http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(12): 1719–1726 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.1003356
ORIGINAL ARTICLE
The physicochemical properties of geraniin, a potential antihyperglycemic agent Sumita Elendran1, Lee Wang Wang1, Richard Prankerd2, and Uma D. Palanisamy1 School of Medicine and Health Sciences, Monash University, Selangor, Malaysia and 2Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
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1
Abstract
Keywords
Context: Natural products play a vital role in the discovery of leads for novel pharmacologically active drugs. Geraniin (GE) was identified as the major compound in the rind of Nephelium lappaceum L. (Sapindaceae), while ellagic and gallic acids have been shown to be its main metabolites. GE and its metabolites possess a range of bioactive properties including being an anti-infective, anticarcinogenic, antihyperglycemic, and antihypertensive. Objective: GE and its metabolites were investigated to establish its gastrointestinal absorption and physicochemical properties. Materials and methods: GE was purified from N. lappaceum rind extract using reverse-phase C18 column chromatography. Lipophilicity (log P) was determined using the 1-octanol/water shakeflask method. Equilibrium solubility of GE and its metabolites (20 mg) was determined in water and four biorelevant media: simulated gastric, simulated intestinal, fasted state-simulated intestinal, and fed state-simulated intestinal after 72 h. Results and discussion: The purification yield was 10.8%; where a 97–99% pure GE was obtained. Log P values for GE, ellagic, and gallic acids were established as 0.73 ± 0.17, 0.11 ± 0.06, and 0.71 ± 0.21, respectively, establishing them as polar compounds. All three compounds were found to exhibit poor solubility in gastric (0.61–8.10 mg/mL) but good solubility in intestinal fluids (3.59–14.32 mg/mL). Conclusion: The above results indicate that the compounds have limited gastrointestinal absorption due to its polarities. To consider these compounds as oral drug candidates, formulation strategies are being developed.
Ellagic acid, gallic acid, geraniin, HPLC validation, lipophilicity, solubility
Introduction Plants have formed the core of traditional medicine systems, among which are the Unani, Chinese, and Ayurvedic that have been in existence for thousands of years and continue to offer novel remedies to mankind (Rates, 2001). Natural products isolated from medicinal plants play a vital role in the discovery of leads for the development of novel and clinically active drugs against various pharmacological targets. Over a 100 naturally derived compounds are undergoing clinical development worldwide, and at least 100 similar projects are in preclinical development (Harvey, 2008). It takes about 10–15 years to develop one new medicine from the time it is discovered to when it is available for treating patients (Dimasi, 2001). The average cost to research and develop each successful drug is estimated to be $800 million to $1 billion. This value includes the cost of the thousands of failures (Dimasi & Grabowski, 2007). Correspondence: Dr. Uma D. Palanisamy, School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway 46150, Selangor Darul Ehsan, Malaysia. Tel: +603 55145840. E-mail: [email protected]
History Received 21 May 2014 Revised 29 October 2014 Accepted 22 December 2014 Published online 8 April 2015
For every 5000–10 000 compounds that enter the research and development pipeline, ultimately only one receives approval (Dimasi et al., 2003). These numbers challenge imagination, but a deeper understanding of the drug development process can explain why so many compounds do not make it and why it is such a lengthy and difficult process to get new medicine to patients. There are various reasons that drug candidates might fail during development. The prominent cause for the failure of new chemical entities (NCE’s) (Kennedy, 1997) in clinical development is associated with poor pharmacokinetics and absorption, distribution, metabolism, and excretion properties (Van De Waterbeemd & Gifford, 2003). Gastrointestinal absorption of NCE’s from natural sources is a concern to pharmaceutical industries because they tend to be larger and bulkier. They have a larger molecular weight and higher lipophilicity leading to both permeation and solubility issues (Kansy et al., 1998). Hence, a detailed study of the physicochemical properties of the naturally derived compounds for gastrointestinal absorption and bioavailability is an important part of the optimization process of potential leads to candidates suitable for clinical trials.
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Figure 1. Chemical structure of geraniin (C41H28O27, mass 952.08) and its metabolites corilagin (C27H24O18, mass 636.46), ellagic acid (C14H6O8, mass 302.19), and gallic acid (C7H6O5, mass 170.12).
Geraniin (GE) (C41H28O27.7H2O) (Figure 1), an ellagitannin, was identified as the major compound in the ethanolic extracts of N. lappaceum L. (Sapindaceae) rind, with yields of almost 30% by weight and a molecular weight of 952 g/mol (Palanisamy et al., 2011b). Corilagin (CO) (C27H24O18), ellagic acid (EA) (C14H6O8), and gallic acid (GA) (C7H6O5) (Figure 1) have been shown to be the main metabolites of GE (Ito et al., 2007; Thitilertdecha et al., 2010). GE and its metabolites have been found to possess a range of bioactive properties, which include antioxidant and free radical scavenging activity (Bala et al., 2006; Palanisamy et al., 2008), anticancer (Yoshida et al., 2005), antimicrobial (Konishi et al., 2004), antiviral (Notka et al., 2003; Yang et al., 2008), and anti-hyperglycemic activity (Palanisamy et al., 2011b). However, the physicochemical properties, permeability, absorption, and metabolism of these compounds are still poorly understood. This study aims to establish the physicochemical properties of GE and its metabolites. The lipophilicity and the solubility of the compounds were investigated to gain an understanding of its in vivo gastrointestinal absorption and bioavailability.
Materials and methods Materials and reagents Davasil (633NC18E) reverse-phase C18 silica (particle size ˚ ) was obtained from Grace (Washington, 50 mm, pore size 60 A CT). Ethanol (absolute and denatured) was purchased from Scharlau Chemicals (Barcelona, Spain). Acetonitrile and dichloromethane were supplied by Mallinckrodt Chemicals (Phillipsburg, NJ). Milli-QÕ water, ethanol, trifluoroacetic acid, analytical grade n-octanol, hydrochloric acid, and glacial acetic acid were obtained from Merck (Darmstadt, Germany). Formic acid, sodium chloride, monobasic potassium phosphate, sodium di-hydrogen phosphate, sodium
hydroxide pellets, sodium taurocholate, and lecithin were supplied by Sigma Aldrich (St. Louis, MO). Instruments A Favorit glass column (250 mm 50 mm i.d.) with a fritted glass filter (porosity 2 mm) and vacuum inlet used for reversephase chromatography were obtained from PLT Scientific (Selangor, Malaysia). High-performance liquid chromatography (HPLC) equipment was a Shimadzu Prominence UFLC-ITTOF equipped with a photodiode array detector (Shimadzu, Kyoto, Japan) using a Merck Chromolith Performance RP-18 (100 4.6 mm2) column (Merck, Darmstadt, Germany). The WisecubeÕ orbital rotary shaker was obtained from Daihan Scientific (Seoul, Korea). Large-scale purification of GE from Nephelium lappaceum rind extract Nephelium lappaceum rind was obtained from Kuala Lumpur, Malaysia, in March 2013, and plants were authenticated by the Forest Research Institute of Malaysia and voucher specimen maintained at their Herbarium. A crude ethanolic extract of N. lappaceum rind was prepared as described by Palanisamy et al. (2008). GE was purified from the crude extract by means of reverse-phase chromatography as described by Perera et al. (2012). Crude extract (20 g) was dissolved in a minimum amount of Milli-QÕ water (Merck, Darmstadt, Germany) (40 mL) and loaded onto a glass column packed with 200 g of reverse phase C18 silica. The solvent flow rate through the open tubular column was maintained by means of a vacuum pump attached to the column vacuum inlet. The column was first eluted with water (300 mL) and then fractions were collected using a step gradient of water and acetonitrile. The solvent system was as follows: water (100%, 400 mL), acetonitrile:water (5:95%, 350 mL), and acetonitrile:water (10:90%, 1000 mL).
DOI: 10.3109/13880209.2014.1003356
Finally the column was eluted with methanol (100%, 500 mL). The silica was cleaned by flushing the column sequentially with dichloromethane (100%, 300 mL), methanol containing a few drops of trifluoroacetic acid (100%, 300 mL), methanol (100%, 300 mL), and finally allowing it to dry completely. Crystallization of GE Fractions collected were analyzed using HPLC in the presence of GE. Fractions containing GE were concentrated using a rotary evaporator and the saturated fractions were allowed to cool at 4 C for 48 h until the formation of yellow crystalline GE. The GE was filtered using a Whatman No. 114 filter paper and crystals were allowed to dry at room temperature. The crystals were collected and weighed.
Physicochemical properties of geraniin
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determinations per concentration. Precision is further subdivided into intra-day and inter-day precisions, which measures precision within a single day and on two different days. Accuracy was calculated as percent recovery by adding known amounts of standard solution in the sample solution. LOD and limit of quantitation (LOQ) were also determined by using the formula below taking into consideration of a signal-to-baseline noise ratio of 3 and 10, respectively: LOD ¼ 3:3 =slope LOQ ¼ 10 =slope where is the standard deviation and slope is the slope of the calibration curve. Octanol–water partition coefficients
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Purity analysis and identification of GE GE purity was determined by means of HPLC using a Merck Chromolith Performance RP-18 column (Merck, Darmstadt, Germany) maintained at room temperature. HPLC analysis was carried out using an Agilent 1200 HPLC system coupled with a multiple wavelength detector (MWD) (Agilent, Santa Clara, CA). Gradient elution was performed at a flow rate of 0.5 mL/ min using a mobile phase consisting of water (Solvent A) and acetonitrile (Solvent B), starting from a gradient of 2–100% solvent B over 30 min, and 100% solvent B for 5 min and finally 100–2% solvent B over 5 min to re-condition the column. The mobile phase was prepared fresh on the day of use, filtered through a 0.45-mm nylon filter, and de-aerated by sonication for 10 min. The peaks were monitored at 254 nm. Quantification and validation of GE, EA, and GA Quantification was carried out using an Agilent 1200 HPLC system coupled with a multiple wavelength detector (MWD) (Agilent, Santa Clara, CA). All separations were performed using a Merck Chromolith Performance RP-18 column, 100 4.6 mm2, (Merck, Darmstadt, Germany) maintained at room temperature. GE, EA, and GA were eluted isocratically at a flow rate of 0.5 mL/min using a mobile phase consisting of acetonitrile and water (30:70%, v/v). The mobile phase was prepared fresh on the day of use, filtered through a 0.45-mm nylon filter, and de-aerated by sonication for 10 min. The absorbance of the eluent was measured at 254 nm. The chromatographic method was validated according to the International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology for parameters such as linearity, precision, accuracy, limit of quantification (LOQ), and limit of detection (LOD). Method validation All validation runs were performed in triplicates on three consecutive days to assess inter-day and intra-day variation. Linear relationship was determined by generating a calibration curve using a minimum of five different concentrations of GE, EA, and GA ranging from 0.001 to 2 mg/mL. The slope, linearity, and other statistics of calibration curves were evaluated by constructing a regression line and analysis of variance (ANOVA). Precision was measured at three concentrations, i.e., low, medium, and high using three
A partition-coefficient measurement was carried out using the following procedure at 25 C. A mixture of octanol and water was shaken for 24 h and was permitted to reach equilibrium to allow the phases to separate. Stock solutions of GE, EA, and GA were prepared to a concentration of 1 mg/mL in the aqueous phase (AP). The octanol phase (OP) was then added and the mixture was equilibrated during several 4–5 min vigorous shaking periods spaced 10 min apart and allowed to settle for at least 24 h. In order to ensure the accuracy of the partition coefficient, triplicate determinations were made under different test conditions, whereby the ratio of AP and OP was varied, i.e., 1:1, 1:2, and 2:1. Upon equilibration, an aliquot from each phase and test condition was taken and centrifuged at 13 200 g for 15 min at 25 C. The concentration of the compound(s) in each phase was analyzed using HPLC. The mole % recovery of the compound(s) in the two phases was calculated. Assays were performed in triplicates. Qualitative solubility estimation To assess the visual solubility of GE, EA, and GA, 1 mg of the compound(s) were added to 5 mL of water, respectively, in a water bath. If the compound was found to dissolve completely, this concentration is recorded as its final visual solubility. However, if it is insoluble, further 5 mL addition of water was made consecutively to the compound(s) until complete solubility is observed. Preparation of biorelevant media Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) without enzymes were prepared according to United States Pharmacopeia (USP 32). The Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF) represent a simplification of the proximal small intestine composition in the fasted and in the fed state, respectively (Dressman et al., 1998). The compositions of the fluids are as summarized in Table 1. The FaSSIF-V2, an updated version of the standard FaSSIF media, was used (Jantratid et al., 2008). Equilibrium solubility determination in biorelevant media The equilibrium solubility of GE, EA, and GA in water, SGF, SIF, FaSSIF, and FeSSIF at two different temperatures
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Table 1. Composition of simulated gastric fluid (SGF), simulated intestinal fluid (SIF), Fasted State Simulated Intestinal Fluid (FaSSIF), and Fed State Simulated Intestinal Fluid (FeSSIF) medium.
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Composition
SGF
SIF
Sodium di-hydrogen phosphate Monobasic potassium phosphate Sodium chloride Sodium hydroxide pellets 0.2 N sodium hydroxide Hydrochloric acid Glacial acetic acid Water (mL) Sodium taurocholate (hydrate, 97% purity) Lecithin
– – 2.0 g – – 7.0 mL – 1000 – –
– 6.8 g – – 77 mL – – 250 – –
1 M sodium hydroxide or 1 M hydrochloric acid pH Osmolality (mOsm kg1) Buffer capacity (mmol l1 DpH1)
1.2 – –
FaSSIF 4.04 g – 6.19 g 0.34 g – – – 1000 1.613 g 0.157 g
Sufficient amount to adjust the pH 6.8 6.5 – 180 ± 10 – 10
FeSSIF – – 11.88 g 4.04 g – – 8.65 g 1000 8.25 g 2.95 g 5.0 670 ± 10 25
(25 C and 37 C) was determined by the shake flask method. An excess of GE, EA, and GA was added into respective solvents (1 mL), sonicated for 10 min, and shaken at 25 C and 37 C at 100 rpm for 3 days. Aliquots were withdrawn at various time points, until equilibrium was attained. The suspension obtained was centrifuged at 13 200 g for 15 min to obtain a clear solution. The concentration of the compound(s) in water, SGF, SIF, FaSSIF and FeSSIF was determined by HPLC. Solubility determination was repeated three times.
Results and discussion Purification and crystallization of GE Crude N. lappaceum rind extract (20 g) was subjected to the reverse-phase chromatography. GE was detected in the fractions that were eluted with acetonitrile:water (10:90%, v/v). The fractions were concentrated by evaporation to onefourth of its volumes and cooled at 4 C to allow the formation of GE crystals. The GE crystals obtained were yellow in color. A total of six purifications were carried out and the total mass of GE purified via crystallization from 120 g of crude N. lappaceum rind extract was 12.95 g, hence the total yield of crystalline GE from the crude extract was 10.8%. Purity analysis and identification of GE HPLC analysis of crystallized GE established that GE crystals had a purity of 99.43%, based on the peak area of GE observed in the HPLC chromatogram, at a retention time of 10.99 min (Figure 2). A minor impurity identified as corilagin was observed at a retention time of 10.57 min, which constituted 0.57% of the sample (Figure 2). Corilagin is an ellagitannin closely related to GE. Corilagin has been isolated previously from extracts of N. lappaceum rind, and has also been identified as a product of GE metabolism in rats (Ito et al., 2007; Thitilertdecha et al., 2010). HPLC method validation for GE, EA, and GA Linearity, precision, accuracy, LOD, and LOQ were evaluated for quantitative purposes. Good linear correlations for GE,
Figure 2. HPLC chromatogram of geraniin crystal.
EA, and GA were obtained between peak areas and concentration in the selected range of 0.001–2 mg/mL. Characteristic parameters for regression equations and correlation coefficients are given in Table 2. The linearity of the calibration graphs was validated by the high value of correlation coefficients of the regression graph. The results of recovery study ranging from 98 to 102% for all the compounds suggested a good accuracy (Table 3). The precision of the proposed method was carried out in terms of the repeatability, inter-day, and intra-day time periods. The low % relative standard deviation values of repeatability for both inter-day (2%) and intra-day (1%) variations for all the compounds reveal that the proposed method is precise (Table 3). Thus, these results demonstrate that the method is reproducible, as the introduced variations in the test have no influence on the experimental results. According to the ICH recommendations, the approach based on the standard deviation (SD) of the response and the slope was used for determining the detection and quantitation limits. The values are shown in Table 2; these
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Table 2. Quantification and validation of geraniin, ellagic, and gallic acid using HPLC. Compounds 1 a
Linearity range (mg mL ) Regression equation Correlation equation (r) Limit of detection (LOD) (mg mL1) Limit of quantification (LOQ) (mg mL1) Standard error F SS (residual) MS (residual) SS (regression) MS (regression) Lower 95% Upper 95%
Geraniin
Ellagic acid
Gallic acid
0.01–2.00 y ¼ 23 824x + 135.48 0.999 0.01 0.03 79.5 8.59E + 04 3.16E + 04 6.32E + 03 5.43E + 08 5.43E + 08 2.48E + 04 2.52E + 04
y ¼ 49 412x + 1450.7 0.997 0.01 0.05 279.6 5.28E + 02 2.34E + 05 7.81E + 04 4.13E + 07 4.13E + 07 6.56E + 04 8.67E + 04
y ¼ 28 489x + 651.06 0.995 0.01 0.05 160.3 3.36E + 04 1.28E + 05 2.57E + 04 8.65E + 08 8.65E + 08 3.11E + 04 3.20E + 04
a
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Linearity established using nine concentrations (n ¼ 3).
Table 3. Intra-day and inter-day precision and accuracy of geraniin, ellagic, and gallic acid. Intra-day Compounds
Concentration (mg ml )
Accuracy (%)
Precision (% RSDb)
Inter-day Precision (% RSDb)
0.01 0.1 1 0.01 0.1 1 0.01 0.1 1
100 99 101 99 98 102 99 100 98
2.81 0.75 1.97 2.52 2.41 0.03 2.18 1.98 1.04
0.83 0.92 1.98 0.55 0.73 0.77 1.03 1.50 0.15
Geraniin Ellagic acid Gallic acid
1 a
a
Linearity established using nine concentrations (n ¼ 3). Relative standard deviation.
b
Table 4. Concentration of geraniin, ellagic, and gallic acid in aqueous phase (AP) and octanol phase (OP). AP Compounds 1
Geraniin (mg mL ) Ellagic acid (mg mL1) Gallic acid (mg mL1)
OP
CONC
Mole % recovery
CONC
Mole % recovery
0.30 ± 0.05 1.19 ± 1.06 0.76 ± 0.10
99 101 98
0.06 ± 0.01 5.69 ± 0.34 0.97 ± 0.01
97 102 101
Data are presented as mean ± SD.
results also validated the sensitivity of the proposed analytical method. Octanol–water partition coefficients The concentration ratio of a compound in aqueous phase (AP) and octanol phase (OP) was determined and results are shown in Table 4. The concentration of GE dissolved in the OP was found to be significantly low compared with the concentration in the AP. However, EA and GA were found to have a higher concentration in the octanol phase. Log P, in contrast, provides an indication of compound permeation across the lipid bilayer membrane. It is, therefore, suggested that partition of a drug into 1-octanol predicts its ability to passively diffuse across biological membranes (Andersson & Schrader, 1999). Thus, the passive diffusion of GE across the lipid membrane is expected to be challenging. Conversely, the
passive diffusion of the less polar EA and GA is expected to be easier. GE, EA, and GA had log P values lower than 1 in all the three solvent ratios [AP:OP (1:1), AP:OP (1:2), and AP:OP (2:1)] examined (Table 5). The final log P values were also found to be lower than 1 while deviation from the mean for all compounds was ± 0.3 log units. This value is considerably lower than that stated by the Organization for Economic Cooperation and Development (OECD) test guideline, indicating that this method has good repeatability. The mole % recovery of the compounds in the two phases was calculated to be within the range of 97–103% (Table 4) indicating that the analytical procedure used is acceptable. As the log P values for GE, EA, and GA are lower than 1, they are categorized into the 1.0 to 1.0 range of log P group as shown in Table 5. The three compounds were established to be polar compounds with good aqueous affinity but poor lipid affinity, hence are
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Table 5. Partition coefficient (P) and log P values of geraniin, ellagic and gallic acid at three different ratios of aqueous phase (AP) and octanol phase (OP). AP:OP (1:1)
AP:OP (1:2)
AP:OP (2:1)
Compound
P
log P
P
log P
P
log P
Final log P
Geraniin Ellagic acid Gallic acid
0.19 ± 0.04 3.11 ± 1.27 1.25 ± 0.09
0.75 ± 0.08 0.48 ± 0.18 0.08 ± 0.04
0.13 ± 0.02 8.67 ± 6.42 1.52 ± 0.19
0.90 ± 0.04 0.87 ± 0.36 0.18 ± 0.06
0.29 ± 0.06 6.04 ± 0.42 1.20 ± 0.14
0.56 ± 0.09 0.78 ± 0.03 0.08 ± 0.06
0.73 ± 0.17 0.71 ± 0.21 0.11 ± 0.06
Data are presented as mean ± SD.
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Table 6. Qualitative solubility of geraniin, ellagic and gallic acid in water. Compounds
Qualitative solubility (mg mL1)
Geraniin Ellagic acid Gallic acid
0.08 0.002 0.4
expected to have poor absorption and distribution. Similar results have been shown for EA and GA by Locatelli et al. (2008) and Ratnam et al. (2006)
Qualitative solubility The qualitative solubility estimation (visual solubility) was carried out for GE, EA, and GA (Table 6). Ellagitannins are classified as hydrolysable tannins and are known for their good water solubility (Okuda et al., 1989). However, water solubility for most ellagitannins decreases with the increasing number of galloyl groups but increases with additional biarylhexahydroxydiphenoyl (HHDP) groups. GE is a typical ellagitannin, as it is composed of entirely common acyl units such as galloyl, HHDP, and dehydrohexahydroxydiphenoyl (DHHDP) groups, giving GE an aqueous solubility of 0.08 mg/mL (Juang et al., 2004; Xu & Howard, 2012). EA, an extremely stable polyphenol, exhibits the least water solubility of 0.002 mg/mL while GA has the highest water solubility of 0.4 mg/mL.
Equilibrium solubility in biorelevant media To forecast the in vivo performance of a drug in the gastrointestinal tract, it is important that in vitro tests mimic the in vivo conditions as closely as possible. In addition, to predict the in vivo performance of weakly acidic compounds such as GE, EA, and GA based on its equilibrium solubility, it is essential to focus on the changing pH conditions in the human gastrointestinal tract and the impact of bile components on drug solubilization in the small intestine. There are a few factors that affect the solubility of compounds such as temperature, pH, and also the presence of bile salts and lecithin. It was observed that an increase in the temperature increased the solubility of GE, EA, and GA, regardless of the solvent used (Table 7). A similar observation was reported by Daneshfar et al. (2008), whereby the solubility of gallic acid in different solvents increased correspondingly with increasing temperature.
Following oral administration, a pH gradient is experienced progressing through the gastrointestinal tract, as such pH has a significant contribution to the solubility of a compound (Dressman et al., 2007). The solubility of GE, EA, and GA was observed to be the highest in intestinal fluids (SIF: pH 6.8, FaSSIF: pH 6.5, and FeSSIF: pH 5) and the least in gastric fluid (SGF: pH 1.2). This could be due to the acidic nature of the compounds (Ascacio-Valdes et al., 2011; Daneshfar et al., 2008; Przewloka & Shearer, 2002). Drug solubility in the small intestine can be enhanced by amphiphilic bile components such as bile salt, lecithin, and monoleins (Charman et al., 1997). Lecithin and cholesterol are normal components of bile, while fatty acids and monoglycerides are the normal breakdown products of fat digestion, have been shown to form mixed micelles with the conjugated bile salt in the small intestine (Gibaldi & Feldman, 1970). When these substances are present in concentrations higher than their critical micelle concentration, micellar solubilization of the drug can occur (Bates et al., 1966). The effect of bile salt solubilization has been studied for many drugs such as griseofulvin, danazol, digoxin, glutethimide, diethylstilbestrol, diazepam, halofantrine, leucotriene antagonists, and gemfibrozil (Charman et al., 1997; Horter & Dressman, 2001). FaSSIF and FeSSIF media simulate the physiological condition in the small intestine before and after food consumption, respectively. SIF media simulates standard intestinal fluid without the composition of bile salts and lecithin. It was observed that GE, EA, and GA have different solubilities in the intestinal fluids examined. On one hand, GE exhibited the highest solubility in FaSSIF followed by FeSSIF and SIF media in both 25 C and 37 C (Table 7). On the other hand, GA was most soluble in FeSSIF followed by FaSSIF and SIF in both temperatures. The same was observed for EA at 37 C but at 25 C it displayed highest solubility in SIF followed by FaSSIF and FeSSIF. The above results indicate that GE is more soluble in the small intestine at fasted condition while EA and GA in fed condition. The stomach, although not the primary site for drug absorption, provides the first site at which an orally administered formulation can quantitatively release its drug (Dressman et al., 2007). For compounds highly soluble in gastric pH, complete dissolution can occur in the stomach. For such compounds, gastric emptying may well limit the subsequent rate of absorption from the small intestine (Klein, 2010; Stegemann et al., 2007). For example, poorly soluble weak acid ibuprofen, little dissolution will occur in the stomach. In contrast, the small intestine with its higher pH offers a more favorable environment for dissolution of acids. For Ibuprofen and similar weak acids, emptying from the
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Table 7. Solubility of geraniin, ellagic and gallic acid in water and biorelevant media at 25 C and 37 C. Biorelevant media pH Bile salt and lecithin Geraniin (mg mL1) Ellagic acid (mg mL1)
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Gallic acid (mg mL1)
25 C 37 C 25 C 37 C 25 C 37 C
Water
SGF
SIF
FaSSIF
FeSSIF
– No 1.40 ± 0.10 4.41 ± 0.00 11.34 ± 0.21 16.35 ± 0.00 10.75 ± 0.25 11.84 ± 0.01
1.2 No 0.61 ± 0.01 1.43 ± 0.03 1.16 ± 0.04 3.11 ± 0.08 5.68 ± 0.01 8.10 ± 0.02
6.8 No 3.59 ± 0.10 6.65 ± 0.16 9.57 ± 0.36 5.89 ± 0.37 6.14 ± 0.01 9.43 ± 0.01
6.5 Present 6.30 ± 0.28 9.06 ± 0.06 4.35 ± 0.07 11.22 ± 0.28 11.01 ± 0.00 12.82 ± 0.02
5 Present 4.20 ± 0.13 6.73 ± 0.14 4.22 ± 0.40 8.57 ± 0.33 13.90 ± 0.30 14.32 ± 0.02
stomach becomes rate limiting to the onset of dissolution, and hence absorption (Dressman et al., 2007; Stegemann et al., 2007). Similar observations can be speculated for GE, EA, and GA as they are weak acids too. Although the compounds examined have relatively low solubility in SGF, their subsequent absorption in small intestine may also be limited. The results obtained are not conclusive as the behavior of compounds do not define the actual situation in the gastrointestinal tract and depends on many other physiological factors including fluid volume, fluid composition, transit, motility, microfloras, and enzymes, which are further influenced by food, gender, and age (McConnell et al., 2008).
Conclusion Purification of GE from Nephelium lappaceum rind waste was achieved in a single reverse phase chromatographic method. The simplicity of this purification method, the easy availability of the raw material, high yield, and purity of GE form the basis of this large-scale purification method. A simple, accurate, and precise HPLC method for the quantification of GE, EA, and GA using the HPLC was developed and validated for its linearity, precision, accuracy, detection, and quantitation limit. Log P of GE, EA, and GA were determined and the compounds were established to be polar compounds. Qualitative solubility reported for GE, EA, and GA provides an estimated solubility of the compounds in water. Based on the results obtained for equilibrium solubility in biorelevant media, GE, EA, and GA were found to exhibit poor solubility in gastric fluid and good solubility in intestinal fluids. When the aqueous solubility of a compound is identified as a problem from in vitro testing, simple and effective formulation strategies are applied to secure the compound deposition in vivo. The solubility results obtained in this study will be useful when running dissolutions alongside formulation development. It is envisaged that GE and its metabolites will face gastrointestinal absorption and bioavailability challenges and as such formulation strategies would need to be developed. Some of the formulations that are currently being investigated are solid lipid nanoparticles, phytosomes, and solvent-and surfactantbased formulations.
Acknowledgements Special thanks to Asiri Perera and Usha Sundralingam from Monash University Malaysia and Thavamanithevi Subramaniam from SIRIM BhD.
Declaration of interest The authors report that they have no conflicts of interest. This research project was funded by Monash University Major Grant BCHH-SM-2-02-2010.
References Andersson J, Schrader W. (1999). A method for measuring 1-octanolwater partition coefficients. Anal Chem 71:3610–14. Ascacio-Valdes JA, Buenrostro-Figueroa JJ, Aguilera-Carbo A, et al. (2011). Ellagitannins: Biosynthesis, biodegradation and biological properties. J Med Plants Res 5:4698–703. Bala I, Bhardwaj V, Hariharan S, Kumar M. (2006). Analytical methods for assay of ellagic acid and its solubility studies. J Pharm Biomed Anal 40:206–10. Bates TR, Gibaldi M, Kanig JL. (1966). Solubilizing properties of bile salt solutions I: Effect of temperature and bile salt concentration on solubilization of glutethimide, griseofulvin, and hexestrol. J Pharm Sci 55:191–9. Charman WN, Porter CJH, Mithani S, Dressman J. (1997). Physicochemical and physiological mechanisms for the effects of food on drug absorption: The role of lipids and pH. J Pharm Sci 86: 269–82. Daneshfar A, Ghaziaskar HS, Homayoun N. (2008). Solubility of gallic acid in methanol, ethanol, water, and ethyl acetate. J Chem Eng Data 53:776–8. DiMasi JA. (2001). Risks in new drug development: Approval success rates for investigational drugs. Clin Pharmacol Ther 69:297–307. DiMasi JA, Grabowski HG. (2007). The cost of biopharmaceutical R&D: Is biotech different? Manage Decis Econ 28:469–79 DiMasi JA, Hansen RW, Grabowski HG. (2003). The price of innovation: New estimates of drug development costs. J Health Econ 22:151–85. Dressman J, Vertzoni M, Goumas K, Reppas C. (2007). Estimating drug solubility in the gastrointestinal tract. Adv Drug Deliver Rev 59: 591–602. Dressman JB, Amidon GL, Reppas C, Shah VP. (1998). Dissolution testing as a prognostic tool for oral drug absorption: Immediate release dosage forms. Pharm Res 15:11–22. Gibaldi M, Feldman S. (1970). Mechanisms of surfactant effects on drug absorption. J Pharm Sci 59:579–89. Harvey AL. (2008). Natural products in drug discovery. Drug Discov Today 13:894–901. Horter D, Dressman J. (2001). Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Adv Drug Deliver Rev 46:75–87. Ito H, Iguchi A, Hatano T. (2007). Identification of urinary and intestinal bacterial metabolites of ellagitannin GE in rats. J Agric Food Chem 56:393–400. Jantratid E, Janssen N, Reppas C, Dressman JB. (2008). Dissolution media simulating conditions in the proximal human gastrointestinal tract: An update. Pharm Res 25:1663–76. Juang LJ, Sheu SJ, Lin TC. (2004). Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by higher performance liquid chromatography and capillary electrophoresis. J Sep Sci 27: 718–24. Kansy M, Senner F, Gubernator K. (1998). Physicochemical high throughput screening: Parallel artificial membrane permeation assay
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in the description of passive absorption processes. J Med Chem 41: 1007–10. Kennedy T. (1997). Managing the drug discovery/development interface. Drug Discov Today 2:436–44. Klein S. (2010). The use of biorelevant dissolution media to forecast the in vivo performance of a drug. AAPS J 12:397–406. Konishi Y, Hitomi Y, Yoshioka E. (2004). Intestinal absorption of p-coumaric and gallic acids in rats after oral administration. J Agric Food Chem 52:2527–32. Locatelli C, Rosso R, Santos-Silva MC, et al. (2008). Ester derivatives of gallic acid with potential toxicity toward L1210 leukemia cells. Bioorg Med Chem 16:3791–9. McConnell EL, Fadda HM, Basit AW. (2008). Gut instincts: Explorations in intestinal physiology and drug delivery. Int J Pharm 364:213–26. Notka F, Meier G, Wagner R. (2003). Inhibition of wild-type human immunodeficiency virus and reverse transcriptase inhibitor-resistant variants by Phyllanthus amarus. Antiviral Res 58:175–86. Okuda T, Yoshida T, Hatano T. (1989). Ellagitannins as active constituents of medicinal plants. Planta Med 55:117–22. Palanisamy U, Cheng HM, Masilamani T, et al. (2008). Rind of the rambutan, Nephelium lappaceum L., a potential source of natural antioxidants. Food Chem 109:54–63. Palanisamy UD, Ling LT, Manaharan T, Appleton D. (2011b). Rapid isolation of geraniin from Nephelium lappaceum L. rind waste and its anti-hyperglycemic activity. Food Chem 127:21–7.
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Perera A, Appleton D, Ying LH, et al. (2012). Large scale purification of geraniin from Nephelium lappaceum L. rind waste using reverse-phase chromatography. Sep Purif Technol 98:145–9. Przewloka S, Shearer B. (2002). The further chemistry of ellagic acid II. Ellagic acid and water-soluble ellagates as metal precipitants. Holzforschung 56:13–19. Rates S. (2001). Plants as source of drugs. Toxicon 39:603–13. Ratnam DV, Bhardwaj V, Kumar M. (2006). Simultaneous analysis of ellagic acid and coenzyme by derivative spectroscopy and HPLC. Talanta 70:387–91. Stegemann S, Leveiller F, Franchi D, et al. (2007). When poor solubility becomes an issue: From early stage to proof of concept. Eur J Pharm Sci 31:249–61. Thitilertdecha N, Teerawutgulrag A, Kilburn JD, Rakariyatham N. (2010). Identification of major phenolic compounds from Nephelium lappaceum L. and their antioxidant activities. Molecules 15:1453–65. Van de Waterbeemd H, Gifford E. (2003). ADMET in silico modelling: Towards prediction paradise? Nat Rev Drug Discov 2:192–204. Xu Z, Howard LR. (2012). Analysis of Antioxidant-rich Phytochemicals. Oxford, UK: Wiley-Blackwell. Yang CS, Sang S, Lambert JD, Lee MJ. (2008). Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol Nutr Food Res 52:S139–51. Yoshida T, Hatano T, Ito H. (2005). Chapter Seven High molecular weight plant poplyphenols (tannins): Prospective functions. Recent Adv Phytochem 39:163–90.
ORIGINAL ARTICLE
The physicochemical properties of geraniin, a potential antihyperglycemic agent Sumita Elendran1, Lee Wang Wang1, Richard Prankerd2, and Uma D. Palanisamy1 School of Medicine and Health Sciences, Monash University, Selangor, Malaysia and 2Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
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1
Abstract
Keywords
Context: Natural products play a vital role in the discovery of leads for novel pharmacologically active drugs. Geraniin (GE) was identified as the major compound in the rind of Nephelium lappaceum L. (Sapindaceae), while ellagic and gallic acids have been shown to be its main metabolites. GE and its metabolites possess a range of bioactive properties including being an anti-infective, anticarcinogenic, antihyperglycemic, and antihypertensive. Objective: GE and its metabolites were investigated to establish its gastrointestinal absorption and physicochemical properties. Materials and methods: GE was purified from N. lappaceum rind extract using reverse-phase C18 column chromatography. Lipophilicity (log P) was determined using the 1-octanol/water shakeflask method. Equilibrium solubility of GE and its metabolites (20 mg) was determined in water and four biorelevant media: simulated gastric, simulated intestinal, fasted state-simulated intestinal, and fed state-simulated intestinal after 72 h. Results and discussion: The purification yield was 10.8%; where a 97–99% pure GE was obtained. Log P values for GE, ellagic, and gallic acids were established as 0.73 ± 0.17, 0.11 ± 0.06, and 0.71 ± 0.21, respectively, establishing them as polar compounds. All three compounds were found to exhibit poor solubility in gastric (0.61–8.10 mg/mL) but good solubility in intestinal fluids (3.59–14.32 mg/mL). Conclusion: The above results indicate that the compounds have limited gastrointestinal absorption due to its polarities. To consider these compounds as oral drug candidates, formulation strategies are being developed.
Ellagic acid, gallic acid, geraniin, HPLC validation, lipophilicity, solubility
Introduction Plants have formed the core of traditional medicine systems, among which are the Unani, Chinese, and Ayurvedic that have been in existence for thousands of years and continue to offer novel remedies to mankind (Rates, 2001). Natural products isolated from medicinal plants play a vital role in the discovery of leads for the development of novel and clinically active drugs against various pharmacological targets. Over a 100 naturally derived compounds are undergoing clinical development worldwide, and at least 100 similar projects are in preclinical development (Harvey, 2008). It takes about 10–15 years to develop one new medicine from the time it is discovered to when it is available for treating patients (Dimasi, 2001). The average cost to research and develop each successful drug is estimated to be $800 million to $1 billion. This value includes the cost of the thousands of failures (Dimasi & Grabowski, 2007). Correspondence: Dr. Uma D. Palanisamy, School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway 46150, Selangor Darul Ehsan, Malaysia. Tel: +603 55145840. E-mail: [email protected]
History Received 21 May 2014 Revised 29 October 2014 Accepted 22 December 2014 Published online 8 April 2015
For every 5000–10 000 compounds that enter the research and development pipeline, ultimately only one receives approval (Dimasi et al., 2003). These numbers challenge imagination, but a deeper understanding of the drug development process can explain why so many compounds do not make it and why it is such a lengthy and difficult process to get new medicine to patients. There are various reasons that drug candidates might fail during development. The prominent cause for the failure of new chemical entities (NCE’s) (Kennedy, 1997) in clinical development is associated with poor pharmacokinetics and absorption, distribution, metabolism, and excretion properties (Van De Waterbeemd & Gifford, 2003). Gastrointestinal absorption of NCE’s from natural sources is a concern to pharmaceutical industries because they tend to be larger and bulkier. They have a larger molecular weight and higher lipophilicity leading to both permeation and solubility issues (Kansy et al., 1998). Hence, a detailed study of the physicochemical properties of the naturally derived compounds for gastrointestinal absorption and bioavailability is an important part of the optimization process of potential leads to candidates suitable for clinical trials.
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Figure 1. Chemical structure of geraniin (C41H28O27, mass 952.08) and its metabolites corilagin (C27H24O18, mass 636.46), ellagic acid (C14H6O8, mass 302.19), and gallic acid (C7H6O5, mass 170.12).
Geraniin (GE) (C41H28O27.7H2O) (Figure 1), an ellagitannin, was identified as the major compound in the ethanolic extracts of N. lappaceum L. (Sapindaceae) rind, with yields of almost 30% by weight and a molecular weight of 952 g/mol (Palanisamy et al., 2011b). Corilagin (CO) (C27H24O18), ellagic acid (EA) (C14H6O8), and gallic acid (GA) (C7H6O5) (Figure 1) have been shown to be the main metabolites of GE (Ito et al., 2007; Thitilertdecha et al., 2010). GE and its metabolites have been found to possess a range of bioactive properties, which include antioxidant and free radical scavenging activity (Bala et al., 2006; Palanisamy et al., 2008), anticancer (Yoshida et al., 2005), antimicrobial (Konishi et al., 2004), antiviral (Notka et al., 2003; Yang et al., 2008), and anti-hyperglycemic activity (Palanisamy et al., 2011b). However, the physicochemical properties, permeability, absorption, and metabolism of these compounds are still poorly understood. This study aims to establish the physicochemical properties of GE and its metabolites. The lipophilicity and the solubility of the compounds were investigated to gain an understanding of its in vivo gastrointestinal absorption and bioavailability.
Materials and methods Materials and reagents Davasil (633NC18E) reverse-phase C18 silica (particle size ˚ ) was obtained from Grace (Washington, 50 mm, pore size 60 A CT). Ethanol (absolute and denatured) was purchased from Scharlau Chemicals (Barcelona, Spain). Acetonitrile and dichloromethane were supplied by Mallinckrodt Chemicals (Phillipsburg, NJ). Milli-QÕ water, ethanol, trifluoroacetic acid, analytical grade n-octanol, hydrochloric acid, and glacial acetic acid were obtained from Merck (Darmstadt, Germany). Formic acid, sodium chloride, monobasic potassium phosphate, sodium di-hydrogen phosphate, sodium
hydroxide pellets, sodium taurocholate, and lecithin were supplied by Sigma Aldrich (St. Louis, MO). Instruments A Favorit glass column (250 mm 50 mm i.d.) with a fritted glass filter (porosity 2 mm) and vacuum inlet used for reversephase chromatography were obtained from PLT Scientific (Selangor, Malaysia). High-performance liquid chromatography (HPLC) equipment was a Shimadzu Prominence UFLC-ITTOF equipped with a photodiode array detector (Shimadzu, Kyoto, Japan) using a Merck Chromolith Performance RP-18 (100 4.6 mm2) column (Merck, Darmstadt, Germany). The WisecubeÕ orbital rotary shaker was obtained from Daihan Scientific (Seoul, Korea). Large-scale purification of GE from Nephelium lappaceum rind extract Nephelium lappaceum rind was obtained from Kuala Lumpur, Malaysia, in March 2013, and plants were authenticated by the Forest Research Institute of Malaysia and voucher specimen maintained at their Herbarium. A crude ethanolic extract of N. lappaceum rind was prepared as described by Palanisamy et al. (2008). GE was purified from the crude extract by means of reverse-phase chromatography as described by Perera et al. (2012). Crude extract (20 g) was dissolved in a minimum amount of Milli-QÕ water (Merck, Darmstadt, Germany) (40 mL) and loaded onto a glass column packed with 200 g of reverse phase C18 silica. The solvent flow rate through the open tubular column was maintained by means of a vacuum pump attached to the column vacuum inlet. The column was first eluted with water (300 mL) and then fractions were collected using a step gradient of water and acetonitrile. The solvent system was as follows: water (100%, 400 mL), acetonitrile:water (5:95%, 350 mL), and acetonitrile:water (10:90%, 1000 mL).
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Finally the column was eluted with methanol (100%, 500 mL). The silica was cleaned by flushing the column sequentially with dichloromethane (100%, 300 mL), methanol containing a few drops of trifluoroacetic acid (100%, 300 mL), methanol (100%, 300 mL), and finally allowing it to dry completely. Crystallization of GE Fractions collected were analyzed using HPLC in the presence of GE. Fractions containing GE were concentrated using a rotary evaporator and the saturated fractions were allowed to cool at 4 C for 48 h until the formation of yellow crystalline GE. The GE was filtered using a Whatman No. 114 filter paper and crystals were allowed to dry at room temperature. The crystals were collected and weighed.
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determinations per concentration. Precision is further subdivided into intra-day and inter-day precisions, which measures precision within a single day and on two different days. Accuracy was calculated as percent recovery by adding known amounts of standard solution in the sample solution. LOD and limit of quantitation (LOQ) were also determined by using the formula below taking into consideration of a signal-to-baseline noise ratio of 3 and 10, respectively: LOD ¼ 3:3 =slope LOQ ¼ 10 =slope where is the standard deviation and slope is the slope of the calibration curve. Octanol–water partition coefficients
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Purity analysis and identification of GE GE purity was determined by means of HPLC using a Merck Chromolith Performance RP-18 column (Merck, Darmstadt, Germany) maintained at room temperature. HPLC analysis was carried out using an Agilent 1200 HPLC system coupled with a multiple wavelength detector (MWD) (Agilent, Santa Clara, CA). Gradient elution was performed at a flow rate of 0.5 mL/ min using a mobile phase consisting of water (Solvent A) and acetonitrile (Solvent B), starting from a gradient of 2–100% solvent B over 30 min, and 100% solvent B for 5 min and finally 100–2% solvent B over 5 min to re-condition the column. The mobile phase was prepared fresh on the day of use, filtered through a 0.45-mm nylon filter, and de-aerated by sonication for 10 min. The peaks were monitored at 254 nm. Quantification and validation of GE, EA, and GA Quantification was carried out using an Agilent 1200 HPLC system coupled with a multiple wavelength detector (MWD) (Agilent, Santa Clara, CA). All separations were performed using a Merck Chromolith Performance RP-18 column, 100 4.6 mm2, (Merck, Darmstadt, Germany) maintained at room temperature. GE, EA, and GA were eluted isocratically at a flow rate of 0.5 mL/min using a mobile phase consisting of acetonitrile and water (30:70%, v/v). The mobile phase was prepared fresh on the day of use, filtered through a 0.45-mm nylon filter, and de-aerated by sonication for 10 min. The absorbance of the eluent was measured at 254 nm. The chromatographic method was validated according to the International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology for parameters such as linearity, precision, accuracy, limit of quantification (LOQ), and limit of detection (LOD). Method validation All validation runs were performed in triplicates on three consecutive days to assess inter-day and intra-day variation. Linear relationship was determined by generating a calibration curve using a minimum of five different concentrations of GE, EA, and GA ranging from 0.001 to 2 mg/mL. The slope, linearity, and other statistics of calibration curves were evaluated by constructing a regression line and analysis of variance (ANOVA). Precision was measured at three concentrations, i.e., low, medium, and high using three
A partition-coefficient measurement was carried out using the following procedure at 25 C. A mixture of octanol and water was shaken for 24 h and was permitted to reach equilibrium to allow the phases to separate. Stock solutions of GE, EA, and GA were prepared to a concentration of 1 mg/mL in the aqueous phase (AP). The octanol phase (OP) was then added and the mixture was equilibrated during several 4–5 min vigorous shaking periods spaced 10 min apart and allowed to settle for at least 24 h. In order to ensure the accuracy of the partition coefficient, triplicate determinations were made under different test conditions, whereby the ratio of AP and OP was varied, i.e., 1:1, 1:2, and 2:1. Upon equilibration, an aliquot from each phase and test condition was taken and centrifuged at 13 200 g for 15 min at 25 C. The concentration of the compound(s) in each phase was analyzed using HPLC. The mole % recovery of the compound(s) in the two phases was calculated. Assays were performed in triplicates. Qualitative solubility estimation To assess the visual solubility of GE, EA, and GA, 1 mg of the compound(s) were added to 5 mL of water, respectively, in a water bath. If the compound was found to dissolve completely, this concentration is recorded as its final visual solubility. However, if it is insoluble, further 5 mL addition of water was made consecutively to the compound(s) until complete solubility is observed. Preparation of biorelevant media Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) without enzymes were prepared according to United States Pharmacopeia (USP 32). The Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF) represent a simplification of the proximal small intestine composition in the fasted and in the fed state, respectively (Dressman et al., 1998). The compositions of the fluids are as summarized in Table 1. The FaSSIF-V2, an updated version of the standard FaSSIF media, was used (Jantratid et al., 2008). Equilibrium solubility determination in biorelevant media The equilibrium solubility of GE, EA, and GA in water, SGF, SIF, FaSSIF, and FeSSIF at two different temperatures
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Table 1. Composition of simulated gastric fluid (SGF), simulated intestinal fluid (SIF), Fasted State Simulated Intestinal Fluid (FaSSIF), and Fed State Simulated Intestinal Fluid (FeSSIF) medium.
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Composition
SGF
SIF
Sodium di-hydrogen phosphate Monobasic potassium phosphate Sodium chloride Sodium hydroxide pellets 0.2 N sodium hydroxide Hydrochloric acid Glacial acetic acid Water (mL) Sodium taurocholate (hydrate, 97% purity) Lecithin
– – 2.0 g – – 7.0 mL – 1000 – –
– 6.8 g – – 77 mL – – 250 – –
1 M sodium hydroxide or 1 M hydrochloric acid pH Osmolality (mOsm kg1) Buffer capacity (mmol l1 DpH1)
1.2 – –
FaSSIF 4.04 g – 6.19 g 0.34 g – – – 1000 1.613 g 0.157 g
Sufficient amount to adjust the pH 6.8 6.5 – 180 ± 10 – 10
FeSSIF – – 11.88 g 4.04 g – – 8.65 g 1000 8.25 g 2.95 g 5.0 670 ± 10 25
(25 C and 37 C) was determined by the shake flask method. An excess of GE, EA, and GA was added into respective solvents (1 mL), sonicated for 10 min, and shaken at 25 C and 37 C at 100 rpm for 3 days. Aliquots were withdrawn at various time points, until equilibrium was attained. The suspension obtained was centrifuged at 13 200 g for 15 min to obtain a clear solution. The concentration of the compound(s) in water, SGF, SIF, FaSSIF and FeSSIF was determined by HPLC. Solubility determination was repeated three times.
Results and discussion Purification and crystallization of GE Crude N. lappaceum rind extract (20 g) was subjected to the reverse-phase chromatography. GE was detected in the fractions that were eluted with acetonitrile:water (10:90%, v/v). The fractions were concentrated by evaporation to onefourth of its volumes and cooled at 4 C to allow the formation of GE crystals. The GE crystals obtained were yellow in color. A total of six purifications were carried out and the total mass of GE purified via crystallization from 120 g of crude N. lappaceum rind extract was 12.95 g, hence the total yield of crystalline GE from the crude extract was 10.8%. Purity analysis and identification of GE HPLC analysis of crystallized GE established that GE crystals had a purity of 99.43%, based on the peak area of GE observed in the HPLC chromatogram, at a retention time of 10.99 min (Figure 2). A minor impurity identified as corilagin was observed at a retention time of 10.57 min, which constituted 0.57% of the sample (Figure 2). Corilagin is an ellagitannin closely related to GE. Corilagin has been isolated previously from extracts of N. lappaceum rind, and has also been identified as a product of GE metabolism in rats (Ito et al., 2007; Thitilertdecha et al., 2010). HPLC method validation for GE, EA, and GA Linearity, precision, accuracy, LOD, and LOQ were evaluated for quantitative purposes. Good linear correlations for GE,
Figure 2. HPLC chromatogram of geraniin crystal.
EA, and GA were obtained between peak areas and concentration in the selected range of 0.001–2 mg/mL. Characteristic parameters for regression equations and correlation coefficients are given in Table 2. The linearity of the calibration graphs was validated by the high value of correlation coefficients of the regression graph. The results of recovery study ranging from 98 to 102% for all the compounds suggested a good accuracy (Table 3). The precision of the proposed method was carried out in terms of the repeatability, inter-day, and intra-day time periods. The low % relative standard deviation values of repeatability for both inter-day (2%) and intra-day (1%) variations for all the compounds reveal that the proposed method is precise (Table 3). Thus, these results demonstrate that the method is reproducible, as the introduced variations in the test have no influence on the experimental results. According to the ICH recommendations, the approach based on the standard deviation (SD) of the response and the slope was used for determining the detection and quantitation limits. The values are shown in Table 2; these
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Table 2. Quantification and validation of geraniin, ellagic, and gallic acid using HPLC. Compounds 1 a
Linearity range (mg mL ) Regression equation Correlation equation (r) Limit of detection (LOD) (mg mL1) Limit of quantification (LOQ) (mg mL1) Standard error F SS (residual) MS (residual) SS (regression) MS (regression) Lower 95% Upper 95%
Geraniin
Ellagic acid
Gallic acid
0.01–2.00 y ¼ 23 824x + 135.48 0.999 0.01 0.03 79.5 8.59E + 04 3.16E + 04 6.32E + 03 5.43E + 08 5.43E + 08 2.48E + 04 2.52E + 04
y ¼ 49 412x + 1450.7 0.997 0.01 0.05 279.6 5.28E + 02 2.34E + 05 7.81E + 04 4.13E + 07 4.13E + 07 6.56E + 04 8.67E + 04
y ¼ 28 489x + 651.06 0.995 0.01 0.05 160.3 3.36E + 04 1.28E + 05 2.57E + 04 8.65E + 08 8.65E + 08 3.11E + 04 3.20E + 04
a
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Linearity established using nine concentrations (n ¼ 3).
Table 3. Intra-day and inter-day precision and accuracy of geraniin, ellagic, and gallic acid. Intra-day Compounds
Concentration (mg ml )
Accuracy (%)
Precision (% RSDb)
Inter-day Precision (% RSDb)
0.01 0.1 1 0.01 0.1 1 0.01 0.1 1
100 99 101 99 98 102 99 100 98
2.81 0.75 1.97 2.52 2.41 0.03 2.18 1.98 1.04
0.83 0.92 1.98 0.55 0.73 0.77 1.03 1.50 0.15
Geraniin Ellagic acid Gallic acid
1 a
a
Linearity established using nine concentrations (n ¼ 3). Relative standard deviation.
b
Table 4. Concentration of geraniin, ellagic, and gallic acid in aqueous phase (AP) and octanol phase (OP). AP Compounds 1
Geraniin (mg mL ) Ellagic acid (mg mL1) Gallic acid (mg mL1)
OP
CONC
Mole % recovery
CONC
Mole % recovery
0.30 ± 0.05 1.19 ± 1.06 0.76 ± 0.10
99 101 98
0.06 ± 0.01 5.69 ± 0.34 0.97 ± 0.01
97 102 101
Data are presented as mean ± SD.
results also validated the sensitivity of the proposed analytical method. Octanol–water partition coefficients The concentration ratio of a compound in aqueous phase (AP) and octanol phase (OP) was determined and results are shown in Table 4. The concentration of GE dissolved in the OP was found to be significantly low compared with the concentration in the AP. However, EA and GA were found to have a higher concentration in the octanol phase. Log P, in contrast, provides an indication of compound permeation across the lipid bilayer membrane. It is, therefore, suggested that partition of a drug into 1-octanol predicts its ability to passively diffuse across biological membranes (Andersson & Schrader, 1999). Thus, the passive diffusion of GE across the lipid membrane is expected to be challenging. Conversely, the
passive diffusion of the less polar EA and GA is expected to be easier. GE, EA, and GA had log P values lower than 1 in all the three solvent ratios [AP:OP (1:1), AP:OP (1:2), and AP:OP (2:1)] examined (Table 5). The final log P values were also found to be lower than 1 while deviation from the mean for all compounds was ± 0.3 log units. This value is considerably lower than that stated by the Organization for Economic Cooperation and Development (OECD) test guideline, indicating that this method has good repeatability. The mole % recovery of the compounds in the two phases was calculated to be within the range of 97–103% (Table 4) indicating that the analytical procedure used is acceptable. As the log P values for GE, EA, and GA are lower than 1, they are categorized into the 1.0 to 1.0 range of log P group as shown in Table 5. The three compounds were established to be polar compounds with good aqueous affinity but poor lipid affinity, hence are
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Table 5. Partition coefficient (P) and log P values of geraniin, ellagic and gallic acid at three different ratios of aqueous phase (AP) and octanol phase (OP). AP:OP (1:1)
AP:OP (1:2)
AP:OP (2:1)
Compound
P
log P
P
log P
P
log P
Final log P
Geraniin Ellagic acid Gallic acid
0.19 ± 0.04 3.11 ± 1.27 1.25 ± 0.09
0.75 ± 0.08 0.48 ± 0.18 0.08 ± 0.04
0.13 ± 0.02 8.67 ± 6.42 1.52 ± 0.19
0.90 ± 0.04 0.87 ± 0.36 0.18 ± 0.06
0.29 ± 0.06 6.04 ± 0.42 1.20 ± 0.14
0.56 ± 0.09 0.78 ± 0.03 0.08 ± 0.06
0.73 ± 0.17 0.71 ± 0.21 0.11 ± 0.06
Data are presented as mean ± SD.
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Table 6. Qualitative solubility of geraniin, ellagic and gallic acid in water. Compounds
Qualitative solubility (mg mL1)
Geraniin Ellagic acid Gallic acid
0.08 0.002 0.4
expected to have poor absorption and distribution. Similar results have been shown for EA and GA by Locatelli et al. (2008) and Ratnam et al. (2006)
Qualitative solubility The qualitative solubility estimation (visual solubility) was carried out for GE, EA, and GA (Table 6). Ellagitannins are classified as hydrolysable tannins and are known for their good water solubility (Okuda et al., 1989). However, water solubility for most ellagitannins decreases with the increasing number of galloyl groups but increases with additional biarylhexahydroxydiphenoyl (HHDP) groups. GE is a typical ellagitannin, as it is composed of entirely common acyl units such as galloyl, HHDP, and dehydrohexahydroxydiphenoyl (DHHDP) groups, giving GE an aqueous solubility of 0.08 mg/mL (Juang et al., 2004; Xu & Howard, 2012). EA, an extremely stable polyphenol, exhibits the least water solubility of 0.002 mg/mL while GA has the highest water solubility of 0.4 mg/mL.
Equilibrium solubility in biorelevant media To forecast the in vivo performance of a drug in the gastrointestinal tract, it is important that in vitro tests mimic the in vivo conditions as closely as possible. In addition, to predict the in vivo performance of weakly acidic compounds such as GE, EA, and GA based on its equilibrium solubility, it is essential to focus on the changing pH conditions in the human gastrointestinal tract and the impact of bile components on drug solubilization in the small intestine. There are a few factors that affect the solubility of compounds such as temperature, pH, and also the presence of bile salts and lecithin. It was observed that an increase in the temperature increased the solubility of GE, EA, and GA, regardless of the solvent used (Table 7). A similar observation was reported by Daneshfar et al. (2008), whereby the solubility of gallic acid in different solvents increased correspondingly with increasing temperature.
Following oral administration, a pH gradient is experienced progressing through the gastrointestinal tract, as such pH has a significant contribution to the solubility of a compound (Dressman et al., 2007). The solubility of GE, EA, and GA was observed to be the highest in intestinal fluids (SIF: pH 6.8, FaSSIF: pH 6.5, and FeSSIF: pH 5) and the least in gastric fluid (SGF: pH 1.2). This could be due to the acidic nature of the compounds (Ascacio-Valdes et al., 2011; Daneshfar et al., 2008; Przewloka & Shearer, 2002). Drug solubility in the small intestine can be enhanced by amphiphilic bile components such as bile salt, lecithin, and monoleins (Charman et al., 1997). Lecithin and cholesterol are normal components of bile, while fatty acids and monoglycerides are the normal breakdown products of fat digestion, have been shown to form mixed micelles with the conjugated bile salt in the small intestine (Gibaldi & Feldman, 1970). When these substances are present in concentrations higher than their critical micelle concentration, micellar solubilization of the drug can occur (Bates et al., 1966). The effect of bile salt solubilization has been studied for many drugs such as griseofulvin, danazol, digoxin, glutethimide, diethylstilbestrol, diazepam, halofantrine, leucotriene antagonists, and gemfibrozil (Charman et al., 1997; Horter & Dressman, 2001). FaSSIF and FeSSIF media simulate the physiological condition in the small intestine before and after food consumption, respectively. SIF media simulates standard intestinal fluid without the composition of bile salts and lecithin. It was observed that GE, EA, and GA have different solubilities in the intestinal fluids examined. On one hand, GE exhibited the highest solubility in FaSSIF followed by FeSSIF and SIF media in both 25 C and 37 C (Table 7). On the other hand, GA was most soluble in FeSSIF followed by FaSSIF and SIF in both temperatures. The same was observed for EA at 37 C but at 25 C it displayed highest solubility in SIF followed by FaSSIF and FeSSIF. The above results indicate that GE is more soluble in the small intestine at fasted condition while EA and GA in fed condition. The stomach, although not the primary site for drug absorption, provides the first site at which an orally administered formulation can quantitatively release its drug (Dressman et al., 2007). For compounds highly soluble in gastric pH, complete dissolution can occur in the stomach. For such compounds, gastric emptying may well limit the subsequent rate of absorption from the small intestine (Klein, 2010; Stegemann et al., 2007). For example, poorly soluble weak acid ibuprofen, little dissolution will occur in the stomach. In contrast, the small intestine with its higher pH offers a more favorable environment for dissolution of acids. For Ibuprofen and similar weak acids, emptying from the
Physicochemical properties of geraniin
DOI: 10.3109/13880209.2014.1003356
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Table 7. Solubility of geraniin, ellagic and gallic acid in water and biorelevant media at 25 C and 37 C. Biorelevant media pH Bile salt and lecithin Geraniin (mg mL1) Ellagic acid (mg mL1)
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Gallic acid (mg mL1)
25 C 37 C 25 C 37 C 25 C 37 C
Water
SGF
SIF
FaSSIF
FeSSIF
– No 1.40 ± 0.10 4.41 ± 0.00 11.34 ± 0.21 16.35 ± 0.00 10.75 ± 0.25 11.84 ± 0.01
1.2 No 0.61 ± 0.01 1.43 ± 0.03 1.16 ± 0.04 3.11 ± 0.08 5.68 ± 0.01 8.10 ± 0.02
6.8 No 3.59 ± 0.10 6.65 ± 0.16 9.57 ± 0.36 5.89 ± 0.37 6.14 ± 0.01 9.43 ± 0.01
6.5 Present 6.30 ± 0.28 9.06 ± 0.06 4.35 ± 0.07 11.22 ± 0.28 11.01 ± 0.00 12.82 ± 0.02
5 Present 4.20 ± 0.13 6.73 ± 0.14 4.22 ± 0.40 8.57 ± 0.33 13.90 ± 0.30 14.32 ± 0.02
stomach becomes rate limiting to the onset of dissolution, and hence absorption (Dressman et al., 2007; Stegemann et al., 2007). Similar observations can be speculated for GE, EA, and GA as they are weak acids too. Although the compounds examined have relatively low solubility in SGF, their subsequent absorption in small intestine may also be limited. The results obtained are not conclusive as the behavior of compounds do not define the actual situation in the gastrointestinal tract and depends on many other physiological factors including fluid volume, fluid composition, transit, motility, microfloras, and enzymes, which are further influenced by food, gender, and age (McConnell et al., 2008).
Conclusion Purification of GE from Nephelium lappaceum rind waste was achieved in a single reverse phase chromatographic method. The simplicity of this purification method, the easy availability of the raw material, high yield, and purity of GE form the basis of this large-scale purification method. A simple, accurate, and precise HPLC method for the quantification of GE, EA, and GA using the HPLC was developed and validated for its linearity, precision, accuracy, detection, and quantitation limit. Log P of GE, EA, and GA were determined and the compounds were established to be polar compounds. Qualitative solubility reported for GE, EA, and GA provides an estimated solubility of the compounds in water. Based on the results obtained for equilibrium solubility in biorelevant media, GE, EA, and GA were found to exhibit poor solubility in gastric fluid and good solubility in intestinal fluids. When the aqueous solubility of a compound is identified as a problem from in vitro testing, simple and effective formulation strategies are applied to secure the compound deposition in vivo. The solubility results obtained in this study will be useful when running dissolutions alongside formulation development. It is envisaged that GE and its metabolites will face gastrointestinal absorption and bioavailability challenges and as such formulation strategies would need to be developed. Some of the formulations that are currently being investigated are solid lipid nanoparticles, phytosomes, and solvent-and surfactantbased formulations.
Acknowledgements Special thanks to Asiri Perera and Usha Sundralingam from Monash University Malaysia and Thavamanithevi Subramaniam from SIRIM BhD.
Declaration of interest The authors report that they have no conflicts of interest. This research project was funded by Monash University Major Grant BCHH-SM-2-02-2010.
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