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  • Nasal lavage or wash the virus away

    I moved my post to this position to make sure everyone interested in alt. methods has a chance to read and comment. <HR SIZE=1>LMonty, you are succinct.
    Quote:
    That one needs no translation! It's the gold standard, IMHO (and Shannons, too, if its ok I point that out and I'm not mistaken!) for preventing cytokine storm-keeping the viral titers low!


    My focus has changed from one limiting inflammatory response to keeping the virus at low levels. It means aggressively and pro-actively going after the viruses.

    I am currently working on a formula for lowering the numbers in the nose and throat through lavage and spray. I invite comment on the herbs chosen.

    The idea is to make a tea using,
    bromelain (eats the spikes off the viral coat)
    one drop of tea tree or eucalyptus oil (viricide)
    garlic (viricide) fresh and very gently simmered in tea
    nettle, (prevents the manufacture of prostaglandins, also useful for coughs)
    lemon balm (viricide and sooths nasal passages)
    thyme oil (viricide)

    I am also considering adding,
    skullcap (scutellaria baicalensis, anti-inflammatory, and antihistamine, aids in treating allergies or allergic rhinitis, particularly when used with stinging nettle)
    olive leaf (anti-viral)
    polygonum cuspidatum ( called Hu Zhang in Chinese, contains resveratrol a neuraminidase inhibitor, difficult to absorb through the gut this should allow it to be taken up directly into the bloodstream hence, bypassing the first pass through the liver)
    yerba santa (one of the best decongestant herbs as it decreases secretions as well as allays inflammation. Also strengthens weak, irritated and leaky membrane capillaries)

    I welcome any thoughts. __________________

    </IMG></HR>
    Last edited by Shannon Bennett; June 12th, 2006, 03:42 PM.
    Please do not ask me for medical advice, I am not a medical doctor.

    Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
    Thank you,
    Shannon Bennett

  • #2
    Re: Nasal lavage or wash the virus away

    Neti pots are great! I use mine whenever I feel my sinusitis is acting up.
    This week I have been thinking a lot about starting habits that would stand me in good stead should a pandemic strike. Some of the habits are as follows:
    1. Handwashing...before eating, after bathroom use, after using public anything(computer keyboards and phones are examples)
    2. Removing shoes outside the house before coming in.
    3. Carrying a hand sanitizer in purse and in car.
    4. I also now think about washing my hands whenever I have shaken hands with others in a social setting such as church.(It might be a good idea if Westerners adopted bowing as a sign of respect instead of shaking hands...lolol

    These are simple habits but if you haven't been doing them on a regular basis they are hard to maintain.....all it takes is one slip up.....
    We all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their color.
    Maya Angelou

    Comment


    • #3
      Re: Nasal lavage or wash the virus away

      Thank you Pam for the neti pot endorsement. I do think flushing the little boogers (sorry I couldn't resist) out before they have a chance to replicate is a valid idea. I think dh and I are going to spring for the water pic kind. We will have to have a neti pot for a back up, however. I think the professional device will help the kids learn the technique. It will take some getting used to but whatever works.

      Now *toe-tapping here* let me hear from some of you re: the herbs selected, or the method, or whatever your thoughts are. Does this seem feasible, reasonable, valid? I really would like to know your thoughts on this. Do you need more research to back up the claim? Read more about neti pots and how doctors are beginning to endorse their use?
      </IMG>
      Please do not ask me for medical advice, I am not a medical doctor.

      Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
      Thank you,
      Shannon Bennett

      Comment


      • #4
        Re: Nasal lavage or wash the virus away

        final list.

        bromelain (herb, available from most bulk suppliers)
        stinging nettle (herb, available from most bulk suppliers)
        polygonum cuspidatum ( called Hu Zhang in Chinese, I order mine from kaylx.com)
        lemon balm (herb, available from most bulk suppliers)
        skullcap (scutellaria baicalensis, [the s. lateraflora is not what I am looking for] I order mine from kaylx.com)
        (* note, olive leaf is now omitted and substituted with verbascum)
        verbascum (herb, available from most bulk suppliers)
        garlic
        Thyme oil
        Tea tree oil
        Last edited by Shannon Bennett; June 14th, 2006, 11:29 AM.
        Please do not ask me for medical advice, I am not a medical doctor.

        Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
        Thank you,
        Shannon Bennett

        Comment


        • #5
          Re: Nasal lavage or wash the virus away

          Originally posted by Shannon
          final list.

          bromelain (herb, available from most bulk suppliers)
          stinging nettle (herb, available from most bulk suppliers)
          polygonum cuspidatum ( called Hu Zhang in Chinese, I order mine from kaylx.com)
          lemon balm (herb, available from most bulk suppliers)
          skullcap (scutellaria baicalensis, [the s. lateraflora is not what I am looking for] I order mine from kaylx.com)
          olive leaf (herb, available from most bulk suppliers)
          Thyme oil
          Tea tree oil
          Are you going to leave off Tumeric (curcumin?)

          Curcumin


          Curcumin is the compound that gives turmeric spice its bright yellow appearance. It has been used in herbal medicine for a variety of inflammatory conditions, including fever, arthritis, and psoriasis. Curcumin not only blocks TNF, but it is an inhibitor of the MAPK p38 system. At present, the Pubmed research database identifies 110 citations when searching for "MAPK curcumin" while the search phrase "TNF curcumin" returns 82 results. Review of these articles makes it clear that curcumin holds great promise as an agent that may reduce the lethal effects of the avian flu cytokine storm.

          Curcumin is quite inexpensive. At Vitacost (where I buy most of my supplements), 60 capsules of NSI Turmeric (standardized 95% curcumin) 900 mg cost just under $14 when I last checked - less than the typical co-pay for a prescription.

          Absorption of curcumin is modest to poor, but can be increased when co-administered with piperine (a compound found in various species of pepper, including the black pepper found in most kitchens). PMID: 9619120 Several commercial formulations of curcumin include piperine (sometimes called bioperine). Piperine itself inhibits TNF, IL-1, IL-6 and other inflammatory cytokines. PMID: 15531295

          While it is best to store it at room temperature, it will not be completely inactivated under the un-refrigerated conditions that would destroy the potency of some expensive pharmaceutical TNF blockers. Unlike the pharmaceutical TNF blockers, curcumin is associated with a reduced risk of many types of cancer. In particular, lab studies have shown that curcumin induces apoptosis (programmed cell death) in several types of lymphoma. ( citation 1, citation 2, citation 3, citation 4 ).
          We all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their color.
          Maya Angelou

          Comment


          • #6
            Re: Nasal lavage or wash the virus away

            Can we confirm that tea tree oil has specific activity to influenza? I know it is anti bacterial and anti-fungal. In Aromatherapy for Health Professionals by Price, it has a great table on p 75 listing Essential oils mentionaed as having antiviral effects. They show tea tree as having activies against herpes simplex, viral enteritis, and viral enterocolitis. I have not re-read PubMed, the research here, nor the FluWiki CAM page.

            I am still working out the other herb stuff. I remember the Olive leaf and Cranberry (seperate) studies mentioning nasal potentials but when I spoke with the head of Ameriden (olive leaf mfgr) about simply using it in a nasal spray he seemed to say it was not that simple. More studies need to be done which requires money. Again, we use what we have. Big drug companies won't invest in saving lives if they can't make a profit.

            Comment


            • #7
              Re: Nasal lavage or wash the virus away

              Curcumin is not on the lavage list. I do not want to introduce more than one compound that will increase the likelihood for bleeding. Both garlic and curcumin increase the possibility. My focus here was to reduce the viral titers, not to reduce inflammation. This preparation is designed to get rid of surface virus. It is primarily focused on mechanical means of eradicating the virus. Garlic is indicated as it has antiviral properties while the curcumin does not. So, since one of them had to be eliminated to prevent excess bleeding, the curcumin was off the list.
              The list includes garlic, tea tree oil, both of which actively kill the virus if it comes into contact with them. Bromelain which removes the spikes on the surface of the virus. And both the olive leaf and scullcap are anti-viral.
              The polygonum cuspidatum was added because it was a good way to increase the serum levels of resveratrol. This method bypasses the stomach and gut which have a poor uptake of the compound. And, the liver removes most of the active ingredient before it can reach the bloodstream.
              The nettle was the only anti-inflammatory included. It specifically inhibits prostaglandins, but does not effect bleeding . Curcumin really has no place here. It should rather be used later in the progression of the disease. The only contraindications for nettle are, some diabetics should not use the preparation, some may be naturally allergic to the herb, and finally it has never been tested on either kids or pregnant women but there has never been a case to support any danger to them either. You may already know this but in many cultures and even here in the US people frequently eat this prepared as a potherb. It is far safer that is curcumin with piperine added.
              Please do not ask me for medical advice, I am not a medical doctor.

              Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
              Thank you,
              Shannon Bennett

              Comment


              • #8
                Re: Nasal lavage or wash the virus away

                Wow! Great research done! Sorry, when I read your post I did not see that the herbs mentioned were for the lavage only. I was thinking the list was just alternative products for avian flu in general.

                Just FYI I found a product that is in use that contains some antivirals for nasal lavage. Yours sounds better.

                Add anti-microbial and anti-viral ingredients to your nasal wash with Neti Wash Plus&#174;. It contains zinc and potent extracts to tone the mucous membranes of your nasal passages and support the health of your sinuses and immune system.

                Neti Wash Plus&#174; also contains extracts of Grapefruit Seed and Goldenseal Root (cultivated, certified organic), found by scientists to inhibit the growth of hundreds of strains of pathogens. Grapefruit Seed Extract has been used by the natural foods industry for over 20 years as an antibiotic, disinfectant, and antiseptic.

                Ingredients: Zinc acetate, cultivated organic Goldenseal root (Hydrastis canadensis), Phellodendron bark (Phellodendron amurense), Coptis root (Coptis chinensis), Barberry root bark (Berberis vulgaris), Grapefruit seed extract, vegetable glycerin and distilled, microfiltered, ozonated water.
                We all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their color.
                Maya Angelou

                Comment


                • #9
                  Re: Nasal lavage or wash the virus away

                  Fredness, there is evidence to support tea tree is antiviral. However the mechanism is as yet not understood. ( btw and fyi It has had a lot of research done on the use and positive results on viruses responsible for cold sores.)
                  The link provides not only some of the current testing of tea tree but it also includes another list member (thyme oil) as having a mechanical ability to destroy the virus. It partially removes the lipid coating on the surface of the virus.

                  http://aac.highwire.org/cgi/content/full/46/6/1914

                  <!-- begin ad tag --><SCRIPT language=JavaScript>var axel = Math.random() + "";var ord = axel * 1000000000000000000;document.write('<SCRIPT LANGUAGE="JavaScript1.1" SRC="http://ad.doubleclick.net/adj/aac.asm.tmus/;pos=1;dcopt=ist;abr=!webtv;tile=1;sz=220x40;ord=' + ord + '?"><\/SCRIPT>');</SCRIPT><SCRIPT language=JavaScript1.1 src="http://ad.doubleclick.net/adj/aac.asm.tmus/;pos=1;dcopt=ist;abr=!webtv;tile=1;sz=220x40;ord=6 20437802307974600?"></SCRIPT><SCRIPT>if ((!document.images && navigator.userAgent.indexOf("Mozilla/2.") >= 0) || navigator.userAgent.indexOf("WebTV")>= 0) {document.write('');document.write('');}</SCRIPT> <NOSCRIPT></NOSCRIPT><!-- End ad tag -->
                  <NOBR> </NOBR><!-- subline logic: standard default --><!-- NOT GUEST -->
                  <HR align=left width=450 color=#000000 noShade SIZE=1><TABLE class=content_box_outer_table align=right><TBODY><TR><TD><!-- beginning of inner table --><TABLE class=content_box_inner_table><!-- citation --><TBODY><TR><TD class=content_box_title_highlight colSpan=2>This Article</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Abstract </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Full Text (PDF) </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me when this article is cited </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me if a correction is posted </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Services</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Similar articles in this journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Similar articles in PubMed </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me to new issues of the journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Download to citation manager </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Cited by other online articles </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Books from ASM Press </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Google Scholar</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Carson, C. F. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Riley, T. V. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles citing this Article </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>PubMed</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>PubMed Citation </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Carson, C. F. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Riley, T. V. </TD></TR></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>Antimicrobial Agents and Chemotherapy, June 2002, p. 1914-1920, Vol. 46, No. 6
                  0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.6.1914-1920.2002
                  Copyright &#169; 2002, American Society for Microbiology. All Rights Reserved.

                  Mechanism of Action of Melaleuca alternifolia (Tea Tree) Oil on Staphylococcus aureus Determined by Time-Kill, Lysis, Leakage, and Salt Tolerance Assays and Electron Microscopy

                  Christine F. Carson,<SUP>1</SUP><SUP>*</SUP> Brian J. Mee,<SUP>1</SUP> and Thomas V. Riley<SUP>1</SUP><SUP>,2</SUP>

                  Department of Microbiology, The University of Western Australia, Crawley,<SUP>1</SUP> Division of Microbiology and Infectious Diseases, The Western Australian Centre for Pathology and Medical Research, Nedlands, Western Australia 6009, Australia<SUP>2</SUP>
                  Received 24 April 2001/ Returned for modification 19 August 2001/ Accepted 6 February 2002
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                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> ABSTRACT </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
                  The essential oil of Melaleuca alternifolia (tea tree) has broad-spectrum<SUP> </SUP>antimicrobial activity. The mechanisms of action of tea tree<SUP> </SUP>oil and three of its components, 1,8-cineole, terpinen-4-ol,<SUP> </SUP>and -terpineol, against Staphylococcus aureus ATCC 9144 were<SUP> </SUP>investigated. Treatment with these agents at their MICs and<SUP> </SUP>two times their MICs, particularly treatment with terpinen-4-ol<SUP> </SUP>and -terpineol, reduced the viability of S. aureus. None of<SUP> </SUP>the agents caused lysis, as determined by measurement of the<SUP> </SUP>optical density at 620 nm, although cells became disproportionately<SUP> </SUP>sensitive to subsequent autolysis. Loss of 260-nm-absorbing<SUP> </SUP>material occurred after treatment with concentrations equivalent<SUP> </SUP>to the MIC, particularly after treatment with 1,8-cineole and<SUP> </SUP>-terpineol. S. aureus organisms treated with tea tree oil or<SUP> </SUP>its components at the MIC or two times the MIC showed a significant<SUP> </SUP>loss of tolerance to NaCl. When the agents were tested at one-half<SUP> </SUP>the MIC, only 1,8-cineole significantly reduced the tolerance<SUP> </SUP>of S. aureus to NaCl. Electron microscopy of terpinen-4-ol-treated<SUP> </SUP>cells showed the formation of mesosomes and the loss of cytoplasmic<SUP> </SUP>contents. The predisposition to lysis, the loss of 260-nm-absorbing<SUP> </SUP>material, the loss of tolerance to NaCl, and the altered morphology<SUP> </SUP>seen by electron microscopy all suggest that tea tree oil and<SUP> </SUP>its components compromise the cytoplasmic membrane.<SUP> </SUP>
                  <!-- null -->
                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> INTRODUCTION </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
                  The essential oil derived by steam distillation from the leaves<SUP> </SUP>of Melaleuca alternifolia is also known as tea tree oil (TTO)<SUP> </SUP>or Melaleuca oil. TTO is well characterized and contains approximately<SUP> </SUP>100 terpenes and their related alcohols (6). The physical and<SUP> </SUP>chemical properties of commercial TTO are regulated by an international<SUP> </SUP>standard (23). TTO has antibacterial (8, 17), antifungal (18,<SUP> </SUP>19), antiviral (4), and anti-inflammatory (5) properties in<SUP> </SUP>vitro, suggesting that it may have a role in the treatment of<SUP> </SUP>cutaneous infection. Clinical trials have demonstrated that<SUP> </SUP>TTO may be efficacious in the treatment of acne (2) and oral<SUP> </SUP>candidiasis (24) and in the decolonization of methicillin-resistant<SUP> </SUP>Staphylococcus aureus carriers (7). Although the in vitro antimicrobial<SUP> </SUP>activity and in vivo efficacy of TTO have been reported, less<SUP> </SUP>is known about its mechanism of action. Since this will have<SUP> </SUP>implications for its spectrum of activity, selective toxicity,<SUP> </SUP>and the development of resistance, we examined the mechanism<SUP> </SUP>of action of TTO and its components against S. aureus.<SUP> </SUP>
                  <!-- null -->
                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> MATERIALS AND METHODS </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
                  TTO and components. TTO (batch 971) was kindly provided by Australian Plantations<SUP> </SUP>Pty. Ltd., Wyrallah, New South Wales, Australia. The levels<SUP> </SUP>of the components, determined by gas chromatographic analysis<SUP> </SUP>according to the international standard (23), were as follows:<SUP> </SUP>41.5% terpinen-4-ol, 21.2% -terpinene, 10.2% -terpinene, 3.5%<SUP> </SUP>terpinolene, 2.9% -terpineol, 2.5% -pinene, 2.1% 1,8-cineole,<SUP> </SUP>1.5% -cymene, 1% aromadendrene, 1% -cadinene, 0.9% ledene, 0.9%<SUP> </SUP>limonene, 0.6% globulol, 0.4% sabinene, and 0.3% viridiflorol.<SUP> </SUP>Terpinen-4-ol and -terpineol (Aldrich Chemical Co. Inc., Milwaukee,<SUP> </SUP>Wis.) were 97 and 98% pure, respectively. 1,8-Cineole (Sigma<SUP> </SUP>Chemical Co., St. Louis, Mo.) was at least 99% pure. Terpinen-4-ol<SUP> </SUP>and -terpineol were chosen on the basis of their antimicrobial<SUP> </SUP>activities (10). 1,8-Cineole was selected since it has some<SUP> </SUP>antimicrobial activity and its presence in TTO has been contentious,<SUP> </SUP>due mainly to its erroneous reputation as a skin irritant (10).<SUP> </SUP>
                  Bacterium. S. aureus ATCC 9144 was used in all experiments and was obtained<SUP> </SUP>from the culture collection of the Department of Microbiology,<SUP> </SUP>The University of Western Australia, Crawley, Western Australia,<SUP> </SUP>Australia.<SUP> </SUP>
                  Inoculum effect. The MICs and minimal bactericidal concentrations (MBCs) of TTO<SUP> </SUP>for S. aureus were determined by a previously described broth<SUP> </SUP>microdilution method (8), except that the test medium was Mueller-Hinton<SUP> </SUP>broth (Oxoid Ltd., Basingstoke, United Kingdom), the Tween 80<SUP> </SUP>concentration was reduced to 0.001% (vol/vol), and several inoculum<SUP> </SUP>concentrations were used. Final inoculum concentrations were<SUP> </SUP>5 x 10<SUP>3</SUP>, 5 x 10<SUP>5</SUP>, 5 x 10<SUP>7</SUP>, and 5 x 10<SUP>9</SUP> CFU/ml.<SUP> </SUP>
                  Preparation of bacterial suspensions. Unless stated otherwise, suspensions of organisms in the stationary<SUP> </SUP>phase of growth were prepared by inoculating two colonies of<SUP> </SUP>S. aureus from overnight cultures on blood agar into 400 ml<SUP> </SUP>of Mueller-Hinton broth, which was incubated at 37&#176;C for<SUP> </SUP>18 h with shaking. After incubation, the bacteria were separated<SUP> </SUP>from the growth medium by centrifugation at 10,000 x g for 12<SUP> </SUP>min at 4&#176;C, washed twice with phosphate-buffered saline<SUP> </SUP>(PBS; pH 7.4), and resuspended in PBS supplemented with 0.001%<SUP> </SUP>Tween 80 (Sigma) (PBS-T). Suspensions were adjusted so that<SUP> </SUP>the optical density at 620 nm (OD<SUB>620</SUB>) of a 1-in-100 dilution<SUP> </SUP>was 0.3, which was 3 x 10<SUP>10</SUP> CFU/ml.<SUP> </SUP>
                  General experimental conditions. Experiments were conducted at room temperature (22&#176;C). The<SUP> </SUP>concentrations of TTO and its components used for treatment<SUP> </SUP>were derived from MICs previously determined by broth microdilution<SUP> </SUP>methods and ranged from one-half to two times the MICs. The<SUP> </SUP>MICs of TTO, terpinen-4-ol, and -terpineol for S. aureus were<SUP> </SUP>0.25%; and the MIC of 1,8-cineole was 0.5% (9, 10). Stock concentrations<SUP> </SUP>of TTO or its components were prepared in PBS-T at 10-fold the<SUP> </SUP>desired final concentration (in percent [vol/vol]) and were<SUP> </SUP>added to bacterial suspensions at a ratio of 1:9. PBS-T was<SUP> </SUP>added to the control suspensions. No inactivating agents have<SUP> </SUP>been established for TTO or its components. In the absence of<SUP> </SUP>an inoculum effect, dilution was used to arrest treatment and<SUP> </SUP>reduce carryover. The minimum dilution used was 1 in 10. All<SUP> </SUP>serial dilutions were done in PBS-T. All experiments except<SUP> </SUP>time-kill assays and inoculum effect assays were conducted four<SUP> </SUP>to six times; time-kill assays were done in triplicate, and<SUP> </SUP>inoculum effect assays were done in duplicate. Viable counts<SUP> </SUP>were plated in duplicate by using a spiral plater (Don Whitley<SUP> </SUP>Scientific Ltd., Shipley, United Kingdom) and nutrient agar<SUP> </SUP>(NA) (Oxoid) in all experiments with the exception of those<SUP> </SUP>investigating an inoculum effect. After incubation at 37&#176;C<SUP> </SUP>for 24 to 72 h, the colonies were counted manually with the<SUP> </SUP>aid of a counting template (Don Whitley Scientific). The detection<SUP> </SUP>threshold was 10<SUP>3</SUP> CFU/ml. For inoculum effect assays, 10 &#181;l<SUP> </SUP>of serial 10-fold dilutions of inocula was plated onto blood<SUP> </SUP>agar in quadruplicate, the plates were incubated, and the resulting<SUP> </SUP>colonies were counted. In assays for bacterial killing, bacteriolysis,<SUP> </SUP>and loss of 260-nm-absorbing material, samples taken 5 min before<SUP> </SUP>treatment addition were adjusted to account for dilution and<SUP> </SUP>used to determine the concentrations and ODs at 0 min.<SUP> </SUP>
                  Bacterial killing assays. The activities of TTO and its components against S. aureus were<SUP> </SUP>evaluated by measuring the reduction in the numbers of CFU per<SUP> </SUP>milliliter over 2 h. Bacterial suspensions (5 ml) were prepared<SUP> </SUP>as described above. After a pretreatment sample (0.5 ml) was<SUP> </SUP>taken, TTO or one of its components was added to yield final<SUP> </SUP>concentrations of one-half, one, and two times the respective<SUP> </SUP>MICs. The suspensions were mixed for 20 s with a vortex mixer;<SUP> </SUP>and a sample (0.5 ml) was removed at 30 s, serially diluted,<SUP> </SUP>and plated onto NA. Additional samples were taken at 30, 60,<SUP> </SUP>and 120 min. When rapid killing occurred, samples were also<SUP> </SUP>taken at 3, 6, 9, 12, and 15 min. Samples were diluted, plated<SUP> </SUP>onto NA, and incubated overnight. The mean number of survivors<SUP> </SUP>was determined.<SUP> </SUP>
                  Bacteriolysis. Suspensions of S. aureus were prepared as described above. After<SUP> </SUP>retrieval of the pretreatment sample, the agents were added<SUP> </SUP>so that the suspensions contained TTO or one of its components<SUP> </SUP>at concentrations equivalent to the MIC and two times the MIC.<SUP> </SUP>The suspensions were mixed for 20 s with a vortex mixer, a sample<SUP> </SUP>was removed at 30 s and diluted 1 in 100, and the OD<SUB>620</SUB> was<SUP> </SUP>measured. Additional samples were taken at 30, 60, 90, and 120<SUP> </SUP>min; and the ODs were measured immediately. Corresponding dilutions<SUP> </SUP>of test agents were used as blanks. Preliminary work indicated<SUP> </SUP>that the ODs of undiluted samples and samples diluted 1 in 10<SUP> </SUP>were inconsistent (data not shown). The 1-in-100 dilution reduced<SUP> </SUP>the level of carryover of TTO or its components to levels which<SUP> </SUP>contributed negligibly to the absorbance. The results were expressed<SUP> </SUP>as a ratio of the OD<SUB>620</SUB> at each time interval versus the OD<SUB>620</SUB><SUP> </SUP>at 0 min (in percent).<SUP> </SUP>
                  In two separate experiments, after the initial OD<SUB>620</SUB> measurement,<SUP> </SUP>the samples were retained and the OD<SUB>620</SUB> was remeasured 6.5 or<SUP> </SUP>23 h later. The OD<SUB>620</SUB>s of the control suspensions diminished<SUP> </SUP>over these time intervals; the OD<SUB>620</SUB>s of the samples obtained<SUP> </SUP>at 0 min were measured again 6.5 h later and were 91.5% of the<SUP> </SUP>original OD<SUB>620</SUB>, while the OD<SUB>620</SUB>s measured 23 h later were 84.4%<SUP> </SUP>of the original OD<SUB>620</SUB>. Since there was an inherent progressive<SUP> </SUP>reduction in the OD<SUB>620</SUB> over time, when the OD<SUB>620</SUB>s of the samples<SUP> </SUP>were measured 6.5 or 23 h later, their OD<SUB>620</SUB>s were compared<SUP> </SUP>to the OD<SUB>620</SUB>s of the 0-min samples, which were also remeasured<SUP> </SUP>6.5 or 23 h later.<SUP> </SUP>
                  Loss of 260-nm-absorbing material. Suspensions of bacteria were prepared as described above. A<SUP> </SUP>pretreatment sample was taken, diluted 1 in 100, and filtered<SUP> </SUP>through a 0.2-&#181;m-pore-size filter (Gelman Sciences, Ann<SUP> </SUP>Arbor, Mich.). The treatment agents were added at final concentrations<SUP> </SUP>equivalent to their MICs. Additional samples were removed after<SUP> </SUP>30 and 60 min, diluted, and filtered as described above. Filtrates<SUP> </SUP>of the appropriate dilution of each agent were prepared, and<SUP> </SUP>200-&#181;l volumes were used to blank the wells of a UV-transparent<SUP> </SUP>96-well tray (Molecular Devices, Sunnyvale, Calif.) at 260 nm.<SUP> </SUP>The blanks were discarded, the wells were loaded with the corresponding<SUP> </SUP>test filtrates, and the OD<SUB>260</SUB>s were determined. The OD<SUB>620</SUB>s of<SUP> </SUP>four to eight samples obtained at each time point were measured.<SUP> </SUP>The OD<SUB>260</SUB> at each time point was expressed as a proportion of<SUP> </SUP>the initial OD<SUB>260</SUB>. Mean ratios for each treatment agent and<SUP> </SUP>time were calculated and compared to the means for the corresponding<SUP> </SUP>untreated samples by using the two-tailed Student t test (P<SUP> </SUP>< 0.05).<SUP> </SUP>
                  Loss of salt tolerance. The ability of S. aureus cells treated with TTO or its components<SUP> </SUP>to grow on NA supplemented with NaCl (Merck Pty. Ltd., Kilsyth,<SUP> </SUP>Victoria, Australia) was investigated. In preliminary experiments,<SUP> </SUP>untreated suspensions of S. aureus were plated onto NA and NA<SUP> </SUP>containing NaCl at 5 to 100 g/liter (NA-NaCl) and incubated,<SUP> </SUP>and the resulting colonies were counted. Concentrations of NaCl<SUP> </SUP>that modestly compromised the colony-forming abilities of untreated<SUP> </SUP>organisms were selected. These were 50 and 75 g/liter. Suspensions<SUP> </SUP>of bacteria were prepared as described above and treated with<SUP> </SUP>TTO and 1,8-cineole at one-half, one, and two times their MICs<SUP> </SUP>and with terpinen-4-ol and -terpineol at one-half and one time<SUP> </SUP>their MICs. After 30 min, samples were removed, serially diluted,<SUP> </SUP>and inoculated onto NA and the selective medium NA-NaCl. After<SUP> </SUP>incubation, the numbers of CFU per milliliter on each NA-NaCl<SUP> </SUP>plate were compared to those on the NA plate, and the result<SUP> </SUP>was expressed as a percentage. The mean proportions of survivors<SUP> </SUP>from treated suspensions were compared to the corresponding<SUP> </SUP>means for the untreated controls by the two-tailed Student t<SUP> </SUP>test (P < 0.05).<SUP> </SUP>
                  Electron microscopy of terpinen-4-ol-treated bacteria. Suspensions of S. aureus in the stationary phase of growth were<SUP> </SUP>prepared by inoculating and incubating 30 ml of heart infusion<SUP> </SUP>broth (HIB; Gibco Diagnostics, Madison, Wis.). Organisms were<SUP> </SUP>harvested by centrifugation at 1,500 x g for 10 min, and the<SUP> </SUP>pellet was resuspended in HIB supplemented with Tween 80 (0.5%<SUP> </SUP>[vol/vol]) (HIB-T). Suspensions of S. aureus were treated with<SUP> </SUP>0.3% terpinen-4-ol for 10 min. Controls in HIB-T stood for 10<SUP> </SUP>min. After centrifugation at 1,500 x g for 5 min, the pellets<SUP> </SUP>were fixed overnight in 2.5% glutaraldehyde in 0.1 M cacodylate<SUP> </SUP>buffer at room temperature. Fixed microbial pellets were processed<SUP> </SUP>in graded alcohols, propylene oxide, and araldite and cured<SUP> </SUP>for 48 h at 60&#176;C. Ultrathin sections were stained with uranyl<SUP> </SUP>acetate and lead citrate and were examined with a Philips 410<SUP> </SUP>transmission electron microscope at an accelerating voltage<SUP> </SUP>of 80 kV.<SUP> </SUP>
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                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> RESULTS </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
                  Inoculum effect. Inoculum concentrations over the range 10<SUP>3</SUP> to 10<SUP>9</SUP> CFU/ml did<SUP> </SUP>not significantly alter the MICs or MBCs of TTO. At an inoculum<SUP> </SUP>of 5 x 10<SUP>3</SUP> CFU/ml, the MIC was 0.12% and the MBC was 0.25%.<SUP> </SUP>At inocula of 5 x 10<SUP>5</SUP> and 5 x 10<SUP>7</SUP> CFU/ml, the MICs were both<SUP> </SUP>0.25% and the MBCs were both 0.5%. At an inoculum of 5 x 10<SUP>9</SUP><SUP> </SUP>CFU/ml, the MIC was 0.5% and the MBC was 1%.<SUP> </SUP>
                  Bacterial killing assays. Treatment of S. aureus with TTO at two times the MIC reduced<SUP> </SUP>the viability of S. aureus by 5 log<SUB>10</SUB> over 120 min, while treatment<SUP> </SUP>with TTO at the MIC resulted in an 1 log<SUB>10</SUB> reduction (Fig. 1A).<SUP> </SUP>Treatment with 1,8-cineole at the MIC or two times the MIC resulted<SUP> </SUP>in greater than 3 log<SUB>10</SUB> reductions over 120 min (Fig. 1B). Terpinen-4-ol<SUP> </SUP>or -terpineol at one-half times the MIC had little effect on<SUP> </SUP>the viability of S. aureus. In contrast, treatment with either<SUP> </SUP>of these components at two times the MIC effected reductions<SUP> </SUP>of 7.5 and 6.5 log<SUB>10</SUB> in 9 and 30 min, respectively (Fig. 1C and D).<SUP> </SUP>Treatment with terpinen-4-ol at the MIC consistently<SUP> </SUP>reduced the viability by 6.5 log<SUB>10</SUB> over 120 min, while treatment<SUP> </SUP>with -terpineol at the MIC gave inconsistent results. Representative<SUP> </SUP>results are shown in Fig. 1D.<SUP> </SUP>
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                  </NOBR> </TD><TD vAlign=top align=left>FIG. 1. Time-kill curves of S. aureus ATCC 9144 in control suspensions () and after treatment with TTO (A), 1,8-cineole (B), terpinen-4-ol (C), or -terpineol (D) at one-half the MICs (), the MICs (•), and two times the MICs (). The MICs of TTO, terpinen-4-ol, and -terpineol were 0.25% (vol/vol); and that of 1,8-cineole was 0.5%. The organisms were suspended in PBS-T. Each symbol indicates the means &#177; SEs for at least three replicates. The lower detection threshold was 10<SUP>3</SUP> CFU/ml.
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                  Bacteriolysis. The mean OD<SUB>620</SUB> of untreated S. aureus suspensions after 120<SUP> </SUP>min was 96.9% (n = 9; standard error [SE] = 1.3%) of the original.<SUP> </SUP>Table 1 shows the OD<SUB>620</SUB>s of suspensions treated with TTO or<SUP> </SUP>its components. Treatment with TTO or 1,8-cineole at the MIC<SUP> </SUP>or two times the MIC had no effect (OD<SUB>620</SUB> range, 95 to 97% of<SUP> </SUP>the original). Treatment with terpinen-4-ol or -terpineol reduced<SUP> </SUP>the OD<SUB>620</SUB> slightly. Table 1 also shows the results obtained<SUP> </SUP>when the OD<SUB>620</SUB>s of dilutions of S. aureus suspensions was remeasured<SUP> </SUP>either 6.5 or 23 h after the initial OD<SUB>620</SUB>s were measured. Modest<SUP> </SUP>reductions in the OD<SUB>620</SUB>s of the TTO-treated suspensions were<SUP> </SUP>noted when they were measured again 6.5 h later. In contrast,<SUP> </SUP>large reductions were noted in the OD<SUB>620</SUB>s of the suspensions<SUP> </SUP>treated with the components of TTO, particularly those left<SUP> </SUP>for 23 h, in which only 30% of the original OD<SUB>620</SUB>s remained.<SUP> </SUP>Notably, the OD<SUB>620</SUB>s of the suspensions treated with terpinen-4-ol<SUP> </SUP>or -terpineol at two times the MIC for 30 s were reduced to<SUP> </SUP>44.9 and 67.4%, respectively, when they were read again 23 h<SUP> </SUP>later (data not shown).<SUP> </SUP>

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                  </NOBR> </TD><TD vAlign=top align=left>TABLE 1. Proportion of initial OD<SUB>620</SUB> of suspensions of S. aureus remaining after treatment with TTO or its components for 120 min
                  </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>
                  Loss of 260-nm-absorbing material. The OD<SUB>260</SUB>s of filtrates from control suspensions were not significantly<SUP> </SUP>different after 30 min but were significantly different (P =<SUP> </SUP>0.020) after 60 min (Fig. 2). Significant increases in the OD<SUB>260</SUB>s<SUP> </SUP>occurred after 60 min of treatment with TTO (P = 0.019), 1,8-cineole<SUP> </SUP>(P = 4.3 x 10<SUP>-4</SUP>), terpinen-4-ol (P = 0.038), or -terpineol (P<SUP> </SUP>= 0.005) at the MIC. Only 1,8-cineole treatment resulted in<SUP> </SUP>significant (P = 1.4 x 10<SUP>-7</SUP>) increases after 30 min.<SUP> </SUP>

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                  </NOBR> </TD><TD vAlign=top align=left>FIG. 2. Appearance of 260-nm-absorbing material in the filtrates of S. aureus control suspensions (white bars) and after treatment with the MICs of TTO (0.25%; bars with diagonal stripes), 1,8-cineole (0.5%; grey bars), terpinen-4-ol (0.25%; bars with horizontal stripes), or -terpineol (0.25%; black bars). The organisms were suspended in PBS-T. The means &#177; SEs for at least three replicates are illustrated.
                  </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>
                  Loss of salt tolerance. The results of tests of salt tolerance loss are shown in Fig.<SUP> </SUP>3A to D. The addition of NaCl to NA reduced the colony-forming<SUP> </SUP>ability of untreated S. aureus cells to 81.4% (n = 22; SE =<SUP> </SUP>2.5%) with NaCl at 50 g/liter and 73.3% (n = 22; SE = 2.8%)<SUP> </SUP>with NaCl at 75 g/liter. Treatment with TTO or 1,8-cineole at<SUP> </SUP>two times their MICs significantly reduced the ability of survivors<SUP> </SUP>to form colonies on NA-NaCl (with NaCl at 50 or 75 g/liter),<SUP> </SUP>with only 1.5% or less of the survivors able to form colonies.<SUP> </SUP>1,8-Cineole at the MIC had a similar effect, with 0.7% or less<SUP> </SUP>of the survivors able to form colonies on NA-NaCl. Treatment<SUP> </SUP>with TTO and terpinen-4-ol at their MICs also reduced the ability<SUP> </SUP>of survivors to form colonies on NA-NaCl, but to a lesser extent<SUP> </SUP>(<30%). By treatment with -terpineol at the MIC, the proportion<SUP> </SUP>of survivors able to form colonies on NA-NaCl was not significantly<SUP> </SUP>reduced when NaCl was used at 50 g/liter (P = 0.072) but was<SUP> </SUP>significantly reduced when NaCl was used at 75 g/liter (P =<SUP> </SUP>0.032). Of the four treatments at one-half the MIC, only 1,8-cineole<SUP> </SUP>significantly reduced the colony-forming ability of the survivors,<SUP> </SUP>and this was only when the higher salt concentration was used<SUP> </SUP>(P = 0.030).<SUP> </SUP>

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                  </NOBR> </TD><TD vAlign=top align=left>FIG. 3. Proportion of S. aureus cells able to form colonies on NA (white bars), NA supplemented with 50 g of NaCl per liter (black bars), and NA supplemented with 75 g of NaCl per liter (grey bars) after 30 min of treatment with TTO (A), 1,8-cineole (B), terpinen-4-ol (C), or -terpineol (D) at one-half the MICs, the MICs, and two times the MICs. The MICs of TTO, terpinen-4-ol, and -terpineol were 0.25% (vol/vol); and that of 1,8-cineole was 0.5%. The organisms were suspended in PBS-T. The means &#177; SEs for at least four replicates are illustrated.
                  </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>
                  Electron microscopy of terpinen-4-ol-treated bacteria. Terpinen-4-ol-treated S. aureus cells contained multilamellar,<SUP> </SUP>mesosomelike structures (Fig. 4B and C) that were not seen in<SUP> </SUP>untreated cells (Fig. 4A). In addition, the contents of some<SUP> </SUP>treated cells appeared depleted and amorphous (Fig. 4C).<SUP> </SUP>

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                  </NOBR> </TD><TD vAlign=top align=left>FIG. 4. Electron micrographs of S. aureus cells stained with uranyl acetate after no treatment (A) and after treatment with 0.3% terpinen-4-ol for 10 min (B and C). Magnifications: x11,500 (A) and x14,200 (B and C).
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                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> DISCUSSION </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
                  Interactions with the hydrophobic structures of bacteria play<SUP> </SUP>a key role in the antimicrobial actions of hydrocarbons (32).<SUP> </SUP>Consequently, assumptions regarding the mechanism of action<SUP> </SUP>of TTO have been based on the nature of its components. In the<SUP> </SUP>present investigation, S. aureus cells in the stationary phase<SUP> </SUP>of growth were killed by TTO and its components. Organisms in<SUP> </SUP>this growth phase are generally less sensitive to injury than<SUP> </SUP>those in the exponential phase (11), and this has been shown<SUP> </SUP>for Escherichia coli treated with TTO (16). Since antimicrobial<SUP> </SUP>agents that affect cellular synthetic processes often have little<SUP> </SUP>effect on organisms in the stationary phase of growth (15, 28),<SUP> </SUP>these results suggest that the principal target of TTO is not<SUP> </SUP>a macromolecular synthetic process.<SUP> </SUP>

                  Some antimicrobial agents cause gross membrane damage and provoke<SUP> </SUP>whole-cell lysis (14, 28); and this has been reported previously<SUP> </SUP>for essential oils from oregano, rosewood, and thyme (20). However,<SUP> </SUP>the major components of these oils, including carvacrol, citronellol,<SUP> </SUP>geraniol, and thymol (38), are not found in TTO. The failure<SUP> </SUP>of TTO or its components to lyse S. aureus cells suggests that<SUP> </SUP>their primary mechanism of action is not gross cell wall damage.<SUP> </SUP>Treatment-induced release of membrane-bound cell wall autolytic<SUP> </SUP>enzymes will induce lysis eventually (15), and this may explain<SUP> </SUP>the delayed lysis of S. aureus seen when suspensions were reexamined<SUP> </SUP>after several hours; S. aureus cells treated for as little as<SUP> </SUP>30 s were disproportionately sensitive to lysis. While the activation<SUP> </SUP>of autolytic enzymes may have been responsible for this effect,<SUP> </SUP>the lysis may also have been due to weakening of the cell wall<SUP> </SUP>and the subsequent rupture of the cytoplasmic membrane due to<SUP> </SUP>osmotic pressure (rather than a specific action on the cytoplasmic<SUP> </SUP>membrane).<SUP> </SUP>
                  Marked leakage of cytoplasmic material is considered indicative<SUP> </SUP>of gross and irreversible damage to the cytoplasmic membrane<SUP> </SUP>(21). Many antimicrobial compounds that act on the bacterial<SUP> </SUP>cytoplasmic membrane induce the loss of 260-nm-absorbing material,<SUP> </SUP>including chlorhexidine (21), hexachlorophene (25), phenethyl<SUP> </SUP>alcohol (34), tetracyclines, polymyxin (12), -pinene (1), and<SUP> </SUP>lemongrass oil (27). S. aureus suspensions treated with TTO<SUP> </SUP>or its components lost significant 260-nm-absorbing material,<SUP> </SUP>suggesting that nucleic acids were lost through a damaged cytoplasmic<SUP> </SUP>membrane.<SUP> </SUP>
                  Sublethal injury of microbial cell membranes may alter their<SUP> </SUP>permeability and affect the membrane's ability to osmoregulate<SUP> </SUP>the cell adequately or to exclude toxic materials (15). Consequently,<SUP> </SUP>the loss of tolerance to salts or other potentially toxic compounds<SUP> </SUP>may be exploited to reveal membrane damage (22) in sublethally<SUP> </SUP>injured bacteria. Treatment of S. aureus with TTO or its components<SUP> </SUP>significantly reduced the ability of the survivors to form colonies<SUP> </SUP>on media containing NaCl. This effect was most marked with 1,8-cineole,<SUP> </SUP>which was also the only compound with which treatment with one-half<SUP> </SUP>the MIC resulted in a significant loss of salt tolerance. These<SUP> </SUP>results correlate well with the OD<SUB>260</SUB> results since, in each<SUP> </SUP>case, treatment with TTO or its components at the MICs induced<SUP> </SUP>the loss of salt tolerance and 260-nm-absorbing material. Notably,<SUP> </SUP>treatment with TTO, terpinen-4-ol, or -terpineol at one-half<SUP> </SUP>the MIC had no effect on salt tolerance or actually increased<SUP> </SUP>it. Treatment with these agents at one-half their MICs may have<SUP> </SUP>killed the most susceptible cells, leaving the more robust and,<SUP> </SUP>possibly, more salt-tolerant cells. However, of the three treatments<SUP> </SUP>with which an increase in salt tolerance was noted, only treatment<SUP> </SUP>with TTO at one-half the MIC for 30 min reduced the viability<SUP> </SUP>of S. aureus. Alternatively, the treatments may have induced<SUP> </SUP>the expression of stress tolerance mechanisms.<SUP> </SUP>
                  Electron microscopy of terpinen-4-ol-treated cells corroborated<SUP> </SUP>the inability of terpinen-4-ol to lyse S. aureus and suggested<SUP> </SUP>membrane damage by the appearance of mesosomes and a loss of<SUP> </SUP>cytoplasmic material. These lesions have been reported previously<SUP> </SUP>after treatment with antimicrobial agents including vancomycin<SUP> </SUP>(29), phenethyl alcohol (11), defensins (30), and betane (3).<SUP> </SUP>Similar cytoplasmic losses, as well as bleb formation, were<SUP> </SUP>reported for E. coli treated with lemongrass oil (26), and both<SUP> </SUP>effects were noted when E. coli was treated with TTO (16).<SUP> </SUP>
                  The original premise was that TTO and/or its components act<SUP> </SUP>on microbial membranes. The loss of 260-nm-absorbing material,<SUP> </SUP>increased susceptibility to NaCl, the formation of mesosomes,<SUP> </SUP>and the loss of cytoplasmic material suggest that the cytoplasmic<SUP> </SUP>membrane is compromised by treatment with TTO and its components.<SUP> </SUP>Additional support for this idea comes from work showing that<SUP> </SUP>TTO treatment of E. coli or S. aureus induces the loss of potassium<SUP> </SUP>ions, inhibits respiration, and promotes the uptake of propidium<SUP> </SUP>iodide (13). In other work done with essential oils and their<SUP> </SUP>components, -pinene inhibited respiratory activity in yeast<SUP> </SUP>mitochondria (1) and &#223;-pinene and limonene had similar<SUP> </SUP>effects in intact yeast cells and isolated mitochondria (35).<SUP> </SUP>Collectively, these observations suggest that further work could<SUP> </SUP>examine the effect of treatment on microbial energy transduction,<SUP> </SUP>in particular, the transmembrane proton motive force.<SUP> </SUP>
                  When the mechanisms of action of lipophilic biocides have been<SUP> </SUP>examined, effects on the cytoplasmic membrane and/or the enzymes<SUP> </SUP>embedded in it have been demonstrated (1, 3, 31, 33, 35-37).<SUP> </SUP>The data on TTO and its components support these observations.<SUP> </SUP>However, the possibility remains that sites of action other<SUP> </SUP>than the cytoplasmic membrane exist. Furthermore, it is possible<SUP> </SUP>that other oil components, not examined thus far, may contribute<SUP> </SUP>to the antimicrobial activity of the oil by other mechanisms.<SUP> </SUP>Given the heterogeneous composition of TTO and the antimicrobial<SUP> </SUP>activities of many of its components, it seems unlikely that<SUP> </SUP>there is only one mechanism of action or that only one component<SUP> </SUP>is responsible for the antimicrobial action. Further work is<SUP> </SUP>required to understand fully the mechanisms involved.<SUP> </SUP>
                  <SUP></SUP>
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                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> ACKNOWLEDGMENTS </TH></TR></TBODY></TABLE>
                  This work was supported by a grant (grant UWA-24A) from the<SUP> </SUP>Rural Industries Research & Development Corporation, Canberra,<SUP> </SUP>Australian Capital Territory, Australia.<SUP> </SUP>
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                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> FOOTNOTES </TH></TR></TBODY></TABLE>
                  <!-- null -->* Corresponding author. Mailing address: Department of Microbiology, The University of Western Australia, Crawley, Western Australia 6009, Australia. Phone: 61 8 9346 3288. Fax: 61 8 9346 2912. E-mail: ccarson@cyllene.uwa.edu.au<SCRIPT type=text/javascript><!-- var u = "ccarson", d = "cyllene.uwa.edu.au"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></SCRIPT> .

                  <!-- null -->
                  <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> REFERENCES </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
                  Abstract
                  Introduction
                  Materials and Methods
                  Results
                  Discussion
                  References
                  </TH></TR></TBODY></TABLE>
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Antiviral activity of the essential oil of Melaleuca alternifolia (Maiden and Betche) Cheel (tea tree) against tobacco mosaic virus. J. Essent. Oil Res. 7:641-644.<!-- HIGHWIRE ID="46:6:1914:4" --><!-- /HIGHWIRE --><!-- null --> <LI value=5>Brand, C., A. Ferrante, R. H. Prager, T. V. Riley, C. F. Carson, J. J. Finlay-Jones, and P. H. Hart. 2001. The water-soluble components of the essential oil of Melaleuca alternifolia (tea tree oil) suppress the production of superoxide by human monocytes, but not neutrophils, activated in vitro. Inflamm. Res. 50:213-219.<!-- HIGHWIRE ID="46:6:1914:5" -->[CrossRef][Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=6>Brophy, J. J., N. W. Davies, I. A. Southwell, I. A. Stiff, and L. R. Williams. 1989. Gas chromatographic quality control for oil of Melaleuca terpinen-4-ol type (Australian tea tree). J. Agric. Food Chem. 37:1330-1335.<!-- HIGHWIRE ID="46:6:1914:6" --><!-- /HIGHWIRE --><!-- null --> <LI value=7>Caelli, M., J. Porteous, C. F. Carson, R. 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The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J. Appl. Microbiol. 88:170-175.<!-- HIGHWIRE ID="46:6:1914:13" -->[CrossRef][Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=14>Denyer, S. P., and W. B. Hugo. 1991. Biocide-induced damage to the bacterial cytoplasmic membrane, p. 171-187. In S. P. Denyer and W. B. Hugo (ed.), Mechanisms of action of chemical biocides: their study and exploitation. Blackwell Scientific Publications, Oxford, United Kingdom.<!-- HIGHWIRE ID="46:6:1914:14" --><!-- /HIGHWIRE --><!-- null --> <LI value=15>Gilbert, P. 1984. The revival of microorganisms sublethally injured by chemical inhibitors, p. 175-197. In M. H. E. Andrews and A. D. Russell (ed.), The revival of injured microbes. Academic Press, London, United Kingdom.<!-- HIGHWIRE ID="46:6:1914:15" --><!-- /HIGHWIRE --><!-- null --> <LI value=16>Gustafson, J. E., Y. C. Liew, S. Chew, J. Markham, H. C. Bell, S. G. Wyllie, and J. R. Warmington. 1998. Effects of tea tree oil on Escherichia coli. Lett. Appl. Microbiol. 26:194-198.<!-- HIGHWIRE ID="46:6:1914:16" -->[CrossRef][Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=17>Hammer, K. A., C. F. Carson, and T. V. Riley. 1996. Susceptibility of transient and commensal skin flora to the essential oil of Melaleuca alternifolia (tea tree oil). Am. J. Infect. Control 24:186-189.<!-- HIGHWIRE ID="46:6:1914:17" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=18>Hammer, K. A., C. F. Carson, and T. V. Riley. 1997. In vitro susceptibility of Malassezia furfur to the essential oil of Melaleuca alternifolia. J. Med. Vet. Mycol. 35:375-377.<!-- HIGHWIRE ID="46:6:1914:18" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=19>Hammer, K. A., C. F. Carson, and T. V. Riley. 1998. In vitro activity of essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J. Antimicrob. Chemother. 42:591-595.<!-- HIGHWIRE ID="46:6:1914:19" -->[Abstract]<!-- /HIGHWIRE --><!-- null --> <LI value=20>Horne, D. S., M. Holm, C. Oberg, S. Chao, and D. G. Young. 2001. Antimicrobial effects of essential oils on Streptococcus pneumoniae. J. Essent. Oil Res. 13:387-392.<!-- HIGHWIRE ID="46:6:1914:20" --><!-- /HIGHWIRE --><!-- null --> <LI value=21>Hugo, W. B., and R. Longworth. 1964. Some aspects of the mode of action of chlorhexidine. J. Pharm. Pharmacol. 16:655-662.<!-- HIGHWIRE ID="46:6:1914:21" --><!-- /HIGHWIRE --><!-- null --> <LI value=22>Iandolo, J. J., and Z. J. Ordal. 1966. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142.<!-- HIGHWIRE ID="46:6:1914:22" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=23>International Organisation for Standardisation. 1996. Essential oils—oil of Melaleuca, terpinen-4-ol type (tea tree oil). ISO-4730. International Organisation for Standardisation, Geneva, Switzerland.<!-- HIGHWIRE ID="46:6:1914:23" --><!-- /HIGHWIRE --><!-- null --> <LI value=24>Jandourek, A., J. K. Vaishampayan, and J. A. Vazquez. 1998. Efficacy of melaleuca oral solution for the treatment of fluconazole refractory oral candidiasis in AIDS patients. AIDS 12:1033-1037.<!-- HIGHWIRE ID="46:6:1914:24" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=25>Joswick, H. L., T. R. Corner, J. N. Silvernale, and P. Gerhardt. 1971. Antimicrobial actions of hexachlorophene: release of cytoplasmic materials. J. Bacteriol. 108:492-500.<!-- HIGHWIRE ID="46:6:1914:25" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=26>Ogunlana, E. O., S. H&#246;glund, G. Onawunmi, and O. Sk&#246;ld. 1987. Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells. Microbios 50:43-59.<!-- HIGHWIRE ID="46:6:1914:26" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=27>Onawunmi, G. O., and E. O. Ogunlana. 1985. Effects of lemon grass oil on the cells and spheroplasts of Escherichia coli NCTC 9001. Microbios Lett. 28:63-68.<!-- HIGHWIRE ID="46:6:1914:27" --><!-- /HIGHWIRE --><!-- null --> <LI value=28>Russell, A. D., A. Morris, and M. C. Allwood. 1973. Methods for assessing damage to bacteria induced by chemical and physical agents. Methods Microbiol. 8:95-182.<!-- HIGHWIRE ID="46:6:1914:28" --><!-- /HIGHWIRE --><!-- null --> <LI value=29>Sanyal, D., and D. Greenwood. 1993. An electron microscope study of glycopeptide antibiotic-resistant strains of Staphylococcus epidermidis. J. Med. Microbiol. 39:204-210.<!-- HIGHWIRE ID="46:6:1914:29" -->[Abstract]<!-- /HIGHWIRE --><!-- null --> <LI value=30>Shimoda, M., K. Ohki, Y. Shimamoto, and O. Kohashi. 1995. Morphology of defensin-treated Staphylococcus aureus. Infect. Immun. 63:2886-2891.<!-- HIGHWIRE ID="46:6:1914:30" -->[Abstract]<!-- /HIGHWIRE --><!-- null --> <LI value=31>Sikkema, J., J. A. M. de Bont, and B. Poolman. 1994. Interactions of cyclic hydrocarbons with biological membranes. J. Biol. Chem. 269:8022-8028.<!-- HIGHWIRE ID="46:6:1914:31" --><NOBR>[Abstract/Free Full Text]</NOBR><!-- /HIGHWIRE --><!-- null --> <LI value=32>Sikkema, J., J. A. M. de Bont, and B. Poolman. 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev. 59:201-222.<!-- HIGHWIRE ID="46:6:1914:32" -->[Abstract]<!-- /HIGHWIRE --><!-- null --> <LI value=33>Sikkema, J., B. Poolman, W. N. Konings, and J. A. M. de Bont. 1992. Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes. J. Bacteriol. 174:2986-2992.<!-- HIGHWIRE ID="46:6:1914:33" -->[Abstract]<!-- /HIGHWIRE --><!-- null --> <LI value=34>Silver, S., and L. Wendt. 1967. Mechanism of action of phenethyl alcohol: breakdown of the cellular permeability barrier. J. Bacteriol. 93:560-566.<!-- HIGHWIRE ID="46:6:1914:34" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=35>Uribe, S., J. Ramirez, and A. Pe&#241;a. 1985. Effects of &#223;-pinene on yeast membrane functions. J. Bacteriol. 161:1195-1200.<!-- HIGHWIRE ID="46:6:1914:35" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=36>Uribe, S., P. Rangel, G. Esp&#237;nola, and G. Aguirre. 1990. Effects of cyclohexane, an industrial solvent, on the yeast Saccharomyces cerevisiae and on isolated yeast mitochondria. Appl. Environ. Microbiol. 56:2114-2119.<!-- HIGHWIRE ID="46:6:1914:36" -->[Medline]<!-- /HIGHWIRE --><!-- null --> <LI value=37>Williams, D. E., L. J. Swango, G. R. Wilt, and S. D. Worley. 1991. Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells. Appl. Environ. Microbiol. 57:1121-1127.<!-- HIGHWIRE ID="46:6:1914:37" -->[Medline]<!-- /HIGHWIRE --><!-- null -->
                  2. Windholz, M., S. Budavari, R. F. Blumetti, and E. S. Otterbein (ed.). 1983. The Merck index, 10th ed. Merck & Co., Inc., Rahway, N.J.<!-- HIGHWIRE ID="46:6:1914:38" --><!-- /HIGHWIRE -->
                  <HR>Antimicrobial Agents and Chemotherapy, June 2002, p. 1914-1920, Vol. 46, No. 6
                  0066-4804/02/$04.00+0 DOI: 10.1128/AAC.46.6.1914-1920.2002
                  Copyright &#169; 2002, American Society for Microbiology. All Rights Reserved.



                  <!-- null -->

                  This article has been cited by other articles: (Search Google Scholar for Other Citing Articles)
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                  Please do not ask me for medical advice, I am not a medical doctor.

                  Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
                  Thank you,
                  Shannon Bennett

                  Comment


                  • #10
                    Re: Nasal lavage or wash the virus away

                    Fredness, my addition of olive leaf was based on both the pet studies (imported to the alt. meds research forum in page 3) and to the fluwikie post located http://www.fluwikie.com/pmwiki.php?n=Consequences.Olive . It specifically mentions the efficacy in a nasal wash.
                    Of all the herbs suggested, it has the least research to back up the claim. If then you were to eliminate one of the compunds this would be the one to go.
                    Please do not ask me for medical advice, I am not a medical doctor.

                    Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
                    Thank you,
                    Shannon Bennett

                    Comment


                    • #11
                      Re: Nasal lavage or wash the virus away

                      A good substitute for olive leaf would be verbascum. It has had significantly more studies done on it. It tastes rather nasty however.

                      http://www.fluwikie.com/pmwiki.php?n...uences.Mullein

                      SUPPLEMENT/MEDICATION /COMPOUND NAME: Mullein

                      USE: The lyophilized infusion from flowers of Verbascum thapsiforme Schrad. (FVI) showed anti-viral activity in in vitro studies against Fowl plague virus, several influenza A strains, influenza B strain as well as Herpes simplex virus. Influenza viruses titer decreased by 1–3 log units, while of H. simplex virus by 2.3 log. FVI has shown virucidal activity on H. simplex virus at 300 micrograms/ml, but did not inactivate influenza viruses. (1)

                      POTENTIAL BENEFIT: Potential anti-influenza effects.

                      SUPPORTING RESEARCH:

                      (1)Antiviral activity of Flos verbasci infusion against influenza and Herpes simplex viruses

                      (2) McCutcheon, A.R., Roberts, T.E., Gibbons, E., Ellis, S.M., Babiuk, L.A., Hancock, R.E., Towers, G.H. (1995). Antiviral screening of British Columbian medicinal plants. J. Ethnopharmacol. 49(2):101–10

                      (3) Effects of Herbal Preparations on the Propagation of Influenza Viruses
                      Teresa Skwarek, Dept of Pharmaceutical Microbiology, Institute of Clinical Pathology, Medical Scool in Lubin Poland
                      26 plant preparations were tested with respect to their antiviral activity against influenza A2 (England 1964) and B in cultures of chicken fibroblasts and chicken embryos…Decoction of mullein (Verbascum thapsus) was found to has the strongest antiviral effect.


                      CONTRAINDICATIONS, PRECAUTIONS, ADVERSE REACTIONS:

                      None known
                      Please do not ask me for medical advice, I am not a medical doctor.

                      Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
                      Thank you,
                      Shannon Bennett

                      Comment


                      • #12
                        Re: Nasal lavage or wash the virus away

                        Shannon,

                        How often do you plan to use the nasal wash (how many times a day)?

                        Would you be able to prepare the herbal solution ahead of time or need to prepare before each use?

                        Thanks!

                        CR

                        Comment


                        • #13
                          Re: Nasal lavage or wash the virus away

                          polygonum cuspidatum ( called Hu Zhang in Chinese, I order mine from kaylx.com)
                          skullcap (scutellaria baicalensis, [the s. lateraflora is not what I am looking for] I order mine from kaylx.com)
                          bromelain (herb, available from most bulk suppliers)
                          stinging nettle (herb, available from most bulk suppliers)
                          lemon balm (herb, available from most bulk suppliers)
                          verbascum, flower or leaf (herb, available from most bulk suppliers)
                          (*note, olive leaf removed, replaced with verbascum*)
                          garlic


                          C.R., Once a day if no one was sick, increasing to four times a day if certain of exposure or, illness in either myself or a family member. I could partially prepare this in advance by mixing the herbs that go in after the polygonum and the skullcap were boiled together. Make sure if you lump them together they are well mixed. The polygonum and skullcap need to be put through a coffee mill to partially pulverize them, if they are not already ground. Then one tsp. of each needs to be boiled gently for 25 minutes (this is how you make a decoction) in two 1/2 cups of pure water. Make sure you leave the lid on during the process and don't have the heat up so high you end up with no water left! Remove from heat and add the other herbs at the rate of one teaspoon each, one minced then mashed garlic clove and 1/2 tsp. salt. Stir in and steep in hot water with the lid on for ten minutes. Strain, cool, and use. The extra should be put in the refrigerator to be used later in the day. Do not put it in the microwave to re-heat to body temp. Instead pour the tea into a zip type bag carefully sealing it and, immerse the bag in hot water until the tea comes to a comfortable temperature, shake vigorously before pouring into neti pots. In the case of the spray bottles simply immerse the bottle in hot water to bring to a more comfortable temp or just use it cold remembering to shake vigorously before using.
                          It is important to make sure no one is unlucky enough to be allergic to any of the ingredients. The two most likely candidates for allergic reaction are the verbascum and the nettle but, anyone can be allergic to anything. So before I give this to a family member I am going to test each herb alone on all family members. A simple tea using 1/2 tsp. of herb in 1/2 cup of piping hot water (allow to cool before spritzing up nose) will be sprayed up our noses one person at a time on different days. I am going to make sure we have benedryl on hand should one of us turn out to be allergic. It is also important to make sure everyone knows how to use a neti pot before they get sick. Your six-year-old son may have already mastered this technique with milk. If he can pull milk in one nostril and have it come out the other nostril then he should go first to illustrate the process.
                          Last edited by Shannon Bennett; June 14th, 2006, 05:32 PM.
                          Please do not ask me for medical advice, I am not a medical doctor.

                          Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
                          Thank you,
                          Shannon Bennett

                          Comment


                          • #14
                            Re: Nasal lavage or wash the virus away

                            I would avoid Tea Tree for 2 reasons, it is probably gonna hurt, and we should stick to what we know is effective against influenza. A PubMed search on Melaleuca alternifolia (tea tree) and influenza came up blank. Maybe no one has studied it yet and that does not mean it will not work. There are too many warnings about toxicity related to oral ingestion. It does not sound safe to recommend.

                            Natural Standard "Tea tree oil has been reported to cause cutaneous allergic responses and central nervous system depression when ingested orally. For these reasons, it is not recommended that tea tree oil be administered orally."

                            PDR http://www.pdrhealth.com/drug_info/n...s/102757.shtml
                            "Never take essential oils internally. They are extremely potent and can be poisonous."

                            It may be used as aromatherapy but would be unsafe as a nasal wash, some of which will pass into the throat by gravity.


                            I agree about olive not showing obvious activity.

                            What, no elderberry?

                            Actually your nasal wash could be a throat spray too.
                            Last edited by fredness; June 14th, 2006, 05:06 PM.

                            Comment


                            • #15
                              Re: Nasal lavage or wash the virus away

                              Fredness, I dissagree about the tea tree. I have found and imported some studies here indicating its anti-viral properties, and have seen it used as a nose drop in another.
                              I think we can agree to disagree here.
                              Fredness, did you look in Google Scholar under the heading Melaleuca Alternifolia antiviral?
                              Last edited by Shannon Bennett; June 14th, 2006, 04:52 PM.
                              Please do not ask me for medical advice, I am not a medical doctor.

                              Avatar is a painting by Alan Pollack, titled, "Plague". I'm sure it was an accident that the plague girl happened to look almost like my twin.
                              Thank you,
                              Shannon Bennett

                              Comment

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