*Article Salvia officinalis* **L. Essential Oil: Characterization, Antioxidant Properties, and the Effects of Aromatherapy in Adult Patients**

**Maria-Daniela Mot 1,†, Simona Gavrilas, 2,† , Andreea I. Lupitu 2,† , Cristian Moisa 2 , Dorina Chambre 2 , Delia Mirela Tit 1,3 , Mihaela Alexandra Bogdan 1 , Adina-Maria Bodescu 4 , Lucian Copolovici 2 , Dana Maria Copolovici 2,\* and Simona Gabriela Bungau 1,3**

**Abstract:** The purpose of this study is to reveal the chemical and biochemical characteristics and the potential aromatherapy applications of the essential oil (EO) of *Salvia officinalis* (common sage) within a hospital environment. The chemical composition was determined by gas chromatography with mass spectrometry and ATR-FTIR spectroscopy. Three types of sage EOs were included in this study: two commercial oils and one oil obtained by in-house hydrodistillation. Based on the findings, these EOs were included in different chemotypes. The first two samples were similar to the most common chemotype (α-thujone > camphor > 1,8-cineole > β-thujone), while the in-house sage EO revealed a high content of 1,8-cineole, borneol, α-thujone, similar to the Dalmatian type. The latter sample was selected to be evaluated for its antioxidant and medical effects, as borneol, a bicyclic monoterpene, is known as a substance with anesthetic and analgesic effects in traditional Asian medicine. The study suggests that the antioxidant capacity of the sage EO is modest (33.61% and 84.50% inhibition was determined by DPPH and ABTS assays, respectively), but also that the inhalation of sage EO with high borneol content by hospitalized patients could improve these patients' satisfaction.

**Keywords:** *Salvia officinalis*; essential oil; chemical analysis; antioxidant activity; aromatherapy; inhalation

### **1. Introduction**

Essential oils (EOs) have proven their specific applications in different areas, including in human health, as a treatment for various disorders (migraine [1], skin disorders [2], fatigue [3,4], stroke [5], sleep disorders [6,7], endocrine disorders [8], depressive disorders [9], etc.). In medicine, EOs are considered good candidates as complementary and alternative treatment components due to their antimicrobial properties [10], possible anesthetic [11], or immunostimulatory effects [12]. An EO can be used alone and/or in synergetic associations with other EOs [13,14].

The qualitative composition of essential oils is determined by the genome of the particular plant and the variation caused by several environmental conditions such as daylight, temperature, and light. The conditions mentioned above select a pattern of seasonal fluctuations in plant metabolites. These conditions vary predictably and significantly throughout the vegetation interval, usually recurrent each year [15].

*Salvia officinalis* L. is one of the most widely used sources for EOs in traditional medicine. Even though the chemical composition of *Salvia officinalis* EOs has already been

**Citation:** Mot, M.-D.; Gavrilas, , S.; Lupitu, A.I.; Moisa, C.; Chambre, D.; Tit, D.M.; Bogdan, M.A.; Bodescu, A.-M.; Copolovici, L.; Copolovici, D.M.; et al. *Salvia officinalis* L. Essential Oil: Characterization, Antioxidant Properties, and the Effects of Aromatherapy in Adult Patients. *Antioxidants* **2022**, *11*, 808. https:// doi.org/10.3390/antiox11050808

Academic Editors: Filomena Nazzaro and Dimitrios Stagos

Received: 20 March 2022 Accepted: 18 April 2022 Published: 21 April 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

determined, the composition of EOs is very complex, depending on the plant part, time of harvesting, season, genetic diversity, climate, and meteorological conditions. Different studies have used gas-chromatography techniques [16–19] or FTIR [20] to determine *Salvia officinalis* L. EOs' chemical composition. It is claimed that the antimicrobial properties of EOs are related to an extended alcoholic profile. Also, it was assessed that skin injury restoration is enhanced in the case of using plant extracts containing increased amounts of ketones [2,21]. EOs are rapidly absorbed via the skin into the bloodstream and thus travel to the brain and other organs [1,4,5,9,22–28]. A reduction of migraine discomfort is the result of the combination of the following factors: neurogenic inflammation, as well as a decrease in distress perception, along with blood vessel dilation [1]. The same processes may be responsible for the positive response to aromatherapy and synergic musical therapy obtained for anxiousness and/or suffering states recorded in different post-procedural phases of adult patients [29,30]. For insomnia improvement, the olfactory route is considered based on the comparative results regarding inhalation versus the transdermal pathway [31]. The inhalation of EOs may stimulate the immune and limbic systems responsible for the body's state of well-being and emotional integration. The quality of life in burnout cases, determined by different diseases, such as chronic hemodialysis [32] or long periods of hard work, could be improved through aromatherapy [33,34]. Several reports are focused on evaluating essential oils' toxicity profile [35–37]. For example, sage essential oil toxicity has been assessed on rat hepatocytes [36], and it is not toxic at concentrations below 200 nL mL−1 . A report presents an accidental exposure to a newborn and a toddler to sage EO that lead to generalized tonic-clonic seizures [38]. Not all medical aspects are fully elucidated so far, as relevant elements such as the toxicological profile and different interactions still need to be clarified and studied.

It is used to treat colds, tuberculosis, bronchitis, gastrointestinal diseases, inflammation, and presents antibacterial, antifungal [39], antitumor [37,40–42], and antioxidant properties [43]. With respect to *Salvia officinalis,* different mechanisms of action have been proposed. Most of the research has been focused on the influence of *Salvia officinalis* on mental functions. Memory enhancement is thought to be a consequence of *Salvia officinalis*' stimulating effect on the nicotinic and muscarinic receptors. Studies have also been conducted on the inhibitory effect on acetylcholinesterase, a compound considered to have a major role in Alzheimer-specific symptom progress [44]. At 100 mg/mL concentration, *Salvia officinalis* extract revealed high activity towards *Escherichia coli*, which is ordinary with respect to *Staphylococcus aureus* and *Pseudomonas aeruginosa*, and low in the case of *Bacillus subtilis* [45]. The use of the extracts in the presence of Hp-2-Minh ligand improved their biocidal characteristic even at lower concentrations. Research has proven the antimicrobial potential of EOs. This finding has medical significance, as it justifies reducing the use of synthetic medicinal products and utilizing EOs as a viable alternative in cases of drug-resistant pathogens.

Aromatherapy refers to the use of volatile compounds or fragrances from EOs obtained from plants, typically by inhalation, to prevent or cure diseases, infections, and indispositions. For alleviating pain, nausea, and anxiety, an ancient practice—clinical aromatherapy—is gaining attention in contemporary health services, where the intention of both the consumers and clinicians is to minimize the usage of medications. Thus, using integrative and complementary therapies is increasing, and scientific proof for the integrative therapies continues to flourish [46].

Different EOs were included in complementary and alternative treatment (such as aromatherapy) and were evaluated in a hospital setting in the following instances: peppermint EO for nausea and vomiting in women [25], lavender EO [47], *Salvia officinalis* EO for reducing nausea and vomiting in patients with cancer undergoing chemotherapy [48], *Rosa damascene* EO to decrease anxiety and increase sleep quality in cardiac patients [49], and *Citrus aurantium* to reduce anxiety in patients with acute coronary syndrome [50]. In the case of hospitalized patients, one of the main issues that arise is the decrease of peace of mind and the increase in anxiety during the hospitalization period [51].

Therefore, the present investigation aimed to assess the chemical composition and antioxidant activity of the essential oil of *S. officinalis* obtained from aerial parts of the plant and evaluate aromatherapy's influence on patients in a hospital setup. The results of this study can be a starting point for the development of Aromatherapy Programs in Patient Care Settings in Romania.

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

All the reagents and solvents used in the experimental part were of adequate analytical or chromatographical grade (Sigma Aldrich, Fluka, Switzerland, and Merck, Darmstadt, Germany).

### *2.1. Sample Collection and Preparation*

The EO named L-SEO was obtained by hydro-distillation from the dried aerial parts of *Salvia officinalis* plants grown in Arad County, Romania (coordinates 46◦1003000 N 21◦1804500 E, sample harvested in June 2019). The EO was stored in glass vials at +4 ◦C until further analysis. The samples B-SEO and EG-SEO are sage EOs commercially available on the Romanian market.

### *2.2. GC-MS Determination of the Chemical Composition of the Essential Oil from Salvia officinalis*

The constituents of the EOs were determined by the gas chromatography method, using a gas chromatograph (GC) (Shimadzu2010, Kyoto, Japan) coupled with a triple quadruple mass spectrometer (MS) (TQ 8040, Shimadzu, Kyoto, Japan). The column used was an Optima 1 MS (30 m × 0.25 mm i.d., with a film thickness of 0.25 mm). At a flow rate of 1 mL/min, Helium was used as a carrier gas. The procedure for separating and quantifying EO components is described above [52]. The compounds from the analyzed samples were identified based on their mass spectra using the NIST 14 and Wiley 09 mass spectrum libraries (Scientific Instrument Services, Palmer, MA, USA). The Kovats retention indices for the identified compounds were calculated using the C8-C40 alkane standard. All analyses were performed in triplicate.

### *2.3. ATR-FTIR Spectroscopy*

The ATR-FTIR spectra of *Salvia officinalis* L. EOs (L-SEO, B-SEO, and EG-SEO) were obtained on the wavelength range between 600 and 4000 cm−1 with a Bruker Vertex 70 (Bremen, Germany) spectrophotometer equipped with a Pike Miracle ATR cell. A sample volume of ~10 µL from each EO was placed directly on the surface of the ZnSe ATR crystal in the Teflon depression and covered with a metal cover. To avoid evaporation of the sample, a black outer ring was screwed on to press the Teflon depression tightly on the crystal. At the same time, the upper handle of the ATR cell was rotated so that its slightly concave stainless steel tip applied pressure against the metal cover.

The experimental spectra of the samples were recorded with a resolution of 4 cm−1 , and 32 scans were accumulated per spectrum. The spectra were obtained in duplicate for each sample, and the average spectrum of the two measurements was calculated. Before each ATR measurement, the ZnSe crystal was carefully cleaned with isopropyl alcohol, and an air background spectrum was performed.

The OPUS software, version 6.5 from Bruker (Germany), was employed for spectra acquisition, minimum–maximum normalization, baseline correction, and identifying the wavelength value corresponding to the maximum absorbance of the recorded FT-IR bands.

### *2.4. Antioxidant Activity (DPPH and ABTS Assays)*

The L-SEO sample's antioxidant capacity was evaluated using two spectrophotometric assays (DPPH assay and ABTS assay), as reported earlier [22]. A UV-VIS spectrophotometer Specord 200 (Analytik Jena, Jena, Germany) and a 10 mm quartz cuvette were used in the procedure described above. Briefly, for the DPPH assay, we mixed a 0.1 mL control sample with 3 mL of 0.2 mM ethanolic DPPH• solution. The absorbance was recorded after 60 min of reaction in the dark at λ = 517 nm. Positive controls containing 0.02–4 mM Trolox solutions were prepared. The data are expressed in mg Trolox/L and inhibition (%).

For the ABTS assay, we mixed 0.5 mL sample or control with 1 mL ABTS\* solution, prepared 16 h before from ABTS reagent and 2.45 mM aqueous solution of sodium persulfate. The absorbance was recorded at λ = 734 nm, after 10 min of reaction time in the dark. Positive controls containing 0.02–1.0 mM Trolox solutions were prepared. The data are expressed in mmol TEAC/L (TEAC: Trolox Equivalent Antioxidant Capacities) and inhibition (%). All the experiments were performed in triplicate.

### *2.5. Aromatherapy Effects: Clinical Application*

This study was performed between August 2019 and August 2020, as a randomized, single-blind study involving adult patients at the Lipova City Hospital, Arad County, Romania.

In total, 174 hospitalized patients aged 23–85 years were enrolled, meeting the following inclusion criteria: adult patients with chronic conditions who have had at least one hospitalization in the same section in the past, an unimpaired sense of smell, no psychiatric pathologies, ability to communicate, and minimum 4 days and maximum 5 days hospital stay. Exclusion criteria included surgery intervention and unwillingness to participate.

Patients were randomized into two groups: 50 in the control group and 124 in the aromatherapy group; no patients were excluded, and no patients quit the study. Random allocation of the patients was made by hospital personnel who were not involved in the data collection or data analysis, depending on the number of patients hospitalized in each room. During the hospitalization period, patients admitted to the same ward were included in either the aromatherapy group or the control group (there were no patients from both groups at the same time in a ward). For the patients enrolled in the aromatherapy group, on a daily basis, a hospital staff member prepared the cotton disc with two drops of sage EO (L-SEO sample) that was kept on the patient's pillow for a minimum of 30 min as the patient inhaled the volatile compounds. The patients from the control group received only routine care.

Only one EO (L-SEO) was used in aromatherapy, which was selected after determining the chemical composition.

Descriptive information form: A questionnaire was prepared by the authors of this study, including the patients' related characteristics such as gender, age, weight, educational level, habits, personal evaluation, and health status. The questionnaire was completed by respondents on the last day of the hospitalization and was used to determine the following: (a) the demographic profile of the participants in terms of age, gender, weight, educational level, social status, health status in the last year, marital status, the use of aroma indoor and of perfumes, the use of sedatives/anxiolytics and the presence of some allergies; (b) the evaluation on the quality of in-hospital services in the present hospitalization stay, with seven possible answers: very weak, weak, acceptable, improved, good, very good, and excellent.

The study was approved by the Ethics Committee of Lipova City Hospital (no. 62/1 July 2019) and was conducted following the ethical principles of the Declaration of Helsinki [53]. The patients provided their written informed consent after a verbal and written explanation of the study protocol.

### *2.6. Statistical Analysis*

Data were processed via GraphPad Prism (version 5.0 for Windows, GraphPad Software, San Diego, CA, USA), and F values (at *p* < 0.05) were considered statistically significant. The *t*-test, chi-squared test, Kruskal-Wallis test, Fisher's exact test, Friedman test, and the Mann-Whitney test were used to compare qualitative variables in two groups.

### **3. Results**

### *3.1. Chemical Analyses*

### 3.1.1. GC-MS Analyses

Figure 1 depicts the chromatograms obtained for the three *Salvia officinalis* essential oils samples investigated in this study, and Table 1 shows their composition as determined by GC-MS method. The EOs' constitutive elements are listed according to the elution time. In the liquid phase, 47 compounds were separated.

**Table 1.** *Salvia officinalis* L. essential oil chemical composition (% TIC) determined by GC-MS analyses.

MH—monoterpene hydrocarbons; MO—oxygenate monoterpene; SH—sesquiterpene hydrocarbons; SO—oxygenated sesquiterpene, KI—Kovats retention index.

*2.6. Statistical Analysis*

*3.1. Chemical Analyses*  3.1.1. GC-MS Analyses

**3. Results**

Table 1.

effects [56].

**Figure 1.** GC-MS chromatograms obtained for the investigated *Salvia officinalis* essential oil samples: B-SEO, pink; EG-SEO, black; L-SEO, blue.

Data were processed via GraphPad Prism (version 5.0 for Windows, GraphPad Software, San Diego, CA, USA), and F values (at *p* < 0.05) were considered statistically significant. The t-test, chi-squared test, Kruskal–Wallis test, Fisher's exact test, Friedman test, and the Mann–Whitney test were used to compare qualitative variables in two groups.

Figure 1 depicts the chromatograms obtained for the three *Salvia officinalis* essential oils samples investigated in this study, and Table 1 shows their composition as determined by GC-MS method. The EOs' constitutive elements are listed according to the elu-

The results obtained follow the data presented in the literature [43]. The monoterpenes are the most represented, whilst the differences between the hydrocarbons (1,8-cineole, 17.98%; β-pinene, 10.52%; camphene, 8.73%) and the oxygenated (borneol, 15.86%; β-thujone, 1.34%) ones are modest. The sesquiterpenes have a prevalence lower than 9% (α-humulene, 8.64%; β-caryophyllene, 5.66%; caryophyllene oxide, 0.22%). The analyzed samples have a different composition in comparison to major compounds. B-SEO and EG-SEO have two major compounds (namely α-thujone and camphor), whilst L-SEO has four major compounds (namely 1,8-cineol, borneol, β-pinene, and α-thujone). L-SEO has a closer composition to the marocain one, possibly due to the plant growing conditions. B-SEO and EG-SEO have a composition similar to the EO obtained in Turkey, as shown in

The differences in the chemical composition of the analyzed EOs could be attributed to several factors such as environmental factors, growth conditions, and time of harvesting [19,43,54]. The results obtained in this present study agree with previous research that concluded that 1,8-cineole, camphor, and α- and β-thujone are the main components of *Salvia officinalis* EOs [17–19,37]. As shown earlier, (+)-borneol is the only enantiomer found in the EO of Salvia species [55]; this compound is associated with analgesic and sedative

tion time. In the liquid phase, 47 compounds were separated.

The results obtained follow the data presented in the literature [43]. The monoterpenes are the most represented, whilst the differences between the hydrocarbons (1,8-cineole, 17.98%; β-pinene, 10.52%; camphene, 8.73%) and the oxygenated (borneol, 15.86%; βthujone, 1.34%) ones are modest. The sesquiterpenes have a prevalence lower than 9% (α-humulene, 8.64%; β-caryophyllene, 5.66%; caryophyllene oxide, 0.22%). The analyzed samples have a different composition in comparison to major compounds. B-SEO and EG-SEO have two major compounds (namely α-thujone and camphor), whilst L-SEO has four major compounds (namely 1,8-cineol, borneol, β-pinene, and α-thujone). L-SEO has a closer composition to the marocain one, possibly due to the plant growing conditions. B-SEO and EG-SEO have a composition similar to the EO obtained in Turkey, as shown in Table 1.

The differences in the chemical composition of the analyzed EOs could be attributed to several factors such as environmental factors, growth conditions, and time of harvesting [19,43,54]. The results obtained in this present study agree with previous research that concluded that 1,8-cineole, camphor, and α- and β-thujone are the main components of *Salvia officinalis* EOs [17–19,37]. As shown earlier, (+)-borneol is the only enantiomer found in the EO of Salvia species [55]; this compound is associated with analgesic and sedative effects [56].

### 3.1.2. ATR-FTIR Spectroscopy

The obtained Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectra for the investigated *Salvia officinalis* L. EOs are depicted in Figure 2, and the wavelengths values (in the 600–4000 cm−1 range) for all recorded peaks are presented in Table 2. The different chemical compositions of the investigated EOs samples led to obtaining major differences in the intensity of the peaks located at the following wavelengths (L-SEO/B-SEO/ED-SEO): 3459/3474/3474 cm−1 , 1741/1741/1741 cm−1 , 1642/1637/1638 cm−1 , 1054/-/- cm−1 , -/1045/1045 cm−1 , 982/983/983 cm−1 , and 877/880/879 cm−1 .

44 1593 Unidentified 2.34 ± 0.19 1.88 ± 0.18

45 1594 Unidentified 0.35 ± 0.03 0.61 ± 0.08 0.6 ± 0.09 46 1603 Unidentified 0.08 ± 0.01 47 1607 Unidentified 0.6 ± 0.08

3.1.2. ATR-FTIR Spectroscopy

bons; SO—oxygenated sesquiterpene, KI—Kovats retention index.

1054/-/- cm−1, -/1045/1045 cm−1, 982/983/983 cm−1, and 877/880/879 cm−1.

**Figure 2.** The ATR-FTIR spectra of the *Salvia officinalis* L. essential oil samples. **Figure 2.** The ATR-FTIR spectra of the *Salvia officinalis* L. essential oil samples.

MH—monoterpene hydrocarbons; MO—oxygenate monoterpene; SH—sesquiterpene hydrocar-

The obtained Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectra for the investigated *Salvia officinalis* L. EOs are depicted in Figure 2, and the wavelengths values (in the 600–4000 cm−1 range) for all recorded peaks are presented in Table 2. The different chemical compositions of the investigated EOs samples led to obtaining major differences in the intensity of the peaks located at the following wavelengths (L-SEO/B-SEO/ED-SEO): 3459/3474/3474 cm−1, 1741/1741/1741 cm−1, 1642/1637/1638 cm−1,

### 3.1.3. Antioxidant Capacity

The antioxidant capacity of the sage EO (sample L-SEO) was evaluated by using two methods: DPPH assay (inhibition 33.61 ± 2.12%, antioxidant activity 0.81 ± 0.11 mg Trolox/L) and ABTS assay (inhibition 84.50 ± 2.23%, antioxidant activity 0.81 ± 0.03 mmol TEAC/L). All analyses were performed in triplicate. The data are expressed as mean ± STDEV.

### *3.2. Patients' Characteristics*

The mean age of the patients from the control group was 58.74 years whilst that of the salvia EO group was 52.34 years, with a *p*-value of 0.3530 (*t*-test). There are no significant differences between the two groups with respect to the following characteristics: gender, weight, educational level, social status, health status in the last year, marital status, the use of aroma indoor and perfumes, the use of sedatives/anxiolytics, and the presence of some allergies (Table 3). Between the groups, there is a significant difference in the variables of the consumption of alcohol, smoking, and the presence of chronic disease.

**Table 3.** Patients' demographic and clinical characteristics.

n—number of patients; \* *t*-test; \*\* chi-squared test; \*\*\* Kruskal-Walis test; \*\*\*\* Fisher's exact test.

The patients' perception of the quality of in-hospital services and, thus, their wellbeing after receiving treatment while hospitalized were assessed by answering questions

designed with seven possible answers: very weak, weak, acceptable, improved, good, very good, and excellent. Group comparison was made using the Mann-Whitney U test. No significant differences were observed in the between-group comparison (*p* = 0.8969, U = 23). Most patients in both groups rated in-hospital services as excellent and very good (64% in the control group and 76% in the aromatherapy group) (Figure 3). No adverse effects were reported by patients or observed by medical staff in this study. *Antioxidants* **2022**, *11*, x FOR PEER REVIEW 10 of 16

**Figure 3.** Patients' satisfaction with the treatment they received during their current hospitalization **Figure 3.** Patients' satisfaction with the treatment they received during their current hospitalization (Mann-Whitney U test).

### **4. Discussion**

(Mann–Whitney U test).

**4. Discussion**  Much research has focused on investigating sage EO extracted from sage leaves harvested from diverse parts of the world. The percentage of the constituents varies widely due to geographical region, season, environmental conditions, genetic differences, phenological stages, sampling, and extraction methods. The number of detected constituents varies, being around 14–67 (14 with concentrations >1% [41], 15 [62], 22 [17], 23 [43], 42 [19], 44 [63], 67 [64]). Also, sage EO's composition is dependent on the growing ecosystem conditions, as determined in an exhaustive study reported by Russo et al., where 18 samples were collected in 2008–2009 in the south-central part of Italy and investigated [41]. The EOs obtained from sage leaves contained α-thujone (7.8–20.1%), camphor (8.4–20.8%), borneol (2.5–16.9%), γ-murolene (2.9–13.8%), and sclareol (5.9–23.1%) as the major compounds. Recently, another study identified oxygenated monoterpenes (67.7%) and mon-Much research has focused on investigating sage EO extracted from sage leaves harvested from diverse parts of the world. The percentage of the constituents varies widely due to geographical region, season, environmental conditions, genetic differences, phenological stages, sampling, and extraction methods. The number of detected constituents varies, being around 14–67 (14 with concentrations > 1% [41], 15 [62], 22 [17], 23 [43], 42 [19], 44 [63], 67 [64]). Also, sage EO's composition is dependent on the growing ecosystem conditions, as determined in an exhaustive study reported by Russo et al., where 18 samples were collected in 2008–2009 in the south-central part of Italy and investigated [41]. The EOs obtained from sage leaves contained α-thujone (7.8–20.1%), camphor (8.4–20.8%), borneol (2.5–16.9%), γ-murolene (2.9–13.8%), and sclareol (5.9–23.1%) as the major compounds. Recently, another study identified oxygenated monoterpenes (67.7%) and monoterpenes hydrocarbons (19.1%) in the EO from sage leaves harvested in June 2020 in Tuscany, Italy. The main components identified in this EO batch were 1,8-cineole (30.3%), camphor (17.1%), α-thujone (9.7%), camphene (7.9%), and chrysanthenone (6.8%) [43].

oterpenes hydrocarbons (19.1%) in the EO from sage leaves harvested in June 2020 in Tuscany, Italy. The main components identified in this EO batch were 1,8-cineole (30.3%), camphor (17.1%), α-thujone (9.7%), camphene (7.9%), and chrysanthenone (6.8%) [43]. Due to their chemical composition (shown in Table 1), two of the investigated samples (B-SEO and EG-SEO) were included in the more common C1c chemotype, as classified by Craft et al. [65]: α-thujone > camphor > 1,8-cineole > β-thujone, which was reported Due to their chemical composition (shown in Table 1), two of the investigated samples (B-SEO and EG-SEO) were included in the more common C1c chemotype, as classified by Craft et al. [65]: α-thujone > camphor > 1,8-cineole > β-thujone, which was reported in samples from Turkey [17], Brazil [16], Mexico [65], and Croatia [66]. Meanwhile, the L-SEO sample is a chemotype found in the sage EO from the Dalmatian region, where 1,8-cineole is the main component and borneol is in high quantity [67].

in samples from Turkey [17], Brazil [16], Mexico [65], and Croatia [66]. Meanwhile, the L-SEO sample is a chemotype found in the sage EO from the Dalmatian region, where 1,8- The reported FTIR results of the EOs show that the components found in higher concentrations dominate the resulting vibrational spectra, while the components found in

The reported FTIR results of the EOs show that the components found in higher con-

The GC-MS results (Table 1) obtained for L-SEO sample indicate 1,8-cineol (17.98%) and borneol (15.86%) as their main components, which can influence the features of the recorded ATR-FTIR spectrum and the intensity of the obtained bands (Figure 2). The 1,8 cineole (eucalyptol) can be distinguished by the following key bands: 1371 cm−1 (δsym(CH3-

concentrations do not have significant influence [54,68,69]. Ciko et al. [20] showed that FTIR analysis of the EO obtained from *Salvia officinalis* L. leaves indicated the presence of

cineole is the main component and borneol is in high quantity [67].

monoterpenes such as thujones, camphor, 1,8-cineole, and pinene.

low concentrations do not have significant influence [54,68,69]. Ciko et al. [20] showed that FTIR analysis of the EO obtained from *Salvia officinalis* L. leaves indicated the presence of monoterpenes such as thujones, camphor, 1,8-cineole, and pinene.

The GC-MS results (Table 1) obtained for L-SEO sample indicate 1,8-cineol (17.98%) and borneol (15.86%) as their main components, which can influence the features of the recorded ATR-FTIR spectrum and the intensity of the obtained bands (Figure 2). The 1,8-cineole (eucalyptol) can be distinguished by the following key bands: 1371 cm−1 (δsym(CH3-CO) symmetric in plan bending/scissoring), 1266 cm−1 (ν(C-O) stretching from alkyl ether, 1214 cm−1 (νasym(C-O-C) asymmetric stretching), 1080 cm−1 (νsym(C-O-C) symmetric stretching), and 982 cm−1 (ωasym(CH2) asymmetric out-of-plane bending/wagging vibration). Due to the higher content of 1,8-cineole in L-SEO, the 982 cm−1 band is more intense than in B-SEO and EG-SEO, with 844 cm−1 corresponding to ωasym(C-H) asymmetric out-of-plane bending/wagging vibration [20,70,71].

For borneol, the characteristics bands are 3459 cm−1 (νsym(O-H) stretching from Hbonded alcohols), 1303 cm−1 (δsym(O-H) scissoring vibration), 1166 cm−1 (δsym(C-OH) in-plane bending of alcohol groups), 1054 cm−1 (νasym(C-O) asymmetric stretching from primary alcohols), 1106 cm−1 (νsym(C-O) symmetric stretching deformation), and 642 cm−1 (ω(C-O) out-of-plane bending) [20,30].

Other classes of compounds that occur in high proportions in the composition of L-SEO are bicyclic monoterpenes with =CH2 alkyl groups and sesquiterpenes with C=C bonds (sabinene + camphene, 8.87%; β-caryophyllene + α-humulene, 14.3%), with the key bands located at 3066 cm−1 (νsym(=C-H), Csp2 from the terminal vinyl group) and 1642 cm−1 (ν(C=CH2) stretching vibration). Schultz et. al. reported a band due to the stretching vibration of (C=C) located at 1653 cm−1 for sabinene and 1635 cm−1 for βcaryophyllene [70], while Agatonovik-Kustin et al. mentioned a vinyl group vibration at 1635–1650 cm−1 , although the intensity of this peak was very low [70].

Compared to B-SEO and EG-SEO samples, in the case of L-SEO, the 1642 cm−1 band is more evident due to the higher content of compounds with =CH2 and C=C groups, with 1456 cm−1 corresponding to δsym(C-H) and δasym(C-H) in-plane bending of CH3 and -CH2 groups. The C=CH2 in-plane deformation vibration was not recorded as a separate band near ~1410–1420 cm−1 [20], probably being hidden under the -CH3 and -CH2 absorption bands, with 877 cm−1 corresponding to ν(=CH2) methylene out-of-plane deformation due to the strained ring structure with an exocyclic =CH2 group [70]. The higher intensity of the 877 cm−1 band, for the L-SEO compared with B-SEO and EG-ESO, is due to the higher content of compounds with the exocyclic =CH2 group.

In addition, it is mentioned that in the 2850–3000 cm−1 wavelength range, a group of characteristic bands with strong intensity were recorded (2951 cm−1 , 2926 cm−1 , 2875 cm−1 ) due to the symmetric and asymmetric stretching vibration of (C-H) bond from CH3, CH2, and CH. The intensities of these bands are higher for L-SEO than for B-SEO and EG-SEO.

For the B-SEO and EG-SEO samples, the obtained GC-MS results (Table 2) reveal that thujone and (+) camphor are the major components (50.77%) that influence the absorption band intensity. These compounds are monoterpenes with a carbonyl group (>C=O), belonging to the ketones class. In the obtained B-SEO and EG-SEO ATR-FTIR spectra (Figure 3), the band positioned at 1741 cm−1 is attributed to thujones and camphor being specific for (ν(C=O)) stretching vibration of 5-membered cyclic ketones [18,23]. Compared with the L-SEO sample, in the case of B-SEO and EG-SEO, the intensity of the 1741 cm−1 band is higher due to the different chemical compositions of the EOs.

Other absorption bands that showed changes in intensity compared to L-SEO sample, due to the difference in the chemical composition of B-SEO and EG-SEO, were observed at 3474 cm−1 due to (ν(O-H)) stretching vibration, weak band; 1415 cm−1 due to (ν(C=CH2) inplane deformation vibration; 1241 cm−1 due to (ν(C-O)) stretching vibration; and 1045 cm−1 due to (ν(C-O)) stretching vibration from primary alcohols [69].

The main efficient constituents of *Salvia essential* oils are terpenes and polyphenols, with one major bioactivity being represented by their antioxidant action. The antioxidant capacity of the investigated sage EO (L-SEO) reached 33.61% and 84.50% by DPPH and ABTS analyses, respectively, which are values specific to samples rich in 1,8-cineole (60–70% inhibition in [69–72]).

The use of plant-derived essential oils for wellness and to sustain human health is called aromatherapy. In British herbal encyclopedias, the species of the Salvia family have long been renowned for being characterized as compounds that improve memory. Salvia essential oils can be helpful for the enhancement of cognition and mood, as demonstrated by various studies [73].

In this randomized, controlled study, the participants were adult men and women undergoing hospitalization who received or did not receive EOs for daily inhalation. The demographic and clinical data (shown in Table 3) revealed no statistical differences between the groups.

Our results revealed that the level of patient perception of the quality of in-hospital services, when sage EO is inhaled daily by patients, during hospitalization, is not statistically significant, as compared to that of the patients from the control group. The aromatherapy confirmed its beneficial action as a complementary treatment to increase the well-being of patients during hospitalization, as it showed a significant effect on hospitalized patients who evaluated their level of satisfaction as excellent (Figure 3).

The main limitations of this study consist of the somewhat limited number of patients in each group and a lack of medium/long-term follow-up. Further studies with a more extended follow-up period and more in-depth research on the use of sage EO in massage are needed to confirm our findings. Additionally, it is worth noting that the study did not include post-surgery patients. Therefore, our future goal is to develop randomized clinical trials to evaluate the large-scale efficacy of aromatherapy as an alternative therapy in patients with diverse pathologies. On the other hand, some strengths of this study must be highlighted, including the use of a chemically characterized EO in aromatherapy and the increase in some of the patients' satisfaction regarding their hospitalization experience with this approach.

### **5. Conclusions**

The GC-MS analysis of three samples of *Salvia officinalis* L. EOs indicated the presence of terpenes such as 1,8-cineole, thujones, borneol, camphor, sabinene, camphene, and caryophyllenes as the main components. The FTIR results agree with the GS-MS data of investigated samples, which leads to the conclusion that two commercial samples are part of the α-thujone > camphor > 1,8-cineole > β-thujone chemotype. In contrast, the other, in-house sample is part of the 1,8-cineole, borneol chemotype (Dalmatian type). The chemical analysis of EOs used in aromatherapy mechanism is very important, as different biological outcomes could arise based on the major compounds identified. The present results did not reveal a statistically significant difference between groups that inhaled sage EO during hospitalization versus the group that did not. However, more research is needed to investigate the mechanisms responsible for specific effects attributed to EOs to justify the therapeutic effect of sage EO and identify the side effects related to this product.

**Author Contributions:** Conceptualization, D.M.T. and D.M.C.; methodology, M.-D.M., A.I.L., C.M., D.C. and D.M.C.; software, A.I.L., C.M., D.C. and L.C.; validation, A.I.L., C.M., D.C., D.M.C. and L.C.; formal analysis, A.I.L., C.M., A.-M.B., D.C. and L.C.; investigation, M.-D.M., A.I.L., C.M., D.C. and D.M.C.; data curation, A.I.L., D.C., A.-M.B. and D.M.C.; writing—original draft preparation, M.-D.M., S.G., M.A.B. and D.M.C.; writing—review and editing, D.M.T., S.G.B. and D.M.C.; supervision, S.G.B. and D.M.C.; project administration, D.M.C.; funding acquisition, A.I.L. and L.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS—UEFISCDI, project number PN-III-P1-1.1-PD-2019-0349, within PNCDI III.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of Lipova City Hospital (Spitalul Orasenesc Lipova), Romania (no. 62/1 July 2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors wish to thank Lisa Craiut for the professional editing of this paper.

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

### **References**

### *Article*


**In Vivo Detection of Tetrodotoxin in** *Takifugu obscurus* **Based on Solid-Phase Microextraction Coupled with Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry**

**Hengli Meng 1,2, Shui Jiang 1,2,\* , Yin Zhang 3 , Yun Hu 4,\* and Yuan Liu 1,2**

**Abstract:** Pufferfish is nutritious and delicious, but the tetrodotoxin (TTX) that may exist in its body poses a serious safety hazard. It is important to use scientific and effective methods to detect the TTX in pufferfish, but most of the existing methods require complex pre-treatment steps and have sample lethality. The solid-phase microextraction (SPME) technology can be used for in vivo detection due to its advantages such as no solvent demand, simple operation, and fast detection speed. In this study, the GO-PAN@PNE SPME fibers were made via a dipping method, and their extraction effect was verified in the TTX aqueous and spiked fish. The established method has good reproducibility, and the limit of detection of TTX in pufferfish was 32 ng·g−1, and the limit of quantitation was 150 ng·g−1, which can meet the detection needs of pufferfish for safe consumption. This method was used to in vivo detect the *Takifugu obscurus* exposed to the TTX, to determine the content of TTX in the pufferfish muscle. The detection method established in this study can relatively quickly and easily realize the in vivo detection of TTX in the pufferfish, which can provide theoretical support for improvement in the food safety level of the pufferfish.

**Keywords:** tetrodotoxin; *Takifugu obscurus*; solid-phase microextraction; ultrahigh-performance liquid chromatography–tandem mass spectrometry

#### **1. Introduction**

Pufferfish is a general name for a group of poisonous economic fish species of Pelecypoda, containing more than 200 species, distributed in temperate, tropical, and subtropical regions. Pufferfish is rich in flavoring amino acids, nucleotides, and some flavor peptides, which form the unique flavor of pufferfish [1], so it is welcomed by consumers all over the world, especially in China, Japan and Korea. However, the possible presence of tetrodotoxin (TTX) in pufferfish poses a great threat to the safety of pufferfish consumption. Pufferfish poisoning incidents frequently occur all over the world, especially in Asian countries [2,3].

TTX is generally believed to be produced by marine bacteria and enriched in higher organisms such as pufferfish through the food chain and mostly accumulates in the liver and ovaries of pufferfish [4]. Tetrodotoxin is a neurotoxin that can selectively block sodium channels on the surface of muscle and nerve cell membranes when ingested, thereby blocking action potentials and causing nerve and muscle paralysis, which can lead to death in severe cases [5]. Furthermore, tetrodotoxin has high stability, which is difficult to be destroyed by normal cooking methods, and there is no targeted antidote or antitoxin after poisoning [6]. Therefore, it is very important to detect tetrodotoxin in pufferfish using scientific methods to avoid the occurrence of tetrodotoxin poisoning.

**Citation:** Meng, H.; Jiang, S.; Zhang, Y.; Hu, Y.; Liu, Y. In Vivo Detection of Tetrodotoxin in *Takifugu obscurus* Based on Solid-Phase Microextraction Coupled with Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry. *Molecules* **2022**, *27*, 6122. https://doi.org/10.3390/ molecules27186122

Academic Editor: Francesco Cacciola

Received: 19 August 2022 Accepted: 15 September 2022 Published: 19 September 2022

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

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Traditionally, the detection of TTX relies on the mouse bioassay, immunoassay, and instrumental assay. The mouse bioassay is a traditional assay method that determines the TTX content based on the time of death of mice after the injection of sample extracts. Different mouse strains have different susceptibility to TTX [7]. The limit of detection of TTX using Kunming strain mice is 0.56 μg·g−1 [8]. The mouse bioassay is intuitive and has low costs, but it has a high detection limit, poor reproducibility, and no specificity. The principle of an immunoassay is the qualitative or quantitative analysis of the substance to be measured in the sample by the specific binding of antigen and antibody. The commonly used immunoassay for the detection of TTX is the enzyme-linked immunosorbent assay (ELISA) and the colloidal gold strip test [9]. The immunoassay method is highly sensitive, specific, and easy to operate. In instrumental assays, the level of TTX in the sample is obtained by extracting the TTX from the specimen with a specific solution and then measuring the TTX content with an instrument. The LC-MS method is a commonly used instrumental method for TTX detection, which has a low limit of detection and a good linear range [10,11]. All of the above methods can achieve the detection of TTX, and each has its advantages and disadvantages. However, most of the above methods require complex pre-treatment steps and have sample lethality, which destroys the higher commercial value of pufferfish. Therefore, it makes sense to find a rapid in vivo method for detecting TTX. The pre-treatment of samples often requires the biological sample to be killed and the corresponding solvent to be used for the extraction of the substances to be tested, which is not only a complex and cumbersome procedure but also unethical.

The solid-phase microextraction (SPME) technique, first proposed by Professor Janusz Pawliszyn in Canada in 1990 [12], is a solvent-free extraction method. It is a non-exhaustive extraction method, based on the partitioning equilibrium of the substance to be measured in the sample and the extraction coating, a phase equilibrium process. This technique was selected as one of the six greatest ideas in the field of analytical science in the 1990s, together with electrospray ionization and capillary electrophoresis [13]. Since the sampling process of SPME does not require the use of solvents and is less damaging to the analytical system, it can be applied to in vivo testing (in vivo SPME). In vivo SPME does not require extraction, centrifugation, purification, and other processes; rather, it simplifies these steps into one step, which greatly shortens its detection time and significantly improves the sampling efficiency; moreover, in vivo SPME technology is less damaging to the analyte system and is more suitable for the analysis of high-value or rare plants and animals such as pufferfish. In addition, in vivo SPME can more accurately reflect the contents and changes in analytes in living organisms, which is conducive to the tracking study of individual organisms [14]. Given these advantages of in vivo SPME technology, in vivo SPME has been used in studies about plants [15], animals [16,17], insects [18], and even humans [19].

In this work, we applied the SPME technique to the detection of TTX, constructed a rapid analysis method for TTX with a self-made SPME fiber, and attempted to use the fiber for the in vivo detection of TTX, to realize the hazard identification and risk assessment of TTX and provide theoretical support for improving the food safety of pufferfish.

#### **2. Results and Discussion**

#### *2.1. Preparation and Characterization of SPME Fibers*

GO-PAN@PNE fibers with brown color and uniform coating thickness were successfully prepared via a dipping method (Figure 1). In recent years, many new materials are used as novel SPME fiber coatings, including some carbon materials such as graphene oxide (GO) [20]. GO is an oxide of graphene and has been used for the SPME of various polar substances [21–23]. After oxidation treatment, GO still maintains the layer structure of graphite, and oxygen-containing groups that are highly hydrophilic and have a large specific surface area, such as carboxyl, hydroxyl, and epoxy groups, are introduced on each layer of graphene monoliths [24]. The aforementioned advantages make the GO a potential material with good adsorption capacity to TTX. The PAN is used as a biocompatible polymer in biomedical applications, such as dialysis and ultrafiltration. Its high

levels of chemical and mechanical stability allow PAN to be used as an ideal stationary phase fixative [15]. The NE, formed when N-methyl is removed from epinephrine, is a catecholamine neurotransmitter that shares a similar oxidative polymerization process with the common neurotransmitter dopamine [25]. Polynoradrenaline (PNE) is the product of a polymerization reaction, and it is capable of sheath wrapping on its own and possesses good biointerfacial properties. In this study, PNE was used to improve the extraction performance and corrosion resistance of the SPME fibers.

**Figure 1.** The GO-PAN@PNE fibers made via dipping method (**a**) and the in vivo sampling in the dorsal–epaxial muscle of living pufferfish using the SPME fiber (**b**).

During heat treatment, the carboxyl group on GO sheets nucleophilically attacks the carbon of the cyano group to initiate the anionic cyclization of the PAN molecular chain, which in turn connects GO and PAN to form a more stable membrane structure [26]. Graphene oxide–polyacrylonitrile composites have applications in the fields of electrochemistry, dye removal, and VOC adsorption [27–30] but have been rarely used in direct immersion SPME.

A Nova Nano-SEM 450 field-emission scanning electron microscope (FESEM, FEI, USA) was used to characterize the fibers. The fiber surfaces were observed at magnifications of 400, 800, and 3000 (at higher magnifications, the coating was damaged by the high-energy electron beam). As shown in Figure 2, at lower magnifications, the overall surface of the fiber coating showed a smooth and uniform morphology without large bumps, depressions, and agglomerates, and this uniform distribution facilitated the reproducibility of the fibers. At higher magnification, it can be observed that the surface of the fiber gradually became rougher and appeared folded and porous. The DMF was evaporated from the coating solution, and the remaining PAN and GO were fixed on the surface of the fiber, which formed a stable structure. Such a structure was beneficial to increasing the contact area and improving the loading capacity of the fiber, which enhanced the extraction performance.

**Figure 2.** SEM images of the GO-PAN@PNE fiber in different magnifications: (**a**) 400×; (**b**) 800×; (**c**) 3000×).

#### *2.2. In Vitro Evaluation of SPME Fibers*

The extraction of a 1 μg·mL−1 aqueous solution of TTX and 1 μg·g−1 spiked fish was performed using blank PAN fibers, GO-PAN fibers, and GO-PAN@PNE fibers, respectively,

to evaluate the extraction performance of the self-made fibers. As shown in Figure 3, the extraction performance of the SPME fibers with the addition of GO was significantly improved, compared with the blank PAN fibers, because GO has a large specific surface area and strong adsorption to small molecules. Furthermore, the surface of GO contains oxygen-containing groups that are highly hydrophilic and can produce hydrogen bonding and induce forces on tetrodotoxin molecules during the extraction process. It can also be observed that the extraction amount of TTX in the fish meat was lower than that in the aqueous solution when the fibers were not wrapped with the PNE bionic sheath, while the GO-PAN@PNE fibers with the addition of the PNE bionic sheath had better extraction performance in the fish meat. This is because the fish meat samples not only contained TTX but also biomolecules such as proteins, which may reduce the loading capacity of the fibers by binding to the surface and competing with TTX for the adsorption sites during the extraction process. At the same time, the biomolecules in the elution solvent reduced the ionization efficiency of the sample during the LC-MS/MS analysis. These biomolecules that adhere to the fiber surface are not easily cleaned off, thus reducing the loading capacity of the fibers for the next use. PNE with good resistance to biocorrosion can inhibit the attachment of biomacromolecules such as proteins to the surface and enhance the extraction performance of the fibers in fish. Meanwhile, the interface of the PNE sheath layer is hydrophilic and does not hinder the binding of TTX to the coating.

**Figure 3.** In vitro evaluation of extraction performance of PAN, GO-PAN, and GO-PAN@PNE fibers.

#### *2.3. Method Optimization*

The results of SPME were related to various factors. The extraction time, elution solvent, and elution time were used as variables in turn to investigate the effects of these factors on the extraction amount and to select the best experimental method for subsequent evaluation and in vivo SPME extraction.

#### 2.3.1. Extraction Time

The SPME process is essentially an equilibrium of the analytes between the matrix and the extraction phase, which requires a certain amount of time to reach equilibrium. Extraction was carried out in the fish meat samples with 1 μg·g−1 TTX in the time range of 5–60 min. As shown in Figure 4a, the extraction efficiency increased with the extraction time and had little change after 30 min; thus, the extraction time was set as 30 min for subsequent experiments. This extraction time is close to some previous in vivo SPME studies [15,17,31].

**Figure 4.** Optimization of SPME method using GO-PAN@PNE fibers in spiked fish samples: (**a**) extraction time; (**b**) elution solvent; (**c**) elution time.

#### 2.3.2. Elution Solvent

The elution solvent greatly affects the efficiency of analyte removal from the fibers. Pure water, 50% (*v*/*v*) methanol/water, pure methanol, 50% (*v*/*v*) acetonitrile/water, and pure acetonitrile were selected as elution solvents for the experiments to select the most suitable solvent for elution. It could be observed that pure methanol had the best effect on the elution of TTX, as shown in Figure 4b, so methanol was selected as the best elution solvent, which is consistent with the previous research [31].

#### 2.3.3. Elution Time

Similar to the extraction process, the elution process also needs a certain time to reach equilibrium. As shown in Figure 4c, the best extraction performance was obtained when the elution time was 30 min.

#### *2.4. Method Evaluation*

The proposed in vivo SPME extraction method was further evaluated with the linear range, the limit of detection (LOD), the limit of quantitation (LOQ), and reproducibility. Eight concentration gradients of 5, 10, 50, 100, 250, 500, 750, and 1000 ng·mL−1 (or ng·g−1) were determined in the TTX aqueous and TTX spiked fish meat, respectively. As shown in Table 1, the linear range was good in the concentration range of 150–1000 ng·g−1, and the *R*2 values were 0.9955 in the aqueous and 0.9895 in the spiked fish. The LOD and LOQ of the method were calculated based on the signal-to-noise ratios of 3 and 10, respectively. The LOD and LOQ of the fibers in the TTX aqueous solution were lower than those in the TTX spiked fish meat, which is probably because the biomolecules in the fish samples bound to the fiber surface and competed with TTX for the adsorption sites during extraction, leading to a decrease in the specificity of the fibers for TTX.

**Table 1.** Linear range, limit of detection (LOD), limit of quantitation (LOQ), and reproducibility.

The LOD for the fish analyzed with this method is far lower than that of the traditional mouse biological method (0.56 μg·g−1) [8]. However, the LOD of this method is higher than that of the instrumental analysis using the liquid-phase extraction method [10,11]. Liquidphase extraction could completely extract the TTX in the sample by grinding the sample to minced status and using the solvent. The SPME technology is an in vivo extraction method that belongs to non-exhaustive extraction, and only a small part of the TTX in the sample was extracted by using the SPME fibers. The lethal dose of TTX for humans is about 10,000 MU (about 2 mg) [3]. A concentration of 2 μg·g−1 of TTX in a 1 kg pufferfish is required to reach lethal levels, so the LOQ of 150 ng·g−1 of this method can fully meet the needs of food safety testing for pufferfish.

The reproducibility of the proposed method was determined in a 1000 ng·mL−1 TTX aqueous solution and 1000 ng·g−1 spiked fish. As shown in Table 1, the inter-fiber RSD values of the SPME fibers in the aqueous solution and spiked fish meat were 4.63 and 8.82, respectively, indicating that the uniformity of the SPME fibers' preparation was good. However, the intra-fiber reproducibility was not very ideal, especially in the spiked fish. Although PNE was used as the biological sheath in the fiber preparation process to improve the anti-biological contamination ability of the fibers, the extraction stability of a single fiber in spiked fish meat still decreased with the increase in extraction time, possibly because the PNE sheath was scraped off in the fish flesh. The biocorrosion resistance and service life of the fibers need to be improved.

#### *2.5. In Vivo SPME Extraction*

The results of in vivo SPME are shown in Table 2. On the day of the start of the exposure experiment, one of the pufferfish (No. 1) was detected using SPME extraction, and no TTX was detected, which indicated that the pufferfish was suitable for the exposure experiment. After the pufferfish were exposed to TTX for 7 days, the dorsal muscles of the pufferfish surviving in good condition were extracted in vivo using the self-made SPME fibers to obtain the level of TTX. The results of fish No. 4 and No. 6 were higher than the LOD (32 ng·g−1) but lower than the LOQ (150 ng·g−1), which can indicate the presence of TTX in the fish muscle but cannot be precisely quantified. While the TTX concentration of fish No. 5 was below the LOD, it could not be determined that fish No. 5 contained TTX. During the feeding process of the pufferfish, the phenomenon of fish death occurred due to long transportation time, the replacement of the feeding environment, and changes in the feeding water quality. There were several fish that died 2 days after exposure (No. 2), and 6 days after exposure (No. 3). In order to explore the content of TTX in the body of pufferfish after death, we also performed in vivo SPME extraction on the dead pufferfish, using the method described in Section 3.6 with the anesthesia step omitted.

**Table 2.** In vivo SPME results of TTX.

Pufferfish No. 1, 2, and 3 died before testing, and pufferfish No. 1 had not been exposed to TTX.

The result of the exposure experiment demonstrated that pufferfish could accumulate TTX in the muscles when eating TTX-contained food, and the TTX contents of the different pufferfish individuals were different. Furthermore, TTX could not be detected in the muscle of those pufferfish that were not fed with TTX-contained food. These results of the exposure experiment could be explained by the exogenous theory in the TTX source theory [4]. Generally speaking, when the TTX concentration of pufferfish is lower than 100 MU·g−1 (about 20 μg·g−1), it can be considered that pufferfish are non-toxic or lowtoxic to the human body [17], so all the pufferfish in this research were considered non-toxic to humans.

At the same time, we also found that after the fish died, the level of TTX in the muscles significantly increased, and this increase might even be close to 10 times. As shown in Table 2, the TTX contents in the dorsal muscles of two dead pufferfish (No. 2 and No. 3) were higher than that of the living fish. Even the fish that had been fed TTX for only 1 day (No. 2) had a higher TTX content in the muscle after death than the living pufferfish (No. 4, 5, and 6) that had been fed for 7 days. The reason for this phenomenon may be that, after the fish died, the TTX in the blood, liver, gonads, and other parts with high content of TTX diffused into the muscle, resulting in a substantial increase in the content of TTX in the muscle. Therefore, when cooking pufferfish, it should be handled in time after the death of the fish to avoid the increase in TTX in the muscles.

#### **3. Materials and Methods**

#### *3.1. Chemicals and Materials*

The species of the pufferfish used in this experiment is *Takifugu obscurus*, which was purchased from Jiangsu Zhongyang Company (Nantong, China). The weight of the pufferfish was about 250g. Medical stainless steel wires (0.5 × 2.5 cm) were purchased from Guangdong Longxin Co. (Zhongshan, China). Graphene oxide (GO) powder (>98 wt%) was purchased from 3A Chemicals Co. (Shanghai, China). Polyacrylonitrile (PAN, average

Mw 85000), norepinephrine (NE, 98%), and glacial acetic acid (analytical purity) were purchased from Macklin Co. (Shanghai, China). N,N-dimethylformamide (DMF, analytical purity), tetrodotoxin (≥99%), and methanol (chromatographic grade) were purchased from Aladdin Reagent Co. (Shanghai, China). Tris(hydroxymethyl)amine (TRIS, ≥99%) was purchased from Solarbio Co. (Beijing, China). And ultrapure water was prepared by the water purifier purchased from Chengdu Haokang Technology Co. (Chengdu, China).

#### *3.2. Preparation of SPME Fibers*

The GO-PAN@PNE fibers used in this study were prepared via a simple dipping method, with the stainless steel wires as the substrates.

#### 3.2.1. Preparation of Coating Solution

Firstly, 0.1 g of PAN solid was added to 0.8 g of DMF in a 1.5 mL centrifuge tube, and the suspension was heated at 90 ◦C for 1 h to fully dissolve the PAN solid and form PAN glue. Then, 20 mg of GO powder was added to 250 μL of DMF, and the mixture was ultrasonicated for 1 h to make the GO uniformly distributed; then, it was added to the PAN glue and stirred well to form a coating solution.

#### 3.2.2. Dipping Process of Fibers

The medical stainless steel wires were cut into small segments with a length of 2.5 cm and ultrasonicated in concentrated hydrochloric acid for 10 min to activate the steel wire surface. Additionally, the GO-PAN@PNE fiber was prepared via a dipping method: One end of the treated stainless steel wire was dipped vertically into the coating solution and taken out slowly; then, the fiber was heated at 90 ◦C for 3 min to volatilize DMF and fix the coating. The dipping and heating steps were repeated several times so that the coating was wrapped as evenly as possible on the steel wire, and the final thickness of the coating was about 50 μm.

#### 3.2.3. Addition of PNE Bionic Sheath

A certain amount of NE powder was dissolved in a mixed solvent of Tris buffer (10 mM, pH = 8.5) and methanol (1:1, *v*/*v*) to prepare the NE solution (2.0 mg·mL−1). Afterward, the prepared GO-PAN fibers were thoroughly submerged in the NE solution for 24 h.

#### *3.3. Characterization of SPME Fibers*

The self-made GO-PAN@PNE SPME fibers were cut to the appropriate size (about 1 cm) and attached to the sample stage with a conductive adhesive. A Nova Nano-SEM 450 field-emission scanning electron microscope (FESEM, FEI, Hillsboro, OR, USA) was used to observe the surface morphology of the fibers.

#### *3.4. Extraction Performance Evaluation*

In addition to the final GO-PAN@PNE fibers, blank PAN fibers without GO addition and the GO-PAN fiber without the PNE sheath layer were prepared as references. The TTX aqueous (1 μg·mL−1) and TTX spiked fish (1 μg·g−1) were used as samples to conduct the in vitro SPME extraction. The extracted TTX amount was used to evaluate the performances of these three fibers and to confirm the role of GO and PNE in the extraction. The sample preparation and extraction method for the spiked fish were as follows: A few *Takifugu obscurus* purchased from Jiangsu Zhongyang Company (Nantong, China) were dissected, and 5 g of their dorsal muscles were ground into minced meat and then added into 10 mL glass sample bottles. Then, 5 mL of the aqueous solution of TTX was added to prepare a spiked fish sample of TTX, which was used for the in vitro SPME extraction. The pretreated fibers were inserted vertically into the glass bottles containing the spiked fish sample to ensure that the coating was completely immersed in the samples. The extraction process was controlled for a certain period. After extraction, the fiber was rinsed with ultrapure

water for 3 s and then dried with a Kimwipe paper towel. The extracted TTX was eluted from the fibers with 250 μL of elution solvent for a certain time. The elution solution was analyzed via the LC-MS/MS analysis. The in vitro extraction was repeated to optimize the extraction time, elution time, and elution solvent.

#### *3.5. Exposure Experiment*

The pufferfish were raised in a fish tank containing 40 L of oxygenated dechlorinated tap water and fed with eel meal containing TTX. The exposure dose of TTX was 2 MU·g−1 body mass·day-1 (each pufferfish was fed with about 100 μg of TTX every day; MU: mouse unit, 1 MU represents the average amount of toxin that kills a male mouse weighing 20 g within 30 min after intraperitoneal administration, and 1 MU is equivalent to about 0.2 mg). The pufferfish exposure experiment lasted for 7 days. The feeding condition was controlled during the exposure period. The water temperature was controlled at about 25 ◦C with an air conditioner. The water was continuously oxygenated with an air pump to ensure the oxygen content of the water body. A filter device was used to filter the residual bait and feces. Nitrifying bacteria were added to establish a nitrification system. Additionally, 0.5% of NaCl was added to maintain water osmotic pressure. During the exposure experiment, the in vivo SPME extraction was performed to determine the TTX content in the pufferfish.

#### *3.6. In Vivo SPME Extraction*

The procedure of in vivo SPME extraction in the fish muscle was similar to previous research [17,31]. The pufferfish was picked up from the water and submerged in a 0.1% eugenol (*v*/*v*) aqueous solution until its body was unbalanced. A medical syringe needle was inserted into the side of the dorsal muscle near the fin of the pufferfish and then was withdrawn to obtain an approximately 1.5 cm deep pinhole. Afterward, the SPME fiber was inserted into the pinhole to extract the TTX in the muscle, as shown in Figure 1b. During the extraction process, the pufferfish was placed back into the clean water to make it free to swim. After the extraction, the pufferfish was picked up from the water again, and the SPME fiber was withdrawn from the muscle. Finally, the TTX was eluted by the solvent from the SPME fiber for the instrumental analysis to obtain the TTX content in the pufferfish muscle.

#### *3.7. Instrumentation (UPLC-MS/MS)*

The instrumental analysis was based on a UPLC system coupled to a triple-quadrupole tandem mass spectrometer (Nexera LC30AD&SCIEX SelexION Triple Quad 5500 System). An ACQUITY BEH HILIC column (2.1 × 100 mm, 1.7 μm) was used for separation, and the column temperature was 40 ◦C, and the injection volume is 2 μL. Gradient elution was applied by using water with 0.1% formic acid as mobile phase A and ACN with 0.1% formic acid as mobile phase B. The initial gradient of 5% A was kept unchanged for 0.5 min, then ramped to 50% A in 2.5 min and ramped to 95% A in 1 min, and kept for 2 min, then decreased to 5% A in 1 min, and kept for 1.9 min. The flow rate was set at 0.4 mL·min−1, and the total analysis time for each sample was 8 min.

The mass spectrum parameters were as follows: electrospray ionization (ESI); the positive ionization mode; curtain gas 35 psi; ion spray voltage 5500 V; desolvation temperature 600 ◦C; nebulizing gas 55 psi; auxiliary heating gas 55 psi; analysis of the samples via multiple reaction monitoring (MRM). The transition was monitored with the positive ion mode at *m*/*z* 320.1/162.0, and the detailed mass spectrometry parameters are shown in Table 3.

**Table 3.** Mass spectral parameters of TTX.

In the UPLC-MS/MS results, the characteristic ion and retention time (around 2.43 min) were used for qualitative analysis, and the characteristic peak areas for quantitative analysis. The UPLC-MS/MS chromatograms of the TTX standard are shown in Figure 5.

**Figure 5.** UPLC-MS/MS chromatogram of TTX standard.

#### **4. Conclusions**

In this study, a detection method of TTX in pufferfish was established based on in vivo SPME extraction and UPLC-MS/MS. The GO-PAN fibers were fabricated through a dipping method, followed by biomimetic PNE sheath modification. The self-made SPME fibers could successfully extract TTX in both the spiked fish samples and living pufferfish, and the LOD of the proposed method was 32 ng·g−1, which can meet the requirement for safe consumption of pufferfish. The exposure experiment demonstrated that the pufferfish could accumulate TTX in the muscles when eating TTX-contained food, and the TTX level in the muscles of the dead pufferfish significantly increased up to 10 times that of the living pufferfish. The established method could quickly and easily detect the TTX content in pufferfish in vivo without damage to the living pufferfish. This study provides theoretical support for improvement in the food safety level of pufferfish.

**Author Contributions:** Conceptualization, H.M., S.J. and Y.H.; methodology, H.M. and S.J.; validation, S.J. and Y.L.; formal analysis, H.M.; investigation, H.M.; writing—original draft preparation, H.M.; writing—review and editing, S.J., Y.Z. and Y.L.; supervision, S.J., Y.Z., Y.H. and Y.L.; project administration, Y.H. and Y.L.; funding acquisition, Y.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Key Research and Development Project of Yangzhou in 2020 (YZ2020047), the Open Project Program of State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology (No. SKLFNS-KF-201801), and Shanghai Science and Technology Commission (Grant No. 19DZ2284200).

**Institutional Review Board Statement:** Ethical review and approval were waived for this study due to the fact that the fish used in this study were purchased from the general market and were raised in a normal environment, and the experiment caused little damage to them.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


*<IP_ADDRESS>. Portugal*

The 1755 Lisbon earthquake in combination with subsequent fires and a tsunami almost totally destroyed Lisbon and adjoining areas. Tsunamis as tall as 20 m swept the coast of North Africa, and struck Martinique and Barbados across the Atlantic [18].

Using the actual offshore bathymetry, we simulated a tsunami approaching the Portuguese coastline from the west, results are shown in Video 3, available at http://bit.ly/29l4vCx Bathymetry contours are shown in order to understand the tsunami refraction. The epicenter was located more than 200 km to the west of the map. When the source is so distant, the initial condition for solving the PDE can be taken to be a plane wave, corresponding to a ridge of water traveling eastward. This approximation is reasonable whenever the source is distant from the near-field region and is convenient to model for numerical solutions. The domain for the numerical solution consists of the coastline of interest and the open box edges over the ocean. The coastline was assumed to have a Neumann (reflective) boundary condition.

This region was also selected for study because there are three 13.5-MHz SeaSonde HF radars operating at nearby locations, which are shown by green squares in the video. Tsunami observation software is being installed at these sites. The three radars would not see the tsunami if its propagation followed the line of sight from the source because the coast of southern Portugal would shadow those paths. In fact, the model output shows how the tsunami wave refracts and approaches the sites from the south. Reflection of the wave from the coast is clearly visible.


**Author details**

Jack Denur Electric & Gas Technology, Inc., Rowlett, Texas, USA

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 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.

*Improving Heat-Engine Performance via High-Temperature Recharge DOI: http://dx.doi.org/10.5772/intechopen.89913*


**3. Special types of contact urticaria**

#### **3.1. Occupational contact urticaria**

Skin diseases are the second most common occupational diseases in Europe and occupational contact urticaria (OCU) makes up 1–8% of occupational skin disorders [12]. The most commonly affected professional groups are healthcare employees, food handlers, farmers and hairdressers [24, 25]. Immunologic and non-immunologic contact urticaria types can be seen in OCU. The risk of sensitization against all proteins is high in presence of atopy in OCU [10]. Besides, atopy is also important in OCU associated with NICU [10].

Natural rubber latex is the most commonly identified allergen and this allergy is seen in 1–3% in general population and 5–10% of healthcare workers in Europe [10]. *H. brasiliensis* proteins are the main responsible agents for natural rubber latex allergy [10]. A reaction against modified proteins (wheat, soy and Croetin Q) that are added to shampoo and especially ammonium persulfate is often observed in hairdressers [26, 27]. Reactions against saliva, amniotic fluid, urine and seminal fluid of animals have been defined in animal handlers, farmers and veterinarians. Dyes cause contact urticaria at significant levels in the cosmetic and industrial sectors [4, 6].

#### **3.2. Oral allergy syndrome (food contact dermatitis)**

"Oral allergy syndrome" is used to identify ICU developing in the mucosa [28]. It is characterized by mucosal edema, itching and a burning sensation after contact of the oral mucosa with respiratory allergens [29]. Cross-reactivity between homologous pollen and food allergens is accused in the etiology [29]. The term pollen-food allergy syndrome (PFAS) can therefore also be used [30].

Fruits and vegetables especially apples, carrots, tomatoes, pears, cherries, plums, celery, spices and hazelnuts are the agents that are often blamed for the oral allergy syndrome. The individuals who have oral allergy syndrome frequently suffer from atopy and pollen allergy, therefore a cross allergy against IgE antibodies has been observed [30].

#### **3.3. Physical contact urticaria**

Some physical urticaria cases occur following skin contact with hot, cold, light (UV: solar urticaria), water or as dermographism, pressure hives and vibratory angioedema. A physical agent does not cause a reaction alone but leads to the activation of a chemical product in some cases. It is possible to see this mechanism in induced contact urticaria. Benzophenones, chlorpromazine, methenamine hippurate and formaldehyde are included among the agents that can cause such a reaction [31–33].

#### **3.4. Delayed and prolonged contact urticaria**

Contact urticaria, protein contact dermatitis and allergic contact dermatitis can sometime coexist. The patients can primarily present with an urticarial lesion and the contact dermatitis and eczematous lesions can develop later [32, 34]. Elm, vaseline and castor oil are agents that often cause delayed and prolonged contact urticaria [10].


**1. Introduction**

The concept of cultural heritage is subjective and evolves over the years. It is subjective in the sense that cultural heritage is made up of those goods that the values that prevail in each society at a given time establish must be susceptible of being protected and preserved as a legacy of the past for future generations. This approach is reflected in the definition of cultural heritage proposed in The Charter of Krakow [1] (p. 5): "heritage is that complex of man's works in which a community recognizes its particular and specific values and with which it identifies. Identification and specification of heritage is therefore a process related to the choice of values". Smith [2] defines the concept of cultural heritage as the footprint of memory and oblivion, as a cultural practice involved in the construction and regulation of a spectrum of values and ways of understanding. This footprint is composed of the set of products of human activity, and its remains, intentional and unintentional, tangible intangibles, represent historical and social processes.

Therefore, in the first place it is important to establish the concept of "cultural heritage" and the elements that make it up. First, it is important to refer to the work done by the United Nations Educational, Scientific and Cultural Organization—UNESCO. UNESCO aims to encourage "the identification, protection, conservation, presentation and transmission to future generations of the cultural and natural heritage" throughout the world that is considered of great value to humanity [3]. The work was shown in the international treaty adopted in 1972, the "Convention Concerning the Protection of the World Cultural and Natural Heritage" [3], which lays the foundation for the first definition of the term "cultural heritage".

In this sense, one of the first definitions is the one proposed by UNESCO in the Declaration of Mexico on Cultural Policies [4] (p. 3): "The cultural heritage of a people includes the works of its artists, architects, musicians, writers and scientists and also the work of anonymous artists, expressions of the people's spirituality, and the body of values which give meaning to life. It includes both tangible and intangible works through which the creativity of that people finds expression: languages, rites, beliefs, historic places and monuments, literature, works of art, archives and libraries".

Subsequently, UNESCO approved The Convention for the Safeguarding of the Intangible Cultural Heritage [5] (p. 4) in 2003, which specifically defines what is meant by intangible cultural heritage: "the 'intangible cultural heritage' means the practices, representations, expressions, knowledge, skills—as well as the instruments, objects, artefacts and cultural spaces associated therewith—that communities, groups and, in some cases, individuals recognize as part of their cultural heritage. This intangible cultural heritage, transmitted from generation to generation, is constantly recreated by communities and groups in response to their environment, their interaction with nature and their history, and provides them with a sense of identity and continuity, thus promoting respect for cultural diversity and human creativity ... ". According to Timothy and Boyd [6] (p. 94), the conservation of cultural heritage can be carried out in different ways: by the preservation, restoration, renovation of goods, and urban regeneration.

According to UNESCO, cultural heritage is subdivided into the tangible (movable and immovable property), which can be preserved and restored by some kind of intervention [4], and intangible, which "is manifested inter alia in the following domains: (a) oral traditions and expressions, including language as a vehicle of the intangible cultural heritage; (b) performing arts; (c) social practices, rituals, and festive events; (d) knowledge and practices concerning nature and the universe; (e) traditional craftsmanship" [5] (p. 4).

Continuing with the contextualization of this research work, the next step is to relate cultural heritage and tourism. Thus, cultural heritage, composed of a wide variety of tangible and intangible assets, can be enhanced in order to articulate new development alternatives by the tourism activity. Tourism management of cultural heritage enables it to be transformed into tourist resources.

Thus arises the concept of heritage tourism and cultural tourism. Timothy [7] (pp. 4–5) points out that both terms are often used in the literature as distinct but related concepts. For Timothy and Boyd [6] (p. 14), the terms cultural and heritage tourism overlap and, in their opinion, are interchangeable (see the overlapping of the concepts cultural and heritage tourism in Timothy and Boyd [6] (p. 9)). Along the same lines, Timothy [7] suggests that heritage tourism is based on "old things" and usually occurs in rural areas and in more specific places, while cultural tourism predominates in urban areas and less determined places (see the exhaustive classification of the different attractions offered by heritage tourism in Timothy [7] (p. 49)). For Timothy [7], all definitions of heritage tourism include elements of the human past as a resource, and all collect a variety of reasons why tourists move (tourists move for reasons that may stem from the desire to improve their own personal culture, learn something new, spend time with friends or family, satisfy a curiosity, or simply spend the free time available).

In this sense, there are several studies that show that knowledge of the culture of other spaces is one of the main motivations that drive tourists [8–11]. Therefore, exploiting cultural heritage, being either tangible or intangible, through tourism activity becomes a fundamental means throughout the world for the development of spaces where this heritage is located. According to the World Tourism Organization (UNWTO), it was identified that within the practice of tourism activities related to culture, those related to intangible heritage are the most important, closely followed by tangible ones [12]. In short, tourism becomes an attractive option to take advantage of the local resources available, including heritage [13].

Focusing on cultural wealth as attractions of tourist interest allows for the creation of several hubs through which the cultural heritage and tourism relationship is developed, including: (1) the recovery or conservation of heritage, (2) economic growth focused on the commercialization of goods, (3) the increase of tourist facilities from internal or external investment ad an improved image for the community, and (4) social benefit for communities in order to improve living conditions [14–17]. In this way, cultural heritage becomes an element capable of generating benefits for the development of societies, the relationship with tourism being the most often used to achieve this goal. Thus, cultural tourism becomes an essential strategic feasibility for tourism development. This type of tourism may reduce the seasonality of the sector and favor the opening to new markets.

Developing tourism around cultural heritage is considered an action that is not focused on mass culture that homogenizes, imposing one culture over another, or that mythologizes or fragments the value of heritage. In this sense, it is proposed by the popular culture in which its vital particularities are created and preserved, and it is not based on selling but on participating with the purpose that this is produced and consumed by the human group itself. This approach allows for an increase in the quality of life, which, at the same time, enables the strengthening of the transmission and revitalization networks of elements contained in the collective memory of groups [14,15]. With this, it is argued that each community has something unique to offer that manages to generate considerable tourist flows based on the interests that they arouse for the existence of a vibrant culture.

The participation of the community is essential, and it should not be left out; the empowerment of processes leads to a more efficient development of heritage [18]. This must be the pillar in the process of tourism development and heritage management [19], always supported by local identity, that is, heritage [20,21].

In this context, this research aims to analyze and examine the scientific production developed in relation to exploiting cultural heritage by tourism activities for regional development. To achieve the objective, a bibliometric analysis is first performed, which is complemented by a bibliographic analysis, allowing for a systematic and structured analysis of production. The novelty of the study is based on the fact that there is no research of similar characteristics that provides a complete view of the research carried out in this field of study. The findings will be very useful for researchers as they provide a better understanding of current research and serve as a guide for future research.

This article is structured into five sections. The topic is contextualized, the literature is reviewed, and the objective is presented in the introduction. In the second section, the methodology is introduced. The results are shown in the third section and in the fourth section, the bibliographic analysis. Finally, to conclude, the conclusions and limitations of the investigation are discussed.

### **2. Methodology**

The methodology used in this research is an exploratory quantitative bibliometric and bibliographic analysis, focused on identifying and analyzing scientific production on cultural heritage and its use by the tourism activity for regional development. This methodology is the most adequate to organize scientific information; on the one hand, it enables the identification and analysis of bibliographical sources of interest, while on the other hand, it provides an evaluation structure for this scientific production [22].

In the bibliometric analysis, indicators are applied [23] that arise from different mathematical models that are based on the relationship of two or more variables [24], which enables the measurement of the bibliographic material in quantitative terms. The indicators that will be used in this research are classified according to two criteria: activity indicators that provide information on quantity, productivity, dispersion, and collaboration, among others, and impact indicators, which provide information on the citation level of the documents through the impact factor or immediacy index, the H index, among others [25].


*5.3. Consequences of the EEDI*

#### 5.3.1. EEDI Candidate 1

Figure 10 shows the attained EEDI results of the ships with varied R/G fractions. Each graph includes the required EEDI phase 3 line of the LNG carrier with the same volumetric capacity. As shown in Equation (10), the calculation results obtained using the same R/G fraction for each ship tend to decrease as the volume capacity of the ships increase. This indicates that as the volumetric capacity of the ship increases, the attained EEDI of the LH2 carriers tends to decrease and becomes more similar to EEDI candidate 1. As the R/G fraction increases, the BOH utilized in the PEMFC decreases and the required power from the main engine increases. The more power the main engine generates using the LNG fuel, the more CO2 the ship emits.

Table 10 shows the permittable R/G fractions according to EEDI candidate 1, indicating that only a small amount of BOH is permittable for re-liquefaction. In the cases of Ships #1 and #2, whose capacity is relatively smaller than the other LH2 carriers, additional hydrogen is required to satisfy the EEDI candidate 1. Additionally, in the cases of Ships #3 to #5 with larger capacities, less than 15% of the generated BOH is permittable for re-liquefaction.

**Table 10.** Permittable R/G fractions of the LH2 carriers according to EEDI candidate 1.

**Figure 10.** *Cont*.

**Figure 10.** Attained EEDIs for (**a**) Ship #1, (**b**) Ship #2, (**c**) Ship #3, (**d**) Ship #4, and (**e**) Ship #5 using EEDI candidate 1.

#### 5.3.2. EEDI Candidate 2

Figure 11 shows the energy-based EEDI calculation results defined for EEDI candidate 2. Each graph for Ships #1 to #5 presents the results of this energy-based EEDI with varied R/G fractions. The graphs also exhibit the required EEDI phase 3 for LNG carriers with the same rescaled deadweight as each LH2 carrier. Similar to the attained EEDI shown in Figure 10, as the R/G fraction increases, the energy-based EEDI increases. Moreover, the energy-based EEDI tends to decrease as the volumetric capacity of the ships increases. However, unlike the attained EEDI results shown in Figure 10, every ship is able to reliquefy a ratio of BOH between 25% and 33% such that the energy-based EEDI is less than the required EEDI phase 3 of LNG carriers. These results were obtained due to the rescaled deadweight that was increased from the original deadweight considering the differing heating values of LH2 and LNG. Table 11 shows the permittable R/G fractions of Ships #1 to #5. The permittable R/G fraction tends to increase as the volumetric capacity of the ships increases.

**Table 11.** Permittable R/G fractions of the LH2 carriers according to EEDI candidate 2.

The differences between Tables 10 and 11 indicate how the mass and energy densities of LH2 differ from those of LNG. Because LH2 has a lower density but larger heating value than LNG, the permittable R/G fraction is larger in EEDI candidate 2 than in EEDI candidate 1. The cargo of the currently used energy carriers under EEDI regulations is mainly hydrocarbon materials such as oil and LNG. These materials have different densities and heating values compared to hydrogen. The existing EEDI regulation for energy carriers, which is calculated using the mass-based deadweight, is used due to the properties of these hydrocarbons. Therefore, the application of this regulation directly to LH2 carriers without considering the properties of LH2 is inappropriate. The large heating value of hydrogen should be reflected in these regulations such that the energy carrier may carry energy efficiently.

**Figure 11.** *Cont*.

**Figure 11.** Energy-based EEDIs for (**a**) Ship #1, (**b**) Ship #2, (**c**) Ship #3, (**d**) Ship #4, and (**e**) Ship #5 using EEDI candidate 2.

#### 5.3.3. EEDI Candidate 3

EEDI candidate 3 exempts the LH2 carriers from the EEDI regulations. The LH2 carriers deliver LH2 cargo, which emits no CO2, unlike other fuels. In addition, it is highly likely that only CO2-free LH2 will be allowed for international trading. Therefore, although regulations on CO2 emissions may not be imposed on LH2 carriers, LH2 is far less CO2 intensive than other liquefied cargos such as LNG and LPG considering the entire supply chain.

In this case, the BOH R/G fraction is determined mainly via economic motivations. As discussed in Section 5.2*,* the SLCC of the BOH re-liquefaction system decreases as the R/G fraction increases. Consequently, all BOH may be re-liquefied considering the economic results obtained using EEDI candidate 3.

Table 12 shows the SLCCs for the permittable R/G fractions obtained using each EEDI candidate. As described in Section 5.3.1, the permittable R/G fraction indicates the amount satisfying the EEDI restrictions for each candidate. In the case of EEDI candidate 3, this ratio is 100% because there is no EEDI restriction. Compared with EEDI candidate 1, the SLCC for the permittable R/G fraction decreases from 50% to 68% depending on the capacity of the LH2 carriers in EEDI candidate 3. Likewise, the SLCC decreases from 18% to 48% compared to the EEDI candidate 2. These results indicate the economic advantages that may be obtained when LH2 carriers are not subjected to EEDI restrictions. Considering this advantage and the CO2-free characteristic of LH2, the EEDI-free regulation of LH2 carriers can be considered, which exempts LH2 carriers with LNG fuels from the CO2 emissions restrictions.

**Table 12.** SLCCs for the permittable R/G fractions obtained using each EEDI candidate.

#### **6. Conclusions**

This study proposed a partial BOH re-liquefaction system based on the reverse Brayton helium cycles. This system divides the generated BOH into two streams, one of which is to be re-liquefied and the other is utilized to generate electricity in PEMFC stacks. Various evaluations for the system were performed based on an assumed voyage route, five different LH2 carrier specifications, and an assumed LH2 production cost.

The SEC increased from 8.22 to 10.80 kWh/kg as the R/G fraction increased from 10% to 100%. The exergy efficiency was increased from 0.209 to 0.258 as the R/G fraction increased from 10% to 30%, and it converged to 0.258 when the R/G fraction was larger than 30%. The exergy loss in heat transfer occupied the largest portion of all. Due to the excessive cold energy of the BOH heading to the PEMFC stacks, compared to other R/G fraction cases, 58% and 15% more exergy loss occurred in 10% and 20% cases, respectively.

The system economics indicated that the re-liquefied mass of BOH is inversely proportional to the SLCC. The gradient of this decrease became smoother as the re-liquefied mass of BOH increased. When the re-liquefied mass of BOH was larger than 7200 kg/day, the SLCC was almost unchanged from 1.5 \$/kg; this value is much lower than 6.50 \$/kg, which is the assumed LH2 production cost.

Considering EEDI candidate 1, the attained EEDI demonstrated that most of the BOH should not be re-liquefied when the required EEDI was evaluated based on the parameters of the LNG carrier for the required EEDI phase 3 with the same volumetric capacity. However, for EEDI candidate 2, it was shown that the permittable R/G fraction was between 25% and 33% considering energy-based EEDI and required EEDI phase 3. Finally, for EEDI candidate 3, the EEDI-free regulation of LH2 carriers was discussed considering the CO2-free characteristic of LH2. If the EEDI regulation is not used for LH2 carriers, the SLCC of the BOH re-liquefaction system decreases up to 68% compared to LNG carriers with equivalent EEDI regulations.

**Author Contributions:** Conceptualization, M.C., W.J. and S.L.; methodology, W.J. and T.J.; software, M.C.; validation, M.C. and W.J.; formal analysis, M.C.; investigation, M.C.; resources, T.J.; data curation, M.C.; writing—original draft preparation, M.C.; writing—review and editing, W.J.; visualization, M.C.; supervision, W.J. and D.C.; project administration, D.C.; funding acquisition, D.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was a part of the project titled 'Development of Safety and Control Standards for Hydrogen Ships: Cargo Handling and Fuel Gas Supply Systems' (Grant number: 20200456), funded by the Ministry of Oceans and Fisheries, Korea.

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

#### **Nomenclature**


# **Current Advances on Non-Melanoma Skin Cancer**

Constantin Caruntu Edited by

Printed Edition of the Special Issue Published in *Journal of Clinical Medicine*

www.mdpi.com/journal/jcm

**Current Advances on Non-Melanoma Skin Cancer** • Constantin Caruntu


*4.2. Studies Addressing TNF-α and TNFRs in Human Kidney Disease and Related Clinical Outcomes*

In clinical studies, circulating TNFR1 and TNFR2 were shown to be increased in several cohorts of patients, with different CKD etiologies and diverse age-groups and races (Table 2). Despite being responsible for engaging different downstream signaling pathways, the strength of associations with renal function is similar for both receptors.

The first study assessing the serum levels of a TNFR (unidentified either as TNFR1 or TNFR2) in CKD patients was published in 1994, and showed a strong correlation between the receptor levels and serum creatinine, in a group of 26 non-dialyzed CKD patients [109]. TNFR1 and TNFR2 were further associated with eGFR and with albuminuria in several subsequent studies [110–113]. In a prospective cohort that included 984 CKD patients, eGFR was negatively correlated with the serum levels of TNFR1 and TNFR2 [113]. To a lesser extent, both biomarkers were also positively correlated with urinary protein-tocreatinine ratio. Furthermore, in a cohort of patients, with a diverse set of kidney diseases and undergoing native kidney biopsy, TNFR1 and TNFR2 plasma levels were associated with underlying histopathologic lesions and adverse clinical outcomes, such as disease progression and death [114].

In a group of 106 biopsy-proven IgA nephropathy patients, higher serum levels of TNFR1 and TNFR2 were present in patients with more severe renal interstitial fibrosis [112]. Increased circulating levels of TNF receptors were similarly described as prognostic markers of idiopathic membranous nephropathy [115] and contrast-induced nephropathy [116]. Patients with systemic lupus erythematosus (SLE) have also been studied, with urinary TNFR1 [117] and serum TNFR2 [118] levels being elevated in cases of lupus nephritis.

The predictive value of TNFRs was mostly described in diabetic nephropathy, as reviewed by Murakoshi et al. (2020) [119]. Several results from the Joslin Kidney Center studies showed that the TNFRs seem to be candidate biomarkers of renal function decline in both type 1 [120] and type 2 [121] diabetic patients. Moreover, in type 1 diabetic patients, the increased circulating levels of TNFR1 and TNFR2 were the strongest determinants of CKD progression, preceding the onset of microalbuminuria and/or its progression to macroalbuminuria [122]. Higher baseline circulating levels of TNFR1 and TNFR2 were associated with a higher risk of eGFR worsening in patients with both early and established diabetic nephropathy [123]. A systematic review and meta-analysis highlighted the reliability of TNFRs in predicting diabetic kidney disease progression. The results seem to be consistent across different cohorts of diabetic patients [124–127]. A recently published study, evaluated a composite risk score termed KidneyIntelX for predicting the progression of diabetic kidney disease, in a large multinational cohort. KidneyIntelX comprises clinical variables and the circulating levels of three biomarkers, TNFR1, TNFR2, and kidney injury molecule 1 (KIM-1). KidneyIntelX successfully stratified patients for disease progression, showing that, after 1 year, a greater reduction in eGFR was observed in patients with higher changes in KidneyIntelX risk scores, independently of the baseline risk score value and the treatment option [128].

CKD patients have an increased risk of mortality due to CVD, which is independent of the traditional risk factors, possibly due to the chronic inflammatory state. Both circulating TNFRs were described as predictors of CVD risk [113,129] and all-cause mortality [129,130] in CKD populations, independently of eGFR and albuminuria, and irrespective of the cause of kidney disease. Some studies [131–133] have also addressed the prognostic value of circulating TNFRs in HD patients. Despite TFNRs being substantially linked with other inflammatory markers, Carlsson et al. observed no significant connection between either TNFRs and death, in a longitudinal cohort analysis of 207 prevalent HD patients [131]; two more recent studies, including one from our team, reported that circulating levels of TNFR1 and TNFR2 are independent predictors of all-cause mortality in ESKD patients under chronic HD [132,133] (REF 2017 and 2021), although for cardiovascular mortality, the significance was only observed for TNFR1 [132].

In the last 15 years, proteomic and transcriptomic studies have proven useful in discovering new insights into the TNF-α signaling pathway in CKD, as well as the associatedcomorbidities. In a proteomic analysis of human serum from patients with CKD, TNF-α was associated with disease severity [134,135], as well as with vascular changes [134]. The circulating extracellular vesicles of CKD patients showed a pro-inflammatory profile, that included markers of the TNF signaling pathways. Niewczas et al. measured 194 circulating inflammatory proteins using aptamer-based proteomics analysis of different cohorts of diabetic patients [136]. The results showed that, out of the 194 measured proteins, 17 were TNFR superfamily-related, and also that TNFR1 and TNFR2 were strong predictors of renal function decline [136]. Accordingly, Ihara et al. showed that a profile of multiple circulating TNF receptors, including TNFR1 and TNFR2, was associated with early progressive renal decline in type 1 diabetes [137]. Tubular cells of IgA nephropathy patients also overexpressed genes of the inflammatory TNF signaling pathway [138].


**Figure 7.**

*Cytotoxic activity of Organo tin complexes of thiosemicarbazones.*

the ligand [75]. 2-Benzoylpyridine N(4)-phenyl thiosemicarbazone; 2-Acetyl pyrazine N(4)-phenyl TSC and its Diorgano Sn(IV), Antibacterial activity and cytotoxic activity against K562 leukemia cells of the free ligands and complexes were reported [76]. Pyridoxal thiosemicarbazone and its complexes of Diorgano Sn(IV) antitumor

activity were studied and observed that Ethyl, butyl, and phenyl substituted compounds suppress the proliferation of friend erythroleukemia cells (**Figure 7**) [77].