Extraction optimization and microencapsulation of Berberine from Berberis vulgaris incorporated in a functional orange drink: Physiochemical attributes and kinetic release studies

Abstract

BACKGROUND:

Berberine, extracted from Berberis vulgaris, is one of the well-known natural antioxidant sources.

OBJECTIVE:

Optimizing the berberine extraction conditions from the whole Barberry plant and microencapsulation of the optimized extract to be used as a bioactive ingredient in functional orange juice.

METHODS:

Seventeen extraction processes were designed to determine an optimized method for producing an ethanol/water extract with maximum yield, safety, and antioxidant properties. The optimal extract was microencapsulated by complex coacervation using tragacanth/gelatin and then spray-dried. The selected microcapsules based on morphology, particle size, and solubility were added to orange juice, and the physical and sensory properties of the functional drink, as well as the kinetic release models, were analyzed.

RESULTS:

An optimal extract with 82% antioxidant activity was prepared using a 75% ethanol/water ratio and an extraction time of 0.5 h at 22.3°C. Spherical-shaped microcapsules could create a desirable cloudy appearance with good stability in the pH of orange juice. The kinetics of the berberine release revealed an initial burst phase followed by a prolonged one, which would appeal to consumers’ sensory perceptions.

CONCLUSIONS:

The excellent compatibility between berberine and orange juice provides a potential capacity to fortify a high-consumption drink with a phytonutrient presented in a berry fruit.

Keywords

Berberine microcapsules antioxidant capacity ethanolic extract

1 Introduction

In todays’ world, improvement in the health and safety of humans is the primary goal of food scientists and nutritionists, which may be achieved through providing the needed nutrients and boosting human wellness [1]. Fruit and vegetable-based functional drinks are among the healthy products with a fast-growing demand [2]. Barberry is an Asian fruit, usually considered a well-known flavoring compound used to formulate Iranian food products [3]. The Berberidaceae family (order Ranunculales) includes more than 15 genera of flowering plants named the Barberry family. This family is primarily found in the northern hemisphere and is native to the moderate and temperate regions of Europe, Asia, North, South, Central, America, and North Africa [4, 5]. Berberis is the largest genus of this family and contains more than 500 species of evergreen shrubs [6]. The Common Barberry (B. vulgaris) consists of different varieties, including a seedless one known as B. vulgaris L. var. asperma [7]. It is a popular seedless barberry variety in Europe and Asia. Various parameters, such as maturity, growth conditions, and harvest season, are shown to affect the fruit quality and its functional properties [8]. In Iran, especially in Southern Khorasan province, barberry is a well-known medicinal plant [9], with a production yield of about 22000 tons in 2020 [10]. Up to now, many researchers have investigated the therapeutic characteristics of different parts of this plant. These berry fruits have been proved to be effective in diseases of the kidneys, urinary or gastrointestinal tract, liver, lungs and act as a stimulant for the circulatory system [11]. In addition, the results reported by Ghafourian [12] demonstrated the anti-cancer properties of Berberis vulgaris fruits against breast cancer. Various health and therapeutic effects, such as anti-diabetic, anti-hypertensive, anti-histaminic, anti-cholinergic, and anti-neoplastic, have been proved by in-vitro and in-vivo investigations [13, 14].

The health-benefit effects of these crop groups that have been used as traditional medicine are primarily due to complex secondary metabolites such as berberine, which is chemically 2,3-methylenedioxy-9,10-dimenthoxyprotoberberine chloride [15]. Berberine has antioxidant, antifungal, anti-diabetic, anti-tumor, anti-inflammatory, anti-mutagenic activities in addition to beneficial effects on colitis [14, 16]. Furthermore, berberine acts as an oxidative inhibitor and protects low-density lipoprotein (LDL) against oxidation. Besides, it has been proved that berberine might cause a decrease in the level of metal ions, thereby inhibiting metal ions-catalyzed lipid peroxidation [15, 17]. In 2021, Patel [18] reviewed recent studies about berberine, its chemistry, bioavailability, sources, therapeutic applications, and extraction procedures. Barberry juice powder was used as an antioxidative ingredient in the formulation of effervescent tablets [19]. The published studies are from all over the world, predominantly Asian countries, and reveal the potential therapeutic effects on human health and animals [20]. Various parts of the plant (roots, stems, leaves, rhizomes, and fruits) contain berberine [3 , 21–25]. The extraction of these types of bioactive compounds by different classical and modern methods has previously been studied [26]. Solvent extraction is a prevalent method that might be industrially preferred because of its simplicity, convenience, and cost-effectiveness compared to modern processes. A safe product with the best yield may be produced by modifying some processing parameters, taking into account the following factors: (a): Extraction yield and antioxidant activity (b): Solvent type (polar or non-polar) such as acetone, methanol, ethanol, and hexane [5 , 27–29] (c): Toxicity of the residual solvent in the final product and its risk to human health (d): Workers’ health and safety; and (e): Solvent waste disposal problems and environmental impacts [30]. Producing an extract with maximum yield, maximum safety, antioxidant properties, minimum residual solvent, and low environmental pollution problems might encourage farmers and industrial investors. Ethanol is a bio-solvent because it is made from renewable resources and is a recommended solvent from an environmental standpoint [31]. The Food and Drug Administration (FDA) emphasized the need for residual solvent testing in any food exposed to solvents, and ethanol was grouped in class 3 with a very low-risk level for human health based on the United States of Pharmacopeia (USP) [32]. According to directive 2009/32/EC, ethanol may be used in compliance with good manufacturing practices during the processing of foodstuffs [33]. Recent research by Troung [34] has indicated that the type of solvent significantly influences the antioxidant activity of extracts, and, among all, methanol and ethanol are the best. However, Venkatesan [35] reported that a mixture of ethanol/water acts successfully in the extraction of phytochemical compounds, especially alkaloids.

Berberine microcapsules were produced to mask the unpleasant taste of some drugs in oral disintegrating tablets [36]. The complexes of berberine hydrochloride and β-cyclodextrin have been developed to enhance intestinal absorption [37]. Encapsulation of berberine into nanoparticles was also investigated to improve its release properties, solubility, and anti-cancer activities [38, 39]. However, the novelty of the current study, not previously considered by other researchers, is to design a practical approach to achieve the best functionality of these bioactive compounds when added to consumers’ diets. This goal might be achieved by optimizing the extraction processes, preserving the maximum activity after microencapsulating, incorporating the microcapsules into a popular, daily-consumed food product and investigating the kinetics of berberine release from the microcapsules into the product.

Sensitive compounds such as flavorings, antimicrobials, antioxidants, and bioactive nutrients are not usually directly added to the food matrix to be protected against harsh food processing and storage conditions, and to prevent inactivation upon interacting with the other food ingredients [40]. Different types of microencapsulation techniques, including chemical, physical, and physicochemical processes, might be applied to accomplish this goal and to protect these compounds against oxidation and other undesirable reactions, and at the same time, make them releasable at controlled rates with better functionalities over a prolonged period [41 –43]. Complex coacervation is one of the microencapsulation methods based on physicochemical processes. Compared to the other methods, the main advantages of complex coacervation are high efficiency and several types of biopolymers that may be used as microcapsule wall materials [43]. In the coacervation technique, associative phase separation of two or a mixture of hydrocolloids from a suspension leads to forming coacervates around the bioactive compounds suspended in the same reaction media [41 , 45]. Generally, natural polymers such as proteins and polysaccharides may be used in this process as wall materials. A protein/polysaccharide interaction is usually exploited in the complex coacervation process. Gelatine is an odorless, translucent, somewhat tasteless protein. It is also biocompatible, nontoxic, and stable over a wide range of pH with unique gelation properties that may form polyelectrolyte complexes with different polyanionic polymers, including sodium alginate, κ-carrageenan, and gum acacia [43 , 47].

Gum Tragacanth (GT), commonly known as Katira, is an exudate gum derived from many species of Astragalus (family Leguminosae) with a heterogeneous and highly-branched structure [48]. This type of polysaccharide gum structurally exists in two forms: ribbon and flake, with the latter being of lower grade and cost [49]. Up to now, two species of this family, including Astragalus gossypinus and Astragalus compactus, have been used as wall materials for microencapsulation [50], nanoencapsulation [51], and complex coacervation [42]. Astragalus rahensis is one of the known species of the Iranian Astragalus [52, 53], which is relatively low in cost and exhibits good physiochemical behavior in various food products [50 , 54].

The complex coacervates of this type of traditional gum and sodium caseinate have been recently investigated [55]. Since gum tragacanth is an anionic biopolymer, it may be a perfect choice to participate in the complex coacervation process with proteins [49]. In the current research, the gelatine/tragacanth system is intended for use in berberine coacervates, which was not previously reported by other researchers prior to 2021. Nowadays, food product manufacturers are more interested in food fortification to improve consumers’ health and reduce the risk of diseases. Fortified beverages such as fruit juices or smoothies are frequently consumed along with meals and may act as suitable carriers for nutritional supplements. The processing and the characteristics of these types of functional fruit and vegetable juices have been reviewed by Speranza [56]. From a nutritional standpoint, minimally processed juices and smoothies may have become more popular in recent years among health-conscious consumers looking for a well-balanced daily diet. Orange juice is one of the most prevalent fruit juices for all ages; thus, it may be considered a suitable product to be fortified [57 –59].

The main objectives of this research were: (a): to exploit the whole barberry plant (roots, stems, leaves, rhizomes, and fruits) of B. vulgaris L. var. asperma as raw material to minimize agricultural waste. (b): to optimize the berberine extraction conditions from the whole plant, in terms of concentration and antioxidant activity of the extract, based on three processing parameters as follows: time (0.5–24 hrs.), temperature (5–50°C), and solvent concentration (ethanol/water ratio) (0–75%). (c): to microencapsulate the optimized berberine extract with gelatin and tragacanth from Astragalus rahensis (d): to study the physical and morphological properties of the microcapsules and the release kinetics of berberine into the orange juice enriched with microcapsules.

2 Materials and methods

2.1 Materials

The whole plant of Berberis vulgaris L. var. asperma (including fruit, leaves, and stems) was collected in the summer season (from August 15 to September 5, 2017) from South Khorasan Province, Iran. Ethanol (absolute grade, Merck Co., Darmstadt, Germany) was used as an extraction solvent. Berberine chloride (B3225, purity > 90%) as an analytical standard, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), and ascorbic acid were purchased from Sigma Aldrich, Austria. Gelatin (bovine skin (type B), Sigma Co. (St. Louis, USA)) and Gum Tragacanth (Astragalus rahensis, flake form, Esfarayen, Iran) were used as wall materials for the microcapsules. The Transglutaminase enzyme (TEGEN 220 DM) is provided by Benosen Food Additives Marketing Ltd. (Istanbul, Turkey). The fresh/unprocessed orange juice used in this research was one of the primary products of the Tackdaneh Co. (Marand County, East Azarbaijan, Iran).

2.2 Raw material preparation

The whole plant of B. vulgaris L. var. asperma as a berberine source was first cleaned and dried by the shade-drying method for seven days, and then was ground with a lab grinder, IKA A11 analytical mill, into mixed powders with a measured size in the range of 0.2–0.5 mm. Finally, the powdered sample was dried in a vacuum oven (75°C for 2 hours) until constant moisture was obtained [5].

2.3 Berberine extraction process

Box-Behnken design (Design-Expert software, trial version 7, Stat-Ease Inc., Minneapolis, MN, USA) was used to create seventeen aqueous ethanolic extraction processes with three levels of ethanol concentration (0, 37.5, 75%), processing time (0.5, 12.25, 24 hrs.), and temperature (5, 27.5, 50°C) (Table 1). Three grams of the powdered mixture were suspended in an ethanol/water solvent (pH = 7) [60], mixed at 100 rpm by a shaker incubator (Fan Azma Gostar), and then centrifuged for 10 min at 9000 rpm. The berberine extracts were prepared after filtering the supernatants through Whatman filter paper (grade 41) [25].

Table 1
Berberine Concentration and antioxidant activity for Berberis vulgaris extracts using the different levels of extraction variables of the Box-Behnken design criterion

Sample No. Temp.¹ (°C) Time (hrs.) Ethanol/Water (%) Berberine Con.² (μg/ml) Antioxidant activity (%)

1 27.5 12.25 37.5 7.32±0.20 68.22±1.02

2 27.5 12.25 37.5 7.21±0.02 68.43±1.21

3 27.5 24 75 12.68±0.01 64.33±1.31

4 5 24 37.5 1.75±0.01 58.05±2.21

5 50 24 37.5 13.05±0.01 66.25±1.40

6 27.5 12.25 37.5 7.25±0.02 68.94±1.23

7 5 12.25 75 9.25±0.01 73.43±1.21

8 27.5 0.5 0 1.36±0.01 70.82±1.32

9 5 0.5 37.5 8.15±0.03 76.14±1.24

10 27.5 12.25 37.5 7.12±0.02 67.41±1.31

11 27.5 12.25 37.5 7.27±0.02 68.23±1.02

12 50 0.5 37.5 7.03±0.01 72.21±1.41

13 50 12.25 75 17.29±0.01 78.21±1.04

14 5 12.25 0 0.50±0.01 59.44±2.02

15 27.5 0.5 75 13.77±0.03 82.91±1.01

16 27.5 24 0 2.08±0.01 62.33±1.22

17 50 12.25 0 2.76±0.01 73.43±1.12

Sample No.	Temp.¹ (°C)	Time (hrs.)	Ethanol/Water (%)	Berberine Con.² (μg/ml)	Antioxidant activity (%)
1	27.5	12.25	37.5	7.32±0.20	68.22±1.02
2	27.5	12.25	37.5	7.21±0.02	68.43±1.21
3	27.5	24	75	12.68±0.01	64.33±1.31
4	5	24	37.5	1.75±0.01	58.05±2.21
5	50	24	37.5	13.05±0.01	66.25±1.40
6	27.5	12.25	37.5	7.25±0.02	68.94±1.23
7	5	12.25	75	9.25±0.01	73.43±1.21
8	27.5	0.5	0	1.36±0.01	70.82±1.32
9	5	0.5	37.5	8.15±0.03	76.14±1.24
10	27.5	12.25	37.5	7.12±0.02	67.41±1.31
11	27.5	12.25	37.5	7.27±0.02	68.23±1.02
12	50	0.5	37.5	7.03±0.01	72.21±1.41
13	50	12.25	75	17.29±0.01	78.21±1.04
14	5	12.25	0	0.50±0.01	59.44±2.02
15	27.5	0.5	75	13.77±0.03	82.91±1.01
16	27.5	24	0	2.08±0.01	62.33±1.22
17	50	12.25	0	2.76±0.01	73.43±1.12

¹Temperature, ²Concentration.

2.4 Identification and quantitative determination of berberine

Identification and quantitative determination of berberine were accomplished by a high-performance liquid chromatography system (Knauer, advanced scientific instrument, Berlin, Germany) equipped with a 25 cm×4.6 mm Eurospher 100-5 C18 analytical column and a pre-column provided by Knauer (Knauer company, Berlin, Germany). A Jet Stream 2 Plus Oven has been used to control the temperature of the column. The mobile phase was a mixture of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution with a flow rate of 1 mL/min. Twenty μl of the sample was injected at 25°C through a 3900 Smartline auto-sampler equipped with a 100μl loop. A 1000 Smartline Pump, a 5000 Smartline Manager Solvent Organizer, and a 2800 Smartline photo-diode array detector were used in this analysis. Peaks were monitored at a 232 nm wavelength. EZChrom Elite software was used to collect data and integrate it [22]. A series of standard berberine solutions in the range of 0.7 to 25μg/mL were prepared. Quantification was done using external standard calibration.

2.5 Antioxidant activity analysis

In brief, 1 ml of each sample was added to 3 ml of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) solution (0.1 mM ethanol solution) and then incubated in the dark for 30 minutes at 30°C. The absorbance was measured against pure ethanol at 517 nm, and the percentage of DPPH radical scavenging activity was calculated using the following equation: $Antioxidant Activity (%) = \frac{A control - A sample}{A control} \times 100$

Ascorbic acid (prepared as described above at 1.5μg/ml) and DPPH solutions were used as positive and negative controls (A control), respectively [61].

2.6 Preparation and characterization of berberine microcapsules

The microcapsules preparation method was complex coacervation using a gum tragacanth/ gelatin (1 : 2) bio-polymeric system. Gelatin (3.5% w/w, 100 mL) and gum tragacanth (GT) solutions (1.75% w/w, 100 mL) solutions in deionized water were prepared at 50°C by continuous stirring (Heidolph Heater/Stirrer, Germany) at 600 rpm until completely dissolved. The T25 digital Ultra-Turrax® homogenizer (IKA, Germany) was then used to mix and homogenize 100 mL of these biopolymer solutions for 3 minutes at 10,000 rpm. Ten mL of berberine extract was mixed with the polymer suspension, and the pH was adjusted to 4.0±0.1 by adding an aqueous solution of acetic acid (10% (w/w)) and stirring continuously at 600 rpm for 2 hrs. Transglutaminase (120 U/g gelatin) powder was gradually added to this mixture as the cross-linking agent. The suspension of microcapsules was then cooled to 10°C and kept in an incubator for 18 hrs. A Büchi mini spray-drier (B-290 Advanced 230V/50–60 Hz) was used to dry the suspension and prepare the coacervate microcapsules. The operating conditions were as follows: Inlet temperature of 125–130°C, outlet temperature of 70–75°C, and airflow rate of 50 m³/hr [62, 63].

The morphological characteristics of microcapsules were studied before and after drying. The moist particles were evaluated using an optical microscope (Motic® BA300, Barcelona, Spain) equipped with Motic® Image Plus 2.0 software (Causeway Bay, Hong Kong). The dried microcapsules were taped to aluminum stubs and coated with a 400°A layer of gold using an SEM sputter coater before being scanned by an electron microscope (ESEM XL30, Philips, the Netherlands) with a voltage acceleration of 20 kV and magnifications of 2000 and 100000 [42].

One mg of the microcapsules was dispersed in 10 mL of deionized water by stirring continuously at room temperature for 30 min. The particle size distributions were analyzed using a laser diffractometer equipped with a Hydro 2000S small volume automated wet dispersion accessory (Malvern Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK) and were reported in terms of the volume mean diameter D [4,3, 4,3] [53].

The microencapsulation yield was calculated as the final weight of dried microcapsules divided by the total weight of materials used to prepare the microcapsules [42].

Microencapsulation efficiency was investigated according to the method of Rocha [64] with minor modifications. The sample was diluted with aqueous ethanol to prepare the standard solutions at different concentrations (5, 10, 25, 50, 75, and 100μg/mL). These solutions were analyzed by a double beam UV-Visible spectrophotometer (Cecil 7200, UK) at 470 nm. The standard curve was prepared by plotting absorbance at 470 nm against concentration, and the regression equation was obtained as y = 0.0081x–0.0365 (R² = 0.9996). Three mg of microcapsules were added to 3 mL of methanol in a falcon tube. The mixture was shaken at 100 rpm (Heidolph Heater/Stirrer, Germany) for 5 min and stored in the dark for 4 hrs., followed by centrifugation at 9000 rpm for 10 min. The supernatant was used for spectrophotometric determination of free berberine content, as described above. The following equation gives the encapsulation efficiency [64]. $EE % = (Total berberine - Total free berberine) / (Total berberine) \times 100$

The water solubility of the microcapsules was measured using a gravimetric method [65].

2.7 Preparation method of functional orange juice

Three different functional juices, named A, B, and C, were formulated from 4% (W/V) orange concentrate (60 Brix), 8.5% (W/V) sugar, 0.1% (W/V) citric acid, 700 mL of deionized water, and 1, 2, and 4% of berberine microcapsules, respectively. All of the mixtures were first homogenized by a magnetic stirrer at 600 rpm for 15 min and then pasteurized for 10 min at 80°C [66].

2.8 Particle size, color, and turbidity measurements of orange juice samples

The particle size distributions of the juice samples at room temperature (Malvern Mastersizer 2000 particle size analyzer; Malvern Instruments Ltd., Worcestershire, UK) were reported based on d (0.1), d (0.9), and the span (width of distribution) [53].

The color characteristics (L^*, a^*, and b^*) were analyzed in triplicate by using a Color-Eye 7000A reflectance spectrophotometer (CPS Color Pty Ltd., Parramatta, NSW, Australia) [52, 53]. The turbidity was determined by a turbidity meter system (Aqualytic Model 100-IR90-250 VAC., Germany) after keeping the product for a specific time (0, 7, and 30 days) at room temperature [67]. The results are reported in nephelometric turbidity units (NTU).

2.9 Berberine release and kinetic models

Study of the kinetics of berberine release from the microcapsules into the orange juice was necessary to predict the availability of this nutritious compound for improving consumers’ healthy diets. This investigation was accomplished at the following temperature/time conditions: (5°C for 0, 3, 5, 7, 10, 20, and 30 days, and 80°C for 10, 20, 40, 60, 80, and 100 min). After each test, the sample solution was filtered by a syringe filter with a pore size of 0.45μm, and then the released berberine concentration was measured spectrophotometrically at 470 nm as described above. Orange juice without microcapsules was considered as a blank for the standard curve [68].

The release data were fitted to the three following models that are usually considered the best to explain the compound release from polymeric microcapsules: $Zero - Order model : Qt = K_{0} t + C$

C and Qt are the amounts of berberine in the solution before release (which is actually zero) and the amount of berberine released over time, respectively. K₀ is the zero-order release rate constant. $Higuchi model : Qt = K_{H} t^{0.5} + C$

K_H is the Higuchi rate constant. $Modified Korsmeyer - Peppas model : Q_{t} / Q_{\infty} = K_{K - P} t^{n} + b$

Q_t and Q_∞ are the amounts of berberine released over time and at the equilibrium state (infinite time), respectively. K_K - P is the Korsmeyer-Peppas constant; b is the y-axis intercept when Q_t/Q_∞ is plotted against t and characterizes the burst effect (indicating an abrupt increase in initial active compound release). The burst release may be a favorable situation for encapsulated flavors, which is most probably related to the morphology and porous structure of the polymeric matrix of microcapsules [69]. In the above equation, n is the release exponent to interpret the release mechanism. If n < 0.45, then the release is mainly governed by the Fickian diffusion mechanism, and when 0.5 < n < 1, both Fickian and swelling may be considered [70 –72]. The adjusted coefficient of determination (adjusted-R²) was used to compare the theoretical and experimental data, and the maximum value was considered the best-fitted model.

2.10 Sensory analysis of the orange juice samples

Twenty-five panelists (18 females and 7 males, aged 22 to 37), including graduate students, faculty members from the food science and technology department of Islamic Azad University, and workers from the fruit juice production line, were trained according to ISO 8586 : 2012 [73] to perform sensory tests. All of the panelists were volunteers for testing this product and gave informed consent to tests on a non-standard/natural food with no hazard to human health to set the ethical and professional practices for sensory evaluation [74]. On a 5-point hedonic scale, the panelists assessed the sensory attributes of appearance, color, odor, consistency, taste, and overall acceptability. The scores ranged from 5 = very good to 1 = undesirable. The laboratory was equipped with five individual sensory booths, and orange juice samples were served along with mineral water for mouth rinsing between tests [53].

2.11 Experimental design and statistical analysis

The optimal combination of berberine extraction variables was determined using the Box-Behnken design (Design-Expert software, trial version 7, Stat-Ease Inc., Minneapolis, MN, USA) [75]. Three independent variables were ethanol/water ratio (0, 37.5, and 75%), extraction time (0.5, 12.5, and 24 hrs.), and temperature (5, 27.5, and 50°C). Berberine concentration (g/mL) and antioxidant activity (%) were the dependent variables. The complete design consisted of 17 runs, including five central points [25 , 77].

Each analytical experiment was carried out in triplicate on microcapsules. SPSS® 22 (IBM® Co., Armonk, NY, USA) was used to conduct an analysis of variance (ANOVA) followed by Duncan’s test.

3 Results and discussion

3.1 Berberine extraction

The HPLC chromatogram of the standard solution of berberine is presented in Fig. 1. A peak corresponding to the berberine compound was observed at RT = 24.243 min, and a linear response (y = 52197×–46125: R2 = 0.9958) was obtained for the standard solutions of berberine concentrations in the range of 0.2 to 25μg/mL. The effects of extraction conditions on berberine extracted concentrations and antioxidant activities are reported in Table 1. The minimum berberine concentration belonged to the aqueous extracts where the ethanol concentration was zero (runs 8, 14, 16, 17). The antioxidant activities of these extracts were at a high level (59.4–73.4%) despite the low yields (0.5–2.76μg/mL). This result may confirm that some water-extractable bioactive ingredients are available in Berberis vulgaris. The extraction yields are significantly improved by increasing the ethanol concentration. It may be expected, as berberine is a polar alkaloid, extractable by polar solvents such as ethanol [28]. Lu [80] investigated the solubility of berberine in different organic solvents, including ethanol, 1-octanol, 2-propanol, and 1-butanol, and reported the highest solubility by using ethanol. On the other hand, methanol frequently provides better extraction yields than ethanol [29 , 81–84]. Nevertheless, an aqueous-ethanolic solvent was preferred to methanolic solvents, considering human health and safety expected by the food and pharmaceutical industries [85]. The extraction time (0.5, 12.5, 24 h) was another factor that positively affected the berberine extracted concentration (Table 1). Wu [25] has previously reported this effect. Generally, berberine is a quaternary protoberberine alkaloid (QPA), widely found in various plants in the form of protoberberine salts [26, 86]. These groups of alkaloids were revealed to be enzymatically biosynthesized by tyrosine. Several enzymes, including berberine bridge enzyme and tetrahydroprotoberberine oxidase, participate in this bioconversion [86]. Actually, the isolation and extraction process of the protoberberine salts involves interconverting the salts to their base forms, which are more soluble in organic solvents such as ethanol [87]. In the current research, an unexpected result was obtained when the extraction time was increased from 0.5 h to 24 h at a constant temperature (5°C) with a solvent concentration of 37.5%. By comparing run 4 with run 9 (Table 1), one can observe that the berberine concentration decreased more than four times when extraction time increased. It may be emphasized that this result was observed only at low temperatures (5°C) and after a long processing time. This lower yield appears to be caused by the free base forms of protoberberine becoming unstable and converting to their disproportionated derivatives over time (more than 12 hours) [86, 87]. However, higher solvent concentrations, processing at higher temperatures, and shorter time might rapidly facilitate berberine extraction (Fig. 2) due to softening and disintegrating of the plant tissues [76, 88].

Fig. 1

HPLC chromatogram of the standard solution of berberine.

Fig. 2

3D surface plot showing the effect of extraction condition on berberine yield at 5°C.

As shown in Table 2, the experimental data for berberine yield and antioxidant activity were fitted to four different models, including linear, 2F1 (interactions), quadratic, and cubic, and the p values for all of the models except the cubic model were significant (p < 0.05). The polynomial models with the highest R² were quadratic and cubic (R² > 0.995) according to the data of both of the responses. However, the former was suggested, while the latter was aliased. Meanwhile, the lack of fit was used to predict the models’ fitness. The quadratic model for both of the responses had p values greater than 0.05. That means an insignificant lack of fit and also reveals that the quadratic model is suitable for predicting berberine yield and antioxidant activity [89].

Table 2

Model fitting for berberine yield and antioxidant activity

Source	Sum of squares	DF	Mean squares	F- value	P value	Std. Dev.	R²	R²_Adj	R²_pred	Press	Remarks
Sequential-model sum-of-squares tests and summary statistics for berberine yield
Mean vs Total	928.85	1	928.85
Linear	321.31	3	107.10	28.37	< 0.0001	1.94	0.8675	0.8369	0.7230	102.59
2Fl	48.86	3	16.29	760.20	< 0.0001	0.15	0.9994	0.9991	0.9986	0.51
Quadratic	0.18	3	0.059	11.33	0.0045	0.072	0.9999	0.9998	0.9993	0.26	Suggested
Cubic	0.014	3	0.0047	0.83	0.5413	0.075	0.9999	0.9998		^*	Aliased
Residual	0.023	4	0.0056
Total	1299.23	17	76.43
Lack-of-fit tests for berberine yield
Linear	49.05	9	5.45	968.08	< 0.0001
2Fl	0.191	6	0.032	5.68	0.0574
Quadratic	0.014	3	0.0047	0.83	0.5413						Suggested
Cubic	0.000	0									Aliased
Pure error	0.023	4	0.0056
Sequential-model sum-of-squares tests and summary statistics for antioxidant activity
Mean vs Total	81715.80	1	81715.80
Linear	560.88	3	186.96	23.07	< 0.0001	2.85	0.8419	0.8054	0.6793	213.68
2Fl	93.12	3	31.04	25.42	< 0.0001	1.11	0.9817	0.9707	0.9483	34.41
Quadratic	8.91	3	2.97	6.30	0.0213	0.69	0.9950	0.9887	0.9469	35.38	Suggested
Cubic	2.09	3	0.70	2.30	0.2180	0.55	0.9982	0.9927		^*	Aliased
Residual	1.21	4	0.30	23.07
Total	82382	17	4846.00	25.42
Lack-of-fit tests for antioxidant activity
Linear	104.13	9	11.57	38.29	0.0016
2Fl	11.00	6	1.83	6.07	0.0513
Quadratic	2.09	3	0.70	2.31	0.2180						Suggested
Cubic	0.000	0									Aliased
Pure error	1.21	4	0.30

The polynomial models for the berberine yield (Y1) and antioxidant activity (Y2) were as follows:

$Y 1 = 7.230 . 12 A + 2 . 58 B + 5 . 79 C + 3 . 15 AB - 0 . 45 AC + 1 . 44 BC + 0.12 A^{2} + 0.0 97 B^{2} + 0.12 C^{2}$ $Y 2 = 65.257 . 12 A + 2 . 86 B + 3 . 36 C + 1 . 58 AB - 2 . 52 AC - 3.80 BC + 0.92 A^{2} + 0.45 B^{2} + 0.93 C^{2}$

where A, B, and C represent time, temperature, and solvent percentage, respectively. Processing temperature (B) and ethanol concentration (C) were the parameters with the highest positive effect on both responses, i.e., the berberine extraction yield and the antioxidant activity. Temperature showed a positive/boosting effect on the berberine extraction yield at a constant level of solvent (Table 1, Fig. 3a). The yield increased even after a short processing time for extraction processes with maximum ethanol concentration. A higher solvent level (75%) was required to achieve the maximum yield (Fig. 3b, c). Figure 3d showed that a berberine extract with maximum antioxidant activity might be produced using the highest solvent ratio (75%) and the lowest extraction time at 22–23°C. According to the results of statistical optimization analysis, an optimum berberine extract with maximal antioxidant activity (82.9%), berberine concentration (13.5μg/mL), and desirability of 88.1% may be obtained at optimum conditions of temperature: 22.3°C, time: 0.5hrs, and solvent level: 75% (Fig. 3d, e, and f). This statistically optimized extraction process was validated by producing a berberine extract under these conditions and determining the experimental data. These results are compared in Table 3 and indicate that the predicted and experimental values for both responses were not significantly different (p > 0.05).

Fig. 3

3D plots of the effects of extraction parameters (time, temperature and solvent ratio) on Berberine yield (a, b, and c) and antioxidant activity (d, e, and f).

Table 3

Predicted and experimental berberine yield and the antioxidant activity in optimal extraction conditions

Parameters	Predicted	Experimental	p-value
Berberine yield (μg/ml)	13.54	16±0.16	0.063
Antioxidant activity (%)	82.10	85.3±0.21	0.054

Optimal conditions: 75% ethanol percent percentage, 0.5 h extraction time, and 22.29°C temperature.

3.2 Microcapsules characteristics

The optical microscopy image of the wet berberine microcapsules (Fig. 4a) demonstrates a uniform/spherical shape with a wide range of sizes. The scanning electron microscopy (SEM) image of spray-dried microcapsules with a 1 : 2 polysaccharide /protein (Ps/Pr) ratio is presented in Fig. 4b. The microcapsules are composed of a dense, rigid polymeric network with no porous structure, suggesting that the drying process does not cause surface crack formation. However, some irregularities are formed on some of the particles (shown by arrows in Fig. 4b); these protrusions may improve the release properties of the microcapsules due to slightly increased surface area [42]. According to the particle size analyzer data, the mean diameter size of the microcapsules, D [4,3], was 37.5μm, which was ideal for those produced by complex coacervation [65].

Fig. 4a

Optical microscopy image of berberine wet microcapsules (40x).

Fig. 4b

Scanning electron micrograph of gelatin/ tragacanth complex coacervate berberine microcapsules (10000X). The arrows show the surface irregularities.

As shown in Table 4, the berberine microencapsulation yield and efficiency for Ps/Pr of 1 : 2 were 50% and 67%, respectively. These results are approximately similar to the data reported by Jain [42], at a 1 : 2 Ps/Pr and a 0.25% total biopolymer concentration. Thus, the Ps/Pr ratio may change the biopolymer surface charge balance, influencing complex coacervates. In addition, the water solubility of microcapsules was relatively low at room temperature (Table 4). This characteristic is another crucial parameter that affects the behavior and rehydration ability of the microcapsules when used in the aqueous medium of food products. Previous research has reported that the most influential factors in the reconstitution properties of spray-dried microcapsules are the inlet drying air temperature and carrier agent type and concentration [90]. Tragacanth gum is composed of an anionic, water-soluble fraction of tragacanthin (20–30%) and the insoluble-but-swellable-in-water bassorin (60–70%) [91]. Bovine gelatin contains hydrophilic groups that are well soluble in hot water (40°C) [92, 93]. However, complex coacervation of protein/polysaccharide such as gelatin/anionic polysaccharide leads to the formation of coacervates with poor water solubility. Since the current research uses berberine microcapsules to incorporate into an orange juice formulation with a pH of 3.40±0.05, which has not been previously reported, it will be necessary to investigate the release kinetics in acidic media to define the functionality.

Table 4

Characteristics of the complex coacervated berberine microcapsules with Ps/Pr¹ of 1 : 2²

Yield (%)	Efficiency (%)	Solubility (%)	Span (–)	D[4,3, 4,3] (μm)
50±0.52	67±1.04	8±0.71	1.76±0.11	37.5±1.1

¹Ps/Pr: Polysaccharide/ protein ratio ²Results are expressed as mean value±standard deviation.

3.3 Orange juice characterization

3.3.1 Particle size distribution, color attributes, and turbidity

A bimodal particle size distribution curve was obtained for the original orange juice (Fig. 5a). As filtered orange juice has been used, this curve particularly corresponded to the serum phase, including the colloidal pulp particles < 100μ, and emulsified oil droplets (> 2μ) [94]. The addition of 1–4% of microcapsules with a volume-based mean diameter of 37.56μm (Table 5) slightly (< 10%) increased the frequency of these sized particles (Fig. 5b). However, increasing the parameters d (0.9), d (0.1), and the span in the samples A–C compared to the control (Table 5) suggested the possibility of some aggregations of the microcapsules, which may be confirmed by the optical microscopic image of the wet microcapsules (Fig. 4a) [95, 96]. The data given in Table 5 showed that the diameter of 90% of the particles suspended in control and sample C was between (2.47–180.78μ) and (3.39–274.75μ), respectively. Thus, the suspended particles are shifted to larger sizes, which may increase the turbidity of orange juice. The turbidity or cloudy appearance is one of the crucial physical characteristics of orange juice, positively influencing the overall acceptability [97]. Table 6 shows the turbidity values for three orange juice samples by adding 1–4% microcapsules during storage for one week and one month. As the large pectic particles were removed from the orange juice by primary filtration, its initial turbidity was very low compared to the results reported for this product in previous studies [98]. Nevertheless, incorporating 1–4% of coacervated microcapsules caused a 5.5–40% increase in turbidity on the first day (Table 6). Speranza [56] reviewed the characteristics of fruit juices supplemented with microcapsules of active compounds and reported that some types of physical changes, especially related to turbidity, may occur due to the stratification of the microcapsules. Because of the very low solubility of complex coacervated gelatin microcapsules in an acidic medium (pH < 4.5) [99], the microcapsules may be kept stable in the functional orange juice before consumption; and the increased turbidity of the samples, especially during storage, is mainly due to the flocculation of the suspended particles, including microcapsules (Table 6). This desirable cloudy appearance may decrease the lightness level of the final product (Table 5). Furthermore, the two other color attributes (a^*, b^*) obtained for the samples were slightly higher than the control. This change may have corresponded to the yellowish/reddish color of the gelatin microcapsules [100].

Fig. 5a

Particle size distribution curves of the original orange juice.

Fig. 5b

Particle size distribution curves of the sample C with 4% berberine microcapsules.

Table 5

Particle size distribution parameters and color characteristics for orange juice samples containing berberine microcapsules^1,2

Samples	Span (–)	d (0.9) (μm)	d (0.1) (μm)	L^*	a^*	b^*
Control	3.41±0.22^d	180.78±0.14^c	2.47±0.46^c	31.01±0.41^a	4.40±0.20^a	26.12±0.51^a
A	3.59±0.12^c	185.97±0.13^b	3.01±0.25^b	30.35±0.12^b	4.27±0.21^b	25.83±0.30^b
B	4.79±0.24^b	220.42±0.14^b	3.12±0.37^a	24.37±0.20^c	3.75±0.40^c	23.50±0.22^c
C	5.76±0.14^a	274.76±0.23^a	3.39±0.41^a	23.32±0.61^d	3.52±0.50^d	22.28±0.31^d

A, B, and C: orange juice samples with the addition of 1, 2, and 4% of berberine microcapsules, respectively. ¹Values with different superscript letters in each column are significantly different (p < 0.05). ²Results are expressed as mean value±standard deviation.

Table 6

Turbidity (NTU) values for orange juice samples containing berberine microcapsules during 1,7, and 30 days of storage^1,2

Samples	Turbidity (NTU)³
	1	7	30
Control	418±3.2^d	422±1.5^d	430±3.1^d
A	441±1.3^c	498±3.2^c	550±2.5^c
B	498±1.2^b	565±2.4^b	648±1.6^b
C	541±2.1^a	600±3.3^a	680±1.2^a

3.3.2 Release kinetics of Berberine into the orange juice at two different temperatures

Berberine release patterns from the microcapsules into orange juice (Fig. 6a, b) indicated that the release pattern was generally similar at both refrigerator (5°C) and pasteurization (80°C) temperatures, including the following phases: 1) The dissolution and diffusion of the accessible berberine close to the surface of the microcapsules resulted in a rapid release phase at first. 2) A slow and prolonged phase occurred due to berberine being entrapped within the inner parts of the microcapsules and diffusing out gradually through the channels of a network of pores [101, 102]. The difference was in the rate and amount of released berberine. A burst effect initially appeared in the release profile and has been previously reported where (a) the active compound is highly soluble, (b) high-loading dose of microcapsules, and (c) the polymeric matrix exposed to the surrounding medium might be hydrated and become highly porous, leading to the rapid diffusion of the active compound [96 , 103]. The values of the kinetic models’ parameters are given in Table 7. According to the results, the zero-order and Higuchi models were inappropriate (R2 < 0.9) to describe the release of berberine. However, the modified Korsmeyer-Peppas model fitted well with the release results (R² ≈0.97). The exponent release value (n) was 0.34, which specifies the berberine release mechanism is the Fickian diffusion (n < 0.45) for a polydispersed spherical system [71, 104]. Fig. 6a, b represent an initial burst phenomenon for berberine release from microcapsules into orange juice at both temperatures (5°C and 80°C), where the burst constant (b) was approximately 9% bigger at the lower temperature. Thus, it may be concluded that the swelling properties of the polymeric matrix might also influence the release mechanism, and this effect occurs slightly less at 80°C than at 5°C. Despite the fact that good thermal stability of the gelatin coacervates has been reported (T > 300C) [105], this minor difference may be due to structural changes in the polymeric matrix occurring at temperatures close to 100C, resulting in lower hydration and swelling capacity [106]. However, Devi [105] reported that the cross-linked gelatin complex coacervates are moderately swellable in acidic media. This aspect is critical in our research because it allows the encapsulated berberine flavor to release gradually in orange juice.

Fig. 6a

Berberine release rate from microcapsules into orange juice at 5°C.

Fig. 6b

Berberine release rate from microcapsules into orange juice at 80°C.

Table 7

Parameters of the kinetic release models of berberine from the microcapsules at 5°C and 80°C

Model	Kinetic parameters	5°C	80°C
Zero-order	K₀	0.425	0.121
Q₀= K₀ t + C	C	48.59	41.84
	R²	0.673	0.708
Higuchi	K_H	3.17	1.73
Q_H = K_H t^0.5 +C	C	43.83	36.35
	R²	0.833	0.827
Modified Korsmeyer-Peppas	K_K - P	44.51	1.24
Q_t/Q₀ = K_K - P tⁿ + b	n	0.34	0.53
	b	43	39.2
	R²	0.965	0.935

3.3.3 Sensory properties

The sensory parameters for the orange juice samples are listed in Table 8. According to these results, sample C, which included 4% of the microcapsules, achieved the highest scores for the sensory characteristics of appearance, flavor, odor, consistency, and color. There was no significant difference between this sample and the others in terms of overall acceptability. This finding indicates that the sensory properties of the newly developed functional orange juice successfully meet consumers’ expectations. The perfect compatibility of berberine with the taste and color of orange juice could provide a potential capacity to fortify a popular, high-consumption drink with a beneficial natural compound.

Table 8
Sensory evaluation Parameters for orange juice samples containing berberine microcapsules¹

Samples Appearance Flavor Odor Consistency Color Acceptability

A 3.60^c 3.04^c 3.04^c 3.60^c 3.56^c 3.20^b

B 3.96^b 3.40^b 3.32^b 3.88^b 3.92^b 3.40^a

C 4.12^a 3.70^a 3.88^a 4.20^a 4.28^a 3.40^a

Samples	Appearance	Flavor	Odor	Consistency	Color	Acceptability
A	3.60^c	3.04^c	3.04^c	3.60^c	3.56^c	3.20^b
B	3.96^b	3.40^b	3.32^b	3.88^b	3.92^b	3.40^a
C	4.12^a	3.70^a	3.88^a	4.20^a	4.28^a	3.40^a

4 Conclusion

Berberis vulgaris has been identified as a potential native berberine source that might be used to make a fruit-based/antioxidant-rich beverage. The Berberine extraction process has been optimized based on three processing parameters (time, temperature, and solvent ratio). The extract produced under optimized conditions was subsequently microencapsulated as tragacanth/gelatin complex coacervates to preserve from the acidic medium of orange juice. The microcapsules showed good stability and were slightly swellable in this drink, which is desirable for providing a controlled release mechanism of the bioactive compounds. This functional orange juice with good sensory scores may be a healthier alternative to high-calorie beverages. Since the presence of small aggregates of wet microcapsules may affect the rheological properties of this product, including its consistency, more research is needed to understand these features as well as the clinical effects of drinking of this novel type of orange drink on the human body.

Author contributions

Maryam Keshtkaran: Investigation, writing original draft, and visualization

Maryam Mizani: Supervisor, conceptualization, methodology, writing the review, and editing the manuscript

Seyed Mohammadali E. Mousavi: Supervisor, conceptualization, methodology

Mahammad A. Mohammadifar: Conceptualization, methodology

Reza Azizinezhad: Software and investigation

Footnotes

Acknowledgments

The Tehran University and the National Nutrition and Food Technology Research Institute of Shahid Beheshti (Tehran, Iran) are appreciated for providing technical assistance.

Funding

The authors report no funding.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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Extraction optimization and microencapsulation of Berberine from Berberis vulgaris incorporated in a functional orange drink: Physiochemical attributes and kinetic release studies

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Materials and methods

2.1 Materials

2.2 Raw material preparation

2.3 Berberine extraction process

2.5 Antioxidant activity analysis

2.6 Preparation and characterization of berberine microcapsules

2.7 Preparation method of functional orange juice

2.8 Particle size, color, and turbidity measurements of orange juice samples

2.9 Berberine release and kinetic models

2.10 Sensory analysis of the orange juice samples

2.11 Experimental design and statistical analysis

3 Results and discussion

3.1 Berberine extraction

3.3.1 Particle size distribution, color attributes, and turbidity

Table 8 Sensory evaluation Parameters for orange juice samples containing berberine microcapsules1 Samples Appearance Flavor Odor Consistency Color Acceptability A 3.60c 3.04c 3.04c 3.60c 3.56c 3.20b B 3.96b 3.40b 3.32b 3.88b 3.92b 3.40a C 4.12a 3.70a 3.88a 4.20a 4.28a 3.40a

Author contributions

Footnotes

Acknowledgments

Funding

Conflict of interest

References

Table 8
Sensory evaluation Parameters for orange juice samples containing berberine microcapsules¹

Samples Appearance Flavor Odor Consistency Color Acceptability

A 3.60^c 3.04^c 3.04^c 3.60^c 3.56^c 3.20^b

B 3.96^b 3.40^b 3.32^b 3.88^b 3.92^b 3.40^a

C 4.12^a 3.70^a 3.88^a 4.20^a 4.28^a 3.40^a