Selected latest applications of molecular spectroscopy in natural product analysis

The field of molecular vibrational spectroscopy applied to natural products is advancing extremely fast. Traditionally applied separation techniques (LC-, μ-LC–MS, GC, CE-MS) offer the advantages of high selectivity/sensitivity, but their application for routine quality control is limited due to long analyses times. Therefore, molecular spectroscopy in combination with multivariate analysis (MVA) enjoys excellent reputation, because of the fast and non-invasive measurement enabling the analysis of several physical and chemical parameters simultaneously. Near infrared (NIR; 4.000–10.000 cm⁻¹), attenuated total reflection (ATR; 400–4.000 cm⁻¹), Raman and far ultraviolet (FUV;120–200 nm) spectroscopy have permanently increased their efficiencies for quality control of predominantly food stuff, but also of other natural products including mainly medicinal plants. All four techniques enable not only a quantitative analysis of potent ingredients, but also qualitative fingerprint analysis for the discrimination of, e.g., species and/or geographic origin, respectively. Thereby, each individual spectroscopic technique possesses its specific strength. Powerful miniaturized portable spectrometers based on linear variable tuneable filter (LVTF) or micro-electro-mechanical systems (MEMS) are helpful in order to prevent consumers from deception on one hand, on the other hand they represent powerful analytical instruments for measurements in the field. 2-Dimensional correlation spectroscopy (2DCOS) represents a powerful technique for monitoring the dynamics of a system including temperature stability, extraction procedures etc. Imaging and mapping spectroscopy using infrared radiation and/or Raman scattering are not only suitable for classification of food stuff including e.g. maize kernels and/or coffee beans, but also for localizing the distribution of ingredients down to a resolution of 4 μm.In the present contribution, the latest progresses of the different techniques are introduced and their applicability in the fields of natural product analysis will be discussed in detail by distinct selected applications.     

Novel cancer-targeting SPECT/NIRF dual-modality imaging probe 99mTc-PC-1007: Synthesis and biological evaluation

Synthesis, characterization, in vitro and in vivo biological evaluation of a heptamethine cyanine based dual-mode single-photon emission computed tomography (SPECT)/near infrared fluorescence (NIRF) imaging probe ^99mTc-PC-1007 is described. ^99mTc-PC-1007 exhibited preferential accumulation in human breast cancer MCF-7 cells. Cancer-specific SPECT/CT and NIRF imaging of ^99mTc-PC-1007 was performed in a breast cancer xenograft model. The probe uptake ratio of tumor to control (spinal cord) was calculated to be 4.02 ± 0.56 at 6 h post injection (pi) and 8.50 ± 1.41 at 20 h pi (P < 0.0001). Pharmacokinetic parameters such as blood clearance and organ distribution were assessed.     

Properties of antibacterial bioboard from bamboo macromolecule by hot press

Bamboo macromolecules were pretreated with bamboo vinegar, which has an antibacterial property, and processed into antibacterial bioboard (ABB) by hot pressing. The ABB was then analyzed by conducting Fourier-transform infrared spectroscopy, thermogravimetric analysis and differential thermal analysis. Results showed that ABB samples had average density of 1.0 g/cm³, which is appropriate for application. The physical and mechanical properties were best for the ABB sample pretreated with bamboo vinegar and hot pressed at 165 °C for 10 min. Fourier-transform infrared spectroscopy revealed that the optimum conditions for hot pressing were a temperature of 165 °C, duration of 10 min, and the addition of bamboo vinegar. Thermogravimetric analysis/differential thermal analysis curves indicated that the thermal degradation of the ABB was less than that of bamboo, revealing that hot pressing increased the thermal stability of ABB samples. Analysis revealed that pretreatment with bamboo vinegar improved the antibacterial property of the ABB.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.     

Profiling the chemical content of Opuntia ficus-indica flowers by HPLC–PDA-ESI-MS and GC/EIMS analyses

The qualitative and quantitative analysis of flavonoids from Opuntia ficus-indica flowers methanol extract from the Mediterranean area is described. On the basis of HPLC–PDA-ESI-MS/MS analysis seven compounds have been identified as kaempferol, quercetin, and isorhamnetin glycosylated derivatives. The total amount of flavonoids of O. ficus-indica flowers was 81.75 mg/1 g of fresh plant material, with isorhamnetin 3-O-robinobioside being the major component (52.22%). The plant flowers volatiles composition was also characterized and a total of 18 components were identified. The main constituents were found to be germacrene D (12.6%), 1-hexanol (12.3%), n-tetradecane (9.1%) and decanal (8.2%).     

Diterpenoids and triterpenoids with potential anti-inflammatory activity from the leaves of Aglaia odorata

Chemical investigation of the leaves of the oriental medicinal plant Aglaia odorata resulted in the isolation of five compounds: two dolabellane diterpenoids, two dammarane triterpenoids and a protostane triterpenoid, along with twenty known compounds. Their structures were elucidated on the basis of extensive spectroscopic analysis and by comparison of their NMR spectroscopic data with those reported in the literature. The anti-inflammatory activities of all compounds were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Eleven compounds possessed potent nitric oxide inhibitory activity with IC₅₀ values ranging from 2.1 to 14.2 μM, these being better than that of the positive control, indomethacin (IC₅₀ = 14.5 μM). In addition, three compounds exhibited significant activity against PGE₂ release with IC₅₀ values of 2.6, 16.1 and 23.0 μM.     

Rapid determination of tacrine and other drug metabolites in microsomal incubate by newly developed targeting algorithm on UHPLC/TOFMS

A rapid simultaneous determination method for in vitro Cytochrome P450 (CYP) activity assay of 1,2,3,4-tetrahydroacridin-9-amine (tacrine) metabolites using ultra high performance liquid chromatography (UHPLC) coupled with computer-assisted in-source collision induced dissociation (CID) monitoring was investigated. In general, enzyme inhibition assays require quantitative analysis of incubates with drugs using various concentrations of substrates/inhibitors. The assay of CYP isozyme inhibition is an important informational step in the drug discovery process and, with the many substrates listed by the FDA, high-throughput qualitative and quantitative analyses are desired. Based on sub-2-micron packing material with reversed phase chromatography combined with a single liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), a less than 1 min analysis time is presented for two additional drugs. We successfully determined seven of the eight potential isomer metabolites for the drug tacrine in 2.5 min using a 2 mm internal diameter × 100 mm length column and applying in-source CID with our newly developed chromatographic peak deconvolution technique. Although two of the peaks were heavily fused at a peak width of less than 300 ms, we could clearly identify these peaks by monitoring the chromatographic intensity difference of their fragment peaks on the mass spectrum.     

Bioactive 2-pyrone metabolites from an endophytic Phomopsis asparagi SWUKJ5.2020 of Kadsura angustifolia

Five new 2-pyrone metabolites, phomaspyrones A–E (1–5) along with three known compounds, macommelin-8,9-diol (6), macommelin-9-ol (7), and macommelin (8), were isolated from the culture broth of an endophytic fungus Phomopsis asparagi SWUKJ5.2020 of medicinal plant Kadsura angustifolia by sequentially purification over silica gel, Sephadex LH-20 column chromatography and preparative HPLC. Their structures were elucidated on the basis of spectroscopic analysis, including two-dimensional (2D) NMR and HRMS (ESI-TOF) data. Among them, compounds 3–5 exhibited notable cytotoxicity against HL-60 and K562 cells with IC₅₀ values of below 10 μg/mL better than those of the other isolated metabolites (IC₅₀ = 10.6–26.6 μg/mL). The results showed that the keto or hemiketal functionality might play an important role in cytotoxic activity of this class of compounds.     

Green synthesis of silver nanoparticles using Andean blackberry fruit extract

Green synthesis of nanoparticles using various plant materials opens a new scope for the phytochemist and discourages the use of toxic chemicals. In this article, we report an eco-friendly and low-cost method for the synthesis of silver nanoparticles (AgNPs) using Andean blackberry fruit extracts as both a reducing and capping agent. The green synthesized AgNPs were characterized by various analytical instruments like UV–visible, transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The formation of AgNPs was analyzed by UV–vis spectroscopy at λ_max = 435 nm. TEM analysis of AgNPs showed the formation of a crystalline, spherical shape and 12–50 nm size, whereas XRD peaks at 38.04°, 44.06°, 64.34° and 77.17° confirmed the crystalline nature of AgNPs. FTIR analysis was done to identify the functional groups responsible for the synthesis of the AgNPs. Furthermore, it was found that the AgNPs showed good antioxidant efficacy (>78%, 0.1 mM) against 1,1-diphenyl-2-picrylhydrazyl. The process of synthesis is environmentally compatible and the synthesized AgNPs could be a promising candidate for many biomedical applications.     

Synthesis and QSAR study of novel α-methylene-γ-butyrolactone derivatives as antifungal agents

Thirty-six new α-benzylidene-γ-lactone compounds based α-methylene-γ-butyrolactone substructure were prepared and characterized by spectroscopic analysis. All compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi and the half maximal inhibitory concentration (IC₅₀) against Botrytis cinerea and Colletotrichum lagenarium were investigated. Compounds 5c-3 and 5c-5 with the halogen atom exhibited excellent fungicidal activity against B. cinerea (IC₅₀ = 22.91, 18.89 μM). The structure-activity relationships (SARs) analysis indicated that the derivatives with electron-withdrawing substituents at the meta- or para-positions improves the activity. Via the heuristic method, the generated quantitative structure-activity relationship (QSAR) model (R² = 0.961) revealed a strong correlation of antifungal activity against B. cinerea with molecular structures of these compounds. Meanwhile, the cytotoxicity of 20 representative derivatives was tested in the human tumor cells line (HepG2) and the hepatic L02 cells line, the result indicated that the synthesized compounds showed significant inhibitory activity and limited selectivity. Compound 5c-5 has the highest fungicidal activity with IC₅₀ = 18.89 μM (against B. cinerea.) but low cytotoxicity with IC₅₀ = 35.4 μM (against HepG2 cell line) and IC₅₀ = 68.8 μM (against Hepatic L02 cell line). These encouraging results can be providing an alternative, promising use of α-benzylidene-γ-lactone through the design and exploration of eco-friendly fungicides with low toxicity and high efficiency.     

Lemairones A and B: Two new antibacterial tetraflavonoids from the leaves of Zanthoxylum lemairei (Rutaceae)

Zanthoxylum lemairei is widely used in African folk medicine for its pharmacological relevance. Chemical investigation of the ethanol extract from the leaves of this plant lead to the isolation of two new tetraflavonoids, lemairones A (1) and B (2), along with three known compounds, lupeol, sitosterol, and sitosterol 3-O-β-d-glucopyranoside. The antibacterial screening of the leaves of this plant, characterization of compounds 1 and 2, and their antibacterial activity are reported for the first time. The isolation of the compounds was performed using different chromatographic methods while their structures were elucidated by spectroscopic techniques including MS and NMR, and by comparison of data with those of similar flavonoids reported in the literature. The isolated compounds and the crude extract were tested against ten Gram negative multi-resistance bacterial strains including clinical isolates using a broth dilution method. The crude ethanol extract showed weak activity against the tested bacteria strains with a minimal inhibitory concentration (MIC) ranging from 512 to 1024 μg/mL. Among the isolated metabolites, only the new tetraflavonoids were tested. Lemairone A displayed weak activity while lemairone B had moderate activity against the resistant Escherichia coli AG100 with MIC values of 128 μg/mL and 64 μg/mL respectively. In addition, both molecules displayed weak activity against Klebsiella pneumoniae KP55 (MIC 128 μg/mL).     

Effect of dioxane on the structure and hydration–dehydration of α-chymotrypsin as measured by FTIR spectroscopy

A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 °C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic α-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities (a_w < 0.5) during hydration. At a_w about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At a_w < 0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low a_w. At a_w > 0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given a_w. This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm^?1 that was assigned to the intermolecular β-sheet aggregation. Changes in the intensity of the 1628 cm^?1 band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of α-chymotrypsin depend markedly on how enzyme has been hydrated — whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity-a_w profiles.     

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.     

Labdane diterpenoids from Marrubium vulgare

Phytochemical investigation of the whole plant of Marrubium vulgare L. (Lamiaceae) resulted in isolation and characterization of two new labdane diterpenoids, 12(S)-hydroxymarrubiin and 3-deoxo-15-methoxyvelutine C and 11 known compounds, marrubiin, peregrinin, thessaline D, marrubinone B, deacetylvitexilactone, verbascoside, leucosceptoside A, martynoside, anisofolin A, terniflorin, and apigenin. Structure elucidation of the isolated compounds was determined by using spectroscopic techniques and HRESIMS. 12(S)-Hydroxymarrubiin, marrubiin, peregrinin, and marrubinone B, exhibited no antimicrobial activity up to 20 μg/mL against ten strains.     

Bioactivity-guided isolation of cytotoxic constituents from stem–bark of Premna tomentosa

A bioassay-guided fractionation and chemical investigation of the stem bark of Premna tomentosa resulted in the isolation and characterization of four new icetexane diterpenes (1–4), along with the known compounds coniferaldehyde (5), syringaldehyde (6), lupeol (7), betulin (8), and 2-(4-methoxyphenyl)-2-butanone (9). Their structures were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by comparison with the spectroscopic data reported in the literature. The new compounds exhibited diverse functionalities on a common icetexane diterpene skeleton. In addition, cytotoxic activities of the icetexanes (1–3) were evaluated by determining their inhibitory effects on the human cancer cell lines (MCF-7, HT-29, Hep-G2, A-431, and A-549). Compounds 1 and 3 showed selective inhibitory activity against MCF-7 (15.96 μg/mL and 15.84 μg/mL) and HT-29 cell lines (16.21 μg/mL and 14.57 μg/mL), respectively.     

Synthesis,characterization and cytotoxic activities of the [RuCl2(NO)(dppp)(L)]PF6 complexes

The synthesis and characterization of ruthenium compounds of the type [RuCl₂(NO)(dppp)(L)]PF₆ [dppp = 1,3-bis(diphenylphosphino)propane; L = pyridine, 4-methylpyridine, 4-phenylpyridine and dimethyl sulfoxide] are described. The complexes were characterized by elemental analysis, UV/Vis and infrared spectroscopy, cyclic voltammetry, and X-ray crystallography for the complexes with the pyridine and 4-methylpyridine ligands. In vitro evaluation of these nitrosyl complexes revealed cytotoxic activity from 7.1 to 19.0 μM against the MDA-MB-231 breast tumor cells and showed that, in this case, they are more active than the reference metallodrug cisplatin. The 1,3-bis(diphenylphosphino)propane and the N-heterocyclic ligands alone failed to show cytotoxic activities at the concentrations tested (maximum concentration utilized = 200 μM).     

Analysis of aminoglycoside residues in bovine milk by liquid chromatography electrospray ion trap mass spectrometry after derivatization with phenyl isocyanate

A derivatization procedure using phenyl isocyanate was adapted to liquid chromatography ion trap mass spectrometry (LC–MSⁿ) for confirmation and quantification of aminoglycoside residues in milk. Aminoglycoside residues were extracted from milk with acid and isolated from the matrix with a weak cation exchange solid-phase extraction cartridge. After isolating the compounds from the milk, derivatives of gentamicin, neomycin, and tobramycin were formed by reacting the drugs with phenyl isocyanate in the presence of triethylamine. The analytes were separated using a dilute formic acid/acetonitrile gradient on a reversed-phase LC column. The derivatized compounds were analyzed using positive ion electrospray LC–MSⁿ with ion trap detection. Product ion spectra were generated from the derivatized protonated molecules. Specific ion transitions were evaluated for quantitative determination and qualitative confirmation of residues in milk. Using this procedure, residues were qualitatively confirmed in milk samples fortified with gentamicin and neomycin at levels ranging from 15 to 300 ng mL^?1. Gentamicin has four major components that were successfully separated and confirmed independently; for quantitative determination the peak areas from the four analogs were summed. Tobramycin was added as an internal standard for quantitation to mitigate the effects of matrix ion suppression and variable recoveries. Overall recoveries for this method ranged from 80% to 120% with relative standard deviations of less than 25%. The method detection limits are 9.8 ng mL^?1 for NEO and 12.8 ng mL^?1 for total GEN residues.     

A new benzil derivative from Derris scandens: Structure-insecticidal activity study

Bioactivity-directed investigation of root extract of Derris scandens has led to the isolation and characterization of a new benzil derivative (11), along with ten known compounds (1–10). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. The insect antifeedant activity and growth inhibitory studies of these compounds were investigated against castor semilooper pest, Achaea janata using a no-choice laboratory bioassay. Several of the isolates displayed potent feeding deterrence and were also toxic or caused developmental abnormalities following topical administration. The new compound, derrisdione A was moderately active with an antifeedant index of 58.6 ± 1.7% at 10 μg/cm³ against A. janata.     

Two new rotenoids from the roots of Millettia speciosa

Two new rotenoids, named millettiaosas A–B (1–2), together with four known compounds were isolated from the roots of Millettia speciosa. Their structures were elucidated on the basis of spectroscopic analysis including 1D and 2D NMR techniques and HRESIMS. Evaluation of the two new compounds for cytotoxicity against four human cancer cell lines (MCF-7, HCT-116, A549 and HepG-2) showed moderate activities (10 μM < IC₅₀ < 26 μM).