Zamberlan, Amaral, Arantes, Machado, Mizdal, Campos, and Soares (2016) conducted a study to see if rosemary was connected to anti-aging.  They used Caenorhabditis elegans which is a non-parasitic nematode to model this as it seems to modulate aging and stress.  DAF-2 is an insulin receptor that is expressed with C. elegans and mutations have shown links to longevity because it increases the resistance to oxidative stress and heat for C. elegans.

The dose of 25 micrograms per mL was effective at increasing the life span of the worms in both the oxidative stress test and the heat-induced test. 

They also mutated some of the pathways in the worms and in these mutants, the rosemary oil was not successful.  They did succeed in extending the lifespan of the worms by 2 days through the DAF-2 pathway but not through the mutated pathways.  

The prevalent compounds found in the essential oil tested here were the flavonoids quercetin, rutin, and kaempferol and the phenolic acids chlorogenic acid, caffeic acid, rosmarinic acid, and carnosic acid.

While this study proves the extract protected the worms against heat and oxidative stress, it further points out the importance of the other pathways which help the body communicate to itself to modulate healing.  When those pathways were cut off due to the mutation, healing was not possible.  These pathways were HAF-1 (guides DAF-16, helps in the process to protect against heat shock), DAF-16/FOXO (development, stress resistance, heat tolerance, metabolism), and SKN-1/Nrf (regulates resistance to oxidative stress).

When these mutations were induced, the worms were more susceptible to oxidative stress.  This indicates these pathways are required for rosemary to protect against oxidative stress. 
 
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Zamberlan, D.C, Amaral, G.P., Arantes, L.P., Machado, M.L,. Mizdal, C.R., Campos, M.M.A., and Soares, F.A.A. (2016). Rosmarinus officinalis L. increases Caenorhabditis elegans stress resistance and longevity in a DAF-16, HSF-1 and SKN-1-dependent manner. Brazilian Journal of Medical and Biological Research, 49(9): e5235, doi:10.1590/1414-431X20165235

Cardoso, Dantas, Sousa, and Peron (2014) looked for cytotoxicity of rosemary using the roots of an onion, Allium cepa.  The made rosemary infusions at .02, .04, .06, and .08 mg/mL with .02 being the standard dose recommended.  The onion roots were exposed to the infusions for 24 hours and 48 hours, meaning they were placed in the infusions instead of water.  There were at least 6 roots in each group.  Cell division was inhibited in all groups. 

The authors then summarized several studies of rosemary inhibiting cell growth in various parts of the body such as cancer and other illnesses.  They recommend additional testing to ensure safe levels are utilized.

Have A Great Day!
 
Cardoso, Dantas, Sousa, and Peron. (2014). Cytotoxicity of aqueous extracts of Rosmarinus officinalis L. (Labiatae) in plant test system. Brazilian Journal of Biology, 74(4):886-889. doi: 10.1590/1519-6984.07313

Zheljazkov, Astatkia, Zhalnov, and Georgieva (2015) decided to compare extraction techniques using both dried and fresh rosemary leaves to determine if one technique was better to treat one ailment while another technique is more suited for a second ailment.  In this case, they specifically looked at the length of distillation.  These times were 1.25, 2.5, 5, 10, 20, 40, 80, and 160 minutes.  They grew the plants from the exact same species and in the same soil using raised beds to control soil content.  The dried leaves produced more oil but the yield was not increased after the 10-minute mark. 

The amount of verbenone was not seemingly affected by the length of boiling while camphor and borneol were more prevalent in the 80- and 160-minute samples. 

Myrcene and linalool were not affected by time but the dried material produced more.  Myrcene was most prevalent in the dried herb. 

The short boiling time had more a-pinene, camphene, eucalyptol (1,8-cineole), and b-pinene.

Camphene was not affected by whether the leaves were dry or not dry but eugenol was more prevalent in the oil made from the fresh leaves, as was eucalyptol. 

Borneol and b-caryophyllene were more prevalent from the dried herb at 40 minutes.

Bornyl acetate was more prevalent in the fresh samples. 

A-pinene, camphene, b-pinene, myrcene, eucalyptol, camphor had differing results between the dry and fresh herb. 

The highest amount of a-pinene was at the 2.5-minute mark from dry leaves. 

For b-pinene, it was fresh leaves at the 2.5-minute mark. 

Camphor was the highest at the 160-minute mark from fresh leaves while b-caryophyllene was the most at the 80-minute mark with dried leaves.

They tested some of the oils against various microbes and the oils were not effective.  In the discussion portion, they discuss the optimal time for distilling the oil is 20 minutes but all of the studies they refer to are between 2 and 4 hours. 

The study they conducted didn’t go longer than 2 hours.  The distillation methods for these studies were not necessarily the same (some didn’t mention the method).  They also mention the distillation time had a direct relationship to the concentration of the chemicals in the oil, more so than harvesting stage (not further described). 

They mention the studies where the oil worked against various microbes used a much higher concentration of the oil that that used in this study.  The chemical profile was also different for this study. 
 
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Zheljazkov, V. D., Astatkia, T., Zhalnov, I., and Georgieva, T. D. (2015). Method for attaining Rosemary Essential Oil with Differential Composition from Dried or Fresh Material.  Journal of Oleo Science, 64(5):485-496.  doi: 10.5650/jos.ess14258.

Barbosa, Probst, Andrade, Alves, Albano, Cunha, Doyama, Rall, and Junior (2015) looked at bacteria that are becoming resistant such as Streptococcus pneumoniae, Escherichia coli (15 strains), Pseudomonas aeruginosa (15 strains), Staphylococcus aureus (MRSA) (15 strains of the resistant version and 16 strains of the sensitive version), Salmonella Typhimurium (15 strains) and Salmonella Enteritidis (16 isolates). 

Herbs they looked at were rosemary, Baccharvis dracuncutifolia (alecrim do campo), Vernonia polyanthes (assa peixe), Matrivaria recutita (chamomile), Cinnamomum zeylanicum (cinnamon), Caryophyllus aromaticus (clove), and Eugenia uniflora (pitanga).  All essential oils were distilled using steam. 

There is a lot of data in this study with the number of bacteria and herbs being tested.  For rosemary, the top 5 ingredients identified were camphor, limonene, myrcene, a-pinene, and b-pinene. The authors provide a chart which breaks these out for each herb tested. 

They also tested some combinations.  For the rosemary and alecrim do campo, the top 5 ingredients were limonene, merolidol, camphor, a-pinene, and myrcene. When rosemary was combined with cinnamon, the top 5 were cinnamaldehyde, camphor, limonene, myrcene, and a-pinene.  Combined with clove, the top were eugenol, camphor, ethyl eugenila, limonene, and b-caryophyllene.

Rosemary by itself took 79.35 mg/mL to be effective against all but S. aureus, MRSA, and MSSA. It was only 8.6 mg/mL these.  It took 24 hours to get a full kill ratio for E. coli and S. aureus. The combination of cinnamon and rosemary was also very effective against E. coli and rosemary combined with clove was very effective against S, aureus.

Of the plants tested, rosemary produced a larger amount of oil than most of the others. 

The authors mentioned the antimicrobial activity in the oils tested are a result of the composition of terpenes, stating most of what was extracted was a terpene or a derivative. 

NOTE: This study can be a bit confusing to read as the charts are many and the graphs were probably in color originally but are not in the paper.  Fortunately, I’m on a computer and can use the zoom feature! The rest of the herbs in this study will be broken out when I use this study in the books about each of them. 

Also, the amount needed to kill salmonella and the rest in that group explains why it performed so poorly in the study with chicken.  Putting that much rosemary on the chicken would have made it so strong, no one would have wanted to eat it because rosemary has a very powerful flavor. This rosemary is also from a different region with a different chemical compound, though it was the same species.
 
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Barbosa, L. N., Probst, I. S., Andrade, B. F. M. T., Alves, F. C. B., Albano, M., Cunha, M. L. R. S., Doyama, J. T., Rall, V. L. M., and Junior, A, F. (2015). In Vitro Antibacterial and Chemical Properties of Essential Oils Including Native, Plants from Brazil against Pathogenic and Resisant Bacteria. Journal of Oleo Science, 64(3):289-298. doi: 10.5650/jos.ess14209.

Kahraman, Issa, Bingoli, Basaran, Kahraman, and Dumen (2015) wanted to help solve the problem of food safety and tested rosemary to see if it could be used as a meat preservative in replacement of other methods.  The essential oil they used came from fresh leaves that were air-dried then steam distilled.  The oil was diluted to .05%, .1%, ;2%, .3%, .5%, and 1%.  After being added to broth, they were tested sat the 0, 4, 8, 12, and 24-hour marks.  They were tested against Salmonella Typhimurium and Listeria monocytogenes. 

For the taste portion, they established a panel with four males and two females.  The panel was then trained on terminology such as too sweet and given a scale on 1-5 where 1 was dislike extremely and 5 was like extremely.  The chicken samples were refrigerated for 1, 3, 5, and 7 days prior to cooking. 

The components of the oil used in this study were 1,8-cineole, a-pinene, camphor, 2a-pinene, camphen, and caryophyllene. They reminded the reader the composition of the oil depends on region the plant is from, plant variety and the extraction method.  The sample they used came from Turkey.  

In this study, the bacteria growth was inhibited at concentrations of .2% and higher.  They mentioned another study where the oil was not effective against Salmonella Typhimurium.  They mentioned the strain of bacteria used may account for the difference as well as the reasons mentioned for the differences in the oil composition.  It is believed the anti-bacteria properties are from 1,8-cineole and a-pinene.  1,8-cineole is most prominent in the samples from the region of Turkey containing at least 40% of the oil. 

For the tasting portion, no meat was actually consumed because the bacteria was not deteriorated enough to qualify for consumption despite how effective it was on the broth.  At concentrations higher than .2%, the rosemary scent was stronger and the color decreased in brightness.  The oil was effective at reducing the oxidation of the meat. 
 
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Kahramany, T., Issa, G., Bingoli, E. B., Basaran, B., Kahraman, B. B., and Dumen, E. (2015).  Effect of rosemary essential oil and modified-atmosphere packaging (MAP) on meat quality and survival of pathogens in poultry fillets. Brazilian Journal of Microbiology 46(2):591-599.

Kozlowska, Laudy, Przybyl, Ziarno, and Majewska (2015) investigated the effects of some herbal extracts (70% methanol and ethanol) against gram-positive and gram-negative bacteria.  Plants they looked into were thyme, rosemary, oregano, peppermint and sage. All plant samples were dried and extracted with both fluids mentioned above. 

For each plant, 10 grams were combined with 250 milliliters of the extraction liquid. They were heated for 10 minutes, strained then the liquid was dehydrated.  Of the herbs tested, the ethanol extract of rosemary was the highest and the highest methanol extract was sage.  In comparing the two, the ethanol extracts had higher numbers for all but peppermint and sage. Rosemarinic acid was the most prevalent and the highest amounts were found in the methanol extract of thyme and sage. Oregano and peppermint had the least of the methanol extracts. 

Throughout this portion of the paper, the authors compare their results to the amounts found by other researchers and noted the differences.  They point out the many reasons such as region of the world the plant came from, time of harvest, and extraction techniques.  The concentrations of caffeic acid were about the same regardless of extraction technique. Against the tested bacteria, the methanol and ethanol on their own had no effect on any. 

Rosemary had the broadest spectrum with both extracts, effective against all of the gram-positive bacteria and four of the gram-negative bacteria.  Enterococcus faecalis and E. hirae were the most effected gram-positive bacteria while Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were the more effected gram-negative bacteria.  S. maltophilia was the most sensitive of the gram-negative bacteria to rosemary.  L. monocytogenes was reduced by .25 mg/ml of the ethanol extract and .5 mg/ml of the methanol extract.

NOTE: It would be easiest to put the effectiveness of each extract in a chart with effective doses but this book is specifically about rosemary.  With all the information in this study, I‘ll save the data on those herbs for the books dedicated to that information. The authors also added the findings of similar studies within this one but it would be best to put each study, individually, into this book so the information of each study will be included.
 
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Kozlowska, M., Laudy, A. E., Przybyl, J., Ziarno, M., And Majewska, E. (2015). Chemical Composition and Antibacterial Activity of Some Medicinal Plants From Lamiaceae Family.  Acta Poloniae Pharmaceutica - Drug Research, 72(4):757-767. Polish Pharmaceutical Society

In a world where many think herbal are safe, with no interacts and can be used at will, Raskovic, Milanovici, Pavlovic, Milijasevic, Ubavic, and Mikov (2015) conducted a study to see if there were any interactions between rosemary essential oil and the drugs codeine and paracetamol (acetaminophen) as these can be used to treat renal colic pain, minor muscular pain, minor peripheral circulatory disorders and dysmenorrhea. This experiment involved eight groups with six mice in each group.  The length of the experiment was 7 days.

The control group was given saline. The REO group was given 20 mg/kg of rosemary essential oil. The Cod group was given saline for 7 days and one dose of codeine at the end. The Par group got saline for 7 days with a dose of paracetamol at the end. CodR10 got 10mg/kg of rosemary essential oil for 7 days with a dose of codeine at the end. CodR20 got rosemary essential oil for 7 days with a dose of codeine at the end. ParR10 was treated with rosemary essential oil for 7 days with a dose of paracetamol at the end.  They stated the recommended human dose is 40 mg/day. 

For the induced pain, they used a hot plate and placed each mouse on it and timed how long it took for the mouse to react to the pain caused by the heat. 

The rosemary group performed better than the paracetamol group compared to the placebo group, lasting at least 20 minutes, some as long as 50 minutes. The codeine group lasted the longest in comparison.  The rosemary and paracetamol group pasted 30-50 minutes.  The rosemary and codeine group lasted the longest.  With the rosemary groups combined with a drug, the larger dose performed best. 

The oil the used had 1,8-cineole, camphor, and a-pinene as the main components, what they call the Morocco/Tunisian Type based on the region it comes from. 

The authors concluded rosemary essential oil works in the management of pain and can be combined with other drugs but the correct dose must be determined as rosemary opens the pathways which could exacerbate the hepatotoxic properties of paracetamol, causing damage to one area of the body while trying to treat another.
 
Have a Gteat Day!

Raskovic, A., Milanovici, I., Pavlovic, B., Milijasevic, B., Ubavic, M., and Mikov, M. (2015). Analgesic effects of rosemary essential oil and its interactions with codeine and paracetamol in mice. European Review for Medical and Pharmacological Sciences, 19: 165-172.

I think it would have been better to put the tomatoes on either side of the front of the greenhouse.  Now I know for next time!

Raskovic, Milanovic, Pavlovic, Cebovic, Vukmirovic, and Mikov (2014) evaluated the effects of rosemary essential oil on liver injury. The injury to the rats was induced using carbon tetrachloride (CCl4).  Instead of ordering the oil they made it in their lab using established procedures (hydrodistillation). 

The rats were divided into 6 groups of six. There were two control groups. One was given a saline solution (CON S) and the other was given one dose 1 milliliter per kilogram of weight of CCl4.  There were two rosemary groups with this same dosage. The dosages for rosemary were 5 and 10 milligrams per kilogram. The other two groups were just given rosemary essential oil at these same doses. 

The rosemary was given via a feeding tube for seven days then the CCl4 was administered. The most prominent constituents of the essential oil were 1,8-cineole, camphor, a-pinene, b-pinene, camphene, and b-caryophyllene.

The rosemary essential oil demonstrated a free radical scavenging property where 77.6 microliters per milliliter achieved 50% effectiveness.  Markers they looked at were malondialdehyde (MDA) and glutathione (GSH) levels as well as catalase (CAT), peroxidase (Px), glutathione peroxidase (GPx), and glutathione reductase (GR) activities.  With CCl4, MDA had a 3.5-fold increase. Both groups where the rosemary was introduced before the CCl4 had significantly reduced oxidative stress levels. The authors said the REO10 group was near that of the control. The urea and creatine levels suggested rosemary essential oil repaired some of the damage to the liver excretion function.  

MDA is one of the first indicators that liver function is impaired. Rosemary prevented the increase in MDA. Damaging the liver significantly impaired GSH levels as well. It reduces CAT activity and increases GPx. Rosemary reversed all of the indicators, especially at the higher dose. 

Rosemary essential oil limited cell peroxidation which reduced the damage to the cell membranes. Given the variations in oil composition, the authors can only speak for the composition of the oil tested. Their sample came from Belgrade.

NOTE: One important thing mentioned in this study is the composition of the oil based on the region of origin of the plant.  They named three different regions that, when compared, differed making it important to understand what properties the plant being used have.  This could be vital in regards to people with plant allergies.  They added season of harvest was an important factor.
 
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Raskovic, A., Milanovic, I., Pavlovic, N., Cebovic, T., Vukmirovic, S., and Mikov, M. (2014). Antioxidant activity of rosemary (Rosmarinus officinalis L.) essential oil and its hepatoprotective potential. Complementary and Alternative Medicine 14:225

This one is really long but this study deserved a very detailed look.  I explained my feelings on it at the end.  I started this at 5:30 this morning and am posting it at 2:30 in the afternoon. 

Alfonso, Mara de O Silva, Carvalho, Rivelli, Barros, Rogero, Lottenberg, Torres, and Mancini-Filho (2013) compared two different forms of rosemary extract and their effect on high cholesterol. 

One was a water extract, 20 grams of herb to 100 milliliters of water at room temp for 1 hour.  It was then centrifuged, filtered then 100 milliliters of water added.

The non-estrified phenolic fraction (NEPF)was powdered, defatted leaves. They did this with tetrahydrofuran, 6 one-minute periods of 20ml, at room temperature.  They filtered it with sodium sulfate anhydrous.  They used vacuum evaporation at 30 degrees Celsius. The dried matter was then placed in 5 milliliters of methanol then filtered then the flask was filled to 20 milliliters.  

This study was conducted using 48 mice which were divided into six groups. All groups were fed a high cholesterol (HC) diet except the control group.  The HC group received distilled water. 

There were two water extract groups. One was treated with 70 milligrams of the extract per kilogram of body weight per day, the other with 140 (AQ 70 and 140).  The other extract was given to the last two groups at 7 milligrams per kilogram of body weight per day and 14. 

When they evaluated the phenolic content of the extracts, there was twice as much phenolic compound in the water extract, 15. 67 to 8.59 in the NEPF.  The water extract had 1.87 grams of rosmarinic acid per 100 grams of dry weight.  The NEPF extract had 5.71 grams of carnosic acid per 100 grams of weight. 

The antioxidant activity was ten times higher in the NEPF extract.  The total cholesterol was 39.8% lower in the AQ70 group and the non-high density lipoprotein cholesterol (non-HDL) was 44.4 % lower.  The higher dosage did not have any significant result in comparison.  The triglyceride levels were higher and the HDL were lower but not significantly.  In the AQ70, the thiobarbituric acid reactive substances (TBARS) were reduced from 1.34 to .83 micromoles/milligram.

With NEPF, at 7 and 14 milligrams per kilogram, TBARS in the heart dropped by 24.32% and 16.05 respectively. In renal tissues, levels dropped by 33.61% and 27.29% respectively.  Both rosemary groups showed significant drops in TBARS levels in the brain and liver in comparison with the high cholesterol group. 

In both rosemary extracts, the catalase activity was significantly increased, even higher in the higher dosage groups. For glutathione, the higher dosage of water extract performed the best.  In the kidneys, the NEPF increased catalase and superoxide dismutase activities.  Neither extract helped the catalase and superoxide dismutase activities in the cardiac tissue.

An important note on the catalase activity: The mice fed a high cholesterol diet and no rosemary had poor catalase activity.  Adding the rosemary treatment improved this activity seeming to reverse that negative effect from the diet. This means it seemed to reduce the cardiovascular disease caused by the poor diet by reducing the lipid profile.

            NOTES: I know this is a long one but this is an interesting study that is missing some things.  I want to be fair to it so I’m sharing as much of this data as I can.  The authors say only the water extract significantly reduced total cholesterol and HDL and attributed this to overall phenolic compound levels being more than twice as high in the water extract. 

My issue with this is they don’t break this out in this study, just the rosmarinic acid for the water extract and the carnosic acid for the NEPF extract. This leaves the question of what were the other compounds and how much of each was in each extract.  I also don’t like how they provide numbers in some comparisons and percentages in others which doesn’t provide a “fair” comparison between these two groups unless you can see the graphs they include in the study.  As an analyst, I start questioning why the numbers are not presented in the same form when doing comparisons. 

I do think this form of study is vital, in that the readers need to understand there is a reason to use different types of extraction methods.  When you can see the differences in effectiveness for different issues, it helps bring this concept home.  It helps to understand why we use teas, infusions, decoctions, tinctures and other methods of use, often different methods for each herb. 
 
Have a Great Day!

Alfonso, M. S., Mara de O Silva, A., Carvalho, E. B. T., Rivelli, D. P., Barros, S. B. M., Rogero, M. M., Lottenberg, A. M., Torres, R. P., and Mancini-Filho, J. (2013). Phenolic compounds from Rosemary (Rosmarinus officinalis L.) attenuate oxidative stress and reduce blood cholesterol concentrations in diet-induced hypercholesterolemic rats. Nutrition and Metabolism, 10:19.