Introducing a new active antimicrobial

The tree develops a massive trunk and can live to a great age. The Totara depicted in the Mende-Biotech logo is Pouakani, reputed to be the oldest living Totara, standing 40 metres high and estimated to be over 1800 years old.

Totara can be found throughout New Zealand; in former times it was particularly abundant in the forests of the central North Island. Maoris revered the Totara as a symbol of strength and used the wood for canoe-making because of its length, lightness and durability. The timber of Podocarpus totara is renowned for its resilience against rotting, which made it valuable to early European settlers of New Zealand for uses such as wharf piles, fence posts and foundation blocks. The durability stems from the anti-bacterial activity of totarol which can be extracted from the dead wood, negating the need to cut down live trees. It is also extractable from other podocarps including rimu (Dacrydium cupressinum or Red Pine); some trees in the cypress family (cypress, juniper, thuja); and rosemary2. It is most abundant in Totara trees2. The extraction process2 is called “supercritical extraction”.

This is a process that uses high pressure carbon dioxide under defined conditions of temperature, pressure and gas flow to extract totarol from powdered Totara deadwood (in the form of old fence posts, telegraph poles, house piles, etc). The process ensures that the extract and residual wood has no harmful solvent residues, and that the highest possible quality of extract is obtained. This extraction process results in a product containing approximately 55-70% Totarol by mass.

Totarol is a broad-spectrum antibacterial, being active against Staphylococcus aureus3; methicillinresistant Staphylococcus aureus4 (epidemic, community, and multi-drugresistant strains2); Streptococcus mutans;3,5 penicillin-resistant Streptococcus pneumoniae;6 Erythromycin-resistant Streptococcus pyogenes;2 high-levelgentamicin- resistant Enterococcus faecalis;6 vancomycinresistant Enterococcus faecalis;2 Salmonella menston,5 Eschericia coli;5 Enterobacter aerogenes;5 Pseudomonas aeruginosa;5 Bacillus subtilis;5 Brevibacterium ammoniagenes;5 and Propionibacterium acnes.3 Beta-amino alcohol derivatives of Totarol have demonstrated antiplasmodial properties that have no cross-resistance with chloroquine.7 Totarol has demonstrated activity against Mycobacterium tuberculosis.8

Totarol’s mechanism of antibacterial activity is not known, however some authors have suggested that it compromises the functional integrity of cell membranes.9 Haraguchi et al studied totarol’s actions on Pseudomonas and discovered that it inhibited oxygen consumption and respiratory-driven proton translocation in whole cells, and oxidation of NADH in membrane preparation (by inhibiting a number of NADH-related enzymes).10 Evans et al alternatively suggested that it disrupts bacterial energy metabolism, although they admitted that this action occurs at much higher concentrations than are significant for antibacterial activity.6 Although the exact mechanism of action is not known, all proposed mechanisms are dissimilar to those of macrolides and tetracyclines. Thus cross-resistance of macrolideand tetracycline-resistant bacteria to totarol seems unlikely. Unfortunately researchers have yet to produce a totarol-derived systemic antibiotic.

The potential benefits would seem to be considerable. Totarol has been tested for safety in human applications. A skin irritation/ sensitisation test of 1% to 0.05% totarol solution on 50 human subjects resulted in no evidence of any toxic effects on the skin.11 Furthermore, it is not cytotoxic at concentrations required for antibacterial and antioxidant activity in cosmetic applications.2 However, above these concentrations it can be.

It therefore has potential for use in current practice as a topical agent for treatment of acne vulgaris. Since totarol has antibacterial activity against a bacterium that is related to the development of acne, Priopionibacterium acnes, its potential use for aiding patients with acne is obvious. However a PubMed search revealed no papers on totarol’s application to clinical practice.

Method of extraction and production
Industrial Research Limited, New Zealand (IRL) was commissioned to develop a commercial scale extraction process to obtain the highest possible quality totarol extract that is free from any solvent residues, artifacts and oxidation products. The extract is produced using supercritical extraction. Water is then removed from the extract by freeze-drying. Supercritical extraction is a relatively new processing technology that uses high pressure carbon dioxide as the extraction solvent. This process ensures that the extract and residual wood have no harmful solvent residues, and that the highest possible quality of extract is obtained. Heartwood from dead Totara (fence posts, fallen logs, construction timber) is ground to a fine powder. The powder is loaded into extraction baskets, which are placed in a multi-vessel extraction plant. The Totara powder is extracted under defined conditions of temperature, pressure and carbon dioxide flow rate to give totarol. The process conditions can be adjusted to give a desired concentration of totarol. The extraction process has recently been patented. An unavoidable part of the extraction process is the co-extraction of water. This is removed from the extract by freeze-drying and the material ground to a fine powder. The resultant totarol extract is a yellow/brown crystalline free-flowing powder and packed into foil pouches.

Antimicrobial properties

Previous literature data
The antimicrobial properties of totarol against a range of gram-positive bacteria has been determined by the pioneering work of Kubo et al3 and Evans et al.6 A summary of the available data is shown in Table 1, where a comparison is made against tea tree oil, which is one of only a few known broad spectrum antimicrobial natural products.

Testing of totarol was commissioned against a range of gram positive and gram negative bacteria. The tests were as follows:

Testing of extract against the grampositive acne causing bacteria Proprionibacter acnes by AMS Laboratories Pty Ltd, Australia

Testing of extract against gram positive bacteria by Environmental Research Limited (ESR) New Zealand

Testing of extract against selected gram negative bacteria by Agricultural Research New Zealand (AgResearch)

The test results are as follows:

Testing of extract against the grampositive acne causing bacteria Proprionibacter acnes by AMS Laboratories Pty Ltd, Australia Totarol extract was tested against Proprionibacter acnes using a suspension challenge test. Totarol extract was dissolved in isopropyl myristate at 1% w/v, and then diluted to 1:20, 1:200 and 1:2000 using bacteriological peptone water. 1 ml of bacterial inoculum containing 1 x 106 colony forming units (CFU) was then added to 9 ml of the diluted solutions to give an overall concentration of 0.05% w/v, 0.005% w/v and 0.0005 % w/v of totarol. The CFU were then counted after 1, 5, 15, 30 and 60 minutes. Reactions were stopped by dilution into neutraliser and further dilutions made with peptone water. The results are shown in Table 2 Totarol shows remarkable activity against Proprionibacter acnes even at very low dilution levels. A dilution of 0.05 % w/v is sufficient to ensure almost a thousand fold reduction in viable bacteria after a contact time of one hour.

Testing of extract against gram positive bacteria by Environmental Research Limited (ESR) New Zealand Totarol extract was tested for activity against a range of gram-positive bacteria using the agar dilution method. The test samples were dissolved in ethanol, and then further diluted in water to give final concentrations, in a doubling dilution series, from 0.004 to 64 µg/mL.

The agar used was Mueller Hinton agar. An inoculum of approximately 1 x 104 organisms of each bacterial strain was replicated onto the agar plates containing the totarol-containing extract. The MIC for each bacterial strain is shown in Table 3. The results shown in Table 2 demonstrate that the totarol extract has very high antibacterial activity against a wide range of antibiotic-resistant grampositive bacterial strains.

Testing of extract against selected gram negative bacteria by Agricultural Research New Zealand (AgResearch) The MIC against four gram-negative bacteria-Salmonella menston; Escherichia coli; Enterobacter aerogenes; and Pseudomonas aeruginosa – was determined for the totarol extract, tea tree oil and a mixture of the totarol extract and tea tree oil. The results are shown in Table 4. The results in Table 3 and Figure 1 below show that the totarol extract is a very effective antimicrobial against a range of gram-negative bacteria. The MIC for each bacterial strain tested is approximately 0.3-1.25 µg/mL, and surprisingly, this figure is the same for each strain, whether subjected to the totarol extract or tea tree oil (Melaleuca alternifolia). Tea tree oil is known to have activity against gram-negative bacteria, while pure totarol has not been reported to have any activity against gram-negative bacteria. Other components of the totarol extract may be responsible for the activity against gram-negative bacteria.

The antimicrobial effect of the totarol extract is enhanced when combined with tea tree oil only for Enterobacter aerogens. A concentration of 0.3-0.6 µg/mL of the totarol extract was sufficient to stop the growth of the other three gram-negative strains tested. This concentration was not decreased by the addition of tea tree oil, nor was the MIC of tea tree oil affected by the addition of totarol extract. The results obtained for tea tree oil are in accordance with those found in the literature, where the MIC for tea tree oil ranges between 0.2 and 2.0 % (v/v), which equates to between 2.0 and 20 µL/mL.

Antioxidant properties

The remarkable antioxidant properties of totarol have been known for many centuries by firstly the Maori of New Zealand, and then by European settlers. More recent research has shown that the chemical that is responsible for preserving Totara from oxidative degradation is totarol.

Bendall and Cambie note that “In a study of the retardation of drying times of painted Totara boards which exhibited the hard resinous appearance known as ‘greasy heart’, Brandt showed that even small (0.05%) additions of totarol to linseed oil paints would retard the drying time”. The resinous ‘greasy heart’ is a totarol-rich exudate. As paint dries by a polymerisation and oxidation process, totarol acts as a strong antioxidant and prevents the drying process. Antioxidant activity in oil-based systems was confirmed by Haraguchi et al.10 The abstract of their paper is as follows: A diterpenoid, totarol from Podocarpus nagi was evaluated as an antioxidant. This diterpenoid inhibited autoxidation of linoleic acid. Mitochondrial and microsomal lipid peroxidation induced by Fe3+ - ADP/NADH or Fe3+ - ADP/NADPH were also inhibited. Nagilactone E, a noditerpene lactone isolated from the same source, had no antioxidative activity. Furthermore, totarol protected red cells against oxidative hemolysis. This diterpene (totarol) was shown to be effective in protecting biological systems against oxidative stress. The Horticultural Research Institute Limited, New Zealand, was commissioned to determine possible skin cell sensitisation after exposure to totarol with and without UV radiation, and found that totarol had antioxidative effect and mild protective properties against UV radiation.

Toxicity testing

A range of toxicity tests were carried out to demonstrate that totarol is safe for human use. The test regimes were as follows:

Determination of minimum lethal dosage in rats (LDL50)

Skin cell (cell culture) sensitisation with exposure to UV radiation

Skin cell (cell culture) irritation/inflammation after exposure to totarol

Skin cell (human volunteers) irritation/inflammation tests

Cytotoxicity (cell culture) tests Skin cell (human cell culture) sensitisation with exposure to UV radiation

The Horticultural Research Institute, New Zealand (HortResearch) was commissioned to determine the DNA damage, oxidative stress, glutathione and mitochondrial membrane potential of human epidermal keratinocytes that had been exposed to totarol. The cells were treated with concentrations of totarol ranging from 4 to 20 (µ/ml, and were then incubated for 24 hours. The cells were then exposed to UV (except for control cells, also exposed to the same totarol levels) and incubated for a further 24 hours. The cells were then harvested and labelled for flow cytometry testing. Results showed that totarol was not toxic to cells, has an antioxidative effect and even has mild protective properties against UV radiation. It also has mild cell proliferation activity and thus would be useful for cell renewal.

Skin cell (cell culture) irritation/ inflammation after exposure to totarol The Horticultural Research Institute, New Zealand (HortResearch) was commissioned to determine the inflammatory or anti-inflammatory properties of totarol on mouse macrophage cells. Cells were treated with concentrations of totarol ranging from 4 to 20 (µ/ml, and were then incubated for 24 hours. Cells were then exposed to LPS (lipopolysaccharide, a standard inflammatory agent) except for control cells at the same concentrations, and incubated for a further 24 h. Cells were then harvested and labelled for flow cytometry testing, where cytokines were measured to determine inflammatory responses. The results showed that totarol does not induce an inflammatory response in mouse macrophage cells, and is thus unlikely to cause an allergic reaction in humans (although this test does not rule out the possibility). It does not stop the inflammatory reaction induced by LPS and therefore is not an anti-inflammatory for this bacterial toxin.

Cytotoxicity (cell culture) tests AMA Laboratories Inc (USA) were commissioned to determine the in-vitro cytotoxicity of totarol on human cell cultures CH2983, HeLa and MG63 at a range of concentrations from 0.2 to 25 µg ml-1. At concentrations between 5.0 and 25 µg ml-1, totarol exerted cytotoxic or growth inhibitory effects. Further experiments were performed over the narrower concentration range 7 - 21 µg ml-1 to determine the threshold cytotoxic effect. The results are shown in Table 5. Totarol was shown to be cytotoxic towards proliferating human cell cultures, CH2983, HeLa, and MG63, but only at concentrations of 30mM. At concentrations of 30mM or less, the human cells tested grew normally. Cosmeceutical applications will not need concentrations greater than 4 - 8 µg ml-1 to obtain full antibacterial and antioxidant activity.

Product applications developed

Oral care products: totarol possesses potent antibacterial activity especially against gram-positive bacteria including Streptococcus mutans which has been implicated as one of the major cariogenic organisms present in dental plaque. However, in order to make a good oral hygiene products such as toothpaste and mouth wash based on totarol the products need to possess a broad antimicrobial spectrum to prevent various oral related diseases such as gingivitis and periodontal disease. This was done by combining totarol with an aniseed extract.

Acne solution: totarol exhibits potent anti-bacterial activity against a number of gram positive bacteria. Among the active compounds isolated, totarol showed the most potent activity against Propionibacterium acnes. Combining totarol with testosterone 5a- reductase inhibitor (found by Mende-Biotech Limited), shows a 80% successful treatment rate.

Skin care and hair care products: Obvious benefits here are in the antioxidant levels. Totarol is a potent antioxidant for unsaturated fats and oils, and does not lose its activity with time, compared to vitamin E. Totarol strongly inhibits lipid preoxidation and scavenges ROS (reactive oxygen species) and will help regenerate the cells and so has possible application in anti ageing products.

Preservative: totarol was not water soluble but this problem has been overcome and totarol can be produced as an aqueous preservative. Totarol will work at a preservative on its own at 0.4 - 0.5% in application. A new 100% natural version will be ready for the market in April 2007 and will be launched at In-Cosmetics in Paris.

References

1 RCC Ltd. TotarolTM material safety datasheet.

2 Mende Biotech Ltd. TotarolTM information pack.

3 Kubo I., Muroi H., Himejima M. Antibacterial activity of TotarolTM and its potentiation. Journal of natural products 1992; 55(10): 1436-40.

4 Muroi H., Kubo I. Antibacterial activity of anacardic acid and TotarolTM, alone and in combination with methicillin, against methicillinresistant Staphylococcus aureus. Journal of Applied Bacteriology 1996; 80(4): 387-94. Full text not available.

5 Moorhead S.M., Bigwood T., AgResearch. Report on the efficacy of TotarolTM, and TotarolTM in combination with tea tree oil as an antimicrobial against gram-negative bacteria. Client report for Mende-DEK Ltd Nov 2003.

6 Evans G.B., Furneaux R.H., Gainsford G.J., Murphy M.P. The synthesis and antibacterial activity of TotarolTM derivatives, part 3: modification of ring-B. Bioorganic and medicinal chemistry 2000; 8(7): 1663-75.

7 Clarkson C., Musonda C.C., Chibale K., Campbell W.E., Smith P. Synthesis of TotarolTM amino alcohol derivatives and their antiplasmodial activity and cytotoxicity. Bioorganic and medicinal chemistry 2003; 11(20): 4417-22. Full text not available.

8 Constantine G.H., Karchesy J.J., Franzblau S.G., LaFleur L.E. TotarolTM from Chamaecyparis nootkatensis and activity against Mycobacterium tuberculosis. Full text not available.

9 Micol V., Mateo C.R., Shapiro S., Aranda F.J., Villalain J. Effects of TotarolTM, a diterpenoid antibacterial agent, on phospholipid model membranes. Biochimica et Biophysica Acta 2001; 1511(2): 281-90. Full text not available.

10 Haraguchi H., Oike S., Muroi H., Kubo I. Mode of antibacterial action of TotarolTM, a diterpene from Podocarpus nagi. Plata Medica 1996; 62(2): 122-5. Full text not available.

11 AMA Laboratories Inc. 50 human subject repeat insult patch test skin irritation/ sensitisation evaluation (occlusive patch) Jan 2000.