**The Benefits of Plant Extracts for Human Health**

Editor

**Charalampos Proestos**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Charalampos Proestos National and Kapodistrian University of Athens Greece

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### **Contents**

### **About the Editor**

**Charalampos Proestos** (Associate Professor in Food Chemistry) Charalampos Proestos has a BSc in Chemistry, University of Ioannina, Greece, and an MSc in Food Science at Reading University, U.K. He obtained his Ph.D. in Food Chemistry at the Agricultural University of Athens (AUA), Greece, where he continued his post-doc working on natural antioxidants on programs funded by the EU and Greece. After further training at Wageningen University (the Netherlands), he worked as a Research Associate at AUA. He worked as a Chemist for the Hellenic Food Authority (EFET as a food industry auditor and supervisor of the Chemical Laboratory in Athens accredited with ISO 17025. He is currently Associate Professor at the Department of Chemistry, National and Kapodistrian University of Athens, and director of the laboratory of Food Chemistry. He has published more than 70 papers in reputed journals and has been serving as an editorial board member of more than 10 reputed journals. He is Member of the European Committee of the Division of Food Chemistry, European Association of Chemical and Molecular Sciences (EuChemS). His research field focuses on food antioxidants, plant bioactive compounds, foodomics, and food contaminants.

### *Editorial* **The Benefits of Plant Extracts for Human Health**

#### **Charalampos Proestos**

Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece<EMAIL_ADDRESS>Tel.: +30-210-727-4160

Received: 5 November 2020; Accepted: 10 November 2020; Published: 12 November 2020

Nature has always been, and still is, a source of foods and ingredients that are beneficial to human health. Nowadays, plant extracts are increasingly becoming important additives in the food industry due to their content in bioactive compounds such as polyphenols [1] and carotenoids [2], which have antimicrobial and antioxidant activity, especially against low-density lipoprotein (LDL) and deoxyribonucleic acid (DNA) oxidative changes [3]. The aforementioned compounds also delay the development of off-flavors and improve the shelf life and color stability of food products. Due to their natural origin, they are excellent candidates to replace synthetic compounds, which are generally considered to have toxicological and carcinogenic effects. The efficient extraction of these compounds from their natural sources and the determination of their activity in commercialized products have been great challenges for researchers and food chain contributors to develop products with positive effects on human health. The objective of this Special Issue is to highlight the existing evidence regarding the various potential benefits of the consumption of plant extracts and plant extract-based products, along with essential oils that are derived from plants also and emphasize in vivo works and epidemiological studies, application of plant extracts to improve shelf-life, the nutritional and health-related properties of foods, and the extraction techniques that can be used to obtain bioactive compounds from plant extracts.

In this context, Concha-Meyer et al. [4] studied the bioactive compounds of tomato pomace obtained by ultrasound assisted extraction. In this review, it was presented that the functional extract obtained by ultrasounds had antithrombotic properties, such as platelet anti-aggregant activity compared with commercial cardioprotective products. Turrini et al. [5] introduced bud-derivatives from eight different plant species as a new category of botanicals containing polyphenols and studied how different extraction processes can affect their composition. Woody vine plants from Kadsura spp. belonging to the Schisandraceae family produce edible red fruits that are rich in nutrients and antioxidant compounds such as flavonoids. Extracts from these plants had antioxidant properties and had shown also key enzyme inhibitions [6]. Hence, fruit parts other than the edible mesocarp could be utilized for future food applications using Kadsura spp. rather than these being wasted. Saji et al. [7] studied the possible use of rice bran, a by-product generated during the rice milling process, normally used in animal feed or discarded due to its rancidity, for its phenolic content. It was proved that rice bran phenolic extracts via their metal chelating properties and free radical scavenging activity, target pathways of oxidative stress and inflammation resulting in the alleviation of vascular inflammatory mediators. Villedieu-Percheron et al. [8] evaluated three natural diterpenes compounds extracted and isolated from *Andrographis paniculata* medicinal herb as possible inhibitors of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) transcriptional activity of pure analogues. Yeon et al. [9] evaluated the antioxidant activity, the angiotensin I-converting enzyme (ACE) inhibition effect, and the α-amylase and α-glucosidase inhibition activities of hot pepper water extracts both before and after their fermentation. These water extracts were proved to have potentially inhibitory effects against both hyperglycemia and hypertension. The hydrolyzed extracts of Ziziphus jujube fruit, commonly called jujube, were examined for their protective effect against lung inflammation in mice [10]. They contained significant amounts of flavonoids which inhibited cytokine release from

macrophages and promoted antioxidant defenses in vivo. Tran at al. [11] examined the antidiabetic activity of spray-dried Euphorbia hirta L. herb extracts containing high concentrations of bioactive compounds such as phenolics and flavonoids. Li et al. [12] reported that intestinal microbiota is closely associated with the initiation and progression of diabetes mellitus and reviewed bioactive components which exhibited anti-diabetic activity by modulating these intestinal microbiotas. Essential oils have promising activity against antibiotic-resistant bacteria and chemotherapeutic-resistant tumors. This was supported by the study of Viktorová et al. [13] where lemongrass essential oil and especially citral, the dominant component, proved to have potential antimicrobial and anticancer activity. Additionally, Mitropoulou et al. [14] investigated the antimicrobial potential of *Sideritis raeseri subps. raeseri* essential oil against common food spoilage and pathogenic microorganisms and evaluated its antioxidant and antiproliferative activity. Salehi et al. [15] reviewed the Berberis plants, which contain alkaloids, tannins, phenolic compounds and essential oils, and their possible use in the food and pharmaceutical industry. Last but not least, Kiokias et al. [16] reviewed the naturally occurring phenolic acids from plants and their antioxidant activities in o/w emulsions and in vitro lipid-based model systems.

Still more research is needed to explore more and in depth the health beneficial effects of plant extracts, since nature certainly has more to give to humans.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

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#### *Article*

### **Platelet Anti-Aggregant Activity and Bioactive Compounds of Ultrasound-Assisted Extracts from Whole and Seedless Tomato Pomace**

#### **Anibal Concha-Meyer 1,2, Iván Palomo 3,\*, Andrea Plaza 2, Adriana Gadioli Tarone 4, Mário Roberto Maróstica Junior 4, Sonia G. Sáyago-Ayerdi 5 and Eduardo Fuentes 3,\***

Received: 23 September 2020; Accepted: 21 October 2020; Published: 28 October 2020

**Abstract:** Tomato paste production generates a residue known as tomato pomace, which corresponds to peels and seeds separated during tomato processing. Currently, there is an opportunity to use tomato pomace to obtain a functional extract with antithrombotic properties, such as platelet anti-aggregant activity. The aim of this study was to evaluate the yield and inhibitory activity of different extracts of tomato pomace on in vitro platelet aggregation, comparing this activity with commercial cardioprotective products, and quantify bioactive compounds. Aqueous or ethanolic/water (1:1) extracts of whole tomato pomace, seedless tomato pomace, tomato pomace supplemented with seeds (50% and 20%), and only seeds were obtained with different ultrasound-assisted extraction times. The inhibition of platelet aggregation was evaluated using a lumi-aggregometer. The quantification of bioactive compounds was determined by HPLC-MS. From 5 g of each type of tomato pomace sample, 0.023–0.22 g of a dry extract was obtained for the platelet aggregation assay. The time of sonication and extraction solvent had a significant role in platelet anti-aggregant activity of some extracts respect the control. Thus, the most active extracts decreased adenosine diphosphate (ADP)-induced platelet aggregation from 87 ± 6% (control) to values between 26 ± 6% and 34 ± 2% (*p* < 0.05). Furthermore, different ultrasound-assisted extraction conditions of tomato pomace fractions had varied concentration of flavonoids and nucleosides, and had an effect on extract yield.

**Keywords:** tomato pomace; extraction; platelet; ultrasound; functional ingredient

#### **1. Introduction**

Cardiovascular disease (CVD) is the number one cause of death worldwide since more people die annually (≈17 million) from CVD than from any other cause [1]. In Chile, CVD causes 27% of deaths [2]. In this context, the adherence to a diet rich in fruit and vegetables was associated with a decrease of all-cause mortality among individuals with CVD [3–5]. Furthermore, tomato (*Lycopersicon esculentum*) has been one of the most powerful vegetables associated with the reduction of this type of disease [6–10]. Emerging epidemiological and interventional data support the connection between higher tomato consumption and a lower risk of CVD. Additionally, tomato has the lowest uric acid content of any fruit and vegetable [11,12]. The latter is important because a high uric acid level in the body can cause various health issues like arthritis [13].

Tomato is an important vegetable grown in many countries across the world for fresh markets and multiple processed forms. The worldwide production of processed tomato in 2017 was 38 million tons [14]. During the season 2017–2018, Chile produced 918,000 metric tons of industrial tomato and the Maule region represented about 66% of this total [15]. This crop is industrially processed to produce concentrated tomato paste, which is used to formulate products such as ketchup, sauce, puree, and juice. This processing generates a byproduct named tomato pomace, correspondingmostly to peels and seeds that account for 3–5% (wet basis (w.b.)) of fresh tomato [16]. Currently, the tomato pomace has different uses, such as a protein supplement for growing lambs [17], ruminant feeding [18], chemical and nutritional supplementation of crackers [19], and sustainable fabrication of packaging films [20]. Furthermore, if the latter is not possible, this residue is disposed of on agricultural lands causing environmental contamination [21,22]. The results of the proximate composition of tomato pomace, on a dry weight basis, showed a relevant nutritional content of total protein (19.40%, N × 6.25), total fat (12.33%), available carbohydrates (17.15%), total dietary fiber (47.80%), ash (3.32%), sodium (13.20 mg/100 g), and sugars (9.54%) [23]. Meanwhile, tomato seed oil contains high levels of linoleic (54%) and oleic (22%) acids [24]. On the other hand, the main polysaccharides identified in tomato pomace correspond to glucose (40.5 ± 1.2%) and fructose (22.6 ± 2.1%) from total carbohydrates of 382.9 ± 10.3 mg/g dry weight [25]. Tomato pomace is also known as a source of bioactive compounds, such as carotenoids, phenolic compounds, and dietary fiber [26]. Considering the above, there is an opportunity to use tomato pomace to obtain a functional product with antithrombotic properties, such as platelet anti-aggregant activity, that could be useful as an ingredient in healthy foods for CVD prevention [5].

Different extraction procedures have been evaluated to increase the efficiency of tomato pomace compound extraction for pectin, dietary fiber, and carotenoids such as lycopene, including varied solvent extraction by mixing and heating, microwave-assisted extraction, enzymatic extraction, ultrasound-assisted extraction, and ultrasound-assisted treatment combined with subcritical water [27–30]. Ultrasound is a non-thermal technology that has shown to be particularly effective for improving the extraction of heat-labile compounds [31]. Ultrasound equipment is also commonly available in most analytic laboratories and is used to improve the efficiency of any extraction solvent, thus reducing the extraction times [32]. Furthermore, there is a need to improve the extraction of bioactive compounds, such as flavonoids and nucleosides (adenosine and guanosine), that are present in tomato (*Solanum lycopersicum)* and tomato pomace, which have significant platelet anti-aggregant activity [5,6,33,34]. This work aimed to evaluate the chemical profile and platelet anti-aggregant activity of ultrasound-assisted extracts of different tomato pomace fractions obtained using water or ethanol/water (1:1) as the solvent.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

The solvents used were ethanol (Sigma-Aldrich, St. Louis, MO, USA) and type I ultrapure water (18.2 MΩ-cm), which was supplied by the Purelab Classic Elga water system (Labwater/VWS Ltd., London, UK). Adenosine diphosphate (ADP) was used as an agonist in the platelet aggregation assay (Chrono-log, Havertown, PA, USA).

#### *2.2. Plant Material*

Tomato pomace (mainly peels and seeds), which is a byproduct of the industrial production of tomato paste, was obtained from the company Sugal Chile (Talca, Chile) during the 2016 season. Given the production line of the plant, it was not possible to define specific fruit hybrids that corresponded to the tomato pomace; and it was only possible to identify middle (Sun6366, AB3, and HMX7883) and late (H9665, H7709, and H9997) hybrid tomatoes [35]. Tomato pomace was placed

in trays and dried for 48 h at 60 ◦C in a convection oven (VHC-1A, Ventus Corp., Santiago, Chile); after this, a portion of tomato pomace (22% of seeds) was manually separated into seeds and peels for the preparation of the different extracts; and finally, all parts from tomato pomace were milled and sieved through a 425 μm mesh (No. 40) (TRF 300, TRAPP, Jaraguá do Sul, Brazil).

#### *2.3. Preparation of Extracts*

Aqueous or ethanolic/water (1:1) extracts of whole tomato pomace, seedless tomato pomace, tomato pomace supplemented with seeds (50% and 20%), and only seeds were obtained. Briefly, 5 g of each fraction was mixed with 50 mL of water for aqueous extract, and with 25 mL of water and 25 mL of ethanol for ethanolic extracts. An ultrasonic bath of 2.8 L, 220 V, amplitude 100%, and power 90 W (VWR 97043-962, Leicestershire, UK), was used with different sonication time cycles were applied as the extraction process: cycle 1 with one cycle of 20 min, cycle 3 with three cycles of 20 min each with a 10-min pause in between, and cycle 6 with six cycles of 20 min each with a 10-min pause in between. All cycles were controlled each 1 min at a frequency of 35 kHz and a temperature of 45 ◦C. Then, the extracts were centrifuged at 725× *g* (International Equipment Company IEC, Centra MP4R, Boston, MA, USA) for 5 min to obtain the supernatant, which was frozen at −86 ◦C for 48 h, then freeze-dried (Operon, FDU 7024, Gimpo, Korea) for 20 h with a cold trap (−70 ◦C), and stored at 20 ◦C in vacuum airtight packaging in a dark environment. Freeze-dried extracts were resuspended in physiological saline solution at a concentration of 1 mg/mL and filtered (pore filter 0.22 μm) for analysis and platelet aggregation assay.

#### *2.4. Platelet Aggregation Assay*

The inhibition of platelet aggregation was evaluated in a lumi-aggregometer (Chrono-Log) [5]. Thus, 480 μL of plasma rich in human platelets (2 × 105 platelets/μL) was added to the reaction cuvette that was pre-incubated with 20 μL of extract (all extracts at a concentration of 1 mg/mL) or control for maximum platelet aggregation (0.9% saline). After 5 min of incubation, 20 μL of agonist (ADP, 4 μM) was added to initiate platelet aggregation, which was measured for 6 min. The results were expressed as a percentage of platelet aggregation. Platelets were obtained from 20 different donors and were used during a period of 2 h after blood extraction. All volunteers signed informed consent. The maximum platelet aggregation of the controls (without extracts) was 87 ± 6%. This study was conducted following the Declaration of Helsinki and informed consent was obtained for experimentation with human subjects.

#### *2.5. Identification and Quantification of Bioactive Compounds by HPLC-MS*

#### 2.5.1. Phenolic Compounds

Phenolic compounds were analyzed following the method of Torres et al., [36] with modifications. Briefly, 350 mg samples of each freeze-dried extract were weighed, ground, and mixed with 5 mL of 75% methanol and 100 μL of internal standard (naringenin, Sigma-Aldrich). Samples were centrifuged at 11,180× *g* at 10 ◦C for 10 min (International Equipment Company IEC, Centra MP4R, Boston, MA, USA). The supernatant was removed and diluted with 20 mL HPLC-grade water (Merck, Darmstadt, Germany). Simultaneously, extraction column C18 (No. 12102052, Agilent Technologies, Palo Alto, CA, USA) was activated with 1 mL of 75% methanol. Briefly, 5 mL HPLC-grade water was added to the C18 column and allowed to drain until dry to remove solvent residues. Samples were eluted using a vacuum pump (Welch 2545C/02, Mount Prospect, IL, USA) and a vacuum trap (20–40 kPa). Then, samples inside C18 were allowed to elute until completely drained using 2 mL of pure propanol to remove concentrated sample-specific phenolic compounds and collected in 4 mL test tubes. N2 gas was used to dry samples that were reconstituted in 200 μL of pure methanol, mixed, and sonicated (Bandelin Sonorex TK52H, Berlin, Germany) for 1 min. Samples were analyzed using the ultra-high performance liquid chromatography (UHPLC) Dionex UltiMate 3000 chromatography system (Thermo Scientific,

Waltham, MA, USA) equipped with a refrigerated autosampler. The samples were injected into the Hypersil Gold C18 column (Thermo Fisher Scientific, Bremen, Germany) using 1 μL of samples and a gradient sample with 75% (*v*/*v*) acetonitrile, 24.5% (*v*/*v*) water, and 0.5% (*v*/*v*) formic acid (solution A) and 5% (*v*/*v*) acetonitrile, 94.5% (*v*/*v*) water, and 0.5% (*v*/*v*) formic acid (solution B) with a flow rate of 300 μL/min. Gradient elution conditions were set as follows: initial 0–1 min (10% B), 1–5 min (10% B), 5–10 min (30% B), 10–18 min (100% B), and 18–24 min (0% B), with final cleaning and reconditioning of the column. Mass quantification was carried out with an Exactive Plus Orbitrap spectrometer (Thermo Scientific) equipped with an electrospray interface operating with negative ionization mode, and data were processed using the Xcalibur 2.1 software (Thermo Scientific). Mass spectrometry conditions were 2500 spray volts, vaporization temperature of 350 ◦C, sheath gas pressure of 40 arbitrary units (a.u.), auxiliary gas pressure of 10 a.u., and capillary temperature of 35 ◦C. N2 was used as the collision gas and all values were normalized to 350 mg/dry weight. Phenolic compounds were identified and quantified using suitable standards (Extrasynthese, Lyon, France), which were prepared as 1 mg/mL stock solutions in methanol and stored at −80 ◦C for up to 1 month in dark conditions. All results were expressed in mg/100 g dry weight.

#### 2.5.2. Carotenoids Compounds

Fuentes et al. (2013) procedures were followed with modifications [37]. Briefly, 0.71 mL of 2:1 solution (pure acetone and 0.2 M HEPES buffer (pH 7.7)) was mixed with 500 mg of the freeze-dried extract sample and then agitated and centrifuged at 11,180× *g* for 5 min. The supernatant was separated; and the pellet was mixed with 0.71 mL of 2:1 solution (pure acetone and 0.2 M HEPES buffer (pH 7.7)), then stirred, and centrifuged to obtain the supernatant, which was then deposited together with the previous one. After that, 1 mL of pure acetone was added again to the pellet and centrifuged, the supernatant was deposited in the same tube, 1 mL of pure acetone was added to the pellet and it was stirred and centrifuged, the supernatant was deposited in the same tube, and 0.75 mL of pure hexane was added to the pellet, which was stirred and centrifuged. The supernatant was transferred to the same tube and 0.75 mL of hexane was added and centrifuged. The sample was recovered and evaporated with N2 gas; and the obtained sample was reconstituted, microfiltered, and stored in vials. UHPLC-MS was used for quantification with lycopene and β-carotene standards. Results were expressed in mg/100 g dry weight.

#### 2.5.3. Nucleosides Compounds

The method used was performed considering Dudley and Bond recommendations [38]. Briefly, 1 g of the freeze-dried extract was mixed with 20 mL of ultra-pure water and then homogenized in a mortar. The extract was sonicated with ultrasound (VWR 97043-962, Leicestershire, UK) at 35 kHz for 30 min, then centrifuged at 11,180× *g* for 10 min at 10 ◦C, and filtered at 0.22 μL with a microfilter disc (Millex-GN PTFE, Merck Millipore, Darmstadt, Germany). Quantification was performed by UHPLC-MS. An ESI injector with a positive charge at 35 kV, a capillary temperature of 350 ◦C, a flow of 250 μL were used. Mobile phase A was a solution of 10 mM ammonium acetate with 0.8% acetic acid and mobile phase B was a solution of acetonitrile with 0.1% acetic acid. A gradient of 10% at the first 6 min, then 6 min at 50% A, and then return in 6 min at 10% A was used. For quantification, adenosine, guanosine, and inosine standards were used in a curve of 0.05–1.5 μg/μL. Results were expressed in μg/100 g dry weight.

#### **3. Competitive Study**

The platelet anti-aggregant activity was compared with commercial cardioprotective products sold in the Chilean market (M1: CardioSmile, Nutrartis S.A., Providencia, Chile; M2: UltraPure Omega 3, Unicaps, Brea. CA, USA; M3: Eykosacol, Procaps S.A., Barranquilla, Colombia; M4: Benexia, Functional Products Trading S.A., Vitacura, Chile; and M5: Maqui Berry, Nativ for Life, Santiago, Chile). Liquid products (M2, M3, and M4) were evaluated directly on platelet aggregation at 1 mg/mL, while the solid product (M5) was dissolved in physiological serum in a final study concentration of 1 mg/mL. For the M1 product (given its milky consistency that affects the turbidity of the plasma in the platelet aggregation test), an aqueous extract was obtained. Thus, the product was mixed with water in a 1/8 ratio and centrifuged at 11,180× *g* for 5 min to obtain the supernatant that was freeze-dried and kept at −20 ◦C until use. Before the platelet aggregation assay, the aqueous extract of product M1 was resuspended in physiological saline solution at a concentration of 1 mg/mL.

#### **4. Statistical Analysis**

Data were expressed as mean ± standard deviations and analyzed by the Prism 6.0 software (GraphPad Inc., La Jolla, CA, USA). All measurements were made from six different donors. Before performing the statistical analysis, it was necessary to know if the results met with a normal distribution or not. Thus, using a significance level of 5% and according to Kolmogorov statistic with a *p*-value of 0.003, the results of platelet aggregation showed a non-normal distribution. The results of percentage of platelet aggregation were analyzed using non-parametric Kruskal–Wallis test, and subsequently analyzed by Dunn's test, used as a post-test, to establish significant differences between each extract with respect to control (*p*-value < 0.05).

#### **5. Results and Discussion**

#### *5.1. Extraction Yield*

Table 1 shows the extraction yield of different types of tomato pomace extracts. From 5 g of each type of tomato pomace sample, 0.023–0.22 g of a dry extract was obtained. Freeze drying allowed obtaining extracts (e.g., AWTPE3) that are considered microbiologically safe [5,23]. The process of ultrasound extraction produces a phenomenon called cavitation which generates physical, chemical, and mechanical effects responsible for the cellular wall disruption of the vegetal matrix [39,40]. According to many authors (Al-Dhabi et al., 2017; Chemat et al., 2017; Contamine et al., 1995; Delgado-Povedano and de Castro, 2017; Mason et al., 1996; Rastogi, 2011), the association of different ultrasound extraction conditions (e.g., power, temperature, time, and solvent) may change the polarity and viscosity of the system, as well as the interaction between the solute and solvent. Thus, in this study, aqueous extracts (2.8 ± 1.1% m/m) showed higher extraction yields in comparison with ethanolic extractions (1.0 ± 0.5% m/m), and this was due to the polarity and viscosity of these solvents. According to Chemat et al. (2017) and other studies, variations in viscosity, although small, may induce resistance to ultrasound waves and it may affect the extraction efficiency of the solvent system [39,41,42].

The cavitation phenomena also cause a temperature rise with the extraction time, increasing the extraction yield of phenolic and nucleosides compounds by their higher solubility and diffusion. However, when a higher extraction time exposes these compounds at high temperatures for a long time, it may promote its degradation by oxidation mechanisms, consequently decreasing the extraction yield [41–43]. Therefore, in this study, cycle 3 showed promising potential to obtain high extraction yields, since AWTPE3 preliminarily presented 3.45% (m/m). This could be explained by the higher value of soluble solids observed for AWTPE3 (6.00 ◦Brix) compared with whole ground dried tomato pomace (1.67 ◦Brix, performed in 1 g of sample powder dissolved in 10 mL ultrapure water). The latter can be explained due to mechanical effects of ultrasound that triggered the release of water-soluble compounds such as polysaccharides, polyphenols, and nucleosides from their matrices by disrupting them from cellular tissues [5,6,23,33,34,44,45]. Ultrasound is also an effective method to improve fractioning of water-soluble compounds from tomato pomace fiber, since the latter was separated as a precipitate after centrifugation [5,23].

**Table 1.** Extraction yield and platelet anti-aggregant activity of different types of tomato pomace extracts.

Platelet aggregation data were expressed as mean ± standard deviations (SD). Statistically significant difference in platelet aggregation (%) is considered with \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001, analyzed with Dunn's test with respect to control. ND: not determined. Not determined (ND) extracts correspond to extracts that were generated, but obtained at a very low yield, preventing them to be weighed and evaluated. Cycle 1: 20 min, cycle 3: three cycles of 20 min each; and cycle 6: six cycles of 20 min each.

#### *5.2. Platelet Anti-Aggregant Activity*

We observed that the non-ultrasound-assisted aqueous extraction of whole tomato pomace affected the platelet anti-aggregant activity of the extract (64 ± 11% vs. control 87 ± 6%, not significant). The time of sonication and solvent used in the extraction had a significant role in platelet anti-aggregant activity [46]. As is shown in the Table 1, the extracts with significant antiplatelet activity, with respect to control (87 ± 6%), were AWTPE3 (32 ± 9%, *p* < 0.01), EWTPEC3 (32 ± 9%, *p* < 0.01), ASTPEC6 (26 ± 6%, *p* < 0.001), ESTPEC6 (29 ± 7%, *p* < 0.01), ASEC1 (29 ± 8%, *p* < 0.01), ASEC3 (34 ± 2%, *p* < 0.05), AE5TPSC1 (27 ± 12%, *p* < 0.001), and EE5TPSC1 (29 ± 12%, *p* < 0.01). These levels of inhibition of platelet aggregation induced by ADP were different, depending on ultrasound cycle times, tomato pomace composition, and solvent used. Thus, the sonication cycles only improved the platelet anti-aggregant activity in the seedless extracts. Six cycles of sonication of 20 min each significantly improved the platelet anti-aggregant activity of aqueous (ASTPEC6 26 ± 6%, *p* < 0.001) and ethanolic (ESTPEC6, 29 ± 7%, *p* < 0.01) seedless tomato pomace extracts. Tomato seeds are composed with proteins (35.5%), fiber (13.2%), fatty acids (30%), ash (3%), and also essential amino acids (except tryptophan), so they could be used in several applications, for example, as an enrichment for cereal products and foods low in lysine [22,47].

For aqueous seed extracts and aqueous 50% tomato pomace/50% seed extracts, the best activity was achieved using one ultrasound cycle (ASEC1, 29 ± 8%, *p* < 0.01 and AE5TPSC1, 27 ± 12%, *p* < 0.001), while antiplatelet activity decreased with an increase to six sonication cycles (ASEC6, 40 ± 7% and AE5TPSC6, 54 ± 9%, not significant). On the other hand, three-cycle sonication allowed the best significant activity of the extracts obtained from aqueous whole tomato pomace (AWTPE3, 32 ± 9%, *p* < 0.01) and ethanolic whole tomato pomace (EWTPEC3, 32 ± 9%, *p* < 0.05). Although, in a previous study, the ultrasound extraction time used by the authors was 5 min [10], in this study ultrasound cycle was increased to 20 min to achieve a better extraction of compounds responsible for platelet anti-aggregant activity. Furthermore, 5 min of ultrasound extraction achieved an aggregation of 55% in aqueous tomato pomace extract [10], while the 20-min cycle improved the activity of the aqueous tomato pomace extract by lowering maximum aggregation to 46 ± 12% (AWTPE1).

Based on the results of yield and platelet anti-aggregant activity, it seemed that the most suitable extract was AWTPE3 (yield, 3.45% and platelet aggregation, 32 ± 9%, *p* < 0.01). Meanwhile, ASTPEC6 (platelet aggregation, 26 ± 6%, *p* < 0.001) had a poor yield of 0.80% and showed no statistically significant differences in platelet anti-aggregant activity when compared to AWTPE3.

#### *5.3. Identification of Bioactive Compounds*

Table 2 shows the amounts of bioactive compounds by HPLC-MS on aqueous extracts (whole tomato pomace, seed, and seedless) obtained from the same original matrix. Since tomato pomace is mainly composed of peels and seeds, we compared the bioactive compounds of AWTPE3 (aqueous whole tomato pomace), previously reported [5], with two other aqueous extracts obtained with the same sonication cycles—ASTPEC3 (aqueous seedless tomato pomace) and ASEC3 (aqueous seeds).

\* Data reported in Palomo et al., (2019) [5]. *p* < 0.001 indicates concentrations close to the limit of detection. AWTPE3: aqueous whole tomato pomace extract cycle 3, ASEC3: aqueous seed extract cycle 3, and ASTPEC3: aqueous seedless tomato pomace extract cycle 3. \*\* Twenty-one flavonoid molecules were analyzed and sixteen molecules were detected by chromatography. Three nucleoside molecules were analyzed and detected by chromatography. Two carotenoid molecules were analyzed and detected by chromatography.

Several studies have observed that ultrasound is effective in assisting the extraction of bioactive compounds such as nucleosides, polyphenols, and carotenoids from different plant byproducts, including tomato pomace [5,27,48,49]. In comparison with unprocessed tomatoes and on a dry weight basis, tomato pomace contained significantly lower amounts of lycopene and increased amounts of β-carotene, tocopherols, sterols, terpenes, and flavonoids (e.g., naringenin) [50]. Thus, in this study higher concentrations of flavonoids (procyanidin B2, kaempferol-3-*O*-glucoside, and kaempferol) were observed for AWTPE3 in comparison with whole dried ground tomato pomace [5]. These results demonstrated that the ultrasound-assisted extraction and concentration using freeze drying resulted in a greater concentration of flavonoids in AWTPE3 [5]. A similar result was obtained by Navarro-González et al., where the best extraction of phenolic compounds (e.g., rutin, naringenin, and chlorogenic acid derivatives) was with ultrasonic assistance [51]. This concentration of compounds (e.g., kaempferol-3-*O*-glucoside) in AWTPE3 may be due to the fact that tomato pomace is composed of pulp residues, in addition to peels and seeds [52].

ASEC3 and ASTPEC3 had different levels of polyphenols because they were lost simply by removing their peels or seeds. Thus, the removal of the peels represented a loss of lycopene (80%), β-carotene (57%), and phenolic compounds (63%). Meanwhile, the elimination of seeds had a greater impact on polyphenols with a loss of 63% [53]. ASEC3 showed higher concentrations of flavonoid compounds than ASTPEC3, except for quercetin, which was detected in tomato peels in higher concentrations than seeds, according to literature [44]. In this context, it has been described that seed aqueous lyophilized extract from tomato contains 20,657 mg/100 g phenolic compounds, with the highest amounts of quercetin-3-*O*-sophoroside, kaempferol-3-*O*-sophoroside, and isorhamnetin-3-*O*-sophoroside [54]. However, ASEC3 did not present concentrations of kaempferol and quercetin, suggesting differences due to an extraction procedure effect. ASTPEC3 did not show concentrations of coumaric acid, apigenin-7-*O*-glucoside, kaempferol-3-*O*-glucoside, genistein, kaempferol, daidzein, quercitrin, and epicatechin, suggesting that these compounds were not effectively recovered from tomato peels by ultrasound-assisted extraction (Table 2). Chlorogenic acid, which is usually predominant in tomato byproducts, was not detected or quantified by chromatography in any of the samples. The latter could be explained due to low stability of this molecule during storage of dehydrated tomato byproducts [55].

The data available in the literature suggest a potential therapeutic effect of several flavonoids against CVD through the inhibition of platelet aggregation, such as coumaric acid [56], phloretin [57], procyanidin B2 [58], kaempferol-3-*O*- glucoside [59], and epicatechin [60]. The presence of these compounds in greater amounts in AWTPE3 and ASEC3 than in ASTPEC3 correlated with a better capacity to inhibit platelet aggregation. Several mechanisms of antiplatelet action of polyphenols have been reported, including inhibition of the arachidonic acid pathway, suppression of cytoplasmic increase in Ca2+, degradation blockage, αIIbβ3 integrin-mediated signaling, inhibition of secretion of platelet granules, and inhibition of thromboxane formation [61,62]. The ultrasound was able to increase the extraction of adenosine in AWTPE3 when compared with whole dried ground tomato pomace from 6.32 μg/100 g to 42.90 μg/100 g, almost 6 times more [5]. However, the extraction of inosine and guanosine was not affected by ultrasound. The results obtained from the quantification of nucleosides (adenosine, inosine, and guanosine) demonstrated that AWTPE3 presented greater amounts of these compounds compared with ASEC3 and ASTPEC3 [5]. Authors involved in this present work have previously observed that adenosine is concentrated in higher amounts in the aqueous extract from ripe tomato pulp than whole tomato pomace and peels extracts [9]. The presence of inosine in significant concentrations in ASEC3, but not in ASTPEC3, suggests that this compound is available only in tomato seeds [6,63]. In part, this could explain why ASEC3 had better platelet anti-aggregant activity than ASTPEC3, since inosine has been reported to inhibit platelet aggregation, significantly preventing in vivo thrombus formation [34]. Both extracts (ASEC3 and ASTPEC3) presented close concentrations of guanosine and adenosine, the latter had no significant amount detected. The present research group isolated and identified adenosine and guanosine as bioactive compounds in tomato with a potent platelet anti-aggregant activity [9,33,34].

The results showed that ultrasound was not efficient to extract carotenoids in the conditions established. Lycopene was solely quantified in ASTPEC3. Lycopene is found mainly in the peels and acts as a red pigment and, although resistant to thermal processing, it is very susceptible to light degradation [64]. To determine this compound, a prior extraction process must be carried out, which requires conditions of absence of light to avoid degradation, which makes its quantification very complicated and delicate. In the case of β-carotene, a higher concentration was found in whole dried ground tomato pomace powder in comparison with the other samples. According to literature, most non-polar solvents have higher extraction yield of carotenoids than aqueous extracts, since they are lipophilic compounds that are soluble in organic solvents, such as acetone, alcohol, ethyl ether, chloroform, and ethyl acetate. Therefore, large concentrations of carotenoids in the aqueous extract of tomato pomace were not expected.

#### *5.4. Competitive Study*

In the current market, there are cardioprotective products like phytosterols, polyphenols, and omega-3 [65]. Platelet anti-aggregant activity of commercial products was compared with that of AWTPE3 (Figure 1). In the presence of M2 (platelet aggregation, 57 ± 5%), M3 (platelet aggregation, 56 ± 5%), and M4 (platelet aggregation, 54 ± 7%), platelets showed a significant decrease in aggregation compared with the control of maximum platelet aggregation (79 ± 8%). Products M1 and M5 did not show inhibition of platelet aggregation. AWTPE3 presented a significant difference compared with the control and the commercial products M2, M3, and M4.

**Figure 1.** Platelet anti-aggregant activity of commercial products. Different letters indicate a significant difference with a *p* value<0.05. AWTPE: aqueous whole tomato pomace extract cycle 3; M1: CardioSmile, Nutrartis S.A., Chile; M2: UltraPure Omega 3, Unicaps CA, USA; M3: Eykosacol, Laboratorio Procaps S.A., Colombia; M4: Benexia, Functional Products Trading S.A., Chile; and M5: Maqui Berry, Nativ for Life, Chile.

#### **6. Conclusions**

Currently, tomato pomace is considered as waste by the food industry; however, in this study, we showed that ultrasound-assisted extracts from this byproduct had significant platelet anti-aggregant activity. The levels of inhibition of platelet aggregation were different, depending on ultrasound cycle times, tomato pomace composition (whole, seedless, and seed), the solvent used, and the flavonoid and nucleoside content (Figure 2). In this context, the ultrasound-assisted extraction improved extraction yields, showing significant recovery of bioactive compounds from tomato pomace and increased platelet aggregation inhibition.

**Figure 2.** Platelet anti-aggregant activity and bioactive compounds of ultrasound-assisted extracts from whole and seedless tomato pomace.

**Author Contributions:** All authors have read and agreed to the published version of the manuscript. A.C.-M.: Conceptualization, Investigation and Methodology. I.P.: Investigation and Writing—Original draft preparation. A.P.: Writing—Original draft preparation. A.G.T.: Writing—Reviewing and Editing. M.R.M.J.: Writing—Reviewing and Editing. S.G.S.-A.: Writing—Reviewing and Editing. E.F.: Data curation and Writing—Original draft preparation.

**Funding:** This research was funded by Conicyt Regional/Gore Maule/CEAP (R0912001) and Conicyt Regional (R15F10012).

**Acknowledgments:** This work was supported by Proyecto Enlace Fondecyt (Dirección de Investigación/Vicerrectoría Académica/Universidad de Talca). E.F. thanks Conicyt/Fondecyt (Grant No. 1180427) and Conicyt/Redes (Grant No. 170003). I.P. thanks Redes (Grant No. 170002). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) (Finance Code 001), CNPq (403328/2016-0; 301108/2016-1), and FAPESP (2015/50333-1; 2018/11069-5; 2015/13320-9)". E.F., I.P., S.G.S.-A., and M.R.M.J. acknowledges Red Iberomericana de Alimentos Autoctonos Subutilizados (ALSUB-CYTED, 118RT0543). A.G.T. thanks CNPq (140942/2016-5) for the Ph.D. scholarship.

**Conflicts of Interest:** The authors have no conflicts of interest to disclose.

#### **References**

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### **Bud-Derivatives, a Novel Source of Polyphenols and How Di**ff**erent Extraction Processes A**ff**ect Their Composition**

**Federica Turrini 1,\*, Dario Donno 2, Gabriele Loris Beccaro 2, Anna Pittaluga 1, Massimo Grilli 1, Paola Zunin 1 and Ra**ff**aella Boggia 1**

Received: 10 September 2020; Accepted: 20 September 2020; Published: 23 September 2020

**Abstract:** The use of herbal food supplements, as a concentrate form of vegetable extracts, increased so much over the past years to count them among the relevant sources of dietetic polyphenols. Bud-derivatives are a category of botanicals perceived as a "new entry" in this sector since they are still poorly studied. Due to the lack of a manufacturing process specification, very different products can be found on the market in terms of their polyphenolic profile depending on the experimental conditions of manufacturing. In this research two different manufacturing processes, using two different protocols, and eight species (*Carpinus betulus* L., *Cornus mas* L., *Ficus carica* L., *Fraxinus excelsior* L., *Larix decidua* Mill., *Pinus montana* Mill., *Quercus petraea* (Matt.) Liebl., *Tilia tomentosa* Moench), commonly used to produce bud-derivatives, have been considered as a case study. An untargeted spectroscopic fingerprint of the extracts, coupled to chemometrics, provide to be a useful tool to identify these botanicals. The targeted phytochemical fingerprint by HPLC provided a screening of the main bud-derivatives polyphenolic classes highlighting a high variability depending on both method and protocol used. Nevertheless, ultrasonic extraction proved to be less sensitive to the different extraction protocols than conventional maceration regarding the extract polyphenolic profile.

**Keywords:** bud-derivatives; *botanicals*; polyphenols; UV-Visible spectroscopic fingerprint; chemometrics; targeted chromatographic fingerprint

#### **1. Introduction**

In recent decades, food supplements have an important impact on the consumers showing a significant expectation for their health and well-being [1]. They are concentrated sources of nutrients or bioactive compounds endowed with nutritional or physiological effects and, due to their presumed health benefits, they can supplement the common diet [2,3].

In particular, the interest in herbal food supplements (botanicals) is exponentially grown and consequently the relative market has increased in all the world [4]. Botanicals are become among the most popular into the food supplements category, due to the general belief which "natural" is better, healthier and safer than synthetic drugs, although this is not always true [4]. In Italy, more than 20% of the Italian population is considered "regular" consumer of these herbal products, as highlighted from the recent European PlantLibra (Plant Food Supplements: Levels of Intake, Benefit and Risk Assessment) consumer survey [5]. The wide range of herbal food supplements on the market and the non-attendance of effective legislation to guarantee the safety and quality aspects make these products vulnerable for fraud, falsification and adulteration [6,7].

Bud-derivatives (BDs) are a relatively new category of herbal food supplements and they represent one of the supply chains investigated in the FINNOVER project (Innovative strategies for the development of cross-border green supply chains), an European Interreg Alcotra Italy/France project (2017–2020) whose aim is the green innovation of several agro-industrial chains [8]. BDs are conventionally produced, according to the European Pharmacopoeia VIII edition [9], by cold maceration of the fresh meristematic tissues of trees and herbaceous plants (i.e., buds and young sprouts) using as extraction solvent mixtures of water, ethanol and glycerol [10,11]. These natural products are already marketed, and a long history of use as dietary supplements for human well-being and health is reported in traditional medicine. No health claims are yet approved by the European Food Safety Authority (EFSA) and just for some of these botanicals pharmacognostic findings supported their use as adjuvants in several diseases. In fact, some in-vitro/in-vivo biological studies for human and veterinary use have been already reported in the literature. For examples, Allio et al. (2015) investigated whether *Tilia tomentosa* bud extracts affect hippocampal Gamma-aminobutyric acid (GABA) ergic synapses [12]. In other studies, bud extracts from *Salix caprea* L. have been demonstrated to inhibit voltage gated calcium channels and catecholamines secretion in mouse chromaffin cells [13]. Moreover, different patents have also been registered on the veterinary use of bud-extracts (e.g., Composition of *Salix caprea* bud-extract and its use in the treatment of animal endometritis, patent n. TO2015A000193) [14] and several studies are carried out on several bud-derivative biological effects [15–17].

Although gemmotherapy has been used since ancient times because of the peculiar content of buds in bio-active compounds, especially polyphenols, nowadays BDs are still a little studied "niche" production [18,19]. The lack of detailed scientific information and a clear and unique regulation, as well as for the category of herbal food supplements in overall [6,7,20], it makes these products high risk and there is an increase request for efficient quality control to ensure the proper identification of the botanical source and their content [21].

With regards to BDs, a first problem it is accidentally confusing the raw material: fresh buds must be collected, generally from spontaneous grown, in a very limited period in the late winter and/or in the early spring, corresponding to the annual germination of the plant [18]. During this period, plants may not show their distinctive characteristics and sometimes the attribution of the botanical species may be difficult for the collector. A second problem concerns the manufacturing process and the extraction protocols whose parameters are not strictly defined, and production rules are often loose and deficient [22–24].

Polyphenols play key roles in plant development processes and their synthesis increases when plants are under conditions of abiotic stress, thus helping the plant to cope with environmental constraints [25]. They form an integral part of the human diet and they are very abundant in plant-based foods, such as fruits and vegetables, tea, wine, and coffee [26]. Their chemical structure is based on at least one aromatic ring with one or more hydroxyl groups, which explains their known antioxidant and anti-inflammatory properties [27].

In recent years, many health benefits of dietetic polyphenol supplementation have been described in humans i.e., against aging and cardiovascular disease [28,29], to prevent obesity and diabetes [30,31], to modulate human gut microbiota [32] and to improve the brain cognition skills [33,34]. This knowledge guides the choice of consumers not only towards plant foods but also towards herbal food supplements, whose polyphenol content is often even more concentrated and responsible for their bioactivity [12,13]. Nevertheless, polyphenols content is strongly influenced by the manufacturing methods whose parameters are often not strictly defined (e.g., solvent ratios in the extraction mixtures, raw material/extraction mixture ratios, extraction time) and thus they could affect the final compositions [35].

In previous articles, the polyphenolic pattern of some BDs prepared starting from different botanical species have been studied [10,11,21,36].

In this research, eight species spontaneously grown and commonly used to produce BDs, i.e., *Carpinus betulus* L., *Cornus mas* L., *Ficus carica* L., *Fraxinus excelsior* L., *Larix decidua* Mill., *Pinus montana*

Mill., *Quercus petraea* (Matt.) Liebl., *Tilia tomentosa* Moench, have been taken into account as case study. Two different manufacturing methods, one conventional (maceration) and one innovative (direct sonication), as well as two different extraction protocols have been taken into account and the corresponding polyphenolic extracts' profiles have been investigated.

Pulsed Ultrasound-Assisted Extraction (PUAE), according to the six principles of the green extraction [37] and the twelve principles of green chemistry [38], has been employed as an innovative technique to quickly produce BDs comparing to the long conventional maceration [39]. Even if, the positive impacts of the ultrasound-assisted extraction, i.e., reduction of the extraction time, diminution of solvent and energy used, improvement in yield and selectivity, high reproducibility, intensification of diffusion and eliminating wastes, are known in the scientific literature [40–42], this technique is still underused in this sector.

Moreover, two different BDs manufacturing protocols, which used different extraction mixture of solvents and different solid/liquid ratio, have also been studied to evaluate both the proper identification of the botanical species and the traceability of these vegetal products regardless of extraction method and experimental conditions.

A strategy based on the untargeted UV-Visible fingerprinting coupled to chemometrics (Principal Component Analysis—PCA) has been proposed for the screening of the polyphenolic BDs profile in order to obtain a rapid control tool [43]. Finally, HPLC methods were used to obtain a targeted chromatographic profile [7,44] of the main polyphenol classes (i.e., flavonols, benzoic acids, catechins, cinnamic acids). Polyphenols are correlated with their potential health-promoting activity [45], even if they are strongly influenced both by the methods and protocols used [35].

#### **2. Materials and Methods**

#### *2.1. Raw Samples*

Buds, belonging to eight different vegetable species (*Carpinus betulus* L., *Cornus mas* L., *Ficus carica* L., *Fraxinus excelsior* L., *Larix decidua* Mill., *Pinus montana* Mill., *Quercus petraea* (Matt.) Liebl., *Tilia tomentosa* Moench) were collected from plants spontaneously grown in the Turin Province (Italy) and were immediately authenticated by an agronomist. Sampling has been performed in two years (2018–2019), from February to April, during the bud break ("balsamic period").

Table 1 reports the geo-localization coordinates of the different collection sites and the scientific naturalistic illustrations (specifically achieved during the Finnover project) of all the eight vegetable species investigated.

#### *2.2. Chemicals*

MilliQ ultrapure water, obtained by means of a Millipore equipment (Bedford, MA, USA) was used throughout. All chemicals employed for the extract preparations and for the subsequent analysis were HPLC-grade. They were supplied by VWR International S.r.l (Milan, Italy) and Sigma-Aldrich (St. Louis, MO, USA). Purity of all the used standards for HPLC analysis of BDs has been reported in the Supplementary Materials (Table S1).

**Table 1.** The collection sites, the corresponding geo-localization coordinates, and the scientific naturalistic illustrations of the eight different bud species.

#### *2.3. Bud-Derivatives Manufacturing Applying Two Di*ff*erent Methods*

Fresh buds, after their collection, were immediately processed to prepare the corresponding BDs in order to minimize any degradation preserving the peculiar phytocomplex as much undamaged as possible. The manufacturing was performed both in an Italian company (Geal Pharma Turin, Italy) and by the Authors in the analytical laboratory of the University of Genoa (Department of Pharmacy).

The following two different preparation methods of BDs were investigated: the conventional cold Maceration (M) [9], and a more rapid and innovative procedure by Ultrasounds (US) recently described by the Authors [11]. Moreover, for both preparation methods two different extraction solvents and different sample/solvent ratios were investigated too ("Protocol A" and "Protocol B", see Figure 1), in order to evaluate both the proper identification of the botanical species and the traceability of the BDs independently from their manufacturing process (Table 2). Each extraction was performed in duplicate.

**Figure 1.** The global scheme of the experimental manufacturing of BDs: two methods (cold maceration, namely M, and ultrasounds, namely US) have been used. Each method has been applied following two different protocols (A and B).

2.3.1. Conventional Cold Maceration (M) as Traditional Method

BDs were prepared using a cold maceration by an Italian Company of botanicals (Geal Pharma, Bricherasio, Turin) following two different experimental manufacturing protocols, reported in Table 2:

In both methods, after the maceration step, the extracts, namely BDs, have been obtained by a preliminary filtration, a manual pressing and a second filtration after two days of decanting. The so obtained BDs were stored at 4 ◦C in the dark until their further analysis.

**Table 2.** BDs obtained starting from the eight vegetable species (raw materials). Two different methods (cold maceration-M and Pulsed Ultrasound-Assisted Extraction US) and two different experimental protocol (Protocol A and B) are taken into account.

DW: dry weight; FW: fresh weight.

#### 2.3.2. Green Extraction: Pulsed Ultrasound-Assisted Extraction (US) as Alternative Method

Fresh buds were finely ground by a Grindomix 200 M (Retsch, Haan, Germany) for 20 s at 5000 rpm, and then sieved by a 150 μm sieve, in order to improve the efficiency of the following extraction step [46]. PUAE was performed directly by an Hielscher UP200St sonicator (Teltow, Germany) equipped with an ultrasonic titanium sonotrode (7 mm of diameter), at a constant frequency of 26 kHz. The pulsed mode, referring to an alternation of "on" time and "off" time of the sonicator, guarantees a lowering increase

in temperature, which better preserve the phytocomplex, and greater energy savings compared to continuous treatments [47]. The experimental sonication conditions (amplitude 30%, duty cycle 65%, extraction time 20 min) were previously optimized by the Authors on the same raw materials [11].

The same two experimental extraction conditions described in the paragraph 2.3.1 ("Protocol A" and "Protocol B", see Figure 1) were employed ("US\_A" and "US\_B", see Table 2). The extracts obtained were filtered for Buchner (Whatman n. 1 paper), centrifuged at 3000 rpm for 10 min and then stored at 4 ◦C in the dark until analysis.

#### *2.4. Spectroscopic Analysis: UV-Visible Fingerprint*

UV–Visible absorption spectra (200 nm–900 nm) were recorded by a spectrophotometer Agilent Cary 100 (Varian Co., Santa Clara, CO, USA) with 0.5 nm resolution, using rectangular quartz cuvettes with 1 cm path length. BDs, before the spectroscopic analysis, were suitably diluted in the corresponding extraction solvent (glycerol/ethanol 1/1 *w*/*w* or water/glycerol/ethanol 50/20/30 *w*/*w*/*w*) depending on the followed experimental protocol ("Protocol A" and "Protocol B", respectively). Dilution was necessary to avoid signal saturation but was subsequently considered in order to make a comparison between the different spectra achieved. BDs spectra were acquired in duplicate and then averaged. The collection was performed at room temperature (25 ± 1 ◦C), against a blank solution represented by the corresponding extraction solvent.

#### *2.5. HPLC Analysis*

In this study, effective HPLC–DAD methods were used for fingerprint analysis and phytochemical identification of samples. Four polyphenolic classes were considered: benzoic acids (ellagic and gallic acids), catechins ((+)catechin and (-)epicatechin), cinnamic acids (caffeic, chlorogenic, coumaric, and ferulic acids), and flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, and rutin). Total bioactive compound content (TBCC) was determined as the sum of the most important bioactive compounds with positive effects on human organism ("multimarker approach") [48].

The external standard method was used for quantitative determination of bioactive compounds. Stock solutions of cinnamic acids and flavonols with a concentration of 1.0 mg·mL−1 were prepared in methanol: five calibration standards were prepared by dilution with methanol; stock solutions of benzoic acids and catechins with a concentration of 1.0 mg·mL−1 were prepared in 95% methanol and 5% water. In this case, five calibration standards were prepared by dilution with 50% methanol–water.

An Agilent 1200 High-Performance Liquid Chromatograph coupled to an Agilent UV-Vis diode array detector (Agilent Technologies, Santa Clara, CA, USA) was used for the chromatographic analysis. Four chromatographic methods were used to separate the bioactive molecules on a Kinetex C18 column (4.6 × 150 mm, 5 μm, Phenomenex, Torrance, CA, USA). Several mobile phases were used for bioactive compound identification and UV spectra were recorded at different wavelengths, based on HPLC methods, previously tested and validated [10,40], with some modifications: (i) a solution of 10 mM KH2PO4/H3PO4 (A) and acetonitrile (B) with a flow rate of 1.5 mL·min−1 (method A—analysis of cinnamic acids and flavonols, gradient analysis: 5% B to 21% B in 17 min + 21% B in 3 min + 2 min of conditioning time); (ii) a solution (A) of methanol/water/formic acid (5:95:0.1 *v*/*v*/*v*) and a mix (B) of methanol/formic acid (100:0.1 *v*/*v*) with a flow rate of 0.6 mL·min−1 (method B—analysis of benzoic acids and catechins, gradient analysis: 3% B to 85% B in 22 min + 85% B in 1 min + 2 min of conditioning time). UV spectra were recorded at 330 nm (A); 280 nm (B).

Biomarkers were selected for their demonstrated positive healthy properties and antioxidant capacity by literature in relation to the use of this plant-derived products. All single compounds were identified in samples by comparison and combination of their retention times and UV spectra with those of authentic standards in the same chromatographic conditions. Each sample was analyzed in triplicate and results were reported as mean value ± standard deviation to assess the repeatability of the employed methods.

#### *2.6. Data Analysis*

#### 2.6.1. Chemometric Analysis

Multivariate data analysis has been performed by CAT (*Chemometric Agile Tool*) software, one advanced chemometric multivariate analysis tool based on R, developed by the Chemistry Group of the Italian Chemical Society [49].

PCA was applied as common multivariate statistical method of unsupervised pattern recognition. Its aim is extracting important information from the data and decreasing the high-dimensional dataset volume by maintaining the important information [50,51].

#### 2.6.2. Data Matrices Organization

A data matrix A32,601 consisting of 32 rows (corresponding to the BDs analyzed, 4 samples for each of the eight botanical species investigated) and 601 columns (the absorbance values in the range of 200–500 nm of the UV-Visible spectra, with 0.5 nm of resolution) was prepared and further analyzed by PCA. Standard normal variate (SNV) transform and column autoscaling were previously performed on the spectral data to remove multiplicative effects of scattering and to scale the data, respectively [52].

Available sample were divided in two different subsets: a calibration (or training) set and a test (or evaluation) set in order to build and validate the statistical model, respectively [53]. For a reliable validation strategy, it is important that data used as test set were not used to build the model in order to avoid the overestimations of the prediction ability [53]. 32 samples, previously reported in Table 2, were selected for the construction and identification of the model (Calibration set). The representative calibration data set consisted of 4 extracts (M\_A, M\_B, US\_A, US\_B) for each botanical species investigated (*Carpinus betulus* L., *Cornus mas* L., *Ficus carica* L., *Fraxinus excelsior* L., *Larix decidua* Mill., *Pinus montana* Mill., *Quercus petraea* (Matt.) Liebl., *Tilia tomentosa* Moench). Furthermore 16 BDs, obtained both by conventional maceration and ultrasound extraction respectively from the same eight vegetal species, were randomly selected and used as an independent set to test the model and assess its validity (Test set, Table 3).

**Table 3.** External test set. 16 BDs obtained starting from the eight vegetable species using two different methods (cold maceration M and Pulsed Ultrasound-Assisted Extraction US) and two different experimental protocol (Protocol A and B) are taken into account as independent set to test the statistical model.

All the pre-treated UV-Visible absorption spectra, in the range 200–500 nm, are reported in Figure 2. For each species, the four averaged spectral profiles corresponding to the Calibration set (Table 2) are highlighted in grey while in red have been reported the Test set samples (TS/TS2) belonging to the same class.

**Figure 2.** Averaged UV-Visible spectra of the 8 botanical species after SNV pre-treatment of data. For each species, the four averaged spectral profiles of the Calibration set (Table 2) are highlighted in grey while in red are reported the External Test set samples (Table 3).

Then, a data matrix B32,620 consisting of 32 rows and 620 columns was prepared and analogously analyzed by PCA. B32,620 rows correspond to the 32 BDs analyzed (Calibration set), and columns are the absorbance values of the UV-Visible spectra after SNV in the range 200–500 nm coupled to the chromatographic quantifications by HPLC (4 polyphenolic classes and 13 bioactive compounds). The data set was previously scaled by using a block scaling procedure [54], with the aim to give to the spectroscopic and chromatographic variables a comparable influence in the data analysis. In fact, this pretreatment allows to divide variables in different blocks whose values will be scaled to attain the same block-variance after pretreatment. Moreover, the variables belonging to the same block are equally weighted.

#### **3. Results and Discussion**

The quality control of vegetal material is critical both if the botanical product is to be used as a drug or as an herbal food supplement. For consumer safety and the protection of who operate in this industrial field, quality control should be applied throughout the different processing steps, from the raw material to the final product. Scientific-naturalistic illustrations of the most common buds used in BDs production (Table 1) have been realized within the Finnover project by an expert botanical graphic designer, in order to provide a useful first tool for the operators in the BDs manufacturing. In fact, this peculiar raw material is generally spontaneously collected and mistakes in the attribution of some botanical species may be possible. For this, bud illustrations could represent a preliminary control of these vegetable materials after their collection in the point of view of a controlled manufacturing chain of BDs.

Moreover, a strategy based on the untargeted UV-Visible fingerprinting coupled to chemometrics allows rapid screening of the polyphenolic BDs profile to obtain a preliminary control tool to identify the botanical species.

#### *3.1. Bud-Derivatives Identification: UV-Visible Fingerprint*

Figure 2 show the UV–Visible spectral profiles, after SNV pretreatment of the data, recorded for the eight vegetable species investigated: *Carpinus betulus* L., *Cornus mas* L., *Ficus carica* L., *Fraxinus excelsior* L., *Larix decidua* Mill., *Pinus montana* Mill., *Quercus petraea* (Matt.) Liebl., *Tilia tomentosa* Moench. The extracts were obtained by the conventional maceration and the innovative green extraction (M or US) respectively, using the two experimental protocols (A or B) as described in detail in Table 2. Ultrasounds represent one of the innovative processing techniques of officinal plants [39]. In fact, several companies already exploit innovative applications of ultrasound to obtain liquid foods, beverages, and alcoholic drinks [55,56]. Previously, the Authors described PUAE as an alternative time-saving method to the conventional maceration for the extraction of the polyphenolic fraction from buds [11]. Particularly, PUAE on a lab pilot reactor demonstrated to be an excellent approach for a rapid (20 min vs. 21 days or 3 months of maceration, depending on the Protocol applied) and efficient extraction of phenolic compounds.

Looking at Figure 2, the spectra of the different vegetable species are quite different, highlighting as the pattern of absorbances in the UV–Visible region is strictly connected with the botanical origin of the plants. On the contrary, for each botanical species the spectral differences due to the extraction method (M or US) and to the extraction solvent (Protocol A or B), are minimal. The 501–900 nm interval has been preliminarily removed because there were none interesting absorptions in this spectral region at the assayed concentrations.

PCA, an unsupervised pattern recognition technique [50,51], was applied in order to explore and to analyze the data set using a multivariate approach since the analytical information contained in each spectrum was considered as a multivariate fingerprint. Particularly, the data matrix A32,601, whose rows are the extracts (Calibration set) and the columns are the absorbances recorded in the spectral range 200–500 nm, was considered. PCA was performed on the pretreated and autoscaled data matrix. The first two principal components (PCs) of the data set (A32,601), which together explained the 77.9% of

the total information of the data set since they visualize almost the 80% of the total variance, were firstly taken into account. Figure 3a,b shows the PCA score plots on the 1st–2nd principal components (PC1-PC2) obtained from the above-mentioned data matrix. In Figure 3a the extracts are categorized according to the vegetable species and each one is visualized with a different color (*Carpinus betulus* L.: black, *Cornus mas* L.: red, *Ficus carica* L: green., *Fraxinus excelsior* L.: blue, *Larix decidua* Mill.: brown, *Pinus montana* Mill.: light blue, *Quercus petraea* (Matt.) Liebl.: orange, *Tilia tomentosa* Moench: pink). In Figure 3b, for each vegetable class all the extracts belonging to the calibration set were indicated with their identification code (see Table 2). PC1, the direction of maximum variance which explains almost the 60% of the total information, allows good discrimination between the botanical class regardless of the extraction method (M or US) and the experimental preparation protocol (A or B). Particularly, the *Fraxinus* class (blue, lowest scores on PC1) separates from *Ficus* (green) and *Pinus* (light blue) which have higher scores on PC1. PC2, which explains the 21.1% of the remaining variance, allows to mainly separate *Larix* class (brown, highest scores on PC2) from *Quercus* (orange) and *Carpinus* (black, lowest scores on PC2).

Figure 3c,d show the PCA score plots on the PC1-PC3, which explain together the 69.3% of the total variance of the data set. A good separation among the above cited botanical classes is also highlighted except for *Larix* and *Carpinus* ones. In fact, these latter separate on PC2 (Figure 3a,b) and since PCs are orthogonal, they are uncorrelated and no duplicate information are shown in their plots [50].

In Figure 3e,f, the projections of the external test set (red samples) were reported on the PC1-PC2 and PC1-PC3 score plots respectively, showing a good correspondence with the calibration set for each botanical species.

The spectral variables having greater importance (loading values) on the first three PCs are represented by spectral areas near the following absorbances (in ascending order): 200 nm, 212 nm, 240 nm, 275 nm, 310 nm, 360 nm, 420 nm, as highlighted in the Loading plot on PC1-PC2-PC3 (Figure 4).

Several of them could be related to some secondary metabolites largely distributed in plant material (even in buds) such as tannins, whose structural variability depends on the vegetal species and even among organs of the same plant species [57]. The chemotaxonomic values of tannins have been recognized in the literature for several botanical species [58,59] and, the distribution of hydrolysable tannins has been used as chemotaxonomic markers by several authors [60].

It is well known that the different classes of tannins present characteristic absorption bands in the UV spectral region. Particularly as far as hydrolysable tannins are concerned, gallotannins show two characteristic absorption maximums, λ max around 212 nm and λ max around 275 nm, with an inflection point (λ min) around 242 nm; ellagitannins present strong absorption near 200 nm and a shoulder around 277 nm and another absorption near 360 nm. Instead condensed tannins (or proanthocyanidins), chemically defined as flavonoid polymers in which the phenolic hydroxyls are partially or totally esterified with gallic acid, present an absorption around 200 nm, a λ min between 258–259 nm and λ max between 279–281 nm [57]. Nevertheless, also other polyphenols, such as hydroxycinnamic acids and flavonoids, could contribute to the UV-Visible fingerprints, even if some of them are more ubiquitarians and lesser species-specific [61,62]. Furthermore, as far as flavonoids are concerned, it is important to underline that their absorptions in the Visible are almost negligible at the measured concentrations, which are instead useful to avoid saturation of the UV region.

The fingerprint UV-Visible, at least in a preliminary screening step, seems to discriminate the peculiar polyphenols composition of BDs and could be a simple and quick method to confirm the proper identification of the botanical source after the botanic check by a professional botanist.

**Figure 3.** The scores plots of the UV–Visible absorbances data matrix A32,601. Each vegetable species is reported with a different color (*Carpinus betulus* L.: black, *Cornus mas* L.: red, *Ficus carica* L: green., *Fraxinus excelsior* L.: blue, *Larix decidua* Mill.: brown, *Pinus montana* Mill.: light blue, *Quercus petraea* (Matt.) Liebl.: orange, *Tilia tomentosa* Moench: pink). (**a**) PC1-PC2 score plot with BDs categorized according to the vegetable species; (**b**) PC1-PC2 score plot with BDs categorized according to their identification code (Table 2); (**c**) PC1-PC3 score plot with BDs categorized according to the vegetable species; (**d**) PC1-PC3 score plot with BDs categorized according to their identification code (Table 2); (**e**) PC1-PC2 score plot obtained projecting the external test set samples (highlighted in red); (**f**) PC1-PC3 score plot obtained projecting the external test set samples (highlighted in red).

**Figure 4.** Loading plot on PC1, PC2, PC3.

#### *3.2. Bud-Derivatives Identification: UV-Visible and HPLC Fingerprints*

Figure 5 shows the PCA plots of the data matrix B32,620 on PC1-PC2, which together explained the 76.2% of the total variance.

**Figure 5.** The PC1–PC2 plots of the UV–Visible absorbances coupled to the HPLC data (data matrix B32,620): (**a**) Score plot; (**b**) Biplot.

PCA was performed on the pretreated and autoscaled data matrix, after the block scaling treatment in order to consider in the data analysis the same importance for the spectroscopic and chromatographic variables [63]. The PC1-PC2 score plot (Figure 5a) highlights a good separation between the vegetal species. Particularly PC1, which represents the direction of maximum variance explaining the 55.4% of the total information, allows good discrimination between *Fraxinus* class (blue, highest scores on PC1), *Ficus* (green) and *Pinus* (light blue) classes, which have lowest scores on this PC. As highlighted in the Biplot (Figure 5b) the variables having greater importance (loading value) on this separation are represented by total cinnamic acids, caffeic acid, coumaric acid and hyperoside content which are high in *Fraxinus* species and very low in *Pinus* one (as reported in Table 4). Instead PC2, which explains the 20.7% of the remaining information, allows mainly to separate *Carpinus* (black) and *Cornus* (red) classes from all the other ones. These species result particularly rich in tannins (catechins and benzoic acids).

**Table 4.** Bioactive classes and total phenolics in the analyzed samples.

SD: standard deviation; \*\* FW: fresh weight

In the Supplementary materials an example (*Larix decidua*) of chromatographic pattern was reported. As shown in Figure 5, the addition of chromatographic variables does not greatly improve the taxonomic separation previously obtained by the only UV-Visible fingerprint (Figure 3). However, these results show that the main polyphenols evaluated could be useful markers for identifying the botanical species regardless of the extraction method and the experimental preparation protocol.

#### *3.3. Phenolic Composition of BDs*

In this study, the health-promoting compounds were grouped into four different polyphenolic classes in order to assess the contribution of each class to the phytocomplex composition of buds belonging to the eight different species: cinnamic acids (as sum of caffeic acid, chlorogenic acid, coumaric acid, ferulic acid), flavonols (as sum of hyperoside, isoquercitrin, quercetin, quercitrin and rutin), benzoic acids (ellagic and gallic acids) and catechins ((+)catechin and (-)epicatechin). The identification and quantification of each single bioactive compound, expressed in mg/100 g FW, is reported in the Supplementary Materials (Table S2). For a better data visualization, Figure 6 shows the radar plot, made considering for each botanical species the mean values obtained from the 4 different extracts (M\_A, M\_B, US\_A, US\_B) for each marker compound quantified.

**Figure 6.** The mean content of each phenolic marker (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, hyperoside, isoquercitrin, quercetin, quercitrin and rutin, ellagic acid, gallic acid, (+)catechin and (-)epicatechin) for the eight botanical species investigated.

Several markers of cinnamic acids were considered but not detected in all the extracts. *Fraxinus excelsior* BDs showed the highest content in cinnamic acids (ranged from 113.53 ± 6.70 to 829.03 ± 2.26 mg/100 g FW), and as shown in Table S2, ferulic and chlorogenic acids were the most abundant. *Cornus mas* and *Tilia tomentosa* species showed very low amounts of ferulic acid (respectively 12.14 and 11.73 mg/100 g FW), while in the other species it was not detected. In recent years, several physiological functions of ferulic acid have been demonstrated [64]. Particularly, its free radical scavenging activity and its cholesterol-lowering activity, together with the low toxicity, suggested its chemo preventive effects on heart diseases [65]. Chlorogenic acid is also involved in beneficial effects on human health due to its anti-inflammatory, antioxidative, anti-aging and anticancer activities [66]. Chlorogenic acid was detected only in *Fraxinus excelsior* BDs (ranges from 43.88 to 489.94 mg/100 gFW), in all the other species it was not detectable. Li et al. 2013 reported that chlorogenic acid and flavonols may be considered the main phenolic compounds responsible for in vitro anti-cancer property (i.e., against breast, colon, liver and lung cancer) [66]. As regards the total flavonol content, it was highly variable among species. The highest content was quantified in *Cornus mas* species (mean value: 705.71 mg/100 g FW) while the lowest value in *Pinus montana* (mean value: 25.31 mg/100 g FW). As highlighted in Figure 6, quercetin represented the phenolic marker of BDs belonged to *Larix decidua* species (in orange), while hyperoside was more abundant in *Fraxinus excelsior* ones (in light blue).

Benzoic acids are known to be very important in the human diet because of their relation to many biological and functional activities including antioxidative, anti-inflammatory, anticancer and antihepatotoxic properties [67]. Gallic acid, due to its antioxidant activity, has been shown to be effective against oxidative stress (OS), and many other properties have been reported (i.e., anti-mutagenic, anti-carcinogenic, antiviral, antibacterial, anti-inflammatory, antithrombotic and anti-atherosclerotic activities) [68]. A multi-target activity of ellagic acid, mainly ascribed to its antioxidant property and free radical trapping ability, has been reported too. In particular anti-angiogenic, anti-atherogenic, anti-carcinogenic, anti-obesity, anti-inflammatory, antioxidant, anti-thrombotic and anti-neurodegenerative properties have been demonstrated [69]. Ellagic acid was

very abundant in almost all the described species (Table S2) while gallic acid was not detectable in *Ficus carica*, *Fraxinus excelsior*, *Larix decidua* and *Pinus montana* species. The highest content in ellagic acid was identified in *Cornus mas* extracts, followed by *Larix decidua* and *Quercus petraea* BDs (Figure 6, in red).

Catechins have important effects on human health thanks to its antioxidant, anti-inflammatory, antidiabetic, and antimicrobial properties [67]. The intake of foods and dietary supplements rich in catechins could have an important role in the prevention of various diseases (i.e., cardiovascular diseases), inhibition of lipid peroxidation, improvement of blood flow, elimination of several toxins and inhibition of human cancer cell line proliferation and cyclooxygenase enzymes [70]. All the vegetal species considered in this research were a good source of catechins (catechin and epicatechin) as shown in Table 3 and Table S2. Particularly, as highlighted in Figure 6, catechin represented the phenolic marker of *Pinus montana* BDs (in violet), while epicatechin was more abundant in *Carpinu betulus* and *Quercus petraea* extracts (in light green).

All BDs analyzed showed a good content of phenolics although there was a high variability both between the different vegetal species and between the extracts obtained by the different manufacturing method and experimental conditions starting from the same botanical species. Figure 7 showed the radar plots of each botanical species in order to better highlight the phenolic composition of the 4 different extracts (M\_A, M\_B, US\_A, US\_B).

As showed in Figure 7, the manufacturing methods (conventional maceration or sonication) and the experimental conditions used for the preparation of BDs (i.e., extraction solvent, extraction time, solid/ solvent ratio, extraction time) strongly influenced the phenolic extraction yield despite having removed the variability of the raw material (same batch of buds for each vegetal species). Generally US\_A (green line) and US\_B (red line) appears more similar in terms of phenolic composition respect to M extracts (M\_A and M\_B), except for some species, such as *Pinus montana* and *Larix decidua*, in which there is a greater homogeneity in the polyphenolic profile of the final products. In almost all species, the M\_B extract (yellow line) is the most different from the others. In example, the M\_B extract of *Cornus mas* was rich in catechin which was not detected in extracts obtained by different extraction conditions (M\_A, US\_A, US\_B). Analogously, rutin represents a phenolic marker of the M\_B extract of *Fraxinus excelsior*, while it was poorly detectable in the other extracts of the same species. Surely Protocol A, according to the European Pharmacopoeia, provided a higher alcoholic concentration of the extraction solvent than protocol B and it is known that a higher solvent polarity allows a higher phenolic extraction from plant materials [71]. Moreover, Protocol A used an higher solid/solvent ratio because it is evaluated on the dry weight of the raw material while following the industrial Protocol (B), the fresh weight of buds was taken into account. Regarding the effect of ultrasounds, the implosion of cavitation bubbles on the material surface results in micro-jetting which generates several effects such as surface peeling, detexturation, erosion and cell breakdown [40]. Probably, the destruction of vegetal cells allowed to increase the extraction yield making up for the lower alcohol content of protocol B.

Due to the lack of a single regulation and an unique preparation protocol for these botanicals, very different products can be found on the market in terms of their polyphenolic fraction depending on both the raw materials (i.e., taking into account their specific agro-environmental and biological traits) and on the experimental conditions of manufacturing (method of preparation, extraction solvent, solid/solvent ratio, extraction time).

**Figure 7.** For each botanical species the phenolic composition (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, hyperoside, isoquercitrin, quercetin, quercitrin and rutin, ellagic acid, gallic acid, (+)catechin and (-)epicatechin) of the 4 different extracts (M\_A: blue line, M\_B: yellow line, US\_A: green line, US\_B: red line) was reported.

#### **4. Conclusions**

Although BDs have been widely used in traditional medicine because of the peculiar content of buds in phenolic compounds, nowadays they are a category of botanicals still poorly studied. The lack of detailed scientific information and a clear and unique regulation, it makes these products high risk and vulnerable for accidental mistakes in the attribution of the botanical species, but also frauds and adulterations. Moreover, the polyphenols content of BDs is strongly influenced by the manufacturing processes whose parameters are often not strictly defined (e.g., solvent ratios in the extraction mixtures, raw material/extraction mixture ratios, extraction time) and thus they affect their final compositions.

This research, within the Finnover project, aims to answer to the growing demand for efficient quality control in the BDs field to guarantee the proper attribution of the botanical source and their content. Moreover, a manufacturing process specification should be advisable to monitor the bioactive contents.

UV-Visible spectroscopy and HPLC-DAD analysis have been employed to obtain an untargeted and a targeted phytochemical fingerprint of BDs, respectively. UV-Visible coupled with an appropriate chemometric data processing is a simple, rapid and low-cost technique proved to be very useful to identify the botanical source regardless the manufacturing method and the experimental conditions used. Moreover, the targeted phytochemical fingerprint by HPLC-DAD allowed to obtain a detailed screening of the BDs polyphenolic profile which highlighted an high variability due to the different vegetal species and to the manufacturing method and protocol. The ultrasonic extraction of buds compared to conventional maceration proved less sensitive to the different extraction protocols.

The proposed strategy offers to those operating in this industrial sector an untargeted method for the identification of the bud's botanical species and a green extraction strategy (PUAE) which is more robust with respect to the different extractive protocols that can be used. The same approach, described for BDs, could be analogously applied to other botanical productions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/10/1343/s1, **Figure S1**: *Larix decidua* chromatographic pattern. Table S1: Purity of all the used standards for HPLC analysis of BDs. Table S2: Single phenolic compound fingerprint of BDs.

**Author Contributions:** Conceptualization, G.L.B., P.Z. and R.B.; Data curation, F.T. and D.D.; Formal analysis, R.B.; Funding acquisition, G.L.B., A.P. and R.B.; Investigation, F.T., D.D. and M.G.; Project administration, G.L.B. and R.B.; Software, F.T. and R.B.; Supervision, G.L.B., A.P., P.Z. and R.B.; Writing—original draft, F.T. and D.D.; Writing—review & editing, G.L.B. and R.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by an European Union project called FINNOVER (n◦ 1198), http://www. interreg-finnover.com/.

**Acknowledgments:** The authors acknowledge GEAL Pharma (Bricherasio, Torino, Italy) for providing the samples of plant materials and Teresa Fior for the scientific-naturalistic illustrations of buds.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **Phenolic Profiles, Antioxidant, and Inhibitory Activities of** *Kadsura heteroclita* **(Roxb.) Craib and** *Kadsura coccinea* **(Lem.) A.C. Sm.**

### **Varittha Sritalahareuthai 1, Piya Temviriyanukul 1,2, Nattira On-nom 1,2, Somsri Charoenkiatkul 1 and Uthaiwan Suttisansanee 1,2,\***

Received: 4 August 2020; Accepted: 31 August 2020; Published: 2 September 2020

**Abstract:** *Kadsura* spp. in the Schisandraceae family are woody vine plants, which produce edible red fruits that are rich in nutrients and antioxidant activities. Despite their valuable food applications, *Kadsura* spp. are only able to grow naturally in the forest, and reproduction handled by botanists is still in progress with a very low growth rate. Subsequently, *Kadsura* spp. were listed as endangered species by the International Union for Conservation of Nature and Natural Resources (IUCN) in 2011. Two different *Kadsura* spp., including *Kadsura coccinea* (Lem.) A.C. Sm. and *Kadsura heteroclita* (Roxb.) Craib, are mostly found in northern Thailand. These rare, wild fruits are unrecognizable to outsiders, and there have only been limited investigations into its biological properties. This study, therefore, aimed to comparatively investigate the phenolic profiles, antioxidant activities, and inhibitory activities against the key enzymes involved in diabetes (α-glucosidase and α-amylase) and Alzheimer's disease (acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-secretase 1 (BACE-1)) in different fruit parts (exocarp, mesocarp (edible part), seed, and core) of *Kadsura coccinea* (Lem.) A.C. Sm. and *Kadsura heteroclita* (Roxb.) Craib. The results suggested that *Kadsura* spp. extracts were rich in flavonol (quercetin), flavanone (naringenin), anthocyanidins (cyanidin and delphinidin), and anthocyanins (cyanidin 3-*O*-glucoside (kuromanin), cyanidin 3-*O*-galactoside (ideain), cyanidin 3-*O*-rutinoside (keracyanin), and cyanidin 3,5-di-*O*-glucoside (cyanin)). These flavonoids were found to be responsible for the high antioxidant activities and key enzyme inhibitions detected in *Kadsura* spp. extracts. The findings of the present study can support further development of *Kadsura* spp. as a potential source of phenolics and anti-oxidative agents with health benefits against diabetes and Alzheimer's disease. Besides, exocarp and the core of *Kadsura* spp. exhibited higher phenolic contents, antioxidant activities, and key enzyme inhibitory activities compared to the mesocarp and seeds, respectively. This information can promote the use of fruit parts other than the edible mesocarp for future food applications using *Kadsura* spp. rather than these being wasted.

**Keywords:** *Kadsura* spp.; fruit parts; phenolics; antioxidant capacity; in vitro health properties

#### **1. Introduction**

For thousands of years, humans have consumed naturally indigenous plants as green medicines to treat illnesses, including vegetables, herbs, and fruits. However, the development of effective, advanced scientific technology and synthetic drugs has significantly lowered the popularity of the natural medicine approach. Besides several advantages, including ready-to-use therapeutic applications

and the commercial availability of synthetic drugs, severe side effects and economic impacts are other factors of concern. These issues have brought indigenous plants back into focus. Natural products have been considered as better functional food sources for alternative approaches in the prevention of certain non-communicable diseases (NCDs), and have even been used as green medicine to treat particular diseases. Subsequently, the study of medicinal applications and functional food development from beneficial plants are currently in focus.

*Kadsura* spp. in the *Schisandraceae* family produce ovate-elliptical shaped leaves and solitarily unisexual flowers [1]. The globose fruit is between 14 and 20 cm in diameter and forms a hexagonal structured skin developed by each carpel [1]. The fruit shape is similar to that of sugar-apples, but with a sourer and astringent taste. While the ripened fruit is edible [2], other plant parts can also be used as traditional medicine [1]. The stem and root of *Kadsura coccinea* are used to reduce rheumatic pain in the bones, chronic enteritis, acute gastritis, and the immunologic hepatic fibrosis effect [3], while those of *Kadsura longipedunculata* are used in the treatment of rheumatic arthritis, traumatic injury, dysmenorrhea, abdominal pain, irregular menstruation, canker sores, and gastrointestinal inflammation [3]. Moreover, the *Kadsura heteroclita* stem can prevent and treat rheumatic and arthritic diseases with anti-nociceptive and anti-inflammatory effects [4,5]. Phenolics detected in *Kadsura* spp. appear to be responsible for these health properties. Flavonoids identified in *Kadsura oblongifolia*, including quercetin and kaempferol, possess potent antioxidant activities [6]. Additionally, *K. coccinea* has been previously reported to contain gallic acid as a major phenolic acid, which has strongly demonstrated DPPH-radical scavenging activity (the half maximal inhibitory concentration (IC50) = 23.64 μg/mL) compared to commercial antioxidants, ascorbic acid, and 2,6-ditertbuty l-4-methylphenol (BHT) (the IC50 of 48.46 and 975.96 μg/mL, respectively) [7]. However, stressful conditions i.e., aphid feeding [8], light and drought intensity [9,10], or even the processing of biological material [11] can significantly affect the antioxidant properties or the phenolic profile.

The local Thai name for *Kadsura* spp. is noi-na-kreau and was first discovered in northern Thailand in 1972 [12]. *Kadsura* spp. were listed as an endangered species by the International Union for Conservation of Nature and Natural Resources (IUCN) in 2011, and since then only 11 trees have been found in the Thai provinces of Chiang Rai, Chiang Mai, and Mae Hong Son. Due to how rare they are, *Kadsura* spp. were listed by the Plant Genetic Conservation Project under the initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (RSPG) in the same year of 2011, aiming to sustainably conserve and allocate plant resources to optimize beneficial utilizations [13]. Unlike more ubiquitous fruits in Thailand, *Kadsura* spp. are not well known, and due to largely being unrecognized, they are at high risk of extinction. In order to obtain the main goal of RSPG to sustainably conserve *Kadsura* spp. by expanding the knowledge on their biological properties, eventually leading to proper management of the plant, the objective of the present study is, therefore, to examine the phenolic profiles, antioxidant activities, and in vitro key enzyme inhibitory properties of *K. coccinea* (Lem.) A.C. Sm. and *K. heteroclita* (Roxb.) Craib, the two most abundant *Kadsura* spp. found in northern Thailand. These enzyme inhibitory properties are related to medicinal abilities against diabetes and Alzheimer's disease (AD) by inhibiting key enzymes, which control these diseases. Targeting the inhibition of the carbohydrate degrading enzymes, including α-glucosidase and α-amylase, is a key approach for drugs designed to control diabetes. Based on enzyme–inhibitor structural interactions, several phenolics have been proven as effective α-glucosidase and α-amylase inhibitors [5,14]. In addition, neurotransmitter degrading enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and amyloid precursor protein (APP) degrading enzyme, beta-secretase (BACE-1), are the key enzymes, which control the occurrence of AD. It has been previously suggested that the extreme loss of cholinergic markers found in the cerebral cortex is associated with AD, while BACE-1 causes the formation of beta-amyloid peptides or senile plaques, a hallmark of AD [15]. The findings of the present study provide valuable information for the sustainable conservation of the nearly extinct *Kadsura* spp. by describing their advantageous biological properties and potential applications, which can help to establish proper management and prolong the existence of the plants.

#### **2. Materials and Methods**

#### *2.1. Sample Collection, Preparation, and Extraction*

*Kadsura coccinea* (Lem.) A.C. Sm. and *Kadsura heteroclita* (Roxb.) Craib were collected from forests near Hui-Nam-Guen village, Mae-Jedi-Mai sub-district, Weing-Pa-Pao district, Chiang Rai Province, Thailand (19◦11 58.3" N and 99◦31 00.7" E). The fruits of both species could be harvested only once a year at different time periods. The fruits of *Kadsura coccinea* (Lem.) A.C. Sm. were collected in October 2017, while those of *Kadsura heteroclita* (Roxb.) Craib were collected in January 2018. Both samples were identified and authenticated by Assist. Prof. Dr. Chunthana Suwanthada and Assoc. Prof. Dr. Chusri Trisonthi of the Plant Genetic Conservation Project under the royal initiative of Her Royal Highness Princess Maha Chakri Sirindhorn. The voucher specimens, including BK No. 071406 (*Kadsura coccinea* (Lem.) A.C. Sm.) and BK No. 071407 (*Kadsura heteroclita* (Roxb.) Craib), were deposited at the Bangkok Herbarium (BK), Bangkok, Thailand.

All fresh samples were cleaned with deionized water and prepared as exocarp, mesocarp (edible part), seed, and core (Supplementary Table S1). All the samples were cut (approximately 0.3 cm thick) before freeze drying by a Heto Powerdry PL9000 freeze dryer (Heto Lab Equipment, Allerod, Denmark) for 3 days. Dry samples were then ground using a grinder (Philips 600 W series from Philips Electronic Co., Ltd., Jakarta, Indonesia) into a fine powder before packing in a vacuum aluminum foil bag and storing in a freezer at −20 ◦C for further analysis.

A spectrophotometer (ColorFlex EZ, Hunter Associates Laboratory, Reston, VA, USA) was used to analyze the colors of the fresh and dry samples, which were expressed as CIELAB units, where L\* represents dark (0) to white (100) colors, a\* represents green (−) to red (+) colors, and b\* represents blue (−) to yellow (+) colors. The moisture content of the powdered samples was analyzed using a Halogen moisture analyzer (HE53 series, Mettler-Toledo AG, Greifensee, Switzerland). The color data and moisture content data are presented in Supplementary Table S2.

The extraction of *Kadsura* spp. was optimized as described by a previous study [16]. Briefly, the powered samples (100 mg) were dissolved in distilled water (10 mL) and incubated in a 90 ◦C temperature-controlled water bath shaker (WNE45 series from Memmert GmBh, Eagle, WI, USA) for 1 h. The supernatant was then collected by centrifugation at 3800× *g* using a Hettich® ROTINA 38R refrigerated centrifuge (Andreas Hettich GmbH, Tuttlingen, Germany) for 15 min and filtered through a 0.45 μM PES membrane syringe filter. All the extracted samples were stored at −20 ◦C for further analysis.

#### *2.2. Determination of Antioxidant Activity*

The antioxidant activities, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric ion reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays of the *Kadsura* spp. extracts, naringenin, and quercetin were determined using existing, well-established protocols [17–20].

The DPPH radical scavenging assay was performed using DPPH in 95% (*v*/*v*) aqueous ethanol as previously described [17,20]. Trolox solution (0.01–0.64 mM) was used as a standard, and the results were reported as μmol TE/g dried weight (DW).

The FRAP assay was determined using FRAP reagent as previously described [18,20]. Trolox solution (7.81–250.00 μM) was used as a standard, and the results were reported as μmol TE/g DW.

The ORAC assay was examined using fluorescein reagent as previously described [19,20]. Trolox solution (3.12–100.00 μM) was used as a standard, and the results were reported as μmol TE/g DW.

#### *2.3. Determination of Total Phenolic Contents, Total Flavonoid Contents (TFCs), Total Anthocyanin Contents (TACs), and Phenolic Profiles*

The total phenolic contents (TPCs) of the *Kadsura* spp. extracts were determined using Folin-Ciocalteu reagent as previously described [21,22]. Gallic acid (10–200 μg/mL) was used as a standard, and the TPCs were reported as the mg gallic acid equivalent (GAE)/g DW.

Total flavonoid contents (TFCs) of the *Kadsura* spp. extracts were determined using aluminum trichloride as previously described [23]. Quercetin (0–100 μg/mL) was used as a standard, and the TFCs were reported as the mg quercetin equivalent (QE)/g DW.

Total anthocyanidin contents (TACs) of the *Kadsura* spp. extracts were determined using the pH differential method as previously described [24]. Cyanidin 3-*O*-glucoside (2–63 μg/mL) was used as a standard, and the TACs were reported as the mg cyanidin 3-*O*-glucoside equivalent (C3GE)/g DW.

Phenolic profiles were analyzed using high performance liquid chromatography (HPLC) as described previously [25]. The powdered samples (0.5 g) were mixed with 62.5% (*v*/*v*) aqueous methanol containing 0.5 g/L tBHQ (40 mL) and 6 N HCl (10 mL) before incubating in a 80 ◦C temperature-controlled water bath shaker (WNE45 series from Memmert GmBh, Eagle, WI, USA) for 2 h in dark. The mixture was then cooled in ice for 5 min before sonicating in an ultrasonic cleansing bath (Branson Ultrasonics™ M series, Branson Ultrasonics Corp., Danbury, CT, USA) for a further 5 min. The mixture was filtered through a 0.22 μM PTFE membrane syringe filter. Phenolic acids in the filtrate were analyzed by HPLC utilizing a 5 μm Zorbax Eclipse XDB-C18 column (150 × 4.6 mm from Agilent Technologies, Santa Clara, CA, USA) on an Agilent 1100 HPLC system with a photodiode array detector from Agilent Technologies (Santa Clara, CA, USA). The gradient mobile phases comprised Milli-Q water (18.2 MΩ.cm resistivity at 25 ◦C) containing 0.05% (*v*/*v*) TFA (solvent A), methanol containing 0.05% (*v*/*v*) TFA (solvent B), and acetonitrile containing 0.05% (*v*/*v*) TFA (solvent C) with a constant flow rate of 0.6 mL/min, as shown in Table 1. The existence of the phenolic acids were visualized at 280 and 325 nm using the ChemStation software (Agilent Technologies, Santa Clara, CA, USA) by comparing retention time (*tR*) and spectral fingerprint with standards including 4-hydroxybenzoic acid (>99.0% GC, T), caffeic acid (>98.0% HPLC, T), chlorogenic acid (>98.0% HPLC, T), ferulic acid (>98.0% GC, T), *p*-coumaric acid (>98.0% GC, T), sinapic acid (>99.0% GC, T), syringic acid (>97.0% T) from Tokyo Chemical Industry (Tokyo, Japan), and gallic acid (97.5–102.5% T) from Sigma-Aldrich (St. Louis, MO, USA).

**Table 1.** Solvent system of the phenolic acids and flavonoids using high performance liquid chromatography (HPLC) analysis.

Solvent A = Milli-Q water containing 0.05% (*v*/*v*) TFA; solvent B = methanol containing 0.05% (*v*/*v*) TFA; solvent C = acetonitrile containing 0.05% (*v*/*v*) TFA.

Flavonoid identification was performed similar to that of phenolic acids. However, the existence of flavonoids was visualized at 338 and 368 nm and evaluated by comparing *tR* and spectral fingerprint with standards including apigenin (>98.0% HPLC), genistein (>98.0% HPLC), hesperidin (>90.0% HPLC, T), kaempferol (>97.0% HPLC), luteolin (>98.0% HPLC), myricetin (>97.0% HPLC), naringenin (>93.0% HPLC, T), quercetin (>98.0% HPLC, E) from Tokyo Chemical Industry (Tokyo, Japan), and isorhamnetin (≥99.0% HPLC) from Extrasynthese (Genay, France).

Anthocyanidin and anthocyanin identification was performed as previously described [26,27]. Anthocyanidins were extracted using the powdered sample (500 mg) that was dispersed in 50% (*v*/*v*)

aqueous methanol containing 2 N HCl (5 mL). The mixture was incubated in a 100 ± 2 ◦C water bath (TW20 series from Julabo GmbH, Seelbach, Germany) for 1 h before filtering through a 0.22 μM PTFE membrane syringe filter. Anthocyanidins were identified by the HPLC system (an Ultimate 3000 with diode array and multiple-wavelength detectors) from Thermo Fisher Scientific (Dreieich, Germany) and a 5 μm ReproSil-Pur® ODS-3 column (250 × 4.6 mm) from Dr. Maisch GmbH (Ammerbuch, Germany). Milli-Q water (18.2 MΩ.cm resistivity) containing 0.4% (*v*/*v*) TFA (solvent A) and acetonitrile containing 0.4% *v*/*v* TFA (solvent B) were used as an isocratic mobile phase at a ratio of 82% solvent A and 18% solvent B, with a constant flow rate of 1.0 mL/min. The existence of anthocyanidins was visualized at 530 nm using a Chromeleon™ chromatography Data System (CDS) software (Thermo Fisher Scientific, Dreieich, Germany) by comparing *tR* and spectral fingerprint with the standards including cyanidin (≥96.0% HPLC), delphinidin (≥97.0% HPLC), pelargonidin (≥97.0% HPLC), peonidin (≥97.0% HPLC), and petunidin (≥95.0% HPLC) from Extrasynthese (Genay, France).

Anthocyanin identification was performed similarly to the anthocyanidin identification. Anthocyanins were extracted using the powdered sample (500 mg) that was dispersed in 50% (*v*/*v*) aqueous methanol containing 2% (*v*/*v*) HCl (5 mL). The extract was then sonicated using an ultrasonic cleansing bath (Branson Ultrasonics™ M series, Branson Ultrasonics Corp., Danbury, CT, USA) for 20 min and filtered through a 0.22 μM PTFE membrane syringe filter. Anthocyanins were identified utilizing the same HPLC system as anthocyanidins. Milli-Q water containing 0.4% (*v*/*v*) TFA (solvent A) and acetonitrile containing 0.4% (*v*/*v*) TFA (solvent B) were used as gradient mobile phases with a constant flow rate 1.0 mL/min, as shown in Table 2. The existence of anthocyanins was visualized at 525 nm and compared *tR* and spectral fingerprint with the standards including cyanidin 3-*O*-sophoroside (≥95.0% HPLC), cyanidin 3,5-di-*O*-glucoside (≥97.0% HPLC), cyanidin 3-*O*-galactoside (≥97.0% HPLC), cyanidin 3-*O*-rutinoside (≥96.0% HPLC), cyanidin 3-*O*-glucoside (≥96.0% HPLC), and delphinidin 3,5-di-*O*-glucoside (≥97.0% HPLC) from Extrasynthese (Genay, France).

**Table 2.** Solvent system of the anthocyanins using HPLC analysis.

Solvent A = Milli-Q water containing 0.4% (*v*/*v*) TFA; solvent B = acetonitrile containing 0.4% (*v*/*v*) TFA.

#### *2.4. Determination of Enzyme Inhibitory Activities*

The α-glucosidase inhibitory activity was determined as previously described [28]. Briefly, the assay comprising 2 mM *p*-nitrophenyl-α-D-glucopyranoside (50 μL) in a 50 mM phosphate buffer (pH 7.0), 0.1 U/mL *Saccharomyces cerevisiae* α-glucosidase (type 1, ≥10 U/mg protein, 100 μL), and sample extract (50 μL) was visualized at a wavelength of 405 nm using a microplate reader (Synergy™ HT 96-well UV-visible spectrophotometer using the Gen5 data analysis software from BioTek Instruments, Inc., Winooski, VT, USA). The inhibition percentage was then calculated as follows:

$$\% \text{ inhibition} = \left(1 - \frac{B - b}{A - a}\right) \times 100$$

where *A* is the initial velocity of the reaction with the enzyme, *a* is the initial velocity of the reaction without the enzyme, *B* is the initial velocity of the enzyme reaction with the extract, and *b* is the initial velocity of the reaction with the extract but without the enzyme. The efficiency of flavonoids against α-glucosidase was also determined using the half maximal inhibitory concentration (IC50), and analyzed by a dose-response plot of flavonoids versus the inhibition percentage.

The α-amylase inhibitory activity was determined as previously described [29]. Briefly, the assay comprising of 30 mM *p*-nitrophenyl-α-D-maltohexaoside (50 μL) in a 50 mM phosphate buffer (pH 7.0) containing 200 mM KCl, 30 mg/mL of porcine pancreatic α-amylase (type VII, ≥10 unit/mg, 100 μL), and the sample extract (50 μL) was visualized at a wavelength of 405 nm using the microplate reader. The inhibition percentage was then calculated as above.

Acetylcholinesterases (AChE) inhibitory activities were determined as previously described [25]. The enzyme assay consisting of 20 ng of *Electrophorus electricus* AChE (1000 units/mg, 100 μL) in 50 mM KPB (pH 7.0), 16 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, 10 μL), 0.8 mM acetylthiocholine (40 μL) in 50 mM KPB (pH 7.0), and the extract (50 μL) was detected at a wavelength of 412 nm using the microplate reader. Butyrylcholinesterases (BChE) inhibitory activities were determined similarly to AChE. However, 100 ng equine serum BChE (≥10 units/mg protein, 100 μL) in 50 mM KPB (pH 7.0) containing 1 mM MgCl2 and 0.1 mM butyrylthiocholine (40 μL) in 50 mM KPB (pH 7.0) were used as the enzyme and substrate, respectively. The results were expressed as the inhibition percentage and the IC50 value, as mentioned above.

The beta-secretase (BACE-1) inhibitory activity was determined utilizing a BACE-1 activity detection kit (Sigma-Aldrich, St. Louis, MO, USA). The manufacturer's instructions were followed, and the results expressed as a percentage of BACE-1 inhibition, as above.

All the enzymes, chemicals, and reagents in the enzyme inhibitory assays were purchased from Sigma-Aldrich (St. Louis, MO, USA).

#### *2.5. Statistical Analysis*

All experiments were carried out in triplicate (*n* = 3) and expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Duncan's multiple comparison test and unpaired *t*-test were performed to determine the significant differences between values with *p* < 0.05.

#### **3. Results**

#### *3.1. Total Phenolic Contents (TPCs), Total Flavonoid Contents (TFCs), Total Anthocyanin Contents (TACs), and Phenolic Profiles*

The total phenolic contents (TPCs) of *K. coccinea* (Lem.) A.C. Sm. extracts (KCE) ranged between 0.83 and 43.61 mg gallic acid equivalent (GAE)/g dry weight (DW), while *K. heteroclita* (Roxb.) Craib extract (KHE) exhibited the TPCs ranging from 1.13 to 103.72 mg GAE/g DW (Table 3). Among the different fruit parts, the exocarp of KCE exhibited the significantly highest TPCs, followed by the core, mesocarp, and seed, respectively. However, for KHE, its core exhibited significantly higher TPCs than exocarp, mesocarp, and seed, respectively. Furthermore, significantly higher TPCs were observed in all the fruit parts of KHE compared to KCE.

Similarly, the total flavonoid contents (TFCs) in KCE ranged between 1.45 and 60.52 mg quercetin equivalent (QE)/g DW, with the exocarp providing the highest TFCs, followed by the core, while mesocarp and seed exhibited the lowest TFCs (Table 3). The core of KHE exhibited the highest TFCs, followed by the exocarp, mesocarp, and seed, respectively. In line with the TPC results, higher TFCs were detected in all the fruit parts of KHE (ranging from 1.40 to 206.92 mg QE/g DW) compared to KCE, in exception of seed.

Interestingly, the total anthocyanin contents (TACs) of KCE were only detected in the exocarp and mesocarp (0.02–0.27 mg cyanidin 3-*O*-glucoside equivalent (C3GE)/g DW), while TACs in KHE were found in the exocarp, mesocarp, and core (0.14–0.28 mg C3GE/g DW) (Table 3). The mesocarp of KCE exhibited higher TACs than its exocarp. Meanwhile, the exocarp and mesocarp of KHE exhibited insignificantly different TACs, but were significantly higher than that of the core.

Among all the phenolic acids and flavonoids investigated utilizing high performance liquid chromatography (HPLC), quercetin and naringenin were the only flavonoids identified in *Kadsura* spp. extracts (Table 4, Supplementary Figures S1 and S2). The most abundant flavonoid, naringenin,

was detected in all fruit parts of KCE (ranging from 1395.22 to 1972.65 mg/100 g DW) and KHE (ranging from 1739.91 to 1835.46 mg/100 g DW). Meanwhile, quercetin was only found in the exocarp of KCE (17.94 mg/100 g DW), and in the exocarp and mesocarp of KHE (51.19 and 12.59 mg/100 g DW, respectively). Among all the fruit parts, the exocarp and seed of KCE exhibited significantly higher naringenin content than the core and mesocarp, respectively. However, insignificant naringenin content differences were detected in all the fruit parts of KHE.

**Table 3.** Quantification of the total phenolic contents (TPCs), total flavonoid contents (TFCs), and total anthocyanin contents (TACs) in different fruit parts of *Kadsura coccinea* (Lem.) A.C. Sm. extract (KCE) and *Kadsura heteroclita* (Roxb.) Craib extract (KHE).

All data are expressed as mean ± standard deviation (SD) of triplicate experiments (*n* = 3). GAE: gallic acid equivalent; QE: quercetin equivalent; C3GE: cyanidin 3-*O*-glucoside equivalent; DW: dry weight; ND: not detected. Lower case and upper case letters indicate significant differences (*p* < 0.05) in different fruit parts of KCE and KHE, respectively, using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test; \* showed significant difference (*p* < 0.05) in the same fruit part when comparing between KCE and KHE using the unpaired *t*-test.

Anthocyanidins detected in *Kadsura* spp. extracts were identified as cyanidin and delphinidin (Table 4 and Supplementary Figure S3). Only the exocarp, mesocarp, and core of both *Kadsura* spp. extracts were found to contain these anthocyanidins, while none was detected in the seed. KCE exhibited cyanidin contents in the range of 0.34–1.03 μg/100 g DW, with its exocarp and mesocarp containing higher cyanidin content compared to its core. Similar results were observed in KHE (cyanidin content ranging from 0.03 to 1.00 μg/100 g DW), in which its exocarp and mesocarp provided higher cyanidin contents than its core. Delphinidin in KCE ranged between 0.42 and 4.02 μg/100 g DW, while KHE exhibited delphinidin contents in the range of 0.31–0.50 μg/100 g DW. The exocarp in both *Kadsura* spp. extracts provided higher delphinidin content than the mesocarp and core, respectively.

Anthocyanins (or glycosylated anthocyanidins) detected in *Kadsura* spp. extracts were identified as cyanidin 3,5-di-*O*-glucoside (cyanin), cyanidin 3-*O*-galactoside (ideain), cyanidin 3-*O*-glucoside (kuromanin), and cyanidin 3-*O*-rutinoside (keracyanin) (Table 4 and Supplementary Figure S4). Only the mesocarp of KCE contained cyanin (9.94 μg/100 g DW) and ideain (10.16 μg/100 g DW), while none was detected in the other fruit parts. Meanwhile, KHE exhibited ideain (2.11–53.36 μg/100 g DW), kuromanin (0.27–26.06 μg/100 g DW), and keracyanin (4.16–112.54 μg/100 g DW) in the exocarp, mesocarp, and core, while none was detected in the seed. The exocarp contained higher content for all three anthocyanins compared to the mesocarp and core, respectively. Besides, kuromanin and keracyanin were only detected in KHE, while none was found in KCE. On the other hand, cyanin was only found in KCE, while none was detected in KHE.

 KCE and KHE, respectively,comparingbetweenKCEand

 KHE using unpaired *t*-test.

 using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test; \* showed significant difference (*p* < 0.05) in the same fruit part when

*Foods* **2020**, *9*, 1222

#### *3.2. Antioxidant Activities*

Antioxidant activities of *Kadsura* spp. extracts were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric ion reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) assays (Table 5). KCE exhibited DPPH radical scavenging activities in the range of 0.04–2.73 μmol trolox equivalent (TE)/g DW, FRAP activities in the range of 3.14–300.44 μmol TE/g DW, and ORAC activities in the range of 41.93–957.80 μmol TE/g DW. Among all fruit parts, the exocarp provided significantly higher antioxidant activities than the core, mesocarp, and seed, respectively. This data corresponds with the TPCs, in which the highest TPCs were detected in the exocarp of KCE and the lowest in the seed. Similarly, KHE exhibited DPPH radical scavenging activities in the range of 0.04–6.48 μmol TE/g DW, FRAP activities in the range of 2.91–900.60 μmol TE/g DW, and ORAC activities in the range of 15.84–1330.23 μmol TE/g DW. Among all fruit parts, the core exhibited significantly higher antioxidant activities, followed by the exocarp, mesocarp, and seed, respectively. This data corresponds to the TPCs and TFCs, in which the highest TPCs and TFCs were detected in the core of KHE and the lowest in the seed. Interestingly, considering the abundant flavonoids detected in *Kadsura* spp., naringenin and quercetin, the results suggested that quercetin is a stronger antioxidant with higher DPPH radical scavenging activity of 8.07 μmol TE/g DW, FRAP activity of 13,519.54 μmol TE/g DW, and ORAC activity of 30,112.47 μmol TE/g DW than naringenin with lower DPPH radical scavenging activity of 0.02 μmol TE/g DW, FRAP activity of 30.98 μmol TE/g DW, and ORAC activity of 25,592.45 μmol TE/g DW.

**Table 5.** Antioxidant activities in the different fruit parts of *Kadsura coccinea* (Lem.) A.C. Sm. extract (KCE) and *Kadsura heteroclita* (Roxb.) Craib extract (KHE) and their detected flavonoids, naringenin and quercetin.

All data are expressed as mean ± standard deviation (SD) of triplicate experiments (*n* = 3). TE: trolox equivalent; DW: dry weight. Lower case and upper case letters indicate significant differences (*p* < 0.05) in different fruit parts of KCE and KHE, respectively, using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test; \* showed significant difference (*p* < 0.05) in the same fruit part when comparing between KCE and KHE using unpaired *t*-test.

#### *3.3. In Vitro Enzyme Inhibitory Activities*

*Kadsura* spp. extracts inhibited the key enzymes relevant to diabetes, including α-glucosidase and α-amylase, with different degrees of inhibition (Table 6). The α-glucosidase inhibitory activities in both *Kadsura* spp. extracts were detected in all fruit parts except for the seed. KCE exhibited α-glucosidase inhibitory activities in the range of 60.04–98.23% inhibitions using an extract concentration of 0.5 mg/mL, while those in KHE ranged between 89.72% and 91.84% inhibitions with the same extract concentration. It is also found that the exocarp and core of both *Kadsura* spp. extracts exhibited significantly higher α-glucosidase inhibitory activities than mesocarp, as indicated by the half maximal inhibitory concentration (IC50) (Table 6). Lower IC50 values indicated greater effectiveness of enzyme inhibitions. The exocarp and core of KCE exhibited significantly lower IC50 values (0.13 and 0.45 mg/mL, respectively) than mesocarp (0.56 mg/mL). Similar results were observed with KHE, in which its exocarp and core exhibited significantly lower IC50 values (0.06 mg/mL) than its mesocarp (the IC50 value of 0.64 mg/mL). Interestingly, the exocarp and core of KHE exhibited significantly lower IC50 values compared to those of KCE, while insignificantly different IC50 values were observed in the mesocarp of both *Kadsura* spp. extracts.

Likewise, KCE exhibited α-amylase inhibitory activities in the range of 12.18–51.85% inhibitions using an extract concentration of 2.5 mg/mL, while those in KHE ranged between 33.93% and 44.75% inhibitions when using the same extract concentration. When comparing the different fruit parts of KCE, its exocarp exhibited significantly higher inhibitory activities than its core, mesocarp, and seed, respectively. Similar results were observed in KHE, in which its exocarp and core possessed significantly higher inhibitory activities than its mesocarp, but no inhibitory activity was observed in its seed. Due to low inhibitory activities, the IC50 value against α–amylase of *Kadsura* spp. extracts is unavailable.

*Kadsura* spp. extracts were able to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-secretase (BACE-1), the key enzymes relevant to AD with different degrees of inhibition. The AChE inhibitory activities in KCE ranged between 20.40% and 87.44% inhibitions, while those of KHE ranged between 29.98% and 91.74% inhibitions using the extract concentration of 2.0 mg/mL. The exocarp and core of both *Kadsura* spp. extracts possessed significantly higher AChE inhibitory activities than mesocarp, while no inhibitory activities were detected in the seed. Due to the low inhibitory activities in the mesocarp, the IC50 values against AChE were only detected in exocarp and core. KCE exhibited the IC50 values of 0.88 mg/mL in the exocarp and 1.54 mg/mL in the core, while KHE exhibited the IC50 values of 0.52 mg/mL in the exocarp and 0.41 mg/mL in the core (Table 6). It is also suggested that the KHE exocarp and core exhibited significantly lower IC50 values than those of KCE.

Similar results were observed with BChE inhibition, in which only the exocarp, mesocarp, and core of both *Kadsura* spp. extracts possessed inhibitory activities, while none was detected in the seed. KCE exhibited BChE inhibitory activities in the range of 41.27–96.81% inhibitions, while those of KHE ranged between 63.67% and 99.15% inhibitions using the same extract concentration of 2.0 mg/mL. The exocarp and core of KHE exhibited significantly lower IC50 values (0.20 and 0.16 mg/mL, respectively) than its mesocarp (1.32 mg/mL) (Table 6). However, KCE exhibited significantly lower IC50 values in its exocarp (0.49 mg/mL) than its core (0.67 mg/mL). Interestingly, the exocarp and core of KHE exhibited significantly lower IC50 values than those of KCE.

Additionally, KCE exhibited BACE-1 inhibitory activity in the range of 4.72–56.34% inhibitions, while those of KHE ranged between 12.70% and 35.26% inhibitions using the same extract concentration of 2.0 mg/mL. In KCE, the mesocarp exhibited a significantly higher BACE-1 inhibitory activity than the exocarp, core, and seed, respectively. Similar results were observed in KHE, in which the mesocarp exhibited the significantly higher BACE-1 inhibitory activity, followed by the exocarp and seed, respectively. No inhibitory activity was observed in the core of KHE.

concentration = 2.0 mg/mL; N/A: not available; ND: not detected. The extract concentrations in each enzyme assay was chosen in attempt to differentiate the inhibitory activities among different fruit parts. Lower case and upper case letters indicate significant differences (*p* < 0.05) in different fruit parts of KCE and KHE, respectively, using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test; \* showed significant difference (*p* < 0.05) in the same fruit part when comparing between KCE and KHE using unpaired *t*-test.

#### **4. Discussion**

Two species of *Kadsura* spp., *K. coccinea* (Lem.) A.C. Sm. and *K. heteroclita* (Roxb.) Craib, are found abundantly in Thailand. *Kadsura* spp. has been reportedly used as a folk medicine for several decades. Despite their valuable medicinal applications, *Kadsura* spp. can only be naturally populated in the forest, which makes them unrecognizable for outsiders, while knowledge of their biological properties is also limited. The present research is the first comparative and comprehensive study of two *Kadsura* spp. in terms of their phytochemicals (total phenolic contents (TPCs), total flavonoid contents (TFCs), total anthocyanin contents (TACs), and phenolic profiles), and their health properties (in vitro antioxidant, anti-diabetic, and anti-Alzheimer's properties). The researchers found that (i) *Kadsura* spp. extracts were rich in flavonol (quercetin), flavanone (naringenin), anthocyanidins (cyanidin and delphinidin), and anthocyanins (cyanidin 3-*O*-glucoside (kuromanin), cyanidin 3-*O*-galactoside (ideain), cyanidin 3-*O*-rutinoside (keracyanin), and cyanidin 3,5-di-*O*-glucoside (cyanin)); (ii) the antioxidant activities of *Kadsura* spp. extracts were related to their phenolic contents; (iii) *Kadsura* spp. extracts exhibited strong inhibitory activities against key diabetic enzymes, including α-glucosidase and α-amylase; (iv) *Kadsura* spp. extracts provided effective in vitro anti-Alzheimer properties through acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and beta-secretase (BACE-1) inhibitions. This information supports the further development of *Kadsura* spp. as a potential source of phenolics with health benefits against the occurrence of diabetes and AD.

Flavonoids are diphenylpropanes-structured phytochemicals, which are mostly found in plant-based diets. The HPLC outcomes reveal that the identified flavonol and flavanone in *Kadsura* spp. extracts were quercetin (12.59–51.19 mg/100 g DW) and naringenin (1395.22–1972.65 mg/100 g DW), respectively. Naringenin is the most abundant flavonoid in *Kadsura* spp. extracts and is also widely distributed in citrus fruits such as kumquats, grapefruits, yuzu, and pomelo [30]. Quercetin is the most common flavonol and is found ubiquitously in mulberry, apricot, apple, and onion [31]. The amounts of quercetin and naringenin in the *Kadsura* spp. extracts corresponded with the TPCs and TFCs, in which the exocarp appeared to exhibit higher TPCs and TFCs than mesocarp. Similar results were observed in *K. coccinea* collected in China, which suggested that the TPCs of exocarp was double those detected in mesocarp [7]. Besides, anthocyanidins, the pigmented water-soluble compounds widely found in red to purplish-blue colored plants, were also identified in *Kadsura* spp. extracts as cyanidin and delphinidin. This data corresponds to the anthocyanins found in most vegetables and fruits, in which cyanidin (50%) is the most abundant anthocyanidin, followed by delphinidin (12%), peonidin (12%), pelargonidin (12%), malvidin (7%), and petunidin (7%) [32]. Furthermore, glycosylated anthocyanidins, namely anthocyanins, were identified in *Kadsura* spp. extracts as kuromanin, ideain, keracyanin, and cyanin. These findings correspond to a previous study, suggesting that since cyanidin is the most abundant anthocyanidin found in plants, its glycosylated form, kuromanin, is also the most common form of glycoside derivatives in colored plants [32]. Besides *Kadsura* spp., these anthocyanins are also abundantly detected in most berries. Cyanin is mostly found in mulberries, pomegranate, and wild blackberry [26,27,33,34], while ideain is found in blueberries and cranberries [35,36]. Keracyanin is detected in mulberries, raspberries, blackcurrants, and redcurrants [26,27,35]. In addition, kuromanin is abundantly found in strawberries, mulberries, blackcurrants, and raspberries [26,27,35,37].

These flavonoids appear to be responsible for the high antioxidant activities detected in *Kadsura* spp. extracts. The results on antioxidant activities of individual flavonoid suggested that quercetin is a stronger antioxidant than naringenin. However, due to high content of detected naringenin, this flavonoid might be responsible for high antioxidant activities in *Kadsura* spp. extracts. When comparing fruit parts, the exocarp and core of *Kadsura* spp. extracts exhibited higher antioxidant activities than the mesocarp and seed, respectively. These results correspond with the previous study, finding that antioxidants detected in exocarp of *K. coccinea* collected in China were two times higher than its mesocarp [38]. Comparably, the ORAC values of our *Kadsura* spp. extracts (675–2943 μmol TE/100 g FW) are similar to those of high antioxidant containing fruits, including fuji apple (2589 μmol TE/100 g FW), gala apple (2828 μmol TE/100 g FW), apricot (1110 μmol TE/100 g FW), hass avocado