Abstract
Since monolaurin, a monoglyceride formed in the human body in small quantities, has proven effective both in vitro and in vivo against certain strains of Staphylococcus aureus, an important question arises whether consuming a substance high in lauric acid content, such as coconut oil could increase intrinsic monolaurin production to levels that would be successful in overcoming staphylococcal and other microbial invaders. Both a cup plate method and a microdilution broth culture system were employed to test bacteriostatic and bactericidal effects of the test agents in vitro. To test effectiveness in vivo, female C3H/he mice (10–12 per group) were orally administered sterile saline (regular control), vancomycin (positive control), aqueous monolaurin, or two varieties of coconut oil (refined, bleached, deodorized coconut oil and virgin coconut oil) for 1 week before bacterial challenge and 30 days after. A final group received both monolaurin and vancomycin. In contrast to monolaurin, the coconut oils did not show bactericidal activity in vitro. In vivo, the groups receiving vancomycin, monolaurin, or the combination showed some protection—50–70% survival, whereas the protection from the coconut oils were virtually the same as control—0–16% survival. Although we did not find that the two coconut oils are helpful to overcome S. aureus infections, we corroborated earlier studies showing the ability of monolaurin to do such.
Introduction
M
We designed the current study to examine the ability of two different coconut oils given orally to protect mice against a fatal form of S. aureus. The two forms were refined, bleached, deodorized (RBD) coconut oil (containing lauric acid approximating 50%), and a virgin coconut oil containing lauric acid at roughly the same level. As a first approximation, we also examined, the influences of the two coconut oil preparations in vitro on two strains of S. aureus, one claimed to be multidrug resistant, before launching in vivo experiments. We found that in contrast to pure monolaurin the two brands of coconut oil by in vitro means and given orally to living mice were essentially ineffectual against the Staphylococcal organism and the infection caused by it.
Materials and Methods
In vitro studies
Initially a cup plate method was employed to screen all the test agents, including the antibiotics. 7 Molten nutrient agar cooled to 45°C was seeded with exponentially growing S. aureus cultures and poured into Petri dishes. After the media was hardened, cups were made into the agar using a sterile borer. The wells were filled with the test reagents—0.l mL containing various concentrations of material under examination or the solvent control. The plates were incubated at 37°C for 24 to 48+ h. Antibiotics examined included; amoxicillin, penicillin, streptomycin, methicillin, vancomycin, as well as test agents. RBD and virgin coconut oil and monolaurin (glycerol ester of lauric acid) were the test agents. Absolute ethanol was the solvent control. Test organisms were; S. aureus ATCC strain #BAA42 and ATCC strain #14154.
The zone of inhibition was measured to assess the minimum inhibitory concentration (bacteriostatic) at 24 h and minimum bactericidal concentrations required at the end of 48+ h of incubation. Virgin coconut oil was obtained from North American Herb and Spice (NAHS), Waukeegan, IL, and RBD from San Pablo Manufacturing Corporation, San Pablo City, Philippines.
A microdilution broth culture system (1 mL) was also employed to test the bacteriastatic (24 h) and bactericidal (48+ h) effects of the test agents. 6,8 Serial dilutions of antibiotics, the two oils under investigation and monolaurin were made in nutrient broth and inoculated with exponentially growing (18 h old) bacteria (6×106 cfu). Appropriate solvent controls were also included. Cultures were incubated at 37°C on a rotary shaker for 5 days. Aliquots from the culture were drawn after 24, 48, and 72 h, and plated on nutrient agar plates. Plates were incubated in a 37°C incubator for the appearance of colonies.
In vivo studies
Female C3H/hej mice (15–18 g) were obtained from Taconic Farms (Germantown, NY, USA). The animals, maintained in a controlled environment at 25°C with a 12 h light and 12 h dark cycle, were acclimatized for 3–5 days before use. The mice were housed in groups of five, fed commercial rodent pellets, and given water ad libitum throughout the experiments. The protocol for the in vivo investigation was approved by the Animal Welfare Board at Georgetown University Medical Center (Washington, DC, USA).
The optimal conditions for murine bacteremia model and its treatment by vancomycin were established previously. 8 Twelve animals in each group (single exception was group receiving both monolaurin/vancomycin containing 10 mice) were orally administered with doses of aqueous monolaurin (5.00 mg in 0.75 mL of sterile saline) and coconut oil (0.75 mL) for a week before infection, and this regimen was to continue until the termination of the experiments (30 days later). The control groups were gavaged with either 0.75 mL sterile saline (Regular Control) or 400 μg of vancomycin in 0.75 mL sterile saline (Positive Control). All groups were eventually injected intraperitoneally with 25×106 bacterial particles in 0.1 mL of sterile PBS containing 5% mucin. A new needle and syringe were used for each injection.
The mice were monitored twice daily postinjection with the bacteria until the completion of the experiment. The body weight of each mouse was recorded daily. Animals exhibiting extreme distress, such as, 15% weight loss, severe dehydration, and poor overall appearance at any time during the experimental period, were euthanized. Thirty-day survivors were euthanized at the end. Culturing aliquots of kidney homogenates on SG agar plates further tested the renal burden of S. aureus.
Results
In vitro
Using the cup plate method, 3 monolaurin produced a clear area in culture in a dose-concentrated manner (data not shown). The RBD showed virtually no area of clearing, while the NAHS coconut oil showed some clearing (data not shown).
Using the microdilution method, initial screening of the panel of antibiotics against the two strains of S. aureus revealed that the strain 14154 is in general more resistant and required higher concentrations of antibiotics and monolaurin to be effective (Table 1).
MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; RBD, refined, bleached, deodorized.
Figure 1 depicts the ability of monolaurin at different concentrations (starting at 2.5 μg/mL) to destroy staphylococcus in vitro, whereas no similar effects were seen with RBD coconut oil even at higher concentrations (Fig. 2).
Depict the microdilution broth culture system (1 mL) employed to test the bactericidal (48+ h) effects of monolaurin on Staphyloccoccus aureus BA 42. Serial dilutions of monolaurin and RBD coconut oil were made in nutrient broth and inoculated with exponentially growing (18 h old) bacteria (6×106 cfu). RBD, refined, bleached, deodorized. Color images available online at
Depict the microdilution broth culture system (1 mL) employed to test the bactericidal (48+ h) effects of RBD coconut oil on Staphyloccoccus aureus BA 42. Serial dilutions of monolaurin and RBD coconut oil were made in nutrient broth and inoculated with exponentially growing (18 h old) bacteria (6×106 cfu). Color images available online at
In vivo
Six groups of 12 mice were injected with 0.75 mL sterile saline containing the BAA 14154 strain. The only exception in numbers was the group receiving the combination of monolaurin and vancomycin that contained 10 mice. In the 12 receiving olive oil as baseline, the mice died within 8 days with the exception of one mouse (Fig. 3). Seven of those receiving vancomycin survived 30 days and upon gross inspection and culture of the kidneys were found free of infection. Examining the results of the mice receiving the natural products, six receiving monolaurin survived, as did seven ingesting the combination of monolaurin and vancomycin. In contrast, none survived that received RBD coconut oil and two survived that got concentrated virgin coconut oil. Again, all survivors were free of staphylococcus in their kidneys after 30 days.
The number of surviving mice on the different regimens after 30 days.
Discussion
Herbal products, such as medium chain fatty acids and essential oils either used as nutritional supplements or as food preservatives are known to possess antimicrobial properties. 1 –6,8 –10 For example, we demonstrated earlier that monolaurin, oregano oil, and some other essential oils possess antimicrobial and antifungal activity in vitro and in vivo. 5,6,8 Monolaurin is composed of a glycerol unit and a single lauric acid. Lauric acid is a major component of virgin coconut oil, but virgin coconut generally contains low amounts of monolaurin. However, orally administered or taken as a food additive, some coconut oil is hydrolyzed by pancreatic lipase into the monoglycerate of lauric acid. 11 The RBD coconut oil essentially contains only lauric acid, because many short and medium chain carbon metabolites, such as monolaurin are removed. 12
However, a therapeutic efficacy has been attributed to RBD oil and virgin coconut oil based on the hypothesis that lauric acid can be hydrolyzed in vivo to yield therapeutically effective monoglycerates (monolaurin) and other medium chain fatty acid- esters. 11,13 Similarly, many coconut oils, used as a food-flavoring agents, have been postulated to possess a broad spectrum of antimicrobial activity due to their high content of lauric acid that can be converted into monolaurin. 4 Even though it is generally accepted that coconut oils containing high concentrations of lauric acid possess potential antimicrobial effects, little actual investigation has been performed using these natural product to treat superficial or systemic infections due to bacteria, viruses, or fungi.
Why do many investigators seek effective natural antibiotics? There are at least three good reasons. The first is the possibility of avoiding so-called “drug resistance” whereby the agent is no longer effective in killing organisms. 14,15 In addition to the possibility of avoiding resistance not uncommon with the use of drugs, many of the synthetic antimicrobials are expensive (the second reason) and have shown serious toxic adverse effects (the third reason). 16,17 Accordingly, the development of an inexpensive, natural product free of deleterious health side effects and not prone to the development of resistance would aid in the prevention, amelioration, and/or cure of various frequent, severe infectious diseases.
Our study was designed to examine the ability of RBD coconut oil and virgin coconut oil containing ∼50% lauric acid on a fatal form of S. aureus infection in mice. S. aureus strains (obtained from ATCC) exhibit varying degrees of sensitivity to antibiotics. The strain BAA 42 appeared to be sensitive to the antibiotics tested. The strain BAA 14154 was comparatively more resistant to all the antibiotics, including methicillin. While the coconut oils produced little bactericidal effect in vitro, monolaurin showed significant antibacterial activity, demonstrating bacteriostatic and bactericidal effects. The concentrations at which monolaurin exhibits bactericidal actions on these two strains is indicative of their biological characteristics. The sensitive and less virulent strain, BAA42 required far less antibiotic and monolaurin. It is important to keep in mind that food products by nature are not toxic; hence, often require higher concentrations to bring about the effects exhibited by the antibiotics (which are require much lower concentrations but can be quite toxic). Suffice it to say, the above described features of the natural products could make for good prophylactic agents.
In contrast to concentrated monolaurin, the two coconut preparations did not show any significant effect on S. aureus. These negative results with the two coconut oils in the initial in vitro studies were not totally unanticipated. We expected this, since we had previously postulated that the coconut oil, dense in lauric acid, needed to be converted into monolaurin to be effective. Thus, it could only be effective in vivo where the production of monolaurin could take place. The virgin coconut oil preparation in vitro showed some but only minor bactericidal capability consistent with the presence of low levels of monolaurin known to be present. The lack of any apparent effect from the RBD coconut oil might be ascribed to the methodology that by refining, bleaching, and deodorizing removes many fatty acids that might include monolaurin from the preparation.
What was not fully characterized is whether one can ingest enough of precursor lauric acid from the two coconut preparations to make enough monolaurin to be effective. 4 It is not been established from the literature with certainty that coconut oils containing lauric acid (50% of the total fatty acid present in the coconut) can be metabolized in vivo to yield sufficient quantities of monolaurin, the glycerol salt of lauric acid. 7 Nevertheless, it seemed possible that coconut oil could effectively inhibit bacterial growth in vivo when administered orally. Similar to previous studies, 5 vancomycin and monolaurin protected roughly one half the mice from infectious death (vancomycin 7/12; monolaurin 6/12). Percentage-wise the combination of the two did even slightly better (7/10), but larger numbers are needed to decide whether a combination would be a further improvement. The survival patterns with the RBD (0/12) and the coconut oil (2/12) were essentially similar to that of the control group (1/12). Based on these findings, we were not able to show that ingesting large amounts of lauric acid will produce sufficient monolaurin to be effective.
Perhaps, we did not give the mice enough coconut oil to be effective. Based on calculations of the amount of lauric acid found in Mother's milk, Enig suggested a successful lauric acid diet for the average human adult would contain ∼24 g of lauric acid daily contained in about 3.5 tablespoons of coconut oil. 18 How does this relate to what we gave the mouse? A tablespoon is roughly 15 mL, therefore, a reasonable human daily dose calculates to 52.5 mL. Each mouse weighed ∼33 g. Comparison on a weight basis would require that one multiply the mouse dose by roughly 2100 to approximate the weight of a 70 Kg person. Taking into account surface area, 19,20 the equivalency factor for a mouse (1/12) would make that adjusting ratio closer to 175. Accordingly, the mouse receiving 0.75 mL daily would take in a dose equivalent to a human of 131 mL, roughly 2–3 times human daily dose mentioned above (on the basis of weight alone, more than 20 times). While we believe starting the feeding 1 week in advance is sufficient, still to be determined is whether the metabolism of mice can be compared to men in this respect.
While we did not unearth evidence that two different coconut oils can be helpful to overcome S. aureus infection in vivo, we corroborated as shown earlier that direct monolaurin ingestion is virtually as effective as vancomycin. 5
Footnotes
Author Disclosure Statement
No competing financial interests exist.