Dried sample of Veronica officinalis (whole plant) were supplied from Royal Botanical Garden Scotland, in November 2012. The plant material was air-dried at room temperature, milled, extracted with ethanol (70%) overnight, and filtered, and the process was repeated three times. The resulting ethanol extract was concentrated at reduced pressure in a rotatory evaporator at 40˚C. A portion of the ethanol extract (410 g) was dissolved in distilled water, and placed in a separator funnel, and washed with n-hexane (200 mL, 15 times). The n-hexane phases were then combined and concentrated under reduced pressure. An identical process was repeated with chloroform, ethyl acetate, and butanol leaving a residual mixture of ethanol-water.

Ethyl acetate extract (56.1 g) was fractionated by open CC using silica gel. Elution was carried out with addition of methanol to hexane-ethyl acetate mixtures in different ratios of increasing polarity until 100% methanol was reached. All fractions were analyzed by TLC. Verbascoside was obtained as the major compound by successive washes with CH₂Cl₂-MeOH (7:3) (5.02 g, 8.94% of the ethyl acetate extract). This compound was purified further by silica gel TLC using CH₂Cl₂-MeOH. The chemical structures of verbascoside was determined by comparison of spectroscopic and chromatographic data with those of authentic samples and were reported previously [7] [8] .

Two percent of Veronica officinalis extract was mixed with a formulation containing water, Carbomer, Glycerine, Disodium EDTA (ethylene diamine tetra-acetic acid), Methylparaben, Trithanolamine, Tocopheryl acetate, Polysorbate 60, Stearyl alcohol, PEG-100 (polyethylene glycol-100) stearate, Sorbitan stearate, caprylic/capric triglyceride, Dimethicone, Mineral oil, Propylparaben, Butylene Glycol, Beeswax, and Fragrance. The placebo (control) was identical in composition, except plant extract.

Fibroblast cell was purchased from the Korea cell line bank, Seoul, Korea were cultured in 96-well plates containing Dulbecco’s Modified Eagle Medium (DMEM, 200 µl/well) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin sulfate (100 µg/ml) in a humidified atmosphere of 5% CO₂. The cell viability assay of the Speedwell extract was performed with MTT (3-(4,5-dimethylthiaol-2-yl)-2-5- diphenyltetrazolium bromide) reagent following the protocol of Mosmann et al. [9] .

Procollagen type 1 protein synthesis test was performed according to the protocol of Tanayama et al. [10] with slight modification. 5 × 10⁴ cell/well were seeded in a 24 well plate with DMEM (containing 10% FBS and 100 unit/ml penicillin-streptomycin). The plates were incubated overnight at 37˚C in a humidified incubator, 5% CO₂. After incubation, the test extract or compound was added to the plate in serum free media. After incubation the plate for 24 hour at 37˚C in a humidified incubator, 5% CO₂, the culture supernatant was collected after centrifuging at 13,000 rpm in 4˚C for 20 min. The resultant supernatant was measured in duplicate according to the supplier’s instructions (Takara, MK101). Negative control was performed with buffer and substrate but without enzyme. All assays were performed independently in duplicate.

Statistical analyses were carried out using SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA). The differences among samples were statistically evaluated via one-way analysis of variance (ANOVA) followed by Dunnett’s posthoc test or Wilcoxon’s test when appropriate. The level of significance was set at p < 0.05. Data are expressed as means ± standard errors.

The antioxidant efficacy of ethanolic extract of speedwell and isolated single compound verbascoside (Figure 1) is given in Figure 2. The isolated compound verbascoside showed a higher free radicle (DPPH) scavenging activity with the IC₅₀ value of 36.24 ± 1.81 µg/ml. Similarly, the ethanolic extract of speedwell also showed good inhibition to DPPH radicles in dose dependent manner whose IC₅₀ value was 103.50 ± 2.43 µg/ml.

The single compound verbascoside isolated from speedwell did not show significant result on Collagen synthesis activity in Fibroblasts (data not shown). However, the significantly increased in procollagen type 1 protein synthesis was observed due to the ethanolic extract of speedwell in fibroblast cell without cytotoxic effect (Figure 3). At a concentration of 2%, extract showed 44.6 % increase in collagen synthesis compare to the control (without extract). This activity of extract on PIP could be due to the interactions and synergistic effect of phytochemicals other than verbascoside present in the sample [12] -[14] .

We examined the effect of topical formulated Scoti-speedwell cream on the wrinkles of crow’s feet site of the eyes (Figure 4). The wrinkle area and length difference at the base line were analyzed to identify the differences at sample treated sites. Treatment with formulated cream for 28 days did not show any significant difference with the placebo. However, treatment for 56 days reduced significantly the percentage of wrinkle area (Figure 5) and wrinkle length (Figure 6) by 18.0% and 16.05% respectively compared with the placebo. The dermatological scores of the sides treated by the extract containing cream decreased significantly on 56 days with 66% lower than that of placebo treatment (Figure 7).