The increased HDL-C levels noticed in groups 3 and 4 indicates a possibly lower risk of developing coronary heart diseases and other related cardiovascular events. Nichols et al. [20] reported that a moderate rise in HDL-C levels, resulting from the use of statin drugs, has been linked to a corresponding decline in the risk of developing coronary atherosclerosis. Stocker et al. [21] also reported that lowering LDL-C was a more effective method of reducing the risk of developing cardiovascular diseases than surgical methods. However, the abundance of antioxidants in the plant (*V. calvoana*) could prevent the oxidation of LDL-C within the blood vessels. It is a known fact that LDL-C is virtually harmless till it is oxidized by reactive species in the blood vessels, resulting in atherosclerosis [22]. The reduced atherogenic indices of the extract-treated groups implies a decreased risk of developing atherosclerosis since the atherogenic index of plasma is considered a reliable indicator of the onset of cardiovascular events [23,24]. Our finding is in agreement with the hypolipidemic effect of *V. calvoana* extracts in a diabetic rat model as reported by Iwara et al. [9] which implicated flavonoids and other bioactive principles for the effects. They are also in agreement with the report of Johnson et al. [25] where *V. amygdalina* decreased levels of MDA and prevented peroxidation of lipids in PC-3 cells.

#### **5. Conclusions**

From the results obtained in this study, it is safe to further confirm that the ethanolic leaf extract of *V. calvoana* possesses a lipid-lowering and cardioprotective effects and is a candidate in the management of paracetamol-induced toxicities, however, further studies on the molecular mechanism underlying the effect of *V. calvoana* on functional lipids is being investigated in our laboratory.

**Acknowledgments:** The authors are grateful to Victor Udo Nna of the Department of Physiology for his critical suggestions during the manuscript preparation.

**Author Contributions:** G.E.E. designed and supervised the work, O.D.O. carried out the work, I.J.A. and V.N.N. provided some literature information and also read and approved the final manuscript.

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

#### **References**

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## **Anti-Lipase Potential of the Organic and Aqueous Extracts of Ten Traditional Edible and Medicinal Plants in Palestine; a Comparison Study with Orlistat**

#### **Nidal Jaradat \*, Abdel Naser Zaid, Fatima Hussein, Maram Zaqzouq, Hadeel Aljammal and Ola Ayesh**

Department of Pharmacy, Faculty of Medicine and Health Sciences, An-Najah National University, P.O. Box 7, Nablus 00970, Palestine<EMAIL_ADDRESS>(A.N.Z<EMAIL_ADDRESS>(F.H<EMAIL_ADDRESS>(M.Z<EMAIL_ADDRESS>(H.A<EMAIL_ADDRESS>(O.A.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +970-599739476

Academic Editor: Gema Nieto Received: 21 October 2017; Accepted: 6 December 2017; Published: 8 December 2017

**Abstract: Background:** Herbs have played a fundamental and essential role in the humans life since ancient times, especially those which are used as food and/or folk medicinedue to both their nutritive and curative properties.This study aimed to investigate new antilipase agents from tentraditional Palestinian edible and medicinal plants through inhibition of the absorption of dietary lipids. **Methods:** The anti-lipase activity for ten plants was evaluated and compared with the reference compound Orlistat by using the porcine pancreatic lipase inhibitory test which was conducted by using a UV-visible spectrophotometer. **Results:** The aqueous extracts of *Vitis vinifera* and *Rhus coriaria* had the highest antilipase effects with IC50 values 14.13 and 19.95 mcg/mL, respectively. Meanwhile, the organic extract of *Origanum dayi* had an IC50 value 18.62 mcg/mL. *V. vinifera* showed the highest porcine pancreatic lipase inhibitory effects when compared with Orlistat, which has an IC50 value 12.38 mcg/mL. **Conclusions:** According to the obtained results, *V. vinifera*, *R. coriaria*, and *O. dayi* can be considered a natural inhibitors of the pancreatic lipase enzyme as well as new players in obesity treatment. In fact, these plants can be freely and safely consumed in a daily diet or can be prepared as nutraceutical formulations to treat or prevent of obesity.

**Keywords:** anti-obesity; anti-lipase; traditional medicine; folkloric food

#### **1. Introduction**

The results from the WHO global survey on traditional, complementary/alternative, and herbal medicines showed that the market for these kinds of medicines is growing steadily worldwide. In fact, the usage of phytopharmaceuticals and nutraceuticals is rapidly and continuously expanding. Recently, many people have been using these formulations in the treatment or prevention of various diseases and disorders in different national healthcare systems. Moreover, many patients often use herbal medicines to complement treatment with conventional medicines [1–3]. Herbals and phytopharmaceuticals for the treatment of excess weightand obesity were among the most used remedies, especially in developing and developed countries, since these metabolic disorders became very prominent [4,5]. In fact, obesity poses a worldwide concern, not only for the harm which excess weight alone may cause, but also due to associated health problems such as endocrine, metabolic, and cardiovascular disorders [6–8].

Accordingly, various therapeutic protocols are utilized globally to control excess body weight and hyperlipidemia in obese patients. In fact, many drugs which have recently become available in pharmaceutical markets are recommended for use hand-in-hand with diet and exercise changes for the reduction of body weight and decreasing lipid levels in the plasma [6,9].

Orlistat is prototype weight loss drug. It is a gastrointestinal lipase inhibitor that competes with dietary fats for sites on the lipase molecules and has been shown to block the absorption of around 30% of dietary fat at a therapeutic oral dose of 120 mg, three times a day. Orlistat inhibits the hydrolysis of dietary triglycerides and thus reduces the subsequent intestinal absorption of the products of lipolysis (monoglycerides and free fatty acids), and does not demonstrate any efficient effect on appetite [10].Therefore, one of the most important screening strategies in the discovery of anti-obesity formulations is to search for potent lipase inhibitors from natural plant extracts [10].

Traditional medicinal plants have been used for obesity and body weight control in many countries. In fact, their consumption, along with appropriate dietary changes, is becoming one of the most popular complementary and alternative medicine strategies for the control of obesity and weight gain [11,12].

In this study, the organic and the aqueous extracts of various edible and traditional medicinal plants, which were collected from different regions of Palestine, were screened as potential anti-obesity agents by monitoring their anti-lipase activity. A total of 10 plants belonging to nine families from Palestine, which are used both as folk medicine and food, were studied, including: *Arum palaestinum* Boiss., *Crataegus azarolus* L., *Malva parviflora* L., *Taraxacum syriacum* Boiss., *Rhus coriaria* L., *Rosmarinus officinalis* L., *Psidium guajava* L., *Origanum dayi* Post, *Brassica nigra* (L.) K. Koch, and *Vitis vinifera* L. The first nine plants grow wildly in the mountains of Palestine and most of them are used in folk medicine to control weight gain [13,14].

In this study, the tested plants were evaluated for their antilipase activity by using a simple, fast, efficient, and reliable spectrophotometric method, in an attempt to investigate these new agents for their ability to impair the of digestion and assimilation of dietary fats. In addition, they were compared with Orlistat in order to assess their potential use as an alternative to this chemical agent.

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

#### *2.1. Instrumentation*

Shaker device (Memmert shaking incubator, Buchenbach, Germany), UV-visible spectrophotometer (Jenway 7135, Staffordshire, UK), grinder (Moulinex, model LM2211, UNO, Shanghai, China), balance (Rad wag, AS 220/c/2, Radom, Poland), freeze-dryer (Mill rock technology, model BT85, Danfoss, Shanghai, China), filter paper (Machrery-Nagel, Bethlehem, PA, USA; MN 617 and Whatman no.1), and rotary evaporator (Heidolph OB2000, VV2000, Schwabach, Germany).

#### *2.2. Chemicals*

From Sigma-Aldrich (Schnelldorf, Germany) the following were purchased: dimethyl sulfoxide, p-nitrophenyl butyrate, Orlistat, and tris-HCl buffer; while from Sigma (St. Louis, MO, USA) we purchased porcine pancreatic lipase type II (100–500 units/mg protein (using olive oil (30 min incubation))) and 30–90 units/mg protein (using triacetin)); from Lobachemie (Mumbai, India). We purchased ethanol, acetone, hexane and acetonitrile from SPF (Gurugram, India).

#### *2.3. Preparation of Plants Extracts*

The required parts from *A. palaestinum*, *C. azarolus*, *M. parviflora*, *T. syriacum*, *R. coriaria*, *R. officinalis*, *P. guajava*, *O. dayi*, *B. nigra*, and *V. vinifera* were collected in May 2016 from different regions of Palestine during the flowering time, except *C. azarolus* fruits which were gathered during the fruiting period of the plant. Botanical identification was carried out at the Pharmacognosy and Herbal Products Laboratory at An-Najah National University, and three samples of each plant were taken for the identification process as well as the voucher specimen codes, including: Pharm-PCT-246, Pharm-PCT-712, Pharm-PCT-1506, Pharm-PCT-2396, Pharm-PCT-2037, Pharm-PCT-2732, Pharm-PCT-2720, Pharm-PCT-1727, Pharm-PCT-408, and Pharm-PCT-2665, respectively. The required parts used from the 10 plants were washed and then dried in the shade at a controlled temperature (25 ± 2 ◦C) and humidity (55 ± 5 RH). It took about two weeks until all the plant parts became well dried. After drying, the plant materials were well ground into a fine powder by using a mechanical blender and transferred into airtight containers with proper labeling for future use.

#### *2.4. Preparation of Plant Extracts for Pancreatic Lipase Inhibition Assay*

A total of 25 g of the powdered plant was weighed and then exhaustively extracted by adding 100 mL of n-hexane and 150 mL of 50% ethanol into triply-distilledwater. The mixture was then shaken for 48 h at room temperature using a shaker that was set at 200 rpm. Afterwards, the mixture was filtered using a suction flask and Buchner funnel filtration. The obtained filtrate was separated individually by a separatory funnel into 2 phases—a lower aqueous phase representing the first aqueous extract and an upper organic phase representing the organic extract. The aqueous extract was dried using a freeze-dryer for 48 h. Meanwhile, the organic extracts were placed in a hood at 25 ◦C to evaporate leftover organic solvents until completely dried. The crude organic and aqueous extracts were stored at 4 ◦C for further use [1].

#### *2.5. Pancreatic Lipase Inhibition*

The porcine pancreatic lipase inhibitory assay was adapted from Zheng et al., 2010, and Bustanji et al., (2010) [2,3], with some modifications. 1 mg/mL (1000 μg/mL) plant extract stock solution in 10% DMSO was used, from which five different solutions were prepared with the following concentrations: 50, 100, 200, 300, and 400 μg/mL. 1 mg/mL stock solution of pancreatic lipase enzyme was prepared immediately before being used. This procedure was carried for the ten studied plants species. A stock solution of PNPB (p-nitrophenyl butyrate) was prepared by dissolving 20.9 mg of PNPB in 2 mL of acetonitrile. 0.1 mL of porcine pancreatic lipase (1 mg/mL) was added to test tubes containing 0.2 mL of the various concentrations (50, 100, 200, 300, 400 μg/mL) of plant extract. The resulting mixtures were then made up to 1 mL by adding Tri-HCl solution (pH 7.4) and incubated at 25 ◦C for 15 min. After the incubation period, 0.1 mL of PNPB solution was then added to each test tube. The mixture was again incubated for 30 min at 37 ◦C. Pancreatic lipase activity was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm using a UV-visible spectrophotometer. The same procedure was repeated for the aqueous and organic extracts and for Orlistat (a positive control) using the same concentrations as mentioned above.The established tests were performed in triplicates.

#### **3. Results**

Twenty crude aqueous and organic extracts were prepared from ten plant species found in the West Bank area of Palestine and their anti-lipase activity was investigated at a concentration of 1000 μg/mL for porcine pancreatic lipase inhibition. The inhibitory activities towards pancreatic lipase are reported in Table 1.

The inhibitory effects of the reference drug (Orlistat) and plant extracts aredose-dependent. Different solutions of the Orlistat and plant extracts were prepared in escalating doses as shown in Table 1. The activity of lipase decreased by increasing the concentration of Orlistat and the plant extracts. TheIC50 values for the drug and plant extracts were calculated and the degree of lipase inhibition was plotted as shown in Figures 1–10. The IC50 values represent the concentration of the inhibitors at which 50% of the enzyme is inhibited and it is generally used to express the inhibitory effect of the lipase enzyme.

**Table 1.** Porcine pancreatic lipase inhibitory properties, expressed as IC50 (μg/mL), and yield percentages of the aqueous and organic extracts of 10 plantwerecollectedfromdifferentregionsinPalestine.

 species

Among the 20 plant extractsexamined, seven crude extracts at a concentration 100 μg/mL significantly inhibited porcine pancreatic lipase activity in vitro as demonstrated using a p-nitrophenyl butyrate-based assay. Throughout the investigated results, the aqueous extracts of *V. vinifera* and *R. coriaria,* with IC50 values of 14.13 and 19.95 μg/mL, respectively, showed the highest porcine pancreatic lipase inhibitory effects of all the studied aqueous extracts. Meanwhile, the organic extract of *O. dayi*, with an IC50 value of 18.62 μg/mL, showed the highest porcine pancreatic lipase inhibitory effects of all studied organic extracts.

In addition to that, the studied extracts' IC50 values were compared with the standard antilipase compound Orlistat, which has an IC50 value of 12.38 ± 2.3 μg/mL. These results, and the results of all of the 10 studied plants, are well clarified in Table 1 and in Figures 1–10.

**Figure 1.** The inhibitory effects of the aqueous and organic extracts of *A. palaestinum* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 2.** The inhibitory effects of the aqueous and organic extracts of *B. nigra* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 3.** The inhibitory effects of the aqueous and organic extracts of *C. azarolus* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 4.** The inhibitory effects of the aqueous and organic extracts of *M. parviflora* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 5.** The inhibitory effects of the aqueous and organic extracts of *O. dayi* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 6.** The inhibitory effects of the aqueous and organic extracts of *P. guajava* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 7.** The inhibitory effects of the aqueous and organic extracts of *R. coriaria* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 8.** The inhibitory effects of the aqueous and organic extracts of *R. officinalis* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 9.** The inhibitory effects of the aqueous and organic extracts of *T. syriacum* and Orlistat on the activity of porcine pancreatic lipase.

**Figure 10.** The inhibitory effects of the aqueous and organic extracts of *V. vinifera* and Orlistat on the activity of porcine pancreatic lipase.

#### **4. Discussion**

In this study, ten traditional edible medicinal plants, including *T. syriacum, O. dayi, M. parviflora, B. nigra, V. vinifera, C. azarolus, R. coriaria, A. palaestinum, P. guajava*, and *R. officinalis*, were assessed for

their activity as porcine pancreatic lipase inhibitors. In many countries such as Palestine, Jordan, Iraq, Greece, and Pakistan, most of these plants were reported as being used as traditional medicines for the treatment of obesity [4–16].

Obesity is a chronic metabolic disorder caused by an imbalance between energy intake and expenditure. It is a major risk factor for cancer as well as endocrine, metabolic, and cardiovascular disorders [17–19]. Accordingly, the use of functional foods, such as the consumption of edible plants, would be a great and safe medicinal alternative in the treatment of obesity. They have been targeted to promote beneficial health effects, especially for the prevention of pathophysiological conditions such as obesity, dyslipidemia, diabetes, hypertension, and cancer [20,21].

The lipolytic pancreatic lipase enzyme is synthesized and secreted by the pancreas, plays a key role in the efficient digestion of lipids, and is responsible for the hydrolysis of 50–70% of total dietary lipids. The anti-lipase effect is one of the most widely studied mechanisms in determining the potential efficacy of natural products as anti-obesity agents [22,23].

Recently, global attention has been focused on the effects of plants, especially those that classified as traditional medicinal and edible types for the treatment of obesity and for controlling of overweight due to their safety issue, as well as these plants have been consumed from ancient times and their toxic and other side effects have been observed and documented if present [24].

In addition to this, Palestinian territory is covered with a huge number of plants and from this huge biodiversity, there are large numbers of them considered traditional medicines or foodssinceancient times [25]. From these plants, *A. palaestinum*, *C. azarolus*, *M. parviflora*, *T. syriacum*, *R. coriaria*, *R. officinalis*, *P. guajava*, *O. dayi*, *B. nigra*, and *V. vinifera* were investigated for their efficacy as natural anti-obesity products and compared with Orlistat, which considered the anti-lipase drug of choice [26].

Orlistatis a potent inhibitor of pancreatic lipase enzyme isolated from bacteria *Streptomyces toxytricini*. This prescription drug is designed to treat obesity by lowering lipid digestion and is produced under the trade name Xenical® by Roche [27].

Our obtained results showed that the IC50 values of the aqueous extracts of *V. vinifera* and *R. coriaria* have high porcine pancreatic lipase inhibition potential with IC50 values of 14.13 and 19.95 μg/mL, respectively. The IC50 value of the organic extract of *O. dayi* was 18.62 μg/mL, in comparison with the reference compound (positive control) Orlistat which has an IC50 of 12.38 μg/mL.

Moreover, the *R. coriaria* (Sumac) fruit's aqueous extract showed potential antilipase effects with an IC50 value 19.95 μg/mL, and this plant is an edible one and grows wildly in tropical and temperate regions worldwide, often growing in agriculturally-in viable regions. *R. coriaria* belongs to the Anacardiaceae family and is used in Turkey as a folk medicine for the treatment of diabetes due to its ability to cure various diabetes complications [28].

A study performed by Golzadeh et al., (2012), showed that Sumac reduced total cholesterol, triglyceride, low-density lipoprotein, and blood sugar levels in animal studies [29].

Accordingly, these results were confirmed in our in-vitro study which confirmed its activity as an anti-lipase agent.

In addition to these results, the organic extract of *O. dayi* also showed potential anti-lipase effects with an IC50 value 18.62 μg/mL. It is a perennial, endemic, culinary plant, and is considered a desert plant that is mainly distributed in African and Mediterranean regions [30,31]. It is used as a tea, a spice, and boiled with meat also used in salads in many countries, reflecting its therapeutic effect of reducing lipid absorption in the gastrointestinal tract [32,33].

*Vitis vinifera* belongs to the Vitaceae family, whose origin is Mediterranean, and is considered one of most common plant species cultivated worldwide and one of the most important economical plants because it is used to produce wine, table grape juice, and raisins [34,35].

The leaves of *V. vinifera* consumed with rice and meat and considered one of the most popular culinary dishes in Arabian countries, as well as other countries such as Turkey and Italy [36–38]. This plant's leaves showed a powerful anti-lipase effect in comparison with Orlistat and have an IC50 value 14.13 μg/mL.

In a study which was conducted on the aqueous extract of *V. vinifera* leaves by Fernandes et al., (2013), it was found that the plant's leaves are rich in phenolic compounds, including phenolic acids and flavonoids such as trans-caffeoyl tartaric acid, trans-coumaroyl tartaric acids, myricetin-3-*O*-glucoside, quercetin-3-*O*-glucoside, quercetin, kaempferol-3-*O*-glucoside, quercetin-3-*O*-galactoside, kaempferol, and isorhamnetin [34].

In addition, *R. coriaria* fruits have high concentrations of phenolic compounds, including caffeoylquinic acid, quercetin, rhamnetin, myricetin, kaempferol, gallic acid, ellagic acid, methyl gallate, m-digallic acid, amenthoflavone, agathisflavone, hinokiflavone, and sumaflavone [39,40].

*Origanum dayi* is an endemic subshrub plant species that grows in the West Bank area of Palestine. The leaves of *O. dayi* contain a mixture of volatile oils such as terpinen-4-ol, α-terpineol, 1,8-cineole, (E)-sabinene hydrate, (E)-sabinene hydrate acetate, and linalyl acetate [30,31]. To the best of the authors' knowledge, there were no previous studies in the literature about the chemical constituents of *O. dayi*, except on the volatile oils. However, many phenolic compounds were isolated from other species of *Origanum* (*O. vulgare*), including protocatechuic acid, caffeic acid, 2-caffeoyloxy-3-[2-(4-hydroxybenzyl)-4,5-dihydroxy]phenylpropionic acid, and phenyl glucoside [41].

In fact, in many previous studies, it was reported that flavonoids and other phenolic compounds act as porcine pancreatic lipase enzyme inhibitors by binding to the enzyme−substrate complex, reducing the lipid absorption [42,43].

To the best of the authors' knowledge, there were no previous studies on the effects of the aqueous and organic extracts of *V. vinifera*, *R. coriaria* and *O. dayi* on the lipase enzyme and our study is the first one on these plants. Further pharmacological in-vivo studies are required to confirm these findings and to identify the key chemical elements in these plants, using chromatographical isolation of the bioactive molecules, responsible for these pharmacological effects.

#### **5. Conclusions**

Obesity has reached epidemic proportions and is becoming a public health concern of the highest order. Several methods are now available to treat obesity and Orlistat is the most used one. Several plants are used in folk medicine to treat obesity and the use of edible plants is of great importance since they have few of the adverse effects that may be encountered with chemical or drug treatments. Our obtained results showed that some edible plants could replace Orlistat in the treatment of obesity. According to the obtained results, *V. vinifera, R. coriaria,* and *O. dayi* can be used as natural inhibitors of pancreatic lipase and so are new players in obesity treatment. In fact, these plants can be freely and safely consumed in the daily diet, be prepared as natural supplements to treat or prevent obesity and control weight gain, and can be used for the treatment of hyperlipidemia.

**Acknowledgments:** The authors acknowledge the assistance of the technicians Mohamad Arar and Linda Isa.

**Author Contributions:** N.J. wrote the manuscript. N.J., A.N.Z., F.H., M.Z., and H.A. carried out the experiments. N.J., A.N.Z., and F.H. supervised research work and O.A. drafted the manuscript. All authors read and approved the final manuscript.

**Conflicts of Interest:** The authors declare that they have no financial and/or non-financial competing interests.

#### **References**

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Antioxidant and Antimicrobial Properties of Rosemary (***Rosmarinus officinalis,* **L.): A Review**

#### **Gema Nieto 1, Gaspar Ros <sup>1</sup> and Julián Castillo 2,\***

Received: 1 June 2018; Accepted: 31 August 2018; Published: 4 September 2018

**Abstract:** Nowadays, there is an interest in the consumption of food without synthetic additives and rather with the use of natural preservatives. In this regard, natural extracts of the *Lamiaceae* family, such as rosemary, have been studied because of its bioactive properties. Several studies have reported that rosemary extracts show biological bioactivities such as hepatoprotective, antifungal, insecticide, antioxidant and antibacterial. It is well known that the biological properties in rosemary are mainly due to phenolic compounds. However, it is essential to take into account that these biological properties depend on different aspects. Their use in foods is limited because of their odour, colour and taste. For that reason, commercial methods have been developed for the preparation of odourless and colourless antioxidant compounds from rosemary. Owing to the new applications of natural extracts in preservatives, this review gives a view on the use of natural extract from rosemary in foods and its effect on preservative activities. Specifically, the relationship between the structure and activity (antimicrobial and antioxidant) of the active components in rosemary are being reviewed.

**Keywords:** rosemary; phenolic compounds; antioxidant; antimicrobial

#### **1. Introduction**

Because consumers are concerned about the negative effect of synthetic chemicals in food, there is a need to find "clean label products". Therefore, there is a growing interest in using natural extracts as alternatives for synthetic additives because of (a) their synergy with other preservation methods (b) they are considered safe, and (c) their specific properties as antioxidant, antidiabetic, antimutagenic, antitoxigenic and antibacterial [1].

In general, herbs and plants are rich in compounds with antioxidant properties, such as vitamins (E and C), glutathione, enzymes and phenolic compounds [2]. Several spice extracts have shown their properties to prevent the autoxidation of unsaturated triacylglycerols [3]. Specifically, the natural extract from the *Lamiaceae* family (thyme, sage and rosemary) has been reported in several studies for its antioxidative activity [1,4].

*Rosmarinus officinalis*, L. originating from the Mediterranean region is an aromatic plant from the *Lamiaceae* family. The province of Murcia (Southeast Spain) is one of the major processors and importers of rosemary. In the United States and Europe, rosemary is a unique spice commercially available for use as an antioxidant [5]. Rosemary extracts have been used in the treatment of diseases, due to its hepatoprotective potential [6], therapeutic potential for Alzheimer's disease [7] and its antiangiogenic effect [8]. On the other hand, they have been used in food preservation, because they prevent oxidation and microbial contamination [9–13]. Therefore, rosemary extract could be useful for replacing or even decreasing synthetic antioxidants in foods. As preservatives, rosemary extracts offer several technological advantages and benefits to consumers.

EFSA (European Food Safety Authority) has reviewed the safety of rosemary extracts [14]. It concluded that there are high-intake estimates ranging from 0.09 (the elderly) to 0.81 (children) mg/kg per day of carnosol and carnosic acid. Nowadays, in the European Union, rosemary extracts are added to food and beverages at levels of up to 400 mg/kg (as the sum of carnosic acid and carnosol).

#### **2. Preparation of Antioxidants from Rosemary**

When a new rosemary extract is tested, the most important aspect to take into account is the method of extraction and the sort of solvent used, as this will affect the antioxidant properties.

Various extraction methods for the selective extraction of rosemary leaves have been identified in the scientific literature. This includes both solvent extraction using vegetable oil or animal fat, mechanical pressing methods, water at alkaline pH and organic solvents (e.g. hexane, ethyl ether, chloroform, ethanol, methanol, dioxane and ethylene dichloride).

Previous extraction processes are obsolete and unusual in the industry. Chang et al. [15] and Bracco et al. [16] demonstrated the possibility of separating an active fraction of the rosemary extract by molecular distillation. The process realized by Chang et al. [15] for the extraction of antioxidant involves the extraction with ethyl ether under refluxing conditions. The crude material is washed with water and the solvent is removed. The crude material is dissolved in methanol and activated with carbon at 60 ◦C for 15 min. Another process reported by Bracco [16], involves reducing the particle size to 600 μL and suspending in peanut oil. The finely antioxidant components have a molecular weight range lower than the triglyceride components of the oil used (peanut oil) and can therefore be physically separated by molecular distillation on either a fall-film or centrifugal system.

Nowadays, rosemary extracts are usually prepared from dried rosemary leaves. In all the new methods, the extraction process is often accompanied by a step involving partial deodorisation and/or extract decolourisation. Considering the methods used, in general, the yield of rosemary extract indicated by various authors varies between 2% and 26% based on the raw material used.

#### **3. Composition of Rosemary Extract and Essential Oil**

The polyphenolic profile of rosemary has been widely described in the scientific literature [17–20]. The polyphenolic profile of these plants is characterized by the presence of carnosic acid, carnosol, rosmarinic acid and hesperidin, as major components [21].

Among the most effective antioxidant constituents of rosemary, the cyclic diterpene diphenols, carnosolic acid and carnosol have been identified. In addition, its extract contains carnosic acid, epirosmanol, rosmanol, methylcarnosate and isorosmanol [21–23].

Rosemary oils are obtained by steam distillation of twigs and fresh leaves. Sienkiewicz et al. [24] reported that rosemary essential oil contains mainly 1,8-cineole (46.4%), camphor (11.4%) and α-pinene (11.0%). The composition of the rosemary essential oil used by Jiang et al. [25], was composed mainly by 1,8-cineole (26.54%) and α-pinene (20.14%). Bendeddouche et al. [26], observed that the main constituents of the tested essential oil were camphor (37.6%), 1,8-cineole (10.0%), p-cymene-7-ol (7.8%) and borneol (5.4%).

*Rosmarinus officinalis*, L. is a rich source of phenolic compounds and their properties are derived from its extracts [27] and essential oils [28]. Both are used for the treatment of illnesses and in the food preservation.

In addition to the volatile constituents, extracts of rosemary also contain several antioxidant components, which belong mainly to the classes of phenolic acids, flavonoids, and diterpenoids. Del Baño et al. [2] studied the composition of seven flavonoids in rosemary leaves, flower, roots and steam. They studied the presence of 7-*O*-glucoside, hispidulin, diosmin, hesperidin, 3 -O-β-D-glucuronide, genkwanin and isoscutellarein 7-*O*-glucoside. These authors concluded significant presence of diosmin and hesperidin in the vascular system. Of the other identified compounds found in extracts of rosemary, rosmarinic acid and hydroxyhydrocaffeic acid, also exhibit some complementary antioxidant activity.

The extract of rosemary also contains other caffeic acid derivates. These compounds react with present metal ions, so chelates are formed; they consequently react with peroxide radicals and in that way, stabilise these free radicals.

Rosemary oil is used as a food seasoning [29], due to its chemical compound constituents responsible for the antibacterial, antifungal and antioxidant properties. Traditionally, rosemary oil has been shown to possess a number of applications in managing or curing many diseases such as inflammatory diseases [30] and diabetes mellitus [31].

On the other hand, the bioactivities of rosemary extracts include properties such as anti-inflammatory [32], antidiabetic [33], hepatoprotective [34] and antimicrobial activity [35]. These bioactivities are related to the phenolic compound constituents (mainly caffeic acid, rosmarinic acid and carnosic acid).

#### **4. Mechanism of Antioxidant Action**

The mechanism of action of these compounds has been widely covered in several publications. For example, Höulihan et al. [36] and Wu et al. [37] determined that the antioxidant properties of rosemary are attributed to its richness in isoprenoid quinones, which act as chain terminators of free radicals, and as chelators of reactive oxygen species (ROS). In addition, Gordon [38] indicated that the phenolic compounds existing in the commercial extracts of rosemary act as primary antioxidants when reacting with the lipid and hydroxyl radicals to turn them into stable products. Subsequently, Fang and Wada [39] pointed out that these compounds could act as metal ion chelators (Fe+2 fundamentally), therefore reducing the formation ratio of the reactive species derived from oxygen.

According to Löliger [40], carnosic acid and carnosol act as potent scavengers of peroxyl radicals. This fact explains the conclusions obtained by Chen et al. [41], who confirmed that the effect of both compounds on peroxidation of membrane lipids is higher than the effect reported by artificial antioxidants such as BHA, BHT and propyl gallate [42].

The antioxidative activity of rosemary extracts has been evaluated using different solvents. In this regard, Inatani et al. [43] reported that rosmanol, showed an antioxidant capacity four times higher than BRT and BRA (synthetic antioxidants) in both linoleic acid and lard. In addition, this study reported the antioxidant activity of carnosol and rosmanol by TBA and ferric thiocyanate methods. They reported the correlation between activity and chemical structure as an antioxidant. Aruoma et al. [44] studied the antioxidant and pro-oxidant properties of rosemary. The main constituents with antioxidant properties are carnosic acid and carnosol that are responsible for 90% of the properties. Both are inhibitors of lipid peroxidation in liposomal and microsomal systems, they are good scavengers of CCl3O2 (peroxyl radicals), reduce cytochrome c and scavenge hydroxyl radicals. Specifically, carnosic acid scavenges H2O2, but could also act as a substrate for the peroxidase system.

The antioxidant properties depend on fruiting stages: the increase in concentration of polyphenols, which include carnosol, rosmarinic acid and hesperidin, during the fruiting stage, is directly related to the improvement of the extract antioxidant capacity. This statement is supported by scientific papers previously published by Cui et al. [45] and Kontogianni et al. [20], who consider lactone carnosol (Figure 1) as the main property responsible for this activity. Likewise, rosmarinic acid and hesperidin have been cited in literature as important free radical scavengers [46,47].

One of the most significant aspects of the antioxidant activity of rosemary is the relationship between diterpenes and radical-scavenging activity. In this regard, the study by Munné-Bosch and Alegre [48] describes the antioxidant capacity of diterpenes in rosemary. The most important elements in the rosemary structure are the aromatic ring (C11–C12) in the catechol group together with the conjugation of the three basic rings.

The catechol group is responsible for scavenging the radical electron formed as result of oxidation. The skeleton formed by the three rings allows the delocalization of the charge. The presence of the carboxylic group (in the case of carnosic acid) increases this conjugation, especially in aqueous

systems. However, in slightly polar media, such as fats, it is the lactone structure that seems to confer greater stability.

**Figure 1.** The chemical structure of carnosol.

Moreover, activation of redox-dependent signalling pathways such as Nrf2-dependent transcriptional regulation is known to take part in the antioxidant response of rosemary. The main metabolites carnosol and carnesolic acid have been reported to mediate antioxidant activities by differential mechanisms. Carnosic acid, carnosol, rosmanol and epirosmanol are the major phenolic diterpenes responsible for the antioxidant properties of rosemary (the first proposed oxidation pathway of carnosic acid was reported by Wenkert et al. [49] as shown in Figure 2). In the same way, Wijeratne and Cuppett [50] suggest that the antioxidant activities of carnosic acid are due to their ability to increase or maintain superoxide dismutase and glutathione peroxidase activities. These authors reported that carnosic acid and carnosol inhibited lipid peroxidation by 88–100% and 38–89%, respectively, under oxidative stress conditions.

Generally, the antioxidant effectiveness of natural extracts is higher than synthetic antioxidants, independent of the medium, which is different in water or oil. Table 1 shows the relative antioxidant effectiveness of spices and herbs.

**Table 1.** Antioxidant effectiveness of herbs and spices, evaluated as complete plant materials in different foods.

For reasons explained in past years, the use of new rosemary extracts, mainly refined extracts with antioxidant properties, is an interesting strategy as a food preservative, especially those with animal and/or vegetable fats [16].

There are different applications of rosemary in foods. For example, it has been added to animal products and oils. Different studies have demonstrated the potent activity of rosemary by reducing the colour loss of carotenoids and delaying lipid oxidation in oils [55] and meat products [56–58].

Several studies have shown rosemary properties to achieve good sensory results and the reduction of lipid oxidation after addition into foods: Stoick et al. [59] used 500–1000 ppm of rosemary extracts in beef; Shahidi et al. [60] used rosemary at 200 and 1000 ppm in different foods; Huisman et al. [61] used a concentration of 0.05% in pork; Sánchez-Escalante et al. [62] used a combination of 500 ppm vitamin C and 1000 ppm of rosemary in beef burgers; Formanek et al. [63] used 0.25% of rosemary in beef burgers. In general, all these authors showed that rosemary inhibited the formation of hydroperoxides [64].

**Figure 2.** The first proposed oxidation pathway of carnosic acid. Reproduced from [49]. Copyright 1965, American Chemical Society, Washington, DC, USA.

On the other hand, rosemary extract could be applied through animal diet. In this regard, delayed lipid oxidation was reported in broilers with the administration of sage and rosemary [65]. This agreed with the results of Moñino et al. [66], indicating that antioxidant stability improved with 10% of rosemary leaves into the ewes feed.

The antioxidant activity of rosemary through animal diet has been reported in additional studies by: Lopez Bote et al. [65] with 500 mg kg−<sup>1</sup> in broiler diets; Descalzo et al. [67] in feed of cattle; Petron et al. [68] in lamb, in pork meat [69,70], turkey meat products [71,72], chicken meat [65], hen's meat [73,74], cooked sausages [75]. Generally, the addition of rosemary extract into the meat products or through animal feed improved the meat lipid stability. On the contrary, O'Grady et al. [76] and Galobart et al. [77] concluded that feeding animals with rosemary did not improve the lipid stability of meat or eggs.

As previously reported, the antioxidant effect of rosemary is due to the polyphenols present in the leaves (mainly rosmarinic acid, carnosol and carnosic acid), which accumulate in the fatty membranes of cells where the antioxidant effect is required [72].

Other studies reported the effectiveness of rosemary oil to control oxidation in frankfurters [78] and to protect protein oxidation of meat patties [79]. This behaviour is very important to control colour changes of meat products mainly caused when the haem pigments are oxidized. In this regard, lipid oxidation and their consequences are reduced through the addition of rosemary.

In addition, rosemary has been added as an antioxidant in oils: Tohma et al. [80] studied the effect of the rosemary plant, its alcoholic extracts and essential oil on the oxidative stability of hazelnut oil during deep frying. This study showed that rosemary plant, or its extracts, could be used to extend the usage life of hazelnut oil for frying. The rosemary additives considerably inhibited the formation of oxidation products. The total phenolic compounds and profile of the enriched oils might cause increased frying stability. In addition, Reblova et al. [3] and Taha et al. [81] reported that rosemary extracts improved the sensory characteristic of fries, increased the oxidative stability of the oil, inhibited the decomposition of polyunsaturated triacylglycerols and the formation of polar substances in rape-seed oil. Urbancic et al. [82] reported that rosemary extract reduced acrylamide formation during potato frying. This effect was due to the transfer of phenolic compounds from rosemary into the oil.

#### **5. Mechanism of Antimicrobial Action and Food Applications**

The antibacterial activity of rosemary has been determined in various assay types based on either MIC or MBC. In this regard, Sienkiewicz et al. [24], demonstrated the antibacterial activities of basil (*Ocimum basilicum*, L.) and rosemary (*Rosmarinus officinalis*, L.) These authors reported the inhibition of microbial growth by both essential oils, presented as MIC values. Antibiotic susceptibility was carried out using disc diffusion. The results showed that both essential oils tested are active against all the clinical strains from *Escherichia coli*. Mihajilov-Kristev et al. [83], showed that essential oils containing mainly carvacrol (67.0%) and γ-terpinene (15.3%) were effective against Gram-negative strains, including *Escherichia coli*, with MIC values from 0.025 μL/mL to 0.78 μL/mL according to the broth microdilution method. Probuseenivasan et al. [84], confirmed that rosemary essential oil strongly inhibits *E. coli* ATCC 25922. The minimal inhibitory concentration for rosemary oil against *E. coli* was >6.4 mg/L.

Other studies have shown the antibacterial activity of rosemary oil against *E. coli*, *Bacillus cereus*, *Staphylococcus aureus* [85], *Staphylococcus aureus*, *Clostridium perfringens*, *Aeromonas hydrophila*, *Bacillus cereus* and *Salmonella choleraesuis*. This essential oil was incorporated into meat reporting antibacterial activity against *Brochothrix thermosphacta and Enterobacteriaceae* [86].

The inhibitory effect of rosemary is the result of the action of rosmarinic acid, rosmaridiphenol, carnosol, epirosmanol, carnosic acid, rosmanol and isorosmanol. They interact with the cell membrane, causing changes in genetic material and nutrients, altering the transport of electrons, leakage of cellular components and production changes in fatty acid. In addition, it also produced an interaction with the membrane of proteins that produced the loss of membrane functionality and its structure [87].

Vegara et al. [88] reported that the effectiveness of carnosic acid against pathogenic bacteria is superior to that of any other major extract component, including rosmarinic acid. In contrast, several scientific publications disagree about the possible relationships that may exist between the composition of the polyphenolic extract and its antimicrobial activity. Such is the case of Moreno et al. [89] and Ivanovic et al. [90] who demonstrated that the effectiveness of rosemary is related to a possible synergy between the rosmarinic phenolic acid and the carnosic acid diterpene. Bernardes et al. [91], however, state that there is a close relationship between the concentration levels of the carnosic acid, carnosol diterpenes and the antimicrobial activity of these extracts.

Zaouali et al. [92] reported that, compared with *S. aureus*, antimicrobial activity improves with the presence of α-pinene as a major component. This effect can be correlated with the fact that terpenes can disorganize the cell membrane, and therefore promote the lysis, as stated by Bjapai et al. [93]. The effectiveness of the essential oil of rosemary against *E. coli*, is related to the combined action of the different minority components present in its volatile fraction and should not be associated with the action of any particular component, agreeing with the conclusions published by Zaouali et al. [94]. There are numerous authors who claim that *E. coli, L. monocytogenes* and *S. aureus*, are very resistant bacteria and highlight the importance of chemical composition and proportion between the oil components on their antimicrobial efficacy [94,95].

The antibacterial effect of rosemary has been widely demonstrated in several food studies: beef meatballs [96], cooked beef [97] and in pork sausage [98]. Gomez-Estaca et al. [99] reported that rosemary oil inhibited the growth of common food bacteria contributing to food spoilage. Burt [85] also showed the antibacterial activity of rosemary essential oil against *E. coli*, *Bacillus cereus* and *S. aureus*. In addition, Sirocchi et al. [86] showed that rosemary essential oil inhibited the growth of *Brochothrix thermosphacta* and *Enterobacteriaceae*. Govaris et al. [72] reported an inhibitory effect of dietary supplementation of turkeys with rosemary (5 and 10 g/kg) on the growth of bacteria responsible for spoilage (psychrotrophs, mesophilics, enterobacteria and lactic acid bacteria). Camo et al. [56] also observed an inhibitory effect of the use of rosemary extract added to lamb meat packed in a modified atmosphere in the growth of psychrotrophic bacteria, compared with control meat. This same effect was observed by Quattara et al. [100], however these authors studied pure rosemary essential oil. On the contrary, Ismail et al. [101] showed that rosemary had no antibacterial effect in chicken.

Fernández-López et al. [96], evaluated the antibacterial activity of rosemary extracts in veal meatballs. The results showed a higher antibacterial activity in rosemary extracts, compared to other extracts studied. Only rosemary extracts were able to inhibit the 11 bacteria tested (such as *Lb. lactis FMRD*, *Br. thermosphacta CRA*, *Lb*. *carnosum*, *Br*. *thermosphacta CRA*, *L*. *innocua*, *Lb*. *sake*, *Br*. *thermosphacta CRA*, *Lc*. *mesenteroides* subsp *mesenteroides*, *L*. *monocytogenes*, *Lc*. *mesenteroides* subsp *dextranicum*, *Lb*. *curvatus*).

#### **6. Synergistic Effect**

It is important to note the possible synergistic effect between rosemary extract and other natural antioxidants. The results shown in the scientific literature offer contradictory conclusions. Resurreccion and Reynolds [102], observed that the joint addition of rosemary extract and tocopherols in meat products failed to increase the antioxidant efficacy of these individually used compounds. On the contrary, Wong et al. [103], concluded that the components of rosemary participate in the regeneration of α-tocopherol, which can be used as substitutes for vitamin C to enhance the stability of vitamin E. Wada and Fang [104], proposed that the synergism between both antioxidants is due to the capacity of the rosemary extract to yield hydrogen atoms to the tocopheryl radicals. This fact justifies the results obtained by Fang and Wada [37], who observed that the antioxidant activity of the α-tocopherol-rosemary mixture on a model fish system was significantly higher than that exhibited by the individually added products. In addition, these authors found that the α-tocopherol molecule remained stable for 10 more days when administered together with rosemary extract.

In this same line, Lai et al. [105] and Stoick et al. [59] showed the existence of a synergistic effect between sodium tripolyphosphate (STTP) and rosemary oleoresin (OR). The combination of OR/STTP was as effective as the application of the BHT-BHA or STPP/TBHQ mixtures during the prevention of WOF (warmed-over flavor) in pre-cooked beef and pork meats respectively [58].

#### **7. Conclusions**

The health problems derived from lipid oxidation have attracted the attention of consumers and researchers. Numerous diseases, such as aging, cancer, ischemia and atherosclerosis are linked to dietary and biological lipid oxidation products. In this regard, antioxidant compounds present in rosemary extracts and essential oils, delay lipid oxidation in biological systems and food. However, it is essential to consider that the antioxidant and antimicrobial activity of rosemary depend on the fruiting stage, nature of the extracts, mode of extraction, presence of an inhibitor, presence of a synergistic effect with other components, and the concentration of active extract components. If these aspects are taken into account, the application of this natural extract can be complimented in different food systems such as meat, oils and dressing. In view of its application, rosemary extracts could be used in functional foods, pharmaceutical products, plant products and food preservation. Because rosemary is a cheap, available, and a non-toxic herb, these considerations warrant the introduction of rosemary extracts or essential oils, with high phenolic compound contents, into the food industry.

**Author Contributions:** G.N., G.R. and J.C. wrote the article.

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

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

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Bioactive Compounds and Extracts from Traditional Herbs and Their Potential Anti-Inflammatory Health Effects**

#### **Antonio Serrano, Gaspar Ros \* and Gema Nieto**

Department of Food Technology, Nutrition and Food Science, Veterinary Faculty University of Murcia, Campus de Espinardo, Espinardo, 30100 Murcia, Spain<EMAIL_ADDRESS>(A.S<EMAIL_ADDRESS>(G.N.) **\*** Correspondence<EMAIL_ADDRESS>
Received: 10 May 2018; Accepted: 11 July 2018; Published: 16 July 2018

**Abstract:** The inflammatory processes associated with several chronic illnesses like cardiovascular disease and cancer have been the focus of mechanistic studies of the pathogenicity of these diseases and of the use of different pharmacological and natural methods to prevent them. In this study we review the current evidence regarding the effectiveness of natural extracts from as-yet little-studied traditional botanical species in alleviating the inflammation process associated with several chronic diseases. Additionally, the intention is to expose the known pathways of action and the potential synergistic effects of the constituent compounds of the discussed extracts. It is noted that the here-studied extracts, which include black garlic rich in S-allylcystein, polyphenols from cat's claw (*Uncaria tomentosa*), devil's claw (*Harpagophytum procumbens*), camu-camu (*Myrciaria dubia*), and blackcurrant (*Ribes nigrum*), and citrus fruit extracts rich in hesperidin, have similar or greater effects than other, more extensively studied extracts such as tea and cocoa. The combined use of all of these extracts can give rise to synergetic effects with greater biological relevance at lower doses.

**Keywords:** anti-inflammatory; medicinal plants; chronic diseases; *Uncaria tomentosa*; *Harpagophytum procumbens*; *Myrciaria dubia*; *Ribes nigrum*; hesperidin

#### **1. Introduction**

Inflammation represents a biological response of the organism to a series of mechanical, chemical, or infectious stimuli. Its mission is to isolate, destroy, or dilute, as a form of localized protection. Inflammation can be chronic or acute depending on the characteristics of humoral response and the molecules involved. When inflammatory balance is altered, with excessive pro inflammatory signals (for example in the cyclooxygenase pathway), physiological damage can occur [1].

Chronic inflammation is a status derived from physiopathogenic situations such as metabolic syndrome or inflammatory bowel diseases that involve prolonged exposition to a number of potential pathogenic substances. Those substances are mainly inflammatory mediators like tumor necrosis factor alpha (TNF-α), and are linked to cancer initiation [2]. The combination of these factors leads to an unbalanced inflammatory status with an increment in markers like inflammatory cytokines, including TNF-α, interleukin (IL)-6, and IL-1β, which are also associated with cardiometabolic diseases [3].

Thus, a way to prevent inflammation which can lead to carcinogenesis or cardiovascular diseases is through the use of botanic extracts of spices and herbs which show both antioxidant and anti-inflammatory properties. For this reason, anti-inflammatory phytochemicals could represent an exogenous aid crucial for the prevention of chronic diseases mediated by inflammatory processes.

#### **2. Natural Extracts and Compounds with Anti-Inflammatory Properties**

#### *2.1. Allium nigrum (Black Garlic)*

Garlic is a commonly used condiment with many biological activities due to its sulfur compounds [4] which have antioxidant and antimicrobial properties [5]. Black garlic is obtained through fermentation at a controlled high temperature (60–90 ◦C) and high humidity (80–90%). It has distinct bioactivity with respect to fresh garlic, conferring numerous benefits like anti-inflammatory, anticancer, and antiobesity activity [6]. This nutritional variation is characterized by a decrease in fructan, which is linked to an increase of fructose due to a Maillard reaction that subsequently impacts the color and taste [7]. Changes in the profile of volatile compounds provide black garlic with higher concentrations of S-alk(en)-yl-L-cysteine derivatives, while the quantity of ascorbic acid decreases due to thermal treatment [8]. Meanwhile, the concentrations of some flavonoids, pyruvate, total phenol, and the main antioxidant compound of black garlic, S-allylcystein, are increased [9] (Figure 1).

**Figure 1.** Chemical structure of S-allylcystein.

Some studies have isolated bioactive compounds from black garlic in order to evaluate their bioactivity. When black garlic aqueous extract is compared to raw garlic aqueous extract at similar dose range (between 31.25 μg/mL and 250 μg/mL), higher inhibition of tumor necrosis factor alpha (TNF-α) and prostaglandin E2 (PGE2) is reported for the aged garlic extract [10]. Kim et al. [11] demonstrated that even concentrations as low as between 5 μg/mL and 10 μg/mL of black garlic ethanol extract have anti-inflammatory effects as they inhibit nitric oxide (NO) and PGE2 production in lipopolysaccharide-stimulated (LPS-stimulated) RAW264.7 cells, and also decrease nitric oxide synthases and prostaglandin-endoperoxide synthase 2 expression. Choi et al. [12] showed that antioxidant power increased between day 0 and 21 of fermentation, reaching its limit at day 21 and keeping most of its properties at day 35 even though the levels of ascorbic acid were lower, as shown by Martínez-Casas et al. [8].

#### *2.2. Uncaria tomentosa ("Cat's Claw")*

*Uncaria tomentosa (*UT*)* is a climbing vine from Peru commonly known as cat's claw from the Spanish "*Uña de Gato*" because of its thorns. It has been used over time in traditional medicine to treat diseases like rheumatism and cancer [13]. It has up to 32 phenolic compounds, including hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols monomers, procyanidin dimers and trimers, flavalignans, and propelargonidin dimers [14]. One of the major bioactive compounds of UT is mitraphylline (Figure 2), which was isolated and evaluated by Rojas-Duran et al. [15] using a dose of 30 mg/kg/day for 3 days in mice, resulting in cytokine modulation providing fewer inflammatory signals.

**Figure 2.** Chemical structure of mitraphylline.

The anticancer properties of UT are due to the synergistic activity of alkaloids with its non-polar compounds [16] like isopteropodine, pteropodine, isomitraphylline, uncarine F, and mitraphylline. These compounds also have exhibited anti-apoptotic properties in lymphoblastic leukemia [17]. The antioxidant properties of UT are attributed to its capacity to scavenge radicals such as superoxide anions and hydroxyl radicals, and also prevent lipid membrane oxidation [18]. Allen-Hall et al. [19] used human THP-1 monocytes (human monocytic cell line derived from an acute monocytic leukemia patient), reporting how UT affects the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, inhibiting inflammatory cytokines such as TNF-α and interleukin 1 beta (IL-1β). UT aqueous ethanol extract also has the potential to treat T-helper 1 immuno-mediated disorders with no cytotoxic or immunotoxicity at concentrations of 100 μg/mL and 500 μg/mL on murine splenocytes [20]

Another study suggested that both aqueous and alkaloid-enriched extract of UT act over the wnt-signaling pathway which influences degenerative diseases, diabetes, and cancer, modulating it to a less pathogenic environment [21].

#### *2.3. Harpagophytum procumbens (Devil's Claw)*

*Harpagophytum procumbens* (HP), commonly named devil's claw because of its fruits, is a perennial herbaceous plant which has roots that are traditionally used as anti-inflammatory agents for symptomatic treatment in arthritis and rheumatism. It grows mainly in the Kalahari Desert in the south of Africa. It had been linked with healthy properties like antimalarial, anticancer and uterotonic activities [22].

Its bioactivity is conferred by iridoids, a family of monoterpenoids. The glycoside fraction (containing mainly harpagoside; Figure 3) in devil's claw has been proven to be antimutagenic against environmental injury [23].

**Figure 3.** Chemical structure of harpagoside.

In addition, anti-inflammatory properties have been widely evaluated. The extract of HP influences the synthesis and release of pro-inflammatory factors, inhibiting transcription factor activator protein 1 (AP-1) activity in murine macrophages and cytokine expression such as TNF-α and interleukin 6 (IL-6) when used at concentrations between 100 μg/mL and 200 μg/mL. It decreases COX-2 mRNA levels at concentrations between 50 μg/mL and 200 μg/mL [24]. In any case, devil's claw's effectiveness is not only based on its harpagosides but also on synergistic activity with other compounds, as seen by Hostanska et al. [25], who observed less cytokine TNF-α, IL-6, and interleukin 8 (IL-8) production at concentrations of 250 μg/mL of HP ethanolic extract.

Hapargophytum extract also exhibits the capacity to prevent oxidative stress or loss of cell viability against common oxidants [26]. The phytochemicals responsible for these effects are primarily verbascosides, followed by phenylethanoid-containing fractions from methanolic extract [27].

#### *2.4. Myrciaria dubia (Camu Camu)*

*Myrciaria dubia* (MD) is a shrub from the Amazon rainforest which produces a round red-colored berry with a strong acid flavor. It is characterized by its content in vitamin C and polyphenols which confer it antioxidant, anti-inflammatory, and antimicrobial activities [28]

Camu camu juice, due to its vitamin C content, has been demonstrated to have physiological antioxidant power, decreasing total reactive oxygen species and anti-inflammatory activity and reducing circulating C reactive protein in human at a dose of 70 mL/day [29]. However, polyphenols present in camu camu such as proanthocyanidins, ellagitannins, and ellagic acid derivatives also have antioxidant and anti-inflammatory activities [30]. Separately, higher antioxidant activities were reported for stachyurin and casuarinin together with other tannins present in the fruit [31]. Other authors focused on antigenotoxic effects linked to the antioxidant capacity of the compound mixture present in camu camu, demonstrating that regardless of whether the processes are acute, sub-acute, or chronic, the administration of juice at a concentration of 25%, 50%, and 100% by injection of 0.1 mL/10 g reduces significantly genotoxic damage induced by hydrogen peroxide in mice blood cells [32].

#### *2.5. Citrus Fruits Rich in Hesperidin*

Hesperidin (Figure 4) is a flavanone from the flavonoid family and is mainly found in the epicarp, mesocarp, and endocarp of oranges, lemon, lime, and other citrus fruits [33].

**Figure 4.** Chemical structure of hesperidin.

Hesperidin has been widely studied due to its health benefits against cardiovascular diseases and cancer through its anti-inflammatory properties. It induces apoptotic cell death in gastric, colon, breast, lung, and liver cancer [34]. Its presence at a dose of 25 mg/kg a week before colon carcinogenesis induction and during the next 3 weeks in mice has been proven to enhance antioxidant-inhibiting reactive oxygen species and downregulate expression of inflammatory markers such as NF-κB, iNOS (a gene on chromosome 17q11.2-q12 that encodes inducible nitric oxide synthase), and COX-2 [35]. At a dose of 50 mg/kg 1 h before carrageenan injection it also exhibits antioxidative potential, enhancing endogenous antioxidants and decreasing inflammatory markers such as TNF-α, total leukocytes, neutrophils, lymphocytes, and nitrite concentrations [36]. Hesperidin's anti-inflammatory properties have been observed in mice with induced cognitive impairment, showing that a pretreatment of 100 mg/kg to 200 mg/kg administered intraperitoneal once daily for 15 days modulates neuronal cell death, inhibiting the overexpression of inflammatory markers and providing neuroprotective effects [37].

Similar studies were conducted in rats. Those rats that were supplemented with doses of 50 mg/kg, 100 mg/kg, and 200 mg/kg prior to lipopolysaccharide-induced endotoxicity showed an improvement in endothelial status with respect to those that were not treated [38]. The capacity of hesperidin to down-regulate inflammatory status has been reported to improve rat postoperative ileus in a dose-dependent manner between 5 and 80 mg/kg [39]. A dose of 160 mg/kg in rats reduced lipid peroxidation and improved the activity of endogenous antioxidant enzymes, palliating effects of rheumatoid arthritis [40].

Clinical assays performed in 75 patients with myocardial infarction at a dose of 600 mg/day for four days showed an improvement in inflammatory markers and lipid profiles [41].

#### *2.6. Ribes nigrum (Blackcurrant)*

*Ribes nigrum* (RN) is a woody shrub natural from central and Eastern Europe. Its fruit, the blackcurrant, is traditionally used for the treatment of rheumatic disease. It contains significant concentrations of vitamin C and some polyphenols, mostly anthocyanins. As seen in other extracts, synergistic activities are key in natural extracts but in the blackcurrant, the most remarkable compounds are prodelphinidins [42]. Rutinosides and glucosides of delphinidin and cyaniding are the major compounds in blackcurrant extract, but also other compounds have also been found, such as myiricetin and quercentin glucosides, at lower concentrations [43]. Considering the presence of those compounds, it is not surprising that different studies found anti-inflammatory and antioxidant activities in blackcurrants.

Blackcurrant extract obtained from freeze-dried blackcurrants at a concentration of 1 mg/mL applied to cell culture supernatant down-regulates the expression of inflammatory mediators through the action of intestinal cells and macrophages stimulated with lipopolysaccharides [44]. Another study which also used RAW264.7 macrophages stimulated with LPS showed a reduction in mRNA levels of TNF-α, IL-1β, and iNOS when blackcurrant extract was added at a concentration of 0.2 mg/mL to cultured supernatant. [45]. Further studies with a mix of berries (blueberries, blackberries, and blackcurrants) support these results, using lower concentrations of between 10 and 20 μg/mL in cell cultures and obtaining lower levels of IL-1β messenger RNA [46].

Proanthocyanin-enriched blackcurrant extract suppressed IL-4 and IL-13 secretion from alveolar epithelial cells in a study performed with concentrations of 0.5–10 mg/mL total blackcurrant polyphenolic extract [47]. Concentrations of 5 μg/mL to 25 μg/mL of blackcurrant extract and cyanidin-3-O-glucoside on supernatant were used with monocyte-derived macrophages (U937), showing higher cell viability against nicotine and lower levels of IL-6 secretion when inflammation was induced through lipopolysaccharides [48].

Animal studies were also performed, obtaining anti-inflammatory results due to a depletion in the contents of TNF-α, IL-1β, IL-6, and IL-10 on Wistar rats pretreated with an intraperitoneal administration of proanthocyanidins from RN leaves at concentrations of 10 mg/kg, 30 mg/kg, 60 mg/kg, and 100 mg/kg [49]. In addition, the chemopreventive effects of anthocyanins from RN were tested against hepatic carcinogenesis in Sprague–Dawley rats using a dose between 100 mg/kg and 500 mg/kg of extract during four weeks, obtaining as a result antihepatocarcinogenic effects [50].

#### **3. Comparing Emergent Extracts with Classics**

There is a great complexity in the modes of action of the phytochemical species present in the extracts described above, and while most are used traditionally to palliate a broad spectrum of diseases, just a few natural extracts have been widely popularized.

One of these is green tea (*Camellia sinensis*) for which its phytocompounds have been proven to have anticancer properties [51]. It provides protection against environmental factors that affect homeostasis, such as genotoxic substances and free radicals [52]. Green coffee also exhibits a number of health benefits, such as protection of hepatic cells from oxidative damage [53]. Its chlorogenic acids inhibit LDL (low-density lipoprotein) peroxidation and COX-2 production, helping to prevent colon cancer and cardiovascular diseases [54], and down-regulate cytokines and proliferative factors [55]. Cocoa, from *Theobroma cacao*, has also been studied because of its antioxidant and anti-inflammatory characteristics [56]. The fruit from *Vitis vinifera*, commonly named grape, is also a source of antioxidant and anti-inflammatory compounds in both the seeds and the skin [57,58]. It is widely studied due to its involvement in wine production, which also gives rise to the presence of stilbenoids [59].

World famous foods such as tea, coffee, and cocoa have been studied extensively due to interest not only as foods that promote health but also for their commercial potential given high levels of production and exportation. For this reason, other botanical species potentially beneficial for health have been relegated to the background and should be studied more thoroughly

#### **4. Conclusions**

Nowadays, cardiovascular disease and different types of cancers represent major challenges to public health, and because identification is crucial. Growing interest in health can be an incentive when producing food and nutraceuticals that promote research in this field.

In this review, which includes a summary of the dose–response activity of selected extracts, we can see how different lesser-known extracts have an effectiveness similar to those of more widely used extracts. It is important to bear in mind that botanical species such as UT and HT at low doses have anti-inflammatory, anti-oxidant, and anti-carcinogenic effects when evaluated in cell and animal studies. Even species such as *Allium nigrum* are able to enhance their beneficial effects or add new ones after processing. Hesperidin has also been studied as a representative of citrus fruits given its anti-inflammatory and cardioprotective effects. To conclude, MD and RN suppose a source of antioxidants that are present in the fruit of these botanical species that can not only act as such but also enhance the beneficial effect of the extracts described above.

As we have seen in the selected extracts, synergistic activity is one of the factors that potentiates the effects of natural extracts against purified compounds. This way, it is assumable that the joint use of different sources of bioactive compounds could lead to higher effectiveness. However, the safety of these mixtures should be evaluated and the action pathways identified.

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

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

#### **References**

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Hydroxytyrosol: Health Benefits and Use as Functional Ingredient in Meat**

#### **Lorena Martínez, Gaspar Ros and Gema Nieto \***

Department of Food Technology, Nutrition and Food Science, Veterinary Faculty University of Murcia, Regional Campus of International Excellence "Campus Mare Nostrum" (Economy based on agri-food), Campus de Espinardo, 30100 Espinardo, Murcia, Spain<EMAIL_ADDRESS>(L.M<EMAIL_ADDRESS>(G.R.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +34-868-889624; Fax: +34-868-884147

Received: 27 December 2017; Accepted: 20 January 2018; Published: 23 January 2018

**Abstract:** Hydroxytyrosol (HXT) is a phenolic compound drawn from the olive tree and its leaves as a by-product obtained from the manufacturing of olive oil. It is considered the most powerful antioxidant compound after gallic acid and one of the most powerful antioxidant compounds between phenolic compounds from olive tree followed by oleuropein, caffeic and tyrosol. Due to its molecular structure, its regular consumption has several beneficial effects such as antioxidant, anti-inflammatory, anticancer, and as a protector of skin and eyes, etc. For these reasons, the use of HXT extract is a good strategy for use in meat products to replace synthetics additives. However, this extract has a strong odour and flavour, so it is necessary to previously treat this compound in order to not alter the organoleptic quality of the meat product when is added as ingredient. The present review exposes the health benefits provided by HXT consumption and the latest research about its use on meat. In addition, new trends about the application of HXT in the list of ingredients of healthier meat products will be discussed.

**Keywords:** hydroxytyrosol; antioxidant; antimicrobial; meat; preservative; health

#### **1. Introduction**

Meat and meat product consumption provides high-quality proteins (20–25%), minerals (Feheme, Mg, K, Zn and Se) and vitamins (A, thiamine, riboflavin, niacin, retinol, B6, folic acid, B12, D and K) necessary for a balanced diet. However, these products usually are rich in saturated fatty acids, and recently, the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) has classified processed meat as a carcinogen (Group I) and red meat as possible carcinogen (Group 2A) (October 2015) [1]. In fact, carcinogenic compounds in meat could be added during their processing (synthetic additives), but they also can be formed during their storage through lipid and protein oxidation, or during cooking through the Maillard reaction [2,3]. In this way, synthetic additives such as sulphites, BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole) are added in meat product formulation to preserve them. The use of these synthetic additives has given rise to social concern by consumers, due to studies that correlates their consumption with disease development (asthma, hyperactivity, cancer, etc.) [4–6]. On the other hand, lipid peroxidation in meat and meat products happens through the radical chain reaction mechanism, although oxygen presence accelerates this process. This oxidation is due to several factors such as polyunsaturated fatty acids concentration (PUFA), the deficit of antioxidants in animal feed (tocopherol, rosmarinic acid) and a high concentration of prooxidants, free radicals or added salt (NaCl). At the same time, these reactions produce reactive oxygen species (ROS) like hydroxyl radical, superoxide anion, ferryl and perferryl species, lipid peroxyl radical and secondary products like reactive carbonyl species (MDA (malondialdehyde) and 4-HNE (4-hydroxynonenal)) responsible for the rancid flavour in aged meat.

Although protein oxidation has received less attention, it has a huge influence on quality of meat [7]. Protein oxidation has been defined as a covalent modification of protein induced either directly by reactive species or secondary products of oxidative stress. The same oxidants that induce the lipid peroxidation produce this alteration, and carbonyl formation is a common reaction in protein oxidation. Furthermore, proteins can react with secondary products of lipid peroxidation like aldehydes and ketones to produce complexes between proteins, proteins and carbonyls or proteins and lipids. In muscle fibres, hydroxyl radical (OH) in presence of Fe or Cu or ROS causes modifications of amino acids, like methionine, lysine, arginine, histidine, tryptophan, valine, serine and proline. This reaction increases proteolytic enzymes and protein polymerization, which produces soluble aggregates, that promotes gelation and emulsification that modifies the texture and toughens the meat [8–10]. But this not only is critical for organoleptic quality, but it might have an impact on human health and safety. For example, during cooking it increases free radical generation while it decreases the antioxidant compounds in meat, which contribute to protein oxidation.

Therefore, natural antioxidants can prevent lipid peroxidation on different ways: preventing chain inhibition by scavenging initiating radicals, breaking chain reaction, decomposing peroxides, decreasing localized oxygen concentrations and binding chain initiating catalyst such as metal ions. Therefore, the use of natural preservatives to keep the shelf life of meat has exhibited similar antioxidant properties compared to some synthetic additives. For this reason, it is a promising tool due to many fruits (grapes, grape seed, pomegranate, date, kinnow mandarin), vegetables (broccoli, potato, drumstick, pumpkin), herbs (olive leaf, acerola, grape seed, cocoa, green coffee, *Ginkgo biloba*, etc.) and spices (rosemary, green tea, black pepper, garlic, oregano, cinnamon, sage, thyme, mint, ginger, clove) reported antioxidant properties in meat products [11–14].

One of most potent natural antioxidant extracts is hydroxytyrosol (or 4-(2-dihydroxyphenyl) ethanol) (HXT)), just below gallic acid (Figure 1). This compound is ten times more antioxidant than green tea and two times more than coenzyme Q10 [15], additionally HXT scavenging ability is comparable to oleorupein and catechol. HXT is a phenylethanoid with demonstrated antioxidant properties in vitro, it is found in olive leaf and oil from this fruit, responsible for intense flavour and aroma, being oleuropein precursor [16,17]. In addition, it has demonstrated this capacity in vivo in several studies in rats, such as Merra et al. or Lemonakis et al. [18,19], that showed the power of HXT to reduce the risk to suffer metabolic syndrome. In its chemical structure, this compound has an additional OH group in its benzene ring, compared to the tyrosol (TYR) (Figure 2). Therefore, it obtains a greater function as a free radical scavenging, increasing its antioxidant power, as well as its efficacy under stress conditions.

In this way, this extract has previously demonstrated its antioxidant capacity in meat products rich in unsaturated fatty acids like sausages and frankfurters with added HXT, nuts and extra virgin olive oil [20,21]. Moreover, HXT is an antioxidant compound linked to certain minerals, such as gluconate Fe (II) in black olives, which catalyzes the oxidation of this compound, so it is possible that HXT influence on biological bioavailability of some minerals and trace elements [17].

The objective of this paper is to review the latest literature about HXT consumption benefits, its extraction from olive leave and other sources and its used as natural antioxidant in meat and meat products as substitute of synthetics additives, with emphasis on new trends and future perspectives in investigation and meat industry.

**Figure 1.** Graphical abstract of the use and health benefits of Hydroxytyrosol to prevent lipid and protein oxidation and substitution of synthetic additives in meat products.

**Figure 2.** Chemical structures of TYR and HXT: phenolic compound from olive leave and olive oil. TYR: tyrosol (left); HXT: hydroxytyrosol (right).

#### **2. The Role of HXT in Diet**

Since many years ago it is known that several aspects about the Mediterranean diet have been associated with a minor risk of cardiovascular diseases and cancer (colorectal, breast, prostate or pancreas, between others) [22] (Figure 1). In particular, olive oil as principal component of this dietary model plays a key role in these benefits, due to its composition of fat, richness in monounsaturated fatty acids (oleic acid) and another micronutrient from non-saponifiable fraction such as squalene, phytosterols, tocopherols and secoiridoids.

HXT is a secoiridoid and a potent antioxidant that acts as main component from phenolic fraction of extra virgin olive oil (EVOO). This compound is derivate from hydrolysis of oleuropein and its concentration in EVOO and table olives can change according to several factors: altitude and latitude of olive tree harvest, the variety of olive, the collection time and the processing conditions [23,24]. For example, the HXT concentration in EVOO is 14.32 ± 3.01 mg kg−1, while in refined virgin oil is 1.74 ± 0.84 mg kg−<sup>1</sup> [25]. In the same way, the concentration in Greek black olives is 100–340 mg kg<sup>−</sup>1, in Spanish green olives is 170–510 mg kg−<sup>1</sup> and 250–760 mg kg−<sup>1</sup> in Greek kalamata olives [26]. Taking into account that the average consumption of EVOO and olives in Spain are 15 g/day−<sup>1</sup> and 7 g/day, respectively [27], it can be estimated that the HXT consumption per day is 5.6 mg (0.3 mg from EVOO and 5.3 mg from olives), approximately.

In addition, HXT has a bioavailability of 99%, so this compound is easily integrated by human body [28]. In the same way, it was corroborated by Khymenets et al. [29] in their study about human HXT absorption and excretion from a nutraceutical, that HXT is a phenolic compound resistant to gastric juices and to passing through the digestive system, consequently it is bioavailable and is recovered in the urine chiefly as 3 -sulphate. Therefore, it can be confirmed that the consumption of 5.6 mg day−<sup>1</sup> of HXT can bring great benefits on an organism's functioning.

#### **3. The Role of HXT in Health**

HXT has a great bioactive power due to its great antioxidant capacity due to its protective function in cells, its structural affinity to some compounds (for example, with dopamine, it replaces its amine group (NH2) by hydroxyl group (OH)), it has a simple molecular structure and it is present in organism (e.g., iris of the eye) so it is easy to assimilate by the human body, reaching blood plasma in 15 or 20 min until its elimination 6–8 h later by the renal or digestive system, so it does not present accumulation or toxicity problems. In addition, HXT is an amphipathic, water-soluble and fat-soluble molecule, because of it has a lipophilic end and another hydrophilic end, which makes it a good transporter of substances through the human body, therefore, it can penetrate the cellular membrane easier. These structural and molecular features of HXT make its consumption provide many beneficial effects in the organism [30].

Firstly, in 2012, EFSA (European Food & Safety Authority) accepted that HXT acts as protector of the cardiovascular system, avoiding oxidation of LDL cholesterol by free radicals, maintaining normal blood HDL cholesterol concentrations and preventing atherosclerosis [31]. In addition, HXT consumption can regulate glutathione concentration and provides antioxidant enzymes to adipose tissue [32]. Moreover, in other in vivo study with experimentally induced diabetes mellitus in rats, it was shown that the HXT consumption influenced the major biochemical processes leading to diabetic vasculopathy and reduced cell proliferation in the vascular wall [33]. It is confirmed the prevention against damage by oxidative stress induced by this compound, which acted as regulator of cell protection and damage induction, controls the intracellular redox state. This could influence the prevention of diseases such as cancer, diabetes, inflammation or cardiovascular and neurodegenerative diseases, which aetiology and progression has been linked to the production of ROS on damaged tissues. So, the regular consumption of HXT can help to avoid cardiovascular diseases and diabetes mellitus [34].

Secondly, HXT has the capacity to inhibit COX (cyclooxygenase) and LOX (lipoxygenase) enzymes of arachidonic acid (AA), reducing the oxidative deterioration characteristic of inflammations [35]. At the same time, HXT stimulates the production of chondrocytes by regeneration and repairing the articular cartilage. During physical exercise, HXT helps to increase the production of glutathione and to reduce the production of lactic acid and the consequent muscular atrophy [36].

Because of the connection between cellular oxidation, inflammation and the formation and development of a tumour, it can have a clear idea of the anticancer ability of this extract. Moreover, HXT alters a tumour's eicosanoid biosynthesis and shows an inhibition of tumour cell proliferation [37]. At the same way, the daily consumption of 50 μM–100 μM of HXT, through the intake of EVOO and table olives, has showed antiproliferative activity, apoptotic activity and inhibition of metastasis of leukemia cells (HL60), adenocarcinoma cells (HT29), human colon cancer cells (Caco-2 and HT115) [38,39] and in breast cancer cells (MCF10A, MDA-MB-231 and MCF7) [40].

On the other hand, HXT is protective against neurodegenerative damage and cognitive decline associated with age or diseases like Alzheimer or Parkinson. It is due to the fact that HXT protects brain cells from lipid peroxidation because it is able to cross the blood–brain barrier [41].

Another notable property of HXT is its antimicrobial capacity and acts by inhibiting the growth rate of bacteria in humans, as demonstrated in the research of Medina et al. [42], (against *Clostridium perfringens*, *Escherichia coli*, *Staphylococcus aureus*, *Salmonella enterica*, *Yersinia sp*. and *Shigella sonnei*) and Brenes et al. [43] (against *Helicobacter pylori*). There are also studies with its antifungal properties against *Mycoplasma hominis* and *Pneumoniae fermentans* [44], and against *Fusarium sambucinum*, *Vericillium dahliae* and *Alternaria solani* [45]. Therefore, HXT consumption has an antimicrobial effect that can avoid infections in the respiratory, intestinal and genital systems and it strengthens the immune system. In addition, HXT can act against virus like HIV. Lee-Huang et al. [46] demonstrated that HXT and oleuropein inhibit the entry and integration of virus, as inside as outside cells.

Finally, HXT also helps to prevent osteoporosis because its consumption has positive effects on the formation and growth of bones [47]. In addition, the retina is also protected by HXT and it is beneficial for ocular health specifically in the regeneration of the retinal pigment epithelium, macular degeneration and glaucoma, caused by oxidative stress [48]. Furthermore, HXT has dermoprotective effects: it protects against UV rays, it reduces the pigmentation of the skin, it protects against oedema and erythema caused by excessive sun exposure, it may even be effective in treating psoriasis, it has anti-aging effect and it stimulates the production of cell-survival promoting proteins [49] (Figure 1).

#### **4. Preparation of HXT Extract**

The highest concentration of HXT is found in olive leaves. However, it is lost during oil manufacturing after the waste of the pomace and vegetable water as residues of EVOO, or refined olive oil production. In order to take advantage of this waste and minimize losses to the oil industry, these residues are used by ingredient companies to obtain natural extracts used by the cosmetic, pharmaceutical and food industries (Figure 1).

HXT can be obtained from vegetation water or from olive leave using different ways. It is based in three phases: first, a phenol-rich liquid is obtained as the raw material for the extraction and purification of phenolic compounds; a second phase enables a HXT enriched extract and a HXT with 3,4-dihydroxyphenylglycol (DOPEG) mixture to be obtained, and HXT acetate is produced. In the third phase, highly pure HXT-DOPEG and HXT acetate are obtained. These phases comprise extraction, reaction, concentration, adsorption and desorption, using mixtures of ion exchange resins, adsorption in nonionic resins and a polymer phenolic fraction, membranes of reverse osmosis and evaporators. This methodology is an example of extraction method for HXT from vegetation waters and *Olea europaea* L. subproducts that it is under intellectual property of the Spanish Research Council (Consejo Superior de Investigaciones Científicas, CSIC) [50].

From the industrial point of view, the extraction method depends on the source used for HXT [21]. The first one obtains HXT from olive waters during fruit processing (separating the oil from wet centrifugation). It is used a solvent extraction and purification process, including, crystallization and clarification steps. For that, the original plant material (vegetation water) is dried and suspended in ethanol. The filtered hydroalcoholic solution is concentrated under vacuum until obtain a syrup with a HXT percentage around 20–25%. Among the characteristic polyphenolic compounds from olive oil that this extract contains are large quantities of fulvic acids. HXT can be also obtained from olive leaves (dehydrated) by hydroalcoholic extraction and subsequent hydrolysis. The hydroalcoholic solution obtained contains as main active compound the oleuropein that is present in the leaves.

Another form to obtain HXT extract can also be through olive waters (fruit processing) by liquid–liquid extraction with ethanol. For that purpose, the original plant material (vegetation water) is concentrated in vacuum at temperature of 50–60 ◦C until a syrup of 65–70% ◦Bx (*w*/*w*) is obtained. This syrup is suspended in ethanol and the supernatant is removed, which is concentrated in vacuum and finally a hygroscopic solid.

#### **5. Use of HXT Extract as Functional Ingredient in Meat**

Firstly, AECOSAN (Spanish Agency for Consumer Affairs, Food Safety and Nutrition) accepted using of HXT extract as functional ingredient in 2015 [51], while in 2017, EFSA (European Food & Safety Authority) has confirmed that its use does not provided negative effects on health of consumers [52].

Among all of the literature on this topic, it has been selected as the most innovative and current (Table 1). For example, Rounds et al. [53] researched the antimicrobial activity of HXT (10,000 and 30,000 ppm) in ground beef patties inoculated with *E. coli.* They showed that the cell count was reduced 3% more than the control at the same conditions and the amine formation was 50.6% lower than control.

 **1.** Results on the applicability of HXT on meat and its effect on product quality.

**Table**

However, the antimicrobial capacity of HXT has already been demonstrated in several matrices, so after this researcher focused on studying the shelf life of meat products in which synthetic additives have been replaced by compounds derived from olive: HXT, tyrosol, oleorupein, verbascoside and pinoresinol. Such as, in the work of Chaves-López et al. [55], they elaborated fermented sausages with HXT (100.23 ppm) and proved that lipid oxidation was lower (12–38%), while volatile compounds were reduced and colour redness was increased. On the other hand, Muíño et al. [56] analysed lamb meat patties enriched with omega 3 (fish oil) and HXT (100, 200 and 400 ppm). They found that antioxidant activity of patties increased with HXT presence and lipid oxidation was lower at day 3, 6 and 9 of storage compared with control samples. In addition, patties with HXT kept the colour and texture stable while the odour and flavour were modified by the extract. Similar results were obtained in pork sausages enriched with HXT (75 and 150 ppm) and stored for 14 days, even though Balzan et al. [57] also showed how this extract had an inhibitory effect on lipolytic activity and microorganism growth. Finally, on chicken frankfurters, Nieto et al. [20,21] proved that a small quantity of HXT (50 ppm) is enough to maintain the colour and reduced the lipid and protein oxidation until 21 days if it is combined with other ingredients like EVOO (extra virgin olive oil) and/or walnuts. At the same way, they compared three extracts: HXT 23% from olive waters, HXT 7% from olive leaves and HXT 7% from olive waters; and they reported that HXT 7% from olive waters was more sensory acceptable than the others.

In addition, meat can be enriched exogenously through the use of natural extracts to replace synthetics additives or endogenously through the animal diet. It is the case of Branciari et al. [58] whose research was based on feeding 297 chickens during 21 days with three treatments: basal diet, diet supplemented with 82.5 g/kg of "paté cake" rich on olive phenols (4.6 ppm HXT), and diet supplemented with 165 g/kg of the same product (9.5 ppm HXT).  It is important to remark that the sensory quality of meat was not modified by olive feed.

Taking into account previous studies, HXT can be used as exogenously as endogenously to report benefits to the meat and meat products.

#### **6. New Trends**

New trends in the use of HXT in meat focus on the maintenance of its antioxidant power and eliminating its intense flavour and smell, as shown in the studies of Cofrades et al. [54] or Freire et al. [59]. At first, it was proved that HXT added to the ground pork meat in W1/O/W2 emulsions reduced lipid oxidation by more than 50%. In the same way, Freire et al. showed that using cold-set gelled double emulsions enriched with perilla oil (*Perilla frutescens* as a source of n-3 fatty acids) and HXT as animal fat replacer during 30 days of storage, it was found HXT maintained the stability of emulsions and increased its antioxidant capacity while it was reduced lipid oxidation and bacterial growth. That kind of emulsion has the characteristic of encapsulating the extract to avoid the strong flavour and odour of HXT on meat. However, there are no studies about sensorial changes after the application of the HXT extracts.

On the other hand, Moudache et al. [60] have studied another application of HXT: an antioxidant food packaging material applied to fresh minced pork and whose plastic film contained olive leave extract. This researcher showed that active films with olive leave extract, rich in HXT, had a positive effect on the oxidation stability of meat fat. In addition, this active packaging does not need being in contact with meat, so it does not alter the sensorial quality of the product.

#### **7. Conclusions and Perspectives**

As reviewed, the beneficial effects of HXT has been extensively investigated during the last twenty years, so many researchers of pharmaceutical industry have been focused on use of this compound due to its nutraceutical power. In parallel, researchers in the food and meat industries have been focused on the elimination of preservatives and dyes in order to achieve the 'clean label'. However, HXT has a strong flavour so cannot be added directly to the meat. For this reason, researchers have currently been focused on the encapsulation of this extract and the production of emulsion gels to prevent the sensorial alteration of meat products. However, nothing has been concluded in this field about the organoleptic quality of the product. Therefore, it can be concluded that the best way to obtain its benefits on meat is endogenously, through the animal diet, or through its application in new packaging systems.

**Author Contributions:** Lorena Martínez collected the information and drafted the article. Gema Nieto and Gaspar Ros interpreted and reviewed the article.

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

#### **References**

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