Preparation of Curcuma comosa tablets using liquisolid techniques: In vitro and in vivo evaluation

Accepted Manuscript Preparation of Curcuma comosa tablets using liquisolid techniques: In vitro and in vivo evaluation Napaphak Jaipakdee, Ekapol Limpongsa, Bung-orn Sripanidkulchai, Pawinee Piyachaturawat PII: DOI: Reference:

S0378-5173(18)30766-X https://doi.org/10.1016/j.ijpharm.2018.10.031 IJP 17849

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

2 August 2018 7 October 2018 10 October 2018

Please cite this article as: N. Jaipakdee, E. Limpongsa, B-o. Sripanidkulchai, P. Piyachaturawat, Preparation of Curcuma comosa tablets using liquisolid techniques: In vitro and in vivo evaluation, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.10.031

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Title. Preparation of Curcuma comosa tablets using liquisolid techniques: In vitro and in vivo evaluation Author names and affiliations. Napaphak Jaipakdee1,2, E-mail address: [email protected] Ekapol Limpongsa1,2*, E-mail address: [email protected] Bung-orn Sripanidkulchai1,2, E-mail address: [email protected] Pawinee Piyachaturawat3 E-mail address: [email protected] 1 Center

for Research and Development of Herbal Health Products, Faculty of Pharmaceutical

Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand 2 Division

of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Khon Kaen

University, Khon Kaen, 40002, Thailand 3

Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400,

Thailand Corresponding author*. Asst. Prof. Dr. Ekapol Limpongsa, E-mail address: [email protected] (E. Limpongsa) Division of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand Tel: +66818024006, Fax: +6643362092

Abstract Curcuma comosa (C. comosa) is a Thai medicinal herb that provides numerous pharmacologic activities due to its estrogen-like action. This study aimed to investigate the use of liquisolid technique to prepare tablets containing oleoresin-like crude extract of C. comosa, and to improve the dissolution profiles of its major compounds, diarylheptanoids (DAs). Free flowing powders of C comosa extract were obtained by adsorption onto solid carriers, microcrystalline cellulose, with colloidal silica as coating material. FTIR results ruled out possible interactions between C. comosa extract and excipients. The results indicated that all of liquisolid tablets met the USP requirements. The release of DAs were significantly increased through liquisolid formulations, compared to crude extract. By decreasing the ratio of carrier to coating from 20 to 10, an improvement in dissolution rate was observed. Addition of additives - namely polymer (polyvinyl pyrrolidone) and/or nonvolatile liquid (propylene glycol) affected tablet properties which involved longer disintegration time and lower DA dissolution. Optimized C. comosa liquisolid formulation was prepared in a carrier to coating ratio of 10 without additives. Stability studies showed that physical properties of liquisolid tablet were not affected by aging, but percent remaining and dissolution profiles of DAs were influenced by storage temperature. In vivo pharmacokinetic behavior of the optimized C. comosa liquisolid tablets was investigated following a single oral administration to rabbits. The results proved that the method used for preparation of liquisolid led to C. comosa tablets with low variation in content uniformity and tablet properties, as well as enhanced dissolution behavior. Keywords: Curcuma comosa; Diarylheptanoids; Liquisolid tablets; Dissolution enhancement; Pharmacokinetic

1. Introduction Phytoestrogens constitute a promising alternative in the treatment of diseases associated with menopause. Phytoestrogens are naturally occurring plant non-steroidal compounds that are structurally and/or functionally similar to estrogens (Poluzzi et al., 2014). Simultaneously, phytoestrogens can induce biological responses in vertebrates, and mimic or modulate the actions of endogenous estrogens, usually by binding to estrogen receptors (Winuthayanon et al., 2009a). Recently, diarylheptanoids (DAs) isolated from Curcuma comosa Roxb., have been identified as phytoestrogens as they exhibited, selectively through ERα, estrogenic-like functions activity both in vitro and in vivo (Suksamrarn et al., 2008; Winuthayanon et al., 2009a; Winuthayanon et al., 2009b). Curcuma comosa (C. comosa), a plant belonging to the family Zingiberaceae, is an indigenous Thai medicinal herb and has traditionally been used for treatment of reproductive disorders in women, and for relief of unpleasant menopausal symptoms among postmenopausal women (Bhukhai et al., 2012; Weerachayaphorn et al., 2011). The extract of C. comosa rhizomes exhibited an estrogenic-like activity and induced cornification of the vaginal epithelium in the smear and keratinization of the mucosal surface of the vagina (Piyachaturawat et al., 1995a, 1995b). Additionally, reported pharmacologic activities of C. comosa extract included hypocholesterolemic effect (Piyachaturawat et al., 1997, 1999), antiinflammatory effects (Sodsai et al, 2007; Sornkaewa et al., 2015), osteoporosis prevention (Tantikanlayaporn et al., 2013; Weerachayaphorn et al., 2011), and improvement of learning and memory (Su et al., 2010, 2011). Amongst several DAs purified from C. comosa extract, two specific molecules, (4E,6E)-1,7-diphenylhepta-4,6-dien-3-ol (DA1) and (6E)-1,7diphenylhept-6-en-3-ol (DA2) (Fig. 1a), were reported as the major compounds, and their pharmacological effects were confirmed (Bhukhai et al., 2012; Su et al., 2012; Suksamrarn et al, 2008; Winuthayanon et al., 2009a, 2009b). The previous report on oral administration of C. comosa hexane extract in rats revealed that DAs, depending on the administration dose, had low oral bioavailability (approximately 17–35%) and fast elimination characteristics. The bioavailability of DA1 and DA2 (125 mg/kg) in rats were only 24.0% and 34.6%, respectively (Su et al., 2012). Based on their chemical structures as shown in Fig.1a, DAs are considered to have low water solubility, and lipophilic molecules with the log P values of 4.36 and 4.87 for DA1 and DA2, respectively (Su et al., 2013). The poor water solubility of these DAs was of concern when considering an appropriate formulation for sufficient systemic delivery of these active phytoestrogens. When given orally, the active compound must be presented in solution form

for absorption through gastrointestinal tract. Therefore, solubility is one of the most important factors, and dissolution is the rate-limiting step in absorption process (Hoerter and Dressman, 2001). To improve the dissolution rate and/or bioavailability of these compounds, drug delivery systems or formulation techniques are needed. Su and coworker (2013) had developed the oil-in-water nanoemulsion C. comosa extract which was composed of C. comosa hexane extract (0.83 mg/mL loading), nonionic surfactants, olive oil, and water. The in situ intestinal absorption study revealed that absorption rates of DAs in nanoemulsions were five to ten times faster than the rates of those prepared as oil-based formulations. Nevertheless, manufacturing of nanoemulsion is an expensive process which requires special process and instruments. Additionally, nanoemulsion formulations present low loading capacity, which make them not suitable for crude extracts, and may potentially pose longterm stability problems. Another major concern facing the formulation designed for crude extracts is that C. comosa extract requires high loading dose in order to boost plasma concentration level of active constituents after oral administration. However, a recent report of in vitro permeation data of DAs has suggested the feasibility of transdermal delivery (Tuntiyasawasdikul et al., 2017). Oral route has been considered as the most preferred and frequently employed means of drug administration route amongst the others due to its ease of administration, formulation design flexibility, cost effectiveness, high patient compliance, and least sterility restrictions during manufacturing. One of the promising methods to enhance dissolution, and in turn improve bioavailability of poorly water-soluble drugs is liquisolid technique. The concept of liquisolid systems was developed from the powdered solution technology that could be used to formulate liquid medication (Elkordy et al., 2013). A “liquisolid system” refers to formulations formed by conversion of liquid medicaments into dry, nonadherent, free-flowing, and compressible powder mixtures by mixing the liquid medicaments with appropriate carriers and coating materials. The term “liquid medication” implies oily liquid drugs and solutions or suspensions of water insoluble solid drugs carried in suitable nonvolatile systems (liquid vehicles) (Javadzadeh et al., 2007a; Lu et al., 2017b; Spireas and Sadu, 1998). Liquisolid formulations offer tablets or powders, to be filled into capsules, dosage forms. The enhanced drug dissolution was attributable to increased surface area and aqueous solubility, and improved wettability of drug particles (Khadka et al, 2014). Aging studies with liquisolid systems of numerous compounds revealed that different conditions of storage neither had an effect on their flow nor release properties (Adibkia et al., 2014; Javadzadeh et al., 2007b, 2008; Lu et al., 2017a, 2017b; Singh et al. 2012). Additionally, because of its advantages, including simplicity, cost effectiveness and

industrial production capability, liquisolid technology had been used in several studies to improve dissolution rates of poorly water soluble drugs, including hydrocortisone (Spireas et al., 1998), indomethacin (Nokhodchi et al., 2005), carbamazepine (Javadzadeh et al., 2007a), piroxicam (Javadzadeh et al., 2005), famotidine (Fahmy and Kassem, 2008.), naproxen (Tiong and Elkordy, 2009), curcumin (Sharma and Pathak, 2016), itraconazole (Gong et al., 2016), tadalafil (Lu et al., 2017a), and risperidone (Khames A. 2017). Liquisolid techniques have been successfully applied to chemical drug compounds in most cases, and only a few literatures have reported the application of these techniques with herbal extracts. Nevertheless, up until now, none of these studies has revealed the effect of liquisolid technique application on the dissolution or in vivo plasma blood concentration of herbal active markers. Given its definition and advantages, the use of liquisolid technique to prepare C. comosa extract and improve its dissolution rate should be of interest. In addition, similar to other crude extracts from plant rhizomes, C. comosa extract are oreoresin-like, viscous liquid in nature as shown in Fig. 1b. Therefore, this study aimed to utilize the liquisolid to prepare tablets containing crude extract of C. comosa. In vitro and in vivo characteristics of C. comosa tablets were evaluated for the first time. In the present study, two major active compounds, DA1 and DA2, were selected to be the markers. The effect of nonvolatile liquid, propylene glycol (PG), and polymer, polyvinyl pyrrolidone (PVP), on the C. comosa liquisolid systems was investigated. Interaction between the C. comosa extract and excipients was studied using FTIR technique. The effects of aging on the tablets and dissolution properties of C. comosa liquisolid tablets were also investigated. 2. Materials and methods 2.1. Materials Fresh C. comosa rhizomes were harvested from Nakon Pathom Province, Thailand. Standard major compounds of diarylheptanoids (DAs), namely DA1 and DA2, were obtained from Professor Apichart Suksamrarn, Ramkhamhaeng University, Thailand. Microcrystalline cellulose (MCC; Avicel® PH102) was provided by Onimax Co., Ltd (Bangkok, Thailand). Colloidal silicon dioxide (colloidal silica, Aerosil® 200) and polyvinyl pyrrolidone K30 (PVP; Plasdone K-29/32) were purchased from Maxway Co. Ltd (Bangkok, Thailand). Croscarmellose sodium (Ac-Di-Sol®) and magnesium stearate were received from S. Tong Chemicals, Bangkok, Thailand. Sodium carboxy methyl cellulose was provided by Ashland® (Switzerland). Sodium lauryl sulfate (SLS) was purchased from LOBA CHEMIE PVT.LTD (Mumbai, India). Ethanol was sourced from the Liquor Distillery Organization

(Chachoengsao, Thailand). Propylene glycol (PG) was purchased from Ajax finechem (Auckland, New Zealand). Absolute ethanol and HPLC grade methanol were purchased from RCI Labscan (Bangkok, Thailand) and Fisher® Scientific (Loughborough, England), respectively. 2.2 Animals Female New Zealand white rabbits (weighing 2.0-2.5 kg) were used as the animal models for in vivo pharmacokinetic experiments. Three healthy rabbits were obtained from the National Laboratory Animal Center (Mahidol University, Nakhon Pathom, Thailand). The animal handling was under supervision of the certified veterinarian of the Northeast Laboratory Animal Center, Khon Kaen University, Thailand. The rabbits were individually housed under standard conditions for one week prior to the experiments. The temperature of animal room was maintained at 23 ± 2 °C and 45 ± 5% relative humidity (RH) with a 12 h light/dark cycle. Except for the final 12 h before experimentation, the rabbits had access to food and water, ad lib. The experimental protocol for animal handling and treatment was reviewed and approved by the Animal Ethics Committee for Use and Care of Khon Kaen University, based on the Ethic of Animal Experimentation of National Research Council of Thailand (AEKKU 70/2556). 2.3 Preparation of C. comosa extract C.

comosa

ethanolic

crude

extract

was

prepared

using

maceration

method

(Tuntiyasawasdikul et al., 2017). The powders of C. comosa dry rhizome were macerated in 95% ethanol for 7 days with frequent stirring. After filtering through Whatman® No.1 paper, the liquid filtrate was collected and evaporated under rotary vacuum evaporator (SB-1000, Eyela®, Japan) to a constant weight. The obtained extract was collected and kept at -40 °C until used. 2.4 Calculation of the loading factor (Lf) In the present study, MCC and colloidal silica were used as carrier and coating material, respectively. The loading factor or binding capacity of carrier coating system for the C. comosa extract, defined as the capacity of powder excipients to hold liquid while maintaining acceptable flow and compression properties, was determined using the method modified from Javadzadeh and coworkers (2007a). Under this method, a constant weight of 2 g of the C. comosa extract was put into a glass mortar, and powder excipients (binary mixture of carrier– coating materials) were added in increments of 0.1 g. The mixture was triturated after each addition to help distribution of the liquid throughout the powder particles, and blended for 10 min. Addition of powder and the trituration were continued until the contents started to look

like dry powder and the powder flow rates, determined using a flow through an orifice (glass funnel) method (USP 39-NF 34), were higher than 10 cm3/s (Adibkia et al., 2014; Javadzadeha et al., 2008). To investigate the effect of PG and PVP on the liquid loading, PG alone or PG-PVP (20% and 30% w/w PVP in PG solution) was firstly mixed with C. comosa extract in a glass mortar. The powder excipients were added in increments until the dry powder mixture obtained was as described earlier. By using Eq. (1), where 𝑊 is the amount of C. comosa extract and 𝑄 is the amount of carrier material, the values of liquid loading factor (𝐿𝑓) (Spireas et al., 1998) were calculated and used to determine the amounts of carrier and coating materials in each formulation. 𝐿𝑓 =

𝑊

𝑄 (1)

2.5 Pre-compression studies 2.5.1 Flow properties of liquisolid powders Carr’s compressibility index (CI) was used to evaluate the flow properties of powders. A weighed quantity of the prepared powder admixture was carefully poured into 100 mLgraduated cylinder. The volume occupied by the powder was read, and the bulk density was calculated in g/mL. The tapped density was calculated using tapped volume which was obtained after sufficient taps. CI of each powder mix was calculated using Eq. (2) (Carr, 1965). 𝐶𝐼 (%) =

(𝑇𝑎𝑝𝑝𝑒𝑑 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 ‒ 𝐵𝑢𝑙𝑘 𝑑𝑒𝑛𝑠𝑖𝑡𝑦) × 100 𝑇𝑎𝑝𝑝𝑒𝑑 𝑑𝑒𝑛𝑠𝑖𝑡𝑦

(2)

2.5.2 Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) ATR-FTIR spectra for C. comosa extract, MCC, colloidal silica, PVP, PG, DC and liquisolid formulations were measured using Fourier transform infrared spectrometer (TENSOR 27, BRUKER, England) under ambient conditions. The detector was deuterated triglycine sulfate (DTGS) with a single-reflection diamond ATR sampling module. ATR-FTIR analysis was conducted over a frequency range of 4000–600 cm-1. All spectra were recorded at a resolution of 4 cm-1. The spectra of liquisolid powder mixtures were compared with those of C. comosa extract to detect interactions between them. 2.5.3 Scanning electron microscopy (SEM) SEM was used to examine the shape and surface of MCC, colloidal silica particles and C. comosa liquisolid systems. Prior to examination, samples were mounted on an aluminum stub, using double adhesive carbon films, and sputter coating was applied under vacuum to render the samples electrically conductive. SEM images were recorded using a field emission

scanning electron microscope (FESEM) with a focused ion beam (FIB) microscope (FIBFESEM) (FEI Helios Nanolab G3 CX DualBeam, USA). 2.6 Preparation of C. comosa liquisolid tablets Several C. comosa liquisolid tablets, symbolized as CLS1 to CLS6 (Table 1) were prepared in 50 tablet batches and compressed into 13-mm diameter flat-faced tablets. Depending on the formulation compositions, different liquid loading factors ranging from 0.26-0.55 were employed. In order to ensure the homogeneity of content, C. comosa extract was firstly dissolved in absolute ethanol, with the ratio of 1:3 of C. comosa extract to absolute ethanol. The C. comosa ethanolic solution was then added to the carrier materials with application of continuous mixing in a glass mortar. The obtained mass was ambient dried for 15 min, and subsequently oven-dried at 50°C for 45 min. Colloidal silica, i.e. the coating, was then added to convert the sticky mixture into dry powder with application of continuous mixing. Finally, the C. comosa liquisolid powder was mixed with 4 %w/w croscarmellose sodium, and then with 0.5 %w/w magnesium stearate for a period of 10 min each. The final mixture was filled manually into the punches and compressed at a compression pressure of 100 kgf/m2 with a hydrostatic press (Model 3126, Shimadzu, Kyoto, Japan) with no holding time. For the formulations containing PVP-PG mixture, the required quantities of PVP and PG were dissolved with C. comosa extract ethanolic solution. The resulting mixture was then added into the carrier material. After being oven-dried, the sticky powder mass was mixed with colloidal silica, croscarmellose sodium, magnesium stearate and compressed into the punches as described earlier. 2.7 Evaluation of C. comosa liquisolid tablets After a relaxation time of at least 24 h after tablet manufacture, tablets were assessed through tests for weight variation (homogeneity of weight), thickness, hardness, friability, disintegration, content uniformity and dissolution. 2.7.1 Weight variation Twenty tablets were randomly selected from each batch and individually weighed using an electronic balance (GF-600, AND®, Japan). The average weight of all tablets and percentage deviation from the mean value for each tablet were determined. 2.7.2 Thickness The thickness of the tablets was determined using a thickness gauge (SM-112, Teclock®, Japan). Six tablets from each batch were used and average values were calculated. 2.7.3 Tablet hardness

The hardness expressed as the force in Newton required to crush the CLS tablets was evaluated using a VanKel hardness tester (VK200, Germany). 2.7.4 Friability test A sample of 10 tablets was taken and carefully dedusted prior to testing. The tablets were accurately weighed and placed in the drum of the friabilator (VanKel friabilator, Germany). The drum was rotated 100 times at 25 rpm, and the tablets were removed, dedusted and accurately weighed. The percentages of friability were calculated using the following equation: 𝑓 = 100 (1 ‒

𝑊

𝑊0) (3)

where 𝑊 = final weight of tablets and 𝑊0 = original weight of tablets. 2.7.5 Disintegration time The disintegration test was performed at 37 ± 0.5 °C in distilled water for six tablets from each formulation, using a basket-rack assembly disintegration test apparatus (Model QC-21, Hanson Research, Northridge, CA). The average disintegration time was calculated. 2.7.6 Content uniformity Each tablet was crushed and grinded to fine powders in a mortar. The powders were transferred to erlenmeyer flasks, and 200 mL of methanol was then added to each tablet. The mixture was ultrasonicated for 60 min and then shaken for 6 h to dissolve and completely extract DA1 and DA2. After filtration (0.45 μm Millipore membrane, CNW® technology, China) and appropriate dilutions were performed, the samples were analyzed using HPLC assay. The percentages of individual DA1 and DA2 contents were calculated and compared to the theoretical loading content. 2.7.7 Dissolution studies The in vitro dissolutions of DA1 and DA2 from C. comosa liquisolid tablets were measured using USP paddle method (Hanson Research, USA). The 500 mL medium was kept at 37 ± 0.5 °C and the rotating speed was 100 ± 2 rpm. Distilled water containing 1% (w/v) SLS was used as dissolution media. At selected time intervals for a period of 120 min, aliquots of 5 mL each was withdrawn from the dissolution medium through a 0.45 μm Millipore membrane (CNW® technology, China) and replaced with an equivalent amount of the fresh dissolution medium. The DA1 and DA2 contents were measured using HPLC with UV detector at 260 nm. The dissolution of DAs from C. comosa crude extract was also confirmed. C. comosa extract, 125 mg, was manually filled into the hard gelatin capsule (size 1) and tested for the

dissolution of DAs using the method and condition applied to C. comosa liquisolid tablets. The DA content was determined using HPLC method. Dissolution parameters: In order to assess the comparative extent of the dissolution enhancement from C. comosa liquisolid formulations, the mean dissolution time or MDT (min) and dissolution efficiency (DE) were calculated. The MDT is defined as the time required for 50% DAs dissolved. The DE is defined as the area under the dissolution curve up to the time, t, expressed as the percentage of the area of the rectangle described by 100% dissolution over the same time period (Nokhodchi et al., 2010). The DE values after 120 min (𝐷𝐸120) were calculated using the following equation: ∫𝑡 𝑦 × 𝑑𝑡

𝐷𝐸120 =

0

𝑦100 × 𝑡

× 100% (4)

where 𝑦 is the percent of DAs dissolved at the time 𝑡, 120 min. 2.8 Stability studies Stability testing of the selected C. comosa liquisolid formulation was performed at long term (30 ± 2 °C/75 ± 5 %RH) and accelerated (40 ± 2 °C/75 ± 5 %RH) conditions for six months (International Conference on Harmonisation, 2003). The DA1 and DA2 contents, as well as the physical appearance, hardness, disintegration time, and dissolution characteristics of aged tablets were evaluated and compared with those of freshly prepared tablets. For dissolution profile assessment and comparison, the similarity factor (𝑓2) was calculated (Javadzadeh et al., 2007a). Results were quoted as significant when 𝑓2 value was less than 50. 2.9 HPLC determination of DA1 and DA2 The quantities of DA1 and DA2 were determined using a validated HPLC assay as reported by Tuntiyasawasdikul et al., 2017. Chromatography was performed on a C18 reversed phase column (Agilent ZORBAX Eclipse Plus column, 100 mm × 4.6 mm, 3.5 μm) attached to a model 1200 series HPLC system (Agilent®, Germany). The analytical system included a quaternary pump with 1260 Infinity VWD detector set at 260 nm. The column temperature was set at 30 °C. The mobile phase consisted of a mixture of water and delivery of methanol (30:70 to 40:60) at 1.0 mL/min, with the injection volume at 20 µl. The method exhibited good linearity (R2 > 0.9999) over the assayed concentration range (1-50 μg/mL), high precision (RSD < 2%) and acceptable recovery (96.02–100.78%, RSD < 2%). The limit of detection and limit of quantification of DA1 and DA2 were 0.2 and 0.5 µg/mL, and 0.4 and 1.0 µg/mL, respectively.

2.10 In vivo pharmacokinetic study A single dose pharmacokinetic study of the selected C. comosa liquisolid tablets was conducted in New Zealand white rabbits. The animals were fasted overnight prior to drug administration and 4 h thereafter, with water allowed. Each rabbit was administered a single oral dose of C. comosa liquisolid tablet, equivalent to 125 mg/kg of C. comosa extract (containing DA1 and DA2 at doses of 15.1 and 11.1 mg/kg) (Huang et al., 2013; Ishikawa et al., 2000). Blood samples were collected from the marginal ear vein before the dose administration, and at 0.5, 1, 2, 3, 6, 12, 24 and 48 h after the dose administration, and transferred into heparinized Eppendorf tubes. Plasma sample processing: The whole blood was extracted and prepared for HPLC analysis as previously described (Su et al., 2013). One mL of whole blood was added with 3.0 mL of ethyl acetate to extract the DA1 and DA2. The mixture was vortexed for 1 min and then ultrasonicated for 10 min, followed by centrifugation at 7,000 rpm for 10 min. The supernatant was withdrawn and transferred to the evaporating disc. The extraction process was repeated three times. All of the supernatants were pooled and air-dried. The remaining solid residue was reconstituted with 200 μL of methanol and 20 μL was injected onto the HPLC for analysis as mentioned in 2.9. Pharmacokinetic analysis: The pharmacokinetic parameters, namely maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were obtained directly from the plasma concentration-time data. The area under the plasma concentration-time curve from 0 to 48 h (AUC) was calculated using the trapezoidal rule. 2.11 Statistical analysis Each experiment was repeated at least three times. All the data generated are expressed as the mean ± S.D. One-way analysis of variance was used to test the statistical significance of differences among groups. Statistical significance of the differences of the means was determined using independent sample t-test. All statistical tests were run using the SPSS program for MS Windows, release 19 (SPSS (Thailand) Co. Ltd., Bangkok, Thailand). The significance was determined with 95% confident limits (α = 0.5) and was considered significant at a level of P <0.05. 3. Results C. comosa ethanolic crude extract was a dark red-brown oreoresin-like, viscous extract, and composed of 12.1 ± 0.2 and 8.9 ± 0.2 % of DA1 and DA2, respectively. 3.1 The loading factor

It is observed that the Lf of DC formulation was the lowest (0.06), whereas those of CLS systems (CLS1-CLS6) were in the range between 0.26 and 0.55 (Table 1). When the coating amount increased, the R ratio decreased from 20 to 10 in CLS1 to CLS2, resulting in the increased Lf. The addition of inert nonvolatile liquid (PG) at low and high amounts (CLS4 and CLS3) presented comparable results on the Lf when compared to CLS2. However, the addition of polymer (PVP) into the liquid additive at different concentrations (CLS5 and CLS6) resulted in the increased Lf. 3.2 Pre-compression studies Flow properties The flow characteristics of the C. comosa liquisolid powder formulations are presented in Table 1. It was found that DC formulation showed fair flow with the highest Carr’s index. However, all of the CLS formulations showed good flow with the Carr’s index of less than 15. SEM The FIB-FESEM photomicrograph of MCC (Fig. 2a) showed rod-like structures with furrowed surface, while colloidal silica (Fig. 2b) appeared loose and fluffy agglomerates formed by the nanometer-sized primary particles. The photomicrograghs of all C. comosa liquisolid powder formulations have shown the adsorbed colloidal silica on the surface of MCC. Fig. 2c (CLS2) represents the photomicrograghs of liquisolid formulations containing C. comosa extract, MCC and colloidal silica. Effect of addition of PG as well as PG-PVP into C. comosa liquisolid formulations was revealed in Fig. 2d (CLS4) and Fig. 2e (CLS6), respectively. There was no obvious difference between the surface morphology of the C. comosa liquisolid powders containing with or without PG (CLS4 and CLS2, respectively). The disappearance of furrowed surface of MCC was observed in the liquisolid powders containing PVP, as shown in the case of CLS6 in Fig. 2e. This indicates the coating of PVP polymer on the surface of MCC particles. ATR-FTIR Spectra ATR-FTIR was performed to examine possible interactions between components in C. comosa extract and excipients in the formulations. The ATR-FTIR of C. comosa extract, MCC, colloidal silica, and C. comosa DC as well as liquisolid systems are shown in Fig. 3. The ATR-FTIR spectrum of C. comosa exhibited a band at 3600-3200 cm-1 due to O−H stretching, and also characteristic peaks at 2931, 1604 and 1055 cm-1, indicating the presence of alkane (C−H), aromatic (C=C) and primary alcohol (C−O), respectively (Suksamrarn et

al., 2008). MCC exhibited an intense band at 3600-3200 cm-1 due to intramolecular O–H stretching and hydrogen bond, and characteristic peaks at 2908, 1639 and 1028 cm-1 due to C−H and CH2 stretching, O−H from absorbed water, and C−O/C−C stretching (Carillo et al., 2004; Lu et al., 2017a; Rojas et al., 2011;). The spectrum of colloidal silica contained a strong, broad band at 1078 cm-1 due to the asymmetric stretch of the Si−O−Si bonds. The spectrum also contained a less intense band at 816 cm-1, which was likely due to the Si-OH bond. The spectrum of PG contained the absorption bands at 3284 cm−1 and the bands around 2900 cm−1 due to the O−H stretching band and the C−H stretching, respectively. The two bands at 1107 and 1029 were assigned to C–O stretching. The spectrum of pure PVP produced a characteristic absorption band at 1649 cm-1 which could be attributable to the carbonyl group (C=O) and the band at 2950 cm−1 was due to the aliphatic C−H stretching (Sethia and Squillante, 2004). In the spectra of C. comosa DC and liquisolid systems (CLS1-CLS6), the band characteristics to the excipients were present at almost the same positions, whereas the bands corresponding to the C. comosa extract were also present, but at a reduced intensity of absorption, indicating the trapping of extract inside the carrier matrix. None of the spectra showed any peaks other than those assigned to C. comosa extract and excipients, indicating that there was no difference between the IR patterns of the DC, CLS formulations and extract. 3.3 Evaluation of CLS tablets Tablet properties As the very low Lf resulted in extremely high and unable to prepare unit dose, together with the fair flowability of DC formulation, only tablets of CLS formulations were prepared. All CLS tablets prepared had a smooth and shiny surface without any sticking and picking. The results of weight variation, content uniformity, thickness, hardness, friability and disintegration time of the C. comosa liquisolid tablets are shown in Table 2. The weight variation for different CLS formulations showed satisfactory results as per the United States Pharmacopeia (USP 39-NF 34) limit. None of the tablets deviated from the average value by more than 5%. DA content assay studies in all CLS tablets were performed and the results revealed the uniformity of content. The percentage of DA1 in all prepared CLS tablet formulations ranged between 14.3 ± 0.7 and 14.7 ± 0.4 mg (94.3 ± 4.9 and 97.4 ±

2.4 %), whereas those of DA2 ranged between 10.6 ± 0.4 and 10.7 ± 0.4 mg (95.2 ± 3.8 and 98.1 ± 3.4 %) with the standard deviation of less than 5%. The thickness of the CLSs was found to be ranged from 2.87 ± 0.01 to 5.94 ± 0.02 mm. It is obviously shown that the tablet thickness was directly related to the tablet weight. The hardness of the CLSs was found to be in the range of 45.0 ± 1.0 to 164.3 ± 1.5 N. The decrease in carrier ratio (from CLS1 to CLS2) resulted in the decreased hardness. At the same R value, the addition of non-volatile liquid (PG) at low and high amounts (CLS4 and CLS3) resulted in increases of hardness by approximately 2 and 4 times, respectively, when compared to CLS2. The formulations containing PVP in liquid additive (PG) showed the increased Lf value, and in turn the lower amount of MCC was noted. On the contrary, the formulation containing 20% PVP in liquid additive (CLS5) showed decreased hardness. However, the formulation containing 30% PVP in liquid additive (CLS6) showed comparable hardness with CLS4. The friability was found to be in the range of 0.02 to 0.05 % which was below 1% for all formulations, indicating the good mechanical resistance of the tablets. The disintegration time was found to be in the range of 2.5 ± 0.0 to 5.5 ± 0.1 min. These results met the limit of disintegration time (less than 30 min) for uncoated dietary supplement tablets specified by United State of Pharmacopeia (USP 39-NF 34). The high amount of PVP (CLS6) may be the reason for the longest disintegration time when compared to other formulations. In vitro dissolution The solubility of DA1 and DA2 in water were found to be 6.4 ± 1.6 and 14.7 ± 3.4 μg/mL, respectively. In simulated gastric fluid, the solubility of DA1 was <1 and that of DA2 was 3.8 ± 0.1 μg/mL, respectively. For simulated intestinal fluid, the solubility of DA1 and DA2 in water were found to be 8.3 ± 0.5 and 12.8 ± 1.2 μg/mL, respectively. According to the definition of solubility by USP, DAs can be considered insoluble compound in water, simulated gastric fluid and simulated intestinal fluid. The solubility of DAs considerably increased in the presence of SLS. In 1% SLS solution, 79.7 ± 7.8 and 211.5 ± 13.0 μg/mL of DA1 and DA2 could be dissolved, respectively. In order to obtain the sink condition, 1% SLS solution was used as dissolution medium. The dissolution profiles of DA1 and DA2 presented in the CLS tablets were shown in Fig. 4. It was found that both DAs initially showed fast dissolve, followed by the plateau within 30 min. DA1, CLS2, CLS3 and CLS4 showed the higher dissolution profiles while CLS1 showed the lowest dissolution profile. DA2, CLS2 and CLS3 showed the higher dissolution

profiles, while CLS1, CLS5 and CLS6 showed the lower dissolution profiles. The dissolution profiles of DA1 and DA2 from C. comosa crude extract (125 mg) were also studied. It was found that both DAs dissolved very slowly. At 120 min, dissolutions of DA1 and DA2 from C. comosa extract were only 1.1 ± 0.4% and 1.8 ± 0.4%, respectively. These cumulative dissolution percentages were lower than those obtained from liquisolid formulations by 58 83 folds and 73 - 88 folds for DA1 and DA2, respectively. The dissolution parameters, including MDT and DE120, were calculated and shown in Table 3. The shortest MDT and highest DE120 values of both DAs were observed from CLS2. The MDT values of both DAs from CLS2 were significantly lower than those from other CLSs (p<0.05). For both DAs, the DE120 values from CLS2 were significantly higher than those from other CLS formulations (p<0.05). MDT and DE120 of DAs from CLS3 and CLS4 were comparable (p<0.05). MDT and DE120 of DAs from CLS5 and CLS6 were lower than that from CLS4 (p<0.05). MDT of DAs from CLS5 was higher than that from CLS6 (p<0.05), whereas their DE120 were similar. In case of C. comosa extract, the DE120 values of both DAs were negligible. Overall, taking into consideration the tablet weight, flow property and the results of quality control tests, CLS2 was considered to be the optimized C. comosa liquisolid formulation and therefore selected for stability and in vivo pharmacokinetic studies. Stability studies Stability study of CLS2 was performed under 2 conditions, at long term (30 ± 2 °C/75 ± 5 %RH) and accelerated (40 ± 2 °C/75 ± 5 %RH) conditions. Samples were withdrawn after 90 and 180 days and investigated for their tablet properties, percentages of remaining DAs and dissolution test. The results showed no significant difference between weight uniformity, hardness, friability, and disintegration time of the freshly prepared and aged CLS2 tablets (p>0.05) (data shown in supplementary section). This indicated that the tablet properties of C. comosa liquisolid tablets were not affected by aging. The percentages of DA1 and DA2 remaining in CLS2 tablets after storage are shown in Fig. 5. It was found that, storage under long term condition for 180 day, the percentages of both DAs did not significantly change (p>0.05). On the other hand, under accelerated condition, the percentages of DA1 was decreased significantly after 90 day storage (p<0.05), while those of DA2 did not change significantly after 90 day storage (p>0.05) but increased significantly after 180 day storage. The dissolution profiles of DA1 and DA2 from CLS2 tablets after storage are shown in Fig. 6. The similarity factor (f2) was calculated and used to find dissimilarities between

dissolution profiles of aged and fresh tablets (data shown in supplementary section). It was found that, the f2 values of both DA1 and DA2 dissolution profiles from 180-day accelerated aged tablets were statistically changed (f2 < 50), while those from 90-day accelerated aged tablets, 90-day and 180-day long term aged tablets were comparable with those of fresh tablets (f2 > 50). 3.4 In vivo pharmacokinetic studies The mean plasma concentration time curves after administration of test preparation were shown in Fig. 7. Pharmacokinetic parameters of DA1 and DA2 were determined and summarized in Table 4. The average peak concentration (Cmax) of DA1 (60.8 ± 14.2 µg/L) was lower than that of DA2 (118.0 ± 25.4 µg/L). The Tmax values were 2.3 ± 1.8 h and 6.3 ± 3.2 h for DA1 and DA2, respectively. AUC of DA1 and DA2 were 1,041.7 ± 265.1 µg⋅h/L, and 1,979.2 ± 925.0 µg⋅h/L, respectively. 4. Discussion It has been suggested that liquisolid technology may have the potential to be used for preparation of tablets containing oreoresin-like viscous crude extract. In this study, the applicability of the liquisolid technique in preparing tablets containing C. comosa crude extract was investigated. The total weight of each individual C. comosa tablet was designed to incorporate 125 mg of C. comosa extract. In order to obtain the optimal liquisolid formulation, two components, namely carrier and coating selection are crucial. For a material to be considered good carrier, it should have a large surface area in order to absorb/adsorb a large amount of liquid medication, and offer hygroscopicity with good flow properties, while retaining the liquid medication, and compressibility (Lu et al., 2017; Vraníková et al., 2015). According to Spireas et al. (1999), a carrier should be a particle with a porous surface which allows higher absorption. The preliminary study on the C. comosa extract binding capacity (capacity of powder excipients to hold liquid while maintaining their flow properties) of several carrier materials, namely MCC, lactose, and dicalcium phosphate, revealed that MCC possessed the highest binding capacity (data not shown) because MCC had the larger specific surface area compared to that of other carriers (Javadzadeh et al., 2007b). The result of this study was in line with other studies (Javadzadeh et al., 2007b; Naveen et al., 2012). In fact, MCC is the most employed carrier particle in liquisolid systems. It was reported that the porous structure of MCC enabled it to adsorb a large amount of liquid (Hentzschel et al., 2011). Therefore, MCC was selected as the carrier material for C. comosa liquisolid systems. The selection of coating

material was based on the ability to uniformly cover the wet carrier particles and produce a dry, free-flowing powder admixture through adsorption of excess liquid medicament (Spireas and Sadu, 1998; Tiong and Elkordy, 2009). As colloidal silica possessed all of the properties recommended for an efficient coating material, it was simultaneously selected as coating material in this study. The ATR-FTIR results ruled out the possibility of any chemical interaction between the components in C. comosa extract and the excipients used in the formulations. The SEM results showed that in CLS formulation, the MCC particles covered with colloidal silica and the extract. These suggested that C. comosa extract was physically adsorbed and/or absorbed onto the carrier in liquisolid powders. According to Spireas and Bolton hypothesis (Spireas and Bolton, 1999), upon liquid medicament, as C. comosa extract was added to the porous and closely matted interior structure of fibers in the carrier material, both absorption and adsorption phenomena occurred. After being absorbed in the carrier’s fibers until saturated, C. comosa extract would be adsorbed onto the internal and external surfaces of the carrier. The incorporation of a coating material, like colloidal silica, is necessary to impart the desirable flow characteristics to the designed liquisolid system due to its high adsorption capacity and large specific surface area (Fahmy and Kassem, 2008). The uniform distribution of DAs in all CLS formulations indicated by the uniformity of both DA1 and DA2 may relate to the process of liquid extract adsorption onto the carriers (Fahmy and Kassem, 2008). The high percentage of the carrier material in the unit dose (range between 58 to 73% in CLS1 to CLS6 formulations) offered a large surface area into which C. comosa extract was absorbed. Additionally, diluting the C. comosa extract with absolute ethanol before mixing it with the carriers would increase the quantity of liquid extract, but reduce its viscosity, resulting in the homogeneous distribution of C. comosa extract within the carrier and coating materials. The optimal flow properties of powder material is critical to the production of solid dosage forms such as tablets. In order to ensure consistency of tablet weight and active ingredient content, a uniform feed, as well as reproducible filling of tablet dies must be achieved. Poor flowability powder led to dose variations as there was variability in the amount of powder filled into the die cavity through the hopper. It is known that the amount of carriers and coating materials determine the flowability of liquisolid formulations (Spireas et al., 1992). An acceptable flow and compactible C. comosa extract liquisolid systems can be prepared only if a maximum amount of retained liquid on the carrier material is not exceeded; such a characteristic amount of liquid, termed liquid load factor (Lf), was therefore evaluated. Furthermore, the flow properties of the C. comosa liquisolid powder were evaluated by

determining Carr’s Index, which is indirectly related to the cohesiveness, and consequently, the flowability of powders. To determine the ability of the liquisolid powder to flow, this study investigated the amount of powder materials applied to ensure that the acceptable flow rate of C. comosa powder was obtained, i.e. approximately 10 cm3/s (Adibkia et al., 2014; Javadzadeha et al., 2008). According to United State Pharmacopeia (USP 39-NF 34), powder is considered to have acceptable flowing properties if its Carr’s index is lower than 15%. In DC formulation, the carrier (MCC) was used without the coating. The very low Lf resulted in an extremely high unit dose, making tableting unachievable. In CLS formulations, both the carrier (MCC) and the coating (colloidal silica) were used. Because of the much larger specific surface area of colloidal silica (201 ± 7 m2/g) compared to that of MCC (1.1 m2/g) (Hentzschel et al., 2011), the addition of colloidal silica increased the Lf, resulting in the lower unit dose. The low Carr’s index of all C. comosa liquisolid powders indicated that all liquisolid powders exhibited good flow property and compressibility. All CLS tablet formulations obtained had the satisfactory tablet properties with low variations in the weight and thickness, as well as the optimum hardness and disintegration time. Tablets should generally have sufficient hardness to resist against mechanical shock during transport and handling, but the hardness should not hinder the disintegration time as it would affect the dissolution rate of the loaded compounds. It is known that the maximum liquid load on the carrier material is closely related to the ratio of the amounts of carrier and coating materials (R) used in a liquisolid system (Spireas and Sadu, 1998). It was found that the carrier to coating ratio, i.e. R value, affected the Lf and consequently the tablet weight, thickness, hardness, disintegration time and dissolution behaviors of C. comosa liquisolid systems. The Lf, unsurprisingly, increased with the decrease of R value from 20 (CLS1) to 10 (CLS2). As a result, the total amount of MCC and colloidal silica necessary for making the dry and free flow powder decreased, and consequently, the tablet weight and the thickness also decreased. CLS2 tablets with lower Rvalues (R = 10) contained relatively smaller amounts of carrier material, MCC, and larger quantities of coating material, colloidal silica. These may be the reason for the decreased hardness and disintegration time of tablets with the lower R-value. MCC has been used as the tablet binder and disintegrant due to its excellent compressibility and swellability. Moreover, it has the plastic deformation nature upon compression, leading to extremely large contact surface area, which in turn increases the hydrogen bonding between adjacent cellulose molecules. These hydrogen bonds may account almost exclusively for the strength and cohesiveness of tablets according to Shangraw (1989). As the disintegrant, MCC acts as a

swellable disintegrant, facilitating water uptake and disintegration of the tablets. On the contrary, colloidal silica has poor compressibility and hydrophobic property. The carrier to coating ratio, R value, was also show to have a significant effect on the DA dissolution rates in the liquisolid formulations. CLS tablets with lower R-value (R = 10) exhibited the faster and greater DA dissolution compared to that of the tablets with higher Rvalue (R = 20). In the formulation with lower R-value, the ratio of C. comosa extract load per powder materials is relatively higher than that of the tablets with higher R-value. As a result, the drug diffusion through the primary particles produced after the disintegration of the liquisolid tablets with lower-R value may be rapid. Additionally, the large surface area of colloidal silica will expose more of C. comosa extract to the dissolution media, and then faster and higher DA dissolution could be obtained. This is in line with Javadzadeh and coworkers (2007a) which reported that the dissolution rate of carbamazepine was improved through the reduction of R ratio from 20 to 10. The much higher DA dissolution rates observed in all CLS formulations compared to that of C. comosa crude extract indicated the significantly larger surface area of C. comosa extract available for dissolution medium. For C. comosa crude extract, the dissolution barely occurred as a result of poor surface area where hydrophobic extract was exposed to the dissolution media. According to the Noyes-Whitney equation, the rate of dissolution is directly proportional to two factors, i.e. the surface area available for dissolution medium and the concentration gradient of compound in stagnant diffusion layer (Noyes and Whitney, 1897). Since the dissolution rates of both C. comosa extract and CLS formulations were studied under the same condition and medium, the identical stagnant diffusion layer thickness and the DA diffusion coefficients can be assumed. Therefore, the remarkable improvement of the in vitro dissolution of DAs in liquisolid formulations may be attributable to the tremendously enhanced effective surface area of C. comosa extract from its adsorption onto the surface of the carrier (Komala et al. 2015; Lu et al., 2017a; Spireas and Sadu, 1998). Nonvolatile liquid, PG, was used as water-miscible solvent miscible with C. comosa extract. It may act as a cosolvent with the dissolution medium (Nokhodchia et al., 2010; Spireas and Sadu, 1998). When PG was added, it was found that the higher amounts of carrier and coating were necessary for converting liquid medication to dry-looking and non-adherent powder. Although the addition of PG had no effect on the Lf, it increased the amount of liquid in the formulations and also the unit dose. The greater the amount of PG resulted in the larger amount of unit dose. The effect of PG was observed in CLS3 and CLS4, compared to CLS2. It was found that the addition of PG resulted in increased tablet weight, thickness, hardness,

and disintegration time. The higher liquid content in formulation resulted in the increases in total amounts of MCC and colloidal silica which were necessary for the making of dry and free flow powder. The weight and thickness of tablets increased in proportion to the amount of PG added. The percent of PG used in formulations CLS2, CLS4 and CLS3 were significantly different (0%, 4.7%, and 11.8 %, respectively), whereas the percent of MCC used in these formulations were comparable (65.2%, 65.5% and 65.8 %, respectively). Therefore, the amount of PG could contribute to the increased hardness and disintegration time. PG may function as the binder by stick, and increase the cohesiveness and strength of the tablets. It is known that in a low concentration of liquid vehicles, namely propylene glycol, glycerin, PEG 400 can act as a binder, which contributes to the compactness of liquisolid tablets (Lu et al., 2017b). The reason may lie in the presence of hydroxyl groups in the molecular structure of liquid vehicles, leading to hydrogen bonding between solvents and other excipients in liquisolid formulations (Lu et al., 2017b). PG is composed of 2 hydroxyl groups in the molecule (Rowe et al., 2009). The increased disintegration time of tablets containing PG may be due to the retardation of water penetration into the tablets. The higher the PG found in CLS formulations, the higher retardation was observed. During disintegration, the water penetrates the powder and builds capillary bridges, resulting in the force of attractive between adjacent particles. A force has to be generated during disintegration, which surpasses the interparticulate forces and disrupts the bonds (Markl and Zeitler, 2017). The significantly higher MDT but lower DE120 of CLS tablets containing PG might relate to the increased weight and thickness, which involved the longer disintegration time of the tablets. Moreover, PG also retarded the disintegration of tablets into fine particles, resulting in slower DA dissolution of the tablets. The mechanisms of dissolution enhancement for liquisolid systems reported are attributed to the nonvolatile liquids. The liquid vehicle existed in the liquisolid system, though in a relatively small amount, might act as a co-solvent, thereby increasing the drug solubility in the diffusion layer microenvironment. The interfacial tension between tablet surface and dissolution medium might be decreased because of the surface activity of liquid vehicles, thereby improving the wettability of hydrophobic drug. Additionally, for liquisolid systems of low dose insoluble drugs, the drug was presented either in a solubilized or dispersed state in liquid vehicle, which contributed to the increase in wetting properties and thereby enhancing the dissolution rate (Lu et al., 2017a, Lu et al., 2017b, Naveen et al., 2012). In the case of C. comosa extract liquisolid systems, surprisingly, nonvolatile liquid addition had no beneficial effect on the DA dissolution. The enhanced DA dissolutions of C. comosa

liquisolid systems seems to be purely attributed to the enhanced surface area of C. comosa extract availability to the dissolution medium. A polymer (PVP) was added into the liquisolid formulations to increase the liquid adsorption capacity (Hentzschel et al., 2011). Therefore, the smaller amounts of carrier and coating materials are needed to produce dry, free-flowing liquisolid powder admixture, resulting in the lower unit dose. This is in agreement with the findings from previous studies of liquisolid technology, in which binders such as PVP or hypromellose were added into the liquid portion (Javadzadeh et al., 2007). The effects of PVP in CLS5 and CLS6 were observed, and the results were compared to those of CLS4. The addition of PVP resulted in the decreased weight and thickness of CLS tablets. PVP had an effect on Lf, and the amounts of MCC and colloidal silica were affected as well. As the amount of MCC in CLS5 was lower compared to that of CLS4, the tablet hardness significantly decreased. The greater hardness of CLS6 may be attributable to the higher amount of PVP which can act as a binder (Rowe et al., 2009). Addition of PVP resulted in a longer disintegration time, which may due to the decrease in tablet porosity and retardation of water sorption by PVP. Consequently, the higher concentration of PVP in PG had greater effects on the tablets. The effect of PVP on MDT and DE120 might be attributable to the lower tablet weight and thickness, which allowed the short path length of water to penetrate into the tablet core. Additionally, PVP is a hydrophilic polymer (Rowe et al., 2009), which may help to accelerate the water sorption speed of the tablets. Both DAs could be dissolved during the disintegration process of the tablets. Stability studies Aging by storage under long term condition (180 days) had no effect on the tablet properties, percent remaining and dissolution profiles of both DAs from C. comosa liquisolid tablets. These results were in agreement with previous studies on the liquisolid formulations of numerous drugs such as piroxicam (Javadzadeh et al., 2009), carbamazepine (Javadzadeh et al., 2007a), griseofulvin (Elkordy et al., 2012), and diltiazem HCl (Adibkia et al., 2014). These studies also used MCC and colloidal silica as carrier and coating material and found no aging effect on the tablet properties and dissolution profiles when storage under room temperature or ambient conditions for up to 180 days. Under accelerated condition (180 days), the percent remaining and dissolution behaviors of both DAs from C. comosa aged tablets were significantly changed. These indicate the instability of DA compounds under high temperature. The similar instability results had been reported with curcumin, a similar molecular structure compound to the DAs. Curcumin was deteriorated when exposed to high

temperature, light, pH and oxygen (Wang et al., 2009). Nevertheless, the stability of DA1 and DA2, as well as other DA compounds should be further studies and clarified. In vivo pharmacokinetic studies Generally, the higher dissolution rates of DAs displayed by liquisolid tablets may also imply enhanced oral bioavailability due to the larger surface area of C. comosa extract available for dissolution. Nevertheless, the pharmacokinetic behavior of C. comosa liquisolid tablet in this study was not comparable to those of C. comosa extract in the previous study. In 2012, Su et al. reported the pharmacokinetic behavior of C. comosa hexane crude extract (in olive oil) in rats (125 mg crude extract containing 20 and 38 mg of DA1 and DA2, respectively/kg body weight). They found that both DAs were quickly absorbed and reached their peak concentration in blood at 2 h after administration and then gradually declined. The Cmax were 0.17 and 0.53 mg/L for DA1 and DA2, respectively. The AUC of DA1 and DA2 were 0.85 mg⋅h/L, and 3.57 mg⋅h/L, respectively. It should be noted that the ethanolic extract of C. comosa with the lower DA contents was used in this study. Moreover, the difference of dosage forms and physiologic conditions may affect the dissolution and absorption of DAs. Based on the preliminary study, it was found that DA1 was practically insoluble, while DA2 was slightly soluble in the simulated gastric fluid. Furthermore, both DAs showed higher solubility values in simulated intestinal fluid. The solubility of DAs could be the reason why Cmax of DA2 was higher than that of DA1 from both studies. The gastric pH of rabbit was around 1 (Kohri et al., 1998) while that of rat was 3.2-3.9 (McConnel et al., 2008). The lower gastric pH of rabbit may have influence on the dissolution and absorption of both DAs, resulting in the lower Cmax and AUC compared to those of the previous study. Typical pH values in the human stomach in the fasted state were within the range of 1.4–2.1. In the fed state, the pH of gastric fluid increased to 3.0–7.0 immediately following a meal, depending on its composition (Dressman et al., 1998; Hamed et al., 2016). Therefore, the C. comosa liquisolid tablet is suggested to be taken after meals. In addition, as both DAs have less solubility in gastric pH and long Tmax, the enteric coated liquisolid tablet might be the appropriate dosage form for delivery of C. comosa extract. 5. Conclusion In this study, an attempt was made to prepare the tablets of C. comosa extract using the liquisolid technique. Free flowing powders of C comosa were obtained through adsorption onto solid carriers - MCC - with colloidal silica as a coating material. The effects of carrier to coating (R) ratio, liquid additive (PG) and polymer (PVP) on the tablet properties and DA

dissolution were clarified. Optimized C. comosa liquisolid formulation was compose of MCC and colloidal silica, carrier to coating ratio of 10, without liquid additive or polymer. The results proved that the liquisolid preparation method produced the formulation of blends with good flow properties and the tablets with low variation in content uniformity and tablet properties. The enhancement of dissolution behavior of DAs from C. comosa liquisolid tablets over that from crude extract was emphasized. The potential capability of this technique as a tool to produce a tablet containing viscous and oleoresin-like natural crude extract, and to enhance dissolution behavior was disclosed. A simple process with great potential for large-scale industrial manufacturing based on this approach is also revealed. Overall, the liquisolid tablets of C. comosa extract developed using this technique has a potential for commercialization once its clinical efficacy has been established. Acknowledgements This study was funded by the Agricultural Research Development Agency (ARDA) (CRP5705020430), Thailand. The authors wish to thank Professor Apichart Suksamrarn, Ramkhamhaeng University, Thailand, for his generous gift of standard diarylheptanoids, namely DA1 and DA2. Appreciation is also extended to Professor Thaned Pongjanyakul, Khon Kaen University, Thailand, for his advice and invaluable assistance. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References Adibkia, K., Shokri, J., Barzegar-Jalali, M., Solduzian, M., Javadzadeh, Y., 2014. Effect of solvent type on retardation properties of diltiazem HCl form liquisolid tablets. Colloids Surf. B. Biointerfaces. 113, 10-14. Bhukhai, K., Suksen, K., Bhummaphan, N., Janjorn, K., Thongon, N., Tantikanlayaporn D., Piyachaturawat, P., Suksamrarn, A., Chairoungdua, A., 2012. A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3β proteindependent activation of the Wnt/β-catenin signaling pathway. J. Biol. Chem. 287, 36168-36178. Carr, R.L., 1965. Evaluating flow properties of solids. Chem. Eng. 72, 163–168. Carrillo, F., Colom, X., Suñol, J.J., Saurina, J., 2004. Structural FTIR analysis and thermal characterization of lyocel and viscose-type fibers. Eur. Polym. J. 40, 2229–2234. Dressman, J.B., Amidon, G.L., Reppas, C., Shah, V.P., 1998. Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms. Pharm. Res.

15, 11–22. Elkordy, A.A., Bhangale, U., Murle, N., Zarara, M.F., 2013. Combination of lactose (as a carrier) with Cremophor® EL (as a liquid vehicle) to enhance dissolution of griseofulvin. Powder Technol. 246, 182–186. Elkordy, A.A., Essa, E.A., Dhuppad, S., Jammigumpula, P., 2012. Liquisolid technique to enhance and to sustain griseofulvin dissolution: Effect of choice of non-volatile liquid vehicles. Int. J. Pharm. 434, 122-132. Fahmy, R.H., Kassem, M.A., 2008. Enhancement of famotidine dissolution rate through liquisolid tablets formulation: in vitro and in vivo evaluation. Eur. J. Pharm. Biopharm. 69, 993-1003. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2003. ICH Harmonised Tripartite Guideline: Stability testing

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Fig. 1. Chemical structures of the major diarylheptanoids from C. comosa extract [a] and C. comosa crude extract [b]. Fig. 2. Scaning electron photomicrograph of (a) MCC (1000X), (b) colloidal silica (left-1000X, right65000X), and the C. comosa liquisolid systems (1000X) (c) CLS2, (d) CLS4, (e) CLS6. Fig. 3. ATR-FTIR spectra of C. comosa extract, MCC, colloidal silica, PG, PVP, DC and the C. comosa liquisolid systems (CLS1- CLS6). Fig. 4. In vitro dissolution profiles of DA1 [a] and DA2 [b] from C. comosa liquisolid tablets and crude C. comosa extract. Data represents mean value of 6 replications ± standard deviation. Fig. 5. Percent of DA1 and DA2 remaining in C. comosa liquisolid tablet, CLS2, after storage at under long term (30 ± 2 °C/75 ± 5 %RH) and accelerated (40 ± 2 °C/75 ± 5 %RH) conditions. Data represents mean value of 6 replications ± standard deviation. Fig. 6. Effect of aging on dissolution profiles of DA1 and DA2 from C. comosa liquisolid tablets, CLS2, after storage under long term (30 ± 2 °C/75 ± 5 %RH) and accelerated (40 ± 2 °C/75 ± 5 %RH) conditions. Data represents mean value of 6 replications ± standard deviation. * Significantly changed as compared to fresh tablets (p<0.05). Fig. 7. Plasma concentration versus time profiles of DA1 and DA2 following a single oral dose administration of C. comosa liquisolid tablets in rabbits. Data represents mean value of 3 replications ± standard error.

Table captions Table 1 Formulation composition and flow characteristics of C. comosa liquisolid (CLS) and direct compression (DC) systems. Table 2 Physical characteristics of C. comosa liquisolid (CLS) tablets. Table 3 Dissolution parameters of C. comosa liquisolid (CLS) tablets and C. comosa crude extract. Data represents mean value of 6 replications ± standard deviation. Table 4 Pharmacokinetic parameters of DA1 and DA2 following a single oral dose administration (125 mg/kg BW) of C. comosa liquisolid tablets in rabbits. Data represents mean value of 3 replications ± standard error.

Table 1 Formulation composition and flow characteristics of C. comosa liquisolid (CLS) and direct compression (DC) systems. Rx Code

C. comosa extract (mg)

Carrier, Q (mg)

Coating, q (mg)

R

Liquid additive (mg)

PVP concentration in liquid additive

Unit (mg)

DC

125

2000

0

-

0

0

2220

CLS1

125

475.3

23.8

20

0

0

652.1

CLS2

125

340.6

34.1

10

0

0

522.1

CLS3

125

692.5

69.3

10

125

0

1057

CLS4

125

440.1

44.0

10

31.25

0

669.2

CLS5

125

284.1

28.4

10

31.25

20

489.8

CLS6

125

284.1

28.4

10

31.25

30

489.8

R: carrier and coating material (𝑄 𝑞) ratio; Lf or liquid load factor is a capacity of powder excipients to hold liquid while maintaining acceptable flow property, about 10 cm3/s. All formulations contain 4% croscarmellose sodium and 0.5% magnesium stearate.

Table 2 Physical characteristics of C. comosa liquisolid (CLS) tablets. Rx Code

Tablet weight (mg) a

DA content (mg) b DA1

DA2

Thickness (mm) b

Hardness (N) b

CLS1

652.6 ± 0.4

14.7 ± 0.4

10.9 ± 0.4

3.93 ± 0.03

60.0 ± 1.0

CLS2

523.5 ± 0.3

14.6 ± 0.5

10.9 ± 0.3

3.18 ± 0.02

45.0 ± 1.0

CLS3

1058.2 ± 0.2

14.6 ± 0.5

10.7 ± 0.4

5.94 ± 0.02

164.3 ± 1.5

CLS4

671.6 ± 0.3

14.5 ± 0.3

10.7 ± 0.3

3.90 ± 0.03

80.7 ± 1.5

CLS5

491.4 ± 0.2

14.3 ± 0.7

10.7 ± 0.5

2.88 ± 0.01

47.3 ± 1.5

CLS6

488.9 ± 0.4

14.6 ± 0.7

10.6 ± 0.4

2.87 ± 0.01

82.7 ± 0.6

All values represent mean ± SD, a n = 20, b n = 6.

F

Table 3 Dissolution parameters of C. comosa liquisolid (CLS) tablets and crude C. comosa extract. Data represents mean value of 6 replications ± standard deviation. Rx Code

DA1

*n/a:

DA2

MDT (min)

DE120 (%)

MDT (min)

DE120 (%)

C. comosa extract

n/a

0.4 ± 0.1

n/a

0.8 ± 0.2

CLS1

8.7 ± 0.0

59.6 ± 2.4

6.3 ± 0.1

76.5 ± 1.1

CLS2

4.0 ± 0.4

86.7 ±3.0

3.4 ± 0.3

92.0 ± 2.4

CLS3

8.5 ± 0.3

81.4 ± 3.5

8.0 ± 0.3

85.0 ± 3.1

CLS4

8.3 ± 0.0

82.6 ± 1.0

7.9 ± 0.1

82.1 ± 0.3

CLS5

7.6 ± 0.1

77.8 ± 1.4

7.2 ± 0.1

74.1 ± 0.3

CLS6

7.1 ± 0.2

77.6 ± 3.4

6.4 ± 0.4

77.0 ± 1.9

not applic able

Table 4 Pharmacokinetic parameters of DA1 and DA2 following a single oral dose administration (125 mg/kg BW) of C. comosa liquisolid tablets in rabbits. Data represents mean value of 3 replications ± standard error. Pharmacokinetic parameters

DA1

DA2

Cmax (µg/L)

60.8 ± 14.2

118.0 ± 25.4

Tmax (h)

2.3 ± 1.8

6.3 ± 3.2

AUC (µg·h/L)

1,041.7 ± 265.1

1,979.2 ± 925.0