Raft-forming gastro-retentive formulations based on Centella asiatica extract-solid dispersions for gastric ulcer treatment

Journal Pre-proof

Raft-forming gastro-retentive formulations based on Centella asiatica extract-solid dispersions for gastric ulcer treatment Saowanee Wannasarit , Sirima Mahattanadul , Ousanee Issarachot , Panupong Puttarak , Ruedeekorn Wiwattanapatapee PII: DOI: Reference:

S0928-0987(19)30477-4 https://doi.org/10.1016/j.ejps.2019.105204 PHASCI 105204

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

5 August 2019 5 December 2019 19 December 2019

Please cite this article as: Saowanee Wannasarit , Sirima Mahattanadul , Ousanee Issarachot , Panupong Puttarak , Ruedeekorn Wiwattanapatapee , Raft-forming gastro-retentive formulations based on Centella asiatica extract-solid dispersions for gastric ulcer treatment, European Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.ejps.2019.105204

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

1

Raft-forming gastro-retentive formulations based on Centella asiatica extract-solid dispersions for gastric ulcer treatment Saowanee Wannasarita,b, Sirima Mahattanadulb,c, Ousanee Issarachotd , Panupong Puttarakb,e, Ruedeekorn Wiwattanapatapeea,b* a

Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of

Songkla University, Hat Yai, Songkhla, 90112, Thailand. b

Phytomedicine and Pharmaceutical Biotechnology Excellence Research Center, Faculty of

Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand. c

Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Prince of Songkla

University, Hat Yai, Songkhla, 90112, Thailand. d

Pharmacy Technician Department, Sirindhron College of Public Health of Suphanburi, 77

moo4, Tubteelek sub-district, Mueang district, Suphanburi, 72000, Thailand e

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical

Sciences, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand.

*

Corresponding author: Assoc. Prof. Ruedeekorn Wiwattanapatapee, PhD.

Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand. Phone: + 66

74

288915, e-mail

address: ruedeekorn. w@psu. ac. th

2

Abstract Liquid raft-forming formulations comprising solid dispersions of glycoside-rich Centella asiatica extract and Eudragit ® EPO (GR-SD) were developed to achieve prolonged delivery of the glycosides, asiaticoside (AS) and madecassoside (MS) in the stomach and thus increase the effectiveness of gastric ulcer treatment. Solid dispersions of GR extract and Eudragit® EPO (GR-SD, weight ratio 1:0.5) resulted in the highest solubility of AS (41.7 mg/mL) and MS (29.3 mg/mL) and completed dissolution of both glycosides occurred in SGF within 10 min. The optimized raft-forming formulation was composed of alginate (2%), HPMC K-100 (0.5%), GR-SD (1.2%), and calcium carbonate (0.5%) as a calcium source and carbon dioxide producer. The formulation provided sufficient raft strength (> 7.0 g), rapid floating behavior in SGF (~30 sec), and sustained release of AS (more than 80%) and MS (85%) over 8 h. GR-SD-based formulations administered once daily to rats for two days at a dose of 10 mg AS/kg reduced the severity of gastric ulcer induced by indomethacin with a greater curative efficacy than those of unformulated GR extract and a standard antiulcer agent: lansoprazole (p<0.05). These findings demonstrate that GR-SD-based raft-forming systems offer significant promise for improving the treatment of gastric ulcers induced by non-steroidal antiinflammatory drugs.

Keywords: Centella asiatica; asiaticoside; madecassoside; solid dispersion; Eudragit ® EPO; raft forming system; gastric ulcers

3

1. Introduction Gastric ulcers present as open sores in the mucosal lining of the stomach. The symptoms may range from mild abdominal discomfort to gastrointestinal perforation and bleeding. The frequent or long term use of non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen and indomethacin is the most common cause of gastrointestinal mucous membrane injury in western or industrialized countries [1], [2]. The most common gastrointestinal adverse effects of NSAIDs are mainly associated with COX-1 and COX-2 inhibition. COX-1 is associated with protection of gastric mucosa while COX-2 enzyme is involved in wound healing. Prostaglandins generated by COX-2 also play a role in stimulating the release of growth factors (bFGF and EGF), which are important in epithelial cell proliferation and new blood vessel growth (angiogenesis) [3]. Several studies have reported the protective and healing effect of Centella asiatica extract and their active pentacyclic triterpenoid compounds, especially asiaticoside (AS) and madecassoside (MS) on gastric ulcers. Cheng and Koo (2000) found that oral administration of aqueous C. asiatica extracts significantly inhibited gastric lesion formation in rats (induced by ethanol) by decreasing mucosal damage, and improving the integrity of the gastric mucosal lining in ex-vivo models [4]. Decreased mucosal myeloperoxidase (MPO) activity was observed indicating an involvement of an antioxidant and anti-inflammatory properties of AS in the C. asiatica extract [5]. Oral administration of AS was also found to accelerate wound healing in rats with acetic acid-induced gastric ulcers by inhibiting MPO activity including enhancing epithelial cell proliferation and angiogenesis through an upregulation of bFGF

4

expression at the ulcerated area [6]. In addition, Guo et al. (2004) reported the healing mechanism of AS via inhibition of iNOS expression and activity, which provided a favorable environment for ulcer healing [7]. Moreover, the anti-inflammatory effect of both AS and MS has been reported in the model of collagen-induced arthritis in mice. Their mechanism of actions might be related to the reduction of COX-2 expression and inflammatory cytokines or inhibition of lymphocyte proliferation [8], [9]. Although the active compounds: AS and MS contained in centella extract appear to be highly beneficial for gastric ulcer treatment, their low aqueous solubility presents a major obstacle to clinical application. AS and MS are crystalline solids of high molecular weight (959.1 g/mol for AS and 975.1 g/mol for MS). Moreover, AS is classed as a sparingly water soluble compound (0.305 mg/L), which is difficult to convert into the ionized form [10]. There are numerous approaches to increase the solubility of poorly water-soluble compounds. In particular, the solid dispersion technique has been widely exploited for enhancing drug solubility by reducing the particle size of drugs to nearly a molecular level, improving wettability, and converting the crystalline form of drug substances to the amorphous state [11]. Eudragit® EPO, the synthetic copolymer of dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate (ratio 2:1:1), has several pharmaceutical applications mainly in the solid oral dosage forms, including moistureprotective coating [12], taste-masking [13], and immediate-release enhancer in acidic media [14]. Recently, this tertiary amine copolymer has been used as an solubility- or a bioavailability-enhancing excipient for many compounds, such as mefenamic acid [15], tacrolimus [16], and curcumin [17]. According to the apparent positive charge of the polymer

5

in pH lower than 8, the improvement in drug solubility was evidently related to the ionic interaction between the deprotonated functional group of the acidic drug and the protonated amino alkyl group of the Eudragit® EPO. The solubility enhancement by Eudragit® EPO was not only beneficial for the lipophilic acidic drugs, but also the basic drugs in solid dispersion forms [18,19]. In the present study, the Eudragit ® EPO was selected as a carrier component in the preparation of glycoside-rich C. asiatica extract-Eudragit® EPO solid dispersion (GR-SD). This cationic carrier was also anticipated beneficial for stabilizing AS and MS in the basic liquid formulation by reducing contact with water due to the insoluble property of Eudragit® EPO in basic solution. Oral drug delivery systems which remain in the stomach for a prolonged period of time have been investigated to improve drug efficacy at reduced doses. Among the gastroretentive drug delivery systems, the floating raft-forming designs have attracted much interest for treatment of gastrointestinal disorders including gastric ulcers. The devices remains buoyant in the stomach without affecting the gastric emptying rate [20], [21]) and mainly consist of a gel-forming hydrophilic polymer and a gas-forming agent. On contact with gastric fluids, the liquid suspension swells to form a viscous, continuous cohesive gel which floats on the surface of gastric fluids due to formation and entrapment of carbon dioxide bubbles in the gel. The divalent cations (Ca2+, Mg2+ ) dissolved in the gastric fluid are employed to crosslink the polymer chains, producing a more rigid raft structure. Recently, the novel concept of raft forming formulation incorporating solid dispersion of poorly natural compound such as curcumin has been reported [17]. By this strategy, the active compound in solid dispersion form could be prolong released from the raft, and showed a superior curative effect on the gastric ulcer treatment. To date, the incorporation of

6

the crude plant extract into the raft forming system has not been reported yet. The richglycoside extract (derived from C. asiatica, GR extract) used in the present study has its own purity of the active compounds (~ 40% AS and 30% MS). Therefore, the solubility of this crude GR extract was improved by solid dispersion technique, and incorporating into the alginate raft formulation. The main aims of this study were to develop and characterize raft forming systems incorporating the GR-SD in order to prolong the release of the glycoside compounds, thereby improving the treatment of gastric ulcers. The therapeutic potential of optimal raft forming formulation was finally assessed on indomethacin induced acute gastric ulcers in rats. The raft-forming system was also considered advantageous as a physical barrier to control gastric acid reflux and provide relief of gastroesophageal reflux disease (GERD) symptoms [22]. 2. Materials and Methods

2.1 Materials Glycoside-rich extract powder derived from Centella asiatica (~40% asiaticoside and 30% madecassoside) was provided by X-Kev Food Additive Co., Ltd (Guangdong, China). Asiaticoside and madecassoside standard compounds (purity >98%) were purchased from Chengdu Biopurify Phytochemicals Ltd. (China). Eudragit® EPO was purchased from Evonik industries AG (Essen, Germany). Medium viscosity sodium alginate (viscosity of 2% solution ~2,000 cps, at 25°C) was purchased from High Science Ltd. (Songkhla, Thailand). Sodium bicarbonate and calcium carbonate were obtained from RCI Labscan (Bangkok, Thailand). Hydroxypropyl methyl cellulose (HPMC) Methocel K100LV; viscosity of 2% solution ~80-

7

120 cps, at 25°C was a gift from Colorcon Asia Pacific Pte Ltd. (Singapore). Indomethacin, lansoprazole and low viscosity carboxymethylcellulose sodium (CMC) were purchased from Sigma-Aldrich Co., Ltd. (USA). Tween80 and propylene glycol were sourced from Vidhayasom Co., Ltd (Songkhla, Thailand). All other reagents were of analytical grade. 2.2 Animals

The animals used in this study were 7-week, male Wistar-strain rats (180-250 g), provided by the Nomura Siam International Co., Ltd, Bangkok, Thailand. They were accommodated under suitable laboratory conditions at 24 ±2 °C with relative humidity of 55 ±5%, and a controlled 12 h light-dark cycle. The standard rodent chow was routinely given with tap water ad libitum. All rats received care in compliance with the guidelines of the Animal Care and Use Committee of Prince of Songkla University (PSU). The experimental procedures were approved by the Institutional Animal Care and Use Committee, PSU. A certified number (MOE) 0521.11/1088 was issued for the research. 2.3. Quantitative HPLC analysis of the pentacyclic triterpenoid glycosides in Centella asiatica extract Pentacyclic triterpenoid glycosides including AS and MS were analyzed using an HPLC system (Agilent 1100, Agilent, USA) equipped with a photodiode array detector as previously described with minor modification [23]. Samples were dissolved in methanol and filtered through a 0.45 µm polypropylene membrane filter (VertiPure™) before injection in triplicate into an ODS-100V, 5 µm column (250 × 4.6 mm, TSK-gel®) via the auto-injector. The gradient mobile phase comprised acetonitrile and water with different elution times and

8

acetonitrile/water ratios as follow: 0-5 min, 20:80; 5-10 min, 30:70; 10-20 min, 65:35; 20-30 min, 70:30. The flow rate and sample injection volume were set at 1 mL/min and 20 µL respectively. The detection of glycoside compounds was performed by monitoring the UV absorbance at 210 nm. The content of active compounds was quantified by comparison of the elution peak area with a standard calibration curve of asiaticoside (AS) and madecassoside (MS) over the concentration range of 12.5-1000 µg/mL. 2.4 Preparation of GR extract-Eudragit® EPO solid dispersions (GR-SD)

Eudragit® EPO, a hydrophilic polymer, was chosen for preparation of solid dispersions using the solvent evaporation technique. GR extract and Eudragit ® EPO at the weight ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2 were dissolved in 75% ethanol (100 ml) and stirred until clear solutions were obtained. The solvent was removed using a rotary evaporator under vacuum (model Hei-VAP value digital, Heidolph Instruments GmbH, Germany) at 40 °C, following extracting moisture residues using vacuum desiccator at room temperature for 10 h. The obtained GR extract-Eudragit® EPO solid dispersions (GR-SD) were removed from the reservoir and the particle size was reduced by pulverizing in a mortar followed by sieving through 250 µm mesh. The samples were stored in air-tight, light-resistant containers at 25 °C until further use. 2.5 Solubility studies of GR-SD solid dispersions

The solubility of AS and MS incorporated in GR-SD solid dispersions was determined using the shake flask method with minor modification [24], [25]. An excess amount of GRSDs (1:0.1, 1:0.5, 1:1, 1:1.5, 1:2 GR: Eudragit® EPO ” weight ratio), physical mixtures (GR-PM)

9

and unformulated GR extract was added to 0.1 N hydrochloric acid, pH 1.2 (1.0 ml). All samples were incubated at 37 °C in a shaking water bath (model SW22, Julabo Technology Co., Ltd, China) at a speed of 150 rpm until equilibrium was achieved (24 h). The supernatants were withdrawn after centrifuging at 6000 rpm for 10 min (Hettich Zentrifugen, model 16R, Germany), then diluted and filtered through a 0.45 µm membrane filter. The concentration of dissolved glycoside compounds was analyzed using HPLC at 210 nm as described in section 2.3.

2.6 Physicochemical characterization of GR-SD solid dispersions Powder X-ray diffraction (PXRD) X-ray diffractograms were generated at a wavelength of 0.154 nm (CuKα) using an Xray diffractometer (X’Pert MPD, Philips, Netherlands) equipped with a Cu tube operated at 40 kV and 30 mA. All samples were measured over a scan range (2Ɵ) of 5-90°, with a step size of 0.05° at a scan speed of 3°/min. Fourier transform-Infrared (FT-IR) spectroscopy The possible interactions between AS, or MS and Eudragit ® EPO in the solid dispersion were identified using a FT-IR spectrometer (Perkin-Elmer, USA). A deuterated triglycerine sulfate was used as a detector. Samples were mixed with KBr powder and compressed into discs. The IR spectra of transmission or absorption were recorded over the range 450-4000 cm-1. 2.7 In vitro dissolution studies of GR-SDs

10

The release behavior of AS from GR-SDs containing 40 mg of asiaticoside was studied using a USP30 dissolution tester (model EDT-08Lx, Electrolab®, India) equipped with rotating paddle apparatus [17]. The samples were placed in dissolution medium (200 mL, 0.1 N HCl) at 37± 0.5 ºC and agitated using a paddle rotation speed of 50 rpm. Release samples (5 mL) were manually collected via the sampling cannula connected with the 35 micron-dissolution filter at 5, 10, 15, 30, 45, 60, 90 and 120 min and replaced with an equal volume of fresh medium. The samples of release media were adjusted to pH 5 using 1.0 M NaOH solution and filtered through a 0.45 µm membrane filter prior to analysis of the concentration of AS and MS using HPLC at a wavelength of 210 nm. The release profiles were presented as a plot of cumulative release (% w/w) versus time (min). Each of the formulations was tested in triplicate and the data were reported as a mean ± S.D. 2.8 Preparation of gastro-retentive, GR-SD-based-raft forming systems

The composition of the raft forming systems investigated is shown in Table 1. The raft formulation was prepared by dissolving 1.5 g of sodium bicarbonate (1.5% w/v) in deionized water (total volume 100 mL) with continuous stirring until the solution was clear. Various concentrations of sodium alginate solution (1-2 % w/v) and 0.5 g of HPMC K100 (0.5 %w/v, as a release retardant) were added to the sodium bicarbonate solution and stirred homogenously (68 h) at room temperature. Insoluble calcium carbonate (0.5-3 g) was added to the resultant polymer solution, followed by GR-SD powder (30-120 mg, ratio 1:0.5). The uniform suspension obtained was stored in tight, light-resistant containers until further use. 2.9 Physicochemical characterization of raft-forming systems

11

Physical appearance The visual appearance of the suspensions used for raft formation and the resulting raft structure were recorded. The pH of the formulation was measured using a pH meter (FiveEasy F20, Mettler-Toledo GmBH, Switzerland) at 25 ±1°C. Viscosity The viscosity of liquid formulations (100 mL) was measured in triplicate using a Brookfield viscometer (DV-III ultra, USA) with spindle no.64 (LV 4) operated at a speed in the range of 30-180 rpm. The temperature was maintained at 25 ±1°C. Raft density The raft density was measured following addition of the liquid raft formulations (10 mL) into a 100 mL-measuring cylinder containing 75 mL of 0.1 N HCl solution (pH 1.2). Briefly, the measuring cylinder was placed on the scale, and the weight was adjusted to zero. Then, the acidic medium was filled in the cylinder and its weight (Wa) was recorded. The liquid raft formulation was then introduced into the medium, and the floating raft gel was allowed to form for 10 min. In the meantime, the decreasing weight could be observed because of the release of CO2. In order to calculate the raft density, the weight of the raft gel (Wr) was indirectly obtained by subtracting the weight of the acidic medium from the total weight between the weight of acidic medium and raft gel (Wa + Wr). The final volume of the raft gel (Vr), in which being observed from the scale on the cylinder, and the final weight of the raft gel, were finally used to calculate the raft density (Wr/Vr). Raft strength

12

The raft strength was investigated using a texture analyzer (TA.XTplus, Haslemere, UK) fitted with a 5-kg load cell. Liquid raft formulation (20 mL) was added to 150 mL of SGF maintained at 37 °C in a 250 ml beaker. The suspension was gently introduced around an Lshaped wire hook (20 x 90 mm) which was held upright in the medium for 30 min. After raft development, the wire hook, in which connected with the adapter and the probe, was attached to the loading arm of the texture analyzer, and pulled vertically upwards through the raft at a rate of 5 mm/s. The resulting force-displacement graph was recorded and the maximum applied force (g) was taken as the strength of the raft [26]. Floating behavior Raft floating behavior was observed by introducing liquid raft formulation (20 mL) into 150 mL of SGF maintained at 37 °C in a 250 mL glass beaker. The time taken for the formulation to float to the surface of the medium (floating lag time), and the time the formulation floated on the medium surface (duration of floating) observed for more than 8 hours were recorded [17]. 2.10 In vitro release behavior of GR-SD -based raft forming systems The release behavior of AS and MS from GR-SD-based raft forming systems was investigated using a USP30 dissolution test apparatus II (model EDT-08Lx, Electrolab®, India) [17]. The release medium (0.1 N HCl solution pH 1.2, 200 mL) was maintained at 37 °C. A paddle rotation speed of 50 rpm was applied so as to mimic the movement of liquid or content inside the stomach but not to disrupt the gel structure. In order to avoid the disturbance of the structure of the raft, the sampling canula was introduced into the

13

dissolution medium prior to the addition of the formulation. The liquid raft formulation (10 mL) containing 30 mg of GR-SD (F1-F6) was directly injected into the dissolution medium using a syringe. Release samples (5 mL) were manually collected via the sampling cannula connected with the 35 micron-dissolution filter after 30 min and then every hour for 8 h, and replaced with 5 mL of fresh medium at each predetermined time interval. Each formulation was tested in triplicate and the concentration of AS and MS in the release medium was quantified by HPLC as described in section 2.3. The % cumulative release (w/w) of AS and MS respectively was plotted against time (h) in order to construct the release profiles. 2.11 Release kinetics of GR-SD-based raft forming systems The release kinetics of AS and MS were investigated by fitting the cumulative release (mean ±S.D.) versus time (h) data to the kinetic models: zero-order, first-order, Higuchi, HixsonCrowell and Weibull. The Korsmeyer-Peppas model was analyzed using the first 60% drug release data. The add-in program, ‘DDSolver’, in Microsoft excel was used to predict the drug release behavior of AS and MS and its underlying mechanism [27]. A coefficient of determination (R2) closer to 1 indicated the best fit of release data to a particular kinetic model.

2.12 Stability study The stability of GR-SD -based raft-forming systems was evaluated according to ICH guidelines (2003) relating to topic Q1A (R2), stability testing of new drug substances and products [28]. Testing under two conditions is specified; intermediate conditions (30 °C ± 2 °C/65% RH ± 5% RH, 6 months) and accelerated conditions (45 °C ± 2 °C/75% RH ± 5% RH, 6

14

months). The appearance, pH, floating behavior and AS and MS content of GR-SD -based raft forming systems were evaluated. 2.13 Determination of ulcer healing efficacy on acute gastric ulcers induced by indomethacin in rats Wistar rats (180-250 g) were randomly divided into 7 groups (at least 6 animals/group) and fasted for 18 h with free access to water, prior to ulcer induction. Animals were orally administered indomethacin at a dose of 30 mg/kg to generate acute gastric ulcers, as previously described by Kuadkaew S (2019) [29]. The animals in each experimental groups were orally administered the respective control or test samples beginning at 5 h after ulcer induction (day 1) and repeated at 24 h (day 2) after ulcer induction. The rats were then sacrificed (day 3) after the last dose using a high dose of pentobarbital sodium (150 mg/kg; I.P. injection). The stomachs were removed and soaked in 10% formaldehyde for 5 min before opening along the greater curvature and washing with normal saline. Ulcerated areas (mm2) of both erosion and haemorrhagic ulceration types were measured using ImageJ software and expressed as mean ± SEM. Ulcer healing rates (%) were calculated from the total ulcerated areas in treatment groups compared with those of the vehicle controls. CMC (1 % w/v solution) was used as a vehicle control for animals administered lansoprazole (a standard antiulcer agent) or GR extract equivalent to 10 mg/kg AS. Blank rafting formulation was used as a control for animals administered rafting formulations containing GR-SDs (equivalent to 5 and 10 mg of AS).

2.14 Statistical analysis

15

The in vitro study data were reported as the mean ± standard deviation (S.D.) and statistical analysis was carried out using Student’s t-test and one-way analysis of variance (ANOVA). For the in vivo study, the data were reported as mean ± standard error of the mean (SEM), and statistically significant of the differences between the treatment and the control groups were identified by ANOVA followed by the Dunnett’s test for multiple comparisons. Statistical probability (p) values less than 0.05 or 0.01 were regarded as significantly different. In order to compare the in vitro release profiles, the different factor (f1) and similarity factor (f2) were calculated. The f1 and f2 values should be between 0-15 and 50-100, respectively to be considered similar for two release profiles [30] 3 Results and discussion 3.1 Solubility study Asiaticoside (AS) has a low aqueous solubility (0.305 mg/mL) at 25 °C due to its high molecular weight (959.1 g/mol) and the difficulty of ionization. The solubility of AS and MS in the unformulated GR extract, GR-SDs and their physical mixtures with Eudragit ® EPO (GR-PM) in different weight ratios is shown in Fig 1. AS and MS in GR-SDs showed a greater solubility compared with unformulated GR extract (5-9 fold for AS, and 1.5-3 fold for MS). However, the physical mixtures of GR extract and Eudragit® EPO did not significantly improve the solubility of AS and MS. The maximum solubility of AS and MS when formulated as GR-SDs was found at the ratio of 1:0.5 (41.7 mg/mL and 29.3 mg/mL for AS and MS respectively). The solubility of both glycosides gradually decreased at GR-SD w/w ratios higher than 1:0.5, similar to the behavior of tacrolimus [16], curcumin [17], and

16

pentacyclic triterpene-rich centella extract- Eudragit® EPO solid dispersions [31]. A reduced solubility at high polymer loads might be contributed to steric hindrance of the interactions between the glycosides and Eudragit ® EPO as previously reported by Saal et al. (2017) [19]. A decrease in molecular interactions (e.g. hydrophobic interaction) might also occur according to the polymeric conformational changes at high concentration of the Eudragit ® EPO, resulted in a reduced solubility.

3.2 Physicochemical properties of GR-SDs PXRD study The crystallinity of the GR extract, solid dispersions of GR and Eudragit ® EPO (GRSDs) and physical mixtures of GR and Eudragit® EPO (GR-PMs) at weight ratios of 1:0.1, 1:0.5, 1;1, 1:1.5 and 1:2 are depicted in the X-ray diffractograms (Fig 2). Several crystalline peaks were detected at 2Ɵ angles within the range of 10°-25° for GR extract and GR-PMs (Fig 2a). The prominent characteristic peaks in the diffractogram of the GR extract were present at 10.3°, 11.7°, 13.0°, 14.3°, 15.4°, 16.0°, 16.6° and 19.5°, in which could be observed in the diffractograms of all PMs. The similarity of the diffractograms indicated the existence of the drug crystallinity in the GR-PMs. In contrast, Eudragit® EPO showed a ‘halo pattern’ as a broad background, representing an amorphous nature of the polymer. Complete disappearance of the crystalline peaks occurred for GR-SD samples, indicating transformation of AS and MS from the crystalline state into an amorphous form. This behavior may be related to the molecular property of the polymer, which could enhance the degree of supersaturation of the glycosides, leading to inhibition of the crystal growth formation [32]. Similar behavior has

17

been reported for solid dispersion of Eudragit® EPO with pentacyclic triterpene-rich C. asiatica extract [31], indomethacin [33] and curcumin [17]. FT-IR study FT-IR analysis was performed to elucidate the possible interaction between GR extract and Eudragit ® EPO in GR-SDs, which might explain the enhance solubility of AS and MS. The FT-IR spectrum of GR extract (Fig 3) features three main absorption peaks at 1728 cm-1, 2914 cm-1 and 3404 cm-1 which relate to stretching of carbonyl ester (C=O), aliphatic C-H and broad polyhydroxy (-OH) in AS and MS. The spectrum of 1:0.5 GR-PM combined the spectra of GR extract and Eudragit ® EPO. On the other hand, the IR-spectrum of 1:0.5 GR-SD showed a reduced intensity of the peak at 1730 cm-1 (carbonyl ester group) compared with the physical mixture. The peak intensity of the hydroxyl group at 3423 cm-1 (assigned to stretch of OH in GR extract) was also reduced. This behavior indicates H-bonding between the C=O groups from Eudragit ® EPO and the OH groups from the glycoside compounds (AS and MS). A similar interaction between curcumin and Eudragit ® EPO was also reported by J. Li et al (2015) [34] and F. Meng et al (2015) [35]. In the present study, the peaks at 2762 and 2814 cm-1, (recognized as C-H stretching of the dimethylamino group of Eudragit ® EPO) almost disappeared from the IR-spectrum of GRSD. The non-covalent cation -π interaction between the cationic dimethylamino group of Eudragit® EPO and the π-cloud, an electron donor, of the olefin group (C12) in AS or MS may be responsible for this behavior. 3.3 In vitro dissolution study of GR-SDs

18

The release behavior of AS and MS from unformulated GR extract and GR-SDs in 0.1 N HCl (pH 1.2) are shown in Fig 4. The glycoside compounds in GR extract dissolved rapidly in the acidic medium, resulting in around 83-85% release in 15 min and maximum release of 90% over 2 h. The dissolution of AS and MS from solid dispersions was more rapid, resulting in complete release within 10 min for 1:0.5 GR-SD and in 30 min for 1:1 GR-SD. Interestingly, the different release profiles of AS and MS from GR-SD at ratio 1:0.5 were also observed in comparison with GR extract (f1 and f2 values = 21.75 and 34.95 for AS, 18.02 and 38.50 for MS, respectively). Therefore, the solid dispersion of GR extract and Eudragit ® EPO at ratio of 1:0.5 was selected for loading into gastro-retentive raft formulation. 3.4 Physicochemical characterization of raft forming systems

Physical appearance The pH of all liquid raft-forming compositions was in the range of 9.03-9.55, due to the presence of basic sodium bicarbonate and calcium carbonate. Raft density The density of all raft formulations following contact with the acidic medium (pH 1.2, density 1.06 g/mL) was in the range of 0.52-0.75 g/mL (Table 1). The main factor that affected raft density was the alginate content (formulations F1-F3). Increasing the concentration of alginate resulted in higher raft density and increasing floating lag time up to 30s. All formulations floated within 30s except F4 (without HPMC) that exhibited an increase in floating lag time by a factor of 16. This behavior indicates that HPMC facilitated fluid movement into the liquid raft formulation through its swelling action, followed by expanding

19

the surface of the raft gel. The produced CO2 might therefore be entrapped more within the raft gel, thereby increasing the buoyancy of the raft. Viscosity of liquid raft formulations The viscosity of the liquid raft formulations is reported in Table 1. Major increases in viscosity from approximately 360 to 2400 mPa“s were obtained on increasing the content of alginate in the formulation, suggesting that polymer chain entanglement is contributing to a higher resistance to liquid flow. Besides, adding a 0.5% HPMC K100 into 2% of alginate solution also increased the viscosity from 1598.0 (F4) to 2457.8 mPa“s (F3). However, neither increasing calcium carbonate (F5-F7) or GR-SD content (F5, F8 and F10) significantly affected the viscosity of the liquid formulation. The rheograms of liquid raft- forming formulations (Fig.5 and 6) reveal the significant increase in viscosity with increasing alginate content and the minor effect of calcium carbonate and GR-SD content. However, all liquid raft-forming compositions displayed a decrease in viscosity with an increase in shear rate, indicative of pseudoplastic flow or shear thinning behavior. Raft strength According to British Pharmacopoeia (BP) 2011, an acceptance raft strength corresponds to a force of 7 g or more, which represents the capability of resisting peristaltic movements of the gastrointestinal tract [26]. All raft formulations exhibited strength greater than 7 g, which are acceptable according to BP 2011 recommendations (Table 1). Changes in GR-SD content of the formulation had no significant effect on raft density and strength (F5, F8-F10). Raft strength increased with alginate content (F1-F3) which may be explained by the

20

higher density of carboxylic and hydroxyl groups available for ionic crosslinking Ca2+ ions. Similar results were reported for the alginate-based floating raft systems containing metronidazole [36] and curcumin[17]. Increasing the content of calcium carbonate in the formulation was found to produce a stronger raft (F5-F7), indicating more extensive crosslinking of alginate chains due to the higher concentration of calcium ions dissolved in the acidic medium. Although inclusion of HPMC in raft formulations beneficially reduced the floating time from 8 to 0.5 minutes, the raft strength was decreased by around 25% (F3 and F4, Table 1) indicating disruption of the alginate gel structure.

3.5 In vitro release behavior of GR-SD-based raft formulations

The release behavior of AS and MS from raft forming systems are shown in Fig 6-8. HPMC functioned primarily as a ‘buoyancy control agent’ to reduce the floating lag time to less than 1 min (Table 1). The concentration of HPMC in the raft formulation was fixed at 0.5% w/w to avoid interference with the alginate structure which may result in a decrease in gel strength. All raft formulations resulted in gradual and highly efficient release of at least 80% of the AS and MS load in simulated gastric fluid (SGF) in 8 h (Fig 6-8). Increasing the alginate content from 1 to 1.5% in formulations containing 0.5% of HPMC K100, was found to reduce the initial release from around 60 to 30 % in 30 minutes, due to restricted diffusion of the glycosides. Removal of HPMC K100 from the raft formulation resulted in an initial increase in AS and MS release (F3 and F4, Fig 6). This HPMC acted as an effective release retardant

21

due to a high degree of polymer chain entanglement which inhibited diffusion of AS and MS from the raft [37]. The effect of CaCO3 content on the release profiles of AS and MS is shown in Fig 7. A low calcium carbonate content (0.5-1%w/w) in the formulations resulted in a similar sustained release pattern over 8 h, with an initial release (30 min) of 25-30% for both AS and MS. Higher CaCO3 concentrations (2-3%w/w) caused a significant burst release (>45%) within 30 min, indicating that the effect of CO2 generation was more influential than the effect of increased Ca2+ concentration on alginate crosslinking and gel density. The behavior may be explained by the increase in gas bubbles inside the raft gel, which produces a more porous structure. This in turn allows the aqueous medium to penetrate the raft forming system, and facilitate AS and MS diffusion from the raft gel [17]. These findings are in agreement with an observation of Rosenzweig O et al.(2013) who reported the effect of gas generating agents on the erosion/release rate of gallic acid from the floating controlled release tablet [38]. No significant effect of GR-SD content on AS and MS release from the raft formulations was measured (Fig 8). The optimized raft formulation was designed on the basis of physicochemical analysis as: of 2% alginate, 0.5% HPMC K100, 0.5% CaCO3 , 1.5% NaHCO3 and 1.2% GR-SD. 3.6 Release kinetics and mechanism of release of AS and MS from GR-SD-based raft systems The zero order, first order, Hixson-Crowell, Higuchi, Korsmeyer-Peppas and Weibull kinetic models were applied to the in vitro release data presented in Fig. 8 in order to explore the mechanism of AS and MS release from raft formulations. The Korsmeyer-Peppas and

22

Weibull model provided the best fit of release data (correlation coefficient; R2, close to 1 as shown in Table 2). The n values in the Korsmeyer-Peppas’s equation varied from 0.323 to 0.468 for AS and from 0.319 to 0.458 in the case of MS release, implying that the release mechanism was controlled by Fickian diffusion [39]. In the same way, the exponent b calculated from Weibull model was less than 0.75, indicating Fickian diffusion of the glycosides from the polymeric network in the raft structure [40]. The release kinetics of the raft forming systems are in agreement with the kinetic models analyzed by Kerdsakundee et al [17] and El Nabarawi et al [41]. 3.7 Stability studies

The physicochemical properties of raft forming systems incorporating GR-SD after storage for six months at different temperatures are shown in Table 3 and Fig 9. The percentage of the active compounds AS and MS remaining in the liquid rafting formulations following storage are shown in Fig 9. Almost 100% of the AS and MS content of liquid raft formulations remained stable when stored at 4°C for 6 months and around 90% of the initial content was measured after 6 months storage at 30°C. Thus liquid raft-forming gastroretentive formulations based on GR-SDs maintain high stability of active glycosides during storage at room temperature and below. However, the stability of AS and MS declined significantly on storage of liquid raft formulations at 45°C resulting in 31 and 40% remaining at 6 months (Fig 9). This behavior suggests that temperature is the main factor that accelerates degradation of the glycosides contained in the liquid rafting formulation. In addition, the water content of the formulation could hydrolyze the glycoside structures at the ester bonds,

23

forming sugar molecules and aglycones (asiatic acid or madecassic acid) as evidenced by HPLC assay (data not shown). 3.8 Ulcer healing efficacy on acute gastric ulcers induced by indomethacin in rats

The ulcer healing efficacy of unformulated GR extract and GR-SD-based raft-forming systems on acute gastric ulcers induced by indomethacin are summarized in Table 4. Following oral administration of indomethacin to rats (30 mg/kg), the stomachs of untreated animals (water control) showed ulcerated area of 64.82 mm2 with severe ulcerations including deep streaks and hemorrhagic lesions. Likewise, animals administered vehicle controls (1% CMC) or blank raft formulations revealed unhealed gastric ulcers with ulcerated area of 57.02 and 59.92 mm2 respectively. In contrast, the GR-SD -based raft forming systems administered once daily for two consecutive days, significantly reduced the severity of the ulcer and promoted ulcer healing (p<0.05). The ulcerated areas were reduced significantly from 59.92 mm2 in the vehicle control group to 42.43 mm2 and 22.20 mm2 (representing healing rates of 29% and 63%) for

the raft forming systems containing 5 and 10 mg/kg AS, respectively,

Moreover, the number of animals presenting severe hemorrhagic lesions significantly decreased in the GR-SD 10 rafting formulation group (1 out of 9 rats) compared to the nontreated group (5 out of 7 rats) (p<0.05). The GR-SD-based raft forming system significantly gave rise to improved ulcer healing rate (63%) compared with those of unformulated GR extract (26%) containing the same amount of AS and a standard antiulcer agent: lansoprazole (50%) (p<0.05). This ulcer healing improvement is considered to be resulted from an increase in AS and MS solubility in gastric fluid due to their inclusion in the raft as a solid dispersion

24

with Eudragit® EPO, together with a prolongation of glycoside released in the rat stomach. The present findings are in agreement with a previous study which demonstrated the ulcer healing efficacy of AS on acetic acid-induced chronic gastric ulcer in Sprague-Dawley rats [6]. The antiulcer mechanisms might be accounted from the promotion of the angiogenesis and epithelial cell proliferation. Besides, the reduction of the myeloperoxidase (MPO) enzyme activity, along with the up-regulation of protein bFGF expression at the ulcer tissues might also facilitate the recovery of the gastric wounds.

4. Conclusions

Novel gastro-retentive raft-forming systems incorporating solid dispersions of glycoside-rich Centella asiatica extract and Eudragit ® EPO (GR-SD) were developed. The aqueous solubility and dissolution rate of the glycosides AS and MS present in the solid dispersion were improved due to their conversion to the amorphous form. The AS and MS contents of the liquid rafting formulations were found to be highly stable on storage at 4°C and 30°C for 6 months. The raft-forming systems floated in SGF within 30 sec and gradually released more than 80% of the AS and MS content over 8 h. GR-SD-based raft forming systems, administered orally at a dose of 10 mg AS /kg was superior to unformulated GR and a standard antiulcer agent: lansoprazole in accelerating the healing of acute gastric ulcers induced by indomethacin in Wistar rats. These findings demonstrate the potential of gastroretentive, GR-SD-based raft-forming systems for treatment of gastric ulcers induced by NSAIDs. . Acknowledgments

25

The authors would like to acknowledge the financial support from the Thailand Research Fund under the Royal Golden Jubilee Ph.D. programme (PHD/0180/2556) and Prince of Songkla University (PSU), Thailand. We would like to thank Professor Allan Coombes for his assistance with English editing of the manuscript and scientific advice. References [1]

Y. Yuan, I.T. Padol, R.H. Hunt, Peptic ulcer disease today, Nat. Clin. Pract.

Gastroenterol. Hepatol. 3 (2006) 80-9. [2]

M. Dufon, The pathophysiology and pharmaceutical treatment of gastric ulcers,

Council for Pharmacy Education. 12 (2012) 747-71. [3]

B.T. Kalish, M.W. Kieran, M. Puder, D. Panigrahy, The growing role of eicosanoids in

tissue regeneration, repair, and wound healing, Prostaglandins Other Lipid Mediat. 104–105 (2013) 130-138. [4]

C. Cheng, M. Koo, Effects of Centella asiatica on ethanol induced gastric mucosal

lesions in rats, Life Sci. 67 (2000) 2647-2653. [5]

A. Shukla, A.M. Rasik, B.N. Dhawan, Asiaticoside‐induced elevation of antioxidant

levels in healing wounds, Phytother. Res. 13 (1999) 50-54. [6]

C.L. Cheng, J.S. Guo, J. Luk, M.W.L. Koo, The healing effects of Centella extract and

asiaticoside on acetic acid induced gastric ulcers in rats, Life Sci. 74 (2004) 2237-2249. [7]

J.S. Guo, C.L. Cheng, M.W. Koo, Inhibitory effects of Centella asiatica water extract

and asiaticoside on inducible nitric oxide synthase during gastric ulcer healing in rats, Planta Med. 70 (2004) 1150-1154. [8]

H.Z. Li, J.Y. Wan, L. Zhang, Q.X. Zhou, F.L. Luo, Z. Zhang, Inhibitiory action of

asiaiticoside on collagen-induced arthritis in mice, Acta pharmaceutical Sinica. 42 (2007) 698703.

26

[9]

M. Liu, Y. Dai, X. Yao, Y. Li, Y. Luo, Y. Xia, Z. Gong, Anti-rheumatoid arthritic effect

of madecassoside on type II collagen-induced arthritis in mice, Int. Immunopharmacol. 8 (2008) 1561-1566. [10]

C.Z. Zhang, J. Niu, Y.S. Chong, Y.F. Huang, Y. Chu, S.Y. Xie, Z.H. Jiang, L.H. Peng,

Porous microspheres as promising vehicles for the topical delivery of poorly soluble asiaticoside accelerate wound healing and inhibit scar formation in vitro & in vivo, Eur. J. Pharm. Biopharm. 109 (2016) 1-13. [11]

Y. Lu, N. Tang, R. Lian, J. Qi, W. Wu, Understanding the relationship between

wettability and dissolution of solid dispersion, Int. J. Pharm. 465 (2014) 25-31. [12]

O. Bley, J. Siepmann, R. Bodmeier, Characterization of Moisture‐Protective Polymer

Coatings Using Differential Scanning Calorimetry and Dynamic Vapor Sorption. J. Pharm. Sci. 98 (2009) 651-664. [13]

A. Gryczke, S. Schminke, M. Maniruzzaman, J. Beck, D. Douroumis, Development and

evaluation of orally disintegrating tablets (ODTs) containing Ibuprofen granules prepared by hot melt extrusion. Colloids Surf. B Biointerfaces 86(2011) 275-284. [14]

K. Pietrzak, A. Isreb, M.A. Alhnan, A flexible-dose dispenser for immediate and

extended release 3D printed tablets. Eur. J. Pharm. Biopharm. 96 (2015) 380-387. [15]

T. Kojima, K. Higashi, T. Suzuki, K. Tomono, K. Moribe, K. Yamamoto, Stabilization

of a Supersaturated Solution of Mefenamic Acid from a Solid Dispersion with EUDRAGIT ® EPO. Pharm. Res. 29 (2012) 2777-2791. [16]

T. Yoshida, I. Kurimoto, K. Yoshihara, H. Umejima, N. Ito, S. Watanabe, et al,

Aminoalkyl methacrylate copolymers for improving the solubility of tacrolimus. I: Evaluation of solid dispersion formulations. Int. J. Pharm. 428 (2012) 18-24. [17]

N. Kerdsakundee, S. Mahattanadul, R. Wiwattanapatapee. Development and evaluation

of gastroretentive raft forming systems incorporating curcumin-Eudragit® EPO solid

27

dispersions

for

gastric

ulcer

treatment. European

Journal

of

Pharmaceutics

and

Biopharmaceutics. 2015;94:513-20. [18]

W. Saal, A. Ross, N. Wyttenbach, J. Alsenz, M. Kuentz, Unexpected solubility

enhancement of drug bases in the presence of a dimethylaminoethyl methacrylate copolymer. Mol. Pharm. 15 (2018) 186-192. [19]

W. Saal, A. Ross, N. Wyttenbach, J. Alsenz, M. Kuentz. A systematic study of

molecular interactions of anionic drugs with a dimethylaminoethyl methacrylate copolymer regarding solubility enhancement. Mol. Pharm. 14 (2017) 1243-1250. [20]

V.D. Prajapati, G.K. Jani, T.A. Khutliwala, B.S. Zala, Raft forming system-an upcoming

approach of gastroretentive drug delivery system, J. Control. Release. 168 (2013) 151-165. [21]

A.Y. Kaushik, A.K. Tiwari, A. Gaur, Role of excipients and polymeric advancements

in preparation of floating drug delivery systems, Int. J. Pharm. Investig. 5 (2015) 1-12. [22]

K.G. Mandel, B.P. Daggy, D.A. Brodie, H.I. Jacoby, Review article: alginate-raft

formulations in the treatment of heartburn and acid reflux, Aliment. Pharmacol. Ther. 14 (2000) 669-690. [23]

P. Puttarak, P. Panichayupakaranant, Factors affecting the content of pentacyclic

triterpenes in Centella asiatica raw materials, Pharm. Biol. 50 (2012) 1508-1512. [24]

N. Kaewnopparat, S. Kaewnopparat, A. Jangwang, D. Maneenaun, T. Chuchome, P.

Panichayupakaranant, Increased solubility, dissolution and physicochemical studies of curcumin-polyvinylpyrrolidone K-30 solid dispersions, World Acad. Sci. Eng. Technol. 55 (2009) 229-234. [25]

S. Setthacheewakul,

S. Mahattanadul,

N. Phadoongsombut,

W. Pichayakorn,

R.

Wiwattanapatapee, Development and evaluation of self-microemulsifying liquid and pellet formulations of curcumin, and absorption studies in rats, Eur. J. Pharm. Biopharm. 76 (2010) 475-485.

28

[26]

F. Hampson, A. Farndale, V. Strugala, J. Sykes, I. Jolliffe, P. Dettmar, Alginate rafts and

their characterization, Int. J. Pharm. 294 (2005) 137-147. [27]

Y. Zhang, M. Huo, J. Zhou, A. Zou, W. Li, C. Yao, S. Xie, DDSolver: An add-in

program for modeling and comparison of drug dissolution profiles, AAPS J. 12 (2010) 263271. [28]

International Conference on Harmonisation (ICH). Harmonised Tripartite Guideline.

ICH Expert Working Group. Stability Testing of new drug substances and products Q1A(R2). London: ICH. 2003. pp.1–20. [29]

S. Kuadkaew, ‚Effectiveness of chitosan-curcumin preparation in management of oral

and gastric ulcers in animal models‛ Ph.D. dissertation, Dept. Clin. Pharm. Prince of Songkla university, Songkla 2019. [30]

United States Food and Drug Administration (1997) Guidance for industry: dissolution

testing of immediate release solid oral dosage forms. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). http://www.fda.gov/cder/guidance/.pdf. Accessed 7 Oct 2019 [31]

S. Wannasarit, P. Puttarak, K. Kaewkroek, Wiwattanapatapee R. Strategies for

Improving Healing of the Gastric Epithelium Using Oral Solid Dispersions Loaded with Pentacyclic Triterpene–Rich Centella Extract. AAPS PharmSciTech. 2019;20(7). [32]

S. Baghel, H. Cathcart, N.J. O'Reilly. Polymeric amorphous solid dispersions: a review

of amorphization, crystallization, stabilization, solid-state characterization, and aqueous solubilization of biopharmaceutical classification system class II drugs. J. Pharm. Sci. 105 (2016) 2527–2544. [33]

T. Xie, W. Gao, L.S. Taylor, Impact of Eudragit® EPO and hydroxypropyl

methylcellulose on drug release rate, supersaturation, precipitation outcome and redissolution rate of indomethacin amorphous solid dispersions, Int. J. Pharm. 531 (2017) 313-323.

29

[34]

J. Li, I.W. Lee , G.H. Shin, X. Chen, H.J. Park, Curcumin-Eudragit® EPO solid

dispersion: A simple and potent method to solve the problems of curcumin, Eur. J. Pharm. Biopharm. 94 (2015) 322-332. [35]

F. Meng, A. Trivino, D. Prasad, H. Chauhan, Investigation and correlation of drug

polymer miscibility and molecular interactions by various approaches for the preparation of amorphous solid dispersions, Eur. J. Pharm. Biopharm. 71 (2015) 12-24. [36]

N.A.H.A. Youssef, A.A. Kassem, M.A.E. EL-Massik, N.A. Boraie, Development of

gastroretentive metronidazole floating raft system for targeting Helicobacter pylori, Int. J. Pharm. 486 (2015) 297-305. [37]

J.J. Escudero, C. Ferrero, M.R. Jiménez-Castellanos, Compaction properties, drug

release kinetics and fronts movement studies from matrices combining mixtures of swellable and inert polymers: Effect of HPMC of different viscosity grades, Int. J. Pharm. 351 (2008) 6173. [38]

O. Rosenzweig, E. Lavy, I. Gati, R. Kohen, M. Friedman, Development and in vitro

characterization of floating sustained-release drug delivery systems of polyphenols, Drug Deliv. 20 (2013) 180-189. [39]

S. Dash, P.N. Murthy, L. Nath, P. Chowdhury, Kinetic modeling on drug release from

controlled drug delivery systems, Acta. Poloniae. Pharmaceutica. 67 (2010) 217-223. [40]

V. Papadopoulou, K. Kosmidis, M. Vlachou, P. Macheras. On the use of the Weibull

function for the discernment of drug release mechanisms, Int. J. Pharm. 309 (2006) 44-50. [41]

M.A. El Nabarawi, M.H. Teaima, R.A. Abd El-Monem, N.A. El Nabarawy, D.A. Gaber,

Formulation, release characteristics, and bioavailability study of gastroretentive floating matrix tablet and floating raft system of Mebeverine HCl, Drug Des. Devel. Ther. 11 (2017) 1081-1093.

30

List of tables Table 1 Composition and physical characteristics of GR-SD-based raft forming systems

Table 2 Values of the correlation coefficient for fit of in vitro release data to zero-order, firstorder, Hixson-Crowell, Higuchi, Korsmeyer-Peppas and Weibull kinetic models Table 3 Properties of raft forming systems incorporating GR-SD (AS content 10 mg/mL) after storage for six months at different temperatures Table 4 Effect of unformulated GR extract and GR-SD-based raft formulations on gastric lesion number and healing rate in Wistar rats

31

Figure captions Figure 1 Solubility of asiaticoside (AS) and madecassoside (MS) in unformulated GR extract compared to solid dispersions and physical mixtures of AS and MS with Eudragit ® EPO at the weight ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2. Data represent the average values ± standard deviation of three replicates. Figure 2 Powder X-ray diffractograms of (a) physical mixtures of GR extract and Eudragit ® EPO (GR-PM) and (b) solid dispersion of GR and Eudragit ® EPO (GR-SD) at w/w ratios of 1:0.1 to 1:2 Figure 3 FT-IR spectra of GR extract, Eudragit ® EPO, physical mixtures of GR extract and Eudragit® EPO (GR-PM, ratio 1:0.5) and solid dispersion of GR and Eudragit ® EPO (GR-SD, ratio 1:0.5) Figure 4 In vitro release profiles of asiaticoside (AS) and madecasoside (MS) from unformulated GR extract and SDs (weight ratio 2:1 and 1:1) in 0.1 N HCl (pH 1.2). Bars represent mean ±S.D. (n=3).

Figure 5

Effect of a) Sodium alginate concentrations on the viscosity of liquid raft

formulations incorporating GR-SD at 1%(w/v) calcium carbonate concentration b) Calcium carbonate content on the viscosity of liquid

raft-forming systems, 2% (w/v) alginate

concentration, 0.3% (w/w) GR-SD concentration c) Effect of GR-SD content on the viscosity of liquid raft- forming systems, 2% (w/v) alginate concentration, 0.5% (w/v) CaCO3 concentration. Bars represent mean ± S.D. (n=3).

32

Figure 6 The effect of sodium alginate content (1.0-2.0 %w/w) of raft formulations on AS and MS release profiles in 0.1 N HCl (pH 1.2) within 8 h. Data reported as mean ±SD (n=3). Figure 7 The effect of calcium carbonate content (0.5-3%w/w) of raft formulations on AS and MS release profiles in 0.1 N HCl (pH 1.2).). Data reported as mean ±SD (n=3). Figure 8 The effect of AS and MS content (10-40 mg) of raft formulations on MS and AS release profiles in 0.1 N HCl (pH 1.2) within 8 h. Data reported as mean ±SD (n=3). Figure 9 Percentage AS and MS remaining in liquid raft-forming systems following storage at a) 4°C, in tightly capped bottle and protected from light b) 30oC/65% RH and c) 45oC/75%RH for one, three and six months.

graphical abstract

33 Table 2 Composition and physical characteristics of GR-SD-based raft forming systems

Raft strength (g)

Density

Floating

Viscosity

(g/mL)

lag time

at 90 rpm

(s)

(mPa“s)

0.520 ±0.013

4

362.6 ±12.1

7.2 ±0.3

0.3

0.645 ±0.009

9

904.2 ±5.4

7.8 ±0.2

1

0.3

0.734 ±0.002

30

2457.8 ±3.3

9.8 ±0.5

-

1

0.3

0.686 ±0.006

480

1598.0 ±6.4

13.7 ±0.6

2

0.5

0.5

0.3

0.713 ±0.010

21

2242.9 ±3.8

9.3 ±0.6

F6

2

0.5

2

0.3

0.737 ±0.003

21

2336.2 ±3.9

11.0 ±0.2

F7

2

0.5

3

0.3

0.750 ±0.002

18

2379.5 ±0.0

12.0 ±0.3

F8

2

0.5

0.5

0.6

0.720 ±0.009

30

2194.6 ±3.3

8.9 ±0.2

F9

2

0.5

0.5

0.9

0.722 ±0.004

24

2204.8 ±4.3

9.0 ±0.3

F10

2

0.5

0.5

1.2

0.724 ±0.016

22

2192.9 ±0.0

8.7 ±0.4

Sodium alginate (% w/v)

HPMC K100 (% w/v)

CaCO3 (% w/v)

F1

1

0.5

1

0.3

F2

1.5

0.5

1

F3

2

0.5

F4

2

F5

No.

*NaHCO3 1.5 % w/w

GR-SD (% w/v)

34 Table 2 Values of the correlation coefficient for fit of in vitro release data to zero-order, first-order, Hixson-Crowell, Higuchi, Korsmeyer-Peppas and Weibull kinetic models Zeroorder (R2)

Firstorder (R2)

HixsonCrowell (R2)

Higuchi (R2)

F5

0.8527

0.9692

0.9490

F8

0.8890

0.8812

F9

0.8943

F10

MS release

AS release

Korsmeyer-Peppas

Weibull

R2

n

R2

B

0.9538

1.0

0.468

0.9975

0.638

0.9691

0.9730

1.0

0.326

0.9941

0.545

0.8752

0.9531

0.9763

0.9994

0.323

0.9984

0.484

0.8891

0.9530

0.9479

0.9741

0.9980

0.441

0.9992

0.569

Zeroorder (R2)

Firstorder (R2)

HixsonCrowell (R2)

Higuchi (R2)

Korsmeyer-Peppas R2

n

R2

B

F5

0.8567

0.9583

0.9666

0.9554

0.9952

0.445

0.9967

0.666

F8

0.8613

0.8829

0.9544

0.9589

0.9993

0.319

0.9973

0.522

F9

0.8864

0.8961

0.9617

0.9724

0.9999

0.354

0.9978

0.539

F10

0.8622

0.9552

0.9528

0.9596

0.9988

0.458

0.9994

0.615

Weibull

*Raft formulations F5, 8, 9 and 10 contained GR-SD with equivalent AS and MS content of 10, 20, 30 and 40 mg respectively. R2 refers to the correlation coefficient; n and b refer to the release exponent and the constant exponent respectively in the Korsmeyer-Peppas and Weibull kinetic models.

35 Table 3 Properties of raft forming systems incorporating GR-SD (AS content 10 mg/ mL) after storage for six months at different temperatures Formulations

pH

Floating lag time (s)

Floating duration (h)

Viscosity (at 90 rpm)

Raft strength (g)

Freshly prepared

9.6±0.1

21±5

>8

2242.9 ±3.8

9.3 ±0.6

Storage at 4 ±2°C

9.7±0.2

23 ±2

>8

2106.2 ±5.4

7.7 ±0.3

Storage at 30 ±2°C / 65% RH

10.0±0.1

20 ±4

>8

1313.1 ±0.5

6.7 ±0.6

Storage at 45 ±2°C /75%RH

10.3±0.1

63 ±6

>8

178.3 ±6.4

5.7 ±0.4

Table 4 Effect of unformulated GR extract and GR-SD-based raft formulations on acute gastric ulcerations induced by indomethacin in Wistar rats Group No.

Experimental group

1

Water control

2

1% CMC

3

Blank raft formulation Lansoprazole

4 5

GR10 extract

Dose (mg/kg)

No of rat

Total ulcerations (mm2 ± SEM)

Ulcer healing rate

7

No of rats presenting hemorrhagic ulcerations 5

-

64.8 ±2.7

7

4

57.0 ±2.7

7

4

59.9 ±3.2

-

6

1

28.5 ±3.9*

50.1

10

9

6

44.4 ±2.7*

25.8

#

1 #

(%)

6

GR-SD 5 rafting formulation

5

8

3

42.4 ±2.8*

29.2

7

GR-SD10 rafting formulation

10#

9

1

22.2 ±2.4*

63.0

Water, 1% CMC and lansoprazole were used as ulcerated, vehicle and positive controls, respectively . Blank rafting formulation was used as a control for drug-loaded rafting formulation groups.

#

Equivalent dose of asiaticoside content.

* p-value

< 0. 05 ; statistically different compared to vehicle control

formulation.

( 1% CMC) or

blank raft

36

35.0

Solid dispersions 41.72

45.0

Physical mixtures

Solubility of MS (mg/mL)

Solubility of AS (mg/mL)

50.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0

4.69

Solid dispersions 29.30

Physical mixtures

30.0 25.0 20.0 15.0 9.46

10.0

5.0

5.0 0.0

0.0 GR extract

1:0.1

1:0.5

1:1

1:1.5

1:2

GR extract

Weight ratios

1:0.1

1:0.5

1:1

1:1.5

1:2

Weight ratios

Figure 1 Solubility of asiaticoside (AS) and madecassoside (MS) in unformulated GR extract compared to solid dispersions and physical mixtures of AS and MS with Eudragit® EPO at the weight ratios of 1:0.1, 1:0.5, 1:1, 1:1.5 and 1:2. Data represent the average values ± standard deviation of three replicates.

GR-extract GR-PM 1:0.1 GR-PM 1:0.5 GR-PM 1:1 GR-PM 1:1.5 GR-PM 1:2

a 14000

12000

b

Eudragit EPO GR-SD 1:0.1 GR-SD 1:0.5 GR-SD 1:1 GR-SD 1:1.5 GR-SD 1:2

14000

12000

Intensity counts

Intensity counts

10000

10000

8000

6000

8000

6000

4000 4000

2000

2000

0 0

5

10

15

20

25

30 2Ө

35

40

45

50

5

10

15

20

25

2Ө

30

35

40

45

Figure 2 Powder X-ray diffractograms of (a) physical mixtures of GR extract and Eudragit® EPO (GRPM) and (b) solid dispersion of GR and Eudragit® EPO (GR-SD) at w/w ratios of 1:0.1 to 1:2

50

37

300.000 0

250.000 0

200.000 0

GR-extract

150.000 0

Eudragit EPO

GR-PM 1:0.5

100.000 0

GR-ESD 1:0.5 50.0000

0.0000

4000

3600

3200

2800

2400

2000

1600

1200

800

400

Figure 3 FT-IR spectra of GR extract, Eudragit® EPO, physical mixtures of GR extract and Eudragit® EPO (GR-PM, ratio 1:0.5) and solid dispersion of GR and Eudragit® EPO (GR-SD, ratio 1:0.5)

120

100 80 60 40

SD 2:1 SD 1:1 GR extracts

20 0 0

20

40

60

80

Time (min)

100

120

140

% Cumulative release of MS (w/w)

% Cumulative release of AS (w/w)

120

100 80 60 40

SD2:1 SD1:1 GR extracts

20 0 0

20

40

60

80

100

120

140

Time (min)

Figure 5 In vitro release profiles of asiaticoside (AS) and madecasoside (MS) from unformulated GR extract and SDs (weight ratio 2:1 and 1:1) in 0.1 N HCl (pH 1.2). Bars represent mean ±S.D. (n=3).

38 F3: Alg 2% F4: Alg2%, no HPMC F2: Alg 1.5% F1: Alg 1%

a 4000 Viscosity (mPa.s)

3500 3000 2500 2000 1500

1000 500 0 0.0

10.0

20.0

30.0

40.0

Shear rate (1/s) b

F5: 0.5% Calcium carbonate

4000

c

F6: 2% Calcium carbonate 3000 2500 2000 1500 1000 500

F10: GR-ESD 1.2% F8: GR-ESD 0.6% F5: GR-ESD 0.3%

3500

F7: 3% Calcium carbonate

Viscosity (mPa.s)

Viscosity (mPa.s)

3500

4000

3000 2500 2000 1500 1000 500

0

0 0.0

10.0

20.0

30.0

40.0

Shear rate (1/s)

Figure 5

0.0

10.0

20.0

30.0

40.0

Shear rate (1/s)

Effect of a) Sodium alginate concentrations on the viscosity of liquid raft

formulations incorporating GR-SD at 1%(w/v) calcium carbonate concentration b) Calcium carbonate content on the viscosity of liquid

raft-forming systems, 2% (w/v) alginate

concentration, 0.3% (w/w) GR-SD concentration c) Effect of GR-SD content on the viscosity of liquid raft- forming systems, 2% (w/v) alginate concentration, 0.5% (w/v) CaCO3 concentration. Bars represent mean ± S.D. (n=3).

100 90 80 70 60 50 40 30 20 10 0

F1: Alg 1% F2: Alg 1.5% F3: Alg 2% F4: Alg 2% without HPMC

0

1

2

3

4 5 Time (h)

6

7

8

% Cumulative release of MS (w/w)

% Cumulative release of AS (w/w)

39

100 90 80 70 60 50 40 30 20 10 0

9

F1: Alg 1% F2: Alg 1.5% F3: Alg 2% F4: Alg 2% without HPMC

0

1

2

3

4 5 Time (h)

6

7

8

9

Figure 6 The effect of sodium alginate content (1.0-2.0 %w/w) of raft formulations on AS and

110 100 90 80 70 60 50 40 30 20 10 0

% Cumulative release of MS (w/w)

% Cumulative release of AS (w/w)

MS release profiles in 0.1 N HCl (pH 1.2) within 8 h. Data reported as mean ±SD (n=3).

F5: Calcium carbonate 0.5% F3: Calcium carbonate 1% F6: Calcium carbonate 2% F7: calcium carbonate 3%

0

1

2

3

4 5 Time (h)

6

7

8

9

110 100 90 80 70 60 50 40 30 20 10 0

F5: Calcium carbonate 0.5% F3: Calcium carbonate 1% F6: Calcium carbonate 2% F7: Calcium carbonate 3%

0

1

2

3

4 5 Time (h)

6

7

8

9

Figure 7 The effect of calcium carbonate content (0.5-3%w/w) of raft formulations on AS and MS release profiles in 0.1 N HCl (pH 1.2) within 8 h. Data reported as mean ±SD (n=3).

40

100 % Cumulative release of MS (w/w)

% Cumulative release of AS (w/w)

100

90 80 70 60 50 40 F5: AS 10 mg

30

F8: AS 20 mg

20

F9: AS 30 mg

10

F10: AS 40 mg

90 80 70 60 50 40 F5: AS 10 mg

30

F8: AS 20 mg

20

F9: AS 30 mg

10

F10: AS 40 mg

0

0 0

1

2

3

4 5 Time (h)

6

7

8

9

0

1

2

3

4 5 6 Time (h)

7

8

9

Figure 8 The effect of drug content (10-40 mg of AS) of raft formulations on AS and MS release profiles in 0.1 N HCl (pH 1.2) within 8 h. Data reported as mean ±SD (n=3).

Figure 9 Percentage AS and MS remaining in liquid raft-forming systems following storage at 3 conditions including 1. 4°C, in tightly capped bottle and protected from light 2. 30oC/65%RH and 3. 45oC/75%RH for one, three and six months.