Principal component analysis (PCA) of bioactive compounds and antioxidant activity of various sample particle sizes of sea urchin shells from coastal area of Lombok Island

Legis Ocktaviana Saputri¹*, Nurhidayati Nurhidayati¹, Herpan Syafii Harahap²,

Fitriannisa Faradina Zubaidi³, Arina Windri Rivarti⁴, Lina Permatasari⁵

¹Department of Pharmacology, Faculty of Medicine and Health Sciences,

The University of Mataram, West Nusa Tenggara, Indonesia.

²Department of Neurology, Faculty of Medicine and Health Sciences,

The University of Mataram, West Nusa Tenggara, Indonesia.

³Biomedical Department, Faculty of Medicine and Health Sciences,

The University of Mataram, West Nusa Tenggara, Indonesia.

⁴Department of Physiology, Faculty of Medicine and Health Sciences,

The University of Mataram, West Nusa Tenggara, Indonesia.

⁵Pharmacy Study Program, Faculty of Medicine and Health Sciences,

The University of Mataram, West Nusa Tenggara, Indonesia.

*Corresponding Author E-mail: legisocktavia@unram.ac.id

ABSTRACT:

The most common technique in increasing pharmacological activity is by reducing sample particle size. This study aims to investigate the impact of different particle sizes on the bioactive composition and antioxidant activity of sea urchin shells collected from the coastal areas of Lombok Island. Sea urchin shell powders ranging in particle sizes <45 µm, >45 µm, <125 µm, >250 µm, and >2000 µm were extracted using 70% ethanol via the cold maceration method. The composition of bioactive compounds was analyzed using GC-MS, while antioxidant activity was evaluated using the DPPH free radical scavenging assay. Principal Component Analysis (PCA), conducted using Minitab software, was employed to summarize the interrelationships among all variables in the study. The findings reveal that each particle size exhibits a distinct composition of bioactive compounds. The highest concentrations of bioactive compounds and the greatest antioxidant activity were observed in samples with particle sizes >45 µm. PCA identified several bioactive compounds, such as lanolin, palmitic acid, myristic acid, stearic acid, heptadecene-(8)-carboxylic acid, 5,8,11,14-eicosatetraenoic acid, and 9-hexadecanoic acid, contributing significantly to this antioxidant effect. Reducing the particle size was found to alter the composition of bioactive compounds and enhance antioxidant activity. These bioactive compounds show promise for further exploration in the development of new drugs derived from marine sources.

KEYWORDS: Bioactive compound, Sea urchin shell, Antioxidant, Reducing sample particle size, Principal component analysis.

INTRODUCTION:

Lombok is an island within the Indonesian archipelago, with a sea area of 59,13 % of its total¹. The sea surrounding Lombok has a diversity of marine biological resources, including genetic diversity².

With the extent of the existing sea area, Lombok has enormous maritime and fishery potential¹, including the potential as a material source for drug development^3-6, one of the species being sea urchins. This invertebrate belongs to the phylum Echinodermata which resides on coastal land⁷. They are recognized for their high content of antioxidants, particularly polyphenols, as well as other bioactive compounds with various pharmacological activities^8-9.

Antioxidants are molecules, either natural or synthetic, that help prevent or reduce oxidative damage generated by free radicals or the overproduction of oxidants such as reactive oxygen species (ROS) in the human body. By removing free radicals in the human body, antioxidants are called miracle workforce which will determine how long a person will survive¹⁰. Moreover, oxidative stress has been reported to be responsible for the pathogenesis of diseases, including cardiovascular conditions, including hypertension, atrial fibrillation, heart failure, and atherosclerosis, as well as neurodegenerative ailments such as Parkinson's disease and Alzheimer's disease^11-14. Oxidative stress has been reported as a hallmark that plays an important role in Alzheimer's disease-associated type 2 diabetes mellitus patients¹⁴.

Enhanced solubility increases the biological activity of a compound. Several techniques are commonly used including physical change-based methods and chemical methods^15,16. Sample particle size reduction is a preferable physical change method used to increase solubility¹⁷. This study is the first to determine the bioactive profile and antioxidant capacity of different particle sizes of sea urchin shells from the coastal area of Lombok Island. Multivariate analysis by chemometric techniques, namely principal component analysis (PCA) was carried out to see the relationship among variables in this study. Therefore, it is possible to determine the effect of decreasing sample particle size on the profile of bioactive compounds and antioxidant capacity. Bioactive compounds that seem to play a role in providing antioxidant activity through PCA can be further explored for their potential in developing new drugs derived from marine sources.

MATERIALS AND METHODS:

Sample Preparation:

Sea urchins were obtained from the coast of Sekotong, West Lombok (8°44'35.7"S 115°57'28.2"E), and transported using open containers with seawater and ice cubes to maintain the freshness of the samples. In the laboratory, the preparation procedure involved using sharp-tipped surgical scissors to remove all external parts such as thorns, pedicellaria, and tube feet. The peristome membrane, including the mouth or teeth, was carefully extracted using clean tweezers. The shell, which is the part used in this study, was split towards the aboral side in the direction of the ambulacral parts¹⁸. The shells were then washed and dried in a 57°C oven set for 24 hours. Following this, the particle size of the sample was reduced using a food-grade grain miller, then sifted with a sieve shaker (Retsch As 200, USA) mesh number 325, 250, 140, 50, and 10, producing dry powder samples with particle diameters <45 µm, >45 µm, <125 µm, >250 µm, and >2000 µm respectively. Particle size variations used in this study refers to research conducted by Prasedya et al¹⁹.

Sample Extraction:

Extraction by maceration method was conducted for 24 hours on the dry powder samples of each particle size using ethanol 70% with sample to solvent ratio of 1:10 (w/v). Periodic stirring was done twice. The supernatants were subsequently gathered through filtration using a filter cloth. This extraction process was conducted 3 times, each with new solvent. Filtrates from the 3 extractions were accumulated. Rotary evaporator (Heidolph Germany) at 40°C was used to obtain thick pastes of condensed extracts, which were stored at 4°C refrigerator for further use.

Bioactive Compound Identification:

Type Shimadzu QP-2010 of Gas Chromatography-Mass Spectrometer (GC-MS), column type Rtx 5Ms, with column length 30 meters was used to identify the chemical compounds in the samples^20-21. The temperature of the column was adjusted to range from 40 to 260°C with the following conditions: 1) set at 40°C for the first 5 minutes, 2) increased until it reached 260°C within the next 7 minutes, and 3) kept constant at 260°C for the final 7 minutes. Therefore, the total GC-MS run time for each sample was 19 minutes. The detector temperature was 200°C and the carrier gas used was He 10 with a split ratio of 51,0.

Assessment of Antioxidant Activity:

The antioxidant activity of each sample was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay^20-23. Initially, a 0.1 mM DPPH solution was prepared under dark and airtight conditions. Sample solutions at concentrations of 200, 400, 600, 800, and 1000 µg/mL were prepared in 2 mL volumes and mixed with an equal volume of DPPH solution in 5 separate test tubes. Each tube was vortexed using a VM-300 vortex mixer, then incubated for 30 minutes in darkness. This procedure was performed for each of the 5 sample particle sizes. The absorbance of each sample solution was measured at a wavelength of 516 nm using a UV-Vis spectrophotometer (SPECORD 200 Plus, Analytik Jena, Germany). A mixture of 70% ethanol and DPPH solution served as the blank, while a 1:1 v/v mixture of ascorbic acid solution and DPPH solution was used as the positive control. The free radical scavenging activity was quantified using the following equation:

(Blank absorbance – Sample absorbance)

Scavenging effect (%) = ------------------------------ x 100

Blank absorbance

Multivariate Analysis Through PCA:

PCA was employed to analyze the data on bioactive compounds and antioxidant activity across various particle sizes of the samples. Minitab software was utilized for the calculations. PCA was conducted with Pareto scaling, which helps to minimize the influence of compounds with low peaks or those inconsistently present across different particle sizes²⁴.

RESULT:

The Composition of The Bioactive Compound Identified at Various Sample Particle Sizes:

The difference in bioactive composition of each sample particle size was examined in this study. Figure 1 presents a comparison of the types and percentages of chemical compounds found in the five samples. The types of compounds detected by GC-MS in this study were 27 compounds in total.

The Antioxidant Activity in Various Sample Particle Sizes:

The antioxidant activity of sea urchin shell 70% ethanol extract in different sample particle sizes was tested with DPPH. Figure 2 illustrates the percentage of free radical scavenging activity for each sample particle size across a series of concentrations.

Principal Component Analysis (PCA):

There were 27 bioactive compounds in the sample as shown in Figure 1, but we chose only the 10 compounds that appeared most frequently with the highest percentage values for further analysis using PCA. These compounds consist of palmitic acid, heptadecene-(8)-carbonic acid, stearic acid, myristic acid, 5,8,11,14-eicosatetraenoic acid, 9-hexadecanoic acid, 1,2-benzenedicarboxylic acid, lanol, pentadecyclic acid, and margaric acid. The structure of these dominant compounds can be seen in Figure 3. PCA is a method that is able to reduce variables while maintaining the information in it, making the data easier to interpret. Biplot provides information regarding the proximity between research objects, variable variance, correlation between research variables, and variable values in research objects. Figure 4 shows which sample particle size shows the greatest antioxidant effect and what bioactive compounds are involved.

Figure 2. Comparison of DPPH free radical scavenging percentage for samples at different concentrations across each sample particle size.

Figure 3. The Structure of dominant compounds detected in sea urchin shell extract.

Figure 4. The PCA biplot of bioactive compound and sample particle sizes datasets.

DISCUSSION:

Figure 1 shows that sample particle size >2000 µm, >250 µm, <125 µm, >45 µm, and <45 µm had 8 compounds, 12 compounds, 12 compounds, 14 compounds, and 16 compounds detected, respectively. As the sample particle size decreased, a greater variety of chemical compounds were detected. This is attributed to the increased total surface area of the sample, which enhances interaction with the solvent and facilitates greater dissolution^17,19. Consequently, a larger number of chemical compounds dissolve in the solvent as the sample particle size decreases.

Apart from the variety of types, the percentages of compounds detected also vary between samples (Figure 1). For sample particle sizes >250 µm, >45 µm, and <45 µm, the most abundant compound was Lanol, found as much as 24.75%, 34.95%, and 18.68% respectively. Lanol or cholest-5-en-3-ol is a cholestanoid (a type of cholesterol). Cholesterol and its derivatives exhibit diverse pharmacological properties, including antioxidant, anti-inflammatory, antimicrobial, anticancer, antipsychotic, and cardioprotective among others²⁵. However, there are no studies that state the biological and pharmacological activities of Lanol specifically. Further studies are needed in this regard.

For sample particle size <125 µm, margaric acid is the most abundant compound with a percentage of 24.35%. This compound was only detected in the 3 smallest sample particle sizes, that is <45 µm, >45 µm, and <125 µm. An odd-chain saturated fatty acid (FA) called margaric acid^26-27 has varying mechanisms against non-small cell lung carcinomas in vitro²⁶. It also can be used against skin cancer protein²⁶. Several studies have found that the antioxidant effect of various plant extracts seem to be caused by the presence of polyunsaturated FA along with saturated FA like margaric acid and palmitic acid²⁸.

Meanwhile, for sample particle size >2000 µm, palmitic acid was the dominant compound (27,54%). Palmitic acid has been reported to possess many pharmacological effects such as antioxidant, antimicrobial, anti-inflammatory, and cholesterol-lowering activities^11,29-30. A study proved that palmitic acid has an anti-inflammatory capacity by competitively inhibiting phospholipase A2³¹. It was also mentioned that palmitic acid is one of the significant antioxidants found in sea urchin extracts⁹.

Overall, it can be noted that the highest proportion of compounds was found in sample particle size > 45 µm (34.95%) with total accumulative compounds of 97.28%. The chemical compounds detected were Lanol (34.95%), Palmitic acid (20,48%), Heptadecene-(8)-carbonic acid (11,96%), Myristic acid (6,27%), 9-hexadecanoic acid (CAS) (5,46%), and other compounds in smaller amounts, all of which have antioxidant properties^9,32-36.

Figure 2 illustrates that the DPPH free radical scavenging activity increases with higher concentrations and smaller sample particle sizes. This indicates that higher sample concentrations result in greater DPPH radical scavenging activity. Regarding particle size, reducing the size enhances the DPPH free radical scavenging activity of the sample. However, for sample particle size >45 µm the activity showed the highest numbers compared to other particle sizes. This corresponds to the highest total bioactive compounds (97.28%) found in sample particle size >45 µm (Figure 1.B).

This study also found that the DPPH free radical scavenging activities of sea urchin shell 70% ethanol extract in all concentrations (200-1000 µg/mL) were below 50%. It can be concluded that the antioxidant activity is classified as very weak³⁷compared to ascorbic acid (y = 8,3804x + 10,225, R2 = 0,9568). In a study by Khalil et al. (2022), it was reported that extracts from the spine and shell of sea urchin (Diadema savignyi) exhibited notable total antioxidant activity and free radical scavenging activity (DPPH), surpassing the effectiveness of both ascorbic acid and butylated hydroxyanisole⁸. Another study indicated that sea urchin spine extracts possess potent antioxidant properties, suggesting their potential applications as gastroprotective, hepatoprotective, and anti-diabetic agents⁸. Meanwhile, in this study, the spine was removed during the sample preparation process. For this reason, this can be an input for subsequent research, by optimizing various processes that will be carried out starting from the sample preparation stage, selecting an appropriate extraction method, and adding the different types of antioxidant activity assays to strengthen the results obtained. Fractionation techniques, such as extraction using organic solvents, differential precipitation, and ultrafiltration, can be employed to enhance the antioxidant properties of a sample^38-40.

Figure 4 presents the PCA biplot of PC1 and PC2 (principle components) of bioactive compounds and sample particle sizes datasets to show how the variables are related. The cumulative proportion of PC1 and PC2 is 75.4% for all variables. This means that both bioactive compounds and sample particle sizes can influence antioxidant activity by 75.4%. Figure 4 indicates that the sea urchin shell extract with a sample particle size >45 µm exhibits the highest antioxidant activity, attributed to compounds forming an angle of less than 90 degrees with the line of antioxidant activity. These bioactive compounds include lanolin, palmitic acid, myristic acid, stearic acid, heptadecene-(8)-carboxylic acid, 5,8,11,14-eicosatetraenoic acid, and 9-hexadecanoic acid. Palmitic acid and stearic acid are recognized for their effective antioxidant properties in treating complex conditions such as atherosclerosis, stroke, diabetes, and cancer^27,40. Meanwhile, myristic acid combined with several FA additives shows antibacterial activity against Staphylococci bacteria in various species^27,41. A study reports that 5,8,11,14-eicosatetraenoic acid has an anti-allergic effect²⁷. There is no data yet to show the pharmacological activity of lanol and heptadecene-(8)-carbonic acid, so this could be a gap in efforts to develop new drugs. Thus, these seven bioactive components can be further researched to develop drugs for diseases related to oxidation processes.

CONCLUSION:

To explore potential marine sources abundant in the coastal areas of Lombok Island for drug development, this study investigated the bioactive composition and antioxidant capacity of different particle sizes of sea urchin shell samples. The findings indicate that samples with particle sizes >45 µm exhibited the highest percentages of major chemical compounds (such as lanolin, 34.95%) and total cumulative compounds (97.28%). Additionally, the DPPH free radical scavenging activity was most pronounced in this particle size (>45 µm), showing 26.77% activity at a sample concentration of 1000 µg/mL. This antioxidant activity is attributed to several compounds including lanolin, palmitic acid, myristic acid, stearic acid, heptadecene-(8)-carboxylic acid, 5,8,11,14-eicosatetraenoic acid, and 9-hexadecanoic acid, all of which hold promise for further exploration in the development of new drugs derived from marine sources.

CONFLICT OF INTEREST:

The authors declare no conflicts of interest about this publication.

ACKNOWLEDGMENTS:

The author acknowledges the Faculty of Medicine and Health Sciences, Mataram University, for their support throughout the research process.

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