**5. Comparison of theobromine and fluoride**

#### **5.1 Microhardness test**

The unique roles of theobromine were accidentally discovered during the caffeine study. Because previously only fluoride has been known to affect the enamel, the next steps were to investigate the comparative studies between theobromine and fluoride on the effects of the enamel in a similar environment using the in vitro system.

If remineralization occurs, then the increased enamel is associated with increased microhardness [33]. The microhardness test was conducted using theobromine [34]. An in vitro study confirmed that theobromine increased the microhardness of the enamel [35–37].

The enamel of human teeth with varying concentrations of theobromine vs. sodium fluoride vs. control groups (distilled water) was performed. Scanning electron microscopy (SEM) and Knoop Microhardness Testing (NMT) were conducted. A Knoop microhardness instrument was used. Knoop Microhardness tests were performed every day for a period of 8 days on each sample and the data were recorded. Day 1 represents the baseline hardness.

On day 8, it was not tested to avoid interfering with the surface of the tooth before taking the scanning electron photomicrographs. The hardness data are shown at the end of the 8th day and the results were published [26]. **Figure 10** shows SEM photomicrographs of the enamel of control, fluoride and theobromine.

In general, the teeth that were coated with theobromine appeared to be cleaner, smooth and excellent mineralization was observed under the electron microscope especially when compared with control group teeth.

#### **5.2 Acid dissolution study**

Samples were covered with small uniformly cut circles of double-stick tape. The tooth was covered in nail polish and the tape was removed to expose a fairly uniform enamel surface on each tooth.

*The Contrasting Effects between Caffeine and Theobromine on Crystallization… DOI: http://dx.doi.org/10.5772/intechopen.101116*

#### **Figure 10.**

*Upper left: control group. Upper right: 0.15% w/v fluoride ion. Bottom: theobromine group: 500 mg/L.*

Each tooth was immersed in either 0.15% w/v fluoride ion solution or theobromine 100 mg/L solution for 30 minutes. Then, each tooth was exposed to a 0.001 N HCl acid solution for 10 minutes to determine how much calcium is released from the enamel. The amount calcium (ppm) released from the surface of the enamel in the F-0.15% was an average of 0.930 whereas theobromine-100 mg was 0.848. The fluoride group of teeth released 9.66% more calcium compared to the theobromine group, suggesting that the pre-exposures by theobromine solution is more effective to make the enamel surface of teeth more resistant than fluoride solution. Theobromine is reported as an effective remineralizing agent [38].
