Mickael Bonnan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63201

#### **Abstract**

Multiple sclerosis (MS) is characterized by the intrathecal synthesis (ITS) of immunoglo‐ bulins (Igs), which, although nonspecific, is the strongest biological marker. Since no specific target has been elucidated, this synthesis is considered to be disease‐irrelevant. We demonstrate that this synthesis provides pertinent information about the pathophy‐ siological processes involved. Quantification of ITS is based on an approximation intrinsically underestimating its level and it remains constant in MS, albeit sometimes at a low level. B‐cell maturation seems to be initiated within the cervical lymph nodes and B‐cells traffic on both sides of the blood‐brain barrier by rounds of bidirectional traffic. During this process, they undergo somatic hypermutation, which is the hallmark of antigen‐driven antibody maturation, suggesting that most of the ITS is probably directed against as yet unknown targets. Alternatively, examining"non‐disease‐relevant" ITS in the light of meningeal tertiary lymphoid organs provides new insights into the patho‐ physiology of MS. Although no specific target has yet been identified in MS, recent developments in the search for targeted antigens point to non‐conventional antigens (posttranslationally modified proteins or oxidized products) of which a few are promising for future research.

**Keywords:** multiple sclerosis, meningeal inflammation, intrathecal synthesis, immu‐ noglobulins, autoantibody, epitopes, antigens


*Review* **Spicy and Aromatic Plants for Meat and Meat Analogues Applications**

**Romina Alina Marc (Vlaic) 1, Vlad Mures, an 1,\*, Andru¸ta E. Mures, an 1,\*, Crina Carmen Mures, an 1, Anda E. Tanislav 1, Andreea Pus, cas, 1, Georgiana Smaranda Mar¸tis, (Petru¸t) 1 and Rodica Ana Ungur 2**

**Abstract:** Aromatic and spicy plants are an important factor that contributes not only to improving the taste of meat, meat products, and meat analogues, but also to increasing the nutritional value of the products to which they are added. The aim of this paper is to present the latest information on the bioactive antioxidant and antimicrobial properties of the most commonly used herbs and spices (parsley, dill, basil, oregano, sage, coriander, rosemary, marjoram, tarragon, bay, thyme, and mint) used in the meat and meat analogues industry, or proposed to be used for meat analogues.

**Keywords:** herbs; essential oils; aroma compounds; antioxidant activity; antibacterial activity; bioactive compounds

#### **1. Introduction**

Spicy and aromatic plants have been used in human consumption for thousands of years (since around 5000 BC). Initially, they played an important role in primary care, being used as therapeutic agents in the treatment of various diseases; however, wider applications are reported today [1]. Over time, spicy and aromatic plants began to be used around the world in various foods to flavor them, but also for preservative purposes. These plants are considered an untapped reservoir of valuable substances, also called phytochemicals, phytogenic, phytobiotics, botanicals or spices, although they are not established as essential ingredients [1–4].

Meat is known to be an important source of protein, essential amino acids, vitamins, and minerals. However, most of the meat worldwide is processed. After processing, the meat becomes more perishable and sensitive to oxidation. To improve these characteristics, as well as the aroma, aromatic and spicy plants with aromatizing roles and natural antioxidants are used [5,6]. Synthetic chemicals are also used, but consumers prefer natural antioxidants due to the possible long-term toxic effects of synthetic substances [7].

The growing population around the world has led to the need to increase the number of protein-containing products. Meat and meat products are the most common sources of high protein, but these sources are no longer able to meet all the needs of consumers: an increasing amount is needed, and for a part of the population, these products are not recommended for certain diseases. Along with this need, the interest in meat analogues has risen considerably [8]. The demand for these vegetable meat alternatives is growing, because they have benefits for consumers, but also for the planet, and are recognized as sustainable protein sources. A vegetable-based diet has been shown to reduce the risk of cardiovascular disease, diabetes, high blood pressure, and mortality [9].

**Citation:** Marc, R.A.; Mures,an, V.; Mures,an, A.E.; Mures,an, C.C.; Tanislav, A.E.; Pus, cas, , A.; Mar¸tis, , G.S.; Ungur, R.A. Spicy and Aromatic Plants for Meat and Meat Analogues Applications. *Plants* **2022**, *11*, 960. https://doi.org/10.3390/ plants11070960

Academic Editor: Manuel Viuda-Martos

Received: 13 March 2022 Accepted: 30 March 2022 Published: 1 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

To make these food products tasty, and to have a pleasant appearance, whether referring to meat products or meat analogues, we use herbs and spices. They are used not only for their flavor, but also for the benefits they bring to finished products and the benefits they bring to consumers. In the meat and meat analogues industries, the most commonly used aromatic and spicy plants are parsley, dill, basil, oregano, sage, coriander, rosemary, marjoram, tarragon, bay, thyme, and mint [2,10–28]. They contain chemicals such as polyphenols, flvonoids, quinics, polypeptides, and alkaloids, or their oxygen-substituted derivatives. Some of these substances can act synergistically and improve bioactivity. Additionally, these bioactive compounds have therapeutic value, such as antioxidant and antiseptic activity [1]. Thus, the active components of these plants might have the ability to reduce the risk of cancer, cardiovascular disease, respiratory disease, and stomach or inflammatory disorders, and reduce oxidative stress. They also contain antimicrobial compounds, which delay microbial growth in food [1,4]. This review aims to gather recent information on spicy and aromatic plants used to prepare meat and meat alternatives.

#### **2. Spicy, Aromatic Plants and Their Applications in Meat and Meat Analogues**

The aromatic and spicy plants regularly used in meat preparations and meat analogues are parsley, dill, basil, oregano, sage, coriander, rosemary, marjoram, tarragon, bay, thyme, and mint. Different parts of plants are used, such as stems, seeds, or leaves, in different forms, including extract powder, essential oils, ground leaves, herbal dust, water extract, or powder extract. They are used in different amounts depending on the type of product to which they are added, and have flavoring, coloring, antimicrobial, antioxidant effects, as shown in Table 1.

**Table 1.** The effect of different parts of spicy and aromatic plants in meat products.

#### **Table 1.** *Cont.*

#### **3. Bioactive Compounds and Antibacterial and Antioxidant Activity of Spicy and Aromatic Plants in Meat**

The consumption of fresh and processed meat has an indisputable value for diet as a source of proteins and micronutrients, but in 2016, an International Agency for Research on Cancer (IARC) working group classified processed meat as "carcinogenic to humans" and red meat as "probably carcinogenic to humans for colorectal cancer". On the other hand, fresh and processed meat is an key part of the Western diet, associated with chronic metabolic inflammation and an important group of chronic diseases: obesity, hyperlipidemia, diabetes, gout, high blood pressure and degenerative neurological diseases, including dementia. In this context, it is very important to associate meat and its derivatives with compounds that can counteract their possible negative effects.

Spices can be important factors that contribute not only to improvements in meat and meat product savor, but also to increasing the nutritive value of meat and derivatives, and counteracting their metabolic disadvantages. It has been demonstrated that plants with green leaves utilized as spices for meat and derivatives have important antioxidant and anti-inflammatory effects, antidiabetic, antimicrobial and antimutagenic actions, associated with their chemical composition, i.e., rich in polyphenols, carotenoids, and terpenoids. The use of synthetic antioxidants is restricted due to their carcinogenicity; therefore, natural antioxidants derived from plants, including aromatic plants, are recommended for use in the food industry. The prevalence of digestive disorders in Western populations is currently increasing. Culinary spices used in meat preparations can stimulate digestive processes by increasing bile and digestive enzyme production, and modulating the structure and function of gut microbiota. On the other hand, the antioxidant, anti-inflammatory, antimicrobial, antifungal, and antimutagenic effects of spices protect the digestive system from cancer, gastritis and ulcers, periodontitis, and colitis. Recently, many spices used in meat processing have been proposed as alternative treatments for SARS-CoV-2-infected patients due to their anti-inflammatory properties that can be potentially efficient to combat a cytokine storm [29].

Due to their ability to prevent and slow down the rate of lipid oxidation in food systems, aromatic herbs and their derivatives are potential natural alternative sources for antioxidants and synthetic preservatives. They can be added directly to the product (in fresh or dried states), its constituents can be extracted and added in the form of essential oil (the addition is limited by the intense aroma) or extracts, and in the combination of different plant extracts. The last method can lead to superior antioxidant activity due to the synergistic action of the compounds. Natural antioxidants have the effect of reducing the formation of cytotoxic compounds during the thermal processing of food (for example, in meat) [30].

**Parsley** (*Petroselinum crispum* Hoffm) stems contain many bioactive compounds such as carotenoids, including *β*-carotene, neoxanthin, violaxanthin, lutein, and glycoside apiose. The leaves have a high content of vitamins (K, A, C), folate, niacin, choline, pantothenic acid and also *β*-carotene, lutein. Parsley is used to improve appetite and alleviate indigestion, flatulence and spasms, and may prevent stomach ulcers [31]. Stem and leaf extracts of *Petroselinum crispum* have been identified to exert antioxidant, anti-inflammatory, and antiplatelet activities, and have protective effects against hyperuricemia and hyperglycemia, brain, heart, and liver diseases [32].

It proved to inhibit the bacterial growth of *Bacillus subtilis*, involved in the pathogenesis of digestive anastomotic leak, and of the pro-inflammatory proteobacterium *Escherichia coli*, involved in Crohn's disease, whose overgrowth is facilitated by Western diets [33–35].

According to Zhang et al. [36], parsley essential oil exhibits antioxidant activity due to the compounds myristicin (phenylpropene) and apiol (phenylpropanoid). The flavonoids isorhamnetin, apigenin, quercetin, luteolin, and chrysoeriol represent the predominant compounds in cell suspension cultures of parsley [32]. Parsley apigenin (yellow color) can be used as a pigment in human and animal nutrition [1].

Wong and Kitts [37] studied the in vitro antioxidant activity by DPPH inhibition of free radicals, ion-chelating, and hydroxyl radical assays from aqueous and methanolic extracts prepared from parsley leaves and stems. The methanolic extracts from the leaves showed a significant (*p* < 0.05) radical scavenging activity, attributed to the total phenolic content, whereas the chelating activity of the ferrous ions was significantly (*p* < 0.05) higher in methanol extracts from the stem.

Phenolic compounds are the main class of plant compounds that contribute to their antioxidant capacity [38].

Due to its high nitrate content, different concentrations of parsley extract powder have been used in the formulation of sausages (V3: 1.07 g, V4: 2.14 g and V5: 4.29 g parsley extract/kg meat), in comparison, from a sensory point of view, with traditional sausages with nitrites (V1) and without nitrites (V2). The evaluators did not observe any significant differences between samples V1, V3, V4, and V5 in terms of red color intensity, although sample V2 showed intense grey color (due to lack of nitrate). Parsley extract powder did not show an influence on taste and aroma and no significant differences were found between the sausage variants. Among sausages produced with parsley extract powder, the general acceptance for sample V3 was reduced, due to the lower concentrations of parsley extract, resulting in lower levels of nitrites and lower nitrosomyochromogen contents formed. Thus, depending on the sensory parameters evaluated, samples V4 and V5, produced with 2.14 g and 4.29 g of parsley extract/kg meat, respectively, had characteristics similar to those of the traditional counterpart treated with nitrate [10].

**Rosemary** (*Rosmarinus officinalis* L.) leaves are rich in compounds with health-promoting properties for a variety of diseases. The main bioactive compounds are phenolic diterpenes carnosic acid and carnosol; triterpenes—oleanolic, betulinic, and ursolic acids; flavonoids hesperidin, homoplantaginin, cirsimaritin, genkwanin, gallocatechin, nepetrin, 6-hydroxyluteolin-7-glucoside, luteolin-3 -glucuronide, and luteolin-3 -*O*-(*O*-acetyl)-*β*-Dglucuronide; and typical compounds—rosmarinic acid, rosmadial, and rosmaridiphenol [39]. They confer antioxidant, anti-inflammatory, antihyperlipidemic, hepatoprotective, renoprotective, antithrombotic, antinociceptive, antidepressant, antimicrobial, and anticancer properties. In traditional medicine, *Rosmarinus officinalis* L. has been used to treat gastrointestinal, hepatic, cardiovascular, nervous, respiratory, genitourinary, and skin disorders [40,41]. In experimental models, rosemary extracts improved body weight control, total cholesterol level, and atherogenic index, cardiac remodeling after myocardial infarction, brain tolerance to artificially induced ischemia, and protection against rupture of the blood–brain barrier [42]. Anticancer properties were demonstrated in several cell-line models, for esophageal squamous cell carcinoma (KYSE30), gastric adenocarcinoma (AGS), epithelial colorectal adenocarcinoma (CaCo-2), breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa), lung carcinoma (A549) histiocytic lymphoma (U-937), and human melanoma (A375) [43]. Rosemary extracts also exert antifungal, antiviral, and antibacterial activities [41].

The stability, antioxidant and antimicrobial activity of rosemary essential oil introduced into meat and meat products can be improved by encapsulating it in a nanogel of chitosan and benzoic acid [44]. Krki´c et al. studied the incorporation of oregano essential oil into a chitosan coating, which reduced lipid oxidation, and contributed to the formation of a smaller amount of aldehydes and superior sensory properties in dry fermented sausages [45].

Rosemary extract (0, 250, 500, and 750 mg/kg) combined with sodium nitrite (40, 80, and 120 mg/kg) was used to obtain liver pâté and to study color stability, lipid oxidation, and concentrations of ascorbic acid, α-tocopherol, and carnosic acid. Regardless of the added dose of rosemary extract, it had a significant effect in reducing lipid oxidation and maintaining high levels of antioxidants, while having no effect on color stability. The concentration of carnosic acid increases with the dose of rosemary extract. Low doses of sodium nitrite (80 mg/kg) can be used without adversely affecting color stability, forming

significantly lower nitrite concentrations and slightly reduced lipid oxidation values, while the use of rosemary extract helps maintain lipid stability [46].

Rosemary and marjoram essential oils have been added in different doses to pork sausages in a study conducted by [47]. The results demonstrated the protective effects of essential oils against oxidation. In addition, the increase in TBARS values and the loss of red color was prevented and compared with samples containing synthetic antioxidants; samples with essential oil obtained similar or better results. Mohamed and Mansour evaluated the antioxidation efficiency of rosemary and marjoram essential oils (200 mg/kg) added to frozen beef patties and stored for 3 months at −18 ◦C. Essential oils have been shown to be effective against oxidation. The results of a sensory analysis showed that the addition of essential oils had a positive effect on the samples, being highly appreciated by evaluators [48].

According to the Food Safety and Inspection Service (FSIS) Directive 7120.1: "Safe and suitable ingredients used in the production of meat, poultry, and egg products"; rosemary extract is the most commonly used natural antioxidant in the meat industry, with it explicitly being allowed for use as a component of an antioxidant mixture [49]. The antioxidant properties of rosemary are due to the presence of phenolic diterpenes, namely carnosic acid and carnosol, which act as hydrogen donors in the reaction with free radicals [46]. In the case of sage, the antioxidant capacity is due to the presence of phenolic diterpene (epirosmanol, carnosol, and carnosic acid) [50]. Rosemary and sage extracts can provide antioxidant species in both the polar and non-polar phases of a food product. For example, carnosic acid is on the lipophilic end of the scale, and rosmarinic acid is on the hydrophilic end. Carnosic acid is a superstoichiometric antioxidant because it can act repeatedly as a reducing agent by donating hydrogen atoms through phenolic compounds [51].

**Sage** (*Salvia officinalis* L.) leaves are rich in terpenes, anthraquinone, and flavonoids with antioxidant, anti-inflammatory, and antimicrobial effects. In sage essential oils, the main components are camphor, 1,8-cineole, *α*-thujone, *β*-thujone, borneol, and viridiflorol [52]. In traditional medicine, *Salvia officinalis* has been used to treat mild dyspepsia, ulcers, and gout. The German Commission E has also accepted the use of *Salvia officinalis* for dyspepsia [53].

It was reported that drinking sage tea prevented the initiation phases of colon carcinogenesis in an experimental rat model [54]. Clinical trials reported memory-enhancing and antidementia benefits in healthy adults or patients with Alzheimer's disease [55], and hypoglycemic and hypolipemic effects of *Salvia officinalis* leaves in patients with diabetes and hyperlipidemia and in healthy volunteers [53].

Sage may produces several types of phenolic species as opposed to rosemary, especially in the production of flavonoids and other phenolic derivatives, although rosemary produces higher amounts of carnosic acid and other diterpenoids related to it [51]. In a study by Kontogianni et al., they showed that rosemary extract is twice as rich in diterpornoid and phenolic compounds and contains about 2.7 times more carnosic acid than sage extract [56].

**Dill** (*Anethum graveolens* L.) seeds used in meat products and derivatives are rich in essential oil, of which the major compounds are carvone, limonene, and camphor, characterized by important antioxidant activity [57]. Antioxidant activity also characterizes the flavonoids quercetin and isoharmentin isolated from *Anethum graveolens* L. seeds, which can help to prevent peptic ulcers. This effect has been verified in experimental models in which aqueous and ethanolic *Anethum graveolens* L. extracts had mucosal protective and antisecretory effects, similarly to high-dose sucralfate [58].

The essential oils found in seeds are carminative, improve appetite, aid digestion, and relieve intestinal spasms. D-limonene is a monoterpene that dissolves cholesterolcontaining gallstones. It is chemopreventive and has chemotherapeutic activities [59]. In experimental models, dill seed extracts suppressed hyperlipidemia induced by a high-fat diet [60] and had inhibitory effects on hepatic carcinoma cells [61].

Extracts from dill (*Anethum graveolens*) obtained in organic and conventional agriculture were prepared in n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and ethanol (EtOH), and the radical scavenging activity (RSA) at 2000 μg mL−1 has been studied by DPPH (2,2-Diphenyl-1-picrylhydrazyl), DMPD+ (N,Ndimethyl-p-phenylendiamine), and NO (nitric oxide) methods. Ethanol extracts (both conventional and organic agriculture) had better inhibitory effects, and the NO radical scavenging activity was particularly noted (78.49 ± 1.86% for conventional agriculture and 71.86 ± 5.41% for organic agriculture). No significant differences were observed between organic and conventional agriculture extracts in the RSA tests. Ferric-ion-chelating capacity and phosphomolybdenum-reducing antioxidant power (PRAP) assays were also studied; dichloromethane (CH2Cl2) extracts had the greatest ferric ion chelation effect (74.34 ± 1.40%), and PRAP values from both extracts had better values than the rest of the samples [62].

**Oregano** (*Origanum vulgare* L.) leaves are rich in essential oils. The volatile oil contains phenolic compounds, monoterpenes and sesquiterpenes: thymol, carvacrol, *p*-cymene, *γ*terpinene, and linalool [63], with antioxidant, anti-inflammatory, and antimicrobial activity. In traditional medicine, oregano has been used for gastrointestinal disorders—indigestions, stomachache, and diarrhea; respiratory diseases—asthma and bronchitis; menstrual disorders; and diabetes, due to its anti-bacterial and anti-inflammatory activity [64–67].

A remarkable property of oregano essential oils is their antiproliferative activity on adenocarcinoma gastric cell line [68]. In case–control studies, gastric cancer was associated with red and processed meat consumption [69]. *Origanum vulgare* essential oils demonstrated inhibitory effects on the growth of carbapenem-resistant Gram-negative bacteria [70], and antibacterial activity and synergistic effect with polymyxin B against multidrug-resistant *Acinetobacter baumannii* [71], whose development is favored by extensive antibiotic utilization, including animal treatments [72,73].

Oregano has a high content of antioxidants, according to a study conducted by Zheng and Wang, which makes it suitable for use as a natural antioxidant. Antioxidant compounds are phenolic acids and flavonoids, such as caffeic acid, rosmarinic acid, hispidulin, and apigenin, as well as carvacrol and thymol, components of the essential oil [74].

Fasseas et al. evaluated the antioxidant activity of meat treated with oregano and sage essential oils extracted by hydrodistillation. In this regard, the minced pork and beef were formulated in three samples as follows: homogenization with 3% (*w*/*w*) of either oregano essential oil or sage essential oil, and a control sample which did not contain essential oils. The samples thus obtained were stored at 4 ◦C in raw and cooked states (85 ◦C, 30 min), and the antioxidant activity was evaluated at 1, 4, 8, and 12 days of storage. Essential oils have led to a decrease in lipid oxidation, the role of antioxidants being affected by meat proteins and was significantly more important in cooked meat [75].

Plant extracts and essential oils including thyme, oregano (rich in thymol and carvacrol), rosemary, and sage are used to prevent the oxidation of meat products, in encapsulated form or in edible films, due to their high solubility, and for flavoring, due to their organoleptic properties [76].

The effect of oregano essential oil used in the formulation of an active coating used on fresh pork meat was studied. The essential oil was used as free oil, nanoemulsified or microencapsulated. All formulated samples showed a delay in the oxidation of lipids and oxymyoglobin, and the sensory profile was more appreciated as opposed to the control sample which did not contain oregano essential oil [44].

**Basil** (*Ocimum basilicum* L.) leaves contain many antioxidant and anti-inflammatory flavonoids such as quercetin, quercetin-3-*O*-diglycoside, querce-tin-3-*O*-β-D-galactoside, quercetin-3-*O*-*β*-D-glucoside, quercetin-3-*O*-*β*-D-glucoside-2-gallate, quercetin-3-*O*-(2-*O*galloyl)-rutinoside, querce-tin-3-*O*-*α*-L-rhamnoside, isoquercetrin, kaempferol; carotenoids *β*-carotene, *β*-cryptoxanthin, and lutein–zeaxanthin; polyphenols—rosmarinic acid, and chicoric acid (dicaffeoyltartaric acid); coumarin, aesculetin, and *p*-coumaric acid [77]. In traditional medicine, *Ocimum basilicum* has been used for the treatment of digestive disorders and demonstrates carminative, stimulant, antispasmodic, antidiarrheal, antibacterial,

and anthelmintic effects [31]. It has also been used in treating vomiting, flatulence, dyspepsia, and gastritis [78]. *Ocimum basilicum* leaves contain caffeic acid, which demonstrates antioxidative and cancer chemopreventive properties [79]. *Ocimum basilicum* essential oils exhibited cytotoxic activity against human liver hepatocellular carcinoma cell lines (HEpG2) and nasopharyngeal cancer cell line (KB) [78]. In experimental models, *Ocimum basilicum* demonstrated anti-hyperglycemic potential, and antioxidant and nephroprotective effects in diabetic disease [80–82].

The antioxidant compounds in basil extracts with a role in antiradical activity are chlorogenic, *p*-hydroxybenzoic, caffeic, vanillic, and rosmarinic acids, as well as apigenin, quercetin, and rutin. Teofilovi´c et al. performed various extractions with mixtures of ethanol–water (30%, 40%, 50%, 60%, 96% *v*/*v*), concentrated methanol (95% *v*/*v*), water (in presence and absence of light), dichloromethane, chloroform, and hexane over different periods of time (10 and 30 min), which exhibited antioxidant activity by the DPPH method with IC50 values between 0.22 ± 0.01 and 12.99 ± 0.87 g/mL for polar solvents and from 12.12 ± 0.54 to 20.49 ± 1.54 g/mL for non-polar solvents. Increasing the extraction time and polarity of the solvent improve the quality of the extracts in terms of phenolic compounds and antioxidant capacity [83].

Basil essential oil was added in various concentrations (0.062%, 0.125%, and 0.25%) to a beef burger to evaluate its natural antioxidant effectiveness. The results showed that the essential oil decreased the rate of lipid oxidation, and the effectiveness did not depend on the concentration [13].

**Marjoram** (*Origanum majorana* L.) leaf essential oils have flavonoids and terpenoids as the main active compounds [42,84]. These are excellently summarized in a review published by Bina [85]. Monoterpene hydrocarbons are represented by *α*- and *β*-pinene, *α*- and *β*-phellandrene, camphene, sabinene, limonene, *ρ*-cymene, *β*-ocimene, *γ*-terpinene, *α*-terpinene, terpinolene, carvone, and citronellol. Thymol, carvacrol, and linalool are other monoterpene compounds. Sweet marjoram essential oil contains phenolic compounds such as rosmarinic acid, sinapic acid, vanillic acid, ferulic acid, caffeic acid, and coumarinic acid, and phenolic glycosides such as arbutin, methyl arbutin, vitexin, and orientinthymonin [85]. Flavonoids such as hesperetin, kaempferol, and luteolin were also found in marjoram extracts and have vasoprotective effects, together with carvacrol and thymol [86]. Marjoram is traditionally used to treat respiratory and gastrointestinal diseases, high blood pressure, dysrhythmia, pains, and fatigue. Marjoram extracts demonstrated antioxidant, anti-inflammatory, anti-hyperglycemic, hypouricemic, anticancer, gastro-, nephro-, and hepatoprotective activity [85]. Acetylcholinesterase and tyrosinase inhibitory activities were also demonstrated, and support the antineurodegenerative effects of marjoram [87]. Essential oils and extracts from *O. majorana* exhibit antiparasitic, antifungal and antimicrobial activity against the Gram-positive species *Staphylococcus aureus*, *Enterococcus faecalis*, and *Streptococcus dysgalactiae*, and the Gram-negative species *Klebsiella pneumoniae* and *Pseudomonas aeruginosa* [88–90].

Marjoram essential oil has antioxidant properties through its ability to inhibit hydroxyl radicals. The major compounds of the oil are terpinen-4-ol (21.3%), *trans*-sabinene hydrate (15.5%), *γ*-terpinene (14.0%), and *α*-terpinene (8.9%). At a concentration of 0.05%, marjoram essential oil inhibited the formation of conjugated dienes by 50% and the generation of oxidized by-products of linoleic acid by 79.85% through its addition to an emulsion system with linoleic acid [91].

**Mint** (*Mentha piperita* L.) leaf essential oils mainly contain oxygenated monoterpenes, monoterpene hydrocarbons, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes [92]. Quantitatively, the most abundant constituents are menthol and menthone. Menthofuran, menthyl acetate, iso-menthone 1,8-cineole, and the toxicity of pulegone should be remarked [93]. The traditional use of mint for treating fevers, colds, digestive diseases, infections, and throat inflammation has been supported by experimental studies. Pharmacological activities of mint demonstrated to date include antioxidant, anti-inflammatory, anticancer, antidiabetic, hepatoprotective, neuroprotective, and radioprotective activity [94]. In healthy adults, essential oil rich in menthol/menthone attenuated mental fatigue associated with extended cognitive task performance [95]. Antimicrobial, antiviral, antifungal, biopesticidal, and larvicidal activity has also been reported.

Mint extracts contain a significant number of phenolic compounds, and thus exert an important antioxidant activity. Their antioxidant activity can be compared with that of synthetic antioxidants [96].

In their study, Kanatt et al. treated lamb pulp with mint extract before it was irradiated (2.5 kGy). An amount of 0.05 g/100 g mint extract inhibited oxidation to some extent (0.6 mg MDA/kg as opposed to using a 0.1 g/100 g extract, which led to inhibition of 50% (0.4 mg MDA/kg) [97]. In another study, Biswas et al. treated ground pork meat with mint extract, and it obtained a good color stability compared with samples obtained with sodium nitrite [98].

**Tarragon** (*Artemisia dracunculus* L.) leaf essential oils are rich in phytochemicals including coumarins, isocoumarins, monoterpenoids, sesquiterpenoids, flavonoids, polyacetylenes, and alkaloids. Tarragon increases bile and gastric acid production, stimulates digestion, and has beneficial effects on gastritis [99]. Essential oil concentrated in coumarin derivatives showed remarkable anticoagulant activity, inducing a therapeutic value (2.34) for the international normalized ratio (INR) in vitro [100]. In muscle cell cultures derived from lean, overweight, and diabetic–obese subjects, bioactive compounds of *Artemisia dracunculus* L. improved insulin sensitivity [101]. Essential oils also exhibited strong antifungal activity [102].

The antioxidant capacity of tarragon (*Artemisia dracunculus*) essential oil (0.01–0.9%) was studied by Behbahani et al. and the results showed that a concentration of 0.9% has an antioxidant activity of 78.87%, similar to that of the synthetic antioxidant BHT (butylated hydroxytoluene) [102].

Nimse and Pal present carotenoids, antioxidant vitamins, hydroxycinnamic acids, flavonoids and terpenes as antioxidant compounds that can help prevent oxidation in meat and meat products [103]. The phenolic content of plant-derived materials has the most significant potential in terms of the antioxidant and antimicrobial activity [76].

**Coriander** (*Coriandrum sativum* L.) leaves have high contents of vitamin C, vitamin A, vitamin K, iron, manganese, thiamine, zinc, *β*-carotene, and anthocyanins, and are used for the treatment of iron and vitamin deficiencies or as potent antioxidants. Fruits and leaves of *Coriandrum sativum* are traditionally used for digestive diseases, nausea, vomiting, indigestion, and against worms [104]. In experimental models, coriander seeds prevented gastric mucosal lesions induced by ethanol due to the protective layer formed by its hydrophobic compounds and free radical scavenging activity of its antioxidant constituents such as flavonoids, coumarins, catechins, and terpenes [105]. *Coriandrum sativum* L. leaf essential oils contain natural antimicrobial compounds that can act against *Candida* spp. [106] and *Campylobacter jejuni* found in beef and chicken meat [21]. Previous studies have demonstrated the antioxidant and neuroprotective effect of *Coriandrum sativum* L. extracts on brain [107], the decrease in brain cholinesterase activity and serum total cholesterol levels, and memory improvement [108]. Extracts from the leaves and stems of *Coriandrum sativum* L. exhibited significant antihyperglycemic activity, and seed extracts normalized glycemia and decreased the elevated levels of insulin [109].

Šoji´c et al. [110] effectively investigated the addition of coriander essential oil (0.075–0.150 μL/g) to pork sausages which also contained different levels of sodium nitrite (0, 50 and 100 mg/kg). In addition to nitrite, coriander essential oil contributes to lower lipid oxidation due to its antioxidant potential due to terpenoid compounds. The essential oil contains linalool (835.2 mg/g), camphor (32.9 mg/g), *γ*-terpinene (32.8 mg/g), geraniol (16 mg/g), and (+)-limonene (6.2 mg/g) [111].

**Bay** (*Laurus nobilis* L.) leaf essential oils contain monoterpenes and monoterpenoids, sesquiterpenes and sesquiterpenoids, diterpenoids, phenyl propene derivatives, alcohols, carbonyls, and esters [112]. *Laurus nobilis* L. is used in the treatment of cancer, gastrointestinal disorders, epilepsy, rheumatic conditions, and several infectious diseases [113]. In an

experimental model of ulcers induced by ethanol, bay leaf extracts demonstrated gastric mucosal protection correlated with antioxidant activity [114].

The antimicrobial activity of essential oils tested against *Escherichia coli*, *Staphylococcus aureus*, *Enterococcus faecalis*, *Pseudomonas aeruginosa*, and *Candida albicans* exhibited inhibitory effects and antioxidant activity [112].

Bay leaf essential oil has antioxidant and antibacterial properties in meat due to oxygenated monoterpenes and phenolic compounds, according to the study conducted by Ramos et al. [115]. Bay leaf essential oil has been used to treat packaged chicken in a microaerobic atmosphere where it has reduced oxidation and prolongs the smell of fresh meat, making it suitable for use as a natural preservative. Bornyl acetate, 1,8-cineole, *β*-myrcene, and carvacrol represents the principal components from bay leaf essential oil [116].

**Thyme** (*Thymbra capitata* (L.) Cav) leaf essential oils contain mainly monoterpenes, generally 10% carvacrol and 50% thymol, but also linalool, *α*-terpineol, camphor, caryophyllene, and *γ*-terpinene [117]. Flavonols (quercetin-7-*O*-glucoside), flavanones (naringenin) and flavones (apigenin), phenolic acids (*p*-coumaric, caffeic, rosmarinic, cinnamic, carnosic, ferulic, quinic, and caffeoylquinic acids), saponins, steroids, alkaloids, and tannins have also been described in thyme extracts [117–119]. In traditional medicine, thyme has been used as a sedative, a carminative, an additive for baths, or an infusion for the treatment of skin diseases [117,118]. Thyme extract might be an effective treatment of chronic respiratory diseases accompanied by the inflammation and hypersecretion of mucus [120]. Previous studies have reported that thymol demonstrates anti-inflammatory, anti-carcinogenic, and immunomodulatory properties, decreased serum lipids, visceral fat accumulation, and reduced blood pressure in experimental models [121,122]. Thyme essential oils have exhibited antifungal and antibacterial bioactivity against both food spoilage microflora and pathogenic microflora in vitro, including *B. subtilis*, *S. aureus*, *S. enteritidis*, and *P. aeruginosa* [123]. Thyme essential oils demonstrated antimicrobial activity against *Listeria monocytogenes* in vivo [124].

Due to the carvacrol present in the composition of thyme essential oil, it is distinguished by its antioxidant capacity. Thus, through their study on chicken breast in which thyme essential oil was added in a proportion of 0.5%, Fratianni et al. demonstrated that it reduced radical formation and lipid peroxidation and prolonged the shelf life of products [125]. In another study, Zengin and Baysal added thyme essential oil to minced beef that was stored for 9 days at 4 ◦C. Thyme essential oil showed a delaying effect in the oxidation process of lipids and color, and the sensory quality of the product was not affected by this addition [126].

#### **4. Essential Oils and Aroma Compounds of Spicy and Aromatic Plants**

Chemically, essential oils are a complex mixture of numerous bioactive chemical components, such as terpenes, terpenoids, and phenolics. Essential oils are synthesized by almost all plant organs, particularly the flowers, buds, leaves, seeds, stems, roots, and fruits [127]. Moreover, essential oils exhibit a very characteristic odor, and are therefore responsible for the specific scents that aromatic plants emit. These essential oils can be stored in epidermal cells, cavities, secretory cells of glandular trichomes. Numerous essential oils have the potential to be used as a food preservative for meat and meat products [128–133]. It should be highlighted that essential oils are generally accepted by consumers due to their high volatility and biodegradable nature.

**Rosemary**. Oxygenated monoterpenes (74.1%), represented mainly by 1,8-cineole (33.1%), camphor (18%), and borneol (7.95%), are the major terpenes of the Tunisian rosemary essential oil (Table 2). Monoterpene hydrocarbons constitute 21.6% of the oil, and α-pinene (10.16%) is the major compound of this class. The amount of oxygenated sesquiterpenes, represented only by caryophyllene oxide, was low (0.62%) [134].

According to the literature [44,135–138], the main primary components of the oil are: 1,8-cineole, α-pinene, camphor, verbenone, and borneol, whereas the secondary components are terpinen-4-ol, *α*-terpineol, *β*-caryophyllene, 3-octanol, geranyl acetate, and linalyl acetate. *Rosmarinus officinalis* L. volatile oil can be distinguished according to its 1,8-cineole, *α*-pinene, and camphor content. Additionally, certain essential oil compositions are dominated by myrcene [139].

The volatile fraction of *R. officinalis* differs for many of the chemotypes growing in the countries listed in Table 2, with regard to compounds, in the genus of components and their relative quantity. The observed differences may probably be due to different environmental and different chemotypes and the nutritional status of the plants, as well as other factors that can influence the oil composition. It is of interest to note the presence of *p*-cymene (44.42%) in very high percentages, which was distinctive of *R. officinalis* [140]. Monoterpenes constituted the major compounds of the oil (86.3%), whereas sesquiterpenes amounted to 11% [141].

**Mint**. The essential oil of *M. pullegium* is rich in pulegone (35.1%) and piperitenone (27.4%) (Table 2). However, none of these compounds were present in the essential oil of the other Mentha species, i.e., mint, whose main component is carvone (75.9%). Pulegone (48%) and menthone (41%) are described in the essential oil of the *M. pullegium* species [135]. According to Erich et al., the chemical composition of peppermint oil, as detailed in Table 2, is dominated by menthol (40.7%) and menthone (23.4%) [142]. The odor perceptions of peppermint essential oil include green, herbaceous, bitter, mint, or fresh. The amount of peppermint essential oil is influenced by geographical area, ripening time, or soil type [143].

**Thyme.** The major component identified from wild thyme was carvacrol (56.0%) [135]. Carvacrol (55.1%) and geraniol (43%) were the main components of the oils from *Thymus x citriodorus* "Archer's Gold" and Thymus x citriodorus, respectively (Table 2). Among the other constituents, *p*-cymol, *β*-caryophyllene, geranial, limonene, and *γ*-terpinene were also characteristic of the oils, but smaller amounts were present. Thymol is the main component of oils from *Thymus vulgaris* and *Thymus serpyllum* (37.1% and 17%, respectively) [144].

**Coriandrum**. Essential oils from the fresh herb *Coriandrum sativum* L. accumulate during the growth of the plant. According to Wei et al., it is recommended to harvest the vegetative part prior to flowering because (*E*)-2-decenal, a potential irritant, is present at higher percentages in the preflowering and full flowering stages [145].

There are significant differences in the essential oil types from different parts of coriander. (*E*)-2-decenal is the dominant constituent in essential oils from coriander leaves: 31.28% and 61.86% of the essential oils from coriander stems [145]. Linalool was found to be the main constituent of essential oils from fully mature coriander fruits, seeds, and pericarps. Linalool and citronellol are the main components of coriander inflorescence essential oils from lower latitudes. The young vegetative organs in the seedling stages not only have a grassy odor, peculiar to coriander, but also a fresh green odor, highly suitable for seasoning [146].

**Sage** it is one of the most appreciated herbs due to its rich essential oils, which have antimicrobial, antifungal, and antimutagenic properties [128]. More than 120 components have been characterized in the essential oil prepared from aerial parts of *S. officinalis*. The main components of the oil include borneol, camphor, caryophyllene, cineole, elemene, humulene, ledene, pinene, and thujone [53].

**Basil.** Oxygenated monoterpenes and phenylpropanoids are the main compounds of the *Ocimum* genus. In different *O. basilicum* cultivars and chemotypes, linalool, eugenol, methyl chavicol, methyl cinnamate, methyl eugenol, and geraniol have been reported as the major components [147].

**Parsley.** In terms of essential oils, the major compound in parsley's essential oils is myristicin, which has spicy, warm, and balsamic odors. The combined cleaning process with drying at different temperatures provided a greater reduction in the microorganisms in relation to separate processes. Drying did not change the oil yield in relation to the fresh plant [148].

**Tarragon.** According to Sobieszczanska et al., tarragon essential oil and its major compounds act against food-associated *Pseudomonas* spp. Tarragon essential oils are rich in other compounds, particularly methyl chavicol (tarragon and basil essential oils [149].

**Origanum** essential oils, together with sage, rosemary, marjoram, and thyme essential oils, are mainly composed of oxygenated monoterpenes (>40%) [150]. Generally, the *Origanum* species is characterized by the presence of two major biochemically related groups of compounds (aromatic monoterpenes such as *p*-cymene, thymol, carvacrol, their precursor *γ*-terpinene, and their derivatives; thujanes, such as sabinene, sabinene hydrate, and their derivatives) [151]. Thymol and carvacrol, which are present in high amounts in its essential oils (78–82%), are generally responsible for its antioxidant properties [128].

**Marjoram.** The results of marjoram essential oil analysis by Dimitra et al. gave a large number of constituents. Among them, 3-thujene (2.8%), *γ*-myrcene (3.8%), 2-carene (7.8%), 2-ethyl-mxylene (5.2%), 3-carene (10.4%), terpinen-4-ol (7.8%), sabinene hydrate (6.0%), R-terpineol (4.2%), and thymol (14%) were detected. Two chemotypes of *O. majorana* are reported in the literature: the cis-sabinene hydrate/tepinen-4-ol chemotype and the carvacrol/thymol chemotype [151].

**Bay.** The predominant flavor compound is 46% eucalyptol; essential oils of bay leaf induced human aryl hydrocarbon receptor activity by threefold, and its major constituent eucalyptol (46%) was inactive [152].

**Dill.** Terpenes were the most abundant volatiles detected in dill essential oils. Dill oil had a relatively limitary chemical profile, approximately equal volumes of carvone and D-limonene. Together, these accounted for 97.5% of the compounds identified by gas chromatography/mass spectroscopy [153].

**Table 2.** Variability in the chemical composition of EOs and aroma compounds of spices and aromatic plants used to prepare meat and meat alternatives.

**Table 2.** *Cont.*

**Table 2.** *Cont.*

a Odor descriptions according to the Flavornet (www.flavornet.org(accessed on 3 January 2022) and Pherobase databases (www.pherobase.com(accessed on 3 January 2022)).

#### **5. Conclusions**

The increasing interest of consumers for most natural foods, natural flavors, natural antioxidants, natural antimicrobial substances is often linked to their bioactive compounds; spices and aromatic plants are some of the richest sources of phytochemicals with demonstrated biological activities. The most commonly used spice and aromatic plants in the meat and meat analogues industry are parsley, dill, basil, oregano, sage, coriander, rosemary, marjoram, tarragon, bay, thyme, and mint. As shown in this paper, they improve meat preparation in terms of flavor, as well as antimicrobial and antioxidant activity. Due to their bioactive compounds, they also have beneficial effects on the consumer health. Although studies of the effects of these herbs and spices on meat analogues are limited, their use is highly recommended due to the flavor improvements, as well as consumers health benefits.

**Author Contributions:** Conceptualization, R.A.M., V.M. and A.E.M.; writing—original draft preparation, R.A.M., C.C.M., A.E.T., A.P., G.S.M. and R.A.U.; writing—review and editing, R.A.M., V.M. and A.E.M.; visualization, R.A.M., V.M. and A.E.M.; supervision, R.A.M., V.M. and A.E.M.; funding acquisition, V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI—UEFISCDI, project number PN-III-P2-2.1-PED-2019-5346, within PNCDI III, and the publication was supported by funds from the National Research Development Projects to finance excellence (PFE)-14/2022-2024 granted by the Romanian Ministry of Research and Innovation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**

## *Article* **Cytotoxicity Evaluation and Antioxidant Activity of a Novel Drink Based on Roasted Avocado Seed Powder**

**Andreea Pus, cas, 1, Anda E. Tanislav 1, Romina A. Marc 1, Vlad Mures, an 1, Andrut,a E. Mures, an 1,\*, Emoke Pall 2 and Constantin Cerbu 2**

**Abstract:** The avocado seed is an underused waste resulting from the processing of pulp. Polyphenols, fibers, and carotenoids are present in the seed, which also exhibits prophylactic, fungicidal, and larvicidal effects. Developing food products with avocado seed as an ingredient or spice is highly desired for nutritional, environmental, and economic reasons. The present study proposed its valorization in a hot drink, similar to already existing coffee alternatives, obtained by infusing the roasted and grinded avocado seed. The proximate composition of the raw or conditioned avocado seed and that of the novel drink were determined. The total phenolic content was assessed using the Folin-Ciocâlteu method. The total carotenoids were extracted and assessed spectrophotometrically. Starch determination was performed by the Ewers Polarimetric method. The highest content of polyphenols, 772.90 mg GAE/100 g, was determined in the crude seed, while in the drink was as low as 17.55 mg GAE/100 g. However, the proposed drink demonstrated high antioxidant capacity, evaluated through the DPPH method. This might be due to the high content of the total carotenoid compounds determined in the roasted seed (6534.48 μg/100 g). The proposed drink demonstrated high antiproliferative activity on Hs27 and DLD-1 cell lines.

**Keywords:** avocado seed; valorization; coffee alternative; food waste; bioactive compounds; antioxidant capacity; cytotoxicity

#### **1. Introduction**

The peel and seed of the avocado, resulting as by-products in the processing of the pulp, are waste materials which should have application in the domains of food, nutrition and medicine, since recent studies revealed and characterized the valuable nutrients they contain, demonstrating health promoting effects of some of their extracts [1–4]. The peel is edible, but is not consumed due to its bitter taste, chewiness, and because it is hard to digest. The seed is tough, has an astringent taste, and needs processing prior to consumption; therefore, formulating food products containing these valuable ingredients is challenging.

The pulp of avocado is a great source of proteins and monounsaturated fatty acids (predominantly oleic acid—62.14%, palmitic 17.2%, linoleic 11.11%, and palmitoleic acid 7.34%) and low amounts of stearic acid, 0.63%, protecting consumers against coronary heart disease development [5,6]. Besides fatty acids, the nutritional value of avocado is also due to antioxidants, carotenoids, phytochemicals such as α-tocopherol and β-sitosterol, and vitamins (B6, biotin, folic acid, thiamine, riboflavin, vitamin D and K) [7,8]. Avocado pulp has been used (fresh or dehydrated, defatted) in supplementing food products such as meat alternatives or in replacing wheat flour and butter, in whole grain crackers [9,10].

**Citation:** Pus, cas, , A.; Tanislav, A.E.; Marc, R.A.; Mures,an, V.; Mures,an, A.E.; Pall, E.; Cerbu, C. Cytotoxicity Evaluation and Antioxidant Activity of a Novel Drink Based on Roasted Avocado Seed Powder. *Plants* **2022**, *11*, 1083. https://doi.org/10.3390/ plants11081083

Academic Editor: Petko Denev

Received: 8 March 2022 Accepted: 12 April 2022 Published: 15 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

The processed products of avocado pulp include oil, the paste, puree, and guacamole; each of them is sensible to oxidation processes, so preservation or physico-chemical treatments are mandatory. It was reported that increased consumption levels can reduce adult weight gain [11], so it is no surprise avocado is becoming more and more consumed. Along with this, a high level of waste material is also generated on the industrial scale or in individual households.

The seed represents 13–18% of the weight of the whole fruit and the residues have a significant environmental impact due to the great organic charge they contain. Additionally, avocado seeds generate costs associated with disposing, handling, transport, and storage [7,12,13]. Numerous studies revealed some valuable compounds in the chemical composition of the avocado seed, imparting antioxidant and antimicrobial properties to it, transforming this waste material into a valuable ingredient of food products with the potential for medicinal use [3,14,15]. These studies were conducted on various species of avocado, such as Corillo [16] or Mill [17], including the Hass variety [18]. Phytosterols, triterpenes, fatty acids, furanoic acid, abscisic acid, proanthocyanidines (PACs), and other polyphenols are present in variable amounts in the seed, depending on the maturity, growth condition, and variety of the avocado [15,19,20].

Besides, anti-inflammatory, hypoglycemic, antihypertensive, fungicidal, larvicidal, hypolipidemic, analgesic, amoebicidal, and giardicidal activities of some extracts from the avocado seed have been reported [15,21–25]. The seed contains more soluble fibers than the pulp, so by ingesting it, one could naturally prevent constipation. It is also proved to be effective in the prophylaxis of gastric ulcers, preventing the occurrence of this disease [26,27]. Avocado seed is also rich in tannins, carotenoids, and tocopherols, which inhibited the in vitro growth of prostate cancer cell lines [28].

Extracts from the peel and seed displayed different functionalities when added to food products [17]. Acetone/water (70:10 *v/v*) extracts from the peel and seed were included in raw porcine patties and hindered the oxidative reactions and color deterioration during chilled storage of the product [29]. A natural orange colorant was extracted with water from the avocado seed which was priorly grinded [30].

Due to some antinutritional compounds present in the avocado seed, namely phytate, oxalate and cyanogenic glycosides [31], studies have been carried out in order to evaluate the effect of processing methods against the antinutritional compounds of *Persea americana* seed as a step towards establishing purposeful utilization in the food production area [13,32,33]. Solid-state fermentation of Hass avocado seed with *A. niger* GH1 led to an improved antioxidant activity [4]. Among other antinutritional factors, tannins, phytic acid and alkaloids were determined [1]. Soaking and boiling of the avocado seeds reduced the antinutritional compounds to a large extent. Some research describing novel food products with avocado seed as the ingredient has already been conducted [9,33–35]. Powders obtained from the seeds of avocado and peeled ginger roots were used for the preparation of eight prototypes of candies with sugar or aspartame. The candies were analyzed in terms of moisture content (77.3–92.5%), total sugars (0.10–0.66 mg/100 g) and microbial count. The sensorial analysis were promising, the candies being scored above average in taste, texture, and flavor [36].

Avocado seeds have been proposed to be valorized as flour and it would be of special use in tropical countries, were crops such as wheat, barley, millet, and rye do not lead to quality flours and lead to higher costs for pastry products [37]. For the flour preparation, the washed seeds were chopped and dried in an oven at 60 ◦C for 5 h. The seeds were then grounded in a pulverizer. The avocado seed flour was characterized in terms of yield, proximate analysis, gluten, and falling number and different biscuit formulations were prepared in order to test its applicability. The anthocyanin pigments of the avocado seed led to a darker color for the biscuits, which had an average acceptability in terms of the organoleptic properties. In conclusion, the avocado seed flour, despite the high nutritional value, would not be suitable for baking, having high values for the falling number and not forming the gluten network needed for structure [37]. However, Hass Avocado seed

flour alone or in mixture with corn was studied for obtaining extruded snacks, showing promising results [35].

In the current study, drying and roasting were explored as conditioning treatments aiming to reduce the antinutritional compounds activity and concentration in the avocado seed. The present study proposed the use of the avocado seed powder in a hot drink, which could be consumed as a coffee alternative. Novel usages would increase its consumption and decrease food waste and pollution. The roasted avocado seed powder and thereof novel drink were analyzed, and the results revealed some antioxidant functionality and anti-carcinogenic effects.

#### **2. Results**

#### *2.1. Optimization of Roasting of Avocado Seed and the Drink Preparation*

Since the organoleptic properties of avocado seed were unknown, the temperature setting for the heat treatment was started at 135 ◦C, with a duration varying from 5–90 min. The best results in term of color and flavor were obtained after 90 min, when the seed was dark brown and well flavored. However, in order to reduce the duration of the roasting process, higher-temperature protocols were also explored. Table 1 presents the organoleptic profile of the avocado seed roasted under different time-temperatures protocols, starting from 160 ◦C ± 5 ◦C to 200 ◦C ± 5 ◦C. Given the results, the most suitable time-temperature protocol was: 180 ◦C/25 min, respectively 180 ◦C/30 min and 200 ◦C/5 min.

**Table 1.** Organoleptic profile of roasted avocado seed at different temperature-time intervals.

In order to obtain a product with optimal properties, different percentages of water and avocado powder were studied. The final composition was decided to be composed of 93% water and 7% roasted avocado seed powder. The powder had an intense and slightly astringent aroma, and it is sufficient even in this low percentage for obtaining the novel hot drink.

#### *2.2. Proximate Composition*

To determine the patterns of biologically active compounds accumulation in agroindustrial by-products, it is important to identify their composition and content in separate parts. The proximate composition of the Hess Avocado Seed, in raw, dried, or roasted state is summarized in Table 2. The results indicated that the seed is a good source of dietary protein, with 5.10 g/100 g Fresh Weight (FW) in the raw condition and 5.35 g/100 g FW when the seed is dried or 4.05 g/100 g FW sample when roasted at 160 ◦C for 60 min. The moisture was reduced in the drying or roasting treatments, leading to different proximate

compositions of the samples. The raw avocado seed, besides protein, contains 0.74 g/100 g FW of fat and 49.72 g/100 g FW of carbohydrates and a content of 1.61 g/100 g FW minerals.

**Table 2.** Proximate composition of raw or conditioned Hass avocado seed expressed as g/100 g (FW).

The raw avocado seed will furnish, upon consumption, 225.94 kcal/100 g FW and a higher caloric content was calculated for the dried and roasted samples (275.41/100 g and 395.37 kcal/100 g FW respectively). The currently proposed drink was prepared from 7% of the roasted avocado powder infused with hot water, and thus will have a total caloric amount of 56.48 kcal/100 mL.

The avocado seed is rich in carbohydrates, and thus the further investigation of starch content, which is the most predominant among the carbohydrates, was performed by the Ewers Polarimetric method. In the dried sample, the content is 43.9868 g/100 g FW and in the roasted sample is 48.1192 g/100 g FW. The thermal treatment of the sample might lead to some hydrolysis, increasing the content of starches in comparison with the dried sample. The rest carbohydrates might be represented by dietary fiber [38].

#### *2.3. Total Polyphenol Content by Folin-Ciocâlteu Method*

As can be seen in Table 3, the highest total polyphenol content was recorded in the crude avocado seed, with a value of 772.90 ± 4.09 mg GAE/100 g FW. The total amount of polyphenols was significantly reduced during processing, while in the drink was as low as 17.55 ± 0.70 mg GAE/100 g FW, given that only 7% of the roasted seed was used for the preparation of the drink. The total polyphenol content of the roasted avocado seed (180 ◦C/25 min) was of 179.07 ± 4.09 mg GAE/100 g FW; thus, the thermal treatment required in order to obtain organoleptic profiles similar to the coffee alternatives will significantly affect the biologically active compounds which are known to be thermolabile. However, polyphenols are generally more hydrophilic than lipophilic, and thus the proposed hot drink could be a good delivery system for these compounds.

**Table 3.** The total polyphenol content of raw and conditioned Hass avocado seed and the novel drink.

Identical superscript letters indicate no significant difference (*p* > 0.05).

#### *2.4. The Total Carotenoid Compounds Content in the Avocado Seed*

The total carotenoid compounds in fruits and vegetables are known to vary because of factors such as genetic variety, maturation stage, or processing conditions. The highest total carotenoid compound in the avocado seed was determined for the dried sample 9228.52 ± 21.20 μg/100 g FW. Similar, but statistically different contents, were determined for the crude and roasted samples (6190.56 ± 14.30 μg/100 g FW and 6534.48 ± 28.30 μg/100 g FW, respectively), as seen in Table 4.

**Table 4.** The total carotenoid compounds of raw and conditioned Hass avocado seed.

Identical superscript letters indicate no significant difference (*p* > 0.05).

#### *2.5. Antioxidant Capacity of Avocado Seed and the Drink*

The antioxidant capacity reflects the capacity of bioactive compounds to maintain the nutritional and sensorial quality of the product. The highest capacity was observed for the dried seed (95.66 ± 0.566 RSA% DPPH inhibited), but the results registered for the crude and roasted seed were not statistically different (Figure 1). On the other hand, good antioxidant capacity was registered for the proposed drink; even if the roasted avocado seed powder represents 7% of its composition, its antioxidant capacity was 90.27 RSA% DPPH inhibited.

#### *2.6. The Total Acidity and pH Analysis*

The organic acid content present in the crude avocado seed, determined as the titratable acidity, was 0.0538% malic acid equivalents, while in the dried and roasted samples the values were increased to 0.1361% malic acid equivalents and 0.1223% malic acid equivalents respectively. The novel drink registered a total acidity of 0.0268% malic acid equivalents. A pH value of 5.12 ± 0.13 was determined for the roasted avocado seed based drink, this being characteristic for acid drinks.

#### *2.7. The Cytotoxicity Assay of Avocado Seed Drink*

Cytotoxicity is considered an important aspect of any new food product or beverage which partially anticipates its health benefits upon consumption. Figures 2 and 3 show the cell viability of the exposed cells to increasing concentrations of the novel roasted avocado seed-based drink.

**Figure 2.** Cell viability of Hs27 cells when treated with the novel roasted avocado seed-based drink.

**Figure 3.** Cell viability of DLD-1 cells when treated with the novel roasted avocado seed-based drink.

In vitro cell viability tests demonstrated less than ≈6% loss in cell viability on both cell lines at the lowest concentration (2.5%) investigated. At the highest concentration (40%), loss in cell viability was observed to be ≈57% for Hs27 cells, whereas the value was ≈47% for the DLD-1 cells at the same concentration. These results showed that the novel drink may decrease the viability of both human fibroblasts (Hs27) and human colorectal adenocarcinoma (DLD-1) cell lines. Furthermore, cell viability experiments demonstrated a dose-dependent response on both cell lines.

#### **3. Discussion**

Dried Hass avocado seed powders (65 ◦C/120–180 min) obtained from ripe or unripe samples were analyzed in terms of proximate composition in another study, in order to be used for preparing extruded snacks [35]. The results of the proximate composition for the ripe seed were slightly different of those presented in the present study, probably due to the longer processing time: the moisture was lower—23.79%, 3.18% protein, 3.33% fibers, 65.62% nitrogen-free extract (carbohydrates), 2.6% ether extract, and 1.51% ashes. This study states the presence of antinutritional factors in the avocado seed (hydrocyanic acid, cyanogenic glycosides, condensed polyphenols and some tannins), which can be eliminated by a cooking treatment [35].

The proximate composition of avocado seed and that of the vitamins were determined in another study after boiling or soaking of the avocado seed. Statistically significant differences (*p* < 0.05) occurred for the content of crude fat, minerals, crude fiber, carbohydrates, and vitamins A, C, and E, during different processing protocols [32]. A content of 0.9% fat and 3.10% protein were determined by other authors in the crude (untreated) Hass Avocado seed [39]. The analysis of individual minerals with the atomic absorption spectrometry method was also performed, revealing high amounts of phosphorus (1000 mg/kg), calcium (533 mg/kg), and magnesium (544 mg/kg); an amount of 1.97% ash was determined, slightly higher than the amount determined in our study [39]. In the Algarvian avocado var. "Hass", lower percentages of protein and a higher amount of fat were determined and the acidity of 2.67 ± 0.17 mg of tartaric acid equivalents/100 g [37]. The starch content of the avocado seed was also investigated by other authors with the Ewers polarimetric method, and the study revealed that higher amounts of starch can be determined in the ripe seed than in the over-ripe seed [39]. Another study reported a starch yield of 42.2% extracted with metabisulfite solution and by producing a dough which was filtered and washed to separate starch from *Daisy* variety [40]. This starches can be further hydrolized using acid or enzyme hydrolysis [41]. The parameters of gelatinization and viscosity of extracted from the avocado seed present restricted dilation, which suggests their possible use in food products which must be heated up at 100 ◦C [35]. A comparative analysis of the antioxidant capacity of different varieties of avocado seed samples and different extraction condition was carried out in the study of Segovia et al. [42]. In regard to the analysis of total phenolic compounds, the following results were registered for the *Persea americana var.* Hass seed, extracted with Methanol/Water (80:20 *v/v*), 60 ◦C, namely 9.51 ± 0.16 mg GAE/g [42]. For the ethanolic extracts, the results were of 8.07 ± 0.03 mg GAE/g in the study of Amado et al. [8]. Similar total phenolic compounds were revealed in the Algarvian avocado var. "Hass" (7.04 ± 0.13 mg GAE/g) [43]. Thus, the extraction method applied in the present study is effective in the determination of the total phenolic compounds.

The determination of the total phenolic compounds in a Turkish chicory root, which was roasted for 2 h at 140 ◦C and grinded to be used as a common coffee alternative, revealed lower contents (between 0.943–13.860 mg GAE/g DW) than those determined in the roasted avocado seed (180 ◦C/25 min), which were 179.07 ± 4.09 mg GAE/100 g [44]. In the study of Afify et al., the TPC determined for different coffee or teas prepared in hot or cold water, varied between 1.68 ± 0.06 to 2.28 ± 0.06 g GAE/100 g in teas and 1.87 ± 0.07 for a coffee variety, which is lower than the amount determined for the drink prepared from the roasted avocado seed (17.55 ± 0.70 mg GAE/100 g) [45].

Given the high availability, economic advantages, and the abundance of the biologically active compounds, it would be of use to assess the acceptability of the consumers toward food products or beverages having avocado seed as ingredient or spice. It is also a good source of carotenoid compounds, a total amount of 0.97 ± 0.164 mg/100 g fresh weight basis expressed as β-carotene equivalents being determined for the Algarvian avocado var. "Hass". This result is higher than the results exhibited by the crude sample explored in our study. In the current study, slightly higher amounts of total carotenoid compounds were detected in the roasted sample, in comparison with the raw sample, which might be caused by the increase in different isomers of lycopene which are precursors of β -carotene [46].

Another study explored the individual carotenoid compounds determined by HPLC-MS in the Hass Avocado variety originating from Chile, lutein (131.51 μg/g oil extracted

from the seed or 2.62 μg/g fresh fresh weight basis) and *β*-carotene (111.88 μg/g oil extracted from the avocado seed or 2.22 μg/g fresh weight basis) being determined as major carotenoid compounds [47]. Numerous studies explored and demonstrated the in vitro antioxidant and cancer inhibitory activity of avocado seed extracts [2]. Cell viability was assessed using a modified MTT assay on the seed extracts of Hass and Fuerte varieties, and their capacity to inhibit TNFα was assessed in LPS-stimulated RAW 264.7 macrophage culture. None of the tested extracts exhibited cytotoxicity up to 10 μg/mL and the seed exhibited good anti-inflammatory effects after 4 h [48]. In another study, a methanol soluble fraction of the avocado displayed the capability to induce apoptosis and anti-proliferative effects to MCF-7 cell lines [49].

#### **4. Materials and Methods**

#### *4.1. Materials*

Hass Avocados were purchased from a local market from Cluj Napoca, Romania and were of Columbian origin. All the reagents were of analytical grade.

#### *4.2. Optimization of Roasting of Avocado Seed*

The roasting of the avocado seed was performed at different time and temperature intervals to highlight the aroma and to obtain organoleptic characteristics as close as possible to those of coffee or its replacements. The organoleptic analysis was conducted by analyzing the flavor profile of the seed after each time and temperature protocol by a part of the collective of authors who were instructed in regard to the desired organoleptic properties and with previous experience in this [50]. The roasting process was conducted in a Memmert UF55 (Buechenbach, Germany) oven (135 ◦C for 5–90 min first and for optimization different time temperatures intervals between 160–200 ◦C, 5–90 min were explored). The temperature in the room was 22–23 ◦C and the relative humidity was 40–42%.

#### *4.3. The Avocado Seed Powder and the Drink Preparation*

The fresh avocado seed contains a high amount of water in the composition and the seeds were naturally dried in a warm airy room, for 5–7 days. The seed is a dicotyledonous and it was kept for 5 min in the oven to facilitate the decortication. The seed was passed through a grater prior to roasting at 180 ◦C for 25 min. The grater was of stainless steel grade and the side with small holes (diameter 2 mm) was used. After roasting, it was finely ground into a powder using the Retsch RM200 (Haan, Germany) grinding machine set in position 8 (100 rot/min for 20 min).

For the hot drink preparation, water (90 ◦C ± 5 ◦C) and a French press were involved until the infusion took place (10 min).

#### *4.4. Proximate Composition Analysis*

The chemical compositions including moisture, ash, total carbohydrates, total sugars, crude fat, and protein content were determined for the fresh, dried, and roasted avocado seeds according to AOAC procedures and were expressed in regard to the fresh weight (FW). For moisture analysis, samples were subjected to drying in an oven at 103 ± 2 ◦C for 3 h, the experiment being repeated until the weight was constant. The samples were cooled in a desiccator for one hour and weighed (AOAC, 1999).

The ash content was determined by calcination at 550 ◦C of 2 g of probe until a gray ash was obtained, with the removal of the carbon black spots by splashing with water, then the process was continued until a gray or white ash resulted (after 6 h).

The crude protein content of the samples was estimated by the Kjeldahl method.

The crude fat content of the samples was determined by extracting a known weight of powdered samples (3 g) with petroleum ether as a solvent, using the Soxhlet apparatus.

The amount of total carbohydrate was calculated by difference.

The starch content of avocado seed was determined using the Ewers polarimetric method (ISO 10520: 1997) with some modifications [51].

#### *4.5. Total Phenolic Content*

The total polyphenols content was assessed using the Folin–Ciocâlteu method [52], slightly modified. An amount of 1 g of sample was mixed with methanol and 0.01% HCl. The obtained extracts were filtered and dried at 35 ◦C under reduced pressure (Heidolph Rotary Evaporator, Schwabah, Germany). A quantity of 25 μL sample was mixed with 1.8 mL of distilled water and 120 μL Folin-Ciocâlteu reagent in a glass vial. A 7.5% Na2CO3 solution prepared in distilled water (340 μL) was added 5 min later to assure basic conditions (pH 10) for the Redox reaction between the phenolic compounds and the Folin-Ciocâlteu reagent. The samples were incubated for 90 min at room temperature. Methanol was used as a control sample. The absorbance at 750 nm was measured using a Shimadzu UV-VIS 1700 spectrophotometer (Shimadzu, Kyoto, Japan). The calibration curve was plotted based on the 0.25, 0.50, 0.75, 1 mg ml−1 concentration of gallic acid. The total polyphenol content of the avocado seed was expressed for fresh weight (FW) in Gallic acid equivalents (GAE)—mg GAE·100 g−1.

#### *4.6. Spectroscopic Analysis of the Total Carotenoid Content*

To assess the total carotenoid content, carotenoids were extracted from the crude, dried, and roasted avocado seeds, using ethanol: ethyl acetate: petroleum ether (1:1:1, *v/v/v*). Successive extractions were performed. The extracts were combined, filtered, and washed with distilled water, diethyl ether, and a saturated solution of NaOH. The ethereal phase was recovered and subjected to rotary evaporation at 35 ◦C (Heidolph Rotary Evaporator, Schwabah, Germany). Estimation of carotenoids was spectrophotometrically determined using Shimadzu UV-VIS 1700 set at 450 nm (Shimadzu, Kyoto, Japan).

#### *4.7. The Antioxidant Activity*

The antioxidant activity was determined using the 2.2-diphenyl-1-picrylhydrazyl (DPPH) method, according to Mures, an et al. [53]. 10 μL methanolic extract from the avocado seed was mixed with 3.9 mL DPPH methanolic solution (0.025 g/L) and 90 μL distilled water. The mixtures were stirred and maintained properly in the dark for 30 min. The absorbance of the samples was measured at 515 nm (Shimadzu 1700 UVVIS, Kyoto, Japan) against a methanol blank. The positive control was prepared using a gallic acid solution (0.5 mg/mL). The negative control was prepared using methanol. Results were expressed as percent over standard DPPH absorbance according to the following equation:

$$\text{RSA} \left[ \% \right] = \frac{\text{A}\_{\text{DPPH}} - \text{A}\_{\text{P}}}{\text{A}\_{\text{DPPH}}} \bullet 100$$

where: RSA [%]—Radical Scavenging Activity; ADPPH—the absorbance of DPPH solution with methanol; AP—the absorbance of DPPH solution after 30 min incubation with sample.

#### *4.8. The Total Acidity and pH Analysis*

The total acidity was performed by neutralization with sodium hydroxide solution (0.1 N) in the presence of fenolftalein as indicator. The results were expressed in g malic acid equivalents/100 g. Titratable acidity calculation was carried out using the formula:

$$\text{Acidity }\% \text{ (malic acid)} = \frac{\text{V} \bullet 0.0067}{\text{m}} \bullet 100\text{.}$$

where 'V' is the volume of NaOH solution 0.1N, 'm' is the weight of the sample, and '0.0067 g of malic acid corresponds to 1 mL NaOH 0.1 N.

pH analysis was also carried out on the novel drink. The determination was based on the property of indicators contained by the pH paper to change their color in the presence of hydrogen ions. To determine the pH of the novel drink, the pH meter Mettler Toledo (Columbus, OH, USA) was employed.

#### *4.9. Cytotoxicity Assay*

The cytotoxicity assay of the avocado seed drink was performed using human fibroblasts Hs27 (ATCC® CRL-1634™, Manassas, VA, USA) and human colorectal adenocarcinoma DLD-1 (ATCC® CCL-221™, Manassas, VA, USA) cell lines. The cells were cultured according to standard conditions. The potential cytotoxicity of avocado seed drink was assessed with (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In order to obtain cell suspensions, the cells were treated with 0.25% trypsin-EDTA, and after centrifugation (1500 rpm for 5 min), 1 × 104 cells/well were seeded on 96-well plates in 200 μL complete culture medium. After 24 h, 80 μL (40% *v/v*), 60 μL (30% *v/v*), 40 μL (20% *v/v*), 10 μL (5% *v/v*), and 5 μL (2.5% *v/v*) of avocado seed drink were added, while removing the same volume of culture media, resulting in a total final volume of 200 μL/well. Control samples were represented by untreated cells. Each experimental condition was performed in triplicate. Cell proliferation analysis was performed after 24 h. After 24 h, the medium was removed and 100 μL of 1 mg/mL MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added. After 4 h of incubation at 37 ◦C in dark, the MTT solution was removed from each well and 150 μL of DMSO (dimethyl sulfoxide) solution (Fluka, Buchs, Switzerland) was added. Spectrophotometric readings at 450 nm were performed with a BioTek Synergy 2 microplate reader (Winooski, VT, USA). Data are shown as percentage of cell viability.

#### *4.10. Statistics*

Statistical differences were obtained through an analysis of variance (ANOVA) followed by Tukey's multiple comparison test at 95% confidence level (*p* ≤ 0.05).

#### **5. Conclusions**

The avocado seed has been explored lately for various applications due to its composition rich in bioactive compounds. Numerous processing techniques such as boiling, dryingor extrusion, have been explored so far as conditioning methods, due to the presence of anti-nutritive factors in its composition. The current study evaluated different timetemperature protocols for the roasting of the avocado seed, along with drying, and the flavor and color modification were assessed. We proposed the valorization of the roasted (180 ◦C/25 min) avocado seed powder in a hot drink, obtained by creating an infusion with 7% of the powder and hot water.

The raw or conditioned (dried or roasted) Hass avocado seeds were examined in terms of proximate composition and bioactive compounds. The seed possess a high amount of carbohydrates, including dietary fibers and between 4–5% protein, depending on the conditioning process applied (drying or roasting). The total polyphenolic content of the avocado seed was reduced during the conditioning, while the acidity and total carotenoid compound were significantly increased. The novel drink exhibited a high antioxidant capacity of 90.27 RSA% DPPH inhibited, which might be due to the presence of carotenoid compounds or flavonoids.

However, the novel drink exhibited a lower concentration of the total polyphenolic compounds in comparison with the raw or conditioned seed (only 17.55 ± 0.70 mg GAE/100 g in the drink compared with 179.07 ± 4.09 mg GAE/100 g in the roasted avocado seed), mostly because the drink has a roasted avocado seed powder concentration as low as 7%. This is a higher content that what was previously registered for coffee or coffee surrogates in other studies. The cytotoxic properties of the novel drink based on roasted avocado seed were also demonstrated, because when it was applied in a concentration of 40% on DLD-1 cells and Hs27 cells during the MTT cytotoxicity assay, it affected the viability of the cells.

**Author Contributions:** Conceptualization, A.E.M.; data curation, A.P.; formal analysis, A.P., A.E.M., A.E.T., R.A.M., E.P. and C.C.; investigation, V.M. and C.C.; methodology, A.P. and A.E.M.; project administration, A.E.M.; supervision, A.E.M., V.M. and C.C.; writing—original draft, A.P.; writing review and editing, V.M. All authors have read and agreed to the published version of the manuscript. **Funding:** This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI—UEFISCDI, project number PN-III-P1-1.1-PD-2019-1108, within PNCDI III and the publication was supported by funds from the National Research Development Projects to finance excellence (PFE)-14/2022-2024 granted by the Romanian Ministry of Research and Innovation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**