Introduction

Berberine is an isoquinoline alkaloid compound found in the barberry shrub’s roots, rhizomes, stems, and bark [1]. Clinically, plants containing berberine compounds have long been used to treat various diseases such as osteoporosis during menopause, atherosclerosis, type 2 diabetes, blood pressure and heart failure, cancer, diarrhoea, polycystic ovary syndrome, kidney and hepatitis diseases, hyperlipidemia and Metabolic syndrome [2,3,4]. Berberine can improve metabolic syndrome including a set of indicators such as obesity, blood lipid disorders, high blood sugar and high blood pressure [5, 6]. Reducing blood lipids and improving insulin resistance are among the most studied features of berberine in clinical trials [7, 8]. In addition, berberine can induce vascular endothelial nitric oxide production and cause dilation of vascular smooth muscle hence lowering blood pressure [9, 10]. The mechanisms that lead to the anti-diabetic effect of berberine include increasing the sensitivity of the insulin receptor and stimulating its secretion, inhibiting gluconeogenesis in the liver, enhancing the secretion of GLP-1, reducing glucose absorption from the intestine by inhibiting the activity of alpha-glucosidase and activating the protein kinase pathway activated by adenosine monophosphate (AMPK) [11,12,13]. Although berberine has so many beneficial effects, poor water solubility and low oral bioavailability (less than 1%) have limited the use of berberine [14, 15]. The low bioavailability of berberine may be attributed to the presence of a quaternary ammonium cation group in its molecular structure [15, 16]. Another obstacle to the systemic absorption of berberine is the self-aggregation of the drug in the acidic medium of the stomach, which has a negative effect on the dissolution process of the drug in the gastrointestinal tract (GIT) [17]. Furthermore, poor permeability through the GIT wall, hepatobiliary reabsorption, and intestinal first-pass metabolism are other reasons for its low bioavailability [18, 19]. Various methods are used to increase the solubility of drugs, such as the formation of drug salts, formation of prodrugs, particle size reduction, formation of micelles, formation of micro or nanoemulsions, self-emulsifying systems, liquisolid powder, liqui-pellet formulations, and solid dispersion technology (SDT) [20,21,22,23,24,25,26,27,28]. Among these methods, SDT has been one of the most successful techniques to increase the dissolution rate of poorly water-soluble drugs [22]. SDT is a useful method for dispersing the drug substance in the form of molecules or small particles in a carrier matrix [29]. The solid dispersion technique increases the dissolution rate of poorly water soluble drugs by reducing the particle size, formation of co-crystal, creating particles with high porosity, and the increase in the dissolution rate subsequently leads to an increase in bioavailability [30].

One of the most important methods of preparing solid dispersion is solvent evaporation [31]. In this method, the drug and the carrier are dissolved in a common solvent followed by the removal of the solvent by heating and drying under a vacuum [31, 32]. The used carrier should have characteristics such as good solubility in various solvents, pharmacologically neutral, safe for oral use and compatible with the drug substance [32]. The reaction between the drug and the carrier prevents the accumulation of drug particles and the creation of a supersaturated state of the drug in the dissolved medium, which leads to an increase in the rate of absorption [32]. One of the reasons for the enhanced dissolution rate of poorly water-soluble drugs could be due to the reduction of the particle size, the reduction of the degree of crystallinity or the creation of an amorphous state [33].

Nowadays, a large number of different carriers are used in solid dispersion formulations [34, 35]. Depending on the nature of the carriers (hydrophilic or hydrophobic), fast-release or controlled-release solid dispersions can be obtained [34]. Among the different types of carriers used in this method, gelucire® (PEG ester of fatty acids), citric acid and phospholipids have been used [36,37,38]. Several studies showed that the use of these carriers can lead to a significant increase in the solubility of poorly soluble drugs [39,40,41]. For this reason, it is expected that gelucire (due to its surfactant nature), phospholipids (due to their amphiphilic nature) and citric acid (due to the creation of hydrogen bonds between the drug and the acid) have an important role in increasing the dissolution of berberine [36,37,38]. Some studies showed that the prepared solid dispersion matrices could produce a sticky and viscous matrix which is practically impossible to make a suitable dosage form [42]. On the other hand, one of the effective dosage forms for drug delivery purposes is the preparation of pellets [43,44,45]. The advantages of pellets as multi-unit dosage forms include a reduction in wettability, improvement of the appearance of the product, narrow particle size distribution, the capacity to load large amounts of drug without creating large particles, reducing the dose dumping, taste masking, quick transit through the stomach and uniform distribution in the GIT, high surface area for absorption and increasing bioavailability, especially for drugs that accumulate in the acidic medium of the stomach, such as berberine chloride [46,47,48]. Therefore, this study aims to compare and select the best carrier for berberine solid dispersion (SD) based on the maximum increase in berberine solubility. In the next step, the feasibility of preparing pellets with different proportions of the optimal SD complex by extrusion-spheronisation method was investigated.

Materials and Methods

Materials

Berberine hydrochloride was purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China); microcrystalline cellulose (Avicel PH 101), polyvinylpyrrolidone (PVP K30) and lactose were purchased from Darupakhsh (Tehran, Iran). Phospholipon® 90 G was purchased from Lipoid GmbH (Ludwigshafen, Germany). Gelucire (Gattefossé,Saint-Priest, Cedex, France), citric acid (Sigma-Aldrich, Steinheim, Germany), ) potassium dihydrogen phosphate, sodium hydroxide, ethanol, hydrochloric acid and Aerosil Merck (Germany were used. f All chemical reagents used were of pharmaceutical grade.

Preparation of Berberine SD Complex with Phospholipid, Gelucire and Citric Acid

Different SD complexes with the same weight ratio of berberine-phospholipid, berberine-gelucire and berberine-citric acid were made by a solvent evaporation method. For this purpose, each of these mixtures was separately dissolved in ethanol and sonicated for 30 min to obtain a clear solution. The resulting solution was heated by a rotary evaporator device at a temperature of 40 °C to completely remove the solvent. Finally, the contents in the rotary flask were transferred to a beaker and placed under the hood for 24 h to dry and prepare for the next tests.

Saturated Solubility Test

The saturation solubility test was used to determine the maximum solubility of pure berberine (as a control) in comparison with SD berberine complexes. After weighing the amount of 30 mg of berberine powder and the amount of complexes containing 30 mg of berberine, the contents were transferred to microtubes containing 3 ml of distilled water. The microtubes were placed in the rotator at 50 rpm for 24 h and then centrifuged for 10 min at 2600 rpm. The supernatant solution was removed and after filtration with a syringe filter, the absorbance of the samples was read at 349 nm (n = 3).

Evaluation of Properties of Optimal Complexes

FTIR Study

To investigate the possible chemical interactions between the components of the SD complex, each of the basic components of the SD, i.e. berberine, phospholipid, gelucire or citric acid and their complex using an infrared spectrum device was accomplished in KBr discs over a range of 4000–500 cm− 1 using IR spectroscope equipment (Vortex 70, Bruker, Germany).

Differential Scanning Calorimetry (DSC)

The thermal behaviour of the berberine, each of the SD components and also optimized SD complexes were examined using differential scanning calorimetry (DSC; Metler Teldo DSC822, Switzerland). The DSC was run from 0 to 300 ◦C at the rate of 10 ◦C/min under the purge of nitrogen gas with a flow of 50 mL/min.

X-ray Diffraction Studies (XRD)

Berberine, phospholipid, gelucire, citric acid and the optimum complexes were evaluated using an X-ray diffractometer (D8 advance BRUKER Germany) equipped with nickel-filtered Cu Kα radiation (λ = 1.5418 Ao) at 40 kV voltage and 30 mA current at an accuracy angle of 5 ͦ − 45 ͦ (2 θ).

Preparation of Berberine Pellets

The pellet formulation was prepared using the extrusion/spheronization method. All dry components (Table 1), including the drug and excipients were combined by dry-mixing in a kitchen mixer (FUMA, Fu-1877 Hand Mixer, Japan) for 15 min followed by the addition of distilled water to the mixture to form a wet mass (37 mL of distilled water was used for a100-gram batch). The wet mass was then passed through an axial screw extruder (type HC 732, Dorsa, Iran) equipped with a die with 1 mm pore diameter, operating at 100 rpm (The process was carried out at room temperature). To achieve rounded pellets, the extrudates were processed in a spheronizer with a cross-hatched friction plate, running at 1000 rpm for 1 min. Finally, the obtained pellets were dried at 45 °C in an oven for 12 h.

Table 1 The composition of different pellet formulation
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Particle Size Analysis

To evaluate the pelletization yield and choose the appropriate size range for subsequent tests, the pellets were sieved by mesh 14 and 25 (1400 and 710 μm). The pellets with particle size less than 710 μm were considered as fine particles and pellets with particle size greater than 1400 μm were considered as large particles. Also, pellets between 710 and 1400 μm were selected as pellets of appropriate particle size and their percentages were calculated as pelletization yield.

Mechanical Strength of Pellets

The hardness and strength of the pellets were evaluated using a material testing apparatus (Hounsfield, England) with a 1 KN load cell and Q-MAT software. The speed of the compression probe was set to 1 mm/min. A force-displacement curve was drawn for each pellet. Using this curve, the parameters of crushing strength (the hardness of the pellet) and elastic modulus (the elasticity of the pellets) were determined.

Morphology of Pellets

Stereomicroscopic image analysis was used to investigate the morphology of the pellets. For this purpose, about 25 randomly selected pellets were fixed on a dark background and the images were taken using a stereomicroscope equipped with a digital camera (Olympus, DP25, Okura, Japan) at magnification (8X). The images were evaluated using image analyzing software (ImageJ 1.50f for Windows). The sphericity and aspect ratio of pellets were calculated as follows:

$$Sphericity{\rm{ }} = {\rm{ }}4\pi A/{P^2}_m\;$$
$$Aspect\begin{array}{*{5}{c}}{}\end{array}ratio{\rm{ }} = {\rm{ }}{d_{max}}/{d_{min}}$$

Where A is the projected area, Pm is the perimeter, dmax and dmin are the longest and shortest Feret’s diameters of pellets.

Dissolution Studies

The berberine release profile from complexes and/or pellets was examined using dissolution apparatus I (Pharmatest, PTWS, Germany). For this purpose, a certain amount of complexes or pellets was added to each basket and subsequently immersed in 900 mL of the dissolution media at 37 ℃ and rotated at 100 rpm. Sampling was done at different time intervals. To create the sink conditions, after each sample withdrawal, the dissolution medium was replaced with the same volume of fresh medium (the maximum concentration of drug in the dissolution medium was 15% of the maximum solubility of the drug in the same dissolution medium). The absorbance of the samples was read after filtration by a syringe filter (0.45 μm pore-size), at the wavelength of 349 nm (n = 3). The concentration and percentage of the released berberine were calculated using the regression equation obtained for the calibration standard.

Statistical Analysis

The statistical analysis was performed using GraphPad Prism software (GraphPad Prism, version 8, San Diego, CA). A one-way analysis of variance (ANOVA) was conducted with a significance level of 5%. Additionally, the Tukey-Kramer multiple comparison test was employed to identify significant differences between groups.

Results and Discussion

Drug Saturation Solubility Test

The results of the saturation solubility test of berberine powder in free form and the SD complex with phospholipid, gelucire, and citric acid in distilled water are reported in Table 2.

As can be seen in Table 2, the solubility of untreated berberine (plain berberine) in water is about 1.7 mg/mL. But when berberine is converted into a gelucire complex form in a ratio of 1 to 2, its solubility increases to 5.53 which is more than 3-fold. The comparison of the gelucire berberine complex and citric acid berberine complex with berberine alone shows an increase in saturation solubility of approximately 2 and 3 times, respectively. Table 2, also shows that, generally, complex forms of berberine with other excipients such as phospholipid, gelucire, or citric acid have higher solubility compared to the plain drug. The table also shows that the highest solubility belongs to the complexes when the ratio of drug to excipient was 1:2 [49,50,51]. This indicates that the ratio of drug to excipients plays a crucial role in enhancing the solubility of the drug. The results revealed that citric acid exhibits a greater capacity to enhance berberine’s solubility than gelucire and phospholipid. This superiority likely stems from the hydrogen bond established between berberine and citric acid [52].

Table 2 The saturated solubility of berberine SD complexes in distilled water at 25 ℃
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Drug Release Test from The SD Complex

Since the ratio of 1:2 (drug to excipient) showed the highest solubility, this ratio was chosen for further analysis. The dissolution test was carried out for the selected ratio of berberine-phospholipid complex, berberine-gelucire complex and berberine-citric acid complex in simulated media of gastric and intestine. The results of the dissolution test are shown in Fig. 1. The figure shows that the berberine-citric acid complex depicted the fastest dissolution rate at both pHs compared to other SD complexes of berberine which could be due to a complete dissolution of the complex in acidic and buffered dissolution media within one hour. While this is not the case for berberine-gelucire complex as they showed different dissolution behavior at both acidic and buffered conditions. The results showed a higher dissolution rate in a buffered medium than in an acidic medium for berberine-gelucire. This phenomenon is probably related to the esterification of gelucire’s fatty esters and the creation of a surfactant state in it, which has increased the solubility in the buffer medium [54]. The findings revealed that citric acid-berberine has the fastest dissolution rate. This superiority likely stems from the hydrogen bond established between berberine and citric acid [37]. It should be noted that this dissolution was performed only on the SD form of drug-excipient. Further dissolution data of these complexes in the pellet formulations will be presented and discussed later in the article.

Fig. 1
figure 1

The release profile of berberine from the optimum SD complexes at pH 1.2 and 6.8. Data are shown as mean ± standard deviation (n = 3)

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Particle Size Distribution, Morphological Observation and Mechanical Testing of Pellets

The results of the sieve test showed that most of the pellets were in the range of 850 to 1200 μm. The results of the image analysis of the pellets showed that all three series of formulations are ideal in terms of aspect ratio and sphericity. In the case of a completely spherical shape, both aspect ratio and sphericity values should be closer to 1. On the other hand, the increase in the aspect ratio and the decrease in sphericity indicate that the particles deviated from the spherical shape. In general, no significant difference was observed in all three series of pellet formulations. The results of mechanical testing of different formulations showed that with increasing amounts of the complex in the matrix of pellets, crushing strength and elastic modulus decrease (Table 3). Since the complexes have viscous and plastic properties, the crushing strength and elastic modulus decreased by increasing their ratio in the pellet.

Table 3 Image analysis and mechanical strength of pellets
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Infrared Spectroscopy (FTIR)

The results of IR spectra are shown in Fig. 2. As can be seen in Fig. 2A, the OH broadband peak in the region of 3500 –3000 cm− 1 is related to the presence of water molecules in the berberine structure. The C = C bond peak of the aromatic ring, methoxy group peak (O-CH3), C = N+ double bond peak and furyl group peak can be seen in the region 1570, 2845, 1630, 1506 cm− 1 respectively. In the molecular structure of phosphatidylcholine, a quaternary amine group, a phosphate group, a saturated and double carbon-carbon bond and a carbon-hydrogen bond are evident (Fig. 2B). These groups can be seen together with berberine-related groups in the berberine-phospholipid complex (Fig. 2C). Gelucire has mono, di and triglyceride esters of fatty acids, so it has alcohol, ester and aliphatic groups (Fig. 2D). Since the position of the peak related to amine has not changed, there seems to be no interaction between these two functional groups. However, the peaks related to ether and aromatic groups were shifted, which indicates that the interaction between berberine and gelucire was probably with hydrogen bonding (Fig. 2E). In the related spectrum of citric acid, the peaks in the 3500 − 3000 region are related to the OH functional group. The peaks of C = O and C-O bonds are also seen in the region of 1748 and 1138, respectively (Fig. 2F). Citric acid contains 3 carboxyl groups and one hydroxyl group. The shift of the C = C bond peak of the aromatic ring of berberine and the C = O bond peak of citric acid in the spectrum of the complex probably indicates the establishment of a hydrogen bond between the two molecules and the formation of a co-crystal (Fig. 2G).

Fig. 2
figure 2

IR spectrum related to A berberine, B phosphatidylcholine, C berberine-phospholipid complex, D citric acid, E berberin-citric acid complex, F gelucire, G berberin-gelucire complex

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X-ray Diffraction Analysis (XRD)

To identify the solid state of berberine, phosphatidylcholine, gelucire, citric acid and their SD complex, XRD was used. Based on the spectra of Fig. 3, the spectrum related to berberine has sharp and narrow peaks that indicate its crystalline nature (Fig. 3A), while the spectrum of phospholipid has no specific peaks (Fig. 3B), and in berberine-phospholipid complex, the characteristic peaks of berberine are faded or decreased to a great extent (Fig. 3C). Gelucire’s spectrum also shows sharp peaks that indicate its crystalline nature (Fig. 3D). In the spectrum of the berberine-gelucire complex, in addition to gelucire peaks, berberine peaks are also visible (Fig. 3E). The berberine- gelucire spectrum, showed a reduction of the peak related to the angle of 26.0 and an increase of the peak in the angle of 23.4. Citric acid does not have any specific peaks around the 2 theta angle of 10, but it has several sharp peaks of different intensities in the 2 theta angles of 15 to 30 (Fig. 3F). In the spectrum of the berberine-citric acid complex, in addition to the two peaks around 10 degrees which were strongly reduced, several new index peaks can be observed at 2 theta angles less than 15 degrees (Fig. 3G). By looking at the two spectra of berberine and citric acid, it was found out that to evaluate the change of the crystalline nature of berberine in the complex sample, the peaks around the 2 theta angle of 10 degrees are specific. The occurrence of several new index peaks around 10 degrees in the spectrum of the complex can indicate the change in the structure of the berberine crystal network and the formation of the co-crystal phase of berberine-citric acid.

Fig. 3
figure 3

XRD spectrum of A berberine, B phosphatidylcholine, C berberine-phospholipid complex, D gelucire, E berberin-gelucire complex, F citric acid and G berberin-citric acid complex

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Differential Scanning Calorimetry (DSC)

The thermogram obtained from the DSC test for berberine showed three endothermic peaks. The dehydration of the berberine molecule is responsible for the endothermic peak at around 73 °C. The endothermic peaks of the 102 to 121 °C are related to the decomposition and separation of the CO group and the wide endothermic peak of the 195 °C region is also related to the melting point of berberine [54]. The presence of a semi-stable polymorph in the berberine structure can be inferred from the exothermic peak at 201 °C followed by an endothermic peak at 290 °C. Failure to observe the berberine phospholipid complex peak is probably related to the transformation of berberine into an amorphous state. A sharp endothermic peak is observed at 45 °C in the spectrum of gelucire which corresponds to its melting point. Also, In the peak related to berberine-gelucire complex, two separate peaks are observed in the regions of 45 and 195 °C. In the spectrum of citric acid, the sharp endothermic peak of region 154 °C is related to the melting point of the substance, and other peaks in this spectrum are related to the destruction of the structure. In the spectrum related to the berberine-citric acid complex, these peaks are shifted and merged. In other words, the endothermic peak of the melting point of the complex is observed between the melting point region of the two substances (region 181 °C) (Fig. 4).

Fig. 4
figure 4

DSC diagrams of A berberine, B phosphatidylcholine, C citric acid, D berberine-phospholipid complex, E gelucire, F berberin-gelucire complex, G berberin-citric acid complex

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Scanning Electron Microscopy (SEM)

As berberine-citric acid complexes provided the highest saturated solubility, therefore, this sample was selected for further morphology studies by SEM. The physical mixture and the complex of berberine-citric acid were used to evaluate changes in berberine in the complex form (Fig. 5). The SEM images show that the berberine crystal lattice is often triclinic (Fig. 5A) while citric acid is seen in irregular crystal form (Fig. 5B). The triclinic structure of berberine and citric acid crystals are clearly visible in the physical mixture and no significant change was observed (Fig. 5C). This is not the case for the SEM image of the complex (berberine-citric acid complex) as the SEM image triclinic structure of berberine is less visible and most of the triclinic structures with asymmetric frills and quasi-hexagonal structures are evident (Fig. 5D).

Fig. 5
figure 5

Scanning electron microscopy of the A Berberine, B Citric acid, C Berberine-citric acid physical mixture, and D Berberine-citric acid complex

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Dissolution Studies

Figure 6 depicts the berberine release profile from the optimum SD complex pellets in acidic and phosphate buffer media with pH 1.2 and 6.8. The evaluation of the release of berberine from the pellets in acidic and buffered dissolution media shows the difference in the release rate of berberine in these two dissolution media due to the increased solubility of berberine in the buffered medium. In other words, the solubility of berberine at pH 1.2 was lower than that in phosphate buffer pH 6.8. The decrease in solubility in 0.1 M HCl solution may be due to the conversion of berberine to berberine chloride, which is less soluble than berberine. As can be observed, the drug release rate in the simulated gastric medium is slower than in the intestinal medium. Such that the pellets composed of berberine-phospholipid complex released about 30% of their drug content within 2 h, while during the same period in the intestine medium (pH 6.8), almost 60% of their drug content was released. The difference in the release rate in these two media is due to the increase in berberine solubility in the buffer medium. However, despite the low release rate of berberine in the gastric medium, it can be seen that pellets composed of berberine-citric acid complex released more than half of their drug content in this medium. By investigating the drug release profile from the berberine pellet compared to the pellets composed of each of the complexes, it can be seen that all formulations have a higher release percentage than the pure berberine pellet. This shows that the complexation of berberine with phospholipid, gelucire or citric acid has a significant effect on increasing its solubility in the intestine medium. The comparison of the release profile of berberine from the prepared complexes shows that the highest release rate in the medium simulated to the intestine is related to the berberine-citric acid complex. This phenomenon indicates the establishment of more hydrogen bonds between berberine and citric acid and of course the creation of a new crystal network during the complex formation process. Since the maximum absorption of berberine occurs in the jejunum, followed by the duodenum and ileum, as a result, it is better to have a higher solubility of berberine in the simulated pH of the small intestine (pH 6.8) [55].

Fig. 6
figure 6

The release profile of berberine from the optimum complexes pellets at pH 1.2 and 6.8. Data are shown as mean ± standard deviation (n = 3)

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Conclusion

The increase in the incidence of metabolic diseases has a significant impact on human health around the world [56]. Thus, finding effective drugs, especially from natural compounds is very important [57]. According to the results of clinical studies in recent years, berberine has favourable and promising therapeutic effects on the improvement of metabolic syndrome indicators [58, 59]. On the other hand, the poor aqueous solubility and low bioavailability of berberine are also an important challenge for the development of the optimal formulation of this drug. The results of this study showed that creating a 1:2 solid dispersion complex between berberine and phospholipid, berberine and gelucire, and berberine and citric acid significantly increases the saturated aqueous solubility of the berberine. such that, the berberine-phospholipid complex increased the saturated aqueous solubility of berberine to about 2-fold and the berberine-gelucire and berberine-citric acid complexes by more than 3-fold. This three-fold increase is probably related to the surfactant properties of gelucire and the hydrogen binding ability of citric acid. The results of IR, XRD and DSC tests indicate the change in the crystal structure of berberine and the creation of a co-crystal between berberine and the excipients used in making the complex due to hydrogen bonding. Pellets were chosen as a promising solid dosage to incorporate the resulting SD complexes. Desirable pellets were prepared with 30 or 40% SD content. The pellets had appropriate size, mechanical strength and sphericity. Among the examined complexes, the berberine-citric acid complex was able to release all its content within 60 min in the buffer environment. This can lead to a faster onset of drug effects in-vivo. In general, the evaluation of the results showed that the berberine-citric acid complex pellet formulation showed acceptable and excellent results in terms of the investigated parameters, including the characteristics of the release profile, and was chosen as the selected formulation. Therefore, the pellet formulation consisting of 40% berberine-citric acid complex or 40% berberine-gelucire complex can be suitable candidates for clinical studies to evaluate its effectiveness in improving the symptoms of metabolic syndrome.