Enhanced solubility and bioavailability of coenzyme Q10 via co-amorphous system using stevioside

Yingting Luo; Yuxin Li; Xuening Song; Yuzhuo Wang; Simiao Liu; Fazheng Ren; Hao Zhang

doi:10.1038/s41538-025-00465-0

. 2025 Jun 7;9:98. doi: 10.1038/s41538-025-00465-0

Enhanced solubility and bioavailability of coenzyme Q10 via co-amorphous system using stevioside

Yingting Luo ¹, Yuxin Li ¹, Xuening Song ¹, Yuzhuo Wang ¹, Simiao Liu ¹, Fazheng Ren ^1,^2,³, Hao Zhang ^1,^2,^3,^✉

PMCID: PMC12145421 PMID: 40483335

Abstract

Coenzyme Q10 (CoQ10) plays a vital role in aerobic respiration and cardiovascular diseases; however, its application is limited owing to poor water solubility. In this study, equimolar CoQ10 and stevioside (STE) formed a co-amorphous (CM) system by lyophilization, and its solubility was approximately 63 times higher than that of CoQ10. Through crystal, thermodynamic, and morphological characterization of the formula, the formation of the CM system was confirmed. The intermolecular interactions were investigated by spectroscopies. The relationship of 8 intermolecular interaction sites between the two was confirmed via molecular dynamics simulation, firmly indicating the strong intermolecular forces. Further, CM products remained stable even under accelerated storage conditions, equivalent to 1 year at room temperature. Meanwhile, the area under curve (AUC) values increased by 5 times in the in vivo bioavailability study. In conclusion, the CoQ10 was transformed into an amorphous structure by initially employing STE through intermolecular interactions to enhance solubility.

Subject terms: Health care, Chemistry

Introduction

Coenzyme Q10 (CoQ10), a yellow-orange crystalline substance, comprises a central benzoquinone component connected to a 10-unit poly-isoprene tail¹. The long side chain of isoprene provides strong hydrophobicity, imparting fat solubility to CoQ10 but extremely poor water solubility. CoQ10 performs crucial physiological functions, producing energy within the mitochondria², and acts as a good antioxidant, preventing low-density lipoprotein peroxidation in blood circulation, thus exhibiting a direct anti-atherosclerotic effect³. Further, CoQ10 supplementation is beneficial to other cardiovascular diseases, including hypertension, heart failure, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases^4,5.

CoQ10 is produced by the human body; however, the ability to synthesize CoQ10 gradually declines with age. For instance, the CoQ10 in the heart decreases by 32% and 60% at 40 and 80 years, respectively⁶. Further, genetics, diseases, or drugs (such as statins) could increase the demand for CoQ10; therefore, appropriate exogenous supplementation of CoQ10 is required. Currently, CoQ10 is primarily incorporated or dispersed in oil as a crystalline powder; however, it exhibits poor rheology, low melting point, and is easily affected by light, heat, and oxidation⁷. Thus, an improvement of its dissolution properties, oral bioavailability, and stability is required for utilization as food supplement formulations. Several studies have been carried out in this regard, involving nanotechnology^8,9, embedding¹⁰, and solid dispersion technologies. However, nanotechnology and embedding technology target uninvolved crystal transformations and the loading capacity of CoQ10 was not high. Further, the research on CoQ10 solid dispersions involving crystal state transformation and solubility improvement was performed primarily using high molecular weight coformers, such as polyethylene glycol, polyvinylpyrrolidone, and hydroxypropyl methyl cellulose^11–13, which involves several challenges such as high moisture absorption, poor miscibility, and larger final dosage form. In contrast, a co-amorphous (CM) system is a single-phase system with small molecular coformers focused on converting insoluble substances from crystalline to amorphous states¹⁴. The system lacked a crystalline long-range ordered lattice; therefore, there was no need to overcome the lattice energy during the material dissolution process. Utilizing small molecules as coformers could lead to a smaller dose volume of the same quantity of substance, and the strong interaction forces in the system make it more stable¹⁵. Additionally, the preparation process is facile, leading to excellent applicability in improving the solubility, stability, and bioavailability of substances, thus being widely used in the solubilization of drugs^16,17, plants, and food bioactive components^18–21. Currently, commonly used excipients include amino acids, organic acids, sugars, etc., while fewer studies have been conducted using glycosides²². And the appropriate ligands should be identified according to the chemical structure of the compounds to maintain the physical stability of co-amorphous system. Stevioside (STE) is a safe small-molecule natural food additive, exhibiting remarkable surface activity and good two-phase solubility of water and alcohol. STE shows good performance in improving the solubility of hydrophobic active substances like lutein and resveratrol^23–25. Furthermore, STE consists of hydrophobic steviol and hydrophilic glucose and rhamnose on either side, which is more likely to form intermolecular interactions with CoQ10 and has application potential in solubilization technology.

There are no studies on CoQ10 and STE CM for food applications; therefore, this study focused on improving CoQ10 water solubility and identifying the optimal formula via screening three preparation methods with food-grade ingredients such as STE. Additionally, X-ray diffractometer, differential scanning calorimetry, and scanning electron microscopy were used to explore the effect of CM technology on the crystal state, thermodynamics, and morphology of substances. The interactions between substances were investigated via Fourier transform infrared, Raman, and hydrogen nuclear magnetic resonance spectroscopies, along with molecular dynamics simulations. Further, the influence of the CM system on the dissolution, storage, and bioavailability of substances was evaluated.

Results

Solubility studies of CoQ10-STE mixture by different methods

The detected solubility of CoQ10 was 2.3 μg/mL. The solubility of CoQ10 in water could be ignored regardless of the incubation temperature (25 °C or 37 °C), or in the pure or oil forms, which belong to insoluble substances^13,26. RE is a commonly used method in the laboratory, whereas SD and FD are feasible for practical production. STE significantly increased the solubility of CoQ10 compared to that of CoQ10 (p < 0.05), which could be attributed to several hydrogen bond donors and receptors, showing great potential for interaction with CoQ10²⁷. The CoQ10 solubility improved as follows: FD > SD > RE (Fig. 1). Compared to that of CoQ10, the solubility of FD CoQ10 powder was 144.6 μg/mL, which showed a significant increase by 63 times (p < 0.05).

Fig. 1 — Effect of an equimolar CoQ10 and STE mixture on solubility improvement by rotary evaporation (RE), freeze-drying (FD), and spray drying (SD). Data are shown as the mean ± standard deviation of triplicate independent experiments.

X-ray diffraction (XRD)

XRD is an effective tool for detecting crystallinity changes in samples²⁸. The XRD patterns of CoQ10, STE, PM, and the FD products of equimolar CoQ10 and STE are shown in Fig. 2A. The 2θ diffraction peaks of CoQ10 were observed at 11.3°, 18.6°, 20.3°, 22.9°, 24.7°, 27.3°, 28.6°, 29.7°, 30.4°, 32.3°, and 38.2°, with two prominent peaks at 18.6° and 22.9°, consistent with the previously reported studies¹². The XRD pattern of STE exhibited a halo-like diffraction peak at 16.5°, corresponding to STE in an amorphous state, consistent with the previous report²⁴. Two primary peaks of CoQ10 were visible in PM, indicating no interactions between the two substances. The XRD pattern of FD CoQ10 showed a peakless ring halo line, which was different from that of CoQ10 and PM, indicating a co-amorphous state for equimolar CoQ10 and STE after lyophilization.

Differential scanning calorimetry (DSC)

DSC is used to observe the thermodynamic changes between CoQ10 and STE. The DSC maps are shown in Fig. 2B. The melting point peak of CoQ10 was at 52 °C approximately, and that of STE was at approximately 203 °C, consistent with the previously reported studies^29,30. Only one melting point peak (50.8 °C) that shifted marginally to a lower temperature was observed in PM, which was found in PM of apigenin and oxymatrine³¹, owing to an increase in temperature resulting in the melting of individual components and formation of partial aggregates in situ³². However, the melting point peak of FD CoQ10 shifted to approximately 49 °C and was remarkably weakened compared to that of PM, owing to the crystal transition of CoQ10¹³.

Fourier transform infrared spectroscopy (FTIR)

FTIR indicates the interactions between substances. Figure 2C shows the characteristic peaks of CoQ10 at 2916–2963 cm⁻¹ (saturated C-H stretching vibration), 2853 cm⁻¹ (saturated C-O stretching vibration), 1649 cm⁻¹ (C=O bond), 1611 cm⁻¹ (benzene ring), 1448 cm⁻¹ (out-of-plane C-H bending vibration), 1154–1287 cm⁻¹ (out-of-plane C-O bending vibration on benzene ring), 1026–1110 cm⁻¹ (C-O-C stretching vibration), and 796–877 cm⁻¹ (C=H stretching vibration)^12,13. The characteristic peaks of STE were observed at 3400 cm⁻¹ (O-H stretching vibration), 2910 cm⁻¹ (C-H stretching vibration), 1723 cm⁻¹ (C=O stretching vibration), 1021–1082 cm⁻¹ (C-O-C stretching vibration), and 890 cm⁻¹ (C= out-of-plane bending vibration)³³. The absorption peaks showed a remarkable overlap for PM, indicating no intermolecular interaction between the two⁹. Compared to that of PM, the CoQ10-STE CM product exhibited no considerable peak shift; however, the O-H vibration peak at 3400 cm⁻¹ widened and weakened substantially. The peaks at 2910–2963 cm⁻¹, 1649 cm⁻¹, 1611 cm⁻¹, and 1021–1082 cm⁻¹ assigned to the C-H vibration, C=O, benzene ring, and C-O-C vibration, respectively, weakened remarkably.

Raman spectroscopy

Raman spectroscopy complements the FTIR to analyze the structural properties and interactions of samples. Figure 2D shows the Raman peaks of CoQ10 at 490 cm⁻¹ (ring deformation), 888 cm⁻¹ (C-O-C stretching), 1010–1163 cm⁻¹ (C-C vibration), 1292–1448 cm⁻¹ (CH₂ deformation), 1622 cm⁻¹ (C=C stretching), 1675 cm⁻¹ (C=O flex), and 2857–2924 cm⁻¹ (CH₂ and CH₃ flex)³⁴. The Raman peaks of STE were observed at 542 cm⁻¹ (C-C-O bending), 1101–1130 cm⁻¹ (C-O stretching of C-O-C and C-OH), 1457 cm⁻¹ (CH₂ and CH₃), and 1674 cm⁻¹ (C=C stretching), which were consistent with the reported studies³⁵. The absorption peaks of PM overlapped remarkably, consistent with the FTIR results, indicating no interaction between the two. In contrast, only three distinct peaks ascribed to C-H deformation, C=O stretching, and C-H stretching were present in the CM product, and their intensities were weaker than those of PM. Moreover, the C-H deformation peak shifted to 1457 cm⁻¹, and the intensities of the remaining Raman peaks weakened or disappeared substantially.

Scanning electron microscopy (SEM)

SEM is used to observe the surface morphology of the sample. The upper rows in Fig. 3A–D shows that the overall morphology of the four substances was relatively consistent. The lower rows in Fig. 3E–H exhibit flat and large clumps for CoQ10 crystal, and the formation of rod-shaped, ellipsoidal, and other particles with different morphologies for STE. PM encompassed the morphologies of two substances. However, the CM product of FD CoQ10 and STE showed an irregular, flocculent state, and the particle size was considerably reduced.

Nuclear magnetic resonance hydrogen spectroscopy (¹H NMR)

¹H NMR analyzes the chemical shift of hydrogen atoms and deduces the form and location of interactions between substances. Fig. S1 shows the ¹H NMR of CoQ10 exhibiting peaks at 5.05–5.15 ppm, 4.91–4.95 ppm, 3.95–4.00 ppm, 3.10–3.23 ppm, 1.95–2.10 ppm, and 1.55–1.77 ppm. The chemical shift deviation (Δδ) of CoQ10 and CM product was analyzed in combination with ChemDraw analysis. Compared to those of CoQ10, six hydrogen peaks of the methyl group on −O-C=C (Δδ = 0.04), and the two hydrogen peaks on the long chain of methylene near the carbonyl group (Δδ = −0.02) were marginally shifted. Three hydrogen peaks on the methyl group near the carbonyl group shifted marginally (Δδ = 0.02), whereas the remaining peaks did not show any shift. The difference in chemical shifts changed marginally at the second decimal point, indicating no chemical reaction between the two. Therefore, the CoQ10-STE CM product might interact under the lyophilized state via intermolecular forces.

Molecular dynamics (MD) simulation

MD simulation supplements the experimental data to demonstrate the interactions between molecules in a co-amorphous system²⁸. The calculated data of PubChem reveals that CoQ10 has 4 hydrogen bond receptor sites, and STE has 11 hydrogen bond donor sites. The possible interaction between these sites was further explored via radial distribution function (RDF) analysis, wherein the horizontal coordinate represents the distance between material sites, and the vertical coordinate is the relative probability of molecular pairs or atomic pairs at this distance. Intermolecular interaction forces such as hydrogen bonds or van der Waals forces are present within a distance of 3.5 Å³¹. Total 8 pairs of intermolecular interactions were observed in CM products (Fig. 4), including C=O₁ site of CoQ10 between O₈-H, O₉-H, and O₁₀-H of STE, O₂ site of CoQ10 between O₆-H of STE, and C=O₄ site of CoQ10 between O₁-H, O₃-H, O₇-H, and O₁₀-H of STE. Among these, two high probability forces were present between C=O₄ of CoQ10 and O₃-H of STE at 1.74 Å and 2.75 Å, respectively, indicating a large contact possibility with the smallest distance between these two sites.

Fig. 4 — MD simulation of CoQ10 and STE CM product RDF diagram.

Storage stability

The stability of a co-amorphous system is a critical index, and the maintenance or transformation of crystallinity is used to evaluate its stability over time. The possible application scenarios were simulated through storage under three conditions: refrigerated (4 °C), unopened at room temperature (dry at room temperature), and long-term storage opened at room temperature environments. Accelerated environmental storage at 40 °C, 75% RH for 3 months, could be used to simulate room temperature storage at 25 °C, 60% RH for 12 months³⁶. Figure 5A and B reveal that the CM product showed no remarkable crystal transformation in the storage environment at 4 °C and room temperature within 6 months, exhibiting good stability. Further, the appearance and morphology of the powder did not remarkably change during this period. Figure 5C shows that the CM product exhibited a little diffraction peak near 18.6° after accelerated storage for 3 months; however, a remarkable entire crystal transition was not observed, indicating that the CoQ10-STE CM product maintained excellent stability at 25 °C and 60% RH for 12 months.

Fig. 5 — A 4 °C, B dry room temperature, and (C) accelerated storage environment at 40 °C, 75% RH.

In vivo bioavailability study

To study the bioavailability of CoQ10 CM products, rats were given crystalline CoQ10 and CM product, and the changes in plasma CoQ10 concentration in rats within 24 h were recorded and compared, as shown in Fig. 6. Related biological parameters are listed in Table 1. The level of CoQ10 in plasma was extremely low when oral crystalline CoQ10 was used, reaching C_max at approximately 1 h, and the values of C_max and AUC were calculated as 50.283 ± 5.695 ng/mL and 196.456 ± 96.874 ng h/mL, respectively. In contrast, the plasma CoQ10 of oral CM product increased significantly (p < 0.05), and the C_max and AUC values were determined to be 308.389 ± 50.250 ng/mL and 934.563 ± 247.801 ng h/mL, which increased by 6 and 5 times, respectively.

Fig. 6 — Plasma concentration-time profiles of pure CoQ10 and CoQ10-STE CM products after oral administration to rats at 15 mg/kg dose (n = 5).

Table 1.

Pharmacokinetic parameters of CoQ10 after oral administration of pure CoQ10 and CoQ10-STE CM products in rats

Parameters	CoQ10	CoQ10-STE CM
AUC_0-24 (ng h/mL)	196.456 ± 96.874	934.563 ± 247.801*
C_max (ng/mL)	50.283 ± 5.695	308.389 ± 50.250*
T_max (h)	1	2

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Data are expressed as mean ± standard deviation (n = 5, 15 mg/kg).

*p < 0.05, compared with crystalline coenzyme Q10 itself.

Discussion

CoQ10 offers various health benefits due to its essential role in mitochondrial energy production³⁷. However, its poor solubility and high molecular weight limit its application and human absorption and utilization. In this study, the water solubility of insoluble CoQ10 was improved by adding equimolar STE via co-amorphous technology. And three widely used preparation methods were tested to obtain the optimum formulation. RE involves slow evaporation under reduced pressure with constant heating and mixing of substances in solution, whereas SD is rapid atomization of the solution into droplets through the nozzle and their continuous interaction with the hot air flow. The large surface area and high-temperature drive of the droplets are conducive to the interaction between substances during SD³⁸. In contrast, FD involves rapid sublimation of water, which provides higher porosity to the powder, resulting in higher solubility. Compared to that of SD product, the FD product had a lower moisture content, which might allow their even dispersion on the surface, and the larger surface area improves their dissolution property, resulting in a higher dissolution property of hydrocortisone after FD compared to that after SD³⁹. Similarly, our studies revealed that the freeze-dried powder exhibited the best solubility improvement, approximately 63 times higher than that of CoQ10.

In our study, the formation of co-amorphous systems was confirmed through various characterization methods. XRD and DSC analyses revealed that the freeze-dried products of CoQ10 and STE were completely transformed from crystalline to amorphous. There were no characteristic crystalline peaks in the XRD pattern, and the substance lacked a periodic molecular arrangement owing to the absence of a crystal lattice. Thus, no lattice energy needed to be overcome during the dissolution process, resulting in high solubility³¹. Further, the melting point of CoQ10 was considerably inhibited in the DSC thermogram, indicating good miscibility, resulting in a co-amorphous system²⁷. SEM analysis showed remarkable changes in the morphology of freeze-dried products compared to those of CoQ10. The reduction of particle size and the increase of surface area contributed to the improved dissolution properties of the substance in a co-amorphous system⁴⁰. FTIR, Raman, and ¹H NMR spectroscopies inferred an intermolecular force between CoQ10 and STE, which was attributed to the hydrogen bond donor and acceptor sites of both molecules. And our results agreed with those obtained by the FTIR spectrum of the SD product of amorphous CoQ10²⁶. Consistent with the FTIR results, the absence of Raman bands is the Raman characteristic of co-amorphous solids⁴¹. Similar experimental phenomena could be observed in the binary products of piperine and curcumin; lovastatin and irbesartan⁴², which confirmed the results of our spectral speculation. Hence, interactions between substances might occur, thus promoting the CoQ10 water solubility, in agreement with previous studies on amorphous lycopene⁴³. Moreover, the MD simulations further confirmed 8 pairs of intermolecular forces between CoQ10 and STE, with high probability forces observed between specific hydrogen bond donor and acceptor sites. These action sites were consistent with the change groups obtained in the above spectrum results, The strong intermolecular interactions in CoQ10-STE CM products played an essential role in the formation and stability of the system⁴⁴.

The co-amorphous freeze-dried powder formed by equimolar CoQ10 and STE exhibited excellent storage stability. At simulated room temperature and 60% RH, the amorphous state remained unchanged for up to one year. The weaker the diffraction peak, the smaller the change in solubility after the storage period⁴⁵. The XRD pattern of the storage period in a co-amorphous system predicts the stability of the sample and reflects the change in the solubility of the substance. The better stability of the CM product could be attributed to the good miscibility of the two, which dispersed the CoQ10 in this co-amorphous system. Moreover, the CM product exhibited smaller particle sizes and strong intermolecular interaction, which might restrain the molecular mobility of CoQ10, thus limiting the recrystallization⁴⁶.

Additionally, the C_max and AUC values of bioavailability in vivo increased by 6 and 5 times, respectively. However, T_max was marginally delayed to 2 h, and this phenomenon could be observed in the results of CoQ10 emulsion, CoQ10-cyclodextrin inclusions, and CoQ10 solid dispersions^10,47, which could be attributed to the high dispersion of CoQ10 in STE, resulting in a slow-release effect. CoQ10 nanoparticles increased C_max and AUC by 3 and 4 times, respectively⁴⁸, whereas the remarkably increased bioavailability of co-amorphous products was ascribed to the amorphous state. Furthermore, oral bioavailability of lipophilic compounds was reported in the order solution > suspension > powder⁴⁹. STE as the ligand significantly increased the solubility of CoQ10, forming a homogeneous solution, contributing to its higher bioavailability. Additionally, the reduction of CoQ10 particle size and the increase of specific surface area contributed to improved bioavailability. While the observed increase in bioavailability is noteworthy, and co-amorphous systems could offer great potential for food applications, their industrial deployment requires overcoming stability and scalability challenges.

In summary, the co-amorphous system using STE shows significant strength for enhancing the solubility and bioavailability of CoQ10. And these findings facilitate the actual production, processing, application, and expansion of CoQ10 in food. Meanwhile, STE, as a safe, environmentally friendly, and easily biosynthesised substance, presents great potential for the sustainability of the formulation. In the future, additional studies could be needed to scale up the formulation for commercial production as well.

Methods

Materials

CoQ10 (purity ≥ 98%, food grade) was purchased from Shanxi Jin KangTai Biotech Co., Ltd. (Shanxi, China). STE (purity ≥ 98%) was purchased from Shandong Pingju Biotechnology Co., Ltd (Shandong, China). Absolute ethanol was procured from Tianjin Zhiyuan Chemical Reagent Co., Ltd (Tianjin, China). Chromatography-pure acetonitrile and methanol were obtained from Beijing Mairuida Technology Co., Ltd (Beijing, China). N-propanol was purchased from Aladdin Reagent (Shanghai, China). Deuterated chloroform was purchased from Cambridge Isotope Laboratories, Inc. (Massachusetts, America), and deuteroethanol from Shanghai Yien Chemical Technology Co., Ltd. (Shanghai, China). Spectrum-pure potassium bromide was purchased from Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, China). All other reagents used were of analytical grade and obtained from Tianjin Yongda Chemical Reagent Co., Ltd (Tianjin, China).

Preparation of CoQ10-STE mixture

A binary mixture of CoQ10 and STE was prepared at a molar ratio of 1:1 via rotary evaporation (RE), freeze-drying (FD), and spray drying (SD). Specifically, CoQ10 was dispersed in absolute ethanol and heated at 50 °C to ensure complete dissolution, whereas STE was dissolved in water. The two solutions with equal molar ratios were mixed to form a clear solution. The solution was subjected to rotary evaporation at 60 °C, followed by drying in a vacuum drying oven at 50 °C⁴⁵. The spray drying conditions were as follows: inlet temperature of 125 ± 5 °C, outlet temperature of 80 ± 5 °C, feed rate of 15%, and atomizing gas flow rate of 35 mm⁵⁰. The freeze-drying was performed at −80 ± 10 °C for 96–168 h. The obtained samples to be measured were placed at 4 °C. The physical mixture (PM) comprises equal molar powder of two substances obtained by grinding.

Solubility studies by high-performance liquid chromatography (HPLC)

Quantitation of CoQ10. CoQ10 was dissolved in ethanol-acetonitrile-water (55:30:15, v/v, pH 4.5 adjusted by phosphoric acid). The CoQ10 content was determined by HPLC⁵¹. The chromatography was performed on a Waters XBridgeTM C₈ column (4.6 × 250 mm, 5 μm) at 45 °C with 100% methanol as the mobile phase. The flow rate of the mobile phase was 1 mL/min with a 20 μL sample volume. The standard curve was drawn with 0.05–25 μg/mL CoQ10-peak area. A supersaturated solution was obtained by mixing the powder with the same volume of water. The solution was shaken in a 200 rpm water bath at 25 °C for 24 h¹³. The CoQ10 solubility was determined using HPLC. The optimal solubility product was selected for subsequent characterization.