Citation2008). Due to the lack of material, the highest measured concentration for narcissin was 6.3 µM, so it may be that narcissin may have activity toward α-glucosidase at higher concentrations. A previous investigation (Shibano et al., Citation2008) reported an IC50 value for narcissin of 510 µM. Chebulic acid trimethyl ester, a novel compound previously isolated by us (Pham et al., Citation2011b), was found to have good 15-LO inhibitory activity, but was inactive as α-glucosidase inhibitor at the highest measured concentration (50 µM). In our study, shikimic acid was inactive as both 15-LO and α-glucosidase inhibitor. Shikimic acid appears not to have been tested for inhibition of 15-LO or α-glucosidase previously. Methyl gallate showed strong 15-LO inhibitory activity. Methyl gallate has previously been reported to be inactive as 15-LO inhibitor (Ha et al., Citation2010; Kubo et al., Citation2010), but the procedure differed somewhat from ours. At the highest concentration measured, 50 µM, it was inactive as α-glucosidase inhibitor in this study. This is in contrast with the data reported by Wansi et al. (Citation2007), but in good accordance with recent results reported by Wan et al. (Citation2012).
Table 1. Glucosidase and 15-LO inhibition of T. macroptera extracts.
Table 2. Effects of isolated compounds from T. macroptera on α-glucosidase and 15-LO inhibition.
The LC50 values against brine shrimp larvae for the tested extracts (DCM, MeOH, EtOAc, and BuOH) were higher than 100 µg/mL, and the LC50 values for the tested compounds (chebulagic acid, chebulic acid trimethyl ester, corilagin, methyl gallate, narcissin, rutin, and shikimic acid) were found to be higher than 200 µM. The isolated compounds and extracts demonstrated low toxicity against brine shrimp larvae compared to the positive control (87% lethality at 50 µg/mL). The chemical compounds and extracts present are thus not toxic against brine shrimp larvae in the tested concentrations. This assay is considered a reliable general bioassay which parallels other cytotoxicity assays (McLaughlin, Citation1991).
Conclusion
Considering the amount of ellagitannins in the extracts of T. macroptera leaves, the inhibitory activity towards 15-LO and α-glucosidase may be due to the high content of these compounds, especially corilagin and chebulagic acid. Although in vivo studies are needed, T. macroptera may be a potential non-toxic resource of 15-LO and α-glucosidase inhibitors that may be useful for the prevention or treatment of diabetes mellitus, and our results may therefore provide a rationale for its use against this disease by traditional healers in Mali.
Declaration of interest
The authors report no declarations of interest.
Acknowledgements
We are grateful to the healers of Mali for sharing their knowledge and experience with us.
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