Microencapsulation, Physicochemical Characterization, and Antioxidant, Antibacterial, and Antiplasmodial Activities of Holothuria atra Microcapsule

This study provides the design of a microencapsulation formula, physicochemical characterization, and antioxidant, antibacterial, and antiplasmodial activities of Holothuria atra microcapsules. The ethanolic extract of H. atra was microencapsulated with chitosan (CHI) and sodium tripolyphosphate (Na-TPP) with various stirring times: 60 minutes (CHI60), 90 minutes (CHI90), and 120 minutes (CHI120). The microcapsules were then observed for physicochemical properties using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR). The microcapsules were tested for antioxidant activity and antibacterial activity against Staphylococcus aureus and Escherichia coli using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. Antiplasmodial bioactivity was assessed through in silico molecular docking. The CHI60 and CHI120 microcapsules exhibited a smaller size and an irregular spherical shape, while the same FTIR profile was observed in CHI90 and CHI120. The bioactivity tests demonstrated that CHI90 exhibited high antibacterial activity against E. coli and S. aureus, while CHI120 exhibited high antioxidant performance. Calcigeroside B and Echinoside B exhibited antiplasmodial activity against the Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) protein, along with an artemisinin inhibition mechanism. In conclusion, the microcapsules with the CHI90 formula demonstrated the best antibacterial activity, while the CHI120 formula exhibited high antioxidant activity. Two terpenoids, Calcigeroside B and Echinoside B, exhibited the best antiplasmodial activity.


Introduction
Te utilization of organic bioactive substances has experienced signifcant growth in several industries, including food, fabric, skincare products, fragrances, and pharmaceutical products, owing to their exceptional bioactive properties.Te sea cucumber is widely recognized as a very promising marine organism due to its abundant organic bioactive substances and diverse range of biological activity [1].Tis organism is classifed within the phylum Holothuroidea, which is a part of the larger taxonomic group known as Echinodermata.Te aquatic environment harbors a species of organisms that bear a resemblance to cucumbers.Out of an estimated global population of 1,200 species, 66 species under the taxonomic category of Holothuroidea have been classifed as sea cucumbers [2].Te potential health benefts of Holothuria atra biological substances include protection from cardiovascular disease, antidiabetic properties, hypoglycemic efects, antioxidant activity, antiasthmatic properties, antieczema efects, anti-infammatory efects, cholesterol-lowering efects, immunomodulatory efects, cytotoxic properties, antiparasitic activity, antimalarial activity, antiviral efects, antifungal properties, antiangiogenic efects, and anticancer efects [3,4].
However, the inherent instability and suboptimal bioavailability of these substances restrict their ability to exert their biological functions [5].Hence, the utilization of appropriate carriers for encapsulation becomes highly signifcant.Currently, there is a trend toward encapsulating bioactives to create fbrous and capsular structures that have a wide range of applications.Te utilization of encapsulation techniques has been observed to efectively address the limitations associated with the delivery of bioactive substances to specifc target areas [6].
Microencapsulation is a viable technique utilized for the purpose of delivering various types of components in their functional states to specifc regions inside a biological system.Te encapsulation procedure refers to a technological method wherein a substance or combination of substances is enclosed within micro and/or nanostructures by entrapping a bioactive core with another substance known as wall materials [7,8].Tis process serves to safeguard bioactive substances, such as favors and pigments, among others.Te composition of the microcapsules includes the core and shell.Te core is the substance that is encapsulated, while the shell is the polymer utilized for encapsulating bioactive substances.Te wall material exhibits no chemical reactivity with the enclosed substance [9].Efective strategies can be employed to encapsulate active substances, safeguarding them against adverse conditions such as high temperatures, oxidation, alkaline or acidic environments, moisture, or evaporation.Te conversion of liquids into powder form is a viable method for enhancing the process of encapsulating biological substances, as it helps prevent the formation of clumps in the fnal product.Te transition from a liquid to a solid state also serves to limit the interaction of these substances with undesired species that could potentially induce polymerization inside the reaction mixture [10].Te selection of an encapsulating material is contingent upon the maintenance of structural integrity, which is a crucial factor in determining the efcacy of shell materials.Te processing and economic dimensions are also included in this matter.Te selection of microcapsules for encapsulation is infuenced by several pertinent factors, including toxicity degree, efcacy, stability, protective level, and microscopic features [5,9].
Chitosan is a biopolymer with cationic properties that is found in abundance following cellulose.It is characterized by the presence of several amino and hydroxyl groups.Chitosan exhibits notable hydrophilic properties due to the presence of amino groups that carry a positive charge.Te polycationic properties of chitosan have garnered signifcant commercial importance due to its potential use in a wide range of applications [11].Te application of this coating on shells for encapsulating materials has proven to be quite benefcial.Chitosan-coated encapsulated products containing active substances have been demonstrated to possess enhanced functional characteristics [8].Chitosan fnds widespread utilization in microencapsulation across various domains, including biomedical research, the agricultural sector, skin care products, food, and fabrics.
Novel formulations, particularly microencapsulation of H. atra, need to be examined for pharmaceutical purposes, involving the exploration of new drugs, assessment of their binding properties and specifcity, characterization of their molecular structures, and confrmation of their efcacy and safety.Physicochemical characterization is important for predicting constituent behavior, assessing performance, and ensuring formulation stability in the development of new compounds and formulations.Methods such as electron microscopy and spectrophotometry are employed to regulate the function of an ingredient in the formulation and confrm the accurate distribution of ingredients [12].Assessing antioxidant activity in novel materials is crucial for assessing their ability to reduce oxidative stress and protect materials from degradation.Te DPPH (2,2diphenyl-1-picrylhydrazyl) approach is a commonly utilized methodology for assessing the antioxidant properties of diferent materials, including novel ones [13].Antibacterial analysis is conducted on novel substances to assess their capacity to impede the growth of bacteria and avert the transmission of infections.Te evaluation is crucial to ascertain the efcacy of novel substances in avoiding the emergence of resistance to antibiotics and minimizing bacterial infections [14,15].Te disc difusion technique is a commonly employed antibacterial test assay that assesses the sensitivity of bacteria to antimicrobial drugs [16].An antiplasmodial analysis aims to assess the capacity of a substance to hinder the growth of Plasmodium, a parasite causative of malaria.Te test is designed to assess the antiplasmodial action of chemicals, extracts, or substances, playing a crucial role in identifying and creating new antimalarial remedies.Antiplasmodial action can be assessed by in silico, in vitro, or in vivo techniques [17,18].In silico approaches utilize computer simulations and modeling to forecast the prospective antiplasmodial action of substances [18].
Based on these phenomena, this study involved conducting an encapsulation procedure on the H. atra extract using chitosan with diferent periods of condensation.Tis study provided microencapsulation and antioxidant, antibacterial, and antiplasmodial activities of the H. atra ethanolic extract.

Sample Collection and Preparation
. Fresh H. atra specimens were collected from the waters of Sapeken Island, Sumenep, Madura, Indonesia, in April 2023 (dry season).Te internal organs were carefully removed, cleaned, and then sun-dried (32-34 °C) for 3 days.Te dried H. atra, with approximately <10% water content, was collected and ground using a grinder machine.Te powder was fltered using a sieve with 40 meshes to obtain a fne powder of H. atra.Five kilograms of dried whole H. atra yielded 4,358 g of H. atra fne powder.

Extraction and Formulation of Holothuria atra
Microcapsules.Freeze-drying, a prevalent method for extracting water from a substance while maintaining its structure and composition, is a component of the process outlined.Tis method prevents the sample from being damaged by high temperatures, which is especially advantageous when dealing with heat-sensitive substances such as biological samples or fragile microcapsules [19].
Approximately 1 kg of H. atra fne powder was soaked with 2 L of ethanol (Merck, Germany) overnight.Te solution was fltered using flter paper and evaporated using a rotary evaporator at 70 °C and 500 rpm.Te residues were macerated and evaporated, and the dried extract was prepared for encapsulation.Te encapsulation of H. atra was processed by ionic gelation processes with chitosantripolyphosphate [20].Te H. atra extract (0.5 g) was dissolved in 17.5 mL of deionized water.Te extract solution was mixed with 50 mL of 1% chitosan in 2% acetic acid bufer.Te mixture was stirred for various times, specifcally 60, 90, and 120 minutes at 500 rpm.Sodium tripolyphosphate (Na-TPP) at 0.3% (175 mL) was slowly added to the mixture.Te microcapsule solution was subsequently dried using freeze-drying at −55 °C and a pressure of 0.02 mbar for 96 hours.A fne powder of the microcapsules was obtained by grinding the sample using a mortar and pestle.

Physicochemical Characterization.
Physicochemical characterization is of utmost signifcance in the pharmaceutical sector, where it is utilized to guarantee the efcacy, safety, and quality of pharmaceutical products [12].Our study employed scanning electron microscopes (SEM) and Fourier-transform infrared spectra (FTIR) to assess physicochemical properties.FTIR spectroscopy is a method that captures the infrared spectrum of emission or absorption from a material, whether it is a liquid, gas, or solid [21].SEM is a high-resolution imaging method that analyzes the surface features and chemical structure of materials using advanced equipment to observe the microstructure of objects [22].
Te morphology of samples was observed using the TM3000 Olympus Tabletop SEM with 8,000-12,000x magnifcations.Briefy, the microcapsule powder was dropped onto a chip coated with Au (gold) metal, air-dried, and observed on the scanning electron microscope.To identify the functional group of Holothuria atra microcapsule, FTIR was employed [23].Te H. atra microcapsule powder was placed on the ATR diamond crystal.Subsequently, both the sample and crystal were clamped using pressure gauges.Te spectra were recorded using a Shimadzu FTIR spectrophotometer with attenuated total refection (ATR-FTIR) in the range from 4000 cm −1 to 500 cm −1 .Te FTIR diagram was plotted using OriginPro 2021.

Antioxidant Activity of Holothuria atra Microcapsules.
Te DPPH method was selected to assess antioxidant activity due to its simplicity, quickness, and capacity to accurately measure antioxidant capacity.It is advisable to utilize many strategies to thoroughly comprehend antioxidant activity, as there are alternative approaches beyond this one [13].Te antioxidant activity of H. atra microcapsules was determined by the DPPH method with some modifcations [24].One milliliter of 0.1 g/mL microcapsules was mixed with a 50 mM DPPH solution in methanol.Te mixture was incubated at 37 °C in dark conditions for 30 minutes.Te absorbance was observed using a spectrophotometer with a wavelength of 517 nm.Methanol was employed as a blank, and a DPPH solution without the sample was employed as a control.

Antibacterial Activity of Holothuria atra Microcapsules
against Staphylococcus aureus and Escherichia coli.Te antibacterial activity of H. atra microcapsules was tested using the disc difusion method [16].S. aureus and E. coli were selected for targeted bacterial inhibition activities.Te bacterial cultures were obtained from the Faculty of Medicine, Brawijaya University, Malang, Indonesia.S. aureus and E. coli culture cells were inoculated in 0.9% sodium chloride, and then 200 μL was spread on a nutrient agar medium.Sterilized and blank paper disks (6 mm diameter, MN Germany) were prepared and separately soaked in 250 mg/mL of amoxicillin and H. atra microcapsules with a concentration of 100 mg/mL.Te paper disks were placed on the cell culture and incubated overnight at 37 °C.Te antibacterial activity of the microcapsules was determined by measuring the inhibition zone of the samples.

Results and Discussion
Malaria remains a signifcant global infectious disease with a high mortality and morbidity rate [28].Te search for new antimalarial agents has been a recent concern.Tese agents should be recognized for their safety and their ability not to develop resistance.In a previous study, H. atra exhibited antiplasmodial activity against Plasmodium falciparum [3].However, the H. atra extract was not stable.An encapsulation technique was required to protect the compounds from environmental degradation.Tis study utilized Scientifca a natural polysaccharide, chitosan, to microencapsulate the H. atra ethanolic extract.Te mixture of the extract and polymer was stirred for diferent times, specifcally 60, 90, and 120 minutes, to optimize the microencapsulation.Te morphological character of H. atra microcapsules is presented in Figure 1.H. atra microcapsules have a brown color and exhibit a more intense brown with medium dryness at a stirring time of 90 minutes (CHI90).H. atra microcapsules with stirring times of 60 and 120 minutes exhibited similar textures and colors.Morphological characteristic observations with scanning electron microscopy revealed some microcapsules with an irregular shape.According to the SEM observation, H. atra CHI120 has the smallest microcapsules with a size of 2.1 μm, followed by CHI60 and CHI90.Chitosan has been reported as a natural polymer for microencapsulating natural compounds [8,11,18,19].A previous report used chitosan to encapsulate cinnamon leaf oil and demonstrated irregular spherical shapes of microcapsules.Te study also confrmed that the concentration of the extract and polymer afected encapsulation efciency [29].Te microencapsulation steps in microcapsule formulation also afected the microcapsules' physical, chemical, and bioavailability.Adding sodium tripolyphosphate (Na-TPP) in stirring processes is critical to creating a stable emulsion [30].Te size of the microcapsules is also afected by NaCl and chitosan concentrations.Increasing NaCl and chitosan concentrations increased the particle size of the microcapsules.NaCl exhibited particle aggregation and competed against chitosan for association [31].
Te FTIR spectra of the H. atra ethanolic extract with various stirring times exhibited diferent profles (Figure 2).Te H. atra ethanolic extract and microcapsules demonstrated a broad hydroxyl group at 3,300 cm −1 , and at 2,900 cm −1 , the microcapsules exhibited a stretch peak.For a stirring time of 60 minutes (CHI60), closed peaks were observed, resembling those of the ethanolic extract of H. atra.Interestingly, CHI60 exhibited a stretch peak at 1,000 cm −1 that was not identifed in all samples.CHI90 and CHI120 exhibited similar broad peaks at 1700 cm −1 -1400 cm −1 .Chitosan is a cationic polysaccharide of D-glucosamine and N-acetyl-D-glucosamine units linked by β-(1-4)-glycosidic bonds.Chitosan was reported to have a broad, intense hydroxyl group at 3,369 cm −1 , 2,866 cm −1 , 1,655 cm −1 , and 1,550 cm −1 [19].A previous study also reported that chitosan exhibited a strong band in the 3,291-361 cm −1 region, representing N-H and O-H stretching, in the 2,921 cm −1 region representing C-H symmetric stretching, and in the 2,877 cm −1 region representing C-H asymmetric stretching [32].Tis study confrmed that stirring time in the microencapsulation process altered the wavenumber shift, indicating functional groups in the complex.CHI60 exhibited a sharp peak near 1,500 cm −1 that might be predicted as an electrostatic interaction of the ethanolic extract and chitosan.
Antioxidant activities were tested against DPPH as a free radical solution to assess the bioavailability of H. atra microcapsules.As depicted in Figure 3(a), CHI120 exhibited higher antioxidant activity than others.However, CHI60 also exhibited higher antioxidant activity than CHI90 and was close to CHI120.Te antioxidant activity was negatively correlated with the inhibitory concentration (IC50) of H. atra ethanolic microcapsules.CHI120 exhibited the lowest IC50, followed by CHI60, and the last was CHI90 (Figure 3(b)).
Te antibacterial performances of H. atra ethanolic microcapsules against S. aureus and E. coli are presented in Figure 4. CHI90 exhibited high antibacterial activity, inhibiting both S. aureus and E. coli.CHI60 and CHI120 demonstrated low activity against E. coli and high inhibition activity against S. aureus growth.In comparison, amoxicillin at 250 mg/mL demonstrated the highest antibacterial activity.Te antibacterial activity of the H. atra extract has been reported in previous studies.Te methanolic extract of H. atra inhibited Vibrio alginolyticus and Vibrio anguillarum.Moreover, 500 μg/mL of the H. atra extract with ethyl acetate as a solvent demonstrated higher inhibition activity against Vibrio alginolyticus and Vibrio anguillarum than ampicillin at 10 μg/mL [33].In another study, the methanol and hexane fractions of H. atra, containing phenolic, terpenoids, and saponin compounds, exhibited antibacterial activity against Pseudomonas aeruginosa [34].Chitosan, as a microcapsule polymer, has been reported to promote antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, E. coli, and S. aureus [35,36].Terefore, using chitosan as an encapsulation matrix might improve the antibacterial and antioxidant activity of H. atra.
Te antiplasmodial performance of H. atra microcapsules was predicted through virtual prediction, involving the interaction of bioactive compounds with the dihydroorotate dehydrogenase of P. falciparum (PfDHODH) [26].Dihydroorotate dehydrogenase (DHODH) is being considered a therapeutic candidate due to its potency in the pyrimidine biosynthetic pathway.It plays a critical role in pyrimidine biosynthesis by facilitating Flavin mononucleotidedependent formation of orotic acid [37][38][39][40].As the pyrimidine biosynthetic pathway is crucial for cell growth, metabolism, and replication, DHODH has been identifed as an important therapeutic target.

Conclusion
Te morphologies of microcapsules CHI60 and CHI120 exhibited a smaller size and an irregular spherical shape.CHI120 demonstrated high antioxidant activity, while CHI90 exhibited high antibacterial activity against E. coli and S. aureus.Calcigeroside B and Echinoside B exhibited antiplasmodial activity against the PfDHODH protein and bound to artemisinin sites.Furthermore, Calcigeroside B also demonstrated a lower binding energy, indicating a tight interaction between the compound and the PfDODH protein [45].Scientifca

Figure 4 :
Figure 4: Antibacterial activity of the Holothuria atra ethanolic extract tested against S. aureus and E. coli.