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Sweet Berberine Chenguang Wang, Sathyanarayana Perumalla, Ruolin Lu, Jianguo Fang, and Changquan Calvin Sun Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b01484 • Publication Date (Web): 18 Dec 2015 Downloaded from http://pubs.acs.org on December 22, 2015

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Crystal Growth & Design

Sweet Berberine

Chenguang Wang, 1, 2 Sathyanarayana Perumalla, 2 Ruolin Lu, 2, 3 Jianguo Fang,1, * and Changquan Calvin Sun2,*

1

Department of Pharmacy, Tongji Hospital affiliated with Tongji Medical College, Huazhong University

of Science and Technology, Wuhan 430030, PR China 2

Pharmaceutical Materials Science and Engineering Laboratory, Department of Pharmaceutics, College

of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA 3

Department of Chemistry, College of Science and Engineering, University of Minnesota, Minneapolis,

MN 55455, USA

*Corresponding author Changquan Calvin Sun, Ph.D. 9-127B Weaver-Densford Hall 308 Harvard Street S.E. Minneapolis, MN 55455 Email: [email protected] Tel: 612-624-3722 Fax: 612-626-2125 1 ACS Paragon Plus Environment


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Abstract Berberine is a drug with intense bitter taste. The high aqueous solubility of its chloride salt, which is commonly used in commercial drug products of berberine, worsens the challenge of taste masking.

We have approached this drug delivery challenge by forming salts with

sweeteners, acesulfame and saccharine, through the anion exchange reaction. In addition to intrinsic sweetness of the two counter ions, both salts also exhibit reduced aqueous solubility, which further alleviates the problem of bitter taste of the drug by limiting dissolution of berberine.

Moreover, both salts exhibit good tableting performance.

They are also non-

hygroscopic and stable against high humidity and temperature. The stability against humidity variations makes the two sweet salts more amenable for tablet development over the chloride salt, which undergoes complex hydration/dehydration phase changes when relative humidity varies. Collectively, the two novel solid phases of berberine are sweet and exhibit superior properties for developing pharmaceutically elegant drug products.

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Introduction Berberine, an active ingredient extracted from Huanglian (Rhizoma Coptidis), a herb that possesses antimicrobial activity and has been used as an effective non-antibiotic anti-diarrheal drug for thousands of years in traditional Chinese medicine.1 Since 1983, it has also been used to treat non-insulin dependent diabetes mellitus (NIDDM).2 In addition, recent research on this molecule showed that the insulin-resistant diabetes and diabetes combine dyslipidemia patients can also benefit from this compound.3 Further more, it has been shown to lower the cholesterol level through a unique mechanism distinct from statins.4 Berberine is well-known for its intense bitter taste. 5,6,7 Perhaps because of the taste problem of this drug, most of its commercial products are either film/sugar coated tablets or capsules. The film coat or capsule shell prevents the drug from coming in direct contact with the tongue when the product is swallowed. The most commonly used solid form of berberine in drug products is berberine chloride (BbCl, Figure 1). It should be noted that berberine, a quaternary ammonium cation, contains a positive charge without acquiring a proton, unlike amine cations. Unfortunately, the chloride salt is often incorrectly called a hydrochloride salt.8 BbCl undergoes solid state transformations among an anhydrate and two hydrates depending on relative humidity.9

Figure 1. Molecular structure of BbCl. 3 ACS Paragon Plus Environment


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In this work, we seek to address the pharmaceutical issues of this drug, including bitter taste and physical instability, by the means of crystal engineering. A search of the literature, including Cambridge Structural Database (CSD), revealed a total of twelve reported unique crystal structures containing berberine.10 More commonly studied berberine solid forms include BbCl (tetra-hydrate, di-hydrate, and anhydrate), berberine tannate, berberine bisulfate, and berberine sulfate. 11,12 However, none of these solid forms are suitable for addressing the drug delivery issues identified above. An efficient approach in solving these drug delivery problems facing BbCl is the novel crystal forms that are capable of both masking the bitter taste and exhibiting solid state stability. Since the cationic nature of berberine requires the presence of charge equivalent anion in solid state to maintain charge neutrality, berberine salts with acidic sweeteners, such as acesulfame and saccharine, are expected to form. The simultaneous dissolution of potent sweetener is expected to alleviate the bitterness problem of the drug, provided they also exhibit solid-state stability. If so, such sweet salts can be used to develop new tablet products with improved clinical profiles. Materials and methods Material Berberine chloride (West plant extraction factory, Sichuan, China), sodium saccharine salt hydrate (Sigma–Aldrich, St. Louis, MO) and acesulfame potassium (Tokyo Chemical Industry co., Ltd, Japan) were used as received. Methods


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It is expected that reaction between BbCl with acesulfame potassium (AsK) and saccharine sodium (SacNa) can yield berberine acesulfame (BbAs) and berberine saccharine (BbSac), respectively, through anion exchange reaction (Scheme-1). These anion exchange reactions are expected to occur spontaneously provided the new salts have much lower solubility than BbCl. Pure sweet salts are expected as long as a sufficient amount of water remains during the reaction to dissolve the by-products, sodium chloride or potassium chloride (both have solubility of ~360 g per liter of water at 25 oC). Synthesis of BbAs A solution of BbCl was prepared by dissolving 9.09 g of BbCl in 75 mL of hot water. Then 4.23 g of AsK was added to the solution, which caused immediate precipitation. The suspension was stirred by the means of magnetic stirring bar and left for overnight. The solid recovered by vacuum filtration was suspended in ~ 15 mL of methanol for overnight. Approximately 12 g of phase pure BbAs salt was isolated by filtration and drying at 60 oC in an oven for about 12 hr. Synthesis of BbSac Approximately 12 g of phase pure BbSac was prepared by reacting 9.09 g of BbCl with 4.31 g of SacNa in 75 mL of water for overnight under stirring, followed by filtration and oven drying at 60 oC.



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Scheme 1. Schematic representation of the anion exchange reaction to prepare BbAs and BbSac from BbCl. The byproducts KCl and NaCl are not shown in these reactions.

Preparation of single crystals 50 mg of BbAs or BbSac was dissolved in 10 mL of slightly heated methanol. After cooling to room temperature, the vial was left in a fume hood undisturbed with cap removed to allow solvent evaporation. Crystals suitable for single crystal X-ray diffraction experiment were obtained within two days in both cases. Single Crystal X-Ray Diffraction Single crystal X-ray diffraction was carried out on a Bruker diffractometer (Bruker AXS Inc., Madison, Wisconsin) equipped with an Apex CCD area detector. The data collection was performed at 173 K using MoKα radiation (graphite monochromator).

Data analyses were

performed using a suite of software from Bruker, including APEX, SADABS, and SAINT. Crystal structure was solved and refined using Bruker SHELXTL. A direct-methods solution was calculated to locate most non-hydrogen atoms from the E-map.

Full-matrix least6

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squares/difference Fourier cycles were performed to locate the remaining non-hydrogen atoms. All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located from the difference Fourier map and allowed to ride on their parent atoms in the refinement cycles. Crystal-structure visualization was carried out using Diamond (Version 3.2i; Bonn, Germany). Powder X-Ray Diffractometry (PXRD) Powders were analyzed on a Siemens 5005 powder diffractometer with Cu Kα radiation (1.54056 Å), with two-theta pre-calibrated using a silicon standard. Samples were scanned from 5 to 35° two theta with a step size of 0.02° at 1 s/ step. The tube voltage and amperage were at 45 kV and 40 mA, respectively. Differential Scanning Calorimetry (DSC) Powder samples (3∼5 mg) were heated from 25 to 250 °C with a heating rate of 10 °C/min on a differential scanning calorimeter (Q2000, TA Instruments, New Castle, DE, USA) under a continuously purged dry nitrogen atmosphere (flow rate of 50 mL/min). Tzero hermetic sealed aluminum pans were used for all samples. The instrument was equipped with a refrigerated cooling system and pre-calibrated for temperature and enthalpy using indium. Thermal gravimetric analysis (TGA) Samples (∼5 mg) were heated in an open aluminum pan, on a thermogravimetry analyzer (Model Q50, TA Instruments, New Castle, DE, USA) from room temperature to 300 at 10 °C/min under 50 mL/min dry nitrogen purge.



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Hot-Stage Microscopy (HSM) Crystals were observed under a polarized light microscopy (Eclipse e200; Nikon, Tokyo, Japan) equipped with a DS-Fi1 microscope digital camera for capturing digital images. HSM experiment was performed, at a heating rate of 5°C/min, on a hot stage with a temperature controller (Linksys 32; V.2.2.0, Linkam Scientific Instruments, Ltd., Waterfield, UK). Fourier transformation infrared spectroscopy (FT-IR) FT-IR spectra of the powder samples collected using a high resolution FT-IR spectrometer (VERTEX 70, Bruker Optics Inc., Billerica, MA, USA). For each sample, 32 scans were averaged. IR data in the range of 4000-600 cm-1 at a resolution of 4 cm-1 were processed using OPUS software (v5.5, Bruker Optics Inc., Billerica, MA, USA). Dynamic Water Vapor Sorption Isotherm (DVS) Water sorption and desorption profiles of the materials were obtained by using an automated vapor sorption analyzer (DVS 1000, Surface Measurement Systems Ltd., Alperton, Middlesex, UK) at 25 °C. The nitrogen flow rate was 50 mL/min. The sample equilibrated at each step with the equilibration criteria of either dm/dt ≤ 0.003% or maximum equilibration time of 6h. Once one of the criteria is met, the relative humanity (RH) was changed to the next target value, following the 0% - 95% - 0% sorption and desorption cycle with the step size of 5% RH. Intrinsic Dissolution Rate Intrinsic dissolution rate (IDR) was measured using the rotating disc method.13 Each powder was compressed at a force of 1000 lb, using a custom-made stainless steel die, against a flat stainless steel disc for 1 min to prepare a pellet (6.39 mm in diameter). The obtained pellet has a 8 ACS Paragon Plus Environment

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visually smooth surface that was coplanar with the surface of the die. While rotating at 300 rpm, the die was immersed in 500 mL of the dissolution medium at 37 °C in a water-jacketed beaker. An UV−vis fiber optic probe (Ocean Optics, Dunedin, FL) was used to continuously monitor the UV absorbance of the solution at λ=343 nm, which was used to obtain concentration – time profiles based on a previously constructed concentration – absorbance standard curve. Thermodynamic solubility The solubilities of berberine salts in water were determined by equilibrating excess amount of solids (40 mg) in 5 mL of water at 25 °C under vigorous stirring for 72 h, followed by filtration through 0.45 µm membrane filters. The filtrates were then diluted with water to an appropriate concentration for assay using a UV/Vis spectrophotometer (DU 530, Beckman Coulter, Chaska, Minnesota) at λ = 343 nm (n = 3). Tabletability A material testing machine (model 1485; Zwick/Roell, Ulm, Germany) was used to perform bulk powder compaction studies at a speed of 2 mm/s. Prior to compaction, powders were grinded in a mortar using a pestle to reduce particle size. Compaction pressure ranged from 50 to 350 MPa, using a die and flat-faced punches (round, 8 mm diameter). Punch tips and die wall was coated with a layer of magnesium stearate suspended in ethanol (5%, w/v) using a brush and dried using a fan before each compaction run. Tablets were allowed to relax under ambient environment for at least 24 h before measuring diametrical breaking force using a texture analyzer (TA-XT2i; Texture Technologies Corporation, Scarsdale, New York). Tablet tensile strength was calculated from the breaking force and tablet dimensions following a standard procedure.14 Taste assessment 9 ACS Paragon Plus Environment


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Round disc shaped tablets (4 mm diameter, 20-30 mg weight) of each berberine salt, compressed at 100 MPa, were used for the taste evaluation by a panel of ten volunteers (four females and six males). Age was not treated as a factor. The volunteers were randomly divided into three groups of 3, 3, and 4. Volunteers refrained from eating and drinking for at least 1 h before the study. At the beginning of each test, volunteers tasted a training solution (0.01 mg/mL BbCl in water), which was kept in contact with the tongue for 10s before being spat out. After rinsing with water and resting for 5 min, the tablet sample was put onto the tip of the tongue and held against the palate for 10 s before being spat out. This design simulates the amount of time a tablet can potentially be in contact with the tongue before being swallowed. The taste score was recorded immediately. After tasting each sample, volunteers gargled purified water and waited for at least 15 min until no residual taste before starting the next test. Each group of volunteers tasted tablets of different salts in each round to avoid potential bias due to repeated loading of taste buds. A taste scoring system of 0-7 (0 = sweet, 1 = no bitter taste, 2 = much less bitter than the training solution, 3 = less bitter, 4 = same as the training solution, 5 = more bitter than the training solution, 6 = very bitter, 7 = extremely bitter) was employed in this study.

Results and discussion To confirm the complexation, IR spectra of BbCl, AsK, SacNa, and these two new solids are compared. The new solid formed between BbCl and AsK has characteristic peaks of both reactants, confirming it being the expected BbAs (Figure 2a). Similarly, IR spectra (Figure 2b) also confirm the formation of BbSac.


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(a)

(b)

Figure 2. IR spectral data for a) BbCl, AsK, BbAs and b) BbCl, SacNa and BbSac salts.

The ability to prepare new solid form from the BbCl solution by adding AsK or SacNa confirms that the two new salts do have lower solubility than BbCl. The reduced solubility of the new salts would be beneficial for reducing the intensity of bitter taste of this compound because of the lower amount of dissolved berberine, within a given contact time, that can interact with the taste buds.

Structure elucidation Structures of the single crystals from methanol solutions also confirm the formation of BbAs and BbSac salts. Asymmetric units of these two salts are shown in the Figure 3.



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(a)

(b)

Figure 3. ORTEP diagrams of (a) BbAs and (b) BbSac salts.

No classic hydrogen bonds can be found in the crystal structures of both salts. Rather, only weak C-H…O hydrogen bonds (Table 1) are present to stabilize these two structures (Figure 4).

Table 1. Hydrogen bonding table for the two new berberine salts

1 2 3 4 5 6 7 1 2 3 4 5 6 7 8

Berberine acesulfame (BbAs) D-H...A D-H H...A D...A … C3-H3 O26A 0.91(2) 2.41(2) 3.301(2) … C6-H6 O24 0.95(2) 2.27(2) 3.161(2) C12-H12A…O1 1.00(2) 2.45(2) 3.340(2) C15-H15…O26B 0.97(2) 2.56(2) 3.487(2) … C18-H18A O26A 1.00(2) 2.448(19) 3.340(2) C18-H18B…O26B 0.954(19) 2.59(2) 3.461(2) C20-H20…O26B 0.954(19) 2.480(18) 3.406(2) Berberine saccharin (BbSac) C2A-H2C…O13 1.00(2) 2.45(2) 3.310(3) C3-H3…O29A 0.96(2) 2.34(2) 3.285(2) C6-H6…O27 0.98(2) 2.26(2) 3.245(2) C9-H9…O27 0.96(2) 2.47(2) 3.305(2) C15-H15…O29B 0.97(2) 2.47(2) 3.397(2) C18-H18A…O29A 0.96(2) 2.49(2) 3.331(3) C18-H18B…O29B 0.96(2) 2.47(2) 3.352(3) C20-H20…O29B 0.963(19) 2.34(2) 3.265(2)

D-H...A 166.0(17) 155.0(17) 147.1(17) 161.9(16) 148.3(15) 152.5(17) 163.9(15) 143.8(17) 166.6(19) 177.9(13) 146.2(18) 162.3(17) 146.8(17) 154.3(18) 162.1(15)


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(a)

(b)

Figure 4. Crystal structures of (a) BbAs and (b) BbSac, both viewed along a-axis.

Phase purity of the bulk powders Powder X-ray diffractograms (PXRD) of BbAs and BbSac well match calculated PXRD patterns from corresponding single crystal structures (Figure 5). This confirms phase purity of the bulk powders. The characteristic peaks (two theta in degree) of the reactants and products are as following: BbAs (17.04, 19.14, 26.80), AsK (8.64, 17.10, 25.66), BbSac (15.76, 15.44, 7.70), and SacNa (24.6, 6.22, 12.32) .

(a)

(b)

Figure 5. Calculated and experimental PXRD patterns of (a) BbAs and (b) BbSac salts.

Thermal analyses 13 ACS Paragon Plus Environment


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Differential scanning calorimetric (DSC) thermograms of BbAs and BbSac shows single endotherms, with onset temperatures of 229 oC (△Hf = 56.44 J/g) and 225 oC (△Hf =112.4 J/g), respectively (Figure 6a). Thermogravimetry data also suggest negligible weight loss of both salts until near their melting temperatures (Figure 6b). Therefore, both salts are thermally stable for pharmaceutical processing. The endotherms observed in DSC correspond to melting events observed under HSM at 232 oC and 227 oC for BbAs (Figure 7a) and BbSac (Figure 7b), respectively.

(b)

(a)

Figure 6. The thermal analyses of BbAs and BbSac, a) DSC and b) TGA.

25 oC

o

230 C

o

231 C

o

233 C

a o

25 C

o

220 C

o

227 C

o

230 C

b

Figure 7. Thermal behaviors of (a) BbAs crystal and (b) BbSac observed under a hot stage microscope


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Moisture sorption and desorption behavior The anhydrous BbCl gained 10.66 % water in the 10-65% RH range (Figure 8), which is close to the theoretical amount of water in a dihydrate (9.68%). In the 85 – 95% RH range, the weight gain corresponds to the theoretical water content in a tetrahydrate (19.37%, Figure 8). Thus, anhydrous BbCl undergoes progressive phase change to a dihydrate and then tetrahydrate when RH increases from 0% to 95% RH. On desorption, the tetrahydrate appears to convert to the anhydrate without a distinct plateau region corresponding to the dihydrate (Figure 8). This is because that the dihydrate formed from tetrahydrate dehydration at 5% RH is exposed to 0% RH immediately. If the experiment was carried out with a smaller step size, the observation of a plateau would have been possible. The large hysteresis in the sorption-desorption behavior (Figure 8) suggests that the two hydrates of BbCl can be kinetically stable over a wide range of RH. In any case, the hydration or dehydration with changing RH makes BbCl a less desirable solid form for tablet product development. For example, uncontrolled solid phase change during wet granulation and subsequent drying likely make the manufacturing process less robust. Even if the drug product has been successfully manufactured, unexpected phase changes during storage due to uncontrolled humidity conditions can still negatively impact performance of the drug product.9 In contrast, the two new solid forms, BbAs and BbSac, are non-hygroscopic. Both salts absorb less than 0.36 % of moisture even at 95% RH (Figure 8). The solid-state stability of the two salts against high humidity is an important advantage in comparison to BbCl.



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Figure 8. The DVS result of BbCl, BbAs and BbSac. Sorption and desorption lines of BbAs and BbSac are superimposable.

Solubility and intrinsic dissolution rate Solubility and dissolution rate are two key pharmaceutical properties that need to be considered for successful drug delivery. As defined by the Biopharmaceutics Classification System (BCS), if the highest dose of a drug can be dissolved in 250 mL water over the pH range of 1.0–7.5 at 37 °C, it can be treated as soluble, otherwise it was poorly soluble. 15 The thermodynamic solubility of BbCl, BbAs and BbSac at 25 °C (Table 2) are 4.900±0.054, 0.646±0.021 and 0.908±0.003 mg/mL of berberine in water. The solubility at 37 oC should be higher than that at 25 oC for the three salts. For the typical dose of 100 mg of berberine, both new salts are soluble according to the BCS definition. Thus the lower solubility of BbAS and BbSac than BbCl does not impact the bioavailability. However, the lower solubility does lead to lower dissolution rate. The intrinsic dissolution rate of BbCl, BbAs and BbSac at 37 °C are 218.44±2.44, 38.93±1.55, 21.75±0.16 µg·cm-2·min-1, respectively (Figure 9).

The reduced 16

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dissolution rates of the two new salts are advantageous because they reduce the problem of bitter taste of this drug by limiting the amount of dissolved drug when the tablet is swallowed. According to the IDR based classification, where 100 µg·cm-2·min-1, is the boundary between compound with or without solubility challenge, BbCl is soluble but the two new salts are not.16 Table 2. The solubility in water at 25 oC (n=3) and intrinsic dissolution rate of berberine salts in water at 37 oC (n = 3).

Material

Solubility (mg·mL-1)

Intrinsic dissolution rate (µg·cm-2·min-1)

BbSac BbAs BbCl

0.646±0.021 0.908±0.003 4.900±0.054

21.75±0.16 38.93±1.55 218.44±2.44

Figure 9. The intrinsic dissolution rate result of BbCl, BbAs and BbSac at 37 °C.

Tabletability



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Tabletability is another important pharmaceutical property critical to the successful tablet formulation of drugs, especially when drug loading is high.17,18,19,20 Tabletability of the three salts follows the order BbSac ~ BbCl > BbAs (Figure 10). For BbSac, tablets with tensile strength more than 2 MPa can be made over the pressure range of 50 – 350 MPa, which is thought sufficient for surviving stresses during handling of tablets after compression.21,22 Although BbAs exhibits lower tabletability, tablets with > 2MPa tensile strength can still be made at 300 – 350 MPa pressures. Representative tablets of BbSac and BbAs are also shown in Figure 10.

BbAs

BbSac

Figure 10. Tabletability of BbCl, BbAs and BbSac. Tablets compressed at 200 MPa are also shown.

Taste assessment A well-recognized challenge in developing tablet products of berberine is the intense bitter taste. A main motivation to this work is to mitigate this problem by forming novel salts with sweet counterions. The taste assessment of the three salts clearly shows the much improved taste profiles of both BbSac and BbAs over BbCl (Figure 11). No extreme bitterness is reported for BbAs and BbSac. Sweet taste is reported by 40% and 20% of the taste panel members for 18 ACS Paragon Plus Environment

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BbAs and BbSac, respectively. This is in stark contrast to the taste profile of BbCl, where 80% of the taste panel members reported either very bitter or extremely bitter taste and all reported taste scores are 4 or higher. Despite the overall improvement, still 40% and 50% of the taste panel members reported bitterness for BbAs and BbSac, respectively. The biomodal distribution of the two “sweet” salts reflects the complexity in human sensory perception. The sweetners, at the fixed 1:1 molar ratio to berberine, does not perfectly counteract the bitterness of berberine. As such, the use of the two sweet salts may not completely eliminate the problem of bitter taste of berberine in wide populations.

Figure 11. Taste assessment of the BbCl, BbAs, and BbSac.

Conclusions Bitterness is one of the greatest challenges for commercialization of berberine due to a lack of patient acceptability and compliance, especially in pediatric patients. We have shown in this work that the formation of sweet salts by the means of crystal engineering is a powerful strategy to address this problem facing berberine. The simultaneous dissolution of the potent sweeteners, in addition to their reduced aqueous solubility, makes this approach effective. In



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addition, both sweet salts exhibit good solid-state stability and acceptable tabletability, which further improve their potential for use in tablet products of berberine.

Supporting Information The crystal structures of the two salts have been deposited in CCDC, 1417380-1417381. PXRD patterns of BbAs, AsK, BbSac, and SacNa. This material is available free of charge via the Internet at http://pubs.acs.org. #Author Contributions W.CG. and S.R.P. contributed equally to this work. The authors declare no competing financial interest.

Acknowledgements We are grateful for resources from the University of Minnesota through the Minnesota Supercomputing Institute, X-ray Crystallographic Facility, Department of Chemistry. Some of the experiments were performed at the University of Minnesota I.T. Characterization Facility, which receives partial support from the NSF through the NNIN program. Ruolin Lu thanks the support by the University of Minnesota's Undergraduate Research Opportunities Program. Members on the voluntary taste panel are also greatly appreciated.


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Page 22 of 22

For Table of Contents Use Only

Sweet Berberine Chenguang Wang, Sathyanarayana Perumalla, Ruolin Lu, Jianguo Fang, and Changquan Calvin Sun

TOC graphic

Synopsis The formation of sweet salts of berberine with two sweetners, acesulfame and saccharine, is a powerful strategy to address the bitterness problem of berberine. Both sweet salts also exhibit excellent solid-state stability and acceptable tabletability, which further improve their potential for use in tablet products of berberine.


Sweet Berberine - ACS Publications - American Chemical Society

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