TDDFT calculations and photoacoustic spectroscopy experiments used to identify phenolic acid functional biomolecules in Brazilian tropical fruits in natura

Highlights

• TDDFT/ADZP UV–Vis spectra of eight phenolic acids

• Photoacoustic spectroscopy UV–Vis spectra of four Brazilian tropical fruits in natura

• Identification of phenolic acids in the Brazilian tropical fruits

Abstract

Time-dependent density functional theory (TDDFT) calculations of electronic transitions have been widely used to determine molecular structures. The excitation wavelengths and oscillator strengths obtained with the hybrid exchange-correlation functional B3LYP in conjunction with the ADZP basis set are employed to simulate the UV–Vis spectra of eight phenolic acids. Experimental and theoretical UV–Vis spectra reported previously in the literature are compared with our results. The fast, sensitive and non-destructive technique of photoacoustic spectroscopy (PAS) is used to determine the UV–Vis spectra of four Brazilian tropical fresh fruits in natura. Then, the PAS along with the TDDFT results are for the first time used to investigate and identify the presence of phenolic acids in the fruits studied in this work. This theoretical method with this experimental technique show to be a powerful and cheap tool to detect the existence of phenolic acids in fruits, vegetables, cereals, and grains. Comparison with high performance liquid chromatography results, when available, is also carried out.

Introduction

Polyphenols constitute one of the most important families of organic compounds of the plant kingdom and have been of increasing interest due to their roles in plant reproduction and protection. In fact, polyphenols constitute one of the most well distributed groups of bioactive molecules in plant kingdom. They are secondary metabolites synthetized by plants and the whole polyphenols family is composed by > 8000 compounds [1]. In addition, polyphenol compounds commonly located at the cell epidermis of the higher plants work as filter for the ultraviolet radiation protecting the plants against damage effects [2].

Bioactive molecules, such as those belonging to the groups of polyphenols, usually have as host a large variety of fruits, vegetables and cereals. They exhibit several pharmaceutical, medicinal, and biochemical properties that result in several protective effects not only in plants but also in animals. Furthermore, it is well established that most of these bioactive compounds exhibit antioxidant activities and hence can provide natural protection against oxidation of biomolecules such as lipid membranes, proteins, and DNA [3]. As it is well known, antioxidant is any molecular or ionic substance that delays or prevents the oxidation of substrates, mainly when the antioxidant agent are present at low concentration relative to that of the substrate [4]. In other words, it could be said that antioxidant agents inhibit cellular damage by quenching free radicals. The antioxidant activity in terms of radical scavenging and hydrogen-donating is considered the main characteristic of the phenolic compounds object of this study, the phenolic acids.

Free radicals have been reported to strongly affect human health by causing several kinds of disease such as heart diseases, cancer, hypertension, diabetes, atherosclerosis, Parkinson's disease, and Alzheimer's disease [5]. Intensive investigations have shown the relevance of antioxidants in the prevention of various diseases. For instance, it has been reported that plant phenolics, rich in antioxidant compounds, protect the brain against neurodegenerative perturbations caused by ischemia [6].

The main as well as the more investigated polyphenol group of biomolecules are the flavonoids, which have a common fifteen carbon skeleton consisting of two aromatic rings A and B (see Fig. 1) bound together by a oxygenated heterocycle C and arranged in the C₆–C₃–C₆ configuration [7], [8], [9].

As far as polyphenols are concerned, the flavonoid family provides the basic mechanisms for antioxidant actions, especially the free radical scavenging and metal ion chelators [10].

Phenolic acids are another important family of compounds belonging to the polyphenol group. Like flavonoids, they are found in most plant foods such as fruits, vegetables, cereals, legumes, nuts and in some beverages. The phenolic acids are also secondary plant metabolites widely spread throughout the plant kingdom [11]. They are monophenols since possess only one aromatic ring and a carboxylic functional group (COOH) attached to it as shown in Fig. 2. They exhibit two distinct structures: the benzoic acid (chemical formula C₆H₅COOH), that is characterized by one carboxylic group COOH, Fig. 2a, and the cinnamic acid (chemical formula C₆H₅CHCHCOOH) that is characterized by two carbon frameworks, Fig. 2b [8], [9].

Phenolic acid derivatives from these two basic acids are commonly obtained from the substitution of one or more hydroxyl groups [9]. The most common benzoic acid derivatives are p-hydroxybenzoic, gallic, protocatechuic, and vanillic, while the most common cinnamic acid derivatives are p-hydroxycinnamic, p-coumaric, caffeic, and ferulic.

Recent investigations have shown that diets based on plant foods rich in antioxidant compounds like flavonoids and phenolic acids, lead to an effective reduction of the risk of many diseases such as those previously indicated [4], caused by the action of free radicals [12]. For instance, very recent researches have found that almost all phenolic acids exhibit considerable antioxidant potential, free radical scavenging, neuroprotective activities, anti-cancer activities, platelet aggregation inhibitors, low-density lipoprotein, oxidation inhibitors among many other functional activities [11]. Therefore, there is little doubt that the health benefits associated with diets based on fruits, vegetables, cereals, grains and nuts can be attributed to the antioxidant activities of the polyphenol compounds including phenolic acids [13].

Photoacoustic spectroscopy (PAS) has been object of increasing interest due to the fact that instead of directly detect the photons emitted by the sample, as it occurs in the conventional ultraviolet-visible (UV–Vis) spectroscopy, in PAS technique the absorbing power is determined by the transformation of the modulated or pulsed radiation energy in sound wave [14]. In that way, photoacoustic signal is independent of scattering effects that is known to broad absorption bands as is the case of conventional spectroscopy. Unlike UV–Vis spectroscopy, PAS is an excellent technique for investigating opaque systems such as organic compounds. For instance, one of us used PAS to investigate the presence of carotenoid and flavonoid functional molecules in some Brazilian tropical fruits and vegetables [15]. Another important area of biological sciences where PAS technique has been largely applied is that of human systems like skin tissue, blood and in living plants. As illustrative examples, a successful mid-infrared pulsed photoacoustic spectroscopy experiment was carried out to monitoring glucose concentration in human epidermis in vivo [16]. In present days, prospects of photoacoustic tomography are carried out [17]. In condensed matter physics, PAS has been used to investigate, for example, phase-transitions in solids [18] and electron-phonon interaction in semiconductors [19]. PAS was also used to investigate band gap energy in semiconductors [20]. In another very interesting application, PAS technique along with density functional theory (DFT) were used to investigate both energy gap and defect states in nitrogen doped TiO_2 − x N films [21].

Nowadays, the time-dependent DFT (TDDFT) [22], [23] becomes the most used approach on transition energy and excited state structure and property calculations. The reason for its growing popularity is because it gives accurate results at a low computational cost. The precision of the results depends on the exchange-correlation functional used. Nonetheless, the TDDFT has been successfully applied on UV–Vis [24], [25], [26], [27], optical rotation [28], [29], [30], [31], [32], and electronic circular dichroism [33], [34], [35], [36] calculations of various molecules.

The main objective of this article is to introduce the fast, sensitive and non-destructive technique of PAS along with the TDDFT to investigate and identify the presence of phenolic acids in several fresh fruits in natura. Most of them are considered as functional foods and commonly found in the Brazilian tropical region, namely: açai (Euterpe oleracea Mart), cupuaçu (Theobroma grandiflorum), Brazil nut (Bertholletia excelsa), and persimmon (Diospyros kaki L.). Furthermore, it was our intention to show that PAS technique can contribute to select and classify fruits and vegetables according with their nutritive, medicinal or phototherapeutic properties. As far as we know, it should be mentioned here that it is the first time that the TDDFT in conjunction with the PAS are employed in this kind of study on biological compounds.