*Article* **Puree and Juice of Thai Mango and Pineapple Analyzed by High-Performance Thin-Layer Chromatography Hyphenated with Effect-Directed Assays**

**Gertrud E. Morlock 1,\* ,† , Newitchaya Wutthinithisanand 1,2,† and Doris Rauhut 2**

**Abstract:** The requirements for analytical tools are changing due to the global production chain, the increasing cases of adulteration, and the growing trend towards consumption of plant-based food products worldwide. The assessment of bioactivity of natural foods is currently not a quality criterion, and a paradigm shift is postulated. A non-targeted effect-directed profiling by high-performance thin-layer chromatography hyphenated with five different effect-directed assays was developed exemplarily for the puree and juice products of mango *Mangifera indica* L. (Anacardiaceae) and pineapple *Ananas comosus* (L.) Merr. (Bromeliaceae). Several bioactive compounds were detected in each sample. The additional bioactivity information obtained through effect-directed profiles improves, expands and modernizes product control. Non-target effect-directed profiling adds a new perspective to previous target analysis results that can be used not only to ensure health claims based on bioactive compounds, but also to detect unknown bioactive compounds coming from contamination or residues or changes caused by food processing.

**Keywords:** effect-directed analysis; healthy food; food processing; adulteration; authentication

#### **1. Introduction**

Bioactive compounds present in natural plant-based foods and their products attract increasingly both consumers and researchers. Epidemiological evidence indicates a contribution to the preventive effects in reducing the risk of certain common diseases, e.g., cardiovascular disorder, cancer, osteoporosis, infections, cataract and diabetes [1,2]. The spectrum and content of bioactive compounds in fruits vary, among other things, depending on the cultivar and maturity stage of the product [3,4]. The most prominent bioactive compounds found in tropical fruits are ascorbic acid (vitamin C), tocopherols (vitamin E), carotenoids (provitamin A candidates), flavonoids and thiol compounds. In particular, the widely distributed polyphenols are the predominant antioxidative secondary metabolite group [5].

The pineapple *Ananas comosus* (L.) Merr. (Bromeliaceae) and the mango *Mangifera indica* L. (Anacardiaceae) are important tropical fruits popular all over the world. Global pineapple and mango production in 2019 were about 28 million tons and 56 million tons, respectively, of which Thailand accounted for about 6% and 3% [6]. Pineapple is commonly consumed as fresh fruit, or processed to conventional products including canned fruit, jam, concentrated juice, and dried chips, or used as an ingredient in exotic food [7] or as material for pineapple wine production [8]. Thailand is not only a world leader in the export volume of canned pineapple, but many local cultivars are also unique in their taste and aroma [9]. Commercial cultivars of pineapple in Thailand are Pattavia, Phuket,

**Citation:** Morlock, G.E.; Wutthinithisanand, N.; Rauhut, D. Puree and Juice of Thai Mango and Pineapple Analyzed by High-Performance Thin-Layer Chromatography Hyphenated with Effect-Directed Assays. *Molecules* **2021**, *26*, 7683. https://doi.org/ 10.3390/molecules26247683

Academic Editor: Smaoui Slim

Received: 2 December 2021 Accepted: 13 December 2021 Published: 19 December 2021

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

Nanglae and Phulae. Pineapple is considered a very nutritious and functional fruit, as it is rich in vitamin A, vitamin B, and minerals such as calcium, phosphorus and iron [10]. It contains also several proteinases, such as bromelain, comosain and ananain, which support the digestive tract [11]. Moreover, it has several compounds with antioxidant properties, namely ascorbic acid, β-carotene and phenolic compounds such as flavonoids [12–15].

The mango fruit is widely accepted by consumers due to its succulence, sweet taste and exotic flavor. Mango flesh is consumed in both ripe and unripe stages. It is also processed to foods and drinks, such as pickled products, beverages, vinegar, chutneys, and desserts [16]. In Thailand, the major mango varieties, consumed fresh when the fruit is ripe, are Nam Dok Mai and Mahachanok. A small percentage of the production is processed to canned, dried and frozen mango [9]. Mangos contain bioactive compounds, such as ascorbic acid, carotenoids (e.g., β-carotene, violaxanthin, cryptoxanthin, neoxanthin, luteoxanthin and zeaxanthin), phenolic components, and mono-, di- and triterpenoids (e.g., ocimene, myrcene or limonene, terpinolene and careen) [2].

The domestic consumption and the export trade of pineapple and mango provide economic value to Thailand. Nevertheless, reports on bioactive compounds and antioxidant activities of these fruits from Thailand are rare. Since bioactive compounds generally occur as complex mixtures in a plant, it is a major challenge to identify the individual substances responsible for specific effects [17–19]. For such effect-directed profiling, high-performance thin-layer chromatography hyphenated with multi-imaging (UV/Vis/FLD) and effectdirected assays (EDA) was proven to be a good choice [20–24]. In this study, a non-target effect-directed profiling by HPTLC−UV/Vis/FLD−EDA was developed for puree and juice products of different Thai pineapple and mango cultivars to gain information on bioactive constituents. The obtained effect-directed profiles pointed to individual bioactive compounds and allowed a comparative evaluation with regard to their activity potential.

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

#### *2.1. Physico-Chemical Characterization of the Produced Fruit Purees and Juices*

Two cultivars each of mango and pineapple were chosen due to their importance as major commercial varieties and their widespread production in Thailand. The two pineapple cultivars Pattavia (PTV) and Nanglae (NL) were selected as sub-varieties of the smooth cayenne pineapple Pattavia variety. The two mango cultivars Nam Dok Mai (NDM) and Mahachanok (MHC) represent poly-embryonic and mono-embryonic ecotypes, respectively. At the commercial maturity stage, the fruits were harvested from the orchard at the Chiang Rai province in Northern Thailand. The regularity of fruit ripeness was controlled by the peel color observation in our study, i.e., 30–40% yellow for pineapple and fully yellow for mango. A puree and a juice sample were produced from each of the four cultivars. Thus, 8 fruit product samples were obtained. Their ripeness (content of total sugars and total acidity) and compositional values (content of total amino acids and total phenolics) were determined (Table 1). Total soluble solids, pH, and total acidity of pineapple and mango puree in both cultivars were in accordance with the literature [12,13,25–29]. The total sugar of pineapple was higher than that reported by Lu et al. [13]; however, values of natural plant-based food can vary depending on harvest time, climate influence each year, agricultural and soil management, among many other aspects. The sugar content normally increases during fruit ripening.

**Table 1.** Properties of the produced purees and juices of the pineapple (PTV and NL) and mango cultivars (NDM and MHC). Each value shows the mean ± standard deviation from three measurement replicates. Different letters (a–d) derived from the ANOVA and DMRT test indicate significant differences among samples.

### *2.2. Development of the Effect-Directed Profiling*

An effect-directed HPTLC method was developed on HPTLC plates silica gel 60 F254 (for the *B. subtilis* bioassay without F254). For a first impression, the more complex puree sample was chosen first. The methanolic extraction was performed at a solid-liquid ratio of 40%. A highly concentrated extract solution was preferred for the analysis of bioactive secondary metabolites, usually present in trace amounts. This allows the use of small sample volumes (rapid application) and easy adjustment to the different detection capabilities of biological and enzymatic assays (dilution step is faster than concentration step). Application as area (4 mg/area) was preferred due to the expected high load of sample matrix onto the adsorbent. Six different mobile phases (which resulted in successful separations in previous projects) were investigated (Table S1 from Supplementary Materials). Solvent mixture 1 consisting of toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *v*/*v*/*v*/*v*, was suited best, as it distributed the sample components well along the developing distance, especially detectable after derivatization with the anisaldehyde sulfuric acid reagent at FLD 366 nm. A large portion of each methanolic extract remained in the initial start zone area, to which the sugar content contributed most, being as high as 16% in the 8 fruit product samples (Table 1). The individual saccharides of the samples, applied at 200 µg/area, were determined using a more polar mobile phase for separation (Table S1 from Supplementary Materials), mobile phase 6: acetonitrile–water 4:1, *v*/*v*, plus 8 mg diphenylboric acid-2-aminoethylester) and the diphenylamine aniline *o*-phosphoric acid reagent for detection at white light illumination. The 8 saccharide profiles showed glucose, fructose and sucrose being present (Figure 1). Among these, the mango cultivars (Table 2, puree IDs 3,1 and 4,1; juice IDs 7,1 and 8,1) showed a lower glucose and instead a higher sucrose content than the pineapple cultivars (puree IDs 1,1 and 2,1; juice IDs 5,1 and 6,1).

Puree extract Juice extract

Pineapple PTV

Pineapple NL

Mango NDM

Mango MHC

1,2 5,2

2,2 6,2

4,2 8,2

1,1

2,1

3,1

4,1

ID Fruit Cultivar ID Fruit Cultivar

Mango NDM 3,2 7,2

5,1

6,1

7,1

8,1

**Figure 1.** Saccharide analysis: HPTLC-Vis chromatogram of fructose (Fru), glucose (Glc) and sucrose (Suc) present in fruit product extracts (IDs 1–8, Table 2, 0.4 g/mL in methanol, 0.5 µL/area) on HPTLC plates silica gel 60 F254 with acetonitrile–water 4:1, V/V, plus 8 mg diphenylboric acid-2 aminoethylester (Table S1 from Supplementary Materials, mobile phase 6) derivatized with diphenylamine aniline *o-*phosphoric acid reagent and documented at white light illumination. **Figure 1.** Saccharide analysis: HPTLC-Vis chromatogram of fructose (Fru), glucose (Glc) and sucrose (Suc) present in fruit product extracts (IDs 1–8, Table 2, 0.4 g/mL in methanol, 0.5 µL/area) on HPTLC plates silica gel 60 F254 with acetonitrile–water 4:1, *v*/*v*, plus 8 mg diphenylboric acid-2 aminoethylester (Table S1 from Supplementary Materials, mobile phase 6) derivatized with diphenylamine aniline *o*-phosphoric acid reagent and documented at white light illumination.

**Table 2.** Produced fruit products (IDs 1–8) extracted either with methanol (x,1) or ethyl acetate (x,2).

Pineapple PTV

Pineapple NL

Mango MHC

*2.3. Selection of the Extraction Solvent for Effect-Directed Profiling* **Table 2.** Produced fruit products (IDs 1–8) extracted either with methanol (x,1) or ethyl acetate (x,2).

#### and the use of derivatization reagents, the compounds in the extracts were detected via *2.3. Selection of the Extraction Solvent for Effect-Directed Profiling*

the Gram-negative *Aliivibrio fischeri* bioassay (Figure 2C). This assay detects more universally any bioactive chemicals interfering with the energetic metabolism of the bacteria and is commonly used in environmental research [30]. The bioautogram revealed a similar Sample preparation was performed as minimally as possible to minimize any influence (alteration) in the fruit products produced. Different extraction solvents (i.e., methanol, acetone, methanol/acetone 1:1, ethanol, ethyl acetate, and acetonitrile) were comparatively studied for the intended non-targeted effect-directed profiling (Figure 2, all 4 mg/area). For polar extraction solvents, the co-extracted saccharides caused a high matrix loading on the start zone area of the adsorbent. This limited not only the amount of sample that could be applied, but also the development with more polar mobile phases (Table S1 from Supplementary Materials, mobile phases 4 and 6). In comparison, the ethyl acetate extracts discriminated the co-extraction of saccharides, as evident in the absent dark start zone area. This was found to be advantageous, in case higher sample amounts need to be used depending on the assay response. In addition, the ethyl acetate extracts contained a similar pattern of medium polar components that was almost comparable to that of the other solvents. Therefore, to cover different selectivities, not only methanol but also ethyl acetate were chosen as extraction solvent (Table 2). Apart from UV/Vis/FLD detections and the use of derivatization reagents, the compounds in the extracts were detected via the Gram-negative *Aliivibrio fischeri* bioassay (Figure 2C). This assay detects more universally any bioactive chemicals interfering with the energetic metabolism of the bacteria and is commonly used in environmental research [30]. The bioautogram revealed a similar pattern of bioactive compounds within the different samples. As the bioactivity pattern of the applied 10 µL sample (4 mg/area) was faint, it was evident that a higher sample

amount was required to detect the bioactive components present at low concentrations. Therefore, a 5-fold amount of each fruit product extract (solid-liquid extract ratio of 20% in ethyl acetate) was applied (100 µL/area) in the following. the applied 10 µL sample (4 mg/area) was faint, it was evident that a higher sample amount was required to detect the bioactive components present at low concentrations. Therefore, a 5-fold amount of each fruit product extract (solid-liquid extract ratio of 20% in ethyl acetate) was applied (100 µL/area) in the following.

pattern of bioactive compounds within the different samples. As the bioactivity pattern of

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**Figure 2.** Investigation of different solvent selectivities for extraction: HPTLC chromatogram at FLD 366 nm after derivatization with anisaldehyde sulfuric acid reagent (**A**), at FLD 366 nm before the bioassay (**B**) and bioautogram after *Aliivibrio fischeri* bioassay, depicting bioluminescence after 30 min as grey scale image (**C**) of methanol (MeOH), mixture of methanol/acetone 1:1 (Mix), acetone, ethanol (EtOH), ethyl acetate (EtOAc) and acetonitrile (ACN) extracts of puree sample IDs 1 and 3 (Table 2, 0.4 g/mL) applied as area (6.0 × 3.0 mm, 10 µL/area), developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *V/V/V/V,* in twin-trough chamber 20 x 10 cm (\*yellow acetone-soluble contamination from plastic flask material). **Figure 2.** Investigation of different solvent selectivities for extraction: HPTLC chromatogram at FLD 366 nm after derivatization with anisaldehyde sulfuric acid reagent (**A**), at FLD 366 nm before the bioassay (**B**) and bioautogram after *Aliivibrio fischeri* bioassay, depicting bioluminescence after 30 min as grey scale image (**C**) of methanol (MeOH), mixture of methanol/acetone 1:1 (Mix), acetone, ethanol (EtOH), ethyl acetate (EtOAc) and acetonitrile (ACN) extracts of puree sample IDs 1 and 3 (Table 2, 0.4 g/mL) applied as area (6.0 × 3.0 mm, 10 µL/area), developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *v*/*v*/*v*/*v*, in twin-trough chamber 20 × 10 cm (\* yellow acetone-soluble conta mination from plastic flask material).

#### *2.4. Physico-chemical Profiling of the Fruit Product Extracts 2.4. Physico-Chemical Profiling of the Fruit Product Extracts*

The physico-chemical profiling of the ethyl acetate extracts of the 8 different fruit products provided information on the absorbance or fluorescence of individual compounds. Close to the solvent front of the separated mango puree extracts (IDs 3,2 and 4,2), a yellow zone, most likely carotenoids, was visible (Figure 3A). UV-active compounds were comparatively more pronounced in the mango (IDs 3,2; 4,2; 7,2 and 8,2) than pineapple product extracts (Figure 3B). Among the mango products, the puree extracts (IDs 3,2 and 4,2) contained more UV-active compounds than the juice extracts, mainly retained in the start area of the adsorbent. The physico-chemical profiling of the ethyl acetate extracts of the 8 different fruit products provided information on the absorbance or fluorescence of individual compounds. Close to the solvent front of the separated mango puree extracts (IDs 3,2 and 4,2), a yellow zone, most likely carotenoids, was visible (Figure 3A). UV-active compounds were comparatively more pronounced in the mango (IDs 3,2; 4,2; 7,2 and 8,2) than pineapple product extracts (Figure 3B). Among the mango products, the puree extracts (IDs 3,2 and 4,2) contained more UV-active compounds than the juice extracts, mainly retained in the start area of the adsorbent.

**Figure 3.** Physico-chemical profiling: HPTLC chromatograms at white light illumination (Vis, **A**), UV 254 nm (**B**), FLD 366 nm (**C**) and after derivatization with anisaldehyde sulfuric acid reagent (**D**  FLD 366 nm and **E** Vis) of the puree and juice extracts (IDs 1–8, Table 2, 0.2 g/mL in ethyl acetate, 100 µL/area of 8.0 × 6.0 mm) developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate– methanol–formic acid 2.4:1.8:0.7:0.06, *V/V/V/V,* in twin-trough chamber 10 cm × 10 cm. **Figure 3.** Physico-chemical profiling: HPTLC chromatograms at white light illumination (Vis, **A**), UV 254 nm (**B**), FLD 366 nm (**C**) and after derivatization with anisaldehyde sulfuric acid reagent (**D** FLD 366 nm and **E** Vis) of the puree and juice extracts (IDs 1–8, Table 2, 0.2 g/mL in ethyl acetate, 100 µL/area of 8.0 × 6.0 mm) developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *v*/*v*/*v*/*v*, in twin-trough chamber 10 cm × 10 cm.

In the FLD 366 nm chromatogram (Figure 3C), a dominantly yellow fluorescent zone was observed in the solvent front of the mango puree extracts (IDs 3,2 and 4,2). This zone was also visible and UV-active as mentioned. Tentatively, more blue fluorescent zones were observed in the pineapple (IDs 1,2; 2,2; 5,2 and 6,2) than mango products (IDs 3,2; In the FLD 366 nm chromatogram (Figure 3C), a dominantly yellow fluorescent zone was observed in the solvent front of the mango puree extracts (IDs 3,2 and 4,2). This zone was also visible and UV-active as mentioned. Tentatively, more blue fluorescent zones were

observed in the pineapple (IDs 1,2; 2,2; 5,2 and 6,2) than mango products (IDs 3,2; 4,2; 7,2 and 8,2). After derivatization with the anisaldehyde sulfuric acid reagent and detection at FLD 366 nm (Figure 3D) or with white light illumination (Figure 3E), further components of the fruit products were detectable. For example, a strongly pink fluorescent (Figure 3D) or lilac (Figure 3E) compound at *hR*F 80 was detected only by chemical derivatization, proving that the commonly used UV/Vis/FLD detection is not sufficient for quality control. In particular, this compound zone also proved to have multiple bioactivities, as discussed subsequently. As expected by the natural density of the product and verified by all the different detection modes, the puree extract samples (IDs 1,2–4,2) were more complex and rich in natural compounds. 4,2; 7,2 and 8,2). After derivatization with the anisaldehyde sulfuric acid reagent and detection at FLD 366 nm (Figure 3D) or with white light illumination (Figure 3E), further components of the fruit products were detectable. For example, a strongly pink fluorescent (Figure 3D) or lilac (Figure 3E) compound at *hR*F 80 was detected only by chemical derivatization, proving that the commonly used UV/Vis/FLD detection is not sufficient for quality control. In particular, this compound zone also proved to have multiple bioactivities, as discussed subsequently. As expected by the natural density of the product and verified by all the different detection modes, the puree extract samples (IDs 1,2–4,2) were more complex and rich in natural compounds.

#### *2.5. Effect-Directed Profiling of the Fruit Product Extracts 2.5. Effect-Directed Profiling of the Fruit Product Extracts*

*Molecules* **2021**, *26*, x FOR PEER REVIEW 7 of 13

The effect-directed profiling of the ethyl acetate extracts of the 8 different fruit products provided information on bioactive components using five different effect-directed assays (i.e., one chemical, two biological and two enzymatic assays). The DPPH• radical scavenging assay (Figure 4A) showed for both mango products (puree and juice) the strongest radical scavenging (antioxidative) compound zone at the start area and at *hR*F 37 due to the ascorbic acid addition. This addition was required during juice production to prevent the enzymatic browning reaction, and thus, darkening of mango products. The effect-directed profiling of the ethyl acetate extracts of the 8 different fruit products provided information on bioactive components using five different effect-directed assays (*i. e.*, one chemical, two biological and two enzymatic assays). The DPPH• radical scavenging assay (Figure 4A) showed for both mango products (puree and juice) the strongest radical scavenging (antioxidative) compound zone at the start area and at *hR*F 37 due to the ascorbic acid addition. This addition was required during juice production to prevent the enzymatic browning reaction, and thus, darkening of mango products.

**Figure 4.** Effect-directed profiling: HPTLC autograms after DPPH• assay (**A**), *Aliivibrio fischeri* bioassay, instantly and after 30 min (**B**), *Bacillus subtilis* bioassay (**C**), AChE (**D,** here zones shifted upwards by 2 mm) and tyrosinase inhibition assays I detected at white light illumination (except for B: bioluminescence depicted as grey scale image) of puree and juice extracts (IDs 1–8, Table 2, 0.2 g/mL in ethyl acetate, 100 µL/area of 8.0 × 6.0 mm) developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *V/V/V/V*, in twin-trough chamber 10 cm × 10 cm (chromatograms at UV 254 nm and FLD 366 nm before the respective assay detection in Table S2 from Supplementary Materials). **Figure 4.** Effect-directed profiling: HPTLC autograms after DPPH• assay (**A**), *Aliivibrio fischeri* bioassay, instantly and after 30 min (**B**), *Bacillus subtilis* bioassay (**C**), AChE (**D,** here zones shifted upwards by 2 mm) and tyrosinase inhibition assays I detected at white light illumination (except for B: bioluminescence depicted as grey scale image) of puree and juice extracts (IDs 1–8, Table 2, 0.2 g/mL in ethyl acetate, 100 µL/area of 8.0 × 6.0 mm) developed on HPTLC plates silica gel 60 F254 with toluene–ethyl acetate–methanol–formic acid 2.4:1.8:0.7:0.06, *v*/*v*/*v*/*v*, in twin-trough chamber 10 cm × 10 cm (chromatograms at UV 254 nm and FLD 366 nm before the respective assay detection in Table S2 from Supplementary Materials).

Nevertheless, clear differences in the antioxidative response at *hR*F 60 and *hR*F 80 were evident for the two mango cultivars (IDs 3,2 versus 4,2; 7,2 versus 8,2). The comparison of the two mango products also revealed a difference. The enzymatic or heat treatment during juice production degraded the antioxidative compound zone at *hR*F 80 (e.g., IDs 4,2 versus 8,2).

At the same position (*hR*F 37) as the previously mentioned main antioxidative compound zone (ascorbic acid), the Gram-negative *Aliivibrio fischeri* bioautogram (Figure 4B) revealed a dominant bioactive dark zone in both mango products and cultivars which, however, substantially decreased over the 30 min imaging inspection. This indicated an acute transient effect, i.e., the energetic metabolism of the bacteria was immediately but temporarily disrupted by this compound and recovered almost completely after 30 min. In the pineapple cultivars, two further dominant antimicrobial zones at *hR*F 58 and 68 were observed. The similar acute transient effect was observed for the antimicrobial zone at *hR*F 68, whereas the antimicrobial zone at *hR*F 58 increased in activity over the monitored 30 min period. The latter indicated a lasting effect with a delayed increase in the activity response. Over the monitoring period, a new antimicrobial compound appeared at *hR*F 80 mainly in both fruit purees, as evident in the 30-min bioautogram. This indicated a delayed increase in the activity response.

The Gram-positive *Bacillus subtilis* bioassay (Figure 4C) revealed the same antimicrobial compound zone at *hR*F 80 in both fruit puree extracts. This colorless zone at *hR*F 80 acting against *B. subtilis* was also the main inhibitor of the AChE (Figure 4D, IDs 1,2–4,2). Another weaker AChE inhibiting zone was detected at *hR*F 60. Both colorless zones were almost absent in the juice extracts, most likely degraded by the enzymatic or heat treatment during the juice production. Especially, the mango cultivar NDM (IDs 3,2 and 7,2) revealed a further AChE inhibiting compound zone at *hR*F 45.

The tyrosinase inhibition assay (Figure 4E) showed three colorless inhibition zones in the puree extracts (IDs 1,2–4,2) with the main inhibition zone at *hR*F 80. This multipotent compound zone showed already a pronounced activity against *B. subtilis* and AChE. Vice versa, the dark zone above (*hR*F 85) in the mango puree extracts (ID 3,2 and 4,2) triggered the fruit browning. Another weaker browning-promoting dark zone at *hR*F 78 was evident in all puree extracts (IDs 1,2–4,2).

Differences in the activity profiles depending on the fruit processing were evident. Both purees contained a multipotent zone at *hR*F 80 (active against both bacteria, AChE and tyrosinase, Figure 4B–E), which was lost during the production of juice made out of the puree. This was a more general observation across all the different assays, as the more apolar bioactive compounds (evident in the upper autogram part) were partially or totally lost during the production of juice. Although it was expected that puree is richer in bioactive compounds than juice, the severe loss was surprising. All in all, the bioprofiling of food products turned out to be a rich source of information with regard to changes in the activity potential caused by food processing.

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

#### *3.1. Chemicals and Reagents*

Bi-distilled water was produced using a Heraeus Destamat Bi-18E (Thermo Fisher Scientific, Schwerte, Germany). Ethyl acetate (≥99.7%) and ammonia (25%) were obtained from Th. Geyer, Renningen, Germany. All amino acids, lithium citrate, all mineral salts for producing phosphate-buffered saline (PBS), acetone (≥99.5%), acetonitrile (≥99.9%), diphenylamine, aniline, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•, 97%), acetylcholinesterase (AChE) from *Electrophorus electricus* (≥245 U/mg, 10 kU/vial), bovine serum albumin (BSA, fraction V, ≥98%) and tyrosinase from mushroom (≥1000 U/mg, 25 kU/vial) were obtained from Fluka Sigma-Aldrich, Steinheim, Germany. 1-Naphthyl acetate (≥98%) and 2-naphthyl-α-D-glucopyranoside were obtained by AppliChem, Darmstadt, Germany. Toluene, ethanol, 2-propanol (all ≥ 99.9%), formic acid (≥98%), sulfuric acid (96%), diphenylboric acid-2-aminoethylester, 4-methoxybenzaldehyde (anisaldehyde,

≥97.5%), tris(hydroxymethyl)amino¬methane (TRIS, ≥99.9%) and thiazol blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT) were purchased from Carl Roth, Karlsruhe, Germany. Methanol (>99.8%) was purchased from VWR, Darmstadt, Germany. (2S)-2-Amino-3-(3,4-dihydroxyphenyl) propionic acid (levodopa) was obtained from Santa Cruz Biotechnology, Dallas, TX, USA. 1-Butanol was from Alfa Aesar, Karlsruhe, Germany. HPTLC plates silica gel 60 F254 (without F254 for *B. subtilis* bioassay), 20 cm × 10 cm, ascorbic acid, potassium disulfite, sodium hydroxide, Grampositive soil bacteria *Bacillus subtilis* subsp. spizizenii (DSM-618), and Folin-Ciocalteu's phenol reagent were from Merck, Darmstadt, Germany. Gram-negative marine *A. fischeri* bacteria (NRRL-B11177, strain 7151) were bought from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures), Berlin, Germany. Fruit samples were from the orchard at Chiang Rai province, Northern Thailand.

#### *3.2. Production of Puree Samples*

Ripe pineapples were washed with water, peeled, cut into small pieces and crushed to form pineapple puree. Then 50 mg/L of sulfur dioxide was added. Ripe mangoes were also washed, peeled, cut and crushed, but after peeling the seeds were removed. The mango purees were treated with 40 mg/L of ascorbic acid and 50 mg/L of sulfur dioxide. Each fruit puree (1 kg) was placed in a polyethylene pouch and stored at −18 ◦C until further use.

#### *3.3. Production of Juice Samples*

The frozen puree of each cultivar was thawed at ambient temperature and treated with pectinase (400 µL/L; Trenolin®Super DF, Erbslöh, Geisenheim, Germany). The puree was incubated at 45–50 ◦C for 1 h. Then, it was heated at 70 ◦C for 5 min to inactivate the enzymes. The juice was separated from the solid material part by centrifugation at 5000× *g* at 4 ◦C for 10 min. Juice (100 mL) was filled in a polyethylene bottle and stored at −18 ◦C until use.

#### *3.4. Physico-Chemical Characterization of the Produced Purees and Juices*

The standard parameters were established as follows: total soluble solids (TSS) was measured by a digital automatic refractometer (Abbemat, Anton Paar®, St. Albans, Austria). The total saccharide content was enzymatically determined using a Konelab 20XTi analyzer (Thermo Fisher Scientific, Schwerte, Germany) and its proper kits (EnzytecTM fluid, Thermo Fisher Scientific). Total acidity and pH were analyzed by a Schott titrator (Titroline alpha plus, SI-Analytics, Texas City, TX, USA). The content of total phenols was evaluated by the Folin assay (Singleton and Rossi 1965) using an automatic analyzer (Konelab 20XTi, Thermo Fisher Scientific). Amino acids were determined by an Amino Acid Analyzer S433 (Sykam GmbH, Eresing, Germany). All measurements were made in triplicate.


*4.2. Deducing Analytical Surrogate Model for IPM*

As shown in Equation (26), the gradient of functions *F*,*G* and *H* are needed in the process of IPM. The gradient of *F*, *G* and *Hc* can be obtained directly. However, since the surrogate model is a "black-box", the gradient of *Hs* cannot be simply calculated. Based on Equations (22) and (23), the gradient of *Hs* can be transformed into:

$$\begin{array}{l}\bigtriangledown\!\Gamma = \bigtriangledown^{2}\!\Gamma\_{\mathfrak{c}} = \bigtriangledown^{2}\!\Psi\_{\mathfrak{c}}(\mathbf{X})\,,\,\mathfrak{c}\in\mathcal{S}\_{\mathfrak{c}}\\\bigtriangledown^{2}\!\mathbf{H}\_{\mathfrak{s}} = \bigtriangledown^{2}\!\Gamma\_{\mathfrak{c}} = \bigtriangledown^{2}\!\Psi\_{\mathfrak{c}}(\mathbf{X})\end{array}\tag{31}$$

Next, by DBN backwards process, Equation (31) is deduced to get the Jacobin and Hessian matrix, which are also known as sensitivities of transient stability against optimization variables. See Appendix A for the detailed DBN backwards process. The algorithm flow and implementation of the proposed method are shown in Figure 1.

**Figure 1.** The flow chart of the proposed method.


*2.2. Plant Materials*

Banana fingers (*Musa* spp. AAA group, Cavendish subgroup) ripened to "yellow with green tips", "yellow", or "yellow, flecked with brown", unless otherwise stated [20], were produced in the Philippines and purchased from local grocery stores, and experiments were conducted in the Osaka Prefecture University lab. For ripening stage influence experiments, unripe fruit were purchased and ripening was triggered by 100 μL L−1 of ethylene at 20 ◦C for 24 h. For all samples, each replicate contained five fingers, and each treatment had three replicates. For fruit pulp slice samples, cylindrical pulp discs were taken from the central section of a banana finger and further divided into 4 wedges. Pulps were homogenized in some experiments.


2.2.1. Cost Function Composition

#### (1) Vehicle Fixed Cost

The fixed cost of a vehicle is the cost of purchase, maintenance, labor paid to the driver, etc. It only depends on the number of vehicles performing the task. The function was as follows:

$$Z\_1 = \mathbb{C}\_0 \sum\_{k=1}^m \sum\_{i=1}^u X\_{0i}^k \tag{10}$$

### (2) Transportation Cost

In general, the shorter the distance traveled, the lower the cost. Thus, the transportation cost of the vehicle is proportional to the distance traveled. *Cp* represents the transportation cost within a unit distance. The total transportation cost *Z*2 could be expressed as:

$$Z\_2 = \sum\_{k=1}^{m} \sum\_{i=0}^{n} \sum\_{j=0}^{n} d\_{ij} \mathbb{C}\_p X\_{ij}^k \tag{11}$$

#### (3) Loss Cost

Both the transit period and the load of goods incurred a loss cost. The continuous lifetime function decayed according to an exponential rate. The decay function of fresh products was: *E*(*t*) = *E*0 · *e*−*θt* [40]. This formula expresses the quality of the product at different times, where *E*0 is the product's initial quality and *θ* is the sensitivity factor of the product to time. The loss cost of transit without opening the door was as follows:

$$\mathcal{K}\_{\mathcal{S}} = \sum\_{i=0}^{n} \sum\_{k=1}^{m} Y\_{i\mathbf{k}} q\_i [1 - K\_1 e^{-\theta(t\_i - t\_0)}] \mathcal{R}\_1 \tag{12}$$

Temperature changes caused by opening the door during loading could also affect the freshness of the product. The door opening time was determined using the customer service time *si*. The loss cost caused by loading goods while opening the door was:

$$\mathcal{C}\_{I} = \sum\_{i=0}^{\mathcal{U}} \sum\_{k=1}^{m} Y\_{ik} Q\_{i0} (1 - K\_{2} \mathcal{e}^{-\theta S\_{i}}) R\_{1} \tag{13}$$

We could obtain the total loss cost in the whole distribution process as follows:

$$Z\_3 = \sum\_{i=0}^{u} \sum\_{k=1}^{m} Y\_{ik} R\_1[q\_i(1 - K\_1 e^{-\theta(t\_i - t\_0)}) + Q\_{i0}(1 - K\_2 e^{-\theta S\_i})] \tag{14}$$

#### (4) Penalty Cost

In order to maximize the freshness of our products, we needed to arrive within the time frames set by our customers individually. Otherwise, there would be penalty costs. Suppose that the time window required by the customer was (*ETi*, *LTi*). We could use *ti* to represent the time when the delivery vehicle arrives at customer *i*. If *ETi* ≤ *ti* ≤ *LTi*, no additional penalty cost would be incurred. If the goods were not delivered within the time window, the penalty cost *Z*4 would be paid. Arriving earlier than the time requested would incur a waiting cost, and arriving later would pay a delayed service fee. The relation was stated as follows:

$$Z\_4 = \gamma\_1 \sum\_{i=1}^{u} \max\{ET\_i - t\_{i\prime}0\} + \gamma\_2 \sum\_{i=1}^{u} \max\{t\_i - LT\_i, 0\} \tag{15}$$


[39] Bassi C, Salvia R, Butturini G, Marcucci S, Barugola G, Falconi M. Value of regional lymphadenectomy in pancreatic cancer. HPB : The Official Journal of the International Hepato Pancreato Biliary Association. 2005;**7**(2):87-92. DOI: 10.1080/13651820510028855

[40] Wagner M, Redaelli C, Lietz M, Seiler CA, Friess H, Büchler MW. Curative resection is the single most important factor determining outcome in patients with pancreatic adenocarcinoma. BJS. 2004;**91**(5):586-594. DOI: 10.1002/ bjs.4484

[41] Verbeke CS, Gladhaug IP. Resection margin involvement and tumour origin in pancreatic head cancer. The British Journal of Surgery. 2012;**99**(8):1036- 1049. DOI: 10.1002/bjs.8734

[42] Bronstein YL, Loyer EM, Kaur H, Choi H, David C, DuBrow RA, et al. Detection of small pancreatic tumors with

**89**

*Photoacoustic Imaging in Gastroenterology: Advances and Needs*

[49] Harris PL, Rumley TO,

Lineaweaver WC. Pancreatic cancer: Unreliability of frozen section in diagnosis. Southern Medical Journal. 1985;**78**(9):1053-1056. DOI: 10.1097/00007611-198509000-00007

[50] Witz M, Shkolnik Z, Dinbar A. Intraoperative pancreatic biopsy—A diagnostic dilemma. Journal of surgical oncology. 1989;**42**(2):117-119. DOI:

[51] Homan K, Shah J, Gomez S, Gensler H, Karpiouk A, Brannon-Peppas L, et al. Combined Ultrasound and Photoacoustic Imaging of Pancreatic Cancer Using Nanocage Contrast Agents. Proceeding SPIE, Photons Plus Ultrasound: Imaging and Sensing. 2009; 71771M, 6 p. DOI:

[52] Homan KA, Souza M, Truby R, Luke GP, Green C, Vreeland E, et al. Silver nanoplate contrast agents for in vivo molecular photoacoustic

imaging. ACS Nano. 2012;**6**(1):641-650.

[53] Dai X, Qian W, Yang H, Yang L, Jiang H. Targeted molecular imaging of pancreatic cancer with a miniature

endoscope. Applied Sciences. 2017;**7**(12):1241. DOI: 10.3390/

[54] Tummers WS, Miller SE, Teraphongphom NT, Gomez A, Steinberg I, Huland DM, et al. Intraoperative pancreatic cancer detection using tumor-specific multimodality molecular imaging. Annals of Surgical Oncology. 2018;**25**(7):1880-1888. DOI: 10.1245/

[55] Petrova EV, Oraevsky AA, Ermilov SA. Red blood cell as a universal optoacoustic sensor for non-invasive temperature monitoring. Applied Physics Letters. 2014;**105**(9):094103.

app7121241

s10434-018-6453-2

DOI: 10.1063/1.4894635

10.1002/jso.2930420210

10.1117/12.807520

DOI: 10.1021/nn204100n

*DOI: http://dx.doi.org/10.5772/intechopen.86051*

multiphasic helical CT. American Journal of Roentgenology. 2004;**182**(3):619-623.

DOI: 10.2214/ajr.182.3.1820619

nrclinonc.2013.123

[43] Vahrmeijer AL, Hutteman M, Van Der Vorst JR, Van De Velde CJ, Frangioni JV. Image-guided cancer surgery using near-infrared fluorescence. Nature Reviews Clinical Oncology. 2013;**10**(9):507. DOI: 10.1038/

[44] Kruskal J, Kane R. Intraoperative ultrasonography of the pancreas: Techniques and clinical applications. Surgical Technology International. Bellingham, Washington, USA.

1997;**6**:49-57. DOI: 10.1148/rg.307105051

[45] Sun MR, Brennan DD, Kruskal JB, Kane RA. Intraoperative ultrasonography of the pancreas. Radiographics. 2010;**30**(7):1935-1953.

DOI: 10.15557/JoU.2015.0005

[46] Hata S, Imamura H, Aoki T, Hashimoto T, Akahane M, Hasegawa K, et al. Value of visual inspection, bimanual palpation, and intraoperative

ultrasonography during hepatic resection for liver metastases of colorectal carcinoma. World Journal of Surgery. 2011;**35**(12):2779-2787. DOI: 10.1097/01.sla.0000171307.37401.db

[47] Handgraaf HJ, Boonstra MC, Van Erkel AR, Bonsing BA, Putter H, Van De Velde CJ, et al. Current and future intraoperative imaging strategies to increase radical resection rates in pancreatic cancer surgery. Biomed Research International. 2014;**2014**:8. Article ID: 890230. DOI:

[48] Nelson DW, Blanchard TH, Causey MW, Homann JF, Brown TA. Examining the accuracy and clinical usefulness of intraoperative frozen section analysis in the management of pancreatic lesions. American Journal of Surgery. 2013;**205**(5):613-617. DOI: 10.1016/j.

10.1155/2014/890230

amjsurg.2013.01.015

*Photoacoustic Imaging in Gastroenterology: Advances and Needs DOI: http://dx.doi.org/10.5772/intechopen.86051*

multiphasic helical CT. American Journal of Roentgenology. 2004;**182**(3):619-623. DOI: 10.2214/ajr.182.3.1820619

*Photoacoustic Imaging - Principles, Advances and Applications*

2016;**151**(2):238-240. DOI: 10.1053/j.

[36] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2018;**68**(6):394-424. DOI:

[37] Hidalgo M, Cascinu S, Kleeff J, Labianca R, Löhr JM, Neoptolemos J, et al. Addressing the challenges of pancreatic cancer: Future directions for improving outcomes. Pancreatology. 2015;**15**(1):8-18. DOI: 10.1016/j.

[38] Stathis A, Moore MJ. Advanced pancreatic carcinoma: Current

[39] Bassi C, Salvia R, Butturini G, Marcucci S, Barugola G, Falconi

in pancreatic cancer. HPB : The Official Journal of the International

Association. 2005;**7**(2):87-92. DOI: 10.1080/13651820510028855

[40] Wagner M, Redaelli C, Lietz M, Seiler CA, Friess H, Büchler MW. Curative resection is the single most important factor

determining outcome in patients with pancreatic adenocarcinoma. BJS. 2004;**91**(5):586-594. DOI: 10.1002/

[41] Verbeke CS, Gladhaug IP. Resection margin involvement and tumour origin in pancreatic head cancer. The British Journal of Surgery. 2012;**99**(8):1036-

[42] Bronstein YL, Loyer EM, Kaur H, Choi H, David C, DuBrow RA, et al. Detection of small pancreatic tumors with

1049. DOI: 10.1002/bjs.8734

Hepato Pancreato Biliary

bjs.4484

treatment and future challenges. Nature Reviews Clinical Oncology. 2010;**7**:163. DOI: 10.1038/nrclinonc.2009.236

M. Value of regional lymphadenectomy

gastro.2016.05.047

10.3322/caac.21492

pan.2014.10.001

3D endoscopy. Light: Science & Applications. 2018;**7**(1):75. DOI: 10.1038/s41377-018-0070-5

10.1371/journal.pone.0120269

10.1055/s-0043-111790

[32] Ng SC, Shi HY, Hamidi N, Underwood FE, Tang W, Benchimol EI, et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: A systematic review of population-based studies. The Lancet. 2017;**390**(10114):2769-2778. DOI: 10.1016/S0140-6736(17)32448-0

[33] Bhutiani N, Grizzle WE, Galandiuk S, Otali D, Dryden GW, Egilmez NK, et al. Noninvasive imaging of colitis using multispectral optoacoustic tomography. Journal of Nuclear Medicine: Official Publication,

Society of Nuclear Medicine.

jnumed.116.184705

10.1056/NEJMc1612455

2017;**58**(6):1009-1012. DOI: 10.2967/

[34] Knieling F, Neufert C, Hartmann A, Claussen J, Urich A, Egger C, et al. Multispectral optoacoustic tomography for assessment of Crohn's disease activity. The New England Journal of Medicine. 2017;**376**(13):1292-1294. DOI:

[35] Waldner MJ, Knieling F, Egger C, Morscher S, Claussen J, Vetter M, et al. Multispectral optoacoustic tomography in Crohn's disease: Noninvasive imaging of disease activity. Gastroenterology.

[31] Lim L, Streutker CJ, Marcon N, Cirocco M, Lao A, Iakovlev VV, et al. A feasibility study of photoacoustic imaging of ex vivo endoscopic mucosal resection tissues from Barrett's esophagus patients. Endoscopy International Open. 2017;**5**(8):E775-E783. DOI:

[30] Yang JM, Favazza C, Yao J, Chen R, Zhou Q, Shung KK, et al. Threedimensional photoacoustic endoscopic imaging of the rabbit esophagus. PLoS One. 2015;**10**(4):e0120269. DOI:

**88**

[43] Vahrmeijer AL, Hutteman M, Van Der Vorst JR, Van De Velde CJ, Frangioni JV. Image-guided cancer surgery using near-infrared fluorescence. Nature Reviews Clinical Oncology. 2013;**10**(9):507. DOI: 10.1038/ nrclinonc.2013.123

[44] Kruskal J, Kane R. Intraoperative ultrasonography of the pancreas: Techniques and clinical applications. Surgical Technology International. Bellingham, Washington, USA. 1997;**6**:49-57. DOI: 10.1148/rg.307105051

[45] Sun MR, Brennan DD, Kruskal JB, Kane RA. Intraoperative ultrasonography of the pancreas. Radiographics. 2010;**30**(7):1935-1953. DOI: 10.15557/JoU.2015.0005

[46] Hata S, Imamura H, Aoki T, Hashimoto T, Akahane M, Hasegawa K, et al. Value of visual inspection, bimanual palpation, and intraoperative ultrasonography during hepatic resection for liver metastases of colorectal carcinoma. World Journal of Surgery. 2011;**35**(12):2779-2787. DOI: 10.1097/01.sla.0000171307.37401.db

[47] Handgraaf HJ, Boonstra MC, Van Erkel AR, Bonsing BA, Putter H, Van De Velde CJ, et al. Current and future intraoperative imaging strategies to increase radical resection rates in pancreatic cancer surgery. Biomed Research International. 2014;**2014**:8. Article ID: 890230. DOI: 10.1155/2014/890230

[48] Nelson DW, Blanchard TH, Causey MW, Homann JF, Brown TA. Examining the accuracy and clinical usefulness of intraoperative frozen section analysis in the management of pancreatic lesions. American Journal of Surgery. 2013;**205**(5):613-617. DOI: 10.1016/j. amjsurg.2013.01.015

[49] Harris PL, Rumley TO, Lineaweaver WC. Pancreatic cancer: Unreliability of frozen section in diagnosis. Southern Medical Journal. 1985;**78**(9):1053-1056. DOI: 10.1097/00007611-198509000-00007

[50] Witz M, Shkolnik Z, Dinbar A. Intraoperative pancreatic biopsy—A diagnostic dilemma. Journal of surgical oncology. 1989;**42**(2):117-119. DOI: 10.1002/jso.2930420210

[51] Homan K, Shah J, Gomez S, Gensler H, Karpiouk A, Brannon-Peppas L, et al. Combined Ultrasound and Photoacoustic Imaging of Pancreatic Cancer Using Nanocage Contrast Agents. Proceeding SPIE, Photons Plus Ultrasound: Imaging and Sensing. 2009; 71771M, 6 p. DOI: 10.1117/12.807520

[52] Homan KA, Souza M, Truby R, Luke GP, Green C, Vreeland E, et al. Silver nanoplate contrast agents for in vivo molecular photoacoustic imaging. ACS Nano. 2012;**6**(1):641-650. DOI: 10.1021/nn204100n

[53] Dai X, Qian W, Yang H, Yang L, Jiang H. Targeted molecular imaging of pancreatic cancer with a miniature endoscope. Applied Sciences. 2017;**7**(12):1241. DOI: 10.3390/ app7121241

[54] Tummers WS, Miller SE, Teraphongphom NT, Gomez A, Steinberg I, Huland DM, et al. Intraoperative pancreatic cancer detection using tumor-specific multimodality molecular imaging. Annals of Surgical Oncology. 2018;**25**(7):1880-1888. DOI: 10.1245/ s10434-018-6453-2

[55] Petrova EV, Oraevsky AA, Ermilov SA. Red blood cell as a universal optoacoustic sensor for non-invasive temperature monitoring. Applied Physics Letters. 2014;**105**(9):094103. DOI: 10.1063/1.4894635

**91**

**Chapter 6**

**Abstract**

Photoacoustic Imaging of the Eye

Photoacoustic imaging (PAI) is a novel, hybrid, non-ionizing, and non-invasive imaging technology with high-resolution, high sensitivity, high-contrast, and high depth of penetration. Hence, it has particularly useful applications in eye investigations. It can provide both anatomic and functional ocular characterizations. Many eye diseases, including macular degeneration and diabetic retinopathy, involve abnormalities in the vasculature, and thus the ability of PAI to affectively visualize the vasculature can be incredibly helpful to evaluate normal and disease states of the eye. In future research, PAI of the eye can be dramatically improved in terms of its resolution, use of contrast agents for molecular imaging, safety evaluations to develop a clinically approved system, and integration with existing fundus imaging modalities. Multimodality ocular imaging platforms have also been successfully developed by a combination of photoacoustic microscopy (PAM) with other optical imaging such as optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), and fluorescence microscopy (FM). The multimodal images can accurately be acquired from a single imaging system and co-registered on the same image plane, enabling improved evaluation of eye disease states. In this book chapter, the potential application of photoacoustic imaging of the eye in both research and clinical diagnosis are comprehensively discussed as a powerful medical

*Yanxiu Li and Yannis Mantas Paulus*

screening technique for visualization of various ocular diseases.

body), retinal pigment epithelium (RPE) and pigmented tumors.

microscopy, multimodal imaging

**1. Introduction**

**Keywords:** photoacoustic imaging, photoacoustic ophthalmoscopy, photoacoustic

The eye is unique organ in that it is optically transparent to visible light in normal health and thus offers optical access to internal structures. The refractive system of the normal eye includes the cornea, the fluid-filled anterior chamber, crystalline lens, and gel-like vitreous. These optically transparent structures have low absorption and light scattering to visible light, which plays a pivotal role in the entry of light and the formation of visual pathways. In the eye, the major endogenous light-absorbing materials are hemoglobin and melanin. Hemoglobin is present in blood vessels in the iris, choroid, ciliary body, retina, conjunctiva, and pathologic neovascularization. Melanin is present inside the uvea (iris, choroid and ciliary

Photoacoustic imaging (PAI), which converts light energy into sound waves, has been demonstrated to image hemoglobin [1, 2] and melanin [3, 4]. Thus eyes are an ideal system for PAI. PAI can provide an ocular vascular image based on the inherent optical absorbance of hemoglobin. Many eye diseases, including corneal neovascularization, macular degeneration, diabetic retinopathy, sickle cell

## **Chapter 6**


**1. Introduction**

Selective laser trabeculoplasty (SLT), used since 1998 when the first successful protocol was described, has become an established method for lowering the intraocular pressure (IOP) in the treatment of open-angle glaucoma (OAG) and ocular hypertension (OH) [1–5]. Multiple prospective or retrospective studies clear demonstrated the safety and efficacy of SLT in reducing the IOP in eyes with OAG or OH [6–17]. Therefore, topical medical treatment and SLT are stated as initial, first line treatment options, and SLT is also an adjunctive treatment option in the treatment for OAG or OH in the latest 5th edition of *Terminology and Guidelines for Glaucoma* from the European Glaucoma Society [18].

Anti-inflammatory topical medication four times a day for 7 days after SLT treatment is described in many studies, although there is little evidence to support this [1,6–14]. Symptomatic or asymptomatic anterior chamber inflammation after SLT may occur, but usually resolves without treatment [1,5,19–23]. Treatment with topical steroids or nonsteroidal anti-inflammatory agents after SLT in most reports has not shown to cause a significant reduction in inflammation or improved efficacy, but it still remains a controversy in clinical practice [20–25].

This retrospective chart review evaluates whether short-term use of topical steroid therapy affected the efficacy of SLT for primary open-glaucoma (POAG) patients.

**Citation:** Graˇcner, T. Impact of Short-Term Topical Steroid Therapy on Selective Laser Trabeculoplasty Efficacy. *J. Clin. Med.* **2021**, *10*, 4249. https://doi.org/10.3390/jcm10184249

Academic Editors: Miriam Kolko and Barbara Cvenkel

Received: 24 August 2021 Accepted: 16 September 2021 Published: 19 September 2021

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**Copyright:** © 2021 by the author. 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/).