Category: Research Paper
Seizures in a 7-month-old child after exposure to the essential plant oil thuja. Pediatr Neurol. 2007 Dec;37(6):446-8.Stafstrom CE.Section of Pediatric Neurology, Departments of Neurology and Pediatrics, University of Wisconsin, Madison, WI 53792, USA. email@example.com
A previously healthy 7-month-old child was treated with homeopathic preparations of thuja, a potentially convulsant compound, for the purpose of providing a calming effect around times of immunizations. The child developed eight generalized tonic-clonic seizures with no other obvious cause, in the context of normal electroencephalograms and a normal brain magnetic resonance imaging scan. Seizures stopped after discontinuation of thuja and brief treatment with phenobarbital. The epileptogenic potential of plant-derived essential oils and other herbal remedies should be recognized by practitioners providing neurologic care to children.
Plant light interception can be explained via computed tomography scanning: demonstration with pyramidal cedar (Thuja occidentalis, Fastigiata). Ann Bot (Lond). 2008 Jan;101(1):19-23. Epub 2007 Nov 2.Dutilleul P, Han L, Smith DL.Department of Plant Science, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Québec, Canada H9X 3V9. firstname.lastname@example.org
BACKGROUND AND AIMS: Light interception by the leaf canopy is a key aspect of plant photosynthesis, which helps mitigate the greenhouse effect via atmospheric CO(2) recycling. The relationship between plant light interception and leaf area was traditionally modelled with the Beer-Lambert law, until the spatial distribution of leaves was incorporated through the fractal dimension of leafless plant structure photographed from the side allowing maximum appearance of branches and petioles. However, photographs of leafless plants are two-dimensional projections of three-dimensional structures, and sampled plants were cut at the stem base before leaf blades were detached manually, so canopy development could not be followed for individual plants. Therefore, a new measurement and modelling approach were developed to explain plant light interception more completely and precisely, based on appropriate processing of computed tomography (CT) scanning data collected for developing canopies. METHODS: Three-dimensional images of canopies were constructed from CT scanning data. Leaf volumes (LV) were evaluated from complete canopy images, and fractal dimensions (FD) were estimated from skeletonized leafless images. The experimental plant species is pyramidal cedar (Thuja occidentalis, Fastigiata). KEY RESULTS: The three-dimensional version of the Beer-Lambert law based on FD alone provided a much better explanation of plant light interception (R(2) = 0.858) than those using the product LV*FD (0.589) or LV alone (0.548). While values of all three regressors were found to increase over time, FD in the Beer-Lambert law followed the increase in light interception the most closely. The delayed increase of LV reflected the appearance of new leaves only after branches had lengthened and ramified. CONCLUSIONS: The very strong correlation obtained with FD demonstrates that CT scanning data contain fundamental information about the canopy architecture geometry. The model can be used to identify crops and plantation trees with improved light interception and productivity.
Diterpenoids from the pericarp of Platycladus orientalis. Phytochemistry. 2007 Sep 11;Wang YZ, Tang CP, Ke CQ, Weiss HC, Gesing ER, Ye Y.State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China.
Eight labdane-type diterpenes, 7beta,13S-dihydroxylabda-8(17),14-dien-19-oic acid (1), 12R,15-dihydroxylabda-8(17),13E-dien-19-oic acid (3c), 12R,15-dihydroxylabda-8(17),13Z-dien-19-oic acid (3d), 12R,13R,14S-trihydroxylabda-12,15-epoxy-8(17)-en-19-oic acid (4a), 12S,13S,14R-trihydroxylabda-12,15-epoxy-8(17)-en-19-oic acid (4b), 15-hydroxy-12-oxolabda-8(17),13E-dien-19-oic acid (5), 14R,15-dihydroxylabda-8(17),12Z-dien-19-oic acid (7a) and 14S,15-dihydroxylabda-8(17),12Z-dien-19-oic acid (7b), along with 20 known diterpenoids, were isolated from the pericarp of Platycladus orientalis. Their structures were unambiguously elucidated by NMR spectroscopic and single crystal X-ray diffraction analyses, as well as via chemical correlation conversion. NMR spectroscopic data of known isomers 8c and 8d were reported as a supplement to existing data.
Biosorption of a textile dye (Acid Blue 40) by cone biomass of Thuja orientalis: Estimation of equilibrium, thermodynamic and kinetic parameters. Bioresour Technol. 2007 Aug 13;Akar T, Ozcan AS, Tunali S, Ozcan A.Department of Chemistry, Faculty of Arts and Science, Eski?ehir Osmangazi University, 26480 Eski?ehir, Turkey
Biosorption of Acid Blue 40 (AB40) onto cone biomass of Thuja orientalis was studied with variation in the parameters of pH, contact time, biosorbent and dye concentration and temperature to estimate the equilibrium, thermodynamic and kinetic parameters. The AB40 biosorption was fast and the equilibrium was attained within 50min. Equilibrium data fitted well to the Langmuir isotherm model in the studied concentration range of AB40 and at various temperatures. Maximum biosorption capacity (q(max)) for AB40 was 2.05x10(-4)molg(-1) or 97.06mgg(-1) at 20 degrees C. The changes of Gibbs free energy, enthalpy and entropy of biosorption were also evaluated for the biosorption of AB40 onto T. orientalis. The results indicate that the biosorption was spontaneous and exothermic. Kinetics of biosorption of AB40 was analyzed and rate constants were also derived and the results show that the pseudo-second-order kinetic model agrees very well with the experimental data.
Continuous determination of total flavonoids in Platycladus orientalis (L.) Franco by dynamic microwave-assisted extraction coupled with on-line derivatization and ultraviolet-visible detection. Anal Chim Acta. 2007 Jul 16;596(1):164-70. Epub 2007 Jun 8. Chen L, Ding L, Yu A, Yang R, Wang X, Li J, Jin H, Zhang H.College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, PR China.
This paper describes a new method for the determination of total flavonoids in Platycladus orientalis (L.) Franco. The method was based on dynamic microwave-assisted extraction (DMAE) coupled with on-line derivatization and ultraviolet-visible (UV-vis) detection. The influence of the experimental conditions was tested. Maximum extraction yield was achieved using 80% aqueous methanol of extraction solvent; 80 W of microwave output power; 5 min of extraction time; 1.0 mL min(-1) of extraction solvent flow rate. The derivatization reaction between aluminium chloride and flavonoid is one of the most sensitive and selective reactions for total flavonoids determination. The optimized derivatization conditions are as follows: derivatization reagent 1.5% aluminium chloride methanol solution; reaction coil length 100 cm; derivatization reagent flow rate 1.5 mL min(-1). The detection and quantification limits obtained are 0.28 and 0.92 mg g(-1), respectively. The intra-day and inter-day precisions (R.S.D.) obtained are 1.5% and 4.6%, respectively. Mean recovery is 98.5%. This method was successfully applied to the determination of total flavonoids in P. orientalis (L.) Franco and compared with heat reflux extraction. The results showed that the higher extraction yield of total flavonoids was obtained by DMAE with shorter extraction time (5 min) and small quantity of extraction solvent (5 mL).
Anatomical and water physiological plasticity of Grewia biloba var. parviflora leaf and secondary xylem: Ying Yong Sheng Tai Xue Bao. 2006 Oct;17(10):1801-6. Chinese.Shi G, Cheng X, Liu L, Ma C.Department of Biology, Huaibei Coal Industry Teachers College, China. email@example.com
Based on the anatomical observations of leaf and secondary xylem as well as the measurements of leaf water physiological parameters, this paper studied the anatomical and water physiological plasticity of Grewia biloba var. parviflora growing in different succession stage communities. The results showed that G. biloba var. parviflora leaf was characterized by thin bifacial with thin cuticle and few stoma, indicating that it was mesophyte anatomically, while the secondary xylem had typical xeromorphic traits, such as short and thin vessel, high vessel frequency, low percentage single pores, and short fibers and rays. G. biloba var. parviflora had high plasticity in the anatomical structure and water physiological features of leaf and secondary xylem, and the plasticity index was in the order of secondary xylem anatomical structure (0. 24) > water physiological traits (0. 19) > leaf anatomical structure (0. 18). Compared with those growing in mixed forest and Platycladus orientalis forest, the individuals of G. biloba var. parviflora in shrub communities had the xeromorphic traits in the aspects of (1) their secondary xylem had shorter vessel elements, higher vessel frequency, less single porous percentage, lower rays, higher relative conductivity and lower vulnerability index, and (2) their leaf had lower water potential, lower water content and free water content, higher bound water content, high ratio of bound to free water content, and less specific leaf area. The phenotypic plasticity, both anatomical and physiological, made G. biloba var. parviflora tolerate to the drought at earlier succession stages and better adapt to the mesophytic condition at later stages, and consequently, become a widely distributed and dominant species in mixed forests.
Labdane diterpenes from the seeds of Platycladus orientalis. J Asian Nat Prod Res. 2006 Dec;8(8):677-82.Ren XY, Ye Y.Graduate School of Chinese Academy of Sciences, Shanghai, 200031, China.
Two new labdane diterpenes, 14(R),15-dihydroxy-8(17),12(E)-labdadien-19-oic acid (1) and 16-methyl-12,15-epoxy-8(17),13-labdadien-19-oic acid (2), together with four known compounds, were isolated from the seeds of Platycladus orientalis. Their structures were established by spectroscopic methods. The stereochemistry of compound 1 was defined by X-ray crystallographic analysis.
Effects of homeopathic preparations on human prostate cancer growth in cellular and animal models. Integr Cancer Ther. 2006 Dec;5(4):362-72. MacLaughlin BW, Gutsmuths B, Pretner E, Jonas WB, Ives J, Kulawardane DV, Amri H.Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007, USA.
The use of dietary supplements for various ailments enjoys unprecedented popularity. As part of this trend, Sabal serrulata (saw palmetto) constitutes the complementary treatment of choice with regard to prostate health. In homeopathy, Sabal serrulata is commonly prescribed for prostate problems ranging from benign prostatic hyperplasia to prostate cancer. The authors' work assessed the antiproliferative effects of homeopathic preparations of Sabal serrulata, Thuja occidentalis, and Conium maculatum, in vivo, on nude mouse xenografts, and in vitro, on PC-3 and DU-145 human prostate cancer as well as MDA-MB-231 human breast cancer cell lines. Treatment with Sabal serrulata in vitro resulted in a 33% decrease of PC-3 cell proliferation at 72 hours and a 23% reduction of DU-145 cell proliferation at 24 hours (P 25 microM) in vitro antiplasmodial effects against Plasmodium falciparum strain 3D7. At the same time, the compounds caused echinocytic or stomatocytic changes of the erythrocyte membrane curvature, indicative of their incorporation into the lipid bilayer, in the concentration region where the antiplasmodial activity was observed. The antiplasmodial effect of these compounds thus appears to be an indirect effect on the erythrocyte host cell. Weak or moderate antiplasmodial activity observed with many other apolar natural products, in particular those with amphiphilic structures, is also likely to be an indirect effect.
Aerobiological diagnosis of respiratory allergy by a personal sampler: two case reports. J Investig Allergol Clin Immunol. 2003;13(4):284-5.Fiorina A, Scordamaglia A, Fumagalli F, Canonica GW, Passalacqua G.DIMI, University of Genoa, Italy.
We describe two cases of respiratory allergy (asthma), which were difficult to diagnosis from an etiological viewpoint. The routine diagnostic tests were not able to determine the causal allergens, although the clinical history suggested that allergens were confined to restricted environments. Therefore, an aerobiological sampling by means of a battery-powered portable device was carried out. This approach allowed identification of the responsible allergens, which were Alternaria spores in one case and thuja pollen in the other. Once a targeted environmental care had been performed, the patients' symptoms rapidly improved and antiasthma therapy could be stepped down or discontinued. We suggest that, in selected cases of difficult diagnosis the presence of proximity allergens is suspected, aerobiological sampling with a portable device should be considered.
GC analysis of essential oils in the rumen fluid after incubation of Thuja orientalis twigs in the Rusitec system. Res Vet Sci. 2004 Feb;76(1):77-82.Chizzola R, Hochsteiner W, Hajek S.Institute for Applied Botany, University of Veterinary Medicine, Vienna 1210, Austria. firstname.lastname@example.org
Methods for the chemical analysis of toxic plant substances in the rumen of ruminants are of importance for the diagnosis of intoxications with poisonous plants. The present work establishes a method to estimate monoterpene components of the essential oil of thuja (Thuja orientalis, Cupressaceae) in these types of samples. Alpha-thujone, which is regarded as the toxic principle, is present at a concentration of 50-60% in the essential oil. The rumen simulation technique (Rusitec) was used to simulate natural digestion. Chopped twigs of thuja were subjected to rumen content in a closed container with an overflow device. The flow of saliva was simulated by the continuous addition of a buffer solution. Samples for analysis were taken from the overflow at 24 and 48 h. A further sample was taken from the remaining liquid fraction of the rumen content in the container at 48 h. The essential oils were extracted with hexane and concentrated. A quantitative determination was done by capillary gas chromatography. Together in the three fractions analysed this resulted in total mean recoveries of 6.8% for alpha-thujone, 5.3% for beta-thujone, 18.9% for fenchone and 27.8% for camphor. The observation that the thujones were recovered to a lesser extent than other oil components is evidence of their fast decomposition in the rumen medium. Under these circumstances the calculated detection limit is 100-200 g thuja twigs in cows with rumen volumes of 60-100 litres. The main essential oil degradation products found in the rumen fluid of all three fractions in the Rusitec system were discovered to be iso-3-thujanol, neo-3-thujanol, carvomenthol and carvomenthone.
Volatile constituents of the fruit and leaf oils of Thuja orientalis L. grown in Iran. Z Naturforsch [C]. 2003 Mar-Apr;58(3-4):171-2. Nickavar B, Amin G, Parhami S.Pharmacognosy Department, School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. email@example.com
The composition of the hydrodistilled essential oils from the fruits and leaves of Thuja orientalis L. grown in Iran was analyzed by GC/MS. Nineteen and twenty-eight compounds have been identified in the volatile oils of the fruit and leaf, respectively. While the fruit oil contained alpha-pinene (52.4%), delta-3-carene (14.2%), alpha-cedrol (6.5%) and beta-phellandrene (5.1%), the leaf oil contained alpha-pinene (21.9%), alpha-cedrol (20.3%), delta-3-carene (10.5%) and limonen (7.2%) as the main components.
The extract of Thujae occidentalis semen inhibited 5alpha-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse. J Dermatol Sci. 2003 Apr;31(2):91-8.BACKGROUND: The conversion of testosterone to dihydrotestosterone; 5alpha-androstan-17beta-ol-3-one by 5alpha-reductase plays a crucial role in hair baldness and prostatomegaly. Recent approach showed specific inhibitors for 5alpha-reductase type 2 such as finasteride promoted hair growth in male pattern alopecia. OBJECTIVE: In order to search for effective medicinal plant extracts applied topically for androgenetic alopecia, we screened natural plant extracts having inhibitory activities of 5alpha-reductase type 2 and demonstrated its biological function in androgen-related animal models. METHODS: We evaluated the inhibition activities of numerous plant extracts by contact cell based metabolic method using a stable HEK 293 cell line expressing human 5alpha-reductase (type 2). To elucidate the biological activity in vivo, the Thujae occidentalis semen (TOS) extract was topically applied to fuzzy rat and androchronogenetic alopecia (AGA) mouse, respectively. The secreted sebum and the size of sebaceous glands of fuzzy rat were measured after 6 weeks. Also, after the topical treatment with TOS extract and androgen receptor antagonist (cyproterone acetate) simultaneously with subcutaneous injection of testosterone (1 mg/mice/day), hair loss patterns of female B6CBAF1/j hybrid mouse were observed. RESULTS: TOS extract showed higher inhibition activity of 5alpha-reductase type 2(IC(50) value=2.6 microg/ml) than that of gamma-linolenic acid, but lower than that of finasteride. When applied to fuzzy rat, the amount of sebum and sebaceous gland size decreased remarkably. In AGA model, alopecia degrees of two groups, treated with TOS extract (P
♥The article and literature was edited by herbalist of MDidea Extracts Professional .It runs a range of online descriptions about the titled herb and related phytochemicals,including comprehensive information related,summarized updating discoveries from findings of herbalists and clinical scientists from this field.The electronic data information published at our official website www.mdidea.com and www.mdidea.net ,we tried best to update it to latest and exact as possible.
♣ last edit date: April 17th 2014 at 14:30
With natural preservation properties, essential oil from the native Canadian Arborvitae tree acts as a powerful cleansing and purifying agent, while providing a warm, earthy aroma.
Part Number: 49360001
Size: 5 mL
Known as the “tree of life, ” Arborvitae is majestic in size and abundant in unique benefits. Arborvitae essential oil is concentrated in tropolones, such as hinokitiol, which are a group of chemical compounds that protect against environmental and seasonal threats and have powerful purifying properties. These compounds also contribute to Arborvitae’s natural insect repellent properties. Thujic acid, another tropolone found in Arborvitae, has been studied for its ability to protect against common threats in the environment. Native to Canada, all parts of the Arborvitae tree were used extensively by Native Americans for health benefits and for building vessels, totem poles, baskets, and clothing. Because of its natural preserving properties, Arborvitae prevents wood from rotting, which makes it popular in woodcraft and for preserving natural wood surfaces.Uses
Diffusion: Use three to four drops in the diffuser of your choice.
Topical use: Apply one to two drops to desired area. Dilute with doTERRA Fractionated Coconut Oil to minimize any skin sensitivity.
Possible skin sensitivity. Keep out of reach of children. If you are pregnant, nursing, or under a doctor’s care, consult your physician. Avoid contact with eyes, inner ears, and sensitive areas.More Info
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The present study was carried out to develop a new natural product reagent which has antimicrobial and antiviral effect, so we assayed the extract from Korean Arbor vitae (Thuja koraiensis ) on antimicrobial and antiviral in vitro. The antimicrobial activity was assayed at two gram-positive bacteria (Staphylococcus aureus,Bacillus subtilis ); two gram-negative bacteria (Escherichia coli,Salmonella typhimurium ) and the results were measured by the paper disc diffusion assay and minimum inhibitory concentration (MIC). The antiviral activity of T. koraiensis extract was assayed at the Bovine viral diarrhoea (BVD) virus which is a RNA virus replication in Madin-Darby bovine kidney (MDBK) cells and the results were measured by maximum non cytotoxic concentration (MNCC) and maximum non-toxic dose (MNTD). The result of paper disk diffusion assay showed that extract had the high antimicrobial effect at S. aureus strain. The MNCC of extract on MDBK cells was 0.031% and the MNTD of extract was 0.0195% on BVD virus. These results suggested that T. koraiensis extract had antimicrobial and antiviral effect, especially at low concentrations which had a strong antiviral effect at BVD virus. The T. koraiensis extract could also be useful as disinfectant for bacterial. The study of T. koraiensis function perhaps would be the first and more research is needed in the future.
Key words:Thuja koraiensis extract, antimicrobial activity, minimum inhibitory concentration, maximum non cytotoxic concentration, maximum non-toxic dose, antiviral activity.
Aljos F (2005). A monograph of Cupressaceae and Sciadopitys. Surrey: Royal Botanic Gardens. "Flora of China" editorial board (1978). Flora of China. Science 7:318.
Baker JC (1995). The clinical manifestations of bovine viral diarrhea infection. Vet. Clin. North Am. Food Anim. Pract. 11:425-445.
Bauer AW, Kirby WM, Sherris JC, Turck M (1966). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:493-496.
Cassel E, Vargas R, Martinez N, Lorenzo D, Dellacassa E (2009). Steam distillation modeling for essential oil extraction process. Ind. Crops Prod. 29:171-176.
Collett MS, Larson R, Belzer SK, Retzel E (1988). Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus. Virology 165:200-208.
Dubey S, Batra A (2008). Antidiabetic activity of Thuja occidentalis Linn. Res. J. Pharm. Technol. 1:362.
Guleria S, Kumar A, Tiku AK (2008). Chemical composition and fungitoxic activity of essential oil of Thuja orientalis L. grown in the North-western Himalaya. Z. Naturforsch. C. 63:211-214.
Houe H (1999). Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet. Microb. 64:89-107.
Jain RK, Garg SC (1997). Antimicrobial activity of the essential oil of Thuja orientalis L. Anim. Sci. Life 16:186-189.
The natural world is filled with mystery and undefinable beauty. Arborvitae seems to tap into the essence of God’s creation while at the same time utilizing nature’s therapeutic powers. Woodworkers and carpenters have long used it to preserve natural wood while preventing rot. While exploring the outdoors or enjoying a sunset dinner on a patio, try using Arborvitae to keep pesky insects away.
Arborvitae is derived from graceful coniferous trees that extend up to 60 feet in height and produce scale-like leaves and broadly-winged seeds, sometimes planted as hedging. Before the tree is ready to supplant essential oil through steam distillation, it must be at least 15 years old.
As part of their cultural heritage, Native Americans often use the entire Arborvitae tree to craft vessels, totem poles, baskets and clothing. A dynamic essential oil, Arborvitae will prompt you to try new ways to explore its potential.
Botanical Name:Thuja plicata
Extraction Method: Steam Distillation
Synonym: Western Red Cedar, Pacific Thuja, Western Arborvitae
Aroma: Woody, sharp, peppery, tenacious, and earthy
Consistency: Thin - Medium
**Euro droppers are not included with 100 & 250 ml bottles**
This section is being updated with exciting new applications and recipes. All of this will be ready later this spring…we greatly appreciate your patience and loyalty.
Safety & Shelf Life
Safety: Consult your health care provider if pregnant, nursing, taking medication or being treated for a health condition. Dilute with carrier oil before topical application or bath use. Keep out of reach of children. Store in a cool, dark place.
Shelf Life: 4 Years
Comments from GC/MS report: Very complex and elegant aroma that is woody and spicy with notes of cumin, nutmeg, and wormwood. The chemical profile is one of the best I’ve seen for giant arborvitae essential oil, with a methylthujate content of almost 60% and nice complexity.
Arborvitae (Thuja Plicata ), meaning Tree of Life, is steam distilled from the Giant Arborvitae tree, a tree highly revered and used by Native Canadians for canoes, baskets, and so much more. Its long lifespan would normally mean that it's difficult to harvest sustainably, but it's closely watched by Green Peace and replanted in abundance to ensure its sustainability. It's aroma is deep and earthy, usually loved or hated with very little in-between.
Arborvitae essential oil has a high content of tropolones, a group of chemical compounds that protect against environmental and seasonal threats, have powerful purifying properties, and promote healthy cell function. Hinokitiol, one of the tropolones in Arborvitae, protects the body from harmful elements while supporting normal cell activity. This compound also contributes to Arborvitae’s natural insect repellent properties. Thujic acid, another tropolone found in Arborvitae, has been studied for its ability to protect against common threats in the environment.Primary Benefits
Emotionally, Arborvitae has been used for centuries by Natives to promote grounding, calming, and spiritual trust. Like all tree oils, it can help us stand strong and stable. It also helps those of us who feel like everything is or has to be a struggle, or that we must do things ourselves without help (hmm, maybe that's why I didn't care for the aroma!). It can help a person process through control issues, rigidity, trust, fighting with reality, courage, and more.Complimentary Oils: How to use Arborvitae Essential Oil:
Traditionally, arborvitae essential oil uses these application guidelines. Click the banner to understand what each symbol means and to learn how each is used safely and effectively:
Precautions of Arborvitae Essential Oil
Possible skin sensitivity. Keep out of reach of children. If you are pregnant, nursing, or under a doctor’s care, consult your physician. Avoid contact with eyes, inner ears, and sensitive areas.Popular Uses of Arborvitae Essential Oil
Not all oils are created equal . Because of a lack of industry standards and a lack of regulation on terms such as "natural" or "pure", much of what you find at the drug store is NOT a therapeutic grade of essential oil and may lack real quality or even contain contaminants or adulterants (way more common than you'd think).A LOT goes into creating a high quality essential oil. A good brand should follow these guidelines:
1 Department of Infectious Diseases, Virology, University of Heidelberg, 69120 Heidelberg, Germany
2 Yazd Shahid Sadoghi University of Medical Science, Safaieh, Yazd, Iran
3 Department of Biology, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany
Received 22 June 2009; Accepted 15 October 2009
Copyright © 2011 Akram Astani et al. This is an open access article distributed under the Creative Commons Attribution License. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Abstract
Essential oil of star anise as well as phenylpropanoids and sesquiterpenes, for example, trans-anethole, eugenol, β -eudesmol, farnesol, β -caryophyllene and β -caryophyllene oxide, which are present in many essential oils, were examined for their antiviral activity against herpes simplex virus type 1 (HSV-1) in vitro. Antiviral activity was analyzed by plaque reduction assays and mode of antiviral action was determined by addition of the drugs to uninfected cells, to the virus prior to infection or to herpesvirus-infected cells. Star anise oil reduced viral infectivity by >99%, phenylpropanoids inhibited HSV infectivity by about 60–80% and sesquiterpenes suppressed herpes virus infection by 40–98%. Both, star anise essential oil and all isolated compounds exhibited anti-HSV-1 activity by direct inactivation of free virus particles in viral suspension assays. All tested drugs interacted in a dose-dependent manner with herpesvirus particles, thereby inactivating viral infectivity. Star anise oil, rich in trans-anethole, revealed a high selectivity index of 160 against HSV, whereas among the isolated compounds only β -caryophyllene displayed a high selectivity index of 140. The presence of β -caryophyllene in many essential oils might contribute strongly to their antiviral ability. These results indicate that phenylpropanoids and sesquiterpenes present in essential oils contribute to their antiviral activity against HSV.1. Introduction
Herpes simplex virus type 1 (HSV-1) is an important pathogen for humans, and discovery of novel effective antiherpetic drugs without adverse effects is of great interest. The primary symptoms of herpes infection include a prodromal flu-like syndrome with fever, headache, malaise, diffuse myalgias, followed by local symptoms consisting of itching and painful papules. Gingivostomatitis and pharyngitis are the most frequent clinical manifestations of first episodes of HSV-1 infection. After establishing latency, HSV can reactivate, causing frequent recurrent infections in some patients, whereas most people experience few recurrences [1 ]. The clinical manifestation of the disease exhibits different severity in immunocompetent patients and in addition some patients always encounter recurrent attacks [2 ]. However in immunocompromised patients and neonates, herpetic infections can cause serious systemic illnesses. Recurrent herpes labialis is the most frequent clinical manifestation of reactivated HSV-1 infection.
A very effective treatment for HSV is available since the introduction of acyclovir in the 1970s and it is still the most commonly used chemotherapy [3 ]. This antiviral agent can be used to shorten the course and decrease the severity of these clinical symptoms and may suppress the virus itself [2 ]. Antiviral agents licensed currently for the treatment of herpesvirus infections include acyclovir and derivatives, foscarnet and cidofovir, all of which inhibit herpesvirus DNA polymerases [4 ]. Some of these antiviral agents might produce toxic side-effects. In addition, the emergence of virus strains resistant to commonly used anti-herpesvirus drugs is of importance, particularly in immunocompromised patients [5 –7 ]. The development of viral resistance toward antiviral agents enhances the need for new effective compounds against viral infections. Thus, new antiviral agents exhibiting different mechanisms of action are urgently needed.
Medicinal plants produce a variety of chemical constituents with the potential to inhibit viral replication and compounds from natural sources are of interest as possible sources to control viral infection. These plants have been widely used to treat a variety of infectious and non-infectious diseases and represent an abundant source of new bioactive secondary metabolites. Thus plants continue to be a major source of new lead compounds. Besides small molecules from medicinal chemistry, natural products are still major sources of innovative therapeutic agents for various conditions, including infectious diseases. In recent years there has been an increasing interest in the use of natural substances, and some questions concerning the safety of synthetic compounds have encouraged more detailed studies of plant resources. Essential oils, odors and volatile products of plant secondary metabolism, have a wide application in folk medicine as well as in fragrance industries. Essential oils are complex natural mixtures of volatile secondary metabolites, isolated from plants by hydro- or steam-distillation. The main constituents of essential oils, for example, monoterpenes and sesquiterpenes and phenylpropanoids including carbohydrates, alcohols, ethers, aldehydes and ketones, are responsible for the fragrant and biological properties of aromatic and medicinal plants [8 ]. Various essential oils and their components possess pharmacological effects, demonstrating antiinflammatory, antioxidant and anticancerogenic properties [9 –11 ].
Antiherpes screening experiments on medicinal plant extracts and plant-derived secondary metabolites have been reported [8. 12 ]. Antibacterial, antifungal, immunomodulatory, antiinflammatory and antirheumatic activities have been described for essential oils [8. 13 –21 ]. The antiherpes activity of several essential oils of different plant sources as well as of some constituents of essential oils had been demonstrated previously [22 –25 ]. The application of tea tree oil, the essential oil of Melaleuca alternifolia. for the treatment of recurrent herpes labialis has been reported recently [26 ]. The antiherpes activity of eucalyptus oil, Australian tea tree oil [27 ], thyme oil [28 ] and manuka oil [14 ] has previously been published. Other medicinal plant extracts of traditional Thai medicinal plants showed a pronounced antibacterial activity against methicillin-resistant Staphylococcus aureus [29 ]. Christoph et al. [30. 31 ] performed a comparative study on the in-vitro antimicrobial activity of tea tree oil with special reference to the activities of β -triketones. Some phenylpropanes [32. 33 ], triterpenes [34 ] and sesquiterpenes [35 –37 ] had been tested for their antiviral activity against different herpesviruses and rhinovirus. However, sesquiterpenes as important constituents of essential oils, have not been analyzed systematically for their antiviral potential. Only few reports describe the inhibition of viral replication by sesquiterpenes, for example, triptofordin C-2 [35 ]. Thus only limited information about sesquiterpenes concerning the inhibition of the viral replication cycle and their mode of antiviral action is presently available.
The aim of the present study is the evaluation of the antiviral activity of selected sesquiterpenes, important constituents of essential oils, against HSV-1 and the mode of antiviral action of these sesquiterpenes during the viral multiplication cycle.2. Methods 2.1. Star Anise Oil, Phenylpropanoids, Sesquiterpenes, Acyclovir
Star anise essential oil met the standard demands of current pharmacopoeias and literature data [38 ] and was obtained from Caelo (Hilden, Germany). This essential oil is rich in trans-anethole and consists of about 80% of this phenylpropanoid [39 ]. Trans-anethole, eugenol, β -caryophyllene were purchased from Roth GmbH (Karlsruhe, Germany), and β -eudesmol, farnesol and β -caryophyllene oxide were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). All phenylpropanoids and sesquiterpenes met high purity standards. High contents of eugenol in clove oil and trans-anethole in star anise oil have been reported previously [8 ]. Structural formulas of these selected phenylpropanoids and sesquiterpenes constituents are presented in Figure 1. Star anise oil and selected compounds were dissolved in ethanol and further diluted in medium for cell culture experiments, always resulting in an ethanol concentration <1%, which has no effect on cells and viruses [28 ]. Acyclovir, a commonly used anti-HSV synthetic drug, was purchased from GlaxoSmithKline (Bad Oldesloe, Germany), dissolved in sterile water and applied as reference compound.
Figure 1: Structural formulas of phenylpropanoids and sesquiterpenes.2.2. Cell Culture and HSV-1
RC-37 cells (African green monkey kidney cells) were grown in monolayer culture with Dulbecco's modified Eagle's medium (DMEM; Gibco, Karlsruhe, Germany) supplemented with 5% fetal calf serum (FCS; Gibco, Karlruhe, Germany), 100 U mL −1 penicillin and 100
g mL −1 streptomycin (both Gibco, Karlsruhe, Germany). The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated onto 96-well and 6-well culture plates for cytotoxicity and antiviral assays, respectively, and propagated at 37°C in an atmosphere of 5% CO2. HSV-1 strain KOS was used for all experiments. Viruses were routinely grown on RC-37 cells and virus stock cultures were prepared from supernatants of infected cells and stored at −80°C. Infectivity titers were determined by a standard plaque assay on confluent RC-37 cells [40 ].2.3. Cytotoxicity Assay
The effect of star anise oil, phenylpropanoids and sesquiterpenes on the proliferation of RC-37 cells was determined in 96-well tissue culture plates at an initial density of 1 × 10 5 cells mL −1. For cytotoxicity assays, cells were seeded onto 96-well plates and incubated for 24 h at 37°C. The medium was removed and fresh DMEM containing the appropriate dilution of the essential oil or compounds was added onto subconfluent cells in eight replicates for each concentration of the drugs. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls. After 3 days of incubation, the growth medium was removed and viability of the drug-treated cells was determined in a standard neutral red assay [41 ]. Neutral red dye uptake was determined by measuring the optical density (OD) of the eluted neutral red at 540 nm in a spectrophotometer. The mean OD of the cell-control wells was assigned a value of 100%. The cytotoxic concentration of the drug that reduced viable cell number by 50% (TC50 ) was determined from dose-response curves. Additionally the maximum noncytotoxic concentration of each drug was determined.2.4. Dose-Response Assays
The antiviral activity of star anise oil, phenylpropanoids and sesquiterpenes was assayed by plaque reduction assay. A virus suspension of HSV-1 containing 2 × 10 3 plaque forming units (pfu mL −1 ) was incubated with an equal volume of DMEM or various concentrations of star anise oil or phenylpropanoids and sesquiterpenes for 1 h at room-temperature, then virus was allowed to adsorb to the cells for 1 h at 37°C. The residual inoculum was replaced by medium containing 0.5% methylcellulose. After incubation for 3 days at 37°C, monolayers were fixed with 10% formalin. The cultures were stained with 1% crystal violet and subsequently the plaques were counted. Each concentration was performed in three replicates, virus-infected cells in wells containing medium with 1% ethanol but no drug were also included on each plate as controls. Inhibitory concentration (IC50 ) was expressed as antiviral activity, which inhibited plaque numbers by 50% compared with untreated control and was determined from dose-response curves.2.5. Time of Addition Studies
Star anise oil, phenylpropanoids or sesquiterpenes were added to the cells before, during and after virus infection. The maximum noncytotoxic concentration was always used to evaluate the mode of antiviral action. Cell monolayers were pretreated with drugs prior to inoculation with virus by adding the oil or compounds to the medium followed by incubation for 1 h at 37°C. For pretreatment of HSV with drugs, about 2 × 10 3 pfu of HSV were incubated in medium containing the drugs for 1 h at room-temperature prior to infection of RC-37 cells. The effect of essential oil or components against HSV was also tested during the replication period by addition of drugs after cell infection to the overlay medium, as typically performed in antiviral susceptibility studies. Each assay was run in three replicates. Plaque reduction assays were carried out as described above and number of plaques of drug-treated cells and viruses were compared with untreated controls. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls.2.6. Statistical Analysis
The selectivity index (SI) was determined by the ratio of TC50 to IC50. All experiments were performed in triplicate, and three independent experiments were conducted. Data were presented as mean ± SD and t -test was used to evaluate the difference between the test and control. A P -value of < .05 was considered statistically significant.3. Results 3.1. Cytotoxicity of Essential Oil Compounds
Star anise oil and six selected phenylpropanoids and sesquiterpenes (Figure 1 ) were serially diluted in ethanol and added to cell culture medium to examine the effect on the growth and viability of tissue culture cells, always resulting in an ethanol concentration <1%, which had no effect on cells and viruses. After 3 days of incubation, cell viability of RC-37 cells was determined with the neutral red assay (Table 1 ). The maximum noncytotoxic concentrations of these drugs were determined between 9 g mL −1 for β -caryophyllene oxide and β -eudesmol and 100 g mL −1 for star anise oil, the drug which revealed the lowest cytotoxicity. Similar results were found for TC50 values, for example, 18 g mL −1 for β -caryophyllene oxide and 160 g mL −1 for star anise oil.
Table 1: Selectivity indices of anise oil and selected phenylpropanoids and sesquiterpenes against HSV-13.2. Antiviral Activity of Essential Oil Compounds
The potential antiviral effect of star anise essential oil and some selected components from different essential oils was determined against HSV-1 in vitro. HSV-1 was incubated for 1 h at room-temperature with various concentrations of star anise oil, trans-anethole, eugenol, β -eudesmol, farnesol, β -caryophyllene and β -caryophyllene oxide. In all assays untreated virus-infected cells were used as a control. Subsequently, aliquots of each dilution were incubated with RC-37 cells for 1 h, then the cells were washed and overlaid with drug-free medium and incubated for 3 days at 37°C. The 50% inhibitory concentrations (IC50 ) for HSV-1 were determined in a wide range between 0.25 g mL −1 for β -caryophylle and 35 g mL −1 for eugenol (Table 1 ). The results are presented in Figures 2(a) and 2(b) as virus reduction and represent the average of three independent experiments. In plaque reduction assays, star anise oil and compounds exhibited a concentration-dependent antiviral effect, star anise oil with a SI of 160 was the most effective drug and slightly superior to β -caryophyllene with a SI of 140. SIs for tested drugs against HSV were calculated as the TC50 /IC50 ratio. The essential oil of star anise was able to suppress viral multiplication by >99% (Figure 2(a) ). Out of six tested compounds, only trans-anethole, β -caryophyllene and farnesol suppressed herpesvirus infectivity by >90% at the maximum noncytotoxic concentration of these drugs.
Figure 2: Antiviral activity of serial dilutions of (a) star anise oil, phenylpropanoids and (b) sesquiterpenes against HSV-1 in viral suspension assays. Diluted drugs were tested up to the maximum noncytotoxic concentration. Number of virus plaques was determined 3 days after infection and compared to untreated control. Results are presented as percentage of plaque reduction, experiments were repeated independently and data are the mean of three experiments ± SD.3.3. Mechanism of Antiviral Action
For investigation of the inhibitory effect on HSV in detail, all drugs were added at different stages during viral infection. For comparison, all untreated controls contained the same concentration of ethanol as the drug-treated viruses, in order to exclude any influence of ethanol. When host cells were pretreated with drugs prior to infection, none of the tested drugs showed statistically significant effects (P = 0.2–0.4) on viral infection (Figure 3(a) ). On the other hand, pretreatment of HSV-1 with star anise oil, phenylpropanoids or sesquiterpenes prior to infection inhibited herpesvirus infectivity. At maximum noncytotoxic concentrations of the tested drugs, infectivity was reduced by >99% for star anise oil followed by 98% reduction for β -caryophyllene (Figure 3(b) ). All other constituents of essential oils revealed a plaque reduction of HSV between about 60 and 90%. However, the antiviral effect of eugenol was not statistically significant (P = .07). Acyclovir showed the highest antiviral activity when added during the replication period with inhibition of the viral replication of >99% (data not shown). This drug inhibits specifically the viral DNA polymerase during the replication cycle when new viral DNA is synthesized. In contrast, when the oil or compounds were added to the overlay medium after penetration of the viruses into the host cells, plaque formation was not significantly (P = 0.2–0.4) reduced (Figure 2.6 ).
Figure 3: Antiviral activity of star anise essential oil and different compounds of essential oils against HSV in time of addition assays. (a) Pretreatment of cells with drugs, (b) pretreatment of virus with drugs and (c) addition of drugs during intracellular replication of HSV. Number of virus plaques was determined 3 days after infection and compared to untreated control. Results are presented as percentage of plaque reduction and are the mean of three independent experiments ± SD and statistically significant results are marked with asterisk.4. Discussion
The pharmaceutical industry is increasingly targeting medicinal plants with the aim of identifying lead compounds, focusing particularly on suitable alternative antiviral agents. Topical treatment of herpes labialis infection is standard, for the most part carried out not only with acyclovir creams, but also with phytopharmaceuticals containing sage or lemon balm extracts [42 –44 ]. Both plant extracts were shown to be significantly superior to placebo and equivalent to acyclovir [43 ]. Our previous in-vitro experiments revealed similar results for essential oils from eucalyptus, tea tree and thyme [18. 27. 45. 46 ]. In the present study, the inhibitory effect of star anise oil against HSV infection was compared with the antiviral potential of phenylpropanoid and sesquiterpene compounds. Experiments to assess the cytotoxicity of essential oils and monoterpenes for cultured eukaryotic cells indicate a moderate toxic behavior in cell cultures according to Halle and Göres [47 ]. Star anise essential oil and most compounds exhibited high levels of antiviral activity against HSV-1 in viral suspension tests. At maximum noncytotoxic concentrations plaque formation was significantly reduced by >99% for star anise oil, phenylpropanoids and sesquiterpenes were able to suppress viral infection by 60–80% and 40–98%, respectively.
The mode of antiviral action was determined in time of addition assays. Pretreatment of the cells with these drugs had no effect on the production of infectious virus and plaque formation. The same results were found when star anise oil or compounds were added during the replication period of the infection cycle. However, high antiviral activity was observed for star anise oil, trans-anethole, farnesol and β -caryophyllene when herpesvirus was incubated with these drugs prior to host cell infection (Figure 4 ). These results suggest that these drugs directly inactivate herpes virus and might interfere with virion envelope structures or mask viral structures that are necessary for adsorption or entry into host cells. A virucidal activity of Melaleuca armillaris essential oil has been reported recently [27 ] and dissolution of the HSV envelope by treatment with oregano essential oil has been described [48 ]. Thus different mechanisms of antiviral activity of different essential oils and compounds of essential oils seem to be present. De Logu et al. [12 ] reported an inactivation of herpesviruses and prevention of cell-to-cell spread by Santolina insularis essential oil. However, no antiviral effect was observed during the intracellular replication phase, which is in accordance to our results and other essential oils [46 ]. Adenovirus, a virus without envelope, was not affected by eucalyptus essential oil due to the lack of a viral envelope [21 ]. Sesquiterpenes, for example, triptofordin C-2 and sesquiterpene coumarins inhibit cytomegalovirus [35 ], severe acute respiratory syndrome coronavirus [49 ] and rhinovirus [37 ]. Pusztai et al. [36 ] reported a specific inhibition of the CMV immediate early gene expression, whereas other sesquiterpenes are moderately virucidal against different enveloped viruses, for example, HSV, cytomegalovirus, measles virus and influenza virus [35 ]. Eugenol, a phenylpropane that represents about 75% v/v in clove essential oil, delayed the development of herpesvirus-induced keratitis in the mouse model [32 ] and inactivated HSV directly [39 ]. Direct inactivation of virus particles by eugenol represents the same antiviral mechanism as examined for the constituents in this study. Isoborneol, a monoterpene and a component of several plant essential oils, showed virucidal activity against HSV-1 and specifically inhibited glycosylation of viral proteins [50 ]. The application of cineole protects mice against infection with HSV-2 [22 ]. Since essential oils are able to inhibit acyclovir-resistant HSV-1 isolates [28 ], the mechanism of interaction between these compounds and acyclovir with HSV must be different. Acyclovir inhibits virus replication by interference with the DNA polymerase inside the cell, whereas star anise oil, phenylpropanoids and sesquiterpenes probably inactivate HSV before it enters the cell. Viral resistance to acyclovir represents a particular problem, the prevalence of resistance in acyclovir-treated immunocompromised individuals is ~4–7% [51. 52 ]. Therefore other antiherpetic agents that are effective for viral mutants resistant to current antiviral agents are of great interest for topical treatment. The application of tea tree oil, the essential oil of Melaleuca alternifolia. for the treatment of recurrent herpes labialis has been reported recently [26. 53 ].
Figure 4: Star anise oil, phenylpropanoids and sesquiterpenes exhibited antiviral activity by direct interaction with free virus particles. Pretreatment of cells with the drugs had no effect on viral infectivity, neither during intracellular viral replication.
The complex mixture of the essential oil revealed a higher antiviral activity and SI of 160, whereas single constituents revealed lower selectivity indices. However β -caryophyllene revealed a SI of 140, which is in the same range as the index value of 160 for star anise oil and is found in many different essential oils from different plant families. Thus β -caryophyllene might be one of the dominant antiviral agents in different essential oils. The antiviral potential of the test compounds in our study can be traced back to their structural features. The sesquiterpene hydrocarbon β -caryophyllene is the most active antiviral compound with an IC50 of 0.25 g mL −1. The introduction of either an epoxide or hydroxyl function into the sesquiterpene backbone led to a moderate decrease in its antiviral effect. Cos et al. [54 ] recommended IC50 values for promising natural products against infectious diseases, for example, for extracts <100 g mL −1. Star anise essential oil in our study revealed an IC50 values of 1 g mL −1 and is far below the recommended cutoff and presents a promising antiinfective agent according to this recommendation.
In conclusion, medicinal and aromatic plants are widely used today in modern phytotherapy [55 ]. The essential oils and their components are known to be active against a wide variety of microorganisms [56 ]. Phenylpropanoids and sesquiterpenes present in essential oils contribute to their antiviral activity. Drugs with a high SI are preferable for antiviral treatment in patients, thus star anise oil as a complex mixture and β -caryophyllene as single constituent might be applied as topical therapeutic agents in the treatment of recurrent herpes infection.Funding
Grant from Iranian Ministry of Health (to A. Astani).References