Corncobs were obtained on August 2008 by Prof. Dr. Ahmed Nader, Agronomy Department, Faculty of Agriculture, Mansoura University, from plants grown in a field at the university campus.

Corncobs type was identified as Zea mays hybrid individual 3080. They were milled to a particle size of 1.25 μm. A voucher specimen (No. 1536) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University.

This assay was performed as described previously [10] . The cells were placed in two 24-well plastic culture plates (one plate for determining melanin and the other for cell viability) at a density of 1 × 10⁵ cells/well and incubated for 24 h in media prior to being treated with the samples. After 24 h, the media were replaced with 998 µL of fresh media and 2 µL of the test sample at concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg/mL (n = 3). At the same time, a negative control (2 µL DMSO) and a positive control (2 µL DMSO with arbutin at a concentration of 50 mg/mL) were tested. The cells were incubated for an additional 48 h, and then the medium was replaced with fresh medium containing each sample. After 24 h, the remaining adherent cells were assayed. To determine the melanin content (for one plate) after removing the medium and washing the cells with PBS, the cell pellet was dissolved in 1.0 mL of 1 N NaOH. After being kept in the dark overnight, the crude cell extracts were assayed by using a microplate reader at 405 nm to determine the melanin content. The results from the cells treated with the test samples were analyzed as a percentage of the results from the control culture. Cell viability was determined by an MTT assay, which provides a quantitative measure of the number of viable cells by determining the amount of formazan crystals produced by metabolic activity in treated versus control cells. For the other well plate, 50 µL of MTT reagent in PBS (5 mg/mL) was added to each well. The plates were incubated in a humidified atmosphere of 5% of CO₂ at 37˚C for 4 h. After the medium was removed, 1.0 mL isopropyl alcohol (containing 0.04 N HCl) was added, and the absorbance was measured at 570 nm after cells were kept in the dark overnight.

RBL-2H3 cells are the tumor analog of mast cells, which after being sensitized with mouse monoclonal IgE or ionophore A23187 respond by releasing inflammatory mediators such as β-hexosaminidase [11] . As a result, the sample is considered to have anti-allergic activity if it can inhibit mast cells degranulation and produce a significant reduction in β-hexosaminidase release. Firstly, we performed a cell viability assay (using MTT) to ensure that the activity of the sample at the used concentration was related to the inhibition of histamine release rather than to the cytotoxicity of the RBL-2H3 cells. The cell viability assay was done as follows: RBL-2H3 cells (100 µL, 1 × 10⁵ cells/well) were cultured with EMEM in a 96-well plate for 24 h. Then samples (1 µL/well; DMSO 1 µL as control) were added. After 24 h incubation in a CO₂ incubator at 37˚C, MTT (10 µL, 5 mg/mL in PBS) was added to each well, and the plate was incubated for another 4 h. The medium was then removed, acid iso-propanol (100 µL, containing 0.04 N HCl) reagent was added to each well, the plate was incubated overnight at room temperature, and the absorbance was read at 570 nm using a microplate reader.

The anti-allergy assay was determined as previously described [12] with minor modification, as follows: RBL-2H3 cells (1 × 10⁶ cells/well) were inoculated with EMEM in a 96-well plate for 48 h, then EMEM medium was replaced by tyroid buffer [100 µL, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl₂, 1 mM MgCl₂·6H₂O, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 5.6 mM glucose, 0.1% BSA, pH 7.2/well], sample (1 µL/well) was added, and the plate was incubated for 30 min in CO₂ incubator at 37˚C. A23187 (10 µg/mL, 2 µL/well) was added after removal of the sample and the addition of new tyroid buffer (100 µL/well). After 30 min incubation, 50 µL from each well was collected and transferred to another 96-well plate. An equal volume of substrate solution (l mM), p-nitrophenyl-N-acetyl-β-glucosaminide was added to each well, and the plate was left at room temperature on the shaker for 1 h. Finally, the reaction was terminated by adding 100 µL of stopping buffer (Na₂CO₃, 100 mM, pH = 10), and the absorbance was measured at 405 nm using a microplate reader. The statistical difference between the control and each sample was determined by student’s t-test.

This assay was determined as described previously [13] with little modification. Briefly, a single colony of the test strain (Escherichia coli and Staphylococcus aureus) was taken and added to 5 mL of NB medium. This medium was incubated at 37˚C ± 1˚C, 120 rpm for 20 h. It was then added to bacterial suspension to prepare a bacterial concentration at 10⁵ CFU/mL. The bacterial solution was used for the following antibacterial assay. In each well of a 96-well plate, 133.5 µL of NB medium, 15 µL of bacteria suspension, and 1.5 µL of DMSO were added with or without each sample. Also, sorbic acid (400 μg/mL) was used as a positive control. The plate was incubated at 37˚C ± 1˚C, 1160 rpm for 18 h. Finally, bacterial growth was measured by a micro-plate reader (630 nm). The statistical difference between the control and each sample was determined by student’s t-test. The MIC (minimum inhibitory concentration) was the lowest concentration of the test extract that completely prevented growth until 18 h.

Also the activity of citric acid was tested against Propionibacterium acnes using the same procedure as above except the use of GAM broth instead of N. broth, bacterial concentration at 10¹ CFU/mL and finally plate was incubated at 37˚C ± 1˚C, 1160 rpm for 24 h. Also benzalkonoium chloride was used as positive control in case of P. acne.

Scientific studies have shown that HA improves skin hydration, stimulates production of collagen in skin, works as an antioxidant and free radical scavenger, maintains skin elasticity, cushions joints and nerve tissues, has an antibacterial and anti-inflammatory activity and maintains the fluid in the eye tissues, which may help to protect against numerous possible eye concerns [14] .

Citric acid was tested for the effect of the production of HA by the use of HA ELISA assay using Biotech Trading Partners (Encinitas, California) according to manufacture’s instruction.

This kit is an enzyme-linked binding protein assay that uses a capture molecule known as hyaluronic acid binding protein (HABP). After growing of the fibroblast cells, they are incubated in HABP-coated micro well plate. Properly samples and HA reference solution are added to this plate, allowing HA present to react with the immobilized binding protein. After removal of unbound molecules by washing, HABP conjugated with horseradish peroxidase (HRP) solution is added to the microwells to form complexes with bound HA. Following another washing step, a chromogenic substrate of tetramethylbenzidine and hydrogen peroxide is added to develop a colored reaction. The intensity of the color is measured in optical density units with a spectrophotometer at 450 nm. The higher the intensity of the color, the higher ability of the sample to produce hyaluronic acid.

An important concept when selecting bioactive compounds that modulates skin pigmentation that for obvious reasons, they should have minimal effects on cell proliferation and/or toxicity. Taking into consideration about the cytotoxicity to cell lines, the most active concentration of citric acid exhibiting melanin synthesis inhibition (~33%) and at the same time with moderate cytotoxicity (16%) was 200 µg/mL, followed by concentration 100 µg/mL (20% inhibition) with low cytotoxicity (10% cytotoxicity). Citric acids at concentrations of lower than 100 µg/mL have no melanin inhibition effect. Results are shown in Figure 1.

The highest inhibition of β-hexosaminidase release was recorded at both concentration of 1000 µg/mL and 500 µg/mL (82% in A23187 assay). However, citric acid showed high toxicity (75%) at 1000 µg/mL compared to those at 500 µg/mL which showed lower cytotoxicity (29%). Citric acid at 250 µg/mL showed 45% inhibition of β-hexosaminidase release without cytotoxicity. Results are shown in Figure 2.

From the results, it was shown that citric acid completely inhibit the growth of S. aureus, E. coli and P. acne with MIC = 25, 50 and 200 µg/mL, respectively. Results are shown in Figure 3.

It was found that citric acid at both tested concentration have a very good stimulating effect for the production of hyaluronic acid more than N-acetyl glucosamine (NAcG) which is used as positive control and at the same time they showed no cytotoxic to the cell. Results are shown in Figure 4.