Antibacterial Botanical Formulations

Zhijun Liu, Zhang, Jian, Owens, William E.  |  4/13/2017 5:11:22 PM

Plants employ a plethora of molecules as their chemical weapons against bacterial invasion. Bovine mastitis is a bacterial infection of the mammary gland of cows and a major health issue for the dairy industry. Germicidal teat dips commonly containing iodine (Figure 1A) as the active ingredient are often used to disinfect the udder prior to milking to help reduce new infections. However, iodine residues in milk can be problematic. This research is intended to find bioequivalent plant extracts as safer and skin-friendlier alternatives.

Many essential oils, including tea tree oil, are known to be antimicrobial. Essential oils have been widely used since ancient times. The problem is essential oils are not miscible or capable of being mixed with water. This property has made it difficult for water applications such as immersions and sprays. There are many methods for dispersing essential oils in water, but effectiveness and stability, as well as safety issues, have made them less desirable. However, a new method has been identified for overcoming this problem. A water-soluble plant extract and an insoluble essential oil were used to show proof of concept. In this study, plant extracts of sweet leaf tea and tea tree oil were prepared so they were soluble in water and culture media without the use of any organic solvents such as ethanol, dimethyl sulfoxide, propylene glycol or polyethylene glycol. Sweet leaf tea is water-soluble (Figure 1B), but tea tree oil is not (Figure 1C). Dispersion in water was done by using a botanical solubilizer from the steviol glycosides family. The LSU AgCenter holds several patents on the use of some steviol glycosides as solubilizers. The use of the botanical solubilizer created a water-miscible tea tree oil solution (Figure 1D).

The aqueous solutions of the extracts were tested against bacteria known to cause mastitis. The test was run based on a standard protocol. The tested organisms included two gram-positive bacteria, Staphylococcus aureus and Enterococcus fecalis, and two gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa. Small samples of each 24-hour bacterial culture were standardized and added to samples of the test extracts as well as iodine as a positive control. After 30 seconds and again after 10 minutes, samples were removed and plated to create colonies, which were incubated for 24 hours and then counted.

Both sweet leaf tea and tea tree oil showed antibacterial activity. Sweet leaf tea inhibited the growth of S. aureus by 56 percent in 30 seconds and 64 percent in 10 minutes; E. fecalis by 92 percent in 30 seconds and 91 percent in 10 minutes; E. coli by 86 percent in 30 seconds and 90 percent in 10 minutes; and P. aeruginosa by 93 percent in 30 seconds and 99 percent in 10 minutes. Tea tree oil was much quicker in action and more potent; in 30 seconds, it killed 99 percent of the gram-positive bacteria and greater than 99 percent of the two gram-negative bacteria. In 10 minutes, tea tree oil killed essentially all bacteria with a greater than 99 percent reduction of every organism that came into contact with it. Iodine, as expected, killed essentially all the bacteria in contact. It is evident that the tea tree oil formulation has a comparable antibacterial effect to the standard iodine product. The solubilizer used in the tea tree oil formulation alone showed some inhibitory effect against these bacteria, but it is the tea tree oil that acts as the antibacterial ingredient (Figure 2).

The sweet leaf tea showed some antibacterial activity, but in its current form maxed out at 90 percent. Because the sweet leaf tea extract is a mixture of compounds, its antibacterial activity may be further enhanced by optimizing the components. But this remains to be seen.

The tea tree oil formulation, on the other hand, showed excellent antibacterial activity. It killed both gram-positive and gram-negative bacteria instantly and completely upon contact, as did the iodine. Using the bioequivalent botanical formulation instead of the standard antibacterial iodine product opens the door to safer and better products in preventing mastitis and bacterial infection. Stevia extract, which was used as a botanical solubilizer in the tea tree oil formulation, is on the Food and Drug Administration GRAS (generally regarded as safe) list for food sweetening. The formulation consisting of an active essential oil and inactive food sweetener would be much safer to humans than iodine and its formulating ingredients. The combined efficacy and safety offer a better way to control bacteria in food production than is currently available. Iodine is a synthetic antimicrobial agent. Although it has been listed as a material allowable for restricted organic food production, iodine is closely monitored for its residue in milk. An increasing level of iodine in milk has been a concern to human health.

Tea tree oil for inhibiting bacterial growth in a 100 percent botanical formulation is bioequivalent to, and has a safer profile than, iodine. This raises promise in finding alternative methods for preventing mastitis. The laboratory results reported here warrant more tests for preventing surface bacterial infection such as bovine mastitis and postharvest food sanitation. The success in formulating tea tree oil also sets the stage for formulating other antimicrobial essential oils, such as those in cloves, cinnamon and thyme, that are even more potent and safer. Essential oils of cloves, cinnamon and thyme are listed as GRAS and as active ingredients eligible for minimum risk pesticide products by the Environmental Protection Agency.

Zhijun Liu is a professor in the Medicinal Plant Lab in the School of Renewable Natural Resources; William Owens is a professor and resident coordinator at the Hill Farm Research Station, Homer, Louisiana; Yixuan Dong is a former postdoctoral researcher; and Jian Zhang is a former research associate in the Medicinal Plant Lab.
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Zhijun Liu. Photo by Olivia McClure

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Figure 1. A) iodine used to sanitize teats before milking to control mastitis; B) water-soluble sweet leaf tea extract; C) tea tree oil on the top layer, which is not mixable with water; and D) tea tree oil dispersed in the presence of a botanical solubilizer. Photos by Olivia McClure

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Figure 2. A plate on which Staph. aureus is being grown. Paper discs were saturated with samples of A: tea tree oil in water with a botanical solubilizer (TTO+BS), B: tea tree oil solubilized in DMSO (dimethyl sulfoxide) solvent (TTO+DMSO), C: DMSO (dimethyl sulfoxide) by itself, D: a botanical solubilizer as a carrier by itself, and E: saline as negative control. Bacterial growth around the samples can be seen. A and B containing the antibacterial tea tree oil produced a clear inhibitory zone, showing no bacterial growth, whereas C and E produced no zone, showing full bacterial growth. D produced a visible but much smaller zone compared to A and B, each containing tea tree oil. This is a clear indication that tea tree oil is antibacterial and can be solubilized without the use of organic solvents such as DMSO.

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