Ocimum basilicum (kemangi) intervention on powder and microencapsulated  Spirulina platensis and its bioactive molecules

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Revised. Amendments from Version 2

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Abstract

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 49.5% and phycocyanin by 40.7%. This is due to the phenol and azulene compounds in  O. basilicum which have a synergistic effect on phenol and phycocyanin in  S. platensis. Microencapsulation using a maltodexrin and gelatin coating is effective in phycocyanin protection and antioxidant activity with an encapsulation efficiency value of 71.58% and 80.5%.  

Conclusion: The intervention of  O. basilicum on  S. platensis improved the total phenol and phycocyanin content and there is potential for a pharmaceutical product for a functional food and pharmaceutical product.

Introduction

Spirulina platensis is a blue-green microalga 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.

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 by freeze drying (MSB) of S. platensis were soaked with O. basilicum extract for 10 min with ratio of 1:4, w/v. A S. platensis sample with no O. basilicum added was used as a control (SP).

Preparation of the intervention of Ocimum basilicum on Spirulina platensis by freeze drying

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 the intervention of Ocimum basilicum on Spirulina platensis by freeze drying

This Microencapsulation was performed following the methods reported by Castro-Munoz et al. and Dewi et al. ^{28, 29} Ten percent (10%) of coating materials (64 g) a consisting maltodextrin and gelatin at a ratio of 9:1, w/w were used for microencapsulated. Then, S. platensis were soaked with O. basilicum extract were added into the mixture. Homogenization was then performed with a homogenizer (15A HG-wiseTis, Germany). S. platensis treated with microencapsulation freeze-dried 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 UV-Vis 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. The result of the gallic acid calibration curve obtained equation y = 1.0677 x – 0.0022 with a value R ² = 0.9915.

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. The result of the quercetin calibration curve obtained equation y = 0.0185 x + 0.0223 with a value R ² = 0.9995.

Phycocyanin content

40 mg of sample was added into 10 ml centrifugal tube phosphate buffer (pH 7) 100 mM; 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:

$$PC(\%)=\frac{\text{Abs}\text{x}\text{v}}{\mathrm{3,39}\text{x}w\text{x}{w}_{dry}}x100\%(1)$$

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:

$$\mathbf{Inhibition}\mathbf{(}\mathbf{\text{\%}}\mathbf{)}\mathbf{=}\frac{\mathbf{\text{Ab}}\mathbf{-}\mathbf{\text{As}}}{\mathbf{\text{Ab}}}\mathit{x}\mathbf{100}(2)$$

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:

$$EE(\%)=\frac{\text{Total}\text{coated}\text{active}\text{compounds}-\text{Free}\text{active}\text{compounds}}{\text{Total}\text{coated}\text{active}\text{compounds}}x100(3)$$

Where total coated active compounds is the total active compounds such as phycocyanin, phenol, flavonoid and antioxidant in the microcapsule (MSB sample). While free active compounds is the mass of active compounds such as phycocyanin, phenol, flavonoid and antioxidant in the microcapsule (powder) surface.

Free active compounds mass was calculated as follow:

• Phycocyanin (40 mg microcapsule were washed with 10 ml of buffer phosphate)

• Total phenol (1 g microcapsule were washed with 9 ml of aquabidest)

• Flavonoid (50 mg microcapsule were washed with 5 ml of methanol)

• Antioxidant activity (20 mg microcapsule were washed with 2 ml of ethanol)

The solution were filtered using Whatman paper No.42. After filtration, the free active compounds was measured according to the same methods described for active compounds such as (phycocyanin, total phenol, flavonoid and antioxidant activity) determination.

Parameter of separation of the free active compound from the encapsulation is the solubility of the active compounds when washed by strirring for one minute.

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 swiss ADME ( 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.

Results

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). Phycocyanin content was measured in SP, DSB and MSB, and then encapsulation efficiency was measured on total phenol, flavonoid, antioxidant activity and phycocyanin. The DSB sample can increase the total phenol 49.50% ( Figure 1) and antioxidant activity 12.67% of S. platensis ( Figure 2). However, total flavonoid is not significantly different with O. basilicum intervention on S. platensis ( Figure 3). The MSB sample is an effective for protecting phycocyanin and antioxidant activity with higher value an encapsulation efficiency. However, this encapsulation is less effective of polyphenol compounds such as phenol and flavonoid as shown in ( Figure 4). Ocimum basilicum extract was analysed the bioactive compounds such as total phenol, flavonoid, and antioxidant activity for 117.24 ± 8.06 mg GAE/g, 7.04 ± 0.18 mg QE/g and 94.93 ± 2.24%, respectively.

Figure 1. Total phenol content (n=3, mean value ± standard deviation; different superscripts indicate a significant difference).

SP = S. platensis with no treatment, DSB = S. platensis treated with freeze-dried O. basilicum, MSB = S. platensis treated with microencapsulation freeze-dried O. basilicum.

Figure 2. Antioxidant activity (n=3, mean value ± standard deviation; different superscripts indicate a significant difference).

SP = S. platensis with no treatment, DSB = S. platensis treated with freeze-dried O. basilicum, MSB = S. platensis treated with microencapsulation freeze-dried O. basilicum.

Figure 3. Flavonoid content (n=3, mean value ± standard deviation; different superscripts indicate a significant difference).

SP = S. platensis with no treatment, DSB = S. platensis treated with freeze-dried O. basilicum, MSB = S. platensis treated with microencapsulation freeze-dried O. basilicum.

Figure 4. Encapsulation efficiency.

(n=3, mean value ± standard deviation; different superscripts indicate a significant difference.

O. basilicum intervention can increase the levels of phycocynin in S. platensis 40.72% shown in ( Figure 5). 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. Raw absorbance data for bioactive compounds assays are available as underlying data ⁴⁰ .

Figure 5. Phycocyanin content (n=3, mean value ± standard deviation; different superscripts indicate a significant difference).

SP = S. platensis with no treatment, DSB = S. platensis traeted with freeze-dried O. basilicum, MSB = S. platensis treated with microencapsulation freeze-dried O. basilicum.

Discussion

Microalgae are a valuable source of proteins and phenol compounds. S. platensis is a type of microalgae with a high total phenol content ⁴¹ . 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 mg 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 of the total phenol ( Figure 1), phycocyanin ( Figure 3) and antioxidant activity ( Figure 4), except for flavonoid content ( Figure 2). 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 derivative compounds play an anti-inflammatory and antioxidant namely hesperidin and quercetin 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 components as phycocyanin, chlorophyll, carotenoid 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 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. The high contents of total phenol ( Figure 1) and phycocyanin ( Figure 3) had a positive correlation with antioxidant activity ( Figure 4) in S. platensis treated with freeze-dried O. basilicum (DSB). 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 3), 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.

Previous research showed that intervention O. basilicum increase hedonic scale of S. platensis. The O. basilicum intervention treatment (DSB) has the best score in aroma and texture, while S. platensis microcapsules with the intervention of O. basilicum (MSB) has the best score in color and appearance ⁷¹ . Volatile compounds that comtributed to this off-odour in S. platensis are geosmin, 2-Methylisoborneol and medium chain-alkanes. The intervention showed in a decrease in these volatile compounds in S. platensis ⁷² .

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 ⁷³ . 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 ⁷⁴ . The encapsulation efficiency of phycocyanin was in accordance with the results of previous studies ⁷⁵ , 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 ^{76, 77} . 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% ⁷⁸ .

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 1).

Table 1. Pharmacokinetics parameters of the analysis compound predicted by Swiss ADME.

Compound	GI absorption	BBB permanent	Bioavailability radar	Boiled egg
Phenol	High	Yes
Azulene	Low	Yes
Flavonoid	Low	No
Phycocyanin	High	No

Note: GI: gatrointestinal; BBB: blood-brain barrier; LIPO: lipophilicity; FLEX: flexibility; INSATU: unsaturation; INSOLU: insolubility; POLAR: polarity; SIZE: molecular weight.

Table 2 and Table 3 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 ⁷⁹ . 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 ⁸⁰ . The rule of five was developed to set drugability guidelines for new molecular entities (NMEs) ⁸¹ . Therefore, the rule suggests that molecules, whose properties fall outside of these boundaries, are unlikely to become orally bioavailable drugs ⁸² . 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.

Table 2. Physicochemical parameters of the analysis compound predicted by Swiss ADME.

Compound	MW (g.mol -1)	HA	AHA	RB	HBA	HBD	MR	TPSA	L
Phenol	94.11	7	6	0	1	1	28.46	20.23	1.24
Azulene	128.17	10	10	0	0	0	43.06	0.00	2.07
Flavonoid	594.52	42	16	6	15	10	138.73	260.20	2.23
Phycocyanin	526.71	39	5	8	3	3	174.47	86.35	4.24

Note: MW: molecular weight; HA: heavy atoms; AHA: aromatic heavy atoms; RB: rotatable bonds; HBA: hydrogen bond acceptor; HBD: hydrogen bound donor; MR: molar refractivity; TPSA: topology polar surface area (Ų); L: lipophilicity

Table 3. Druglikeness property using Lipinski Rule of Five.

Compound	Molecular mass less than 500 Dalton	High lipophilicity (expressed as LogP less than 5)	Less than 5 hydrogen bond donors	Less than 10 hydrogen bond acceptors	Molar refractivity should be between 40–130	Conclusion
Phenol	Yes	Yes	Yes	Yes	No	Yes
Azulene	Yes	Yes	Yes	Yes	Yes	Yes
Flavonoid	No	Yes	No	No	No	No
Phycocyanin	No	No	Yes	Yes	No	Yes

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 gelatin showed high encapsulation efficiency values. Hence, S. platensis treated freeze-dried O. basilicum has potential as a functional foods and pharmaceutical product.

Data availability

Underlying data

Figshare: Underlying data for ‘ Ocimum basilicum (kemangi) intervention on powder and microencapsulated Spirulina platensis and its bioactive molecules’, https://doi.org/10.6084/m9.figshare.14291069.v3 ⁴⁰

This project contains the following underlying data:

• Data file 1. Flavonoid content from the intervention of O. basilicum on S. platensis with microencapsulation.

• Data file 2. Antioxidant activity from the intervention of O. basilicum on S. platensis with microencapsulation.

• Data file 3. Encapsulation efficiency of phenol, flavonoid, antioxidant and phycocyanin content from the intervention of O. basilicum on S. platensis with microencapsulation.

• Data file 4. Phycocyanin content from the intervention of O. bacilicum on S. platensis with microencapsulation.

• Data file 5. Statistical analysis by SPSS v.22 on bioactive compounds.

• Data file 6. Total phenol content from the intervention of O. basilicum on S. platensis with microencapsulation.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).

Funding Statement

The research was funded by the PMDSU (Program of Master Degree Leading to Doctoral Degree for Excellent Graduates) for the Ministry of Research and Technology of the Republic of Indonesia in 2019 with grant number 642-07/UM7.6.1/PP/2020.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.