Spirulina platensis is a blue-green microalgae that thrives in alkaline water and it has a high potential as a source of bioactive compounds with commercial importance ^{1, 2} . High value compounds with interesting functional properties such as phycobiliproteins consisting of phycocyanins and allophycocyanins, carotenoids, phenolic acids, omega-3 and omega-6 polyunsaturated fatty acids, phenol and flavonoid have been identified in S. platensis ^{3– 5} . Phenolic compounds are a source of bioactive molecules with several beneficial health effects ⁶ due to their ability to act as antioxidants ⁷ , antibacterial ⁸ , and antidiabetes agents ⁹ . Phycobiliproteins, carotenoids and phenol present in S. platensis have anti-inflamatory activities ¹⁰ , thus making them a potential functional food product ¹¹ .

Ocimum basilicum, commonly know as sweet basil or kemangi in Indonesia and called rehan in Arabic ¹² is a popular culinary herb. O. basilicum is added to a variety of foods to impart a specific aroma. O. basilicum contains essential oils such as chavicol, linalool and eugenol, which are widely used in the food and pharmaceuticals industries ¹³ . The essential oils are able to reduce unpleasant odors and replace antioxidants ^{14, 15} . Besides essential oils, basil also contains phenol and flavonoid compounds which have antioxidant properties ^{16– 18} .

Microencapsulation is a technique used to coat a material to protect the material from outside factors, as well as ease handling of the material. The most important factor in encapsulation is the type of coating used. The encapsulated material is referred to as the core, intenal phase-, or filler, whereas the walls are sometimes called shells, layers, material wall, or membranes. A microcapsule can be coated by several coatings, but only one core compound can be coated ^{19, 20} .

To predict the potential for bioactivity, absorption, distribution, metabolism, and excretion of a substance, our research was performed with bioinformatics and in silico approaches. If we do not have special apps, certain internet-based or online resources can be used. DOCK Blaster for molecular docking prediction ²¹ , MDWeb and MDMoby for molecular dynamics analysis ²² , ADMET and DrugBank for drug database creation ²³ , as well as PreADME for ADMET tools ²⁴ , are some of the tools available online.

Various studies have reported the presence of bioactive compounds such as phenols and flavonoid in S. platensis ²⁵ and O. basilicum ²⁶ . The present research aims to evaluate bioactive compounds of O. basilicum intervention on S. platensis. Firstly, total phenol, flavonoid, antioxidant activity and phycocyanin contents were evaluated. Secondly, the success of encapsulation of phenol, flavonoid, antioxidant activity and phycocyanin compounds was evaluated. The addition of these compounds was expected to reduce the amount of volatiles in S. platensis, which cause unpleasant odors. The third was predicting absorption, distribution, metabolism, and excretion (ADME) of phenols, azulene, flavonoids, and phycocyanin.

S. platensis powder was obtained from brackish water Aquaculture Fisheries (BBPBAP) Jepara (Central Java, Indonesia), O. basilicum was bought from a traditional market (Semarang, Central Java). The water used was multilevel distilled water, aquabidest Otsu-WI (PT. Otsuka Indonesia, Lawang, Indonesia). The reagents and chemicals used in this study were of analytical grade (CV. Chemix Pratama, Special Region of Yogyakarta, Indonesia), maltodextrin (CV. Multi Kimia Raya, Semarang, Indonesia) and gelatin (Xian, Biof Bio-Technology, Cina). This research was conducted in the food chemistry laboratory of Diponegoro University, Semarang, Central Java, Indonesia) from January 06, 2020 up to May 29, 2020.

Total phenol, flavonoid and antioxidant activity were measured in S. platensis with no treatment (SP), S. platensis treated with freeze-dried O. basilicum (DSB), S. platensis treated with microencapsulation freeze-dried O. basilicum (MSB) and O. basilicum leaf extract (B), plot of standard calibration of gallic acid (left) quercetin (right) ( Figure 1). Phycocyanin content was measured in SP, DSB and MSB, and then encapsulation efficiency was measured on total phenol, flavonoid, antioxidant activity and phycocyanin. The results of the One Sample Kolmogorov Smirnov Test and Levene’s Test were more than (P > 0.05) thus, the data were normally or homogenously distributed. Therefore the data analysed by the One Way ANOVA were less than (P < 0.05) and are shown in Table 1, followed by the post hoc Tukey HSD ( Table 2– Table 6). The DSB sample can increase the total phenol 49.50% and antioxidant activity 12.66% of S. platensis. However, total flavonoid is not significantly different with O. basilicum intervention on S. platensis ( Figure 2).

O. basilicum intervention can increase the levels of phycocynin in S. platensis 40.72% shown in ( Figure 3). O. basilicum intervention on S. platensis when extracted will make a blue ring on the surface, it is caused by compounds contained in O. basilicum called azulene.

This is encapsulation is less effective in microencapsulation of polyphenol compounds such as phenol and flavonoid ( Figure 4). Raw absorbance data for bioactive compounds assays are available as underlying data ⁴⁰ .

Algae are a valuable source of proteins and phenol compounds ⁴¹ . S. platensis is a type of algae with a high total phenol content. Extraction methods and the solvent used are responsible for the type and yield of phenolic compounds from algae sources ⁴² . In S. platensis, distilled water has been reported as the best solvent for extraction of phenolic compounds with total phenol content of 43.2 ± 1 GEA/g ⁴³ . S. platensis powder prepared via oven drying is reported to have a broad range phenolic profile that includes gallic acid, catechin, caffeic acid, P-hydroxybenzoic acid, P-cumaric acid, ferulic acid, quercein, genistein and kaempferol ⁴⁴ . Variation in total phenol content between algae species is reportedly due to algal type, origin and growth condition of different microalgae ⁴⁵ .

Fresh O. basilicum leaf extract has been reported to have lower total phenol content than that which has been freeze-dried ⁴⁶ . Previous studies have reported that dried O. basilicum leaf extracted with methanol gave high total phenol values ⁴⁷ . The phenol compounds present in O. basilicum include rosmarinic, caftaric, caffeic, chicoric, p-hydroxybenzoic, p-coumaric, and protocatechuic acids. The phenol compounds in O. basilicum play an important role in its antioxidant activity ⁴⁸ . S. platensis microcapsule with the intervention of O. basilicum (MSB) gives low total phenol. Microencapsulation using maltodextrin and gelatin can protect polyphenol compounds ⁴⁹ .

O. basilicum intervention on S. platensis significantly increases bioactive compounds ( Figure 3), except for total flavonoid. The total flavonoid content of the S. platensis treated with freeze-dried O. basilicum (DSB) was not significantly different from the control sample (SP). Previous studies have reported that total flavonoid in S. platensis is less than the total phenols, phenolics (1.73%) and flavonoids (0.87%) ⁵⁰ . Another study reported that the powder of S. platensis, which was dried in an oven at a temperature of ± 50°C, did not effect the phenolic compound quercentin, where the compound was one of the active substances of the flavonoid class ⁴⁴ . The flavonoids are considered as indispensable in a variety of medicines, nutraceutical, pharmaceutical and cosmetic applications ⁵¹ . Flavonoids play an anti-inflammatory and anti-viral role ⁵² . The optimum for the extraction process are dry conditions compared to wet conditions. Extraction using ethanol had a higher total flavonoid content ⁵³ . The total flavonoid content of the S. platensis microencapsulated and freeze-dried tended to be low. Microencapsulation can maintain the stability of flavonoid from processing effects that cause degradation ^{54, 55} .

Spirulina platensis could be considered as a valuable source of bioactive colored and phenolic compounds with potent antioxidant activity ²⁵ . The ABTS method was chosen because it has a high level of sensitivity (99.44%) compared to the 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) method (95.3%) ³³ . Total phenol and flavonoid content showed positive correlation to the antioxidant activity of S. platensis. Phenolic components play an important role in the antioxidant activity ⁵⁶ . Phenolic compounds are good electron donors because the hydroxyl groups can contribute to antioxidant activity ⁵⁷ . The tocopherol and phycocyanin in microalgae have potential as antioxidants in food, so that it acts as a functional food ⁵⁸ . The S. platensis treated with freeze-dried O. basilicum (DSB) showed an increase in antioxidant activity compared to S. platensis with no treatment (SP). Previous research explained that O. basilicum contains essential oils which also have potential as antioxidants ⁵⁹ . According to Asghari et al. ⁶⁰ , the mixture of carotenoid pigments, chlorophyll and blue pigments such as phycocyanin of S. platensis produce strong antioxidants.

Ocimum basilicum contains 65 active compounds, and the compounds with the highest content are namely 31.6% linalool and 23.8% methylchavicol. Essential oils in O. basilicum have the potential as antioxidants ¹³ . The essential oil of linalool significantly prevents the formation of UVB-mediated 8-deoxy guanosine, which causes oxidative damage to DNA. This is because it has the ability to prevent reactive oxygen species (ROS) and restore the balance of oxidative cells ⁶¹ . This research indicates that there is a synergistic interaction between phycocyanin and total phenol in antioxidant activities. S. platensis treated with microencapsulation freeze-dried O. basilicum (MSB) impart smaller values on total phenol, flavonoid, phycocyanin and antioxidant activity. This is in correlation with previous research which showed that the S. platensis microcapsule has antioxidant activity of 49.05% ⁶² . Essential oils that play a role as an antioxidant can last for six months with a slight decrease in antioxidant activity and phenol content after microencapsulation ⁶³ . Treated microencapsulation can control antioxidant capacity and is a promising strategy in extending shelf life ⁵⁵ .

S. platensis cultivated with brackish water had a higher phycocyanin content ( Figure 4), whereas S. platensis cultivated in freshwater only had a 1.74% phycocyanin content ⁶⁴ . S. platensis cultivated with seawater has a maximum phycocyanin content ⁶⁵ . Phycocyanin is a natural blue pigment that functions as an antioxidant, anti-inflammatory and anti-carcinogenic ^{66, 67} . The S. platensis treated with freeze-dried O. basilicum (DSB) impart higher levels of phycocyanin, where a combination of S. platensis and O. basilicum with a ratio of 1:5 detects the presence of azulene using gas chromatography-mass spectrometry (GC-MS) ²⁷ . Azulene is an aromatic compound from essential oils in O. basilicum ⁶⁸ , and it is a blue hydrocarbon compound that has a strong dipole moment ^{27, 69} . Azulene has a small gap between the highest energy molecular orbitals (HOMO) with the lowest energy molecular orbitals that do not have electrons (LUMO) ⁷⁰ . Therefore, the presence of azulene in S. platensis treated freeze-dried O. basilicum can increase phycocyanin levels.

Encapsulation efficiency is used to evaluate the success of a microencapsulation technique. Encapsulation using a combination of polyanion and polycation coatings such as maltodextrin and gelatin has a higher yield. This is due to the stability of the emulsion between maltodextrin and gelatin ⁷¹ . The amount of bioactive content on the surface will reduce the value of encapsulation efficiency. This will cause the amount of bioactive compounds that are wrapped to increasingly shrink because many are attached to the surface. So that this event will damage the oxidative stability of microcapsules ⁷² . The encapsulation efficiency of phycocyanin was in accordance with the results of previous studies ⁷³ , which is encapsulation using an alginate coating has an encapsulation efficiency value of 71.75%. The value of encapsulation efficiency in total phenols and flavonoids in S. platensis is effected by using liposomes or nanoliposomes in encapsulation of bioactive compounds, this is because liposome is stable at low pH and is able to withstand the time of release in the stomach, but it is less consistent in the intestine ^{74, 75} . The encapsulation efficiency of antioxidant has been shown in previous research where antioxidant microencapsulation using the freeze drying method has an encapsulation efficiency value ranging from 73–86% ⁷⁶ .

Intestinal absorption and brain permeation set crucial parameters at their target site of action for any medication for its pharmacokinetics and bioavailability. Consequently, the BOILEDEgg study was used, as previously stated, to predict gastrointestinal (GI) absorption and brain access for phenol, azulene, flavonoid, and phycocyanin. The white region is the physicochemical space of the molecules most likely to be consumed by the gastrointestinal tract, whereas the yellow region (yolk) is the physicochemical space of the molecules most likely to reach the brain. The white and yolk regions are not mutually exclusive ³⁶ . Phenol, azulene, and phycocyanin were found to be among the well-absorbed molecules based on the study ( Table 7).

Tables 8 and Table 9 demonstrate that phenol, azulene, and phycocyanin comply with Lipinski or drug-likeness laws. Drug-likeness is a term used to explain how in vivo molecular properties are influenced by compounds’ physicochemical properties. This research indicates that the substance will spread well to all parts of the body to play an active role as a drug ⁷⁷ . The physicochemical properties obtained from molecular structures are used by most drug-likeness testing laws and compare such properties with the medicines that have been reported. The Lipinski rule is one of the most used rules ⁷⁸ . The rule of five was developed to set drugability guidelines for new molecular entities (NMEs) ⁷⁹ . Therefore, the rule suggests that molecules, whose properties fall outside of these boundaries, are unlikely to become orally bioavailable drugs ⁸⁰ . As drug candidates, phenol, azulene, and phycocyanin have excellent potential. This calculation is based on a molecular weight (MW) value of less than 500 g mol ^-1, an acceptor of hydrogen bonds of less than 10, a donor of hydrogen bonds of less than five, a surface area of topology (TPSA) of less than 140 Å, and a LogP of less than five.

Ocimum basilicum intervention significantly increased total phenol, phycocyanin and antioxidant activity in S. platensis. However, total flavonoid content did not differ significantly in untreated S. platensis controls compared to treated. Bioactive compounds after microencapsulation showed the lowest values. Microencapsulation of phycocyanin with maltodextrin and gelatin showed high encapsulation efficiency values. Hence, S. platensis treated freeze-dried O. basilicum has potential as a pharmaceutical product.