Bioactivity-guided isolation of compounds from the stem bark of alafia multiflora and paper-based microfluidic devices for colorimetric detection of disease biomarkers

Abstract

Despite enormous developments in science and technology that made it possible to produce various natural and synthetic medications since Fleming's in 1829, infectious diseases (IDs) continue to be one of the main causes of death worldwide. These IDs primarily affect people in underdeveloped nations with scarce resources and limited access to healthcare. Most of these infections are linked to bacteria pathogens that can withstand the effects of currently available conventional antimicrobials. Alafia multiflora is a tropical African medicinal plant with a long history of traditional use to treat ulcerous wounds and gastrointestinal pain, which inspired the screening for bioactive compounds in this research study. The aim of this research is to extract, isolate, and characterize the active ingredients from Alafia multiflora stem bark and evaluate their activity against various multidrug-resistant bacteria and antiproliferation activity against breast cancer cells. The phytochemical screening of the extracts generally showed the presence of saponins, flavonoids, polyphenols, tannins, triterpenoids, and glycosides. The water-methanol fraction crude extract demonstrated the highest effectiveness in inhibiting the growth of the various bacteria pathogens tested. For example, a minimal inhibition concentration (MIC) of <0.3 μg/mL was obtained against Staphylococcus aureus. Ethyl acetate extract showed modest activity against Klebsiella pneumoniae with an 8.5μg/mL MIC. Whereas dichloromethane (DCM) extract showed some activity against Klebsiella pneumoniae with an MIC of 60μg/mL. The water-methanol, ethyl acetate, methanol, and DCM extracts demonstrated significant antiproliferative effects against MD-MBA-231 breast cancer cells. These extracts notably reduced the viability of MD-MBA-231 breast cancer cells, with activity obtained in the range of 8-40μg/ml. This range means the extracts are active at 40 μg/mL and inactive at 8 μg/mL. The DCM isolate showed the highest activity compared to the extracts tested. The proportion of living cells at a concentration of 10 μg/mL and 5μg/mL of the DCM isolate were 6.7±1.8% and 50.7±14.8%, respectively. Results from thin layer chromatography (TLC) showed a single spot for DCM isolate based on the solvents used for TLC (60/40 hexane/ethyl acetate). However, the HPLC analysis of the DCM isolate suggests the presence of multiple compounds, as indicated by the peaks obtained at various retention times of 1.25, 3.75, 7.10, 13.50, and 16.16 mins under the chromatographic conditions used (C18 column, 45/55 acetonitrile/water mobile phase, 1mL/min flow rate and UV-detection at 250 nm). This suggests further separation may be needed for the components of the DCM isolate. Based on the anticancer activity of this isolate, an attempt was made to determine the possible functional groups and structural components of the compounds present using nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). The proton NMR data, for example, showed proton signals that are characteristic for aromatic compounds at a chemical shift of 7.7 ppm, aliphatic protons at 1.75pm, and allylic and vinylic-H were observed at 5.24 and 4.09, respectively. The chemical shifts obtained from carbon-13 NMR supported the structural features obtained from proton NMR spectra. The FTIR spectroscopic analysis of the DCM isolate showed various functional groups, such as C-H stretch at 2930 cm-1, a broad peak for OH at 3409 cm-1, and carbonyl at 1700 cm-1. The early diagnosis of infectious diseases, particularly in resource-limited regions, necessitates the availability of affordable, point-of-care devices. In this context, paper has emerged as a promising substrate for diagnostic devices. Its low cost, widespread availability, and white background provide optimal contrast for the colorimetric detection of disease biomarkers. This research study focused on the innovative use of hydrophobic ionic liquids as non-volatile media to create paper-based microfluidic channels and well-test zones, offering a low-cost solution for diagnosing disease biomarkers. These devices will be useful for increasing access to diagnostics in resource-constrained areas because they do not require sophisticated equipment, are simple to use, and read the results of the color change with an unaided eye. The test zones on paper were drawn with an infrared (IR) hydrophobic ionic liquid dye (IR 780) prefilled in a ballpoint pen. The multiplexed paper-based microfluidic devices were developed to simultaneously detect glucose, BSA, and uric acid in artificial urine on a single device. This approach makes it possible to detect several targets with a single sample, which lowers the cost of diagnosis and improves patient compliance. In addition, increased access to timely diagnosis and treatment of diseases in resource-limited regions will save lives and improve the quality of life.