Dr. Ana Najwa Mustapa defended her thesis on 2nd December 2016 at the University of Valladolid. The thesis projects was supervised by Dr Maria Jose Cocero and Dr Angel Martin while the International supervisor was Prof Irina Smirnova from University Hamburg of Technology (TUHH).
The increased public awareness to have safer and healthier therapies has led to a wide use of natural products and drugs derived from plants and, particularly, medicinal herbs. Medicinal plants are a rich source of bioactive compounds that possess significant therapeutic activity such as antioxidant, anti-viral and anti-inflammatory effects. However, the application of the herbal medicines in pharmaceutical industry still faces huge challenges, particularly in terms of the efficiency of the delivery system. Therefore, an innovative study on the enhancement of the extraction and formulation of the herbal medicine is needed.
In this thesis, the valorization of the medicinal plant Clinacanthus nutans (C. nutans) by the extraction of high-value compounds and the evaluation of their potential for pharmaceutical applications is studied. The investigation involved the extraction of phytochemical compounds, the formulation of the herbal extracts in dosage form by loading on porous aerogel matrix, and the determination of their bioavailability.
In the first part, the C. nutans plant was extracted by Soxhlet, supercritical fluid extraction (SFE) and microwave-assisted extraction (MAE) method with the aim of determining the most efficient technique for the extraction of the bioactive compounds present in the plant. The phytochemical compounds were screened to determine the major compounds obtained with each method and other significant marker compounds. Results showed that the C. nutans extract consist of phytol as the major compound, and important amounts of other high value constituents such as phytosterols (2.36 ± 0.15 mg BS/g DM obtained by direct saponification in MAE) and polyphenols. The highest total phenols and flavonoid content (i.e. 11.30 ± 0.39 mg GAE/g DM and 4.66 ± 0.22 mg QE/g DM, respectively) was obtained with MAE using 50%vol ethanol/water in comparison to other extraction methods (Figure 1). MAE was demonstrated as the most efficient method (see Figure 2) for extracting the phytochemical compounds from the C. nutans with reasonable high yield in comparison to SFE and Soxhlet method, yielding valuable compounds and, particularly, polyphenols.
In the second part, the performance of MAE in enriching polyphenols was investigated by studying its kinetic modelling and optimizing the effect of specific energy absorbed, ethanol/water concentration as the solvent and the solvent-to-feed ratio (S/F) on the polyphenols content of extracts obtained from C. nutans. The effectiveness of the MAE pre-treatment was compared to the conventional method, and it was found that the MAE technique increased the concentration of polyphenols by 2–5 times compared to the classical solvent extraction method (Figure 3). A different mechanism of heating in both methods can explain the results. Unlike the conventional method that proceeds through a conductive heating, the extraction of solutes in MAE is driven by electromagnetic waves that heat the whole sample simultaneously. As a result, localised heating occur leads to expansion and rupture of cell walls and improve the release of solutes from the material. The SEM of residual from MAE exhibits damaged cell structure (Figure 4) because of sudden rise of temperature during MAE heating followed by a drop of temperature through fast cooling technique in ice bath. This leads to a generation of internal pressure inside the cells which ultimately accelerates the release of solutes from the materials. Polyphenols yield was found to be the maximum using a 50%vol ethanol-water solvent mixture at 14 mL/g of the S/F best ratio whilst the Patricelli’s model gives excellent profiling kinetics behavior and accurate predictions of polyphenols yield and extraction rate. The microwave pre-treatment has markedly improved the extractability of polyphenols from the medicinal plant and identified as a promising approach to enrich compounds of interest in shorter time.
In the third part, the C. nutans extracts derived from the MAE by different ethanol/water concentrations were impregnated into silica and alginate aerogels. In addition, the major compound identified in the previous studies, phytol, was also employed as a model compound to be impregnated in the aerogels and its loading content and bioavailability were studied. The impregnation was carried out with two methods. In the first method, the drugs and C. nutans extracts were loaded into the gels by liquid adsorption named as Wet Impregnation (WI), between the solutions of ethanol/compounds into gels that immersed in the solution for certain time. The wet impregnated gels were then dried by CO2 under supercritical conditions. With this, supercritical CO2 (SCCO2) extract the ethanol, leaving the compounds deposited in the aerogels during the CO2 depressurization. In principle, poor solubility of the compounds in the CO2 results on a high impregnation yield. On the other hand, the drugs and C. nutans also were impregnated by method called supercritical impregnation (SCI). In this technique, the compounds were loaded into dried aerogels by the saturated solution of supercritical CO2/compounds. High diffusivity and low viscosity of the SCCO2 enabled the saturated solution to penetrate into porous structure of the aerogels. Upon depressurization, the compounds entrapped in the aerogels either by deposition or molecular dispersion mechanism. The different mechanisms governing the two methods results in different behavior of the compounds interaction with host matrix as well as their dissolution efficacy.
Results demonstrated that by impregnation via WI, the C. nutans and phytol have higher loading content in the alginate (9.6 ± 2.1 and 18.5 ± 0.5 wt% of the extracts obtained with 50% ethanol/water and pure ethanol solvents, respectively and 18.9 ± 0.8 of phytol) than in silica aerogels (5.2 ± 1.0 and 13.1 ± 0.9 wt% of the extracts obtained with 50% ethanol/water and pure ethanol solvents, respectively and 17.4 ± 0.5wt%). On the other hand, by SCI, the compounds showed higher loading in the silica (11.5 ± 0.4 and 23.9 ± 1.0 wt% for extracts obtained from 50% ethanol/water and pure (CN50) ethanol solvents (CN100), respectively and 30.1 ± 0.6 wt% of phytol) than in alginate aerogels. The differences of the loading were attributed to the higher specific surface are of the silica compared to alginate aerogels in the SCI, meanwhile the effect of the presence of ethanol in the liquid adsorption (WI) impregnation stimulate the interaction between the compounds and alginate matrix thus increased the compounds loading. This finding was evidenced from the scanning electron microscopy (SEM). The images of the impregnated aerogels are showed in Figure 5.
Both aerogels have green and yellow colors indicating the presence of C.nutans extracts in the aerogels whilst the same characteristics were observed for the exterior and interior of the impregnated alginate aerogels, indicating a homogeneous loading of the whole aerogel monolith. Furthermore, the dissolution tests (Figure 6) revealed that the C. nutans in the alginate demonstrated up to 16 times higher compounds release in 6h compared to the case of the silica aerogel, thereby suggesting that the C. nutans extract can have a good bioavailability when loaded in the alginate material. On the other hand, the phytol loaded in the alginate and silica by both impregnation methods showed poor dissolutions from the matrix. This demonstrates that this compound has extremely poor-water soluble regardless any impregnation method or host matrix used. These results motivated to the next task to develop a host matrix that can improve the solubility of the poorly soluble compounds in water.
In the last part, hybrid of β-cyclodextrin/alginate aerogels were synthesized. In this study, the synthesis was only applied to the alginate material due its simple gelation mechanism and because it was compatible with the β-cyclodextrin (βCD) characteristics. The alginate/β-cyclodextrin was prepared as aerogels beads through physical cross-linking, that is an ionotropic gelation mechanism. Two types of the hybrid aerogels i.e. core and floating beads were produced (Figure 7). In the preparation of the gels, based on the solubility data of the β-cyclodextrin in water, a saturated solution of βCD/alginate and βCD/alginate/CaCO3 mixture was prepared and extruded into two different gelation bath solutions yielding the core and floating beads. The aerogels beads were dried by SCCO2 and subsequently undergone supercritical impregnation with phytol. Results demonstrated that in the presence of βCD the loading capacity of phytol was improved from 54.4 ± 0.5 wt% to 57.3 ± 1.2 wt% in the core beads whereas in the floating beads the loading increased from 56.7 ± 1.5 wt% to 60.5 ± 0.9 wt% of phytol. The release of phytol (Figure 8) from the core and floating hybrid aerogels were significantly improved three-fold and six times higher, respectively, in comparison to the release from non-hybrid aerogels. This indicated that the developed new host matrix successfully improved the solubility of the compounds with poor aqueous solubility.
In overall, this thesis presents the research works on the valorization of the C. nutans plant as herbal drug in order to investigate its applicability in drug delivery systems for pharmaceutical industry. The findings revealed that the medicinal plant has a great potential in pharmaceuticals industry as alternative medicine drugs, promoted by the intensification of extraction provided by microwave technology, the exploitation of supercritical fluids technology, and the use of aerogels as matrix for drug delivery.
Thesis supervised by:
- Dr. Ángel Martín Martínez, Universidad de Valladolid – Spain
- Dr. Mª Jose Cocero, Universidad de Valladolid – Spain
- Prof. Irina Smirnova, University Hamburg of Technology (TUHH) – Germany
Members of the Committee:
- Dr Rafael B. Mato Chain, Universidad de Valladolid – Spain
- Dr. Ana Isabel Morales, Universidad de Salamanca – Spain
- Dr Mohd Azizi Che Yunus, University of Technology of Malaysia – Malaysia
- Dr. Pavel Gurikov, Hamburg University of Technology – Germany
- Dr. Jesus Santa Maria Ramiro, Universidad de Zaragoza – Spain