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A pilot study on the degree of tooth staining caused by pollen water and Chlorhexidine: in vitro study


Almulhim, Khalid (2021), A pilot study on the degree of tooth staining caused by pollen water and Chlorhexidine: in vitro study, Dryad, Dataset,


Background: Chlorhexidine oral rinse has been used as an adjuvant in the treatment of periodontal disease. However, there are draw backs of using chlorhexidine i.e. tooth staining and other side effects including allergy reaction. In light of proven therapeutic properties of pollen water as well as its relatively cheap cost in the market, pollen water has a potential to be an effective alternative to chlorhexidine oral rinse. The aim of this study is to compare the degree of tooth staining influenced by pollen water-based mouthwash to the standard Chlorhexidine mouthwash using spectrophotometer.

Materials and Methods: 24 Specimens from extracted intact human teeth were soaked into the three different solutions, Pollen water, and normal water. Color measurements were carried out by a spectrophotometer devise and recorded at 5 different time intervals. Color change (ΔE), Chroma (C*) and Hue (H*) were analyzed and compared among the three solutions.

Results: Overall mean ΔE was similar in all groups, significant difference between all time points was found only in Pollen water. The change in C* was higher in pollen water as compare to other solutions. There was a subtle increase in H* in the Chlorhexidine samples after week 3. The H* values in pollen water was stable but sudden decrease was observed in week 6. The difference in H* among the three solutions was significant after 3 weeks.

Conclusion: Within the limitation of our study, it can be concluded that Pollen water stained teeth to a lesser extent than did Chlorhexidine. It might be beneficial to use Pollen water as mouthwash however further investigation is needed regarding efficacy of plaque control.


This study was conducted at the laboratory of Imam Abdurahman bin Faisal University College of Dentistry, after gaining ethical approval (EA #2018029) from the Scientific Research Unit. It consisted of the following steps.

1) Preparation of three solutions:

  • Chlorhexidine 0.12 % licensed for use as a mouthwash. (positive control)
  • Pollen water licensed for internal/external use (100% pure, cold-pressed). (test group)
  • Normal water. (negative control)

2) Teeth preparation: 24 extracted intact human teeth were used in this study. Inclusion criteria were: caries-free teeth with no cracks, no previous root canal therapy, and no crowns, which were recently extracted for orthodontic treatment purposes. The teeth surfaces were cleaned using a scaler to dislodge debris and periodontal instruments to remove calculus. This was followed by brushing using a rubber cup. Subsequently, each sample was prepared to be at a size of 8 mm ×11 mm × 2.5 mm using a diamond bur (Iwanson, Ustomed, Tuttlingen, Germany). The total sample size was 24 (8 samples for each solution).

3) Color Measurements: Each prepared sample was placed onto the chamber of a benchtop digital imaging spectrophotometer (X-rite color-Eye 7000A; Gmbh, Regensdorf, Switzerland), which was previously calibrated according to the manufacturer’s specifications. Color measurements were obtained in triplicate, and the mean value was considered as the final measurement.

4) Each specimen was soaked in Chlorhexidine, pollen water, or normal water, and staining was measured weekly for 6 weeks.

5) The following parameters were analyzed:

  • ΔE: indicates the change in color from baseline to that at the measurement time.
  • Chroma (C*): indicates the color intensity or saturation
  • Hue (H*): describes the perception of an object’s color (e.g., red, orange, green, blue).

2.1 Statistical Analysis

The color change (ΔE) was calculated using the following Equation 1:

                          ΔEab* = [(ΔL*) 2 + (Δa*) 2 + (Δb*) 2] ½                                (1)


Δ represents the difference between the object being measured and its reference

L* represents lightness

a* and b* are the chromaticity coordinates

Data normality was checked using the Shapiro-Wilk test with p < 0.05 indicating non-normally distributed data. The color differences for L, a, b, C*, and H* were calculated and analyzed using the Friedman test (non-parametric method of repeated-measure analysis of variance [ANOVA]). Repeated-measure ANOVA was applied separately for each solution. For significant findings, the paired sample t-test was performed to assess for individual significant results. We considered statistically significant at P < 0.05. All analysis was done using SPSS version 22 software (IBM, Armonk, NY).