A PROMISING BIOACTIVE COMPONENT TERPINEN-4-OL: A REVIEW

A PROMISING BIOACTIVE COMPONENT TERPINEN-4-OL: A REVIEW

E. Yadav and R. Rao ^*

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar -125001, Haryana, India.

ABSTRACT: Since, the middle ages, essential oils have been widely used for bactericidal, fungicidal, insecticidal, virucidal, cosmetic and medicinal applications, especially nowadays in agriculture, food, sanitary and pharmaceutical industries. Distillation is the common mode of extraction used for aromatic plants, which results in a variety of volatile molecules such as terpenes, terpenoids, aliphatic components, and phenol derived aromatic components. Extensive research has been done in the last decade to screen and evaluate the pharmaceutical potential of the phytochemical constituent, terpinen-4-ol. It is found in various plants species and has gained attention because of its antiviral, antibacterial, anti-inflammatory and antifungal properties. This monoterpene is a bioactive component of tea tree oil (TTO) present in a plant belonging to the genus Melaleuca. In the present review, an overview of the current status and knowledge on the physicochemical properties, analytical techniques, formulations, pharmacological applications, irritation and toxicity potential of terpinen-4-ol have been discussed.

Antibacterial, Monoterpene, Terpinen-4-ol, Anti-inflammatory, Irritation

INTRODUCTION: At present, approximately 3000 essential oils are known, 300 of which are commercially important, especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries ¹. Major plant-derived secondary metabolites, monoterpenes are widely found in vegetables, fruits, and herbs, and play a vital role in the plant's defense mechanism. The monoterpenes consisting of two isoprene units are found abundantly in essential oils ^2-3. Additionally, many monoterpenes have displayed anticancer potential including leukemia, breast, skin, pancreatic and colon cancers in rodents ⁴.

Terpinen-4-ol, one of the primary active ingredients of the TTO, consists of more than 100 different compounds. It is available in a variety of aromatic plants like New Zealand lemonwood tree, oranges, mandarins, origanum and Japanese cedar and, black pepper in leaves, flowers, and herbs⁶. Recently, it has been extracted from the leaves of Melaleuca alternifolia species (belonging to family Myrtaceae).

This monoterpene is also known by several other names like 4-methyl-1-propan-2-ylcyclohex-3-en-1-ol, 4-carvomenthenol; p-menth-1-en-4-ol; 4-terpineol and 1-terpinen-4-ol ⁷. It is used in artificial pepper and geranium oil, and in perfumery for creating herbaceous and lavender notes.

It is a liquid having various properties. It gained attention due to its antibacterial, antiviral, antifungal and anti-inflammatory properties ^{1, 8-10}.

TABLE 1: PLANTS CONTAINING TERPINEN-4-OL AS ONE OF THEIR CONSTITUENT ⁵

Physicochemical Properties: Besides active constituents present in TTO, terpinen-4-ol is its major constituent (ISO 4370 range: 30-48%) which is responsible for various activities of oil. Fig.1. depicts the chemical structure of this constituent ¹¹. It belongs to monoterpenes. It is slightly colorless to pale yellow liquid ¹² with herbal pepper flavoring ¹³, and pine odor ¹⁴. It is miscible with water 3.87 × 10⁺² mg/l at 25 ºC ¹⁵. Its molecular weight is 154.24932 g/mol. It has a boiling point. 209 ºC, density 0.926 g/cu cm at 20 ºC ¹⁶ and vapour pressure: 0.04 mm Hg at 25 ºC ¹⁷. With Log P log Kow = 3.26, it is highly lipophilic in nature ¹⁸.

FIG. 1: CHEMICAL STRUCTURE OF TERPINEN-4-OL

Pharmacological Applications of Terpinen-4-ol:

Anti-inflammatory: Terpinen-4-ol was able to diminish the production of TNF-α, IL-8, IL-1β, IL-10, and prostaglandin E2 by lipopolysaccharide-activated monocytes. Terpinen-4-ol along with α-terpineol is also found to suppress superoxide production by agonist-stimulated monocytes ¹⁹. In contrast, in similar work, it was found that TTO retards the production of reactive oxygen species by both stimulating neutrophils and monocytes, and that it also stimulates the production of reactive oxygen species by nonprimed neutrophils and monocytes ²⁰.

These studies discovered specific TTO mechanisms which may diminish the normal inflammatory response in-vivo. Topically applied TTO has also been shown to modify the edema associated with the efferent phase of contact hypersensitivity in mice ²¹ but not the development of edema in the skin of no sensitized mice or the edematous response to UV-B exposure. This activity was primarily due to terpinen-4-ol and α-terpineol. Recently, it has been shown that terpinen-4-ol, but not 1, 8-cineole or α-terpineol, modify the plasma extravasation and vasodilation associated with histamine-induced inflammation in humans ²².

Anti-microbial: The inhibitory effect of Melaleuca alternifolia (tea tree) essential oil on the development of antibiotic resistant Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated. Frequencies of single-step antibiotic-resistant mutants were measured by inoculating bacteria cultured with or without sub-inhibitory TTO onto agar (containing 2 to 8 times the minimum inhibitory concentration (MIC) of each antibiotic). Relatively minor differences in resistance frequencies were observed in most cases, only the combination of TTO and kanamycin resulted in 10-fold fewer resistant E. coli mutants in comparison to kanamycin alone.

The development of antibiotic resistance in the presence of terpinen-4-ol or TTO was checked by culturing S. aureus, and E. coli isolates daily (with antibiotic alone, antibiotic with TTO) and antibiotic with terpinen-4-ol (for 6 ds). Increase in median MIC for each antibiotic alone was 4 to 16-fold. Subinhibitory terpinen-4-ol or TTO did not greatly alter results, with d 6 median MICs being either the same as or one concentration different from those for antibiotic alone. For terpinen-4-ol and TTO alone, d 6 median MICs had enhanced 4-fold for S. aureus (n = 18) and 2-fold for E. coli (n = 18) from baseline. Lastly, few remarkable changes in antimicrobial susceptibility were observed for S. aureus and S. epidermidis isolates that had been serially subcultured 14 to 22 times with subinhibitory terpinen-4-ol. Overall, the data exhibited that terpinen-4-ol and TTO have little impact on the development of antimicrobial resistance and susceptibility ²³.

Anti-fungal: TTO has been evaluated as a potential antifungal agent against Botrytis cinerea (B. cinerea). In a study, antifungal activity and mode of action of TTO and its components against B. cinerea were checked. Among the components, terpinen- 4-ol showed the highest antifungal activity, followed by TTO, a-terpineol, terpinolene, and 1, 8-cineole. Terpinen-4-ol treatment led to significant alterations in mycelial morphology, membrane permeability, cellular ultrastructure under the scanning electron microscope, transmission electron microscope and fluorescent microscope, and also retarded the ergosterol amount of fungi. The results confirmed that terpinen-4-ol and 1, 8- cineole act mainly on the organelles and cell membranes of B. cinerea, respectively, and when combined possess antifungal activity similar to TTO ²⁴.

Antiviral: A previous study suggested that TTO had a promising antiviral activity against Influenza A in MDCK cells. When tested TTO and some of its components, it was found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and α-terpineol were the main active components. The results obtained by treating the cells with terpinen-4-ol, terpinolene, and α-terpineol before acridine orange staining exhibited that terpinen-4-ol had a potential role in the anti-influenza virus activity as only in the cells treated with this compound were observed to lack the orange fluorescence. These results were validated by measuring the fluorescence intensity by fluorometry, suggesting that TTO and terpinen-4-ol inhibited acridine orange accumulation in cytoplasmic acid vesicles. Further, the data obtained were consistently by the positive control bafilomycin A1. The acidification of lysosomes in MDCK cells recovered completely when the cells were applied with 0.01% (v/v) TTO for 4h, washed and then incubated for 2 h without the compound showing that the cells can re-acidify after treatment with TTO, and that cell morphology is not affected by treatment. Terpinen-4-ol showed similar results ²⁵.

Anti-cancer: The potential anti-tumoral activity of TTO, from Melaleuca alternifolia, was tested in an in-vitro study against human melanoma M14 WT cells and their drug-resistant counterparts, (M14 adriamicin-resistant cells). Both sensitive and resistant cells were cultured in the presence of TTO (at concentrations ranging from 0.005 to 0.03%). Both complex oil (TTO) and its main active component terpinen-4-ol were able to cause caspase-dependent apoptosis of melanoma cells, and this effect was more profound in the resistant variant cell population. Scanning electron microscopy and freeze-fracturing results suggested that the effect of the oil and terpinen-4-ol was due to their interaction with the plasma membrane and subsequent reorganization of membrane lipids. It was concluded that terpinen-4-ol and TTO can inhibit the growth of human M14 melanoma cells. These appear to be more effective on their resistant variants, which show high levels of P-glycoprotein in the plasma membrane, overcoming resistance to caspase-dependent apoptosis exerted by P-glycoprotein-positive tumor cells ²⁶.

Terpinen-4-ol remarkably improves the effect of several biological and chemotherapeutic agents. It significantly inhibits the growth of pancreatic, colorectal, gastric and prostate cancer cells in a dose-dependent manner (10-90% in 0.005-0.1%). The possible mechanism for its activity involves induction of cell-death rendering this compound as a promising anti-cancer drug alone and in combination in the treatment of numerous malignancies. Terpinen-4-ol restores the activity of cetuximab in cancers with mutated KRAS. Sub-toxic concentrations of terpinen-4-ol synergise anti-CD24 mAb (150 μg/ml)-induced growth inhibition (90%). Considerable retardation in tumor volume was observed following terpinen-4-ol (0.2%) treatment alone and with cetuximab (10 mg/kg) (40% and 63%, respectively) in comparison to the control group ²⁷.

Antibiofilm Study: Li-ming Sun, 2012, carried a biofilm evaluation activity for C. albicans (C. albicans) ATCC 11231. The antibiofilm activity of terpinen-4-ol-loaded nanoparticles was evaluated against C. albicans ATCC 11231 cells by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method. These nanoparticles resulted in better efficacy toward C. albicans ATCC 11231 with a longer duration than did terpinen-4-ol only. The antimicrobial property of the samples against the tested strain was checked with the MIC, MBC, and MBEC. The results exhibited that the mean MIC was 5μg/ml; MBC and MBEC were 10μg/ml. The cellular morphological changes can be observed clearly from SEM images where normal C. albicans ATCC 11231 cells grown in YPD medium showed a typical elliptical shape having a smooth surface. The results showed that 10μg/ml terpinen-4-ol effectively eradicated the biofilm. The retardation of C. albicans ATCC 11231 metabolic activities was observed after treatment with terpinen-4-ol-loaded lipid nanoparticles. The metabolic activity was 21.5% of control activity, for biofilm exposed to lipid nanoparticles (containing 10μg/ml terpinen-4-ol), and only 9.7% of control activity for terpinen-4-ol exposure (20 μg/ml). Increasing terpinen-4-ol concentration up to 40μg/ml or 80μg/ml showed no additional effect on the kinetics of biofilm eradication ²⁸.

Analysis of Terpinen-4-ol: Analysis of terpinen-4-ol from different plant species is carried out using different analytical methods. In a study, the leaf volatile constituents of the essential oils of Artemisia indica Wild and Artemisia vestita Wall were studied using a combination of capillary Gas chromatography with flame ionisation detector (GC–FID), gas chromatography-mass spectrometry (GC–MS) and FT-IR (Fourier Transform Infra-Red) spectroscopy. In Artemisia indica leaves, terpinen-4-ol (0.4%) was detected using mass spectroscopy and retention index. The percentage was obtained by FID peak area normalization without using response factors ²⁹. Terpinen-4-ol is the main bioactive in Phlai oil, extracted by steam distillation from the rhizome of Zingiber cassumunar Roxb. commonly used as topical anti-inflammatory herbal medicine. An analytical method for assessment, terpinen-4-ol in cutaneous microdialysis samples by GC-MS was developed by Charlock et al. The obtained calibration curve demonstrated a linear relationship between the peak area ratio of terpinen-4-ol and methyl salicylate (MeS), which was an internal standard, over a range of terpinen-4-ol 0.36-1.79 ppm. The intra- and inter d precisions at all concentrations tested were less than 1.5% and 4.0% R.S.D, respectively. The recoveries of terpinen-4-ol were in the range of 101.22-111.44%. The analyte was stable in working standard solutions after 40 h at room temperature and in standard stock solutions after three ds at -20 ºC without a relevant loss of signal. The limit of detection and quantification were 0.0294 ppm and 0.0883 ppm, respectively.

The analytical method fulfilled the requirement of method validation followed by International Conference on Harmonisation guideline. However, based on this preliminary evaluation, further method testing of this approach in cutaneous microdialysis should be done. Then, it can be applied to the determination of terpinen-4-ol in topical formulations by using microdialysis model for the dermatopharmacokinetic evaluation ³⁰.

GC-FID or GC-MS was used for analysis of TTO by Mondello et al. Gas chromatography equipment used included a Perkin Elmer Auto System having two fused-silica SPB columns (60 m × 0.25 mm i.d.; film thickness 0.25 μm), mounted in parallel in the same oven, with two detectors: FID and Q-Mass 910 (electron ionization 70 eV electron energy, transfer line 220 °C). Carrier gases were oxygen and moisture-free helium obtained from a SUPELCO High Capacity Heated Carrier Gas Purifier (Sigma-Aldrich, Milan), provided with an OMI-2 indicating tube, at an average flow rate of 1 ml/min. The oven temperature programme was kept 60 °C for 4 min, then 2°C/min until 180 °C was reached, then increased 3 °C/min until 250 °C. The detector and the injector temperature was 280 °C. The volume of injected TTO or pure substance was 0.1 μl, and the split ratio was 1:50. Two distinct data systems were connected to the GC-FID or GC-MS: Turbochrom and Q-mass Analytical Workstation Software (PerkinElmer, Milan) with a NIST/EPA/MSDC Mass Spectral database ³¹.

TTO composition was analyzed by this group by comparing the Kovat's Indices, GC retention times ³², and GC/MS spectra with those of the co-injected reference substances. In the absence of reference substances, the structure of the components was tentatively assigned by the Official NIST/EPA/ MSDL Spectral Library. Quantitative data were based on peak area normalization without using a correction factor. The oil was found to be terpinen-4-ol type according to the European Pharmacopoeia ³³ and the International Standard ISO 4730:1996 ³⁴.

GC/MS, chiral GC/MS, and chemometric techniques were used to evaluate a large set (n = 104) of tea tree oils (TTOs) and commercial products containing TTO. Twenty terpenoids were analyzed in each sample and compared with the standards specified by ISO-4730 (2004). Several ISO compliant oil samples when distilled did not meet the ISO standards in this study. This may be primarily because of the presence of excessive p-cymene and depletion of terpinenes. Forty-nine percent of the commercial products did not meet the ISO specifications. Four terpenes, viz., α-pinene, limonene, terpinen-4-ol, and α-terpineol, present in TTOs with the (+)-isomer predominant were measured by chiral GC/MS. The results clearly showed that 28 commercial products contained excessive (+)-isomer or contained the (+)-isomer in concentrations below the norms. Of the 28 outliers, 7 met the ISO standards. There was a substantial subset of commercial products that met ISO standards but displayed unusual enantiomeric ± ratios. Based on the oils that met ISO standards, a class predictive model was constructed. The outliers identified by the class predictive model coincided with the samples that displayed an abnormal chiral ratio. Thus, chiral and chemometric analyses could be used to assure the identification of abnormal commercial products including those that met all of the ISO standards³⁵.

Formulations for Terpinen-4-ol: A limited number of formulations have been reported with terpinen-4-ol in literature. Despite several biological applications, use terpinen-4-ol is limited due to volatile nature, low stability and water-solubility. A solid terpinen-4-ol/β-CD inclusion complex was fabricated using lyophilization by Yang et al. Resulting in inclusion complexes of this molecule were found to possess higher thermal stability and sustained release of terpinen-4-ol at high temperatures. To analyse the release kinetics of terpinen-4-ol from its β-CD inclusion complex, avrami’s equation was used. The results depicted that the release observed was diffusion based (at 40 °C), and a combination of diffusion and the first-order mechanism was found for other temperatures. Release experiments were carried at various temperatures (40, 60, 80, and 100 °C) with 70 % humidity. The release rate of terpinen-4-ol was remarkably enhanced with an increase in temperature.

Also, better antibacterial activity against S. aureus, Pseudomonas aeruginosa (P. aeruginosa) and E. coli, (with concentration ranging from 1.25 mg/ml to 5 mg/ml) was obtained in its inclusion complex. Characterization and evaluation was done using FTIR, XRD, UV-Vis, 1H-NMR, 2D ROESY and TG. In a vacuum, the complexation process of terpinen-4-ol with β-CD was measured by PM3 and ONIOM (B3LYP/6-31G). A 1:1 complex was found to possess an inclusion constant of 252.6.

The TGA results exhibited a significant enhancement in thermal stability of terpinen-4-ol due to the inclusion complexation. The 1H-NMR and 2D ROESY studies ascertained the inclusion and provided information regarding the geometry of terpinen-4-ol inside the β-CD cavity. The thermal stability sustained release and antibacterial activity were enhanced on the inclusion of terpinen-4-ol into β-CD, as affirmed from the quantum chemical calculations and NMR reports. Briefly, terpinen-4-o/β-CD inclusion complex can be promising in the cosmetics, food and pharmaceutical applications. Further, the weak interactions of hydrogen bonds between terpinen-4-ol and β-CD play a significant role in its properties ³⁶.

Terpinen-4-ol shows broad-spectrum antimicrobial activity. However, highly volatile and nonwettability properties have limited its application. Sun et al., 2012, fabricated novel nanocarriers to deliver and protect terpinen-4-ol. The polyethylene glycol (PEG)-stabilized lipid nanoparticles were synthesized and characterized by scanning electron microscope, differential scanning calorimetry, and zetasizer. These nanoparticles after glycine modification had an average diameter of 397 nm and a zeta-potential of 10. The prepared nanoparticles were having a homogeneous particle size, high drug loading, and stability. Liquid chromatography/mass spectrometry showed a sustained release pattern from terpinen-4-ol nanoparticles. Minimum inhibitory concentration and minimum biofilm eradication concentration were checked against C. albicans ATCC 11231. Experiments on isolated mitochondria showed inhibition of enzyme activity and the blockage of biofilm respiration. The effects can be ascertained to the localization of terpinen-4-ol on the mitochondrial membrane ²⁸.

Absorption, Distribution, Metabolism and Excretion Properties: The terpenes cause a modification in the arrangement of lipid in the intercellular region of the stratum corneum (SC) leading to the enhanced skin permeability. This property is commonly used in transdermal drug forms which depend on physicochemical behaviours of terpenes and their concentrations penetrated to the stratum corneum. In a study, four cyclic terpenes, namely terpinen-4-ol, eucalyptol, beta-pinene, and alpha-pinene applied as a neat substance to correlate skin absorption and elimination kinetics with their physicochemical properties. The terpenes were applied in vitro onto the human skin, and after 1-4 h their content in the stratum corneum layers and epidermis/dermis separated by tape-stripping method was determined using GC. Similarly, the amounts of terpenes in the skin were also analyzed during 4 h following 1 h absorption. Terpinen-4-ol showed the fastest and progressive penetration into all skin layers.

Although all studied terpenes are absorbed in the viable epidermis/dermis, penetration into these layers was observed as a time-dependent process, which constantly increased during 4 h. For stratum corneum, the largest accumulation for terpinen-4-ol in epidermis/dermis was observed. Further, it was seen that the elimination of terpenes from the stratum corneum was speedy, especially in the deeper layers, and it was much faster if the initial accumulation was small. Investigated cyclic terpenes have depleted different penetration and elimination characteristics. These were not found to permeate across the skin to the receptor medium due to large accumulation in the skin. Results indicated that the penetration of terpenes into stratum corneum is greater if their log P value is close to 3 ³⁷.

In the following year, Cal et al., evaluated the in-vitro cutaneous penetration of five terpenes--linalool, linalyl acetate, terpinen-4-ol, citronellol, and alpha-pinene--applied in pure essential oils or in dermatological formulations (o/w emulsion, oily solution or hydrogel) containing the essential oils 0.75% w/w. Variable skin absorption was observed depending on the type of the vehicle and log P values of terpenes. Cutaneous accumulation of terpenes was found several times higher if they were applied as pure essential oils than as topical vehicles. Penetration of terpinen-4-ol through the skin was better from an oily solution (approximately 90 mg/cm (2)) than from an emulsion (60 mg/cm (2)). No penetration of linalyl acetate from topical vehicles into viable skin was seen, but the penetration of this terpene to the upper layers of the stratum corneum was twice higher when an oily solution was used. In contrast, the cutaneous absorption of linalool was observed the same from both vehicles (50-60 mg/cm (2)).

The skin penetration of alpha-pinene was not detectable when it was applied in an oily solution. Only a small amount (approximately 5 mg/cm (2)) of this terpene was measured in viable skin after application as a hydrogel. Citronellol applied as hydrogel penetrated into all skin layers (25 mg/cm) (2), while penetration was absent in viable skin layers after application of an oily solution. The only citronellol permeated into the receptor medium ³⁸.

Another study aimed to explore the effect of excipients conventionally used for topical dosage forms, namely isopropyl myristate or oleic acid or polyethylene glycol 400 or Transcutol, on the human skin permeability of terpinen-4-ol (in the pure TTO) was reported. The effect of such excipients was estimated by evaluating the terpinen-4-ol absorption in human epidermis and the changes of the organization of stratum corneum by ATR-FTIR. Among the tested excipients, oleic acid enhanced the absorption of terpinen-4-ol by modifying the stratum corneum lipid barrier. Other excipients showed a weak permeation enhancement ³⁹.

Chooluck et al., investigated dermal pharmacokinetics of terpinen-4-ol in rats following topical administration of plai oil derived from the rhizomes of Zingiber cassumunar Roxb. Unbound terpinen-4-ol amount in dermal tissue was checked by microdialysis. The dermal pharmacokinetic study of terpinen-4-ol was done under non-occlusive conditions. The oil was topically applied at a dose of 2, 4, and 8 mg/cm² plai oil corresponding to the amount of 1.0, 1.9, and 3.8 mg/cm² terpinen-4-ol, respectively. Following topical application of the oil, terpinen-4-ol gets rapidly distributed into the dermis, and showed linear pharmacokinetics with no changes in the area under the concentration-time curves dose-normalized. The mean percentages of free terpinen-4-ol distributed in the dermis per amount of administered were 0.39 ± 0.06%, 0.41 ± 0.08%, and 0.30 ± 0.03% for 2, 4, and 8 mg/cm² doses, respectively. The dermal pharmacokinetics of terpinen-4-ol could prove valuable for its formulation development ⁴⁰.

To study metabolism, (R)-Terpinen-4-ol was mixed in an artificial diet at a concentration of 1 mg/g, and the diet was fed to the last instar larvae of common cutworm (Spodoptera litura). Metabolites were recovered and analyzed spectroscopically. (R)-Terpinen-4-ol was transformed mainly to (R)-p-menth-1-en-4,7-diol. In a similar way, (S)-terpinen-4-ol was transformed mainly to (S)-p-menth-1-en-4,7-diol. It was observed, that the C-7 position (allylic methyl group) of (R)- and (S)-terpinen-4-ol was preferentially oxidized ⁴¹.

Haigou et al., examined the in-vitro metabolism of (+)-terpinen-4-ol using human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was checked by GC-MS. (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R, 2S, 4S)-1, 2-epoxy-p-menthan-4-ol, (+)-(1S, 2R, 4S)-1, 2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were measured through the relative abundance of mass fragments and retention times on GC-MS. Out of 11 recombinant human P450 enzymes checked, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were observed to be major enzymes responsible for the oxidation of (+)-terpinen-4-ol by human liver microsomes. Firstly, among 11 recombinant human P450 enzymes tested, CYP1A2, CYP3A4 and CYP2A6 catalyzed (+)-terpinen-4-ol oxidation. Secondly, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, specific inhibitors for these enzymes. Finally, a good correlation was observed between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes ⁴².

Toxicity: TTO was extracted from the Australian native plant, Melaleuca alternifolia and its potential anti-inflammatory properties were investigated. The ability of TTO to retard the production in-vitro of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF alpha), IL-8, prostaglandin E2 (PGE2) and IL-10 by lipopolysaccharide (LPS)-activated human peripheral blood monocytes was checked. TTO emulsified by sonication in a glass tube into the culture medium (10% fetal calf serum (FCS)) was toxic for monocytes (at a concentration of 0.016% v/v).

However, the TTO water-soluble components (at concentrations equivalent to 0.125%) remarkably suppressed LPS-induced production of IL-1beta, TNF alpha and IL-10 (by approximately 50%) and PGE2 (by approximately 30 %) after 40 h. Gas chromatography/mass spectrometry confirmed a-terpineol (3%), terpinen-4-ol (42%) and 1,8-cineole (2%) as the water soluble components of TTO. From an individual examination of these compounds, only terpinen-4-ol suppressed the production after 40 h of TNF alpha, IL-8, IL-1beta, PGE2 and IL-10 by LPS-activated monocytes. Hence, the water-soluble components of TTO can suppress pro-inflammatory mediator production by activated human monocytes ⁴³.

The antimicrobial activity of Melaleuca alternifolia (tea tree) oil was compared with some of its components, both individually and in two-component combinations. Time-kill assays and MIC revealed that terpinen-4-ol, the major active component of TTO, was more active on its own than when present in TTO. Combinations of terpinen-4-ol and either gamma-terpinene or p-cymene showed similar activities to TTO. Concentration-dependent retardation in terpinen-4-ol activity and solubility was observed in the presence of gamma-terpinene. Non-oxygenated terpenes present in TTO probably lead to the reduction in terpinen-4-ol efficacy by reducing its aqueous solubility. These findings exhibit why TTO can be less active in-vitro than terpinen-4-ol alone and further suggested that the presence of a non-aqueous phase in TTO formulations may reduce the microbial activity of its active components ⁴⁴.

The regulatory properties of the essential oil of tea tree were investigated in-vitro by human peripheral blood leukocytes activated. The ability of TTO to reduce superoxide production by neutrophils and monocytes stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) was explored. The water-soluble fraction of TTO had no remarkable effect on agonist-stimulated superoxide production by neutrophils, but significantly and dose-dependently suppressed agonist-stimulated superoxide production by monocytes. This suppression was not caused by cell death. Chemical analysis assured the water-soluble components as terpinen-4-ol, α-terpineol and 1, 8-cineole. When individually examined, terpinen-4-ol significantly suppressed LPS- and FMLP- but not PMA-stimulated superoxide production; α-terpineol significantly suppressed LPS-, FMLP- and PMA- stimulated superoxide production, 1,8-cineole showed no effect. The results indicated that TTO components suppress the production of superoxide by monocytes, but not neutrophils, suggesting its potential for selective regulation of cell types during inflammation ⁴⁵.

The combined effect of terpinen-4-ol and capric acid against mycelial growth of C. albicans and murine oral candidiasis was investigated in vitro and in-vivo. Mycelial growth of C. albicans was measured by the crystal violet method. Combination of these compounds should be a potent synergistic growth inhibition. Therapeutic efficacy of the combination was also evaluated microbiologically in murine oral candidiasis and results clearly exhibited therapeutic activity. Based on this study, the combination of terpinen-4-ol and capric acid can be used for oral candidiasis therapy ⁴⁶.

Irritation Study: In addition to increasing reports on therapeutic properties of TTO, several toxicity reviews of TTO have been published. TTO produces local adverse reactions, like contact allergy, irritation, and dermatitis in humans. However, it had been suggested in the literature that level of allergy and skin irritation can be controlled by diluting TTO ^47-49.

A study carried in this concern, several diluted concentrations were explored for acute dermal toxicity of TTO. The results demonstrated that skin irritation was remarkably retarded when TTO concentration was less than 2.5%. The TTO components that caused the skin irritation were then further explored. Terpinen-4-ol, the major component in TTO, and 1, 8-cineole, a limited component in TTO, was checked for skin irritation. Results indicated that TTO caused significant skin irritation at 5%, while the maximum percentage of terpinen-4-ol in TTO was 30%. With different concentrations of terpinen-4-ol, no evidence of erythema, edema, or any other skin reactions were seen at 24 h and 48 h.

Hence, the skin irritation of terpinen-4-ol was checked at a concentration of 1.5% and skin irritation was not there. Further, no document describes possible skin toxicity caused by terpinen-4-ol ⁵⁰.

CONCLUSION: The current trend in research is to promote alternative therapies in addition to conventional therapy as many patients believe these show fewer detrimental side effects. Terpinen-4-ol has a good potential to be developed as an alternative therapy for its promising activities. A wealth of in-vitro data supported its antimicrobial, antiviral, anti-biofilm and anti-inflammatory actions. Novel formulations can be fabricated and investigated in future using this molecule to overcome problems like volatility, solubility, and stability. Future, chemists can explore the synthesis of terpinen-4-ol analogs which may provide more effective and therapeutically effective agents in coming future. Still, there is a great need for phytochemical investigations on this bioactive constituent for development of an effective natural remedy responsible for the wide range of applications.

ACKNOWLEDGEMENT: Nil

CONFLICT OF INTEREST: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

REFERENCES:

1. Bakkali F, Averbeck S, Averbeck D and Idaomar M: Biological effects of essential oils – A review. Food Chemical. Toxicology 2008; 46: 446-475.
2. Gershenzon J and Dudareva N: The function of natural terpene products in the natural world. Nature Chemical Biology 2007; 3: 408-414.
3. Wagner KH and Elmadfa I: Biological relevance of terpenoids. Overview focusing on mono-, di- and tetraterpenes. Annals of Nutrition and Metabolism 2003; 47: 95-106.
4. Gould MN: Cancer chemoprevention and therapy by monoterpenes. Environmental Health Perspectives 1997; 4: 977-979.
5. http://www.ars-grin.gov/duke/ accessed on 6/4/2016.
6. Pino JA, Marbot R and Fuentes V: Characterization of Volatiles in Bullock's Heart (Annona reticulata) Fruit Cultivars from Cuba. Journal of Agricultural and Food Chemistry 2003; 51: 3836-3839.
7. https://pubchem.ncbi.nlm.nih.gov/compound/4-Carvomenthenol#section=MeSH-Synonyms accessed on 7/4/2016.
8. Hammer KA, Carson CF and Riley TV: Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy 2004; 53: 1081-5.
9. Burt S: Essential oils: their antibacterial properties and potential applications in foods-a Int. International Journal of Food Microbiology 2004; 94: 223-53.
10. Hammer KA, Carson CF, Riley TV and Nielsen JB: A review of the toxicity of Melaleuca alternifolia (tea tree) oil. Food and Chemical Toxicology 2006; 44: 616-25.
11. Scientific Committee on Consumer Products (SCCP). Opinion on TTO. European Commission 2008.
12. Burdock GA: Fenaroli's Handbook of Flavor Ingredients. Sixth Edition 2010.
13. Lewis RJ: Sax's Dangerous Properties of Industrial Materials. Wiley-Interscience, Wiley & Sons, Inc, Twelfth Edition 2012.
14. http://www.atsdr.cdc.gov/odors/search_results.html accessed on 6/9/2016.
15. http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm accessed on 6/11/2016.
16. Lide DR, Milne GWA. (Editor.): Handbook of Data on Organic Compounds. CRC Press, Inc, Third Edition 1994.
17. http://www.epa.gov/oppt/exposure/pubs/episuitedl.htmaccessed on 6/5/2016.
18. Griffin S, Wyllie SG and Markham J: Determination of octanol-water partition coefficient for terpenoids using reversed-phase high-performance liquid chromatography. Journal of Chromatography A 1999; 864: 221-8.
19. Brand C, Ferrante A, Prager RH, Riley TV, Carson CF, Finlay-Jones JJ and Hart PH: 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. Inflammation Research 2001; 50: 213-219.
20. Caldefie-Chézet F, Guerry M, Chalchat JC, Fusillier C, Vasson MP and Guillot J: Anti-inflammatory effects of Melaleuca alternifolia essential oil on human polymorphonuclear neutrophils and monocytes. Free Radical Research 2004; 38: 805-811.
21. Brand C, Grimbaldeston MA, Gamble JR, Drew J, Finlay-Jones JJ and Hart PH: Tea tree oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response. Inflammation Research 2002; 51: 236-244.
22. Khalil Z, Pearce AL, Satkunanathan N, Storer E, Finlay-Jones JJ and Hart PH: Regulation of wheal and flare by tea tree oil: complementary human and rodent studies. Journal of Investigative Dermatology 2004; 123: 683-690.
23. Hammer KA, Carson CF and Riley TV: Effects of Melaleuca alternifolia (Tea Tree) essential oil and the major monoterpene component terpinen-4-ol on the development of single- and multistep antibiotic resistance and antimicrobial susceptibility. Antimicrobial Agents and Chemotherapy 2012; 56: 909-915.
24. Yu D, Wang J, Shao X, Xu F and Wang H: Antifungal modes of action of TTO and its two characteristic components against Botrytis cinerea. Journal of Applied Microbiology 2015; 119: 1253-1262.
25. Garozzoa A, Timpanaroa R, Stivalaa A, Bisignanob G and Castro A: Activity of Melaleuca alternifolia (tea tree) oil on Influenza virus A/PR/8: Study on the mechanism of action. Antiviral Research 2011; 89: 83-8.
26. Calcabrini A, Stringaro A, Toccacieli L, Meschini S, Marra M, Colone M, Salvatore G, Mondellow F, Arancia G and Molinari A: Terpinen-4-ol, the main component of Melaleuca Alternifolia (Tea Tree) oil inhibits the in-vitro growth of human melanoma cells. Journal of Investigative Dermatology 2004; 122, 349-360.
27. Shapira S, Pleban S, Kazanov D, Tirosh P and Arber N: Terpinen-4-ol: A Novel and Promising Therapeutic Agent for Human Gastrointestinal Cancers. PLoS ONE 2016; 11: 6.
28. Sun LM, Zhang CL and Li P: Characterization, antibiofilm, and mechanism of action of novel PEG-stabilized lipid nanoparticles loaded with terpinen-4-ol. Arabian Journal of Chemistry 2012; 60: 6150-6156.
29. Rathera MA, Darb BA, Shah WA, Prabhakar A, Bindu K, Banday JA and Qurishi MA: Comprehensive GC-FID, GC-MS and FT-IR spectroscopic analysis of the volatile aroma constituents of Artemisia indica and Artemisia vestita essential oils. Arabian Journal of Chemistry 2014.
30. Chooluck K, Sripha K, Derendorf H and Sathirakul K: Preliminary evaluation of GC-MS for the determination of Terpinen-4-ol in cutaneous microdialysis samples. Drug Metabolism Reviews 2009.
31. Mondello F, Bernardis FD, Girolamo A, Cassone A and Salvatore G: In-vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species. BMC Infectious Diseases 2006; 6: 158.
32. Adams RP: Identification of essential oil components by gas chromatography/mass spectroscopy. Allured Publishing Corporation, 1995.
33. European Directorate for the Quality of Medicines: The 5^th Edition of European Pharmacopoeia and its subsequent supplements 2006.
34. International Organization for Standardization. ISO 4730: Oil of Melaleuca, Terpinen-4-ol type (TTO) Geneva: International Organization for Standardization 1996.
35. Wang M, Zhao J, Avula B, Wang YH, Chittiboyina AG, Parcher JF and Khan IA: Quality evaluation of terpinen-4-ol-type Australian TTOs and commercial products: an integrated approach using conventional and chiral GC/MS combined with chemometrics. Journal of Agricultural and Food Chemistry 2015; 63: 2674-2682.
36. Yang Z, Xiao Z and Ji H: Solid inclusion complex of terpinen-4-ol/ β-cyclodextrin: kinetic release, mechanism and its antibacterial activity. Flavour and Fragrance Journal 2015; 30: 179-187.
37. Cal K, Kupiec K and Sznitowska M: Effect of physicochemical properties of cyclic terpenes on their ex vivo skin absorption and elimination kinetics. Journal of Dermatological Science 2005; 41(2): 137-142.
38. Cal K: Skin penetration of terpenes from essential oils and topical Planta Medica 2006; 72(4): 311-316.
39. Casiraghi A, Minghetti P, Cilurzo F, Selmin F, Gambaro V and Montanari L: The effects of excipients for topical preparations on the human skin permeability of terpinen-4-ol contained in Tea tree oil: infrared spectroscopic investigations. Pharmaceutical Development and Technology 2010; 15(5): 545-552.
40. Chooluck K, Singh RP, Sathirakul K and Derendorf H: Dermal Pharmacokinetics of Terpinen-4-ol Following Topical Administration of Zingiber cassumunar (plai) Oil. Planta Medica 2012; 78(16): 1761-1766.
41. Miyazawa M and Kumagae S: Biotransformation of (R) - and (S)-terpinen-4-ol by the larvae of common cutworm (Spodoptera litura). Journal of Agricultural and Food Chemistry 2001; 49(9): 4312-4314.
42. Haigou R and Miyazawa M: Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes. Journal of Oleo Science 2012; 61(1): 35-43.
43. Hart PH, Brand C, Carson CF, Riley TV, Prager RH and Finlay-Jones JJ: Terpinen-4-ol, the main component of the essential oil of Melaleuca alternifolia (tea tree oil), suppresses inflammatory mediator production by activated human monocytes. Inflammation Research 2000; 49(11): 619-626.
44. Cox SD, Mann CM and Markham JL: Interactions between components of the essential oil of Melaleuca alternifolia. Journal of Applied Microbiology 2001; 91(3): 492-497.
45. Brand C, Ferrante A, Prager RH, Riley TV, Carson CF, Finlay-Jones JJ and Hart PH: 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. Inflammation Research 2001; 50(4): 213-219.
46. Ninomiya K, Hayama K, Ishijima S, Takahashi M, Kurihara J and Abe S: Terpinene-4-ol and medium-chain by the combined use of a fatty acid capric acid Candida albicans therapeutic effect of the mycelial form of inhibitory action and mouse oral Yakugaku Zasshi 2013; 133(1): 133-140.
47. Rutherford T, Nixon R, Tam M and Tate B: Allergy to tea tree oil: retrospective review of 41 cases with positive patch tests over 4.5 years. The Australasian Journal of Dermatology 2007; 48: 83-87.
48. Hammer KA, Carson CF, Riley TV and Nielsenc JB: A review of the toxicity of Melaleuca alternifolia (tea tree) oil. Food and Chemical Toxicology 2006; 44: 616-625.
49. Stonehouse A and Studdiford J: Allergic contact dermatitis from TTO. Consultant 2007; 47:781.
50. Lee CJ, Chen LW, Chen LG, Ting-Lin C, Huang CW, Huang MC and Wang CC: Correlations of the components of tea tree oil with its antibacterial effects and skin irritation. Journal of Food and Drug Analysis 2013; 21: 169-176.
    

    ---

    How to cite this article:

    Yadav E and Rao R: A promising bioactive component terpinen-4-ol: a review. Int J Pharmacognosy 2016; 3(8): 336-45. doi: 10.13040/IJPSR.0975-8232.3(8).336-45.

    ---

    This article can be downloaded to ANDROID OS based mobile. Scan QR Code using Code/Bar Scanner from your mobile. (Scanners are available on Google Playstore)

    ---