Isolation of bioactive compounds and in vitro studies on Pentanisia prunelloides (Klotzsch ex Eckl. & Zeyh.) Walp. used in the eastern Free State for the management of diabetes mellitus
The pravelence of diabetes mellitus is increasing and it is one of the major health problems affecting the world. The challenges with synthetic drugs used in the treatment of hyperglycemia such as acarbose and miglitol include abdominal discomfort, bloating and diarrhoea. The present study isolated and evaluated the active antidiabetic constituents from the roots of Pentanisia prunelloides (Rubiaceae) from the eastern Free State Province of South Africa using in vitro models. The antidiabetic potential of the water, ethanol, aqueous-ethanol and hexane root extracts of P. prunelloides was investigated against the specific activities of α-amylase, α-glucosidase, sucrase and maltase. Furthermore, the antioxidant activity of the extracts was determined using iron chelation, 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radical scavenging assays. For the fractions and the isolated compounds, only α-amylase and α-glucosidase assays were used. Fractionation of the ethanol extract was done by vacuum liquid chromatography (VLC), fractions were combined according to thin layer chromatography (TLC) profiles, and further purification of semi-pure compounds was achieved using preparative thin layer chromatography (pTLC) to obtain pure compounds. Isolated compounds were characterised using nuclear magnetic resonance (NMR) (1D and 2D data) and mass spectroscopy (MS). The ethanol extract displayed significantly higher (p < 0.05) inhibition of α-amylase (18.51 μg/mL), hexane (18.08 μg/mL) and ethanol (19.73 μg/mL) extract exhibited strongest inhibition of α-glucosidase. Water extract demonstrated strong inhibition of sucrase (3.85 μg/mL), and aqueous-ethanol extract (26.03 μg/mL) on maltase. Kinetic studies showed that the mode of inhibition of α-amylase and α-glucosidase by ethanol extract was mixed non-competitive and non-competitive respectively. Water and ethanol extract displayed higher DPPH (75.42 μg/mL) and (77.06 μg/mL) scavenging abilities than other extracts but not higher than gallic acid. Hexane extract demonstrated significantly higher (p < 0.05) superoxide (0.33 μg/mL) and hydroxyl radical (0.51 μg/mL) scavenging abilities while aqueous-ethanol exhibited the strongest iron chelation activity 4.24 μg/mL. Phytochemical analysis of the extract revealed the presence of tannins, terpenoids, alkaloids, saponins, flavonoids and cardiac glycosides. Quantification of phytochemicals revealed total flavonoids of 15.40 mg quercetin equivalent (QE)/g in hexane extract which was not significant (p > 0.05) and from water it was 14.70 mg QE/g. The highest tannin concentration of 45.60 mg gallic acid equivalent (GAE)/g was from aqueous-ethanol which was significantly higher than other extracts (p < 0.05). Total phenol from water and aqueous extracts was 0.07 mg GAE/g; alkaloids and saponins were found to be low in the roots of P. prunelloides, at 0.6 and 13.9% respectively. Of the 21 fractions obtained, acetylated fraction displayed significantly higher (p < 0.05) inhibition of α-amylase 48.06 μg/mL while fraction PP-I-101835BII exhibited strongest inhibition of α-glucosidase (19.53 μg/mL). Three compounds were isolated, two sucrose (acetylated and non-acetylated) and tormentic acid. Tormentic acid inhibited α-amylase and α-glucosidase at 70.45 μg/mL and 28.21 μg/mL respectively. Kinetic analysis revealed that tormentic acid inhibited α-amylase in un-competitive manner and α-glucosidase competitively. The ethanol extract and the isolated tormentic acid exhibited best inhibitory activity on the two enzymes studied, and the presence of phytochemicals in the roots of P. prunelloides in this study may be suggested to have contributed greatly to the biological activities of the plant. Tormentic acid appears to be a potential anti-diabetic drug thus supporting usage of the root extract of P. prunelloides in the management and treatment of diabetes mellitus in the eastern Free State Province.