*4.4. Coating Application on the Samples*

The coating procedure is illustrated in Figure 11. For a single-layer Ch coating, approximately 2 kg of flesh-cut purple sweet potatoes were dipped in 5 L of Ch solution for 2 min and dried at room temperature for 30 min. For SA gel coating, flesh-cuts were dipped in SA solution, rinsed for 30 s to remove the residual solution, and thereafter immersed in calcium chloride solution and dried. For the multilayer gel coating (ChCSA), the fresh-cuts were dipped in Ch solution for 2 min, rinsed for 30 s to remove the residual solution, immersed in calcium chloride for 2 min, then rinsed for 30 s and finally dipped in SA solution for 2 min before air-drying. Lastly, distilled water was used as an immersion solution for uncoated samples.

For each coating treatment, approximately 200 g of coated fresh-cuts were weighed and stored in triplicate in Ziploc bags (5 ◦C). Stored samples were removed at 4-days interval during a 16-day storage period and analyzed for quality parameters.

70 °C under stirring until fully dissolved to produce a 2% chitosan solution. Finally, the

The sodium alginate solution was prepared according to the method described by [11], with some modifications. Sodium alginate powder was dissolved in 100 mL of distilled water to obtain a 2% concentration. Then, the solution was stirred in a 70 °C water bath for 2 h to dissolve completely. Finally, the sodium alginate solution was cooled at

Calcium chloride was used as the cross linking agent to produce gel coatings via layer-by-layer treatment. Calcium chloride was weighed and dissolved in 100 mL of distilled water to obtain a 2% solution. Then, the solution was shaken in the incubator to

Purple flesh sweet potatoes without mechanical injuries or fungal infections were selected and washed in running water. Then, they were peeled and diced to get 1 cm

The coating procedure is illustrated in Figure 11. For a single-layer Ch coating, approximately 2 kg of flesh-cut purple sweet potatoes were dipped in 5 L of Ch solution for 2 min and dried at room temperature for 30 min. For SA gel coating, flesh-cuts were dipped in SA solution, rinsed for 30 s to remove the residual solution, and thereafter immersed in calcium chloride solution and dried. For the multilayer gel coating (ChCSA), the fresh-cuts were dipped in Ch solution for 2 min, rinsed for 30 s to remove the residual solution, immersed in calcium chloride for 2 min, then rinsed for 30 s and finally dipped in SA solution for 2 min before air-drying. Lastly, distilled water was used as an immer-

For each coating treatment, approximately 200 g of coated fresh-cuts were weighed and stored in triplicate in Ziploc bags (5 °C). Stored samples were removed at 4-days in-

pH of the solution was adjusted to 5.6 with 1 N NaOH.

room temperature.

become dissolved entirely.

pieces for flesh-cut coating.

*4.4. Coating Application on the Samples*

sion solution for uncoated samples.

*4.3. Sample Preparation*

**Figure 11.** Illustration of coating procedure for fresh-cut purple flesh sweet potatoes. **Figure 11.** Illustration of coating procedure for fresh-cut purple flesh sweet potatoes.

terval during a 16-day storage period and analyzed for quality parameters.

#### *4.5. Color Measurement 4.5. Color Measurement*

The surface color of the samples was determined by randomly selecting 3 samples and taking 3 readings for each treatment using a chromameter (CR-300, Minolta Co., Osaka, Japan). The L\*, a\*, b\* value (CIE L a b) system was numerically specified in a threedimensional spherical space defined by the three perpendicular axes: the L-axis (brightness) ranged from 0 (black) to 100% (white); the a-axis ranged from − a (green) to + a (red); The surface color of the samples was determined by randomly selecting 3 samples and taking 3 readings for each treatment using a chromameter (CR-300, Minolta Co., Osaka, Japan). The L\*, a\*, b\* value (CIE L a b) system was numerically specified in a threedimensional spherical space defined by the three perpendicular axes: the L-axis (brightness) ranged from 0 (black) to 100% (white); the a-axis ranged from − a (green) to + a (red); and the b-axis ranged from − b (blue) to + b (yellow). Total color difference (∆E) was calculated using L, a, and b values with the following equation [51]:

$$
\Delta \mathcal{E} = \sqrt{(L\_2 - L\_1)^2 + \left(a\_2 - a\_1\right)^2 + \left(b\_2 - b\_1\right)^2} \tag{1}
$$

where subscripts 1 and 2 represent the final and initial readings, respectively at a particular storage interval.


**7. Degradation of endosulfan**

**Figure 2** shows the oxidation pathway of endosulfan; it has been reported that even though some hydrolysis takes place, at environmental conditions, the most common product is endosulfan sulfate [2, 16, 22]. After initial oxidation, four intermediates are detected until endosulfan lactone is reached. In further oxidation, the complete mineralization can be obtained. Endosulfan is relatively resistant to photodegradation but its metabolites, including endosulfan sulfate, are susceptible to photolysis [23]. **Figure 2** shows the main pathways of endosulfan degradation.

In aquatic systems, the α-isomer is converted more readily to endosulfan sulfate than the β-isomer. Various studies that have observed the degradation of endosulfan in aqueous phases identified hydrolysis as the dominating abiotic degradation process resulting in the

**Figure 2.** Degradation of isomers α- and β-endosulfan.

generated in India, but it had agreed to phase out its use in 2017. China produced approximately 5000 tons per year, and the remaining endosulfan was produced by Israel, Brazil, and South Korea [16]. Regarding the consumption of endosulfan, India was the main consumer 113,000 t from 1958 to 2000, followed by the United States with 26,000 t from 1954 to 2000. In China, annual use is estimated to average 2800 t/y during the period 1998–2004 [3]. Endosulfan is prohibited in 80 countries; some countries have registered only a few commercial products containing endosulfan, other countries have registered dozens of commercial products from various formulators. Endosulfan is now still produced and widely used in the crop fields in most of the developing countries, due to its effectiveness and low application cost [17].

Endosulfan is widely distributed in the environment and can be detected in all media, soil, sediments, air, water, and vegetation, over long distances from the application source. Levels of endosulfan in the air samples are very variable depending on the location. Recent measure-

Endosulfan is one of the most abundant organic pesticides in the atmosphere and is capable of reaching remote locations such as the Arctic. The presence of endosulfan in the Arctic at

obtained using an XD-based passive sampler with samples collected in monitoring stations. It has been suggested that the transport of endosulfan occurs when the compound volatilizes from warmer regions, experiences long-range atmospheric transport, and condenses again, permitting an accumulation of the substance in temperate regions, higher mountains, and the Arctic. Endosulfan residues in snow and ocean waters in the Artic indicate that atmospheric deposition of endosulfan is taking place, and it can promote bioaccumulation in zooplankton and various species of fish. For some biota, a biomagnification factor for α-endosulfan from

**Figure 2** shows the oxidation pathway of endosulfan; it has been reported that even though some hydrolysis takes place, at environmental conditions, the most common product is endosulfan sulfate [2, 16, 22]. After initial oxidation, four intermediates are detected until endosulfan lactone is reached. In further oxidation, the complete mineralization can be obtained. Endosulfan is relatively resistant to photodegradation but its metabolites, including endosulfan sulfate, are susceptible to photolysis [23]. **Figure 2** shows the main pathways

In aquatic systems, the α-isomer is converted more readily to endosulfan sulfate than the β-isomer. Various studies that have observed the degradation of endosulfan in aqueous phases identified hydrolysis as the dominating abiotic degradation process resulting in the

has been detected. Endosulfan concentration was

**6. Distribution of endosulfan in the environment**

concentrations between 3 and 8 pg m−3

8 Persistent Organic Pollutants

fish to seal is also occurring [3, 19–21].

**7. Degradation of endosulfan**

of endosulfan degradation.

ments indicate that around 320 d is the half-life in the atmosphere [18].

formation of endosulfan diol. A positive correlation between hydrolysis rate (khydro) and pH, with base-driven hydrolysis, a predominant degradation process in seawater conditions, allows a rapid degradation of endosulfan into endosulfan sulfate, whereas it takes longer in pure water.

Furthermore, hydrolysis of endosulfan sulfate to endosulfan diol is markedly slower compared to the hydrolysis of α-endosulfan.

The biodegradation of endosulfan has been reported in anaerobic conditions; these conditions predominate in sediments and the results can contradict the interpretation of the isomer deposition in freshwater sediments. At neutral pH and aerobic conditions, the degradation of endosulfan by biological oxidation has also been reported, being sulfate of endosulfan, the compound with the heaviest concentration.

It has been reported that the decomposition of endosulfan in aqueous solution can take place, but some natural environmental factors lead to the formation of sulfate indicating that some oxidation pathway is followed. The values are much lower than the persistence criteria designated for a POP, although in the colder marine waters at high latitudes, base-driven hydrolysis half-lives for endosulfan are likely to be greatly extended, compared to warmer waters in temperate or tropical regions [3, 15, 23].

There are relatively few studies that have examined the persistence of endosulfan in soil under field conditions. Degradation rates strongly depend on the soil conditions (particularly soil water content and ambient temperature).

the Conference of the Parties the inclusion of technical endosulfan, its α- and β-isomers as well as the main degradation product, the sulfate of endosulfan in Annex A of the Stockholm Convention for global elimination with possibility of specific and temporary

This exchange under the Rotterdam Convention on "prior informed consent" applies to banned or restricted chemicals and to the severely hazardous pesticide preparations listed in Annex III. Currently, there are more than 30 chemical products to which the PIC procedure applies. At the fifth meeting of the Conference of the Parties to the Rotterdam Convention that took place in Geneva, Switzerland, between June 21 and 24, 2011, it was agreed to include the

Previously, the seventh meeting of the Chemical Review Committee, held in Rome from March 28 to April 1, 2011, recommended the inclusion of a number of new chemicals in this Annex, including the pesticide endosulfan that had recently added to the list of the Stockholm

**11. Recent technologies to treat endosulfan present in water and soil**

Nowadays, there is a concern for the development of alternatives for the elimination of endosulfan in both water and soil. In this context, advanced oxidation processes (AOPs) are considered a highly competitive technology for the removal of those organic pollutants not treatable by conventional techniques. All AOPs produce •OH radicals, they are very reactive, attack most organic molecules, and are not highly selective. The generation of •OH radicals is through different reagent systems, which include photochemical degradation processes

/Fe2+), and electrooxidation (BDD electrodes) [27].

Others treatments for pesticides are biodegradation and bioremediation; both of these are based on the conversion or metabolism of pesticides by microorganisms. The difference between these two is that the biodegradation is a natural process, whereas bioremediation is a technology. One disadvantage of these processes is that they require a long time to degrade

One of the technologies most resorted to eliminate pesticides present in water is adsorption with activated carbon, as it can be applied at a large scale. Good characteristics of activated

**Table 2** shows some technologies that have been developed to degrade endosulfan present in

/UV, photo-Fenton reactives), chemical oxidation pro-

Dissemination of Endosulfan into the Environment http://dx.doi.org/10.5772/intechopen.81094 11

exceptions [15].

(UV/O3

cesses (O3

, UV/H2

, O3 /H2 O2 , H2 O2

the pesticide [28].

water and soil.

O2

**10. Rotterdam Convention**

endosulfan pesticide in Annex III of the Convention.

Convention of chemical substances that must be eliminated [26].

), photocatalysis (TiO2

carbon include large surface area and well distributed porosity [29].

The Environmental Protection Agency (EPA) indicates that concentrations above 0.22 μg L−1 (acute) and 0.05 μg L−1 (chronic) have adverse impacts on the health of aquatic organisms. To avoid intoxication from water consumption, fish or shellfish from water bodies near fields where the pesticide is used, concentration limits need to be established. For air concentrations, the limit recommended by the National Institute for Occupational Safety and Health (NIOSH) is 0.1 mg m−3 [22, 24].


**Mobile Cloud-Based Blood Pressure Healthcare for Education**

Chin-Feng Lin, Shere-Er Wang, Yen-Chiao Lu, Chung-I Lin, Chung-Cheng Chang, Tim Yeh, Candice Lee, Jeffson Huang, Chic-Erh Weng, Sue-Hsien Chen, Bing-Leung Sun, Chao-Sheng Wang, Shiue-Li Cheng, Shiou-Yu Li and Lan-Yu Wu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63471

#### **Abstract**

Mercury, pneumatic, and electronic sphygmomanometers were widely used for traditional blood pressure (BP) measurement. Cloud BP database, and mobile information and communication technology (MICT) do not integrate to these BP measurement methods. Pen and papers were employed to record BP values for nurses and physicians, and recording errors are possible to occur. In the chapter, the cloudbased BP platform solution and advanced wireless hospital BP measurement technol‐ ogies were studied. These cloud-based BT measurement technologies were used as teaching aids to train students of electrical and nursing fields for mobile BP health‐ care and health promotion education, and hence interdisciplinary teaching and learning were conducted. The teachers include professors of electrical and nursing fields, physicians, hospital nurses, and the engineer and health management experts of Microlife. The interdisciplinary teaching and learning of mobile BP healthcare and health promotion for smart aging were conducted in the Department of Nursing Division, Chang Cung Memorial Hospital, Keelung Branch, Department of Nursing Ching Kuo Institute of Management and Health, School of Nursing Chung Shan Medical University, and Department of Electrical Engineering, National Taiwan Ocean University. The students of electrical and nursing fields participated for joint interdis‐ ciplinary learning. The concepts of interdisciplinary mobile BP healthcare learning and teaching involve nursing and technology, healthy aging, BP health care for smart aging, telenursing, BP care for smart aging, community/home telecare, and MICT. The objective of teaching and learning is training the design and making electrical engineers

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to understand BP healthcare and health promotion, and nurses to understand mobile BP healthcare and health promotion system for smart aging.

**Keywords:** BP healthcare, smart aging , cloud-based BP measurement technologies, jointly interdisciplinary learning and teaching, mobile information and communica‐ tion technology

## **1. Introduction**

In this chapter, computers, Internet access, mobile computing, Web page systems, short messaging services, multimedia messaging services, and e-mails were examined with respect to students of medical, nursing, and health science disciplines [1]. The advanced technologies help health science students to access healthcare information and adopt effective methods to develop their skills. Mobile information and communication technology (MICT) has been used in the learning and teaching process of health and medicine science education, and has enabled learning and access to health education knowledge anywhere and at any time [2]. Mazzola et al. [3] demonstrated how physical activity, nutrition, and training through a combination of processes and mobile technologies are related to overweight teenagers and obesity in behavio‐ ral education. The People for Ecosystem-based Governance in Assessing Sustainable develop‐ ment of Ocean and coast (PEGASO) scenarios [3] include motion sensors to detect physical activity, GPS location service, and the smartphone acts as a communication gateway for these sensors with feedback functions and information. The awareness and self-management of obesity risks are important to motivate teenagers to engage in affective learning and trigger a behavioral change. Smartphone applications were exploited to improve participants' healthrelated quality of life and healthcare utilization for rheumatic diseases self-management [4]. Disease-associated self-management strategies can be designed using mobile health (mHealth) technologies. Azevedo et al. [4] reviewed several aims, platforms, and characteristics of smartphone applications for self-management of rheumatic diseases.

mHealth is a "medical and public health practice supported by mobile devices, such as mobile phones, patient monitoring devices, personal digital assistants, and other wireless devices" [5], and is recognized by the World Health Organization. Advanced mobile Web-based and Internet technologies have transformed current healthcare models with regard to monitoring of physical activity or encouraging physiological changes that stimulate positive health behaviors. Zhang et al. [6] illustrated how to extend a mobile Web-based app with multimedia features for psychiatry education in Singapore, and found out through a survey that MICT helped them save a significant amount of time in clinical activities. MICT must ensure that the physiological signals provided within are accurate and credible. Healthy behaviors (e.g., sports lifestyle, healthy eating habits, and blood pressure (BP) monitoring) help in reducing fatal health risk, disability, healthcare use and health-related costs [7]. Weight management, physical activity, smoking cessation, self-management of diabetes mellitus, and hypertension care using MICT were demonstrated [8]. MICT-supported health behavior interventions are designed to prevent or manage illness and lead to fundamental changes in health practices, thereby, providing an opportunity to stimulate and sustain healthy behaviors [6, 7]. Chan et al. [9] described that problem-based learning using multimedia, such as video clips, Web sites, images, or photos, was implemented in the health sciences learning process, and students could use their mobile Internet technology to access the knowledge and enhance their process of learning using laptops, tablets, and smartphone mobile devices on a cloud-based learning platform. Problem identification, problem description, problem exploration, applicability, and integration strategies were recommended in the problem-based learning approach [10].

Elliott et al. [11] implemented a mobile electronic health records (EHR) system for medical education. The functionality, connectivity, ease of use, and usage challenges of the developed EHR system in the hospital environment were investigated for learning. Davies et al. [12] developed a mobile learning model using a personal digital assistant (PDA) loaded with medical resources for undergraduate medical students in the clinical environment. MICT was used to provide new learning methods with respect to problem identification and solving skills. ISO/IEEE 11073 and Health Level 7(HL7) V2.6 protocols were used to develop and evaluate self-management mobile Personal Health Record mobile health application for Android 4.0.3 by the Continua Health Alliance for continuous self-management of chronic disease patients [13]. It has the potential to promote new treatment and medical opportunities as well as to reduce medical costs and time for an aging society. It monitors their vital signs using mobile network technology (MNT); thereby, ensuring better healthcare. The user interface, applications, seamless transmission protocol, personal health record, and database managers were demonstrated for the application.

Wu et al. [14] proposed a cellular and iridium network-based blood pressure and body temperature remote measurement platform for mobile healthcare education. The overview of mobile telemedicine research fields was provided by Lin [15, 16]. In previous studies [17–21], 802.11n and ultra-wideband wireless telemedicine transmission schemes, multicode code division multiple access (CDMA) cellular mobile telemedicine transmission mechanism, and multi-satellites wideband CDMA and orthogonal frequency division multiplexing (OFDM) transport architectures were proposed. In this article, a cloud-based mobile blood pressure (BP) healthcare education program for smart aging is investigated.


**Author details**

Tejas Karnati, Edwin Kulubya, Amir Goodarzi and Kee Kim\* Department of Neurological Surgery, UC Davis School of Medicine, University of California, Davis, California, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Wetland Health in Two Agro-Ecological Zones of Lesotho: Soil Physico-Chemical Properties, Nutrient Dynamics and Vegetation Isotopic N15

*Adesola Olaleye, Regina Mating,Tumelo Nkheloane, Tutu K. Samuel and Tolu Yetunde Akande*


**3. Results**

306 Sex Steroids

1999, Chiumello et al., 2000, van der Sluis et al., 2002, Paterson et al., 2004). However, most of these reports studied populations in Western countries, and almost none investigated Asian children (Wacharsindhu et al., 2006). Furthermore, in these studies, GnRHa was administered at higher doses (Boot et al., 1998, Chiumello et al., 2000, van der Sluis et al., 2002) than are used in the standard gonadotropin-suppressive therapy protocol that is currently in operation in Japan. With some exceptions, all these reports showed that obesity is aggravated during GnRHa therapy (Wacharsindhu et al., 2006, Boot et al., 1998, Chiumello et al., 2000, van der Sluis et al., 2002, Paterson et al., 2004, Oosdijk et al., 1996, http://www.iotf.org/documents/iotfsocplan251006.pdf). Therefore, we assessed the effects of the standard gonadotropin-suppressive therapy protocol that is currently used in Japan

The aims of the present study were to prospectively evaluate whether obesity occurs at a high frequency among Japanese children with CPP and to longitudinally evaluate the body

Eighteen patients participated in the study. At diagnosis, all of the patients had a history of increased growth velocity, breast development of Tanner stage 2 or more, and a bone age that was more than 1 yr above their chronological age. Ten girls had idiopathic central precocious puberty (ICPP), and 8 girls had idiopathic central early puberty (ICEP). The diagnosis of ICPP was made based upon the onset of breast development before the age of 7 yr and 6 mo, the generation of pubic hair before the age of 9 yr, or the onset of menses before the age of 10 yr and 6 mo, according to the diagnostic criteria currently used in Japan. ICEP was defined as the appearance of pubertal signs between 8 -10 yr of age. Furthermore, neither set of patients showed any evidence of hypothalamo-pituitary lesions on magnetic resonance imaging or

The median age at the start of treatment was 8.3 yr (range: 6 to 11). All patients received leuprolide acetate (LUPRON DEPOT, Takeda, Osaka, Japan) at an initial dose of 30 g/kg, which was administered subcutaneously every 4 weeks according to the standard

Standard anthropometric measurements were taken at the baseline and during the 2-year GnRHa treatment period. BMI was calculated as weight (kg)/height (m2), compared with age- and sex-matched reference values, and expressed as a standard deviation score (SDS) according to the method of Inokuchi (Inokuchi, 2009). The percentage of overweight (POW) was calculated as 100 x (the measured weight – normal weight)/normal weight (%). Normal weight data were derived from the 1990 Ministry of Health and Welfare data (Yamazaki et al., 1994). A POW of ≥ 20% was considered to indicate obesity (Asayama et al., 2003). Pubertal development was determined according to the method of Tanner (Tanner &

Pituitary-gonadal axis function was considered to be adequately suppressed during treatment if the concentrations of LH and E2 were maintained within the prepubertal normal ranges of our laboratory; i.e., if a) the basal serum LH level was below 0.5 mIU/ml and b) the basal serum E2 level was below 10 pg/ml. In the patients that demonstrated

on body composition in order to review the optimal dose of GnRHa.

**2. Subjects and methods**

**2.1 Patients**

**2.2 Methods**

Whitehouse, 1976).

composition of Japanese children with CPP before and during GnRHa therapy.

additional conditions that might have affected their body mass index (BMI).

gonadotropin-suppressive therapy currently used in Japan.


*3.7.1 TMS-EEG-*

TMS-evoked surface potentials from any cortical region can be recorded with scalp EEG electrodes and used to estimate the regional excitability of the extra-motor cortex [23]. This increases spatial benefits and also the very high temporal resolution of EEG makes it possible to detect differential effects of brain disturbance on TMSinduced responses. TMS-EEG recording is obtained using specialized amplifiers and electrode caps designed to minimize stimulus artifact. The high sampling frequency and other adjustments in the acquiring of signals permit recording cortical potentials induced with TMS and the spread of such oscillation to the different connected regions from the site of stimulation [35].


**Development and Evaluation of a Reconstitutable Dry Suspension Containing Isoniazid for Flexible Pediatric Dosing**

#### **Oluwatoyin A. Adeleke 1,2,\*, Rose K. Hayeshi 2,3 and Hajierah Davids 4**

Received: 31 December 2019; Accepted: 15 March 2020; Published: 23 March 2020

**Abstract:** Tuberculosis (TB) is a major cause of childhood death. Despite the startling statistics, it is neglected globally as evidenced by treatment and clinical care schemes, mostly extrapolated from studies in adults. The objective of this study was to formulate and evaluate a reconstitutable dry suspension (RDS) containing isoniazid, a first-line anti-tubercular agent used in the treatment and prevention of TB infection in both children and adults. The RDS formulation was prepared by direct dispersion emulsification of an aqueous-lipid particulate interphase coupled with lyophilization and dry milling. The RDS appeared as a cream-white free-flowing powder with a semi-crystalline and microparticulate nature. Isoniazid release was characterized with an initial burst up to 5 min followed by a cumulative release of 67.88% ± 1.88% (pH 1.2), 60.18% ± 3.33% (pH 6.8), and 49.36% ± 2.83% (pH 7.4) over 2 h. An extended release at pH 7.4 and 100% drug liberation was achieved within 300 min. The generated release profile best fitted the zero order kinetics (R2 = 0.976). RDS was re-dispersible and remained stable in the dried and reconstituted states over 4 months and 11 days respectively, under common storage conditions.

**Keywords:** pediatric drug delivery; tuberculosis; reconstitutable dry suspension; isoniazid; polymer-lipid; microparticulate; direct emulsification


*Review* **Nutraceuticals for the Control of Dyslipidaemias in Clinical Practice**

**Peter E. Penson 1,2 and Maciej Banach 3,4,\***

**Abstract:** Dyslipidaemias result in the deposition of cholesterol and lipids in the walls of blood vessels, chronic inflammation and the formation of atherosclerotic plaques, which impede blood flow and (when they rupture) result in acute ischaemic episodes. Whilst recent years have seen enormous success in the reduction of cardiovascular risk using conventional pharmaceuticals, there is increasing interest amongst patients and practitioners in the use of nutraceuticals to combat dyslipidaemias and inflammation in cardiovascular disease. Nutraceutical is a portmanteau term: 'ceutical' indicate pharmaceutical-grade preparations, and 'nutra' indicates that the products contain nutrients from food. Until relatively recently, little high-quality evidence relating to the safety and efficacy of nutraceuticals has been available to prescribers and policymakers. However, as a result of recent randomised-controlled trials, cohort studies and meta-analyses, this situation is changing, and nutraceuticals are now recommended in several mainstream guidelines relating to dyslipidaemias and atherosclerosis. This article will summarise recent clinical-practice guidance relating to the use of nutraceuticals in this context and the evidence which underlies them. Particular attention is given to position papers and recommendations from the International Lipid Expert Panel (ILEP), which has produced several practical and helpful recommendations in this field.

**Keywords:** nutraceuticals; dyslipidaemia; atherosclerosis

#### **1. Introduction**

Cardiovascular diseases (CVD) are responsible for an estimated 17.9 million deaths each year and represent the largest overall cause of mortality worldwide [1,2]. Although CVD are a heterogenous range of pathologies, many share a common atherosclerotic pathophysiology. More than four-fifths of these deaths occur as a result of myocardial infarction and stroke [1,2]. Worryingly, one-third of these deaths occur prematurely in people under 70 years of age [1]. However, the picture is not entirely bleak. It has been estimated that over the thirty years to 2016, life expectancy increased by eight years, and the majority of this increase (at least six years) was attributable to cardiology [3]. However, despite the great success of conventional approaches to therapeutics, an enormous disease burden remains. Moreover, in many highly developed countries we may observe a plateau, or even a slight reduction in life expectancy in recent years, which may only be partly attributable to the coronavirus pandemic [4,5].

Nutraceuticals have a role to play in managing and reducing this disease burden [6–8]. Until recently, high-quality evidence relating to the safety and efficacy of nutraceuticals has not been available to prescribers and policymakers. However, as a result of recent randomised-controlled trials (RCTs), this situation is changing, and nutraceuticals are now

**Citation:** Penson, P.E.; Banach, M. Nutraceuticals for the Control of Dyslipidaemias in Clinical Practice. *Nutrients* **2021**, *13*, 2957. https:// doi.org/10.3390/nu13092957

Academic Editors: Paolo Magni, Andrea Baragetti and Andrea Poli

Received: 4 August 2021 Accepted: 22 August 2021 Published: 25 August 2021

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

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

recommended in several mainstream guidelines relating to atherosclerosis and preventive cardiology. This article will summarise recent clinical-practice guidance relating to the use of nutraceuticals in this context and the evidence which underlies them.

#### **2. Dyslipidaemias, Inflammation, and Atherosclerosis**

The associations between plasma lipoproteins, atherosclerosis and CVD have been topics of intense research since the observations by Gofman [9] and the results of the Framingham research group [10,11] identified associations between low-density lipoprotein cholesterol (LDL-C) and atherosclerotic events. Interventions to reduce LDL-C, including statins [12–15], ezetimibe [16] and inhibitors of proprotein convertase subtilisin-kexin type 9 [17] have demonstrated remarkable reductions in CV risk when used alone or in combination [18]. Increasing evidence is emerging for newer therapies, including inclisiran [19] and bempedoic acid [20], which can be used to further optimise LDL-C reduction and to enable the achievement of clinical goals, which are still achieved only in about 1/3 patients [21].

Furthermore, inflammation, which occurs when dyslipidaemia results in the deposition of LDL-C in the blood vessel wall is increasingly recognised as a therapeutic target [22,23] in the management of atherosclerosis. Additionally, recent research has broadened beyond LDL-C with a renewed interest in the cardiovascular risk conveyed by hypertriglyceridaemia [24–26] and hyper-lipoproteinaemia(a) [27]. A wide range of nutraceuticals have been shown to exert biological actions at targets relevant to these processes, and therefore have the potential to treat dyslipidaemias and ameliorate the severity of their consequences.

#### **3. The Use of Nutraceuticals in Dyslipidaemias and Atherosclerosis**

The term 'nutraceutical' was coined by DeFelice in 1989 as a portmanteau of "NU-TRient" and "pharmACEUTICAL". DeFelice defined nutraceutical as "food, or parts of a food, that provide medical or health benefits, including the prevention and treatment of disease" [1,2] but which are *"produced from foods but sold in pills, powders, (potions) and other medicinal forms not generally associated with food"* [3]. Importantly, nutraceuticals are different from 'functional foods' which are *"similar in appearance to conventional foods* ... *consumed as part of a usual diet"* [3]. Nutraceuticals should also be distinguished from other molecules, which come from nature but are not derived from foods. Synthetic molecules, which closely resemble natural molecules also cannot be considered as nutraceuticals. For example, aspirin (derived from willow bark and used in preventative cardiology for its antiplatelet action [28]) and colchicine (derived from the crocus, and which has seen a remarkable renaissance since it has been demonstrated to be effective at addressing inflammation in atherosclerosis [29]) cannot be considered nutraceuticals. Furthermore, although such definitions are somewhat arbitrary, nutraceuticals are generally micronutrients, thus despite extensive research about the health benefits of modifying the proportion of energy derived from different dietary sources [30,31], such macronutrients cannot be considered as nutraceuticals.

The development of nutraceuticals, therefore, attempts to use biologically active druglike molecules from nature to prevent or treat disease. However, instead of consuming such molecules in the diet (by eating foods known to contain them), nutraceuticals enable control of dose, quality, and composition of formulations, thus enabling the principles of Good Manufacturing Practice (GMP) to be applied, using the same approach as is used in the production of conventional pharmaceutical products.

The International Lipid Expert Panel (ILEP) position paper on lipid-lowering nutraceuticals in clinical practice for the first time provides a comprehensive and detailed overview of a very wide range of nutraceuticals with the potential to elicit favourable effects on plasma lipoproteins. These include inhibitors of intestinal cholesterol absorption (plant sterols and stanols, soluble fibres, chitosan and probiotics); inhibitors of liver cholesterol synthesis (red yeast rice, garlic, pantethine, bergamot and policosanols); compounds that promote LDL-excretion (berberine, green tea extracts, soy and lupin); and a range of other nutraceuticals including ω-3 fatty acids, spirulina and curcumin [32]. Helpfully, the position paper makes clear and detailed recommendations for each agent and classifies the recommendations according to the strength of recommendation and level of evidence supporting it. The experts from the panel that was founded in 2015 [33], aimed in this paper to present only these nutraceuticals that have real beneficial effect on LDL-C based on high quality data; they also discussed in detail their safety (nutrivigilance), and present recommendations that patients might benefit from the most and in what clinical settings [32].

A more recent position paper by the same group takes a very similar approach but focuses instead on the evidence supporting the anti-inflammatory actions of nutraceuticals [34], a topic of great importance during the current pandemic and in a situation where there is a lack of effective drugs to reduce inflammatory markers [29,35]. Taken together, these two documents provide a wealth of useful information relating to the management of plasma lipids and the associated risk of atherosclerotic disease in clinical practice.

It is beyond the scope of the present article to review this evidence again; however, the reader is directed to the ILEP papers (including a supplementary paper on the role of nutraceuticals in heart failure patients [36]). The evidence supporting the clinical use of three well-evaluated nutraceuticals (red-yeast rice, ω-3 fatty acids and phytosterols) is described in the sections below, and brief mechanistic details for these agents are provided in Figure 1.

**Figure 1.** Summary of biological mechanisms involved in the anti-atherosclerotic actions of key evidence-based nutraceuticals. Image Adapted from "Atherosclerosis Progression", by BioRender.com (2021). Retrieved from https: //app.biorender.com/biorender-templates (Accessed date: 23 July 2021).

#### *3.1. Red Yeast Rice*

Red yeast rice (RYR) is a traditional fermented ingredient of Chinese food which contains a bioactive molecule called monacolin K, a naturally occurring inhibitor of 3 hydroxy-3-methyl-glutaryl-coenzyme A reductase, the target of statins. As such, the consumption of red yeast rice and its nutraceutical preparations reduces plasma LDL-C [37]. Remarkably, an RCT that evaluated a nutraceutical extract of red yeast rice in 5000 individuals in China reported a 45% relative reduction in cardiovascular events [38]. If this effect size can be achieved in other populations, it suggests that RYR preparations might have an important contribution to make to CVD risk reduction.

In the past, concerns have been raised about the safety of red-yeast rice preparations. These generally relate to the batch-to-batch variability in monacolin content and the presence of toxic by-products of rice fermentation (citrinin) [39]. These issues can be resolved through the employment of the principles of GMP. Indeed, a recent nutrivigilance study of a high-quality nutraceutical preparation containing red-yeast rice and other lipid-lowering nutraceuticals found that only 0.037% of 2,287,449 consumers reported adverse events [40]. The recent draft recommendations to European Food Safety Authority (EFSA) suggest that RYR preparations should contain no more than 3 mg of monacolin K.

#### *3.2. ω-3 Fatty Acids*

Polyunsaturated ω-3 fatty acids (PUFAs), which are derived from oily fish, have been the subject of intense recent research. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) reduce circulating concentrations of triglycerides [41] and therefore have been evaluated for the potential to reduce the CV risk associated with hypertriglyceridemia. Unusually for nutraceuticals, polyunsaturated ω-3 fatty acids have been evaluated in numerous cardiovascular outcomes trials (CVOTs), however, the results of the trials have not been consistent, resulting in considerable discussion within the scientific and medical community.

The VITAL study [42] and ASCEND trial [43] randomised patients to receive placebo or a preparation of ω-3 fatty acids approved by the American Heart Association (AHA) (460 mg EPA, 380 mg DHA). The trials were similar, although a higher risk population was recruited in ASCEND. Neither trial showed a difference in composite CVOTs over a relatively long period of follow-up (7.4 years in ASCEND, or 5.3 years in VITAL). Recently, STRENGTH, a similar trial (however, with the high dose of mixed omega-3 fatty acids of 4 g/day), was halted based on an interim analysis, which suggested no likely benefit of the intervention a combination of EPA/DHA) [26,44].

Conversely, a high dose (4 g) preparation of pure EPA (prepared as an ethyl ester and known as 'icosapent ethyl') has demonstrated benefit on physiological parameters and cardiovascular outcomes [24,25]. The EVAPORATE trial demonstrated [45] that icosapent ethyl resulted in atherosclerotic plaque regression (measured using coronary computed tomography angiography). However, more importantly, the REDUCE-IT randomised, placebo-controlled trial evaluated icosapent ethyl in 8179 participants and demonstrated a 25% relative reduction in cardiovascular events over five years [24,25]. However, the most recent meta-analysis suggest (similarly to the one presented by authors [46]), that omega-3 FAs reduced CV mortality and improved CVOTs, however, with more prominent cardiovascular risk reduction with EPA monotherapy than with EPA+DHA [47].

There are some safety concerns for omega-3 acids in the available studies. In the REDUCE-IT trial, an increase in the rate of hospitalization for atrial fibrillation and peripheral edema in the icosapent ethyl vs. placebo group were observed [24,25].

#### *3.3. Phytosterols*

Phytosterols and phytostanols have been widely used in nutraceuticals and in functional foods (such as phytosterol-enriched yoghurts and spreads). Unfortunately, there is no randomised-controlled trial data evaluating the effects of these compounds on hard clinical outcomes [48]. As such, the evidence basis for these interventions relies on surrogate outcomes. Nevertheless, such evidence is convincing. A meta-analysis of 113 RCTs demonstrated a dose-dependent effect of plant sterols on LDL (by even 15%), with optimal daily doses above 2 g [49].

Also, in case of phytosterols some safety concerns need to be mentioned. They can lead to hypercholesterolemia and elevated CVD risk in some rare genetic abnormalities of the ABCG5/ABCG8 transporters [50].

#### **4. General Recommendations for Nutraceuticals in International Guidelines**

International guidelines vary in the extent to which they recommend nutraceuticals. The 2019 European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) Guidelines for the management of dyslipidaemia give Class A recommendations to the three classes of nutraceuticals described above (red-yeast rice, ω-3 fatty acids and phytosterols) [48]. The American College of Cardiology/American Heart Association (ACC/AHA) guidelines for the primary prevention of cardiovascular disease [51] and the management of blood cholesterol [52] do not recommend any nutraceutical preparations. In the United Kingdom, the National Institute for Health and Care Excellence (NICE) guidelines do not make any recommendations favouring nutraceuticals [53] and specifically advise against the use of ω-3 fatty acid compounds [53]; however, it should be noted that these guidelines precede the publication of IMPROVE-IT and EVAPORATE, and therefore do not reflect the latest evidence relating to EPA. The guidelines are currently undergoing a process of update and revision.

More specifically, ILEP recommendations, which are focused directly on this issue, have suggested a range of specific situations, in which nutraceuticals may be considered in the management of dyslipidaemias and atherosclerosis. These are discussed briefly below (Section 5, Table 1), and the reader is encouraged to access the relevant ILEP position papers for practical advice on the control of dyslipidaemias in clinical practice.

**Table 1.** Summary of recommendations on the use of nutraceuticals in dyslipidaemias and atherosclerosis.

Abbreviations, CVD, cardiovascular disease; ILEP, International Lipid Expert Panel.

#### **5. Specific Situations in Which Nutraceuticals May Be Considered**

#### *5.1. Statin Intolerance*

Statin intolerance occurs when adverse effects on statin therapy limit the ability of an individual to take statins at guideline-recommended doses [56,57]. Many adverse symptoms reported by patients receiving statin therapy are coincidental and not caused by statins. Therefore, careful diagnostic workup must be undertaken to exclude other causes of symptoms [58,59] (see also Section 5.2 'Nocebo/Drucebo effect', below). However, in the rare cases of complete statin intolerance (usually up to 5% where patients cannot tolerate any statin at any dose) or the more common situation of partial intolerance (where a patient can tolerate a statin, but not at a sufficiently high dose to reach their treatment targets), additional lipid-lowering therapies are necessary. Ideally, such patients may receive PCSK9 inhibitors; however, these are not available or reimbursable in all jurisdictions, and therefore, with other drugs still unavailable and not reimbursed (bempedoic acid and inclisiran), nutraceuticals may be considered in combination with other lipid-lowering drugs. Helpful guidelines, including classification of level and class of evidence, have been published by ILEP relating to the use of nutraceuticals in this context [50].

Nutraceuticals may also be of benefit in patients who are nonadherent to statin therapy (as statin intolerance is the most common reason of statin nonadherence), as well as those who are not willing to use statins, despite indications and the physician's attempts to convince them (statin deniers). Unfortunately, this group (often young people who feel healthy in general) can constitute even 5–7% of patients who should be on statin therapy [54,58,60].

#### *5.2. Nocebo/Drucebo Effect*

In relation to statin intolerance (see Section 5.1 'Statin intolerance' above), it is important to note that most reported muscle pain in statin therapy is attributable to the nocebo/drucebo effect and occurs as a result of the patient's expectation of adverse effects [61]. Whilst the ultimate aim of treatment should be to help patients to use life-saving statins [62], nutraceuticals may be useful in the control of plasma lipids in these patients. Forthcoming ILEP recommendations will address this issue in detail [54].

#### *5.3. Patients Considered 'Low-Risk' by Conventional Risk Scores*

In most jurisdictions, patient eligibility for statin therapy is decided based on their risk of cardiovascular disease, usually calculated over a 10-year period, based on their demographic characteristics and physical and biomarker measurements. Such an approach almost certainly underestimates the life-long risk of cardiovascular disease in younger individuals with dyslipidaemias but without other risk factors. Epidemiological and interventional trials have unambiguously demonstrated that CVD risk is a function of lifelong exposure to LDL-C, and this can be helpfully summarised as 'lower is better for longer'. ILEP recommendations have been produced to enable the optimal management of patients in this situation. Initially, LDL-C should be controlled by lifestyle interventions (including exercise, bodyweight reduction, smoking cessation and adherence to a diet low in saturated fat and high in plant protein and fibre); however, if adherence to this regimen is poor, or LDL-C is not sufficiently reduced, then nutraceuticals (especially in the form of polypills) may be considered [55].

#### *5.4. Optimisation of Therapy in High-Risk Patients*

ILEP has also produced advice about the use of nutraceuticals in patients at high risk of CVD. They summarise the potential uses of nutraceuticals as follows: (1) managing residual risk associated with lipids other than low-density lipoprotein cholesterol (for example, risk mediated by triglycerides may be ameliorated by EPA (see Section 3.2 'ω-3 fatty acids')); (2) managing non-lipid-mediated residual risk (such as inflammatory risk [34]); (3) optimising LDL-C treatment in statin intolerance (see Section 5.1 'Statin intolerance' and Section 5.2 Nocebo/Drucebo effect, above); (4) optimising LDL-C treatment when add-on therapies for statins are not available (e.g., with the limited reimbursement criteria); (5) as adjuncts to lifestyle for individuals at high lifetime risk of atherosclerotic cardiovascular disease (ASCVD) [7].

#### *5.5. Patient-Initiated Nutraceutical Use*

A wide variety of nutraceutical preparations are available for patients to purchase over the counter. Prescribers and physicians should always question patients about nutraceutical use as part of routine history taking. This is important to avoid potential adverse effects relating to nutraceutical use. Whilst most preparations are thought to be generally safe, long-term safety data is often not available (see information above). Harm may also result as a result of batch-to-batch variation in the content of the active ingredient in poor quality preparations or because of contamination of preparations with unwanted ingredients [39]. Both problems can be overcome by adherence to the principles of GMP; however, the relatively light regulatory framework for such products in some jurisdictions means that low-quality preparations may reach the market. When patients do choose to use nutraceuticals, it is important that this occurs as a supplement to evidence-based guideline-directed therapies and not as a replacement for them.

#### **6. Conclusions**

The development of nutraceuticals from the micronutrient components of food presents an opportunity to target dyslipidaemias and atherosclerosis through direct effects on plasma lipids, and through the modification of pathophysiological processes elicited through atherogenic lipoproteins. Until recently, limited evidence has been available

to evaluate the efficacy of nutraceuticals on clinical outcomes and their safety at doses above those usually consumed in the diet. However, recent data from RCTs and nutrivigilance studies has changed this picture. The strongest evidence exists for plant sterols and stanols, eicosapentaenoic acid and red-yeast rice. As such, guidelines and position papers (especially those produced by the ILEP) have recommended roles for nutraceuticals. Whilst many practitioners prescribe or recommend nutraceuticals in their practice, the increasing availability of such preparations and their use by patients makes it imperative that all practitioners treating dyslipidaemias and atherosclerosis are aware of major nutraceuticals, their indications, and the evidence supporting their use. It should be stressed, however, that nutraceuticals should only be used to supplement, not to replace guideline-directed evidence-based therapeutics.

**Author Contributions:** Writing—original draft preparation, P.E.P.; writing—review and editing, M.B. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** P.E.P. owns four shares in AstraZeneca PLC and has received honoraria and/or travel reimbursement for events sponsored by AKCEA, Amgen, AMRYT, Link Medical, Mylan, Napp, and Sanofi, M.B.—speakers bureau: Amgen, Esperion, Herbapol, Kogen, KRKA, Novartis, Polpharma, Sanofi, Servier, Teva, Viatris, and Zentiva; consultant to Akcea, Amgen, Daichii Sankyo, Esperion, Freia Pharmaceuticals, Polfarmex, and Sanofi; grants from Amgen, Viatris, Sanofi, and Valeant.

#### **References**

#### *Article*


**Author details**

### Ali Erdal Gunes

Address all correspondence to<EMAIL_ADDRESS>
Department of Underwater and Hyperbaric Clinical Medicine, Faculty of Medicine, Harran University, Sanliurfa, Turkey


**Interaction of Photosynthetic Source-Sink Balance and Activities of Membrane H+ Pumps in Soybean**

Minobu Kasai and Wataru Takahashi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50544


*3.3. Gene Expression Analysis*

The expression profiles of the selected genes did not follow the same trend among the cultivars. In most genotypes, the mRNA levels of TYRD were quite low and remained almost constant as the fruit development proceeded (Figure 3A). An exception was represented by cultivars Sivigliana and Gentile dell'Aquila, in which this gene was highly expressed at the first drupe sampling (T1) and then drastically dropped at the second sampling, albeit with no significance. On the contrary, in cultivars Semidana and Tortiglione, TYRD mRNA levels remarkably increased from the second to the third sampling, thus reaching the highest values when drupes were almost dark and mature.

In some cultivars, the CuAO expression pattern did not notably change during fruit maturation, while in other genotypes, such as Semidana and Bambina, a slight increase was observed up to the third sampling time. Interestingly, the CuAO expression level was noteworthy in one of the Apulian cultivars, i.e., Cima di Melfi, not for its general trend but specifically for its abundance during the entire drupe maturation process, since it was extremely and significantly different (higher) in comparison to that detected in all other cultivars (Figure 3B).

In the majority of genotypes, ALDH was almost exclusively expressed during the T3 stage of fruit development, with a remarkable increase starting at the turning phase of drupes, in particular in the cases of Gentile dell'Aquila and Cima di Melfi, and with a less pronounced increment observed in the cases of Bambina, Semidana and Sivigliana. On the contrary, ALDH expression levels in the remaining cultivars were very low (Figure 3C).

**Figure 3.** Expression profiles of the analyzed genes involved in the first step of the phenol biosynthetic pathway during fruit development (T1 = yellow-green drupes, T2 = turning drupes, T3 = almost dark drupes). The mRNA levels were determined by qRT-PCR and relatively expressed as ΔΔCt. (**A**) TYRD = tyrosine/dopa decarboxylase; (**B**) CuAO = copper amine oxidase; (**C**) ALDH = alcohol dehydrogenase. Data are means (*n* = 3) ± standard deviation. Different letters indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (*p* = 0.05).

#### **4. Discussion**

The identification of minor olive cultivars with favorable agronomic and nutraceutical features among the available and still uncharacterized Italian germplasm, and their fine characterization with different approaches, could effectively contribute to both the valorization and the spread of these cultivars worldwide. The possibility of their introduction in the olive-growing sector is reinforced by the availability of a molecular fingerprint, that can provide a crucial tool for the identification of these genotypes and the traceability of their oils. The use of microsatellite markers in this study has allowed not only a fine characterization of the considered genotypes, but also clarification of the phylogenetic relationships among them and assessment of an identification key. The Unweighted Neighbor Joining dendrogram mostly separated the genotypes according to their geographic origin as reported in many other studies about the evaluation of the genetic diversity of the Italian olive germplasm [5,64]. Surprisingly, only one of the analyzed genotypes has escaped this kind of clustering; indeed, the Gentile dell'Aquila cultivar, belonging to the autochthonous cultivars of Abruzzo region, grouped with all the Sardinian genotypes instead of with the other cultivars from Central Italy (Figure 1). The explanation of this result can be traced back to the complex migration history of the olive. In 2015, Dìez and colleagues [65] demonstrated the existence of three different gene pools (Q1, Q2 and Q3) in the Mediterranean area and the presence of a broad 'mosaic' group as a mixture of Q1 and Q2. The Italian cultivars considered in that research mainly belonged to the Q2 group. Anyway, the authors postulated that the wide occurrence of the 'mosaic' cultivars might be indicative of admixture events. Thereby, both the outcrossing nature of *O. europaea* ssp. *europaea* as well as the exchanges related to human migration over the centuries [64,66] could explain a certain level of genetic admixture between Gentile dell'Aquila and the Sardinian cultivars or their origin from a common wild ancestor.

Over the last decades, virgin olive oil (VOO) became the symbol of good nutritional habits. This role has been mostly linked to some features such as the high content of MUFA and of phenolic compounds, which allow us to recognize VOO as a functional food [67]. As proof of that, the European Food Safety Authority (EFSA) has permitted the declaration "Olive oil polyphenols contribute to the protection of blood lipids from oxidative stress" when olive oil contains a minimum of "5 mg hydroxy-tyrosol and its derivatives per 20 g of olive oil" [68,69]. However, VOO phenolic content not only plays an important nutritional role but also has a great impact on the organoleptic features and on the stability of the product [60]. Given the wide spectrum of features directly linked to olive phenolics, it is clear how important the understanding of their synthesis and regulation is.

Polyphenol accumulation has been reported to be a complex process considerably varying among genotypes, tissues, developmental stages and in response to different agronomic and environmental conditions [70–72]. In accordance with other studies, there was a component of the variability of the phenolic composition observed among the analyzed monovarietal virgin oils that mostly depended on the genotype. On average, the Apulian cultivars generally showed the highest values of the detected compounds at the complete maturation stage of drupes, followed by the Sardinian cultivars. This could also be related to the different cultivation areas and environmental temperatures of the considered groves, since the climates of Apulia and Sardinia are generally similar to each other and usually milder than that of Central Italy (Abruzzo). As reported by [73], the pathways related to olive phenol compounds could be differentially modulated on the basis of altitude and temperatures, phenols biosynthesis being prolonged in temperate areas.

Quantitative differences in phenolic compounds were highlighted both among the analyzed cultivars and among fruits of the same cultivar at different ripening stages. With regard to total phenols (TPC), a sharp decrease is generally and mainly observed between June and September, as result of the oleuropein hydrolysis during drupe maturation (high β-glucosidase activity) [74,75]. Indeed, the very early stages of drupe formation are dominated by secoiridoids (above all oleuropein), while in the last ripening stages, simple phenols and flavonoids become the major components. In our study, the drupe collecting period between October and December did not allow us to highlight such remarkable differences in total phenol content, this period being characterized by a higher stability of phenol metabolism (Table 3; Figure 2D). Thereby, with the only exception of cultivars Tortiglione and Oliva Rossa, that showed a sharp decrease from T2 to T3 stage, the variation of TPC was minimal in all cultivars during the fruit maturation period considered in this study. An intermediate behavior was registered in oils obtained by Sardinian cultivars, that showed a slight, but significant, decrease only at the overripe (T4) stage.

On the contrary, the content of single compounds was very variable among cultivars and sampling times. Secoiridoid derivatives, i.e., aglycon forms of the secoiridoid glucosides usually formed during oil extraction by the enzymatic hydrolysis of oleuropein, demethyloleuropein and ligstroside [76], were reported to be the most abundant phenols in monovarietal olive oils [72]. Among those, oleacein (3,4-DHPEA-EDA) is an important compound, structurally similar to oleocanthal and with comparable pharmacological properties [77]. In our study, the oleacein levels considerably differed among all genotypes, progressively decreasing as fruit maturation proceeded (Table 3; Figure 2C). Indeed, the higher values were found at the T2 time-point, although with remarkable differences among cultivars (in the range between 2 and 63 mg/kg). As a result of an opposite behavior to this general trend, oils obtained by fully ripe drupes (T4) of the Apulian cultivar Cima di Melfi and of the Abruzzo cultivar Gentile dell'Aquila were those with the highest oleacein concentration. As already mentioned, variation in the VOOs' phenolic composition can be ascribable to several factors, including the technological steps of olive oil extraction, which is far from being merely a mechanical process. Indeed, starting from drupe crushing, several enzymes are set free whose activities are strongly influenced by the genotype, the maturity degree and the environmental conditions (time, temperature, atmosphere) [78,79]. Moreover, secoiridoid derivatives, such as 3,4-DHPEA-EDA, are among the most affected olive phenols during the extraction process. For example, in Tuscan olive oils, oleacein was found only a long time after milling, likely due to the activity of different esterases [80]. Thus, linearity between drupe genetic expression and VOO phenols could be difficult to see and the identification of clear markers of maturity is a complex task [81].

The content of minor phenols such as tyrosol and hydroxy-tyrosol was also evaluated in the considered genotypes, due to their relevance as bioactive molecules with beneficial properties on human health [16,82]. Hydroxy-tyrosol is a simple alcohol conjugated to form oleuropein derivatives, usually highly expressed in young olive fruits [42,43]. In accordance with other reports, the hydroxy-tyrosol amount decreased from semi-green to nearly ripe drupes (with the exception of Corsicana, Oliva Rossa and Gentile dell'Aquila), but then an interesting increase was again observed in the final developmental stage, i.e., in totally black fruits (Table 3; Figure 2B). This result is generally attributed to oleuropein catabolism rather than to new hydroxy-tyrosol biosynthesis, as a consequence of the recycling of all the valuable molecules of complex compounds and their conversion to simple phenols [42,44,83]. Moreover, the oleuropein catabolism in the mature fruit is necessary to reduce the bitter taste typically associated with this compound.

Changes in tyrosol content with ripening did not follow a clear pattern among the studied varieties. In some cases (such as Semidana and Cima di Melfi) an opposite trend with the oleacein content was found. These variations could be supposed to be linked to the genetic expression of the enzymes in the biosynthetic pathway (Figure 3). Indeed, tyrosol could be initially synthesized—and mostly found—in free form, while subsequently it is used as a precursor for oleacein production (Table 3; Figure 2A). However, as already reported [81], finding a straightforward relationship between gene expression and phenotype is very difficult, as the interaction with agronomic and pedoclimatic conditions

should be also taken into account. Moreover, the content of phenolic compounds depends also on the activity of other enzymes such as the olive *β*-glucosidases (not considered in this study) which strongly affect their release in the oily phase. Different patterns of tyrosol accumulation with respect to the cultivar and the maturity degree have been already observed by other authors [84].

When this study started, the exact polyphenol biosynthetic pathway was still not clarified in detail; therefore, among genes annotated in phenol biosynthesis, for expression analysis our choice fell on three genes (TYRD, CuAO and ALDH) on the basis of the involvement of the corresponding encoded enzymes in the first steps of this biosynthetic pathway and in relation to the compounds detected in VOOs under characterization. Tyrosine is referred to as the precursor of the secoiridoid class [85]; in particular, the biosynthesis of tyrosol and hydroxy-tyrosol has been proposed to proceed through the formation of tyramine or, otherwise, it can follow the path including L-DOPA and dopamine as intermediates (Figure 4). Initially, an enzyme similar to a tyrosine/DOPA decarboxylase (TYRD) was recognized to be responsible for the conversion of both tyrosine in tyramine and L-DOPA in dopamine [40,86] (Figure 4A). Recently, these two conversions have been clarified to take place by the action of two different enzymes, i.e., a tyrosine decarboxylase (TDC) in the first way and a DOPA decarboxylase (DCC) in the parallel path (Figure 4B) [44]. Moreover, in the previous proposed version, the subsequent steps leading to the conversion of tyramine to tyrosol and dopamine to hydroxy-tyrosol required two enzymes called CuAO and ALDH [87]. Now, the presence of other intermediates in both paths and the action of an additional enzyme, that is, a phenylacetaldehyde reductase (PAR), have been recognized (Figure 4B) [44]. The reaction of the conversion of tyrosol to hydroxy-tyrosol and vice versa seems to involve a polyphenol oxidase (PPO) [42]. Since most of the genes responsible for secoiridoid formation have been identified in a restricted number of species, some steps of the biosynthesis of oleuropein are still unclear, even though the pathway from geranyl diphosphate to secologanin has been elucidated [42,44].

The relative expression of the selected genes was monitored during three stages of fruit maturation, at T1, T2 and T3 sampling times. The expression analysis showed the general tendency of these mRNA levels to decrease or remain more or less stable in semi-green fruits and then to increase, slightly or significantly in a few cases, in semi-dark drupes (Figure 3). These results are in line with the biochemical characterization of monovarietal oils at T2 and T3 stages, which proved that at these time-points the tyrosol and hydroxytyrosol contents were progressively diminishing, following the same pattern as the genetic expression data. Some exceptions to this trend have been surprisingly observed for few varieties and in particular for TYRD and ALDH genes. Indeed, these two genes were highly expressed, respectively, in the Tortiglione and Semidana genotypes, and in Cima di Melfi and Gentile dell'Aquila, with a remarkable increase in their transcript levels from T2 to T3 time-points. These unexpected but interesting results, not yet reported in other olive cultivars, to our knowledge, certainly require further investigation. In order to clarify the role of TYRD and ALDH genes in these cultivars, their expression profile should be investigated at more developmental stages of drupes during more consecutive years. Protein expression experiments as well as enzymatic activity assays could also help us to understand the involvement of these genes in polyphenol metabolism of these minor cultivars. At the moment, we could hypothesize that these genotypes are effectively characterized by a high level of gene expression even towards the end of drupe maturation, contrarily to that reported in this study and, more in general, in the literature. It is also plausible that the corresponding enzymes are conveyed to other metabolisms or that some post-translational control takes place and makes these enzymes unfunctional. Exceptions apart, the general agreement of qRT-PCR results with the biochemical outcomes confirmed that hydroxy-tyrosol biosynthesis is under a transcriptional control, as also reported by other authors [40,42–44].

**Figure 4.** Simplified representation of the biosynthetic pathway of some important olive polyphenols. The previously identified enzymes and reactions (**A**) as well as the recently identified ones, with additional steps in the biosynthesis (**B**), are reported. Abbreviations: 3,4-DHPEA-EDA: elenolic acid linked to 3,4-dihydroxyphenyl ethanol (3,4-DHPEA); 4-HPA: 4-hydroxyphenylacetic acid; 3,4-DHPA: 3,4-dihydroxyphthalic acid; TYRD: tyrosine/DOPA decarboxylase; PPO: polyphenol oxidase; CuAO: copper amine oxidase; ALDH: alcohol dehydrogenase; DCC: DOPA decarboxylase; PAR: phenylacetaldehyde reductase. Adapted from [40,44].

#### **5. Conclusions**

Unveiling the genetic potentialities of minor cultivars/genotypes could open the doors to their valorization. Besides the safeguarding of local varieties, the information provided in this work could be useful for breeding programs with selection of genotypes that, while being resilient and better adapted to specific environmental conditions, could also exploit their potentiality in terms of accumulation of bioactive compounds with important consequences on the nutritional, sensory and stability properties of VOO. With a view to an eco-sustainable and low-input agriculture, the introduction of minor cultivars may offer interesting features that, together with the extraction technology, could address the needs of a worldwide market with growing interest towards VOOs. Thus, the results and information obtained in this study are encouraging for the valorization of the still poorly explored Italian germplasm, with important both economic and scientific consequences.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/foods10061371/s1, Figure S1: Example of SSR electropherograms, Table S1: Microsatellite markers used in this study, Table S2: List of primers utilized for qRT-PCR analysis.

**Author Contributions:** Conceptualization, W.S., C.M. and F.C.; investigation, S.G., I.M., G.S., F.F., P.C. and A.P.; writing—original draft preparation, W.S.; writing—review and editing, W.S., G.S., F.F., C.D.D.M., P.C., A.P., C.M. and F.C.; supervision, W.S., C.M. and F.C.; funding acquisition, F.C., W.S. and C.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was mainly funded by the Italian AGER 2 Project, grant no. 2016-0105, and it was in part supported by the CNR project NUTR-AGE (FOE-2019, DSB.AD004.271) and the Apulia Region Project "Approcci di Next Generation Sequencing per l'analisi di variabilità e di espressione genica in genotipi di olivo autoctoni pugliesi" L.R. 28 December 2018 n. 67, art. 37.

**Conflicts of Interest:** The authors declare that they have no competing financial or personal interests that could influence the work reported in this paper.

#### **References**


**Author details**

Salem Alghamdi1 , Hussein Migdadi1 \*, Muhammad Khan1 , Ehab H. El-Harty1 , Megahed Ammar2 , Muhammad Farooq1,3,4 and Muhammad Afzal1

\*Address all correspondence to<EMAIL_ADDRESS>
1 Legume Research Group, Plant Production Department, Faculty of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia

2 Rice Research and Training Center, Kafr El Sheikh, Egypt

3 Department of Agronomy, University of Agriculture, Faisalabad, Pakistan

4 Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khoud, Oman