**2. Review on international bodies recommendation**

The International Chamber of Commerce (ICC) [12] after reminding that every exceptional time increase the risk for illicit behavior affecting business such as

corruption, money-laundering and fraud, called for the drivers that businesses and policy makers should follow not to be overcome by the pandemic: transparency and integrity in all procurement stages which are helped by the digitalization of permits and licensing; keeping the due diligence process ongoing to mitigate supply chain's risks; fighting corruption in the context of borders and customs; and, protecting the rule of law.

In such a contest, where states closed upon themselves and made trade extremely difficult, in the last 2 years several international organizations presented different suggestions to mitigate the Covid-19 trade-related effects.

The United Nations Industrial Development Organization estimated in July 2020 that, among the 49 countries which represent almost 87% of world manufacturing value-added, about 81% of countries faced a decrease of an average of 6% in industrial production comparing the dataset in December 2019 and March 2020, and an average of 20% in 93% of nations comparing December 2019 and April 2020 [13]. The situation has not changed afterward, with several and severe economic consequences hitting countries until June 2020 [14] where the index of industrial production kept on decreasing, although with a small improvement: from −5.6% in March 2020 to −2.5% in June 2020.

The export of goods has had a particular trend which is indicated in **Table 1** from the 4th quarterly 2019 to the 3rd quarterly 2021 (Q4-2019–Q3-2021), the tendency is expressed in billions of US dollars, for each member of the Group 20 (G20). **Figure 2**, furthermore, highlights that the export of goods of these countries has marginally been impacted in 2020 Q1-2020, and then has had a strong downshift in Q2-2020. Starting from Q3-2020, G20 countries have seen a positive trend of it and in Q4-2020 they exported more goods than in Q4-2019, the quarterly previous the start of the pandemic. In the following quarterlies (Q1-2021 and Q2-2021), this trend has continuously increased reaching, in Q3-2021, the amount of 4262.96 billion US dollars.

Given the above, in 2020, the United Nations Conference on Trade and Development (UNCTAD) drafted a report suggesting some measures to efficiently manage the pandemic consequences [16]. While the world would have needed higher agility and efficiency, trade experienced extensive disruptions, with medical devices and personal protective equipment firstly affected by the export restriction. Therefore, four main interventions categories were identified:

#### iv.Technology.

With regards to "process optimization", UNCTAD recommended following the criteria specified by art. 7.8 of the WTO Trade Facilitation Agreement introducing, among others, fast tracks lanes, prioritization, implementing the trusted traders such as the AEO and simplifying the declarations. About "cost reduction", the UNCTAD remarked the importance of reducing some tariff, accepting some goods received as gifts by approved organizations free of charges, while on point (iii)—"transparency and cooperation"—it highlighted the necessity of having prompt and transparent information among all countries not only about the pandemic itself but also on the

*Application of Internet of Things in the Movement of Goods at Customs Level during Covid-19… DOI: http://dx.doi.org/10.5772/intechopen.102488*

#### **Table 1.**

*Quarterly (Q4-2019–Q3-2021) of G20 export of goods (value—US dollar, billions).*

transported goods. Lastly, about "technology" it remarks that efficient, rapid, and centralized processing should be further implemented.

Linked to this remark, in 2021 WCO together with WTO, drafted a paper [17] whose objective was to provide an evidence-based picture on three different

#### **Figure 2.**

*Export of goods of G20 members (Q4-2019–Q3-2021)—US dollars, billions. Source: authors' own elaboration on Organization for Economic Co-Operation and Development [15].*

technologies that Customs could utilize as trade facilitators to achieve safety and velocity, fundamental main stones in this period. The suggested tools are blockchain; IoT and Big Data. Specifically, WCO found that blockchain is still enduring an experimental phase, mainly involving private and permissioned blockchains. Many customs faced a certain hesitancy towards this technology, but the WCO paper highlight that there is a common "*reasonably optimistic concern*" towards blockchain's potential as it could lead to greater efficiency and reliability in risk management, revenue collection, and trade facilitation as well as creating an environment where public authorities can cooperate with private industry from the early stage of production and trade. Further recognized benefits were identified in transparency, immutability and accessibility of information, lower transaction and verifications costs, and increased availability of information and quality data from different sources. About IoT, WCO found that these tools are used by more than 50% of the respondent, mostly in the area of X-ray and computed tomography, QR Code and barcode readers, electronic seals and automated license plate readers, and radio frequency identification (RFID) antennas or e-seals to ensure traceability of mean of transport and goods. It has also been highlighted the importance of implementing IoT solutions, as they enable them to have better risk management, greater customs clearance process efficiency and improved analytics. Regarding Big Data, data analytics, artificial intelligence and machine learning even if it is quite commonly utilized, there still are multiple obstacles in common usage. Particularly, benefits linked to these technologies have been identified in risk management, fraud detection, and audits' facilitations, while the barriers were identified in associated costs, and lack of expertise and good practices.

Again in 2021, the United Nations Economic Commission for Europe (UNECE)— United Nation Center for Trade Facilitation and Electronic Business (UN/CEFACT) published a white paper [18] to provide some best practices ensuring efficient trade control together with facilitation measures to be implemented during the pandemic to avoid a total interruption of commercial trade. The measures to mitigate the

#### *Application of Internet of Things in the Movement of Goods at Customs Level during Covid-19… DOI: http://dx.doi.org/10.5772/intechopen.102488*

impact on trade should involve several stakeholders, including the public sector (i.e., customs, and border authorities), public-private partnership (i.e., National Trade Facilitation Bodies), and private sector stakeholders (i.e., logistic, transport, production, and manufacturing sector) and shall be aimed to cross border cooperation with an increase of trust between partners. This could be achieved with special regimes, special economic zones, expedite pre-arrival clearance procedures, reliable exchange of information, and coordination between integrated risk management and sharing and combination of data into governmental databases accessible by other interested agencies. Similarly, further inputs should be given to National Trade Facilitation Body and external coordination within multilateral, regional and bilateral agreements as well as to trusted trader schemes. The last category includes, as known, the AEO together with the trusted trader. Given the strict requirements provided by the regulations governing the two schemes, governments can trust certified companies which shared with their border authority's information on how they are structured, and on the management procedures and internal structures (i.e., compliance functions, supply chain management, and codes of conduct). Another proposed solution is to efficiently involve private sector actors who mainly run logistical supply chains who can control their workforce and share the records collected with the border authorities or by using packaging materials that are less likely to keep the coronavirus alive for long hours. Finally, a strong recommendation is given on the usage of technologies. The UNECE reminds that to reduce contagions it is important to limit human contact which can be done by introducing system-to-system electronic data interchange (EDI) such as the National Single Window1 , the port community systems and single submission portals. All these automated systems may have several usages in commercial, logistics, and transport exchanges as well as in the regulatory field.

## **3. Discussions and implications**

Covid-19 pandemic has presented a never seen before (at least in the last century) threat to international trade, leading to an important economic downturn. From the 1,179 billion dollars value of word exports of commercial services in 1995 to the 4,872 billion dollars value in 2014 [20] to 5,898 billion dollars in 2019 [21], the trend has almost always been upwards.

In 2020 Q2 [22], commercial services plunged by 30% and merchandise trade volume by 14.3% [23] on a year-on-year review and while they are now rising back up (respectively, +26% and +1.6% on a year-on-year comparison [24, 25] it will take time for them to be back at the pre-crisis level.

To reach a faster recovery, technological tools are definitely something States and governments should invest on.

The first one to invest on is blockchain, which has been recognized as an instrument that facilitates trade by helping risk management [17]. Nevertheless, it has only been fully deployed by 2% of WCO Members, while more than 40% do not even yet start planning its introduction [17].

1 The National Single Window is defined as "*a facility providing trade facilitation that allows parties involved in trade and transport to lodge standardized information and documents with a single entry point to fulfill all import, export, and transit-related regulatory requirements. Individual data elements should only be submitted once electronically*" [19].

Introducing a standardized dataset between economic operators and government agencies as well as developing common, interconnected, and interoperability blockchain solutions, shared legal requirements and definition of blockchain technology should help a wider usage of this particular tool.

Nevertheless, the actual usage of blockchain covers different areas in the countries where it has been fully or partially used: it may be found in e-commerce environments, in supply chain interconnectivity as well as in electronic certifications and licensing. Particularly, the EU started both a research program to investigate the usage of blockchain in protecting geographical indications, and certification of product origin and in helping VAT collection [17, 26], and the TRICK project in the context of the textile industry funded by Horizon 2020 [17, 27]. Indonesia is currently experimenting with blockchain to automate documentation, simplify the exchange of goods and increase communication and cooperation between counterparts [17].

Other countries are seeking to link it with AEO and container movements: it has been hypothesized that using blockchain in these contexts may faster registration systems and entrust the system and the relationship between counterparts. Among these, it can be named the Malesia experience [17] trying to make the system more competitive and the example of the CADENA project which is a blockchain solution that enables efficient, automated and secure information sharing on AEOs among the Border and Customs authorities of Costa Rica, Peru and Mexico in the context of further implement the mutual recognition agreements [28].

These experiences shall be of guidance and the positive effects that they proved out shall be replicated in other geographical areas.

A second instrument is IoT, which is wildly used to share information with different stakeholders, leading to better risk management, data analysis and efficiency in the Customs clearance process [17]. For example, the usage of QR Codes for transit, the usage of e-lock, GPS tracking, X-ray scanners, and CCTVs cameras interconnected with national or regional Customs are services that help an automatization of border crossing procedures. For example, the Italian Customs and Monopolies Agency is conducting IoT projects based on IoT\_ID code to completely digitalize the procedures for goods transported by road and rail transiting in Italian ports [29] while Hong Kong has implemented the GPS technology and e-lock connecting their data with the one of China Mainland [17].

Other instruments that stakeholders can implement are linked to big data, data analytics, artificial intelligence ad machine learning technologies. The more common advantages are seen in data quality, predicting future trends and facilitating customs audits [17]. These have been utilized, among other uses, to ameliorate the efficiency of financial and tax data, to respond to the most common questions through chatbots, to find anomalies in high revenue areas, to find illicit traffic and goods, to enhance Customs clearance efficiency.

Almost half of the WCO members already use them [17] and only less than a quarter have not started planning to introduce them.

To implement such technologies would require States to invest money in new technologies, to overcome the lack of knowledge and expertise, to find a common and shared legal background, and to strengthen trust between counterparts, finally leaving aside cautiousness and lack of trust towards technologies. Nevertheless, governments, as highlighted in the literature [30], face other challenges such as handling the complexity arising from a massive data extraction and converting it into a meaningful model parameter.

*Application of Internet of Things in the Movement of Goods at Customs Level during Covid-19… DOI: http://dx.doi.org/10.5772/intechopen.102488*


*Review Lycium barbarum* **Berries (Solanaceae) as Source of Bioactive Compounds for Healthy Purposes: A Review**

**Filipa Teixeira , Ana Margarida Silva , Cristina Delerue-Matos and Francisca Rodrigues \***

REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida, 4249-015 Porto, Portugal **\*** Correspondence<EMAIL_ADDRESS>or<EMAIL_ADDRESS>
Tel.: +351-22-83-40-500; Fax: +351-22-83-21-159

**Abstract:** *Lycium barbarum* L. is a species widely used in dietary supplements and natural healthcare products. The berries, also known as goji or wolfberries, mostly grow in China, but recent reports on their outstanding bioactive properties have increased their popularity and cultivation around the world. Goji berries are a remarkable source of phenolic compounds (such as phenolic acids and flavonoids), carotenoids, organic acids, carbohydrates (fructose and glucose), and vitamins (ascorbic acid). Several biological activities, such as antioxidant, antimicrobial, anti-inflammatory, prebiotic, and anticancer activities, have been associated with its consumption. Hence, goji berries were highlighted as an excellent source of functional ingredients with promising applications in food and nutraceutical fields. This review aims to summarize the phytochemical composition and biological activities, along with various industrial applications, of *L. barbarum* berries. Simultaneously, the valorization of goji berries by-products, with its associated economic advantages, will be emphasized and explored.

**Keywords:** goji berries; pro-healthy effects; phenolics; biological properties; functional ingredients

### **1. Introduction**

Plants have been used for thousands of years as a source of compounds for traditional medicine, aiming to prevent and treat health problems [1]. Due to a broad number of studies that describe the influence of natural products on the human endogenous defense system [2,3] as well as "curative" effects against a wide spectrum of disorders, including cardiovascular and neurodegenerative diseases, obesity, and certain types of cancer [4], multiple natural products have been used for healthcare proposes. Nowadays, the interest in exotic berry-type fruits has expanded worldwide [5–8]. Society has become more concerned with eating habits, mostly due to the reinforcement of a positive relationship between good eating habits and the prevention of disease development, particularly diabetes and cardiovascular and neurological pathologies [9]. Simultaneously, the emergent awareness of the planet and the impact that various types of industries have on it has boosted the population's concerns and demands for greener formulations with bioactive ingredients recovered from natural sources [5,10]. Therefore, the consumption of natural matrices has increased in recent years, not only as supplements for imbalanced diets but also as an integral part of a normal healthy diet [7,11].

Recently, several reports highlighted the impressive bioactive capacities of goji berries [2,4,10,12–16], not only in cell assays [17–19] but also in animal studies [3,14,20] and human trials [21]. Despite the growing numbers of published papers regarding the bioactive composition of goji berries, few of them give particular emphasis to in vivo studies as well as to the by-products generated during berry production. Therefore, the aim of this work is to provide a comprehensive review of the bioactive compounds of goji berries, along with their biological activity, giving a particular focus to in vivo studies and clinical trials. The various industrial applications of *L. barbarum* berries will also be highlighted, as well as the valorization of goji berries by-products.

**Citation:** Teixeira, F.; Silva, A.M.; Delerue-Matos, C.; Rodrigues, F. *Lycium barbarum* Berries (Solanaceae) as Source of Bioactive Compounds for Healthy Purposes: A Review. *Int. J. Mol. Sci.* **2023**, *24*, 4777. https:// doi.org/10.3390/ijms24054777

Academic Editors: Antonio Carrillo-Vico and Ivan Cruz-Chamorro

Received: 26 January 2023 Revised: 26 February 2023 Accepted: 28 February 2023 Published: 1 March 2023

**Copyright:** © 2023 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/).

#### **2.** *Lycium barbarum* **L. 2.** *Lycium barbarum* **L.** *Lycium barbarum* L. is one of the most common species of the Solanaceae family

*Lycium barbarum* L. is one of the most common species of the Solanaceae family [13,22]. The berries traditionally grow in China, Tibet, and other parts of Asia [6,13,23]. China is the primary worldwide supplier [24,25], with about 25,000–30,000 tons of dried fruit annually produced in Ningxia, Xinjiang, Gansu, Qinghai, and Mongolia [26]. Asia is the region with the highest production (71.2%), followed by Africa (15.8%), America (8.3%), Oceania (3.4%) and Europe (1.3%) [27]. Due to the rising reports of the positive correlation between the consumption of natural matrices and health improvement [9], the production of fruit has been increasing in the last 20 years all over the world. This is the case of goji berries, whose production has been increasing in the last decades [28], particularly in Europe (Italy, Romania, Bulgaria, Portugal, Greece, Serbia), Northern America, and Australia. Currently, Romania has the largest cultivated area of *L. barbarum* in the European Union [4,10] [13,22]. The berries traditionally grow in China, Tibet, and other parts of Asia [6,13,23]. China is the primary worldwide supplier [24,25], with about 25,000–30,000 tons of dried fruit annually produced in Ningxia, Xinjiang, Gansu, Qinghai, and Mongolia [26]. Asia isthe region with the highest production (71.2%), followed by Africa (15.8%), America (8.3%), Oceania (3.4%) and Europe (1.3%) [27]. Due to the rising reports of the positive correlation between the consumption of natural matrices and health improvement [9], the production of fruit has been increasing in the last 20 years all over the world. This is the case of goji berries, whose production has been increasing in the last decades [28], partic‐ ularly in Europe (Italy, Romania, Bulgaria, Portugal, Greece, Serbia), Northern America, and Australia. Currently, Romania has the largest cultivated area of *L. barbarum* in the European Union [4,10]

along with their biological activity, giving a particular focus to in vivo studies and clinical trials. The various industrial applications of *L. barbarum* berries will also be highlighted,

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 2 of 19

as well as the valorization of goji berries by‐products.

The goji berries can be divided into different classes according to their ripening stage, dimension, weight, color, firmness, solid soluble content, pH, and titratable acidity [22]. The mature fruit (Figure 1) is between 1 and 2 cm long, presenting an ellipsoid shape and a bright orange-red color, similar to a mature mini-tomato, and contains between 20 and 40 tiny seeds per fruit [6,13,22]. The goji berries can be divided into different classes according to their ripening stage, dimension, weight, color, firmness, solid soluble content, pH, and titratable acidity [22]. The mature fruit (Figure 1) is between 1 and 2 cm long, presenting an ellipsoid shape and a bright orange‐red color, similar to a mature mini‐tomato, and contains between 20 and 40 tiny seeds per fruit [6,13,22].

**Figure 1.** *L. barbarum* L. berries [29]. **Figure 1.** *L. barbarum* L. berries [29].

The berries have a sweet taste [16] and are widely used as a dietary supplement and natural health product [4,23,25]. Although mostly consumed fresh in the regions of culti‐ vation [9], around the world, goji berries are essentially consumed dried [9,12] or trans‐ formed into alimentary products, such as juices, herbal teas, yogurt products, granola, powders, and tablets, among others [9,12,30]. The most commonly sold goji berry‐based products are beverages, wine, juice, tea, and concentrates [4,14,23]. A popular goji berry juice brand, "GoChi", has shown increasing effectiveness as an antioxidant, giving rise to subjective feelings of general well‐being as well as improvements in neurologic/psycho‐ logic performance and human gastrointestinal functions [23], which has led to huge pop‐ The berries have a sweet taste [16] and are widely used as a dietary supplement and natural health product [4,23,25]. Although mostly consumed fresh in the regions of cultivation [9], around the world, goji berries are essentially consumed dried [9,12] or transformed into alimentary products, such as juices, herbal teas, yogurt products, granola, powders, and tablets, among others [9,12,30]. The most commonly sold goji berry-based products are beverages, wine, juice, tea, and concentrates [4,14,23]. A popular goji berry juice brand, "GoChi", has shown increasing effectiveness as an antioxidant, giving rise to subjective feelings of general well-beingas well as improvements in neurologic/psychologic performance and human gastrointestinal functions [23], which has led to huge popularity among consumers.

ularity among consumers. For thousands of years, goji berries have been used as herbal medicine in Asian coun‐ tries [24]. Based on their rich nutritional value and medical properties, such as their anti‐ oxidant, antimicrobial, immunomodulatory, and anti‐inflammatory effects [15,23,31], the fruit has been employed as an anti‐aging treatment, tranquilizer, and thirst‐quenching treatment [2,13]. As a folk medicine, *L. barbarum* fruits have been employed by the local population for blood nourishing, in the treatment of early onset diabetes, tuberculosis, For thousands of years, goji berries have been used as herbal medicine in Asian countries [24]. Based on their rich nutritional value and medical properties, such as their antioxidant, antimicrobial, immunomodulatory, and anti-inflammatory effects [15,23,31], the fruit has been employed as an anti-aging treatment, tranquilizer, and thirst-quenching treatment [2,13]. As a folk medicine, *L. barbarum* fruits have been employed by the local population for blood nourishing, in the treatment of early onset diabetes, tuberculosis, dizziness, and chronic cough, and for the protection of eye health [15].

dizziness, and chronic cough, and for the protection of eye health [15]. All these pro-healthy properties have attracted the attention of consumers to goji juices and fruits, transforming goji berries into one of the most popular functional food ingredients/supplements worldwide [13]. Nevertheless, the consumption of natural supplements needs to be balanced to avoid negative effects related to overuse or interaction with other medical treatments [7]. Therefore, risk/benefit evaluations are urgently needed when used in foods or health-promoting formulations in order to avoid negative impacts [4].

#### **3. Bioactive Compounds and Chemical Composition of** *L. barbarum* **L.**

The bioactive composition of plants is influenced by many factors, such as variety, ripeness, geographic location, and climatic conditions. Since during fruit growth, physiological, biochemical, and molecular changes occur, ripeness might be the most influential factor in the fruit's bioactive composition [31,32]. Therefore, deep knowledge about the ripening stage of goji berries is needed to determine the best stage and obtain the most adequate bioactive compounds. Yet, in general, various researchers have reported that goji berries have a remarkable concentration of antioxidants, fat, dietary fibers, essential amino acids, valuable trace minerals, and vitamins [3,6,13,23,30,33,34]. In the next subsections, the different classes of bioactive compounds present in goji berries will be deeply analyzed.

#### *3.1. Phenolic Compounds*

Phenolic compounds act as a defense mechanism to provide adaptation and survival capacity in adverse environmental conditions to plants, such as protection against ultraviolet radiation (UV), pathogen aggression, parasites, and predators [5,35]. These compounds usually add nutritional and functional value, contributing to the fruit's organoleptic characteristics, such as astringency, bitterness, and aroma. Simultaneously, they guarantee outstanding biological activities and pro-healthy properties against oxidative stress [5,13,31], being capable of delaying, preventing, and inhibiting oxidation by scavenging free radicals and, therefore, reducing oxidative stress [35].

Reactive oxygen species (ROS) naturally occur in living organisms, being involved in processes such as proliferation and apoptosis [25]. However, when the quantities of ROS, such as superoxide radicals (O2 ·−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH·-−), overcome the activity of endogenous antioxidant mechanisms, namely antioxidant enzymes (e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) and non-enzymatic molecules (e.g., glutathione (GSH), ascorbic acid, α-tocopherol), a state of oxidative stress is initiated [10,25,35].

Oxidative stress is implicated in the aging process as well as in several pathologies (e.g., cardiovascular dysfunction, various typologies of cancer, inflammation, rheumatism, diabetes, rheumatoid arthritis, pulmonary emphysema, dermatitis, cataract, neurodegenerative diseases, endothelial cell dysfunction, and several autoimmune diseases linked to degenerative processes of aging) that frequently lead to invalidity or death [3,9,13]. When the endogenous antioxidant mechanisms are not enough to stop and prevent oxidative stress, supplementation is needed to strengthen the antioxidant state and the cell defense mechanisms of organisms [25]. Fruit phenolic compounds are excellent candidates to perform this scavenging effect against ROS and other radical species [35].

Phenolics, flavonoids, and carotenoids are intrinsically related to nutritional and health promotion capacity; therefore, their quantification may help to clarify some activities of natural products [36]. Table 1 summarizes the total phenolic compounds (TPC), total flavonoid content and total carotenoid content (TCC) of goji berries, according to different authors.

**Table 1.** Total phenolic compounds (TPC), total flavonoid content and total carotenoid content (TCC) of goji berries according to different studies.

GAE—gallic acid equivalents; CAE—catechin equivalents; HE—hyperoside equivalent; QE—quercetin equivalents; TPC—total phenolic content; TFC—total flavonoid content; TCC—total carotenoid content; fw—fresh weight; dw—dry weight.

As can be observed in Table 1, the values achieved for the different assays (TPC, TFC, and TCC) are slightly different between studies. For example, the TPC varied between 31.6 mg GAE/100 g dw [2] and 3000 mg GAE/100 g dw [12]. These variations are mostly due to the different postharvest techniques used that mainly affect the bioactive components present, as well as the extraction solvents employed and the samples' geographical origin. Islam et al. [2] studied the phenolic profile, antioxidant capacity, and carotenoid content of dried goji berries harvested in China and extracted with a mixture of acetone/water/acetic acid (70:29.5:0.5). According to the authors, the TPC achieved a value of 31.6 mg GAE/100 g dw, while the TFC was 28.3 mg CAE/100 g dw. These values were considerably lower than the ones reported by Magalhães et al. [12]. Despite the same geographical origin, the authors lyophilized the samples and extracted them for 5 days using methanol (80%), which could justify the huge differences observed.

A study conducted by Donno et al. [13] using goji berries supplied by a farm located in Alzate di Momo, Northern Italy, reported the presence of organic acids (4461.02 mg/100 g fw) and polyphenolic compounds (12,697.90 mg/100 g fw). Meanwhile, in a study [31] using goji berries provided by a planting base in Gansu Province, China, the researchers identified nine phenolic compounds by UPLC-MS/MS, including quercetin, isoquercitrin, chlorogenic acid, ferulic acid, *p*-coumaric acid, caffeic acid, isorhamnetin, cinnamic acid, and rutin, being rutin, isoquercitrin, and chlorogenic acid. Similarly, Pires et al. [30] extracted goji berries supplied by a Portuguese company and reported the presence of nineteen phenolic compounds (71 mg/g dw): eight flavonols (27.6 mg/g dw), seven phenolic acid derivatives (32.7 mg/g dw), one flavan-3-ol (10.4 mg/g dw), and three chlorogenic acids (25.07 mg/g dw). According to the authors, the principal phenolic compounds quantified were quercetin-3-*O*-rutinoside (16.6 mg/g dw) and *p*-coumaric acid (12.3 mg/g dw). Nardi et al. [3] conducted a phytochemical analysis on a methanolic extract of goji berries, revealing the presence of phenolic compounds (142.2 mg/100 g of extract) and flavonoids (74.5 mg/100 g of extract), while the qualitative analysis identified rutin and quercetin as the principal compounds. Wojdyło et al. [36] highlighted the carotenoid content of goji berries from new cultivars in Poland (212.94 mg/100 g dw). The authors identified different types of carotenoids, namely zeaxanthin (84.54 mg/100 g dw), β-carotene (19.35 mg/100 g dw), neoxanthin (16.04 mg/100 g dw) and cryptoxanthin (72.29 mg/100 g dw).

#### *3.2.  In another study, Ili´c et al. [33] reported the presence of moisture (75.32 g/100 g of fw), carbohydrates (16.93 g/100 g fw,), dietary fiber (3.63 g/100 g fw), protein (1.98 g/100 g fw), fat (1.15 g/100 g fw), and ash (0.84 g/100 g fw) in *L. barbarum* berries.  The same authors also reported the presence of soluble sugars (27.9 g/100 g dw), such as fructose (12.7 g/100 g dw), glucose (14.4 g/100 g dw), and sucrose (0.8 g/100 g dw). However, different authors [10,16,20,21,38–41] attested that polysaccharides are the major carbohydrates present in goji berries, being the principal active ingredients isolated from the fruit. Among them, water-soluble polysaccharides, homogeneous polysaccharides, pectin polysaccharides, acidic heteropolysaccharides, and arabinogalactans (composed of arabinose, glucosamine, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid) are the most prevalent.

Regarding organic acids, Pires et al. [30] stated that citric, succinic, and oxalic acids (respectively, 1.29 g/100 g dw, 0.77 g/100 g dw, and 0.010 g/100 g dw) were detected, as well as tocopherols, namely α-tocopherol and δ-tocopherol (0.23 mg/100 g dw and 0.09 mg/100 g dw, respectively). The authors also determined the fatty acids content (4.1 g/100 g dw), more specifically detecting sixteen fatty acids, with polyunsaturated fatty acids being the predominant group, namely linoleic acid (53.4%), oleic acid (16.5%) and

palmitic acid (12.77%). In parallel, Ili´c et al. [33] reported that the most abundant fatty acids were linoleic (52.1%), oleic (23.6%) and palmitic (17.6%) acids, accounting for 95% of the total fatty acids, which is in concordance with Skenderidis et al. [32], who reported concentrations of 37.89–43.96%, 16.71–20.07%, and 15.08–21.79%, respectively.

Concerning the mineral content, numerous studies showed that the principal minerals present in goji berries are potassium, sodium, and calcium. Bora et al. [23] reported that 100 g of goji berries contain 434 mg of potassium, 60 mg of calcium, 5.4 mg of iron, and 1.5 mg of zinc. Llorent-Martínez et al. [6] reported a higher amount of potassium (1460 mg/100 g), sodium (550 mg/100 g), and calcium (50 mg/100 g), while Ili´c et al. [33] quantified potassium (445.12 mg/100 g dw), phosphor (231.52 mg/100 g dw), sodium (74.57 mg/100 g dw), and calcium (29.02 mg/100 g dw).

In what concerns vitamins, ascorbic acid (48.94 mg/100 g fw) and tocopherols (0.33 mg/100 g dw) have been described in goji berries [13,14,30,33,41]. Vitamin E, also known as α-tocopherol, is the major liposoluble antioxidant present in the cells' antioxidant defense system, being able to inhibit membrane lipidic peroxidation [5,42], while vitamin C, also known as ascorbic acid, is an important antioxidant compound of goji berries [10].

Table 2 summarizes the nutritional composition and phenolic composition of goji berries according to the data reported by different authors.

**Table 2.** Phytochemical and nutritional composition of *L. barbarum* berries, according to different studies.

fw—fresh weight; dw—dry weight.

The variations observed between the different studies may be explained by several reasons. As mentioned earlier, a broad number of biological variables interfere with the bioactive composition of fruits, such as ripeness, geographic origin, or climatic conditions [26,31,32,36]. On the other hand, the extraction techniques employed, the extractor solvents used, and the quantification techniques employed, may lead to different results [9,43]. Generally, mature fruits are associated with huge amounts of bioactive compounds with pro-healthy effects.

#### **4. Biological Activities of** *L. barbarum* **L.**

Goji berries have been used for thousands of years as herbal medicines in Asian countries due to their rich nutritional value, medical properties, and biological activities [2,13,23,31]. Several studies highlighted the pro-healthy effects of goji berries, particularly in regarding their antioxidant [2,4,12,13], anti-tumor [4,10,15,40], antimicrobial [10,24], hypoglycemic [10], hypolipidemic [10,14], anti-mutagenic [40], immunomodulatory [16], prebiotic [10,23,24], anti-aging [10], anti-fatigue [10] and neuroprotective activities [12]. These biological activities have been closely related to the fruits' phenolic composition, particularly phenolic acids, flavonoids, carotenoids, and tannins, which are associated with different biological effects [2,5,23,31,42]. This correlation led to reports of health benefits associated with liver, kidney, eyesight, immune system, circulation, and longevity disorders [4,19,24,30,36]. The following sections will discuss each biological activity in detail.

#### *4.1. Antioxidant Activity*

Oxidative stress is a phenomenon that occurs due to an imbalance between prooxidants and antioxidants, being a consequence of the excessive production of reactive species [3]. In some situations, external antioxidant supplementation is required to reestablish the balance, and fruits' phenolic compounds are well-known for this capacity [35]. The main contributors to the antioxidant capacity of food, especially fruits and vegetables, are phenolic compounds, particularly phenolic acids and flavonoids, carotenoids, tocopherol, polysaccharides, ascorbic acid, and condensed tannins [2,5,10,36,42]. These molecules stimulate the antioxidant defenses by delaying, inhibiting, or preventing the free radicals from damaging proteins, DNA, and lipids, as well as by scavenging free radicals by hydrogen atom transfer or electron donation or enhancing endogenous antioxidant defenses, such as antioxidant enzymes (SOD, CAT, GPx, . . . ) [3,10,13,35].

Different assays are usually employed to evaluate the radical and antioxidant scavenging capacity. The most frequently applied include the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH• ), the 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS•+ ), scavenging activity, and ferric reducing antioxidant power (FRAP) [10,43]. Table 3 summarizes the reported data on the antioxidant and antiradical activities of goji berries.

**Table 3.** Antioxidant and antiradical activities of goji berries according to different authors.

TE—Trolox equivalent; VCE—vitamin C equivalent; IC50—half-maximal inhibitory concentration; Fe2+E—Fe2+ equivalent; AAE—ascorbic acid equivalent; fw—fresh weight; dw—dry weight.

A study conducted by Islam et al. [2] assessed the TPC (3.16 mg GAE/g dw), TFC (2.83 mg CAE/g dw), condensed tannin content (CTC; 1.08 mg CAE/g dw), and monomeric anthocyanin content (MAC; 0.24 mg MAC/g dw) of goji berries, along with DPPH (16.65 µmol TE/g dw), ABTS (59.14 µmol TE/g dw), and FRAP (3516.75 mmol Fe2+E/g dw) capacity. The results showed a positive linear correlation between DPPH, ABTS, FRAP, and phenolic compounds (0.786, 0.643 and 0.856, respectively), flavonoids (0.857, 0.714 and 0.786, respectively), condensed tannin (0.429, 0.714 and 0.643, respectively) and anthocyanin content (0.643, 0.786 and 0.857, respectively), supporting the idea that phenolic compounds are the main contributors to the antioxidant/antiradical activities of goji berries, which is in line with previous studies [35]. Another study [13] evaluated the TPC (268.5 mg GAE/100 g fw), FRAP (19.36 µmol Fe2+E/g fw), and total bioactive compound content (TBCC; 5806.80 mg/100 g fw) of a methanolic extract of goji berries and identified and quantified the principal bioactive compounds present, namely polyphenols (12,697.90 mg/100 g fw) and organic acids (4461.01 mg/100 g fw). Afterwards, the authors verified the correlation between the antioxidant activity and the variables evaluated, reporting a strong positive correlation between the antioxidant capacity and phenols (0.8290), organic acids (0.8606), TPC (0.9996) and TBCC (0.8363), which attests to the antioxidant capacity of goji berries.

Similarly, after identifying and quantifying the polyphenols (97.23 mg/100 g dw), carotenoids (212.94 mg/100 g dw), organic acids (24.7%), and flavonols (75.3%), Wojdyło et al. [36] determined the antioxidant/antiradical activities of goji berries by FRAP (1.44– 6.30 mmol TE/100 g fw) and ABTS assays (1.60–6.83 mmol TE/100 g fw). The Pearson

correlation allowed the verification of a linear correlation between ABTS or FRAP and the total polyphenolic compounds (0.523 and 0.038, respectively), phenolic acids (0.277 and 0.328, respectively), and flavonols (0.531 and 0.409, respectively). However, goji berries have a high content of carotenoids; therefore, the Pearson correlation between the ABTS or FRAP and the total carotenoids (0.462 and 0.409, respectively), zeaxanthin (0.381 and 0.315, respectively), β-carotene (0.316 and 0.277, respectively), neoxanthin (0.546 and 0.527, respectively) and cryptoxanthin (0.411 and 0.379, respectively) proved the importance of these compounds for the antioxidant power of this fruit.

Meanwhile, Pehl˙Ivan Karakaçs et al. [20] evaluated the levels of antioxidant enzymes (namely SOD, CAT, and GPx) and malondialdehyde (MDA), a characteristic molecule of the oxidative state, when a *L. barbarum* polysaccharide (LBP) extract was orally administrated to rats for 4 weeks. The LBP extract was prepared by crushing 200 g of dried fruits and performing 2 extractions with 600 mL of a chloroform:methanol (2:1) solution at 80 ◦C. The results showed that the SOD, CAT, and GPx levels increased, while the MDA levels decreased in the blood serum of rats treated with LBP extract, supporting the in vivo antioxidant power of goji berries.

Skenderidis et al. [25] also demonstrated the antioxidant activity of an aqueous extract of *L. barbarum* berries cultivated in Greece and extracted by ultrasound-assisted extraction (UAE). The results of the XTT assay performed in C2C12 muscle cells showed cytotoxicity in concentrations higher than 125 µg/mL. The levels of GSH were also evaluated by flow cytometry after exposing C2C12 muscle cells to the extract (25 µg/mL and 100 µg/mL), and the results revealed an increase of 127.5% and 189.5%, respectively, when compared to the control. As for the thiobarbituric acid reactive substance (TBARS) levels, a marker of lipid peroxidation, the decreases of 21.8% and 9.4% after exposure to 25 µg/mL and 100 µg/mL, respectively, attested to the antioxidant capacity of goji berries extracts.

#### *4.2. Anticancer Activity*

Despite the extensive research and the advances in cancer treatments made in recent years, cancer is still a worldwide problem [17,40]. The knowledge that several polyphenolrich extracts from natural matrices have been suggested as promising anticancer agents with few side effects, being associated with lower risks of cancer and cancer mortality, has increased consumers' interest in fruits and natural matrices [5,6,35].

Kwa´snik et al. [17] performed a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with human natural killer cells (NK-92) against human colon cancer cell line LS180 after 48 h of incubation in the presence or absence of an ethanolic extract of goji berries in different concentrations (1–500 µg/mL). According to the authors, in the absence of the extract, the strongest anticancer effect obtained (with the elimination of 91% of the cancer cells) occurs when LS180 and NK-92 were used in a ratio of 1:1. In the presence of extract, using the same ratio, 94.8% of the LS180 cells were eliminated. Therefore, goji berries extract reduced by 5%, 12.8% and 20.6%, respectively, the proliferation of LS180 using concentrations of 1, 2.5 and 5 µg/mL. When LS180 and NK-92 cells, in a ratio of 2:3, are exposed to goji berries extract in concentrations of 2.5 and 5 µg/mL, the viability of LS180 cells is reduced to 96.5% and 98.1%, respectively. These results support the chemopreventive properties of goji berry extract, depending on the dose and number of lymphocytes used. Probably, this is due to the immunomodulatory properties of goji berries, which increase the viability and proliferation of NK cells and promote their recognition and elimination capacity.

Another study assessed the proliferation and apoptotic and necrotic effects of different concentrations of an ethanolic extract of goji berries in the T47D human breast cancer cell line [44]. The MTT assay results attested to a strong decrease in cell proliferation after exposure to the highest extract concentration (1 mg/mL) (70%, 55.7%, and 51.4% after 24, 48, and 96 h, respectively). The bromodeoxyuridine (BrdU) cell proliferation assay supported these results, showing a sharp decrease in the proliferation of T47D cells, similar to the Neutral Red (NR) cell viability assay, which demonstrated a slight decrease in the T47D cell

viability. The Western blotting analyses employed to evaluate the expression of p21 and p53 proteins, cyclin-dependent kinase 6 (CDK6), and cyclin D1 highlighted the significant increase in p21 and p53 protein expression as well as a slight decrease in CDK6 and cyclin D1 expression. Regarding T47D cells, an increase in the apoptotic percentage was detected (37%, 61.6%, and 88% when treated with 0.1, 0.5, and 1 mg/mL of extract, respectively), as well as a significant necrotic change at 0.5 mg/mL of extract for propidium iodide and Hoechst solution staining. These results attested that the anticancer activity of goji berries extract is due to apoptotic effects through the mitochondrial pathway. To confirm these results using Western blotting, the authors demonstrated a dose-dependent significant increase in pro-apoptotic Bax protein expression and a decrease in anti-apoptotic BclxL protein expression after treatment of T47D cells with the extract for 48 h when compared to the control.

 According to the authors, the fruits' crude polysaccharides were obtained by water extraction, alcohol precipitation and deproteinization, and fractional precipitation with gradient concentrations of ethanol (30%, 50%, and 70%), originating different fractions (LBGP-I-1, LBGP-I-2, and LBGP-I-3). Through an MTT assay, all *L. barbarum* fruit polysaccharide fractions showed a remarkable inhibition of SMMC-7721, HeLa, and MCF-7 cell growth in a dose-dependent manner, with MCF-7 cells being more sensitive to the LBGP-I-3 fraction (with a cell viability reduction to 48.96%). To study the inhibitory mechanism behind this result, the cell cycle of MCF-7 cells treated with LBGP-I-3 at 1000 µg/mL was analyzed by flow cytometry. The results showed a percentage of 72.76%, 24.21%, and 3.04% of cells in the G0/G1, S, and G2/M phase, respectively, suggesting that LBGP-I-3 arrested the MCF-7 cell cycle at the G0/G1 phase. AO-EB and DAPI staining were used to detect basic morphological changes in apoptotic cells, with the results attesting that the LBGP-I-3 fraction induces the MCF-7 apoptosis. The results also supported that the apoptotic effect was caused by the increased expression of pro-apoptotic Caspase-3, 8, and 9 proteins as well as the decrease in the Bcl-2/Bax ratio and mitochondrial membrane potential. A significant decrease in T-SOD and CAT activities, as well as GSH-Px activity and GSH content, coupled with the enhancement of the MAPK signaling pathway, down-regulating p-ErK1/2 levels and up-regulating p-JKN and p-p38 levels, was also observed.

#### *4.3. Antimicrobial Activity*

The phytochemicals present in plants, particularly phenolic acids, flavonoids, and tannins, exert their antimicrobial effects by complexing with extracellular and soluble proteins, leading to the disruption of the microbial membrane, metal ion deprivation, and interactions with enzymes [30,45]. Another way of explaining the antimicrobial activity of polyphenols is their structural features, as well as the pH and sodium chloride concentration, which results in physiological changes in the microorganisms and, eventually, cell death [45].

A study carried out by Kabir et al. [45] confirmed that chlorogenic acid, one of the principal phenolic acids detected in goji berries, exhibited a bacteriostatic and bactericidal effect against *Escherichia coli*. The bacteriostatic effect was assessed by measuring the optical density by determining the growth inhibition of chlorogenic acid when added to a culture of *E. coli*. Regarding the bactericidal effect, it was assessed by using midlogarithmic phase cell cultures (106 cells/mL) at different doses (2.5, 5.0, and 10 mM), treatment times (0, 1, 3, and 6 h), temperatures (20, 37, 45, and 50 0C), and pH conditions (8.0, 7.0, 6.0, 5.0, and 4.0). The overall results demonstrated a synergetic antimicrobial effect expressed by chlorogenic acid and related compounds and a dose, temperature, and time-dependent bactericidal effect. The authors stated that the bactericidal effect shown by chlorogenic acid may be due to the capacity to promote physiological changes on the

microbial cell membrane that result in cell death. This study corroborated the results achieved by Ili´c et al. [33], which evaluated the antimicrobial activity of goji berries from Serbia. According to the authors, the methanolic extract of goji berries presented mild antimicrobial activity against Gram-positive and Gram-negative bacteria as well as yeast, with remarkable activity against *Klebsiella pneumoniae*, *Salmonella abony,* and *Pseudomonas aeruginosa.* In another study, Mocan et al. [46] also explored the antimicrobial activity of *L. barbarum* flowers. According to the authors, the antimicrobial activity was mild against Gram-positive (namely *Staphylococcus aureus*, *Bacillus subtilis,* and *Listeria monocytogenes*) and Gram-negative (*Salmonella typhimurium*) bacteria and lacking against *E. coli*, oppositely to what was observed for goji berries.

#### *4.4. Anti-Inflammatory Activity*

The inflammatory response is initiated by the stimulation of innate immunity, the release of immune effectors, the inhibition of enzymes, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukins (IL), and transcription factor nuclear factor kappa B (NF-kB) [3]. If over-expressed, inflammation can cause numerous complications in patients, being an important hallmark of diseases or pathological conditions, such as cancer, neurodegenerative diseases, respiratory pathologies, and several autoimmune diseases [3,5]. Natural extracts, typically rich in alkaloids, flavonoids, terpenoids and tannins, have been associated with anti-inflammatory effects [5]. A study conducted by Nardi et al. [3] addressed the anti-inflammatory effect of *L. barbarum* berries using paw edema carrageenan, an acute model of inflammation widely employed to evaluate the anti-inflammatory effect of natural products. Briefly, the right paw of mice was injected with 450 µg of carrageenan, and the left paw was injected with the same volume of sterile phosphate-buffered saline (PBS) (control). After established conditions, the animals were orally administered with a methanolic extract of goji berries in doses of 50 mg/kg and 200 mg/kg, 12 h after 12 h, for 10 days. The authors stated that after the carrageenan injection, neutrophils migrate to the inflamed paw and release enzymes, such as myeloperoxidase, which increase ROS production and induce an inflammatory state. The results attested to the anti-inflammatory capacity of the compounds present in goji berries, with the doses of 50 mg/kg and 200 mg/kg leading to a reduction of paw edema in 38% and 63.8% of mice, respectively.

#### *4.5. Immunomodulatory Activity*

Immune response suppression is usually associated with the development of immunological diseases. The actual research is focused on finding immunomodulating agents capable of preventing and treating these disorders [47]. Goji berries have been reported as a support to the immune system [6,47], either by regulating the expression of immune factors, such as cytokines and cell adhesion molecules [16,39], or by promoting the proliferation and activity of immune cells, such as natural killer cells [17] and lymphocytes [47].

Different authors [16,24,39,47] stated that the immunomodulatory effects of *L. barbarum* fruits are greatly related to LBP, one of the major active ingredients isolated from berries. LBP is a mixture of proteoglycans and polysaccharides that mainly consists of arabinose, galactose, glucose, xylose, and a small amount of rhamnose, mannose, and galacturonic acid as its glycosidic part. It was reported that LBP is an adjuvant that improves the immune responses against vaccines and increases humoral immunity [39], probably due to an increased expression of IL-2 and TNF-α, molecules involved in immunomodulation [16]. To assess the immune function alteration during the administration of LBP, Zhu et al. [39] divided fourteen mice into two groups and administered a dose of 0.1 mL/10 g body weight of LBP powder for 14 days in the first group, the experimental group, and the same volume of physiological saline via intragastric administration in the second group, the control group. After the experimental period, blood samples were collected, and the animals were sacrificed to excise the spleen and thymus. The viscera indices were measured (thymus or spleen index = weight of thymus or spleen (mg)/body weight (1000 mg)),

and the concentrations of immune factors (namely, transforming growth factor-β (TGF-β), interferon-γ (IFN-γ), and IL-6) in the serum were detected using a commercial enzymelinked immunosorbent assay (ELISA) kit. The results supported the theory that LBP can enhance the innate immune response since the spleen and thymus index of mice from the experimental group (5.12 mg/1000 mg and 4.12 mg/1000 mg, respectively) were significantly higher (*p* < 0.05) when compared to the control group (namely 4.19 mg/1000 mg and 2.86 mg/1000 mg). Moreover, the concentration of the immune factors TGF-β and IL-6 in the experimental group (namely, > 500 pg/mL and ≈ 100 pg/mL) were also significantly higher (*p* < 0.05) when compared to the control group.

It has also been reported that phenolic amides from the nonpolysaccharide fraction of *L. barbarum* fruits can not only in vitro modulate the proliferation of B and T cells but also enhance the immune cell factors (IFN-γ, IL-2, and IL-10) [47]. Prednisone-induced immunodeficient mice were intragastrically administered with a phenolic amid extract, showing a significant spleen cell proliferation (*p* < 0.01) when compared to the prednisoneinduced immunodeficient mice intragastrically administered with an ethanolic extract of goji berries or an LBP extract. The immunological cytokines (IFN-γ, IL-2, and IL-10) were also significantly enhanced (*p* < 0.01) in mice treated with the ethanolic goji berries extract, LBP extract, and phenolic amide extract when compared to the control. Overall, the total phenolic amides had the strongest immunity-activating effects.

Since natural killer cells are one of the first elements from the innate immune system to act, Kwa´snik et al. [17] studied the cell viability and proliferation of human natural killer cells (NK-92) in the presence of an ethanolic goji berry extract. The authors highlighted that goji berry extract has the capacity to enhance NK cell proliferation by 61.0% in concentrations ranging between 1 and 250 µg/mL.

#### *4.6. Prebiotic Activity*

Prebiotics are non-digestible ingredients that can selectively promote the growth and activity of specific bacteria, modulating the gut microbiota [39]. Goji berries have demonstrated a gut microbiota positive modulating effect [18,39], probably due to phenolic compounds or polysaccharides present, such as LBP [43]. The prebiotic activity of goji berries was attested to by Skenderidis et al. [18], who cultivated strains of *Bifidobacterium* and *Lactobacillus* in the presence and absence of different encapsulated goji berry extracts. According to the authors, the extract with higher amounts of polyphenols and polysaccharides stimulated the growth and proliferation of probiotic strains on a larger scale, mostly *Bifidobacterium* strains, when the cultures were submitted to a gastrointestinal environment (simulated gastric and intestinal juices). Another study [39] assessed the prebiotic activity of LBP in vitro through the growth of *L. acidophilus* and *B. longum*. Both strains were properly cultivated, and different concentrations of LBP powder (experimental group) and glucose (control group) were administered. These experiments highlighted the positive effect of LBP on the growth of both strains when compared to the control group. The authors also evaluated the effects of LBP intake on the composition of cecal gut microbiota [39]. After mice were administered a dose of 0.1 mL/10 g body weight of LBP powder for 14 days, the cecal content samples were collected, and the microbial DNA was extracted and amplified. The results showed a clear modification of the gut microbiota after treatment, with the dominant bacterial communities being *Firmicutes* and *Proteobacteria.* Furthermore, the percentage of beneficial bacteria, such as *Akkermansia*, *Lactobacillus* and *Prevotellaceae*, significantly increased when compared to the control.

#### *4.7. Neuroprotective Activity*

Since neurological damage may occur as a consequence of oxidative stress and inflammation, goji berries have been associated with neuroprotective effects [43]. A study conducted by Fernando et al. [19] assessed the neuroprotective activity of a goji berry powder (GBP) against the development of Alzheimer's disease (AD). The authors observed that diets rich in antioxidants can directly affect amyloid beta levels and influence the

development of AD. To test this theory, human neuroblastoma BE(2)-M17 cells were treated with and without 20 µM of amyloid beta 42 and exposed to different concentrations of GBP (0.6, 0.9, 1.2, 1.5 and 1.8 µg/mL). The GBP significantly increased the cell viability up to 105% (GBP at 1.2 µg/mL), while the Western blot analysis showed a significant reduction in the amyloid beta up to 20% (GBP at 1.5 µg/mL). Furthermore, the ELISA attested to a 17% reduction of the amyloid beta in amyloid beta-induced neuronal cells when compared to the control (GBP at 1.5 µg/mL). Other authors [16,24] also stated that the major active ingredient isolated from goji berries, LBP, has ocular and neuroprotective effects [20]. In another study, Pehl˙Ivan Karakaçs et al. [20] reported that LBP has a neuroprotection effect in female rats by performing behavioral tests and immunohistochemistry analyses. The animals were submitted to ovariectomy and daily treated with oral low and high doses of polysaccharides obtained from *L. barbarum* fruits (20 and 200 mg/kg) for 4 weeks. According to the behavioral test performed, the LBP present in goji berries can decrease stress-induced anxious behavior in rats. The behavioral disruption could be caused by the accumulation of ROS in the brain, which increases oxidative stress, leading to mitochondrial dysfunction in neuronal cells and apoptosis. However, behavioral disruption can also be caused by variations in neurotransmitter levels in some brain regions, such as the hippocampus. The neuroprotective effect of LBP was demonstrated by the increased levels of brain-derived neurotrophic factor, which promotes neuronal proliferation, survival, and maintenance of neuron structure and function in the hippocampus region.

#### *4.8. Antihyperglycemic Activity*

Diabetes is a complex chronic disease caused by metabolic disorders of carbohydrates, lipids, lipoproteins, and increased oxidative stress [43]. It is characterized by the inability to maintain blood glucose levels within healthy ranges, leading to hyperglycemia [36]. Drug treatment is inevitable; however, it is often expensive or unavailable [36]. In these cases, a change in patient lifestyle and eating habits is extremely important [21,38,43]. Therefore, industries are encouraged to find alternative compounds with antihyperglycemic properties, mostly from natural sources, such as goji berries [5,10]. Enzyme inhibitors are good candidates for the treatment of non-insulin-dependent diabetic patients [36]. Since pancreatic α-amylase and intestinal α-glucosidase are enzymes responsible for the hydrolysis of carbohydrates into monosaccharides, such as glucose, the capacity of goji berries to inhibit these enzymes proves the antihyperglycemic effect of this fruit [36]. According to Wojdyło et al. [36], the inhibition of α-amylase and α-glucosidase was, on average, 63% and 10%, respectively.

Most of the experiments that report the antihyperglycemic activity of goji berries have attributed this property to LBP [21,25,38]. Cai et al. [21] performed a clinical trial to study the antidiabetic efficacy of LBP. Sixty-seven patients diagnosed with type II diabetes were divided into two groups and treated twice daily, for three consecutive months, with capsules of 300 mg of microcrystalline cellulose in the control group and capsules with 150 mg of LBP power and 150 mg microcrystalline cellulose in the experimental group. The results of the oral metabolic tolerance test reported a significant decrease in serum glucose levels when compared to the control (−7.86% vs. 1.61%), as well as serum insulin (−56.71% vs. −8.73%). Regarding the insulinogenic index (insulin/glucose), the results attested to a noticeable increase (−0.98% vs. 0.04%, respectively, for the experimental and control groups). During the experimental period, the patients were asked not to change their previous therapies in order to compare the antidiabetic effect of LBP with or without the hypoglycemic medicines. After 3 months of treatment, the patients without hypoglycemic drugs showed a significant decrease in serum glucose, serum insulin, and the homeostasis model assessment index of insulin resistance, while the patients with hypoglycemic drugs did not present significant alterations, demonstrating that LBP has profound hypoglycemic efficacy when disassociated from hypoglycemic drugs.

The study developed by Zhao et al. [38] focused on the effect of LBP extract on diabetic rabbits (induced by Alloxan), evaluating the extract's effect on diabetic nephropathy (DN). The experimental period lasted for 12 weeks, in which twenty-five rabbits were divided into five groups: group (I)—control group; group (II)—DN control group; group (III)—LBP prevention group from developing DN; group (IV)—positive DN control group using Telmisartan drug (10 mg/kg/day in 3 mL); group (V)—LBP treatment group using the LBP (10 mg/kg/day in 3 mL) as DN treatment. The results demonstrated that LBP not only helps after the development of DN but also benefits the prevention of complications since group (V) showed improvements in fasting glucose tolerance when compared to group (II), while group (III) had better results than group (V). This study also highlighted that LBP can be more efficient in the treatment of DN than standard drugs (in group (V), the kidney weight index (KWI) was significantly higher (*p* < 0.01) than in group (IV)). As for kidney function, even though LBP may be able to protect this organ from injuries, it cannot entirely treat it since the serum urea nitrogen and creatinine levels from groups (III), (IV) and (V) were significantly lower (*p* < 0.01) that in group (II). No significant changes were observed regarding group (I).

#### *4.9. Antihyperlipidemic Activity*

In addition to the biological activities described above, the antihyperlipidemic effect of goji berries has also been reported. Pai et al. [14] fed murine models with a high-fat diet (5 g of deoxycholic acid and 300 g of warm coconut oil mixed with 700 g of powdered rat chow per day) for 45 days to induce a state of hyperlipidemia. In the last 30 days of the study, a group of animals was orally treated daily with 10 mg/kg and 20 mg/kg of a 50% hydroalcoholic extract of *L. barbarum* fruits. The results showed a positive antihyperlipidemic activity, with the extract promoting a significant decrease in the triglyceride levels for both doses as well as a significant decrease in very low-density lipoprotein cholesterol at the highest dose, a significant increase in the high-density lipoprotein cholesterol at the lowest dose, and a dose-dependent decrease in the total cholesterol and low-density lipoprotein cholesterol levels. Additionally, the results obtained with the animals treated with the *L. barbarum* fruits and the animals treated with the standard antihyperlipidemic drug atorvastatin were similar, reinforcing the positive antihyperlipidemic activity of goji berries. The authors explained that this effect may be caused by more than one compound present in goji berries, namely polysaccharides and vitamins, such as vitamin C. Additionally, riboflavin, ascorbic acid, and coumarin and their synergistic effects may contribute to the hypolipidemic effect observed.

#### **5. Potential Applications of** *L. barbarum* **L.**

Goji berries can be used in different industries, such as the food, nutraceutical, or cosmetic industries. Regarding the food industry, it has been reported that the aqueous extracts of goji berries can be employed as potentially prebiotic food additives [10,18].

Goji extract has also been successfully used in meat products to improve their sensory properties and oxidative stability during storage [48]. The addition of 1.0% of goji berry extract and 1.0% of buckwheat flour improved the oxidative stability and quality of modified horse-meat products, resulting in a better smell, taste, and surface color after 21 days of storage [49]. Another study determined the effect of the addition of goji berries or goji berry extract on sausages [48]. The addition of 1% of goji berry extracts effectively suppressed lipolysis and protein/lipid oxidation, reducing the microbial count during storage and preserving the sausages' color, aroma, and taste. Antonini et al. [50] also evaluated the addition of goji berries to meet formulas and determined that the addition of chia seeds and *L. barbarum* puree (2.5% and 5%, respectively) to beef burgers promotes a higher TPC, an increase of up to 70% of the total antiradical capacity (through the oxygen radical absorbance capacity (ORAC), ABTS, and DPPH) and a decrease in the lipid peroxidation of up to 50%.

The confectionery and bakery industries are also using goji berries and their compounds to improve the functional, sensory, and texture properties of various products [10]. Muffins and cookies enriched with different amounts of goji berry powder or by-products

showed an increase in the TPC, insoluble, and soluble fiber contents as well as good sensory properties [23].

Nutraceuticals are foods, beverages, or supplements with high concentrations of bioactive compounds with outstanding health-promoting effects on the human body, being incorporated into consumers' daily diets [9]. Due to the bioactive compounds present in their fruits, goji berries are being investigated as a potential nutraceutical ingredient [2,3,5]. As previously mentioned, goji berries have been used in herbal medicine for thousands of years [24], along with hundreds of plants cultivated worldwide for their substances to be used in medicine and pharmaceutical formulations [51]. Based on the long-term traditional use of goji berries, this fruit is now generally recognized as non-toxic [10]. However, adverse effects can occur and, depending on the goji berries' use, a public health risk can be considered [51]. The presence of tropane alkaloids, chemical contaminants, such as pesticides and toxic elements, or some proteins that can cause allergic reactions in sensitive consumers are clearly risks [10]. Therefore, caution and professional guidance should be exercised regarding the safe consumption of natural matrices, such as goji berries, to avoid adverse effects, toxicity, and allergies [52].

Furthermore, the application of this fruit and its components at high levels in different industries, such as the food, pharmaceutical, nutraceutical, or cosmetic industry, can substantially deteriorate the sensory, textural, and overall quality of the final products [23]. More studies need to be conducted to determine to what point the use of this fruit is beneficial for consumers [4]. However, overall, considering the excellent nutritional profile and the positive health effects of goji berries, this fruit can be classified as a "superfruit" [12, 13].

#### **6. Valorization Prospects of** *L. barbarum* **L.**

During the production and processing of food products, a huge amount of waste is generated, in which a significant amount of bioactive compounds may be present [11]. In the European Union (EU), approximately 88 million tons of waste are generated annually, with associated costs estimated at EUR 143 billion [42]. The last data from the Food and Agriculture Organization of the United Nations (FAO) state that fruit and vegetable processing wastes, such as peels, pomace, flowers, stems, leaves, seeds, and pulps, are the 5th highest contributor (8% of total food waste) [42,53].

In the processing of goji berries, significant amounts of wastes are generated (approximately 10 kg of waste per 90 kg of goji beverage) [23]. In addition to the economic costs associated with these wastes' disposal, a serious negative impact on the environment is estimated [23]. As a result, industries face the urgent and necessary challenge of implementing greener, more efficient, sustainable, and eco-friendly processing protocols [5]. Regarding processing protocols, since extraction is a critical step in the isolation and purification of bioactive compounds, industries have focused on overcoming the limitations of conventional extraction methods, such as reduced extraction efficiency, high energy employment, and the use of high amounts of solvent wastes [32]. For this reason, new green extraction technologies have emerged [41], such as hot water extraction (HWE), subcritical water extraction (SWE), supercritical fluids extraction (SFE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE).

Therefore, the valorization of agro-residues and industrial wastes has proved to be an advantage [23,42]. As previously referred, goji berries are ingested in many forms and bring a panoply of beneficial pro-healthy effects to consumers, from nutritional to medicinal and curative approaches. However, their by-products, including leaves, stems, young shoots, and root bark, can also be consumed as part of a traditional diet, and used for medicinal purposes [33]. For instance, a study developed in Portugal [30] compared the composition of goji stems and berries and reported similar values of phenolic compounds (71.9 vs. 71 mg/g dw), organic acids (2.08 vs. 2.07 g/100 g dw), energy (383 vs. 408 kcal/100 g dw), total carbohydrates (78.1 vs. 87 g/100 g dw) and fat (4.6 vs. 4.1 g/100 g dw). In what concerns tocopherols (3.59 vs. 0.33 mg/100 g dw), proteins (7.4 vs. 5.3 g/100 g dw) and

saturated fatty acids (68 vs. 26.1%), the stems revealed a high content. However, stems also had higher antioxidant and antiradical capacities (DPPH scavenging activity (EC50 = 0.28 mg/mL), reducing power (EC50 = 0.23 mg/mL), β-carotene bleaching inhibition (EC50 = 0.26 mg/mL), and TBARS inhibition (EC50 = 0.07 mg/mL), as well as antibacterial activity against Gram-negative (*Escherichia coli*, *Morganella morganii*, *Pseudomonas aeruginosa*, *Acinetobacter baumannii*) and Gram-positive bacteria (*Staphylococcus aureus*, *Listeria monocytogenes*, *Enterococcus faecalis*) when compared to goji berries [30].

Another study determined the chemical composition (TPC, TFC, and HPLC/MS analysis) and evaluated the antioxidant activity (Trolox equivalent antioxidant capacity (TEAC), hemoglobin/ascorbate peroxidase activity inhibition (HAPX) assay, inhibition of lipid peroxidation catalyzed by cytochrome c and electron paramagnetic resonance (EPR) spectroscopy) of an ethanolic extract of *L. barbarum* flowers [46]. The results show its richness in chlorogenic, *p*-coumaric, and ferulic acids, isoquercitrin, rutin, and quercitrin (similar to what was stated in studies concerning goji berries [3,30,31]) and a TPC and TFC very similar to *L. barbarum* fruits (3.75 and 0.61 mg/g dw vs. 1.45 and 0.75 mg/g dw [3], respectively). Regarding the antioxidant capacity, the results attested to their activity, which was positively correlated with their chemical composition. After an ultrasonic extraction with methanol/water (70:30, *v/v*), an extract of goji berry leaves was evaluated regarding phenolic profile and antioxidant capacity (TEAC and EPR spectroscopy) [54]. The phenolic profile revealed the presence of chlorogenic acid and rutin, as previously reported for goji berries [3,30,31]. Regarding the antioxidant activity, the results showed a high antioxidant power (140 mg TE/g dw and 212 mg FSE/g dw, respectively). Along with the enzyme inhibitory activity (cholinesterase, α-amylase, α-glucosidase and tyrosinase), antimicrobial activity (*S. aureus*, *Bacillus cereus*, *Listeria monocytogenes*, *Enterococcus faecalis*, *Pseudomonas aeruginosa*, *Salmonella typhimurium*, *E. coli*, *Fusobacterium nucleatum,* and *Peptostreptococcus anaerobius*) and antifungal activity (*Aspergillus flavus*, *Aspergillus niger*, *Candida albicans*, *Candida parapsilosis,* and *Penicillium funiculosum*), this study confirms the potential of goji leaves to be used as a valuable source of bioactive compounds. Apart from that, polyphenols from goji berries leaves were also encapsulated in liposomes to improve their delivery and successfully serve as polyphenol carriers, demonstrating a cytoprotective effect on L-929 mouse fibroblast cells [10].

Based on the results detailed above, it can be stated that goji berry by-products, such as fruits skins, pulps, stems, and seeds, have high amounts of dietary fiber, vitamins, minerals, phytochemicals, and antioxidants, being a valuable ingredient for the food industry. Therefore, the incorporation of goji berry by-products, for example, in the bakery industry, may provide benefits to consumers as well as manufacturers, not only due to their nutritional value but also due to the economic advantage that comes from the elimination of the costs required for its disposal [23]. Nevertheless, safety and security studies should be previously performed considering these potential applications.

#### **7. Conclusions**

Goji berries can be classified as a "superfruit" due to their outstanding phytochemical composition, which includes phenolic acids (up to 32.70 mg/g), flavonoids (up to 38.00 mg/g), organic acids (up to 44.61 mg/g fw), carotenoids (up to 2.13 mg/g dw), carbohydrates (up to 87.00 g/100 g dw), and vitamins (up to 48.94 mg/100 g fw). The main phenolic compounds reported in this fruit are phenolic acids (chlorogenic acid and *p*-coumaric acid) and flavonoids (rutin and quercetin). Aside from these compounds, vitamins (ascorbic acid), carbohydrates (polysaccharides), and carotenoids (zeaxanthin, β-carotene, and cryptoxanthin) are the main compounds responsible for the incredible biological activity observed, such as antioxidant, anti-tumor, antimicrobial, hypoglycemic, hypolipidemic, anti-mutagenic, immunomodulatory, and prebiotic activities. In this regard, goji berries can be used in the food, nutraceutical, and pharmaceutical industries as a source of functional ingredients. Despite this, *L. barbarum* leaves, flowers, and steams are valuable sources of bioactive compounds that can be explored for use in different industries. Even

though the valorization of goji by-products helps to reduce the food waste produced during food processing, the high energy required, as well as the use of high amounts of solvents in the extraction processes, are still challenges. In the future, the implementation of green extraction techniques is expected at industrial levels, minimizing the wastes produced and maximizing the extraction of bioactive compounds from goji berries and by-products.

**Author Contributions:** Conceptualization, F.R.; methodology, F.T.; validation, F.R.; investigation, F.T.; resources, F.R.; writing—original draft preparation, F.T.; writing—review and editing, A.M.S., C.D.-M. and F.R.; supervision, F.R.; project administration, F.R.; funding acquisition, F.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project EXPL/BAA-GR/0663/2021—Kiwi4Health—Exploring the Eco-Innovative Re-Use of Kiwiberry, supported by national funds by FCT/MCTES and by the projects UIDB/50006/2020 and UIDP/50006/2020 through national funds. This work was also financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Filipa Teixeira is thankful for the scholarship from project EXPL/BAA-GR/0663/ 2021—Kiwi4Health—Exploring the Eco-Innovative Re-Use of Kiwiberry. Ana Margarida Silva (SFRH/BD/144994/2019) is thankful for the Ph.D. grant financed by POPH-QREN and subsidized by the European Science Foundation and Ministério da Ciência, Tecnologia e Ensino Superior. Francisca Rodrigues (CEECIND/01886/2020) is thankful for her contract financed by FCT/MCTES—CEEC Individual Program Contract.

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

#### **References**

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**2. Epidemiology**

The global incidence of TBI is estimated at 939 cases per 100,000 people – which translates to 69 million people sustain a TBI every year [8]. Decreasing incident (4.4%) and increasing prevalence (6.6%) of TBI in the United States during 1990 through 2017 are reported by the Global Burden of Disease Study, with the latest

prevalence and incidence reported are 2.104 and 0.961 million, respectively. TBI incidence is among the highest compared to other neurological disorders, even with age-adjustment at 285 cases per 100,000 people [9]. Pediatric TBI contributes to a global incident range of 47–280 per 100,000 children [10]. Changes in Coronavirus disease 19 (COVID-19) pandemic circumstances since 2020 seem to affect TBI epidemiology as implied by the significant decrease in mild TBI incident for children aged 0–5 years (44%) and 6–17 years (93%) in Canada [11].

The epidemiological characteristics are also invariably affected by geographical and sociodemographic features. Countries with the lowest and highest incidence are Sweden (12 cases per 100,000) and Australia (486 cases per 100,000). Higher incidence, severity, and mortality in pediatric TBI are observed in rural as opposed to urban areas [12]. Bimodal age distribution with peaks at 0–4 years and 14–18 years with male-gender preponderance in the pediatric population is observed [13]. Meanwhile, the role of race and socioeconomic status requires further confirmation [10].

The most common injury mechanisms are falls and motor vehicle accidents, although the relative proportions vary by age distribution. The majority of pediatric TBI cases are mild (70–90%), and severe TBI only accounts for 3–7% of all cases. Consistently, the hospitalization rate is 129 per 100,000 in pediatric population and over 90% recover [10, 13, 14]. Nevertheless, 88% of concussions are left undiagnosed and one-third of properly diagnosed cases may experience ongoing sequelae, which would remain undetected until the development reached frontal lobe maturity [1, 14].


**Lithography-Free Nanostructure Fabrication Techniques Utilizing Thin-Film Edges**

Hideo Kaiju1,2, Kenji Kondo1 and Akira Ishibashi1 *1Research Institute for Electronic Science, Hokkaido University 2PRESTO, Japan Science and Technology Agency Japan*

#### **1. Introduction**

568 Recent Advances in Nanofabrication Techniques and Applications

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Kim, J. H., Oh, H. C., & Kim, S. S. (2008). *Electrohydrodynamic Drop-on-Demand Patterning in Pulsed Cone-Jet Mode at Various Frequencies*. Journal of Aerosol Science, 39, 819-825 Rahman, K., Khan, A., Nam, N. M., Choi, K. H. & Kim D. S. (2011). *Study of Drop-on-Demand*

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Poon, H. F. (2202). *Electrohydrodynamic Printing.* PhD thesis, Princeton University

*forced jets. I. Stability theory*. Physics of Fluid, 13 (8), 2201-2220

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*Hollow Tips for Protein Analysis Based on Nanoelectrospray Ionization Mass*

*Thin LiCoO2 Films Fabricated Using the Electrostatic Spray Deposition (ESD) technique*.

*Superimposing an Electric Pulse to Background Voltage*. Journal of Electrostatic, 67, 562-

*Printing Through Multi-step Pulse Voltage*. International Journal of Precision

*Direct Printing of Colloidal Copper Nanoparticles by Electrohydrodynamic Atomization*.

*Evaluation of a Silicon Based Multi-Nozzle for Addressable Jetting Using a Controlled Flow Rate in Electrohydrodynamic Jet Printing*. Applied Physics Letter, 93, 243114 Khan A., Rahman K., Hyun M.T., Kim D.S., & Choi K.H. (2011). *Multi-Nozzle*

*Electrohydrodynamic Inkjet Printing of Silver Colloidal Solution for the Fabrication of Electrically Functional Microstructure*. Applied Physics A (doi:10.1007/s00339-011-

*Layer deposition through electrospray process for solar cell fabrication*. Current Applied

Poulikakos D. (2010). *On Ejecting Colloids Against Capillarity from Submicrometer Openings: On-Demand Dielectrophoretic Nanoprinting*. Advanced Materials, 22, 4701Fabricating nanoscale patterns with sub-10 nm feature size has been an important research target for potential applications in next-generation memories, microprocessors, logic circuits and other novel functional devices. Typically, according to the International Technology Roadmap for Semiconductor (ITRS) from 2009, an 8.9 nm node device is targeted for the year 2024. To achieve this milestone, liquid immersion lithography and extreme ultraviolet (EUV) lithography can be expected to be among the most commonly used techniques for the fabrication of nanopatterns. With liquid immersion lithography using a wavelength of 193 nm and a high numerical aperture (NA), it has been demonstrated that 32 nm features can be patterned (Finders et al., 2008; Sewell et al., 2009). EUV lithography using a short wavelength of 13.5 nm and 0.3-NA exposure tool has also enabled the printing of 22 nm half-pitch lines (Naulleau et al., 2009).

On the other hand, attractive patterning techniques, such as a superlattice nanowire pattern transfer (SNAP) method (Melosh et al., 2003; Green et al., 2007), a mold-to-mold cross imprint (MTMCI) process (Kwon et al., 2005) and a surface sol-gel process combined with photolithography (Fujikawa et al., 2006), are currently proposed and pursued actively. The SNAP method, which is based on translating thin film growth thickness control into planar wire arrays, has enabled the production of molecular memories consisting of 16 nm wide titanium/silicon nanowires. The MTMCI process using silicon nanowires formed by spacer lithography, in which nanoscale line features are defined by the residual part of a conformal film on the edges of a support structure with the linewidth controlled by the film thickness, has been used to produce a large array of 30 nm wide silicon nanopillars. With the surface sol-gel process combined with photolithography, where the linewidth is determined by the thickness of coating silica layer on the resist pattern, the size reduction and the large area of sub-20 nm silica walls have been achieved.

Recently, we have proposed a double nano-baumkuchen (DNB) structure, in which two thin slices of alternating metal/insulator nano-baumkuchen are attached so that the metal/insulator stripes cross each other, as part of a lithography-free nanostructure fabrication technology (Ishibashi, 2003 & 2004; Kaiju et al., 2008; Kondo et al., 2008). The schematic illustration of the fabrication procedure is shown in Fig. 1. First, the metal/insulator spiral heterostructure is fabricated using a vacuum evaporator including a film-rolled-up system. Then, two thin slices of the metal/insulator nano-baumkuchen

Lithography-Free Nanostructure Fabrication Techniques Utilizing Thin-Film Edges 571

Fig. 1. Fabrication procedure of DNB structures and a schematic illustration of QC devices.

The fabrication method of QC devices is shown in Fig. 2. First, Ni thin films were thermally evaporated on PEN organic substrates (2 mm width, 10 mm length and 25 m thickness) in a high vacuum chamber at a base pressure of ~10-8 torr. PEN organic substrates of TEONEX Q65 were supplied by Teijin DuPont Japan and cut down from 5 to 2 mm width using a slitter in a clean environment. A boron nitride crucible N-1, made by DENKA, and a tungsten filament, made by CRAFT, were used for the thermal evaporation of Ni thin films. A heat-block stainless plate with a hole was inserted between the Ni vapor source and the PEN substrate. The length of the crucible and the aperture size in the stainless plate were designed using a geometrical simulation to evaporate uniform Ni films in-plane to PEN substrates. The temperature near PEN substrates was less than 62 °C, which was lower than the glass transition temperature *T*g of 120 °C for PEN substrates. The pressure during the evaporation was 10-5 torr and the growth rate was 1.0-1.5 nm/min at an evaporation power of 280-350 W. Then, fabricated Ni/PEN films were sandwiched between two polymethyl methacrylate (PMMA) resins using epoxy. The volume of the PMMA resin was 6 × 3 × 3 mm3. The edge of PMMA/Ni/PEN/PMMA structure was polished by chemical mechanical polishing (CMP) methods using alumina (Al2O3) slurries with particle diameters of 0.1, 0.3 and 1.0 m. The polishing pressure was 6.5 psi and the platen rotation speed was 75 rpm. Finally, two sets of polished PMMA/Ni/PEN/PMMA structures were prepared and attached together with their edges crossed in a highly clean environment of ISO class minus 1. The attachment pressure was 0.54 MPa and no glue was used. This is a basic process in fabricating QC devices. When

**2. Fabrication and evaluation method of QC devices**

**2.1 Fabrication method of QC devices**

are cut out from the metal/insulator spiral heterostructure. Finally, the two thin slices are attached together face to face so that each stripe crosses in a highly clean environment (Ishibashi et al., 2005; Kaiju et al., 2005; Rahaman et al., 2008). Utilizing this DNB structure, we can expect to realize high density memory devices, the crossing point of which can be scaled down to ultimate feature sizes of a few nanometers thanks to their atomic-scale resolution of the film thickness determined by the rate of metal deposition, ranging from 0.01 to 1 nm/s. This DNB structure also gives a huge potential impact and importance of uniting bottom-up structures with top-down systems (Ishibashi, 2003). One element of the DNB structure is called a quantum cross (QC) device, which consists of two metal thin films (nanoribbons) having the edge-to-edge configuration as shown in Fig. 1 (Ishibashi, 2004; Kondo et al., 2006; Kaiju et al., 2008). In this QC device, the area of the crossed section is determined by the film thickness, in other words 1-20 nm thick films can produce 1×1-20×20 nm2 nanoscale junctions. Since the vacuum evaporation has good spatial resolution of one atomic layer thickness, the junction size of QC devices could ultimately be as small as a few ångströms square (10-2nm2). This method offers a way to overcome the feature size limit of conventional optical lithography. When molecularbased self-assembled monolayers (SAMs), such as rotaxanes (Chen et al., 2003), catenanes (Balzani et al., 2000) and pseudorotaxanes (Pease et al., 2001), are sandwiched between the two thin metal films, QC devices can serve as novel non-volatile memory devices and switching devices. Moreover, when magnetic materials, such as Fe, Co and Ni, are used for the two thin metal films, QC devices can work as nanoscale spin injectors and tunneling magnetoresistance (TMR) devices. Among these devices, the resistance of the electrodes (thin metal films) can be reduced down to ~k since the width of films can be easily controlled to the one as long as ~mm. This makes it possible to realize a highly sensitive detection for a junction resistance and to apply QC devices to high-frequency devices.

In this chapter, we present structural and electrical properties of Ni/polyethylene naphthalate (PEN) films used as electrodes in QC devices (Kaiju et al., 2009 & 2010) and current-voltage (*I-V*) characteristics for three types of QC devices. The three types of QC devices are as follows: (i) Ni/Ni QC devices (17 nm linewidth, 17×17 nm2 junction area), in which two Ni thin films are directly contacted with their edges crossed (Kondo et al., 2009; Kaiju et al., 2010), (ii) Ni/NiO/Ni QC devices (24 nm linewidth, 24×24 nm2 junction area), in which NiO thin insulators are sandwiched between two Ni thin-film edges (Kaiju et al., 2010) and (iii) Ni/poly-3-hexylthiophene (P3HT): 6, 6-phenyl C61 butyric acid methyl ester (PCBM)/Ni QC devices (16 nm linewidth, 16×16 nm2 junction area), in which P3HT:PCBM organic molecules are sandwiched between two Ni thin-film edges (Kaiju et al., 2010; Kondo et al., 2010). In our study, we have successfully fabricated various types of QC devices with nano-linewidth and nano-junctions, which have been obtained without the use of electron-beam or optical lithography. Our method will open up new opportunities for the creation of nanoscale patterns and can also be expected as novel technique beyond conventional lithography. Furthermore, we present the calculation results of the electronic transport in Ni/organic-molecule/Ni QC devices and discussed their possibility for switching devices. According to our calculation, a high switching ratio in excess of 100000:1 can be obtained under weak coupling condition. These results indicate that QC devices fabricated using thin-film edges can be expected to have potential application in next-generation switching devices with ultrahigh on-off ratios.

Fig. 1. Fabrication procedure of DNB structures and a schematic illustration of QC devices.


Contents

Preface

Software usability should be central to any software development, especially when it comes to software that is going to be used by people. What, if not the *ease of use* of technology, could be a sign of its purpose and intent to help and assist humans?

We can all agree that software usability is not yet at the fore of every development project. For far too long, technology has come first, solving problems indeed, but far too often *forcing* users to do so in certain ways, often designed without the

For this book, we set out to put together a collection of high-quality contributions that could help broaden developers' and non-developers' minds alike when it comes to considering software usability. Approachable and easy to understand, this volume presents novel research and experiences in the field of software usability. We hope readers, who may not be software *makers* but who are undoubtedly software *users*,

**Drs. Laura M. Castro and David Cabrero**

Intercultural User Interface Consulting,

University of A Coruña,

**Dr. Rüdiger Heimgärtner**

A Coruña, Spain

Berlin, Germany

Department of Computer Science and Information Technologies,

will find the information contained herein accessible and useful.

involvement of said users.