Toward a More Sustainable Sample Preparation in Phytochemistry: Case Studies in Four Subclasses of Alkaloids.

The fact that alkaloids are bases has been the most explored chemical feature of their extraction and purification procedures. The main drawback of these procedures is that they employ undesirable chemicals, with HCl and CH2Cl2 probably being the most commonly employed chemicals in their subsequent steps. This work tested the hypothesis that advantages in recovery efficiency support this common practice. Experiments were conducted in three laboratories, monitoring the alkaloids harmine (1), boldine (2), vincamine (3), and mescaline (4) extracted from Banisteriopsis caapi, Peumus boldus, Vinca minor, and Trichocereus macrogonus var. pachanoi, respectively. The research demonstrated that HCl could be replaced with citric acid (CA) without loss or even better extraction performance. The recommended EtOAc could completely replace CH2Cl2 in three out of four study cases and partially in the fourth case without harming the extraction efficiency. In addition, the alternative solvents tert-amyl methyl ether (TAME) and n-butyl acetate (BuOAc) could enhance the extraction of alkaloids. These results might incentivize natural products laboratories to consider sustainability more routinely, thus being closer to current practices in the pharmaceutical industry, which has been replacing solvents and processes with greener ones.

Soya agricultural waste as a rich source of isoflavones.

Soybeans are among the world's major crops responsible for food and biodiesel production, as well as a major source of isoflavones - a class of high value-added bioactive compounds. As estimated 460 million tonnes of soya residues (branches, leaves, roots, and pods) will be produced in the 2018/2019 harvest, and 20-40% of this waste must be removed from the field to ensure soil quality and minimize environmental impacts. This work investigated the potential occurrence and content of isoflavones in soya agricultural waste collected directly from the ground after mechanically harvesting. We also assessed the extraction performances of ethanol and acetone for these materials as an alternative to acetonitrile, a problematic solvent from an environmental point of view. Considerable amounts of isoflavones were found in soya agricultural waste collected directly from the ground when compared to soybeans (2.71 ± 0.27, 0.57 ± 0.1, 0.30 ± 0.05 and 2.09 ± 0.24 kg of isoflavones/tonne of leaves, branches, pods, and soybeans, respectively). The greener ethanol and acetone performed well for a broad range of compounds. This is an example in which appreciable amounts of high value-added compounds are wasted. Since isoflavones are considered phytoestrogens, their recovery from part of this waste might avoid potential contamination of soil and groundwater.

Natural deep eutectic solvents and aqueous solutions as an alternative extraction media for propolis.

Ethanolic extracts of propolis are consumed for their health benefits even though direct consumption of alcoholic extracts is not always ideal. Natural Deep Eutectic Solvents (NADES) can potentially extract similar compounds as alcoholic extracts while being better for direct consumption. Therefore, in this work alternative solvents for the extraction of green propolis including its biomarker artepillin C were examined. Sixteen NADES made from low toxicity chemicals, including the essential amino acid l-lysine, were explored along with twelve individual NADES components and honey, which showed similar physical-chemical properties to NADES. At 50 °C NADES made from choline chloride-propylene glycol or lactic acid proved to be equal or better than the benchmark EtOH:Water 7:3 (v/v). Alternatively, aqueous l-lysine appeared as a potential solvent for the preparation of aqueous propolis extracts. From these findings NADES, honey and aqueous l-lysine solutions all demonstrated the potential to replace ethanol or water for extracting green propolis.

Coupled monolithic columns as an alternative for the use of viscous ethanol-water mobile phases on chromatographic fingerprinting complex samples

ABSTRACT Concepts of sustainability have received attention from people involved in investigation of nature-derived matrices. The effects of concomitant pollutant activities are cumulative and harmful to the environment from which these matrices are obtained. High performance liquid chromatography analyses generate millions of litters of chemical waste worldwide every year. Reduction of organic solvent consumption during the analyses and replacement of harmful solvents with greener options are the main approaches to mitigate this problem. This work explored the strategy of employing monolithic columns when the problematic acetonitrile is intended to be replaced with the greener but more viscous ethanol in fingerprinting a leaf extract of Lippia sidoides Cham., Verbenaceae, by high performance liquid chromatography. Two monolithic columns were coupled in series to test a more critical backpressure condition while doubling the number of theoretical plates, which can be useful to separate the hundreds of compounds present in plant extracts. All work was conducted by employing design of experiments. A mathematical model indicated an optimum point in which ethanol was the only organic solvent of the mobile phase. However, the use of a proper metric, which considered environmental parameters together with separation parameters, evidenced that an experimental condition of the original central composite design should be preferred over the former even if containing 20% acetonitrile in the organic modifier mixture. Flow rates of up to 3 ml/min were accommodated with two coupled monolithic columns without exceeding 250 bar. These findings reinforced that no state-of-the-art instruments are needed to shift from traditional harmful solvents to greener ones, but only require a shift in researchers' approach toward sustainability.

Cluster analysis of commercial samples of Bauhinia spp. using HPLC-UV/PDA and MCR-ALS/PCA without peak alignment procedure.

INTRODUCTION: Bauhinia forficata Link. is recognised by the Brazilian Health Ministry as a treatment of hypoglycemia and diabetes. Analytical methods are useful to assess the plant identity due the similarities found in plants from Bauhinia spp. HPLC-UV/PDA in combination with chemometric tools is an alternative widely used and suitable for authentication of plant material, however, the shifts of retention times for similar compounds in different samples is a problem. OBJECTIVE: To perform comparisons between the authentic medicinal plant (Bauhinia forficata Link.) and samples commercially available in drugstores claiming to be "Bauhinia spp. to treat diabetes" and to evaluate the performance of multivariate curve resolution - alternating least squares (MCR-ALS) associated to principal component analysis (PCA) when compared to pure PCA. METHODOLOGY: HPLC-UV/PDA data obtained from extracts of leaves were evaluated employing a combination of MCR-ALS and PCA, which allowed the use of the full chromatographic and spectrometric information without the need of peak alignment procedures. RESULTS: The use of MCR-ALS/PCA showed better results than the conventional PCA using only one wavelength. Only two of nine commercial samples presented characteristics similar to the authentic Bauhinia forficata spp., considering the full HPLC-UV/PDA data. CONCLUSION: The combination of MCR-ALS and PCA is very useful when applied to a group of samples where a general alignment procedure could not be applied due to the different chromatographic profiles. This work also demonstrates the need of more strict control from the health authorities regarding herbal products available on the market.

Green chromatographic fingerprinting: an environmentally friendly approach for the development of separation methods for fingerprinting complex matrices.

A chromatographic fingerprint is a comprehensive method that reveals the distinctive pattern of peaks across the chromatogram for a given sample. It is considered an effective strategy to assess the identity and quality of herbal materials, as well as for the control of the quality of their derived products. HPLC is the most employed technique for these purposes and it is used routinely for quality control in industry. Hence, its impact on the environment should not be neglected. This work provides a rational and generic procedure to qualitatively fingerprint complex matrices. Resource- and time-saving experimental designs were selected; an alternative safer organic solvent was tested and a time-saving and innovative response entitled the green chromatographic fingerprinting response was developed and employed. This procedure was applied in the development of chromatographic fingerprints for extracts of Bauhinia forficata and Casearia sylvestris. Moreover, the response proposed here can be combined with a complementary metric available in the literature to compare methods using different solvents. According to this, the chromatographic fingerprints developed here using ethanol as the organic solvent provided a performance better than that of reference methods in which more harmful acetonitrile or methanol were employed.

Chemical and antifungal investigations of six Lippia species (Verbenaceae) from Brazil.

The Lippia genus is used in ethnobotany as food, beverages, seasoning and antiseptic remedies, among others. The chemical compositions of fifteen extracts of six Lippia species were investigated comparatively by HPLC-PDA. To avoid data replication of previous works on this genus, Lippia lupulina Cham. root ethanol extract was selected for isolation procedures based on Principal Component Analyses (PCA) of such data. Seven compounds previously unreported in this genus were isolated from this extract (a triterpene, two furanonaphtoquinones, a furanochromone, an isoflavone, a stilbene and an iridoid). The activities of extracts, fractions and pure compounds towards Candida albicans, Candida krusei, Candida parapsilosis and Cryptococcus neoformans were investigated. Two fractions from the extract of Lippia salviaefolia leaves showed marked inhibition of fungal growth, in addition to verbascoside and asebogenin, which showed MICs lower than 15.6µg/ml and may be promising leads for the development of new antifungal agents, especially against C. neoformans.

Interconverting flavanone glucosides and other phenolic compounds in Lippia salviaefolia Cham. ethanol extracts.

Four interconverting flavanone glycosides [(2R)- and (2S)-3',4',5,6-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside, and (2R)- and (2S)-3',4',5,8-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside], in addition to eight known flavonoids [naringenin, asebogenin, sakuranetin, 6-hydroxyluteolin 7-O-ß-D-glucoside, (2R)- and (2S)-eriodictyol 7-O-ß-D-glucopyranoside, aromadendrin and phloretin], three phenylpropanoid glycosides [forsythoside B, alyssonoside and verbascoside] and the epoxylignan lariciresinol 4'-O-ß-D-glucopyranoside were isolated and identified in the EtOH extract of the aerial parts of Lippia salviaefolia Cham. The phytochemical study herein was guided by preliminary antioxidant tests, namely, ß-carotene protection and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity. The crude extracts, their active fractions and the isolated compounds were assayed against intracellular reactive oxygen species (ROS) and human embryonic kidney HEK-293 and human melanoma M14 cancer cell growth. Aromadendrin and phloretin were able to counteract elevation of ROS induced by the oxidant t-butylhydroperoxide (t-BOOH) in HEK-293 cells, whereas phloretin strongly protected HEK-293 cells from ROS damage at 1 µM. Additionally, phloretin exhibited a significant growth inhibitory effect at 20-40 µM in both HEK-293 and M14 cells and induced a concentration dependent apoptosis at 20 µM in M14 cells, suggesting a selective action towards malignant cells. Due to their equilibria, the four interconverting flavanone glycosides were studied using 1D and 2D NMR, HPLC-CD-PDA and HRMS analyses.

Analysis of propolis from Baccharis dracunculifolia DC. (Compositae) and its effects on mouse fibroblasts.

This paper confirms Baccharis dracunculifolia DC. (Compositae) as the main botanical source of the propolis from southeastern Brazil (state of São Paulo) investigated to ascertain specific biological activity in relation to mouse NIH-3T3 fibroblasts, skin cells directly involved in the cicatrization processes. Flavonoid and total phenolic compounds were determined by spectrophotometry, and chemical composition by HPLC; the chromatographic profile, characterized largely by flavonoids and aromatic acids, was found to be qualitatively similar to that of Baccharis dracunculifolia DC. The adsorption of phenolic compounds in the propolis to skin powder was also investigated, and 68% of these compounds adsorbed to the skin powder. At concentrations from 0.12 to 7.81 microg/ml, the propolis revealed no statistical significant differences from its control solutions; however, at concentrations of 31.25 microg/ml or more, the propolis was toxic to NIH-3T3 cells. Thus, the propolis from Baccharis dracunculifolia DC. (Compositae) presents an in vitro concentration-dependent toxicity on mouse NIH-3T3 fibroblasts.