Ocimum basilicum (kemangi) intervention on powder and microencapsulated Spirulina platensis and its bioactive molecules [version 1; peer review: 2 approved with reservations]

Background: Spirulina platensis contains several bioactive molecules such as phenol, flavonoid and phycocyanin pigments. This study unveils total phenol, flavonoid, antioxidant activity, phycocyanin content and evaluated encapsulation efficiency from Ocimum basilicum intervention on S. platensis. O. basilicum intervention aims to reduce unpleasant odors from S. platensis that will increase consumption and increase bioactive compounds. Methods: The intervention was carried out by soaking a S. platensis control sample (SP) in O. basilicum with a ratio of 1:4 (w/v) and it was then dried (DSB) and microencapsulated by freeze drying methods (MSB) using a combination of maltodextrin and gelatin. Total flavonoid and phenolic analysis with curve fitting analysis used a linear regression approach. Antioxidant activity of samples was analysed with the 2,2’-azino-bis-3-3thylbenzthiazoline-6-sulphonic acid (ABTS) method. Data were analysed using ANOVA at significance level (p < 0.05) followed by Tukey test models using SPSS v.22. Results: The result of this study indicated that O. basilicum intervention treatment (DSB) has the potential to increase bioactive compounds such as total phenol, antioxidant activity and phycocyanin, and flavonoid content. Intervention of O. basilicum on S. platensis (DSB) significantly increases total phenol by 48.7% and phycocyanin by 40.7%. This is due to the phenol and azulene compounds in O. basilicum which have a synergistic effect on phenol Open Peer Review


Introduction
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][4][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][17][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.

Methods
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.

Preparation of Ocimum basilicum leaf extract
The O. basilicum was extracted using distilled water (aquabidest) following modified methods reported by Handiani et al. 27 . 2000 g of fresh O. basilicum leaves were added to 400 ml aquabidest and ground. The slurry was then filtered by using filter fabric and the extract result was approximately 1200 ml.

Intervention of Ocimum basilicum on S. platensis
A freeze-dried sample (DSB) and microencapsulation sample (MSB) of S. platensis were soaked with O. basilicum extract at a ratio of 1:4, w/v. A S. platensis sample with no O. basilicum added was used as a control (SP).
Preparation of freeze-dried Ocimum basilicum O. basilicum and S. platensis were freeze-dried using a freeze dryer (Heto Powerdry LL 1500, Germany) at a temperature of -100°C for 48 hours. The O. basilicum extracts were applied to S. platensis (DSB) in the intervention study below.
Microencapsulation of freeze-dried Ocimum basilicum Microencapsulation of O. basilicum was performed following the methods reported by Castro-Munoz et al. and Dewi et al. 28,29 Maltodextrin and gelatin at a ratio of 9:1, w/w were used as a coating. Homogenization was then performed with a homogenizer (15A HG-wiseTis, Germany). Microencapsulated O. basilicum (MSB) was used in the intervention studies below.
Determination of total phenol content Total phenol content was measured using modified Folin-Ciocalteu methods 30 . Samples were sonicated for 30 minutes prior to measurement. Gallic acid was used as standard and was read at λ=739 nm using a spectrophotometer. In the test solution, 0.5 ml of Folin-Ciocalteu reagents and 1 ml of NaCO 3 were added to 1 ml of sample and the solution was mixed. Samples were incubated for 10 minutes at room temperature, then diluted with aquabidest to 10 ml. The measurement results were reported in milligram (mg) and were calculated as gallic acids equivalent (GAE) per gram of sample.
Determination of flavonoid content Measurement of total flavonoid was performed using the slightly modified aluminium chloride method 31 . Modification was through ultrasonic treatment before measurement, the sample was sonicated for 30 minutes and quercentin was used as a standard. In the test solution, 1.0 ml of sample was mixed with 0.3 ml of NaNO 2 (5%, w/v) and the solution was left to stand 5 minutes before 0.5 ml of AlCl 3 (2%, w/v) was added to the test solution. Samples were neutralized with 0.5 ml of 1 M NaOH solution and the samples were incubated for 10 minutes at room temperature. Absorbance was measured at λ=310 nm. The results are presented in milligrams (mg) and calculated as quercentine equivalent (QE) per gram of sample.
Phycocyanin content 40 mg of sample was added to 10 ml phosphate buffer (pH 7); the solution was sonicated for 30 minutes and stored at 4°C overnight. Samples were centrifuged to separate the blue supernatant. Next, samples were measured for absorbance at 620 nm according to the methods described by Setyoningrum & Nur 32 . Phycocyanin content was determined using Equation 1: Where PC is phycocyanin content, Abs is absorbance at 620 nm; v is volume of solvent (ml); 3.39 is the coefficient of C-Phycocyanin at 620 nm; w is weight of sample (mg); and w dry is percentage dry weight of sample.

Determination of antioxidant activity
The antioxidant activity of the sample was measured by 2,2'-azinobis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical according to the methods of Shalaby & Shanab 33 . ABTS was formed by reacting 7 mM ABTS aqueous solution with 2.45 mM phosphate per sulphate in the dark for 4-16 hours at room temperature. Dilute ABTS solution with ethanol absorbance of 0.700 ± 0.05 at 734 nm was used for measurement. The photometric test was carried out with 0.9 mL ABTS solution and 0.1 mL of the tested sample mixed for 45 seconds, measurements were made immediately at 734 nm after 15 minutes. Antioxidant activity was expressed as the inhibition percentage of free radicals by the sample and was determined using Equation 2: = Ab -As Inhibition (%) 100 Ab Where Ab is the absorbance of the control reaction and As is the absorbance in the presence of the extract sample.

Determination of encapsulation efficiency
Encapsulation efficiency (EE) was determined following the methods described by Ong et al. 34 . Encapsulation efficiency was calculated based on total coated active compounds and free active compounds. Percent encapsulation efficiency was determined using Equation 3: Total coated active compounds Free active compounds (%) 100 Total coated active compounds

ADME analysis
The research was performed in two phases, namely: the first stage of accessing the PubChem server (https://pubchem.ncbi. nlm.nih.gov/) to obtain canonical SMILE information; the next step is to use swissADME (http://www.swissadme.ch/) to predict absorption, distribution, metabolism, and excretion 35 . The BOILED Egg (Brain Or IntestinaL EstimateD permeation predictive model) methods are used for the determination of the absorption of the inhibitors in the brain and gastrointestinal tract. BOILED Egg provides a threshold (TPSA ≤ 131.6 and WLOGP ≤ 5.88) and the best representation of how far molecular structure is for well-or poorly absorbed 36 . ADME is based on the Lipinski rule of five 37 . The Lipinski rule of five is generally employed in accessing the drug-likeness of active compounds to prioritize compounds with an increased likelihood of high oral absorption 38 .

Statistical analysis
Data obtained was reported as the mean of triplicates (n=3) ± standard deviation. Parametric data was analyzed using SPSS version 22.0 (IBM, Armonk, NY, USA) 39 . Statistical analysis was preceded by a normality test with One Sample Kolmogorov-Smirnov Test and a homogeneity test with the Levenes Test at significance level (P > 0.05). Parametric tests were carried out with One Way ANOVA at significance level (P < 0.05), followed by post hoc Tukey HSD.  (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).

Total
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 .

Discussion
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 . 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      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 BOILE-DEgg 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. Druglikeness 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.

Conclusion
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     provide any additional insight to the manuscript as matrix-derived combination influences ADME. In vivo study is needed to determine the bioavailability of Ocimum basilicum intervention on freeze-dried and microencapsulated Spirulina platensis. If the authors wish to include this data in the current study, additional discussion in limitations and future studies include the need for in vivo study are needed.
In the method section, it is not clear how the encapsulation efficiency was determined. Do the authors analyze the active compounds in both the free and encapsulated fractions? If yes, please describe the procedure and parameter of separation of the free active compound from the encapsulation.

4.
In the discussion section, it is not clear how this research indicates a synergistic interaction between phycocyanin and total phenols in antioxidant activities (page 9 of 16). No data support this statement.
5. Figure 4 is data on encapsulation efficiency. In the discussion (Page 9 of 16), by using the information in Figure 4, the authors stated the effect of different cultivation methods of S. platensis on phycocyanin content. No data support this statement.

Minor:
In the result section paragraph 2 (page 4 of 16), please add a description of the result of MSB. 1.
In the method section, microencapsulation, please describe what is the total solid content of coating materials used in the study.

2.
In the discussion section paragraph 3 (page 6 of 16), please be clear on what bioactive compounds are the authors referring to and for which treatment.
In the introduction and abstract, O. basilicum intervention to S. platensis is to increase consumption and increase bioactive compounds and their potential for a functional food product. The conclusion state as a potential for a pharmaceutical product. Please be consistent.

5.
In Figure 2, B = O. basilicum leaf extract was analyzed. Please describe B in the method section.

6.
The paper would benefit from a more detailed discussion regarding the organoleptic properties of how the Ocimum basilicum intervention reduces unpleasant odours as outlined in the comments above.
An additional suggestion that will improve the presentation of the manuscript is listed below: The result section should focus on the main finding and not the statistical analysis. Figure 1 and Table 1-6 can be added to the supplemental section. 1.
The authors need to check the accuracy of the statement in the result section's first sentence (page 4 of 16). Figure 1 is a standard calibration curve use to calculate total phenol of samples as gallic acid equivalent (GAE) and total flavonoid of samples as quercetin equivalent (QE) but not for antioxidant activity. Additionally, this information is more 2.
suitable to be in the method section and not the result section. Please also refer to my additional suggestion point 1.