Crude extracts of the dried powder of medicinal plants Centella asiatica (L.), Imperata
cylindrica (L.), Morinda citrifolia (L.) and Sauropus androgynus (L.) were obtained by maceration and Soxhlet extraction techniques. Successive extractions were carried out using a
modified procedure described by Kisangau et al., (2007). Briefly, Soxhlet extraction was carried
out by placing plant material in a thimble which was then inserted inside the Soxhlet apparatus and exposed to four different absolute solvents of different polarities (Methanol, Ethanol, Chloroform and Hexane), the ratio of sample to solvent was 1:10 (w/v). The extraction continued until the solvent appeared clear in the upper extraction chamber (Figure 3.6). For maceration, plant material macerated with four different solvents (methanol, ethanol, acetone and chloroform), solvents were changed daily and the collected solvent stored in closed containers in the fridge, this process continued until the solvent appeared clear. Extracts obtained from both procedures were then filtered twice through a Whatman No.1 under vacuum pressure. Extracts were concentrated by vacuum rotary evaporator (IKA RV 10 digital rotary evaporator, Germany), and then extracts freeze dried to completely eradicate solvents. The crude extracts were stored in glass containers and kept in the freezer at -20°C until further analyses.
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Figure 3.6: Schematic diagram of Soxhlet extraction apparatus
3.2.3.2 Isolation and purification of natural products
Isolation and purification of the active compounds were conducted according to the guidance of the bioassay-guided isolation protocol. Briefly, the active extracts were separated by high performance liquid chromatography (HPLC), followed by bioassay test to determine which fractions were most likely to contain the active substances. Finally, active fractions were analysed by spectrometry techniques to identify their molecular structures. All crude extracts obtained from the previous processes were separated further by (HPLC). High performance liquid chromatography is an efficient technique for the isolation and purification of natural products, due to its ability to separate complex mixtures of compounds in a short time with a high purity. In our study, the reverse phase high performance liquid chromatography (RP- HPLC) was applied. For the preparative reverse phase RP-HPLC analysis, an ACE reverse
73 HPLC work was carried out with Kinetex reverse phase column (5µm, C18, 100Å, LC Column 250 x 4.6 mm, Phenomenex, UK).
Analyses were performed using a Gilson 307 HPLC pump with Varian Prostar 320 UV- VIS HPLC Detector. Rheodyne 7725i injection valve was used to deliver the sample to the system, and was fitted with a 20 µl and 500 µl sample loop for analytical and preparative HPLC respectively. The sample concentration was 10 mg/ml for analytical and 100 mg/ml for preparative HPLC. The output signal was monitored and processed using PicoLog data acquisition software (Pico technology Ltd., Cambridge, UK). Both analytical and preparative RP-HPLC methods were optimised for each extract. Analytical HPLC provides a general indication of the compounds in the crude extracts and monitors the purity of the compounds. Whilst preparative HPLC is used to separate large amounts of crude extracts. The peaks eluted from the preparative column were collected manually.
More than one approach was employed in order to develop a mobile phase system for each plant extract, and to achieve that, isocratic solvent systems were used. The mobile phase which gave a consistent good separation, at room temperature conditions (25 ±2ºC), consisted of 15 %
(v/v) HPLC grade methanol with 0.1 % (v/v) trifluoroacetic acid (TFA) in water.Solvents were
filtered through a 0.45μm filter (Millipore, Bedford, MA, USA) and degassed in an ultrasonic bath prior to use. Samples were dissolved in the mobile phase solvent and filtered with a 0.22μm syringe filter, and then injected to the column. The flow rate was set at 1.0 ml/min for analytical column and 10 ml/min for the preparative column, and 260nm was the best wavelength that showed a good detection.
3.2.3.3 Structure elucidation of active compounds
Structure elucidation and identification is a crucial part of natural products discovery. Spectroscopic methods, such as nuclear magnetic resonance NMR (tell us about the environments of the various elements in a molecule) and mass spectrometry MS (gives the molecular weight and formula, both of the molecule itself and various structural units within it), have become indispensable tools in in this field. Development of computerised techniques has permitted utilisation of these methods for acquisition of spectra and greatly increased the capabilities of these methods (Kind & Fiehn, 2010).
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3.2.3.3.1 Nuclear magnetic resonance (NMR) spectroscopy
The measurement of 1D/2D NMR spectra was carried out at Salford Analytical Services
centre (SAS), (University of Salford, Manchester, UK). Proton (1H) and carbon (13C) NMR
spectra were recorded on Bruker Ultra-shield Avance AMX 400 MHz spectrometer. Samples
were dissolved in deuterated DMSO (DMSO-d6) for NMR measurement. Samples were filtered
through a cotton wool plugged into Pasteur pipette and then placed in Wilmad NMR tubes (5 mm diam., precision, 500 MHz, Sigma-Aldrich, UK). More sophisticated NMR tube was used
as well (Shigemi 5mm Symmetrical NMR Microtube assembly matched with DMSO-d6,
Shigemi INC., USA). Solvent peaks were used as an internal standard and all resonance assigned were relative to them. Spectra of pure compounds were processed using TopSpin software (v 3.5) under automation controlled by “iconnmr”. Structural assignment was based on
spectra resulting from The 1D 1H and 13C NMR experiments. In addition, homo and
heteronuclear 2D NMR experiments were also acquired.
3.2.3.3.2 Purity check of the active compounds
Nowadays, softwares specifically created for analysing primarily 1D quantitative NMR
spectra (1H and 13C) have emerged, in addition to analysis software provided by the respective
NMR instrument manufacturers. For the purity determination of the bio-active compounds
isolated in this study, q1H NMR was employed by using Mnova NMR software (v 10.0.2) for
windows, (Mestrelab Research, Santiago de Compostela, Spain). This software has been developed in the last few years, and it is a multivendor, multiplatform software for visualisation, processing, analysing and reporting of 1D and 2D-NMR data, it has designed to support the specific needs of researchers (Mestrelab, 2015). qNMR plugin has been used, which can interact with other Mnova plugins to offer the user access to a very advanced functionality with an accuracy, which is needed for the purity check of natural products. The qNMR process was done by using the automatic functions available in the software, (phase and baseline correction), whilst the solvent peaks were used as an internal reference. The raw data is preserved in the background to allow more detailed processing.
3.2.3.3.3 Mass Spectroscopy (MS) of the active compounds
Samples of the active small molecules were sent to the Intertek pharmaceutical services laboratories (Manchester, UK) for accurate mass analysis. Waters ZQ detector integrated with
75 Waters 2695 Alliance HPLC system (Waters Corp, Milford, MA) was used for the experiment. Prior to the injection into the HPLC system, the sample was dissolved in 50 % (v/v) acetonitrile in water, and the volume injected was 10 μl of approximately 1 mg/ml. The eluent was 9:1
ACN/water at 0.15 ml/min and 2 second scans were acquired over the range 100 to 1500 m/z in
positive and negative ion modes. Subsequently, samples were introduced into an electrospray (ES) ionization source to ionize the molecules at atmospheric pressure. The ions were separated according to their mass-to-charge ratio. The separated ions were detected and then the signals were amplified. Collected spectra sent to the Waters Empower software for further MS analysis.