Ascorbic acid (drug content 97%) and calcium ascorbate (drug content 76%) were obtained from PHARMCROSS Co., Ltd (Chuncheon, Korea). Methanol for high-performance liquid chromatography (HPLC) was purchased from SKYSOLTECH® (SK Chemicals, Ulsan, Korea). Acetonitrile for sample processing was purchased from Honeywell Burdick & Jackson Labs (Muskegon, MI, USA). Acetic acid (glacial, 99.5%) was purchased from Samchun Pure Chemical (Gyeonggi-do, Korea). All other chemicals were of reagent grade and were used without further purification. The distilled and deionized water (DW) was used after sterilization.

Male Sprague-Dawley rats (300 g) were purchased from Dae Han Laboratory Animal Research Co. (Eumsung, Korea) and were given free access to a normal standard chow diet (No. 322-7-1; Superfeed Co., Wonju, Korea) and tap water ad libitum. Throughout the experiments, the animals were housed (four per cage) in laminar flow cages maintained at 25±2℃ and 50–60% relative humidity under a 12 h light–dark cycle. The rats were acclimated under these conditions for at least 1 week. Each rat was fasted for at least 24 h prior to the experiment. The Committee for Ethical Usage of Experimental Animals in Chungnam National University (Daejeon, Korea) approved all animal study protocols (CNU-00605 and CNU-00609).

SGF was prepared with 0.2 g of NaCl, 0.7 ml of HCl, and 100 ml of DW. To prepare SGF with pepsin (SGFw/P), 0.32 g of pepsin was added to the same SGF composition described above. The prepared gastric fluids were used to simulate the mean volume of gastric juice for human (hSGF & hSGFw/P) [21] and rat (rSGF & rSGFw/P) [22], and ascorbic acid was treated at the recommended daily intake dose. To compare the effects of ascorbic acid and calcium ascorbate on the pH and acidity of the gastric fluids, the changes in pH and acidity of SGFs mixed with DW (as the control), ascorbic acid, or calcium ascorbate were determined. The pH was measured with a pH meter (Orion 3-Star Plus; Thermo Fisher Scientific, Waltham, MA, USA). For acidity determination, 500 µl of SGF or SGFw/P was placed in a beaker with 10 µl of 0.1% phenolphthalein solution according to the method of Raoa and Vijayakumar [23], as the acidity was calculated as the volume of 0.01 N NaOH required to change the solution from colorless to rose color.

The animals were fasted for 24 h before the experiment and then anesthetized by intraperitoneal injection of 60 mg/kg of pentobarbital sodium (Hanlim Pharm. Co., Ltd, Seoul, Korea). Then, ascorbic acid (105.96 mg/kg) and calcium ascorbate (135.24 mg/kg) were orally administered to the rats, respectively. The ascorbic acid and calcium ascorbate doses for the rats (equivalent to 102.78 mg/kg as ascorbic acid) were converted from the human dose (1,000 mg/60 kg). For pylorus ligation, a 2 cm abdominal laparotomy was performed, and the pyloric region was tied using silk thread. After suture, the animals were relieved in the recovery chamber and transferred to their cages.

Experiments were carried out according to the method of Rao and Vijayakumar [23]. The animals were sacrificed after a certain period of time and the gastric juice was collected from the stomach. After centrifugation of the gastric juice at 3,000 rpm for 15 min, the exact volume of the supernatant was measured. This volume of gastric contents from the animals was converted to the kilogram weight of the experimental animals, using following equation:

The pH of the collected supernatant was measured with a pH meter (Thermo Fisher Scientific). The acidity obtained from the titration method was converted to total acid output (µEq) using the following equation.

According to the method of Sairam et al. [24], 250 µl of substrate (2.0 [w/v] hemoglobin solution) was added to the blank and sample tubes, and pretreated for 10 min in at 37℃ water bath. Then, 50 µl of the supernatant from the rat stomach sample was added to the pretreated tube. After gentle vortex-mixing, the solution was incubated for 10 min in the 37℃ water bath. Thereafter, 500 µl of 5% trichloroacetic acid was added to the solution, and the tube was gently vortexed and then incubated for 5 min in the 37℃ water bath. The solution was centrifuged at 6,000 × g for 30 min at 25℃ and the supernatant was collected. The supernatant of the sample and blank tubes was diluted four times with 0.1 N HCl and the absorbance was measured at 280 nm wavelength using a UV spectrometer (UV1800; Shimadzu, Tokyo, Japan).

To prepare the standard solution, pepsin was dissolved in 0.1 N NaOH and the solution was diluted to the concentrations of 0, 12.5, 25, 50, 100, 200, and 400 µg/ml. All reactions were carried out twice using the sample and blank solutions. After completion of the reaction, the absorbance of the supernatant was measured without dilution at 280 nm wavelength using a UV spectrometer (Shimadzu).

The oxygen radical antioxidant capacity (ORAC) values for ascorbic acid and calcium ascorbate were determined using the OxiSelect™ Oxygen Radical Antioxidant Capacity (ORAC) Activity Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's instructions. In brief, the area under curve (AUC) of each Trolox concentration (0, 2.5, 5, 10, 20, 30, 40, and 50 µM) was calculated using the standard curve for ORAC activity. Ascorbic acid and calcium ascorbate were calculated as the mmol Trolox equivalents (TE) by comparison against the standard curve. The ORAC values were expressed as TE/L.

Using an animal feeding needle (oral zonde needle, stainless, 7 cm), ascorbic acid (105.96 mg/kg) or calcium ascorbate (135.24 mg/kg) was administered to the rats. An orbital blood sample (1.0 ml) was collected at 0.5, 1, 2, 4, 6, and 24 h post ingestion [25]. The plasma was separated by centrifugation of the blood sample at 12,000 rpm, 4℃, for 10 min, and then 100 µl was stored in the deep freezer at −70℃ prior to HPLC analysis. The HPLC method used was a modification of previous HPLC quantification methods [26]. To determine the concentration of ascorbic acid in the plasma, standard solutions were prepared by mixing the stock solution with blank plasma. For plasma deproteination, the collected plasma (30 µl) was mixed with acetonitrile (90 µl) by vortex mixing, and the mixture was then separated by centrifugation at 12,000 rpm, 4℃, for 10 min (Micro 17TR, Hanil Science, Korea). To remove impurities, the solutions were filtered through a polytetrafluoroethylene syringe filter (pore size 0.45 µm, diameter 13 mm; Whatman, Maidstone, UK). The concentration of ascorbic acid was determined using an SP-LC model HPLC system (PeakmanSP, Seoul, Korea) equipped with two SP 3101 pumps, an SP 3002 UV/Vis detector, an SP 3004 column oven, an SP 3010 switching valve and a 3023 SI-2 autosampler (Shiseido, Tokyo, Japan). The separation was performed on a Shiseido Capcell Pak MGII column (4.6 mm i.d.×150 mm, 5 µm) maintained at 30℃. The mobile phase was prepared by mixing distilled water, methanol, and acetic acid (91.9:8:0.1, v/v/v) and eluted at a flow rate 0.5 ml/min. After 10 µl samples were injected onto the column, the signals were monitored at a UV wavelength of 240 nm.

All data were expressed as the mean±SEM of three or more independent experiments. Analysis of variance was used to compare parameters among multiple groups (GraphPad, San Diego, CA, USA). If a significant difference between treated groups was found, Dunnett's test was applied. Differences with p<0.05 were considered statistically significant.

It is well known that ascorbic acid aids cell growth and helps the body to stay healthy because of its antioxidant activity, which potentially offers protection from some diseases and degenerative aspects of aging [27]. To ascertain whether the antioxidant activity of calcium ascorbate was still functional, the antioxidant activities of calcium ascorbate and ascorbic acid were evaluated by measuring the fluorescence decrease rate from the production and decay of the peroxy radical by 2, 2′-azo-bis-2-methyl-propanimidamide, dihydrochloride (AAPH) using ORAC assay. Table 1 shows that with the antioxidant activity of ascorbic acid (25.24 mmol TE/L) was not significantly different from that of calcium ascorbate (25.65 mmol TE/L), and their relative percentages of antioxidant activity were also not significantly different (100% vs 101%, Table 1). These data show that calcium ascorbate and ascorbic acid have similar antioxidant activities.

Previously, several studies reported that ascorbic acid could induce gastrointestinal disorders such as indigestion, heartburn, nausea, diarrhea, abdominal cramps/pain, and esophagitis [152829]. Because ascorbic acid is highly acidic and could stimulate the secretion of stomach acids such as pepsin, these excess acids may trigger these gastrointestinal symptoms. To determine the effect of calcium ascorbate on the pH and total acid output of gastric juice, the differences in pH and total acid of SGFs following treatment with calcium ascorbate and ascorbic acid were compared. When treated with ascorbic acid, the gastric fluid pH was not different to that of the DW control in both hSGF and rSGF (Figs. 1A and 2A). In contrast, the pH of SGFs treated with calcium ascorbate was significantly higher than that of fluids treated with ascorbic acid (Figs. 1A and 2A). However, the total acid output was significantly higher in ascorbic acid-treated SGFs than in the DW control (Figs. 1B and 2B), whereas calcium ascorbate treatment resulted in no significant changes relative to the control (Figs. 1B and 2B). These results indicate that calcium ascorbate could act to increase gastric pH without increasing the total acid output, thereby attenuating gastric symptoms.

Because pepsin is one of the major enzymes in gastric fluid and is stimulated by low pH and acid [30], its effect on gastric pH and total acid output was investigated in the SGFs treated with calcium ascorbate or ascorbic acid. The gastric pH and total acid output were not affected by pepsin in the hSGFw/P and rSGFw/P (Figs. 1C, 1D, 2C, and 2D).

The reduction of gastric pH and the secretion of gastric juices both work to exacerbate damage to the gastric wall in gastric disorders [30]. Therefore, the effect of calcium ascorbate was further confirmed in the rat pylorus ligation-induced ulcer model. Although the secreted volume of gastric juice was not significantly different between ascorbic acid and calcium ascorbate treatments (Fig. 3A), the gastric juice pH was significantly increased by calcium ascorbate as compared with ascorbic acid until 30 min after administration (Fig. 3B). In contrast, the total acid output was not significantly increased by calcium ascorbate (Fig. 4A).

Pepsin, secreted by the mucous membrane chief cells, is also present in the gastric juice and shows proteolytic activity at low pH (1.8–2.0) [31]. It is also known that the high activity of pepsin induces gastric damage [32]. Therefore, we evaluated whether the secretion of pepsin was affected by calcium ascorbate treatment. Fig. 4B shows that pepsin secretion was not induced by dosing with calcium ascorbate in the rat pylorus ligation-induced ulcer model, whereas it was immediately induced after ascorbic acid administration. Taken together, these results demonstrate that calcium ascorbate is effective in attenuating ascorbic acid-induced gastric symptoms by increasing the gastric pH and preventing pepsin secretion in the rat pylorus ligation-induced ulcer model.

To investigate whether calcium ascorbate could improve the oral bioavailability of ascorbic acid, a pharmacokinetic study was carried out. Calcium ascorbate administration resulted in significantly higher plasma concentrations of ascorbic acid than dosing of ascorbic acid, at 0.5, 1, 2, 4, 6, and 24 h post oral administration (Fig. 5). The AUC_0-24h value from calcium ascorbate (1277.0 ng/ml) was 1.5-fold higher than that from ascorbic acid (705.7 ng/ml), and the AUC_inf value from calcium ascorbate (5769.6 ng/ml) was 2.8-fold higher than that from ascorbic acid (2024.7 ng/ml). The C_max value of calcium ascorbate (91.0 ng/ml) was also higher than that of ascorbic acid (74.8 ng/ml). However, the T_max values were similar (Table 2). After 24 h post dose, the plasma concentration of calcium ascorbate (45.3 ng/ml) was two times higher than that of ascorbic acid (22.6 ng/ml) (Fig. 5). Therefore, calcium ascorbate was obviously better absorbed and hence present in higher plasma levels than ascorbic acid.