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Organic-PLUS D4.5 Report on the chemical analysis and in-vitro trials page 1

Organic-PLUS - grant agreement No [774340]

Pathways to phase-out contentious inputs from organic agriculture in Europe

Deliverable: 4.5

Report on the chemical analysis and in-vitro trials on alternative natural plant products (in vitro anti-parasitic activity of plant sources, chemical

evaluation of alternative sources of vitamins)

Versions

Version: 1.0 (17 April 2020) Draft led by Federico Righi and Giulio Grandi Version: 1.1 (22 April 2020) Draft led by Federico Righi and Giulio Grandi Version: 1.2 (28 April 2020) Draft led by Carmen L. Manuelian Dennis Touliatos Federico Righi and Giulio Grandi Version: 1.3 (30 April 2020) Final version with feedback by Dennis Touliatos, Judith Conroy and Ulrich Schmutz

Funding

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No [774340]


Project Details:

Programme: H2020, SUSTAINABLE FOOD SECURITY – RESILIENT AND RESOURCE- EFFICIENT VALUE CHAINS

Call topic: SFS-08-2017, (RIA) Organic inputs – contentious inputs in organic farming Project

Title: Pathways to phase-out contentious inputs from organic agriculture in Europe Project

Acronym: Organic-PLUS

Proposal Number: 774340

Lead Partner: Coventry University, Centre for Agroecology, Water and Resilience, UK

Time Frame: 01/05/2018 – 31/04/2022

Authors:

Giulio Grandi, Federico Righi, Angela Marseglia, Afro Quarantelli, Andrea Summer, Rosario Pitino, Carmen L. Manuelian, Giorgia Mantovani, Alberto Sabbioni and Massimo De Marchi

Deliverable Details:

WP: 4 LIVESTOCK

Task(s): 4.4: Chemical analysis and in-vitro trials on alternative natural plant products Deliverable

Title: D4.5 Report on the chemical analysis and in-vitro trials

Lead beneficiary: SLU UNIPR

Involved Partners: UNIPD, SLU, UNIPR Deadline

for delivery: month 24, 30/04/2020 Date of

delivery: 30/04/2020


: Additional information related to the COVID-19 pandemic

As a consequence of the COVID-19 pandemic the laboratories of the University of Parma, Italy, where the chemical analysis of plant products and vitamins/immunostimulators was going to take place, were closed from 8-March 2020 - ongoing, with the following impact on the WP4 of the project. We will deliver this data as soon as the laboratories reopen and these tests can be carried out and analysed.

Task Activity/Deliverable delayed Mitigation

T.4.4: Chemical analysis and in vitro trials on alternative natural plant products (M1-24)

Deliverable D4.5; deadline 30th April; • On-time for antimicrobial

and antiparasitic in vitro testing.

• Delayed for chemical analysis on plant products and vitamins/immunostimulators.

• The methodologies for the above analyses are already established and tested.

Based on the last communications from the UNIPR administration, the laboratories are going to re- start their activities gradually during the month of May. All the analyses are currently expected to be performed by end of September.


Table of Contents

1. Summary ............................................................................................................. 6

2. Introduction ........................................................................................................ 7

3. Antimicrobial activity of essential oils and their nature identical active compounds against two Gram positive and two Gram negative bacteria .................... 8

3.1 Introduction .......................................................................................................... 8

3.2 Methodology ......................................................................................................... 8

3.3 Results................................................................................................................. 10

4. Testing the antiparasitic activity of plant products ............................................ 13

4.1 Introduction ........................................................................................................ 13

4.2 Methodology ....................................................................................................... 13

4.3 Results and discussion ......................................................................................... 14

5. Chemical analysis of plant feed additives, essential oil, essential oil active compounds and evaluation of their antioxidant properties ........................................ 17

5.1 Introduction ........................................................................................................ 17

5.2 Chemical characterisation of natural anti-infective (anti-bacterial and antiparasitic) .......................................................................................................................... 20

5.3 Chemical characterisation of polyphenols and vitamins determination (vit E, vit A, beta-caroten) ...................................................................................................... 22

5.4 Antioxidant capacity evaluation: in vitro test ....................................................... 24

5.5 References .......................................................................................................... 25

6. Conclusions ....................................................................................................... 28


List of Figures

Figure 4.1. Results of the comparison between the previously calculated LDA data and the result of the ddPCR technique adopted applied .......................................................................... 14

Figure 5.1. Some representative bioactive compounds present in essential oils .......................... 16

Figure 5.2. Chemical structures of polyphenols Hydroxybenzoic acids (Hba), Hydroxycinammic acids (Hca), Flavonoids (F), Chalcones (C), Stilbenes (S), Lignans (L). R: H, OH or OCH3 ................................................................................................................................ 17

Figure 5.3. Chemical structure of β-carotene and retinol ............................................................. 18

Figure 5.4. Chemical structure of α-tocopherol ........................................................................... 18

Figure 5.5. Oregano essential oil chromatograms obtained by GC/MS technique, DB-WAX column ....................................................................................................................................... 20

List of Tables Table 3.1. Minimal inhibitory concentration (MIC, %) of essential oils (EO) against bacterial strains of interest. Values are mean ± SD .................................................................................... 9 Table 3.2. Minimal inhibitory concentration (µg/ml) of nature identical compounds against bacterial strains of interest tested without a carrier ................................................................... 10 Table 4.1. Total amount of Haemonchus contortus eggs and larvae at different concentrations of plants extracts ................................................................................................ 14 Table 5.1 List of compounds in oregano essential oil ................................................................... 21 Table 5.2. Phenolic composition of Grape Pomace ...................................................................... 22


1. Summary

The in vitro testing and the chemical characterisation of natural plant products is an essential step

for their proper future use in vivo on animals. In general, both in vitro and in vivo trials results allow for the set of proper protocols to use those plant products from a therapeutic point of view (referring to antimicrobials) or as feed additive to be introduced in the daily ration as substitution of synthetic vitamins.

In the last years, there has been a growing interest in finding new antimicrobial agents from plants to prevent microbial resistance but also to provide natural tools to fight against infections in organic livestock. Many examples are already available concerning the use of natural products from plants (both plant extracts and essential oils) as antibacterial products. However, characterisation of plant secondary metabolites (PSM) is needed to identify active compounds and possible interactions with nutrients.

The antiparasitic properties of plants and plant-derived products are usually evaluated through traditional in vitro techniques including egg hatching/larval development tests after parasite exposure to the herbal product. Nowadays, molecular techniques are begun to be used with the same aim.

This report compares two detection methods: Larval Development Assay (LDA) and a new innovative technique droplet digital PCR (ddPCR) to evaluate the anthelmintic properties against the major parasitic threat to sheep production worldwide (Haemonchus contortus) of some herbal extracts (Malva sylvestris, Chamomilla recutita and Althea officinalis).

A wide variety of EOs are known to possess antimicrobial properties, and in many cases this activity is due to the presence of active constituents, mainly to isoprenes, such as monoterpenes, sesquiterpenes, and related alcohols, along with other hydrocarbons and phenols. The GC–MS with derivatisation to HPLC-UV and HPLC-MS/MS has been used to characterise the several essential oils (Peppermint oil, Oregano Spain oil, Cinnamon Ceylon leaf oil, Cloves oil, Thymus vulgaris leaf oil, Rosmarin Morocco oil, Melaleuca alternifolia leaf oil, Lavender altitude oil) because it provides at the same time a rapid, fully validated and omni- comprehensive fingerprinting method for crude plant material.

Natural antioxidants and plant extracts rich in polyphenols seem to be promising substrates of antioxidants, and have demonstrated their efficiency to prevent lipid oxidation in food products. Moreover, the phenolic profile of grape pomace extracts has been related to antioxidant and anti-inflammatory effects, the prevention of cardiovascular and intestinal diseases, anti-aging and anti-diabetic properties. The method described by Castello et al. (2018) for grape pomace will be used.

The present report describes some insights on the methods to chemically characterise essential oils and plant extracts with potential antimicrobial and antiparasitic capacity.


2. Introduction

Following the literature review on potential plant alternatives to the use of synthetic vitamins and immunostimulators, antibiotics and antiparasitics conducted in the first year of the Organic- PLUS project and reported in D4.2, several plant essential oils and extracts have been selected to be tested in in vitro studies. In particular, the antibacterial properties of several certified essential oils and essential active compounds have been tested for their antimicrobial activity through the minimal inhibitory concentration test (MIC), while the antiparasitic properties of three plant extracts have been evaluated though the classic Larval Development Assay (LDA) and through the innovative droplet digital Polymerase Chain Reaction (ddPCR).

The methodologies and protocols for the chemical analysis of these compounds and of polyphenols and antioxidant molecules within the plant products are discussed and some preliminary results and data are provided. Further analysis and results will be delivered in the near future with some delay as a consequence of the restrictions due to the sanitary emergency of COVID-19 outbreak.

This document is composed of 3 main sections:

i) essential oils and nature identical compounds testing as antimicrobials,

ii) plant extract testing as antiparasitic, and

iii) chemical analysis of essential oils, essential oils active compounds and antioxidant/immunostimulants.

Each section presents of a general introduction linking the work done with the previous report on the same topic (D4.2.), state the experimental issue, the methodology used, and a results and discussion subsection.


3. Antimicrobial activity of essential oils and their nature identical active compounds against two Gram positive and two Gram negative bacteria

3.1 Introduction

In the last decades, there has been a growing interest in researching and developing new antimicrobial agents from various sources both to combat microbial resistance but also to provide natural tools to combat infections in organic farming. Many examples already exist on the use of natural products from plants (both plant extracts and essential oils) as antimicrobial products. Among plant extracts, tannins were able to inhibit S. typhimurium growth in vitro (Van Parys et al., 2010). Quebracho (Schinopsis lorentzii) raw extract showed in vitro bacteriostatic effect on Salmonella Enteritidis and reduced bacterial excretion in broilers (Redondo et al., 2014), and showed in vitro antimicrobial activity against Salmonella enteriditis and S. gallinarum (Prosdócimo et al., 2010). Among essential oils, in vitro studies revealed that various essential oils can disrupt the quorum sensing of pathogenic bacteria reducing their aggressiveness (Bjarnsholt et al., 2005; Choo et al., 2006; Zhou et al., 2013; Kerekes et al., 2013; Alvarez et al., 2014). Carvacrol and eugenol inhibited specific virulence determinants in Pectobacteria (Joshi et al., 2016) and cinnamaldehyde and its derivatives have been successfully employed in acquaculture against Vibrio harveyi in brine shrimp (Niu et al., 2006; Brackman et al., 2008). Moreover, essential oils from oregano, carvacrol, thymol, and trans-cinnamaldehyde presented good antimicrobial activity against pathogens inducing mastitis as well as Manuka honey, even if at high doses (Michigan State University, 2012-2016). Considering the importance of the topic to the animal production sector, a great attention has been paid to antimicrobial activity screening and evaluating methods. Nowadays, there are several methods that are well known and commonly used (bioassays such as disk-diffusion, well diffusion and broth or agar dilution) and other that still need to be further developed (flow cytofluorometric and bioluminescent methods) and are not widely employed. Among the various methods, the minimum inhibitory concentration (MIC) is a laboratory test that measures in vitro the minimum concentration of an antimicrobial product needed to inhibit the growth of a specific microorganism. This parameter differs according to the bacteria species and strains tested and the laboratory conditions. Data are available on commonly used essential oils on several swine bacterial pathogens (Omonjio et al., 2018) but also on the MIC of many pathogenic zoonotic and zootechnical bacteria, including Escherichia coli O157: H7 and Brachyspira spp. (Si et al., 2006; Navarrete et al., 2010; Vande Maele et al., 2016). The present section (section 3) reports the antimicrobial activity of eight essential oils (EO) and three nature identical compounds (NIC) that were tested in a period between November 2019 and March 2020 by the O.U. of Animals Infective Disease of the Department of Veterinary Science of the University of Parma.


3.2 Methodology

3.2.1 Products tested

The following EO and NIC products were purchased from Biotrade di Malavasi Claudio S.n.c. (Mirandola, MO, Italy) and tested:

For EO:

• Melaleuca alternifolia leaf oil (Tea tree oil) – trade code OE5370 • Mentha × piperita oil – trade code OE0228 • O.E Origano Spagna – trade code OE0375 • O.E Cannella Ceylon foglie – trade code OE0576 • O.E Garofano Chiodi – trade code OE0898 • Thymus vulgaris leaf oil – trade code OE0969

For NIC:

• O.E Lavanda altitude – trade code OE0985 • O.E Rosmarino Marocco – trade code OE1318 • Carvacrol – code S0404041 • Cinnamic aldehyde – code S0400710 • Terpineol – code S0400648

The antimicrobial activity of the selected EO and NIC was tested against four reference bacterial strains of veterinary interest, two Gram positive (Staphylococcus aureus ATCC 25923, Methicillin Resistant Staphylococcus aureus (MRSA) ATCC 43300) and two Gram negative (E. coli ATCC 25922 and Salmonella Typhimurium ATCC 14028) through a MIC assay in different media: Müller Hinton broth (MHB), MHB with 0,5% of Tween 20, and MHB with 0.5% of Tween 80 (Cermelli et al., 2008; Shelz et al., 2006; Jiang et al., 2011). A control media (pure MHB) was used to ensure the viability of the bacteria strains used. Only for Staphylococcus aureus assay the concentration of Tween 20 was lowered at 0,25% due to a growth difficulty of the control group. Each assay was done in triplicate in three independent experiments.

3.2.2 Inoculum preparation

Four or five bacterial colonies from solid fresh cultures of each tested strain were inoculated in sterile tubes with Müller Hinton broth and incubated at 37°C for 24 hours. After incubation, the bacterial suspension was centrifuged at 2000 rpm at 4°C for 20 minutes in order to separate the bacterial pellet from the supernatant. Then, the pellet was resuspended in phosphate buffer (PB) 10 mM pH 7. The bacterial suspension was adjusted in PB to obtain an optical density (OD) value at 600 nm in a 1 cm light path cuvette in the range 0.08–0.13, approximately equivalent to a 108 CFU/ml suspension. This suspension was further diluted 1:100 in sterile MHB (depending on the assay, MHB has been added with 0.25-0.5% of Tween 20 or 80.) Fifty microliters of the bacterial suspension containing 106 CFU/ml were inoculated into each well to obtain a final concentration of 5 × 105

CFU/ml. Bacterial suspensions were investigated with a Biophotometer plus (Eppendorf, Hamburg, Germany) spectrophotometer (λ = 600 nm). All the microbiological assays were performed within 30 min after the inoculum standardisation.


3.2.3 Minimal inhibitory concentration (MIC) assay of EO

The MIC assay was evaluated following the CLSI guidelines (CLSI, 2018b) protocol with some modifications. The EO bought was diluted to a concentration of 6.25% in MHB or MHB added to Tween 20 or 80 at 0.25%-0.5% in order to obtain the different stock solutions. Two-fold dilutions 3.125%-0.0061% range of the stock solution were performed in a 96-well microtiter plate (Greiner, Milan, Italy); in each well 50 microliters of the bacterial suspension containing 106 CFU/mL were added to obtain a bacterial concentration of 5 × 105 CFU/ml. Growth and sterility controls were performed for each bacterial strain and for each tested compound. Plates were incubated for 24 h at 37°C in aerobic atmosphere. The MIC is the lowest concentration of the tested compound at which there was no bacterial growth. Each assay was repeated for three replicates in three independent experiments. 3.2.4 Minimal inhibitory concentration (MIC) assay of NIC The MIC assay was evaluated following the CLSI guidelines (CLSI, 2018b) protocol with minor modifications. The NIC bought was diluted to a stock concentration of 102.4 mg/ml in DMSO and subsequently each compound was further diluted to a concentration of 204.8 µg/ml in MHB or MHB added to Tween 20 or 80 at 0.25%-0.5% in order to obtain the different solutions to use. Two-fold dilutions 102.4 µg/ml range of the stock solution were performed in a 96-well microtiter plates (Greiner, Milan, Italy); in each well 50 microliters of the bacterial suspension containing 106 CFU/ml was added to obtain a bacterial concentration of 5 × 105 CFU/ml. Growth and sterility controls were performed for each bacterial strain and for each tested compound. Plates were incubated for 24 h at 37°C in aerobic atmosphere. The MIC is the lowest concentration of the tested compound at which there was no bacterial growth. Each assay was repeated for three replicates in three independent experiments.

3.3 Results

The MIC (%) of EO are summarised in Table 3.1. For all the tested EO, MIC value are lower than 7% and for most of them was lower than 1%. For all the EO and tested bacteria, MIC was lower in presence of Tween 20 For E. coli, MIC average of all EO was 1.02% without emulsifier, 0.98% with Tween 20 and 1.7% with Tween 80. For S. Typhimurium, MIC average of all EO was 1.5% without emulsifier, 0.9% with Tween 20 and 2.28% with Tween 80. For S. aureus, MIC average of all EO was 1.2% without emulsifier, 0.2% with Tween 20 and 2.26% with Tween 80. For MRSA, MIC average of all EO was 1.2% without emulsifier, 0.2% with Tween 20 and 2.4% with Tween 80. The most sensible bacterium to EO was S. aureus, conversely, the most resistant was S. Typhimurium. For what concern the single EOs, lowest MIC average among all the tested strains was found for Oregano oil (0.09% ± 0.05%), followed by Thymus oil (0.19% ± 0.15%), Tea tree oil (0.29% ± 0.15%) and Rosemary oil (0.8% ± 0.48%). On the other hand, the highest MIC average value were found for Lavender oil and Clove oil (2.57% ± 1.01% and 2.57% ± 2.3%, respectively) followed by Cinnamon oil (2.4% ± 2.5%) and Mentha oil (1.75% ± 1.09%).


Table 3.1: Minimal inhibitory concentration (MIC, %) of essential oils (EO) against bacterial strains of interest. Values are mean ± SD.

EO emulsifier E. coli ATCC

25922 S. Typhimurium

ATCC 14028 S. aureus

ATCC 25923 MRSA ATCC

43300

Tea tree oil None 0.2 ± 0.0 0.2 ± 0.0 0.622 ± 0.21 0.4 ± 0.0

Tween 20 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 Tween 80 0.2 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.4 ± 0.0

Rosemary oil

None 0.71 ±0.18 2.1 ± 0.75 0.9 ± 0.28 1.15 ± 0.43 Tween 20 0.44 ± 0.13 1.42 ± 0.35 0.8 ± 0.0 0.2 ± 0.0 Tween 80 0.8 ± 0.0 0.8 ± 0.0 0.8 ± 0.0 0.9 ± 0.28

Oregano oil

None 0.025 ± 0.0 0.1 ± 0.0 0.025 ± 0.0 0.1 ± 0.0 Tween 20 0.1 ± 0.0 0.1 ± 0.0 0.05 ± 0.0 0.025 ± 0.0 Tween 80 0.1 ± 0.0 0.18 ± 0.04 0.18 ± 0.04 0.15 ± 0.05

Thymus oil

None 0.05 ± 0.0 0.1 ± 0.0 0.08 ± 0.16 0.1 ± 0.0 Tween 20 0.14 ± 0.05 0.2 ± 0.0 0.05 ± 0.0 0.05 ± 0.0 Tween 80 0.44 ± 0.13 0.27 ± 0.1 0.4 ± 0.0 0.4 ± 0.0

Cinnamon oil

None 1.07 ± 0.4 1.42 ± 0.35 0.98 ± 0.35 1.5 ± 0.27 Tween 20 0.38 ± 0.07 0.4 ± 0.0 0.11 ± 0.03 0.1 ± 0.0 Tween 80 6.25 ± 0.0 4.18 ± 2.45 6.25 ± 0.0 6.25 ± 0.0

Mentha oil

None 1.6 ± 0.0 3.47 ±1.04 1.77 ± 0.51 1.6 ± 0.0 Tween 20 2.79 ± 1.65 2.44 ± 0.8 0.025 ± 0.0 0.2 ± 0.0 Tween 80 0.98 ± 0.35 3.125 ± 0.0 1.06 ± 0.4 1.94 ± 0.67

Lavender oil

None 2.93 ± 0.5 2.93 ± 0.5 2.76 ± 0.66 3.1 ± 0.0 Tween 20 3.1 ± 0.0 3.1 ± 0.0 0.8 ± 0.0 0.1 ± 0.0 Tween 80 3.1 ± 0.0 3.1 ± 0.0 2.76 ± 0.66 3.1 ± 0.0

Clove oil

None 1.6 ± 0.0 1.6 ± 0.0 2.43 ± 0.79 2.1 ± 0.75 Tween 20 0.75 ± 0.13 0.8 ± 0.0 0.1 ± 0.0 1.11± 0.48 Tween 80 1.6 ± 0.0 6.25 ± 0.0 6.25 ± 0.0 6.25 ± 0.0

The MIC (µg/ml) of NIC are summarised in Table 3.2. Between all the tested NIC, the highest MIC was found for Terpineol against all the tested strains (2048 µg/ml). The lowest MIC was instead found for Cinnamic aldehyde against S. aureus without any emulsifier (128 µg/ml), while against the others tested strains MIC was higher (256 µg/ml). For Carvacrol, MIC without emulsifier was found identical for all the tested strains and equivalent to 256 µg/ml.

Table 3.2: Minimal inhibitory concentration (µg/ml) of nature identical compounds against bacterial strains of interest tested without a carrier.

EO E. coli ATCC

25922 S. Typhimurium

ATCC 14028 S. aureus

ATCC 25923 MRSA ATCC

43300 Carvacrol 256 256 256 256

Cinnamic aldehyde 256 256 128 256 Terpineol 2048 2048 2048 2048

3.3 References Alvarez, M. V., Ortega-Ramirez, L. A., Gutierrez-Pacheco, M. M., Bernal-Mercado, A. T., Rodriguez-

Garcia, I., Gonzalez-Aguilar, G. A., Ayala-Zavala, J. F. (2014). Oregano essential oil-pectin edible films as anti-quorum sensing and food antimicrobial agents. Frontiers in Microbiology, 5(DEC), 1–7. https://doi.org/10.3389/fmicb.2014.00699

Bjarnsholt, T., Jensen, P. Ø., Rasmussen, T. B., Christophersen, L., Calum, H., Hentzer, M., Givskov, M. (2005). Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology, 151(12), 3873–3880.


https://doi.org/10.1099/mic.0.27955-0 Brackman, G., Defoirdt, T., Miyamoto, C., Bossier, P., Van Calenbergh, S., Nelis, H., & Coenye, T.

(2008). Cinnamaldehyde and cinnamaldehyde derivatives reduce virulence in Vibrio spp. by decreasing the DNA-binding activity of the quorum sensing response regulator LuxR. BMC Microbiology, 8, 1–14. https://doi.org/10.1186/1471-2180-8-149

Cermelli C, Fabio A, Fabio G, Quaglio P. (2008). Effect of eucalyptus essential oil on respiratory bacteria and viruses. Current microbiology, 56(1):89-92.

Choo, J. H., Rukayadi, Y., & Hwang, J. K. (2006). Inhibition of bacterial quorum sensing by vanilla extract. Letters in Applied Microbiology, 42(6), 637–641. https://doi.org/10.1111/j.1472-765X.2006.01928.x

Clinical and Laboratory Standard Institute (CLSI) (2018b). Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100. 28th ed.

Jiang Y, Wu N, Fu Y-J, Wang W, Luo M, Zhao C-J, et al. (2011). Chemical composition and antimicrobial activity of the essential oil of Rosemary. Environmental toxicology and pharmacology, 32(1):63-8.

Joshi, J. R., Khazanov, N., Senderowitz, H., Burdman, S., Lipsky, A., & Yedidia, I. (2016). Plant phenolic volatiles inhibit quorum sensing in pectobacteria and reduce their virulence by potential binding to ExpI and ExpR proteins. Scientific Reports, 6(March), 1–15. https://doi.org/10.1038/srep38126.

Kerekes, E. B., Deák, É., Takó, M., Tserennadmid, R., Petkovits, T., Vágvölgyi, C., & Krisch, J. (2013). Anti-biofilm forming and anti-quorum sensing activity of selected essential oils and their main components on food-related micro-organisms. Journal of Applied Microbiology, 115(4), 933–942. https://doi.org/10.1111/jam.12289

Navarrete, P., Toledo, I., Mardones, P., Opazo, R., Espejo, R., & Romero, J. (2010). Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolates. Aquaculture Research, 41(10). https://doi.org/10.1111/j.1365-2109.2010.02590.x

Niu, C., Afre, S., & Gilbert, E. S. (2006). Subinhibitory concentrations of cinnamaldehyde interfere with quorum sensing. Letters in Applied Microbiology, 43(5), 489–494. https://doi.org/10.1111/j.1472-765X.2006.02001.x

Omonijo, F. A., Ni, L., Gong, J., Wang, Q., Lahaye, L., & Yang, C. (2018). Essential oils as alternatives to antibiotics in swine production. Animal Nutrition, 4(2), 126–136. https://doi.org/10.1016/j.aninu.2017.09.001

Prosdócimo, F., Batallé, M., Sosa, N., De Franceschi, M., & Barrios, H. (2010). Determinación in vitro del efecto antibacteriano de un extracto obtenido de quebrancho colorado, Schinopsis lorentzii. InVet, 12(2), 139–143. https://doi.org/DOI: 10.1055/s-2007-969152

Redondo, L. M., Chacana, P. A., Dominguez, J. E., & Fernandez Miyakawa, M. E. (2014). Perspectives in the use of tannins as alternative to antimicrobial growth promoter factors in poultry. Frontiers in Microbiology, 5(MAR), 1–7. https://doi.org/10.3389/fmicb.2014.00118

Schelz Z, Molnar J, Hohmann J. (2006). Antimicrobial and antiplasmid activities of essential oils. Fitoterapia, 77(4):279-85.

Si, W., Gong, J., Tsao, R., Zhou, T., Yu, H., Poppe, C., … Du, Z. (2006). Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. Journal of Applied Microbiology, 100(2), 296–305. https://doi.org/10.1111/j.1365-2672.2005.02789.x

Vande Maele, L., Heyndrickx, M., Maes, D., De Pauw, N., Mahu, M., Verlinden, M., Boyen, F. (2016). In vitro susceptibility of Brachyspira hyodysenteriae to organic acids and essential oil components. Journal of Veterinary Medical Science, 78(2), 325–328. https://doi.org/10.1292/jvms.15-0341

Van Parys, A., Boyen, F., Dewulf, J., Haesebrouck, F., & Pasmans, F. (2010). The Use of Tannins to Control Salmonella Typhimurium Infections in Pigs. Zoonoses and Public Health, 57(6), 423–428. https://doi.org/10.1111/j.1863-2378.2009.01242.x

Zhou, L., Zheng, H., Tang, Y., Yu, W., & Gong, Q. (2013). Eugenol inhibits quorum sensing at sub-inhibitory concentrations. Biotechnology Letters, 35(4), 631–637. https://doi.org/10.1007/s10529-012-1126-x


4. Testing the antiparasitic activity of plant products

4.1 Introduction

Livestock parasites can be classified as external parasites (ectoparasites, usually arthropods) or internal parasites (endoparasites, both protozoa and helminths). Both ecto- and endoparasites have detrimental effects on animals’ performance, production and health, the first being also able to act as vectors for several vector-borne diseases. Despite the emergence of drug-resistant parasites worldwide, control of parasites in conventional farming is still based on synthetic drugs that moreover cannot be used in organic farming. In face of these problems and developments, plant products have become important alternatives for the prevention and treatment of the gastrointestinal nematode infections (Pisseri et al., 2013). The use of plants and plant-derived products as endo- and ectoparasiticides is in fact part of the traditional medicine culture, being the only way to treat animals until mid-20th century (French, 2018).

Small farmers around the world rely on plants to treat parasitic diseases of livestock. For example, in northern Europe they still rely on old “traditional” pastures rich in medicinal herbs and legumes for the perceived anthelmintic qualities of specific wild plants (French, 2018). This traditional knowledge is considered as a branch of ethnoveterinary medicine (Mathias, 2004).

Based on the fact that plants produce several thousand compounds including antiparasitic substances (French, 2018; Wink, 2010), exploiting the diversity and bioactivity of plant secondary metabolites may be a viable alternative to treat ecto- and endoparasites. However, the testing procedures of plants and their compounds have until now been heterogeneous, often leading to the generation of confusing and therefore non-applicable results. Plants and plant products known for their antiparasitic activities can show also some negative (antinutritional, toxic) and positive (immunomodulatory) side effects. For this reason, it is fundamental to identify active compounds in order to avoid the toxic effects related to whole- plant administration (Athanasiadou et al., 2007). For example, the majority of compounds with anthelmintic properties from forages and plants are plant secondary metabolites (PSM).

The antiparasitic properties of plants and plant-derived products are usually evaluated through traditional in vitro techniques including egg hatching/larval development tests after parasite exposure to the herbal product. Considering the development of molecular techniques, innovative, technologically-advanced, and theoretically more accurate tests should also be developed and made available to reduce observational bias. Aims of the present work were to evaluate some herbal extracts (Malva sylvestris, Chamomilla recutita and Althea officinalis) for their anthelmintic properties against the major parasitic threat to sheep production worldwide (Haemonchus contortus) and to compare two detection methods for measuring their anthelminthic effects: a standard method (larval development assay) and a new innovative technique (Droplet digital PCR, ddPCR).

4.2 Methodology

4.2.1 Preparation of plant products and parasites

Plants products were obtained from Malva sylvestris, Chamomilla recutita and Althaea officinalis. Finely milled plant products (2 g) were incubated with boiled distilled water cooled to 90°C for 15 min. Suspensions were centrifuged at 3500 × g for 5 min, then supernatants were filtered (245-μm), stored at 4°C and used within 24 h. Parasite eggs of Haemonchus contortus were obtained from faecal samples collected rectally from experimentally infected lambs. Parasitologically-naïve individuals were infected orally with 5000-6000 third-stage (L3) of an anthelmintic-susceptible strain of H. contortus and reared indoor to prevent other infections. Faecal samples were collected from 28 to 60 days post infection. All


experimental procedures involving animals were previously approved by the Ethical committee of the Institute of Parasitology of the Slovak Academy of Sciences (Kosice, Slovakia) that contributed to part of this study.

4.2.2 Larval development assay (LDA)

Larval development assay (LDA) was performed in 96-well microtiter plates in 12 concentrations. In particular, the concentration tested were 40, 20, 10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, 0.078, 0.039, 0.019 mg/ml of the aqueous extracts obtained as above. Each well contained 10 µl of H. contortus egg suspension (approximately 70-100 eggs) containing Amphotericin B, 20 µl of culture medium (yeast extract plus Earl’s balanced salt solution) and 120 µl aqueous plant extract. Controls (culture medium only) were also included. The plates were incubated for 7 days at 27°C and the incubation was terminated by adding 10 µl of Lugol’s solution in each well. The proportion of eggs (unhatched), as well as L1-L3 larvae, in each well after incubation were counted using an inverted microscope (Váradyová et al., 2018).

4.2.3 Analysis of LDA material with droplet digital PCR (ddPCR)

The same plates were then sent to the Swedish University of Agricultural Sciences (SLU) with the aim of evaluating the correspondence between the quantity of larvae observed in LDA by microscopy with the quantity of Internal transcribed spacer-2 (ITS-2) amplicon DNA copies of H. contortus. This assessment was done for each different concentrations of plant extracts and using the ddPCR (BioRad) for their detection. The DNA was extracted from frozen larval cultures from each well, using NucleoSpin XS Tissue kit according to manufacturer’s instructions (Macherey Nagel, Germany). The ddPCR was run on extracted DNA samples using primers UnivHC (Elmahalawy et al., 2018). Sample were placed in 96-well plates with total volume of 22 μl following the guidelines issued by the manufacturer (BioRad). Droplets were generated and dispensed into a new 96-well plate using an automated droplet generator (QX200, BioRad). The new plate was sealed and transferred into a thermal cycler (MyCyclerTM Thermal Cycler). The PCR conditions were as follows: a single cycle of 95°C for 10 min, and 40 cycles of 94°C for 30 sec followed by 40 cycles of 60°C for 1 min and then a single cycle of 98°C to deactivate the enzyme. After the amplification step, the plate containing the droplets was loaded into the droplet reader (QX200, BioRad) and further analysed using QuantaSoft software (Baltrušis et al., 2019).

4.3 Results and discussion

Results of the effects of the different plant extracts tested at the various dilutions that were recorded through Larval Development Assay (LDA) are reported in Table 1. No differences between the plants extracts themselves and between the plant extracts and the control can be observed at the lower concentrations (i.e. dilutions from 0.019 mg/ml to 10 mg/ml). However, even if no statistical test could be performed on this regard, the mean number of eggs and larvae compared to the control appeared to be sensibly lower in all the three tested extracts at 10 mg/ml. A shown in the Table 4.1, starting from the concentration of 1.25 mg/ml it has been possible to observe an interesting trend where higher numbers of H. contortus stages in Chamomilla recutita treatment in comparison to Malva sylvestris and Althaea officinalis were recorded, witnessing a lower anthelmintic effect of the former plant extract. At the concentration of 20 mg/ml, the difference previously described became statistically significant. The comparison between the standard method based on the larval and egg count after exposure to the treatment and ddPCR technique was tested through the regression method and is presented in Figure 4.1. The best fitting curve was the logarithmic function whose equation (y = 4.7201 ln(x) + 3.7068) showed the higher coefficient of determination (R2=0.385).


The literature is rich in studies on the use of plant and plant-derived substances as anti-parasitics (Várady, 2018). Their use is often documented in traditional medicine. Despite the increasing amount of published studies, standardised procedures to test their activity and effectiveness are lacking, as well as an agreement in the scientific community that could lead to a recognised status of anti-parasitic products for at least some of the plants studied. Malva sylvestris has been indicated to possess considerable bioactivities as antiseptic agent (Razavi et al., 2011). However, no antiparasitic effect has been documented. In the present study we observed a good antiparasitic effect documented by reduction of eggs and larvae form at the dilution of 20 mg/ml. This effect was similar to the one observed from Althaea officinalis and overcame the one of Chamomilla recutita. The two methods tested to evaluate the antiparasitic activity of the extracts appeared not to be strongly related, as indicated by the coefficient of determination of the logarithmic equation describing the relationship between the results of the methods tested. More studies are needed to improve the methods for the detection and assessment of anthelmintic properties of new substances.

Table 4.1: Total amount of Haemonchus contortus eggs and larvae at different concentrations of plants extracts.

P.E. Concentration (mg/ml)*

40

20

10

5

2.5

1.25

0.62

0.31

0.15

0.078

0.039

0.019

Mean number of eggs and larvae

Malva sylvestris 0.0 1.5 a 4.0 31.0 31.5 25.5 34.5 42.5 40.0 37.0 41.3 44.5 Chamomilla recutita

0.0

22.0 b

16.0

46.5

43.0

46.5

36.0

44.5

41.0

37.0

43.5

49.5

Althaea officinalis 0.0 4.0 a 12.5 34.5 36.5 41.5 29.0 37.5 46.5 50.0 38.5 52.0

Significance - 0.014 0.340 0.214 0.428 0.170 0.906 0.725 0.864 0.170 0.656 0.391

Control 34 43 44 40 32 37 47 42 38 45 46 39

* Plant Extract concentration.

Figure 4.1: Results of the comparison between the previously calculated LDA data and the result of the ddPCR technique adopted applied.

12000 10000 8000 6000

Copy number

4000 2000 0

0

70 60 50

f(x) = 4.72 ln(x) + 3.71 40 R² = 0.38 30 20 10

Num

ber o

f par

asite

form


4.4 References

Athanasiadou, S., Githiori, J., & Kyriazakis, I. (2007). Medicinal Plants for Helminth Parasite

Control: Facts and Fiction. Animal 1 (9): 1392–1400. https://doi.org/10.1017/S1751731107000730.

Baltrušis, P., Halvarsson, P., & Höglund, J. (2019). Molecular detction of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach. Parasitology Research, 118, 2901-2907.

Elmahalawy, S.T., Halvarsson, P., Skarin, M., Höglund, J. (2018) Droplet digital polymerase chain reaction (ddPCR) as a novel method for absolute quantification of major gastrointestinal nematodes in sheep. Veterinary Parasitology, 261: 1-8. doi: 10.1016/j.vetpar.2018.07.008.

French, K. E. (2018). Plant-Based Solutions to Global Livestock Anthelmintic Resistance. Ethnobiology Letters 9 (2): 110. https://doi.org/10.14237/ebl.9.2.2018.980.

Mathias, E. (2004). Ethno veterinary medicine: harnessing its potential. Veterinary Bulletin 74(8):27–37.

Pisseri, F., de Benedictis, C., Roberti di Sarsina, P., & Azzarello, B. (2013). Sustainable Animal Production, Systemic Prevention Strategies in Parasitic Diseases of Ruminants. Alternative & Integrative Medicine, 2.

Razavi, S., Zarrini, G., Molavi, G., & Ghasemi, G. (2011). Bioactivity of Malva Sylvestris L., a medicina plant from Iran. Iranian Journal of Basic Medical Sciences, 14, 574-579.

Váradyová, Z., Pisarčíková, J., Babják, M., Hodges, A., Mravčáková, D., Kišidayová, S., Várady, M. (2018). Ovicidal and larvicidal activity of extracts from medicinal-plants against Haemonchus contortus. Experimental Parasitology, 195, 71-77.

Wink, M. (2010). Introduction. In Functions and Biotechnology of Plant Secondary Metabolites, edited by M.Wink, pp. 1–20. Blackwell Publishing Ltd., Oxford, UK.


5. Chemical analysis of plant feed additives, essential oil, essential oil active compounds and evaluation of their antioxidant properties

5.1 Introduction

One of today’s challenges, in line with the One-Health principles, is to reduce the use of drugs and antibiotics in humans and livestock because of the rise of antibiotic resistance (EFSA and ECDC, 2013; Dhama et al., 2013). The reduction of the use of antimicrobials in food-producing animals, replacing them where possible and rethinking the livestock production system, is essential for the future of animal and public health (EFSA, 2012; Murphy et al., 2017). For these reasons, several functional feed additives need to be evaluated in order to increase the health status and reduce the need for antibiotics (Windisch et al., 2008 and Dell’Anno et al., 2020).

In this scenario essential oils can be a very interesting natural source of new compounds with biological activity such as antimicrobial properties that could be used as functional ingredients. The constituents of plant EO fall mainly into two distinct chemical classes: terpenes and phenylpropanoids. Terpene compounds can be divided into two main categories: terpenes with a hydrocarbon structure, mainly the mono-, sesqui-, and diterpenes and their oxygenated derivatives, for instance, alcohols, oxides, aldehydes, ketones, phenols, acids, esters, and lactones. A wide variety of EO are known to possess antimicrobial properties, and in many cases this activity is due to the presence of active constituents, mainly to isoprenes, such as monoterpenes, sesquiterpenes, and related alcohols, along with other hydrocarbons and phenols. In particular, terpene hydrocarbons and oxygenated terpenes exhibit pronounced antimicrobial activity. The p-cymene, limonene, menthol, eugenol, anethole, estragole, geraniol, thymol, γ-terpinene, and cinnamyl alcohol are among the examples of some constituents of EO with antimicrobial activity (Figure 5.1) (Chouhan et al., 2017).

Figure 5.1: Some representative bioactive compounds present in essential oils (Chouhan et al., 2017).


Another issue of this study is focused on the vitamins in animal feed. Vitamin E, β-carotene, and vitamin A are important fat-soluble micronutrients required for animal growth and maintenance of the immune system (Weiss et al., 1997; NRC, 2001). Both vitamin E and β-carotene play a major role in biological systems as antioxidants, preventing oxidative damage caused by reactive oxygen species. The β-Carotene is an important precursor of vitamin A and is converted to retinol in the intestinal mucosa (Chew, 1987 and Ghaffari et al., 2019). Vitamin E is the antioxidant most commonly used in animal nutrition, but it presents some drawbacks, including its synthetic origin, its limited bioefficiency when n-3 PUFA intake is too high, its potential antioxidant prooxidant action, and its nonhomogeneous distribution between tissues (Brenes et al., 2008). Research for new bioefficient antioxidants has particularly focused on natural antioxidants to respect consumer’s concerns over safety and toxicity. Plant extracts rich in polyphenols are good candidates because they are easily obtained from natural sources and they efficiently prevent lipid oxidation in food products. These natural antioxidants from plant materials are mainly polyphenols (phenolic acids, flavonoids, anthocyanins, lignans and stilbenes), carotenoids (xanthophylls and carotenes) and vitamins (vitamin E and C). In plant foods, several hundred polyphenols have been identified, which are classified according to the number of phenol rings and their linkage, as presented in Figure 5.2. Polyphenols are mostly present as glycosides and, partly, also as esters. Their activity depending on the number and position of the OH-groups and on the pH (Belitz et al., 2009).

Figure 5.2: Chemical structures of polyphenols Hydroxybenzoic acids (Hba), Hydroxycinammic acids (Hca), Flavonoids (F), Chalcones (C), Stilbenes (S), Lignans (L). R: H, OH or OCH3 (Belitz et al., 2009).

Carotenoids are polyene hydrocarbons biosynthesised from eight isoprene units (tetraterpenes) and, correspondingly, have a 40-C skeleton. They provide the intensive yellow, orange or red colour of a great number of foods of plant origin. They are synthesised only by plants. However, they reach animal tissues via the feed (pasture, fodder) and can be modified and deposited there. The well-studied and the most important of all carotenoids is represented by β-carotene (Figure 5.3, A) also called pro-vitamin A since it is a precursor for vitamin A (retinol) (Figure 5.3, B) (Belitz et al., 2009).


Figure 5.3: Chemical structure of β-carotene (A) and retinol (B) (Belitz et al., 2009).

Moreover, tocol-related compounds, tocopherols (α, β, δ, γ) and tocotrienols, which belong to the vitamin E family, are particularly important bioactive constituents in vegetable oils mainly due to their antioxidative effects. α-Tocopherol shown in figure 5.4, is the most common form of vitamin E (Seçmeler et al., 2019). A recent report by Gladine et al. (2007) confirmed the ability of plant-rich polyphenols, including grape extract, to exhibit a significant antioxidative protective effect in plasma and liver in rats.

Figure 5.4: Chemical structure of α-tocopherol (Seçmeler et al., 2019)

The European Regulation 2018/848 of May 30, 2018, that stipulates that the animal diet supplementation with vitamins should be done with vitamins derived from raw materials occurring naturally in feedstuffs, lists the food additives (Annex VIII) that could be used in organic livestock farming, such as extracts from plant and animal origin. Annex V lists the non-organic feed materials that could be used under certain conditions, which include food industry by- products from non-organic production. In the Mediterranean region these include by-products from the olive oil, citrus, wine, and carob food industries. The possibility of using food industry by-products is linked to the ideas underlying environmental sustainability and organic farming. It allows the reduction of industry waste; by converting these low value products into high value ones (both from an economical and nutritional point of view), it contributes to the reduction of the feed to food competition in livestock production and the animal feed is produced closer to the farm (Nonhebel et al., 2015; Valenti et al., 2018). Chemical characterisation of algae and food industry by-products as sources rich in vitamins and polyphenols will contribute to solving the availability of natural vitamins to be used in organic livestock farming. Due to the complexity and variety of the molecules previously introduced, a detailed molecular characterisation will be carried out by advanced equipment such as mass spectrometry, in order to investigate and to correlate the bioactivity of alternative plant products with the molecular structures of the examined compounds.


The following alternative natural plant products were selected:

Essential oils (as anti-bacterial):

• Peppermint oil

• Oregano Spain oil

• Cinnamon Ceylon leaf oil

• Cloves oil

• Thymus vulgaris leaf oil

• Rosmarin Morocco oil

• Melaleuca alternifolia leaf oil

• Lavender altitude oil

Synthetic Standards

• Cinnamic aldehyde

• Carvacrol

• Menthol

• Thymol

• Terpineol

Plant extracts (as anti-parasitic)

• Malva silvestrys l. (raw)

• Althea officinalis (raw)

• Matricaria chamomilla L. rumancer (raw)

Plant feed additives (as antioxidants)

• Reisinox 80Q (Grape pomace)

• OLG complex (Citrus)

• Algae Scutellaria baicalensis

• Algae Ascophillum nodosum

• Spirulina platensis

• Echinacea purpurea

• Salix alba

• Vitamine E synthetic supplement

• Vitamine E (α-tocopherol / α-tocopheryl acetate – reference standard)


Chemical characterisation of alternatives natural plant products will be carried out within the Department of Food and Drug in the Food Chemistry laboratories and the Department of Veterinary Medicine in the Laboratory of Phytochemicals in Physiology of the University of Parma.

5.2 Chemical characterisation of natural anti-infective (anti-bacterial and anti-parasitic)

Essential oils are known as complex mixtures of several volatile constituents including sesquiterpenes, monoterpenes, aldehydes, alcohols, esters, and ketones. They are known to be involved in plant resistance against pests, herbivores, fungi, and bacteria (Harkat-Madouri et al., 2015). Due to the complex and volatile nature of essential oils constituents, gas chromatography (GC) coupled with mass spectrometry (MS) is the most widely applied analytical technique for their characterisation (Lebano et al., 2020). Analysis of an essential oil usually involves the separation, identification and quantitative determination of its components. The volatility and polarity of essential oil components make capillary gas chromatography the technique of election for their analysis, because essential oils in general are complex mixtures of components with similar physicochemical characteristics (Rubiolo et al., 2010). For the reasons above mentioned, a GC/MS method was adopted to characterise the selected essential oils. The characterisation will be carried out according to the method described by Caligiani et al., (2013) and Mongelli et al., (2016) with minor modifications. The sample treatment will be adapted based on the chemical-physical characteristics of alternative natural plant products considered to test. Plant materials and essential oils

The essential oils (as anti-bacterial), the synthetic standards and the plant extracts (as antiparasitic) will be diluted in CH2Cl2 in a vial and added of a small amount of anhydrous sodium sulphate. GC/MS conditions

All samples will be analysed with a Thermo Scientific (San Jose, CA, USA) TRACE 1300 gas- chromatograph coupled to a Thermo Scientific ISQ™ Single Quadrupole mass spectrometer. The gas-chromatograph is equipped with Supelcowax 10 (30 m × 0.25 mm, f.t. 0.25 μm) (Supelco, Bellefonte, PA, USA) capillary columns and helium will be used as carrier gas (1 ml min−1). Oven temperature gradient started from 50 °C; this condition will be maintained for 3 min, then the temperature will be raised to 200 °C (5 °C min−1). The final temperature will be maintained for 18 min. The injector will be maintained at 230 °C operating in split modality, ratio 1:20. The mass spectrometer will be equipped with an electron impact source (EI, 70 eV) and the acquisition mode will be full scan (from 40 m/z to 500 m/z). A solvent delay time of 4 min will be applied. The main volatile compounds will be identified on the basis of their mass spectra compared with the reference mass spectra libraries (WILEY275, NBS75K, Adams, 2001) and of their calculated Retention Indexes through the application of the Kovats’ formula (KI) with those reported in the literature. When it will be not possible to find the KI in the literature, a tentative identification will be obtained by matching with mass spectra libraries data: a match quality of 98% minimum will be used as a criterion. In order to determinate the RI of the components, a mixture of alkanes (C8–C20) will be injected in the GC-MS equipment and analysed under the same conditions described above. The gas-chromatographic signals will be manually integrated and the resulting peak areas will be compared with the total sum of area and expressed in percentage (Mongelli et al., 2016).


Preliminary results

A first analysis was performed in January 2020 on a sample of oregano essential oil in order to test the procedure. From that analysis a total of 39 compounds (Figure 5.5) belonging mostly to phenolic and terpenes chemical classes were identified and quantified in oregano’s EO. The list of the compounds is reported in Table 5.1.

Figure. 5.5: Oregano essential oil chromatograms obtained by GC/MS technique, DB-WAX column

Table 5.1 Peak number and corresponding list of compounds in oregano essential oil (peaks are relating to the numbers in Figure 5.5)

Peak number

Compound name

Peak number (cont.)

Compound name

Peak number (cont.)

Compound name

Peak number (cont.)

Compound name

1 α-Pinene 11 trans-o-cymene 21 β-Cubebene 31 α-Farnesene

2 α-Thujene 12 p-Cymene 22 linalool 32 δ-Cadinene

3 β -pinene 13 Terpinolene 23 sabinene cis 33 β-Caryophyllene oxide

4 Sabinene 14 (E.E)-allo-Ocymene

24 α-Terpineol 34 germacradienol

5 Myrcene 15 Octan-3-ol 25 calarene 35 spatulenol

6 α-Terpinene 16 1-Octen-3-ol 26 β-Caryophyllene 36 thymol

7 chloroiodometane (IMP)

17 sabinene trans 27 Terpinen-4-ol 37 t-Cadinol

8 eucalyptol 18 β-Elemene 28 carvacrol methyl eter

39 Carvacrol

9 Cis-o-Cymene 19 α-Copaene 29 α-Humulene 39 α-Farnesene

10 γ-Terpinene 20 β-Bourbonene 30 Germacrene 40 δ-Cadinene


5.3 Chemical characterisation of polyphenols and vitamins determination (vit E, vit A, beta-

caroten)

The polyphenols characterisation and vitamins determination will be carried out according to the methods described by Castello et al. (2018) and Ghaffari et al. (2019) with minor modifications. The sample treatment will be adapted based on chemical-physical characteristics of alternative natural plant products considered to test.

5.3.1 Chemical characterisation of polyphenols

The chemical characterisation of polyphenols will be performed as described by Castello et al. (2018) for grape pomace, the major by-product of wine and grape juice industry. This by-product represents a good example since it is a rich source of phenolic compound.

Plant material and extract powders

Samples will be exacted with a methanol/water (70:30 v/v) mixture, diluted in 0.1% formic acid in water and centrifuged at 12000 rpm for 5 min. Prior to the UHPLC-ESI-MS/MS analysis will be filtered (0.45 μm nylon filter).

UHPLC-ESI-QqQ-MS/MS

Samples will be analysed by UHPLC DIONEX Ultimate 3000 equipped with a TSQ Vantage triple quadrupole mass spectrometer (QqQ-MS/MS, Thermo Fisher Scientific Inc., San Jose, CA, USA) fitted with a heated-electrospray ionisation source (H-ESI-II; Thermo Fisher Scientific Inc.). Chromatographic and ionisation parameters for the analysis of the samples will be set as previously described. Phenols identification will be carried out by comparing the retention time with authentic standards and/or MS/MS fragmentation patterns. Up to 160 compounds related mainly to the metabolism of anthocyanins, flavan-3-ols, flavanols and some phenolic acids, will be monitored in selective reaction monitoring (SRM) mode. Quantification will be performed with calibration curves of standards, when available. When not available, the conjugated metabolites will be quantified with the most structurally similar compound.

Preliminary results

A first analysis was performed in January 2020 and a total of 25 phenolic compounds were identified and quantified in the grape pomace (in the Laboratory of Phytochemicals in Physiology of the University of Parma). The retention times and spectrometric characteristics of the compounds detected are reported in Table 5.2. Anthocyanins were the most abundant class of phenolic compounds (70%), followed by flavan-3-ol monomers (23%) and procyanidins (4%). Small amounts of flavanols, galloyl glucose, and gallic acid were also present in the product.


Table 5.2: Phenolic composition of Grape Pomace

Compounds RT [M-H]-

5.3.2 Vitamins determination

In order to verify animal deficiency, as well as their presence and absorption from feeds, a quantitative determination of vitamins in feed, diet and animal plasma and products may be necessary.

HPLC analyses The HPLC analyses will be carried out using a modified gradient reverse-phase system (Waters, Eschborn, Germany). Vitamin E, β-carotene, and vitamin A will be extracted with n-hexane, dried, dissolved in solvent solution, and separated on a reverse-phase column (ReproSil 70 C18 column,

(m/z) Phenolic acids

Gallic acid 1.48 169 Flavan-3-ols

Catechin 3.35 289 Epicatechin 3.65 289 Gallotannins

Galloyl glucose 2.22 331 Flavonols

Quercetin rhamnoside 4.41 447 Quercetin-3-O-glucoside 4.17 463 Quercetin-3-O-glucuronide 4.13 477 Myricetin hexoside 3.84 479 Syringetin hexoside 4.45 507 Quercetin rutinoside 4.00 609 Procyanidins

Procyanidin dimer B-type 3.04 577 Procyanidin dimer B-type 3.20 577 Procyanidin B2 3.42 577 Procyanidin dimer gallate B-type 3.73 729 Procyanidin trimer B-type 3.62 865 Procyanidin trimer B-type 3.26 865 Procyanidin trimer B-type 2.07 865

Anthocyanins [M]+(m/z)

Cyanidin-3-O-glucoside 4.50 449 Delphinidin-3-O-glucoside 3.24 465 Petunidin-3-O-glucoside 3.46 479 Malvidin-3-O-glucoside 3.67 493 Malvidin-3-O-acetylglucoside 4.20 535 Petunidin-3-p-coumaroylglucoside 4.44 625 Malvidin-3-p-coumaroylglucoside 4.68 639 Malvidin-diglucoside 4.39 655


200 × 3.0 mm; particle size 5 μm; Dr. Maisch GmbH, Ammerbuch, Germany) on an Alliance 2695 separation module (Waters). The solvent system consisted of solvent A with methanol, and solvent B with ethyl acetate, at a flow rate of 0,5 mL/min and a column temperature of 40°C. The gradient was 0 to 3 min, 0% solvent B; 3.1 min, 50% solvent B; 10 min, 50% solvent B; 10.1 min, 0% solvent B, and 15 min stop. Vitamin E (as α-tocopherol) and β- carotene will be identified based on their retention times in comparison to an external standard (Sigma-Aldrich, Munich, Germany) using a photodiode array detector (Model 996; Waters). Vitamin E, β-carotene, and vitamin A will be quantified by measuring the absorption at 290 nm, 450 nm, and 325 nm, respectively (Ghaffari et al., 2019).

5.4 Antioxidant capacity evaluation: in vitro test

The analyses will be carried out according to the methods described by Pellegrini et al. (2003) and Dall’Asta et al. (2013) with minor modifications. The sample treatment will be adapted based on chemical-physical characteristics of alternative natural plant products considered to test.

TAC Test (Total antioxidant capacity)

Several methods were developed recently for measuring the total antioxidant capacity of food and beverages; these assays differ in their chemistry (generation of different radicals and/or target molecules) and in the way end points are measured. Because different antioxidant compounds may act in vivo through different mechanisms, a single method cannot fully evaluate the TAC of feeds and foods. Therefore, three methods, i.e., Trolox equivalent antioxidant capacity (TEAC), total radical-trapping antioxidant parameter trap ferric reducing-antioxidant power (FRAP), and free radical scavenging activity test (DPPH) have been selected.

For low lipid samples, a precisely weighed amount of the homogenised sample (1 g) will be extracted with 10 mL of a methanol/water (70:30 v/v) mixture, homogenised with a blender, extracted on a stirrer at room temperature for 1 h and then filtered on paper filter. The extract will be evaporated, dissolved with 1 mL of a methanol/water (70:30 v/v) mixture and centrifuged at 5040 × g for 15 min at 4 °C. For high lipid samples, the pulp residue was reextracted twice by the addition of 2 mL of chloroform under agitation for 15 min at room temperature, centrifuged at 1000 x g for 10 min and the supernatant collected. All plant extracts will be adequately diluted in the appropriate solvent (depending on their activity) and immediately analysed in duplicate for their antioxidant capacity. Vitamin E synthetic supplement and Vitamin E pure standard (α- tocopherol / α-tocopheryl acetate) will tested as reference standard to evaluate the TAC of plant extracts.

The TEAC assay method is based on the ability of antioxidant molecules to quench the long-lived ABTS*+, a blue-green chromophore with characteristic absorption at 734 nm, compared with that of Trolox, a water-soluble vitamin E analogue. The addition of antioxidants to the preformed radical cation reduces it to ABTS, determining a decolourisation. A stable stock solution of ABTS*+ was produced by reacting a 7 mmol/L aqueous solution of ABTS with 2.45 mmol/L potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. At the beginning of the analysis day, an ABTS*+ working solution was obtained by the dilution in ethanol of the stock solution to an absorbance of 0.70 ± 0.02 AU at 734 nm, verified by a Jasco v-530 spectrophotometer. Results are expressed as TEAC in mmol of Trolox per kg (solid feed/foods and oils) or per L (beverages) of sample.

The FRAP will be assessed using a Jasco v-530 spectrophotometer. The method is based on the reduction of the Fe3+-TPTZ complex to the ferrous form at low pH. This reduction is monitored by measuring the absorption change at 593 nm. Briefly, 3 mL of working FRAP reagent prepared daily was mixed with 100 µL of diluted sample; the absorbance at 593 nm was recorded after a 30-min incubation at 37°C. The FRAP values are obtained by comparing the absorption change in the test mixture with those obtained from increasing concentrations of Fe3+ and expressed as mmol of Fe2+

equivalents per kg (solid food) or per L (beverages) of sample.


The DPPH free radical scavenging activity test will be conducted in triplicate on extract previously obtained, mixed with 2.6 mL of methanol and 2 mL of DPPH. The absorbance of the solution is recorded at 517 nm by a Jasco v-530 spectrophotometer after an incubation time of 30 min at room temperature. Blank is prepared and analysed following the same procedure. The radical scavenging activity is calculated as follows: I % = [(Abs0 - Abs1)/Abs0]*100, were Abs0 was the absorbance of the blank and Abs1 was the absorbance of the sample. The TEAC value (Trolox Equivalent Antioxidant Capacity; mmol Trolox eq./g of d.w.) of samples was obtained from the calibration curve calculated measuring the absorbance at 517 nm of Trolox methanolic solutions at different concentrations. One sample will be analysed (n = 4) and each analysis will be replicated three times. Total polyphenols content

Extractable polyphenols will be determined in GPC and diets by Folin-Ciocalteau procedure (Montreauf, 1972) using gallic acid as standard.

5.5 References Belitz, H.D., Grosch, W., and Schieberle, P. (2009). Food chemistry (4a Ed.), Springer, Berlin,

Germany Brenes, A., Viveros, A., Gon˜ i I., Centeno, C., Sa´yago-Ayerdy, S. G., Arija, I., & Saura-Calixto, F.

(2008). Effect of Grape Pomace Concentrate and Vitamin E on Digestibility of Polyphenols and Antioxidant Activity in Chickens. Poultry Science 87:307–316. https://doi:10.3382/ps.2007-00297

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6. Conclusions

From the analysis of the literature and from the results of the trials performed it appears that the efficacy of the plant products against infective agents (bacteria and parasites) can be easily measured in vitro and their action can be quantified.

Among the antibacterial tested, the lowest MIC average was found for Oregano oil, followed by Thymus oil, Tea tree oil and Rosemary oil.

Highest MIC average were instead found for Lavender oil and Clove oil followed by Cinnamon oil and Mentha oil.

Among the tested NIC, the lowest MIC was found for Cinnamic aldehyde against S. aureus without any emulsifier. The highest MIC was found for Terpineol against all the tested strains.

Concerning the herbal extract tested as antiparasitic, in the present study we observed a good anti-parasitic effect of Malva sylvestris documented by reduction of eggs and larvae form at the dilution of 20 mg/ml. This effect was similar to the one observed from Althaea officinalis and overcame the one of Chamomilla recutita.

The analytical procedures selected will be utilised to characterise essential oils and plant extracts from a chemical point of view and to test plant products for their antioxidant properties, in order to evaluate their attitude as possible alternative to antioxidant.

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