Antibacterial properties and major bioactive components of Mentha piperita essential oils against bacterial fruit blotch of watermelon

Plant-derived essential oil is an alternative to antibiotics, eliminating the concern of developing antibiotic-resistant bacterial strains. In this study, using the half-divided Petri plate assays, 32 volatile essential oils were screened for their antibacterial activity against Acidovorax citrulli (Acc). Sweet basil and peppermint oils were the most effective against Acc, with subsequent trials showing that peppermint oil to be the most active. Using gas chromatography–mass spectrometry, the major compositions of peppermint oil were analysed. Among the various compositions of peppermint oil, menthol, neomenthol, isopulegone and 1,8-cineole were significantly active against Acc and each component at 0.2% concentration inhibited all bacterial growth. This study demonstrated in vitro and in vivo antibacterial activities of peppermint oil and its active components against Acc. These results suggest the use of peppermint oil as a potential antibacterial agent to treat seed with Acc.     

Variations in composition of peppermint oil in relation to production areas

Compositional data obtained for peppermint oil fromMentha piperita L. produced in Tasmania as well as oils produced in other major production areas, were analysed by principal coordinate analysis. The 6 variates included in the analysis were the oil compounds limonene, cineole, menthone, menthofuran, menthyl acetate and menthol. In general, Tasmanian oils were characterised by low menthofuran, low limonene, low menthyl acetate and to a lesser extent high menthone, low cineole and high menthol concentrations relative to most major production areas. When the variation in composition of oil samples from within Tasmania was displayed in 3 dimensions (using the first 3 principal coordinates) it was not possible to achieve any pronounced separation of oils produced at different locations within southern Tasmania. Principal coordinates which were based mainly on cineole and menthol concentrations, respectively, did allow a degree of separation between oils produced in southern and northern Tasmania. Generally, oils produced in northern Tasmania had lower cineole and, in some cases, higher menthol than southern Tasmanian oils. Oil extracted from regrowth herb after commercial harvest was distinguished by having very high menthyl acetate, low menthone, high menthol, high menthofuran, low cineole and low limonene concentrations. The effect of some cultural and environmental factors on oil composition is also discussed.     

Antifungal activities of essential oils applied by dip-treatment on areca palm (Areca catechu) leaf sheath and persistence of their potency upon storage

The antifungal activities of cinnamon oil, clove oil, anise oil, and peppermint oil, and their main components (cinnamaldehyde, eugenol, trans-anethole, and menthol, respectively) against molds identified from areca palm leaf sheath (Mucor dimorphosporus, Penicillium sp., Aspergillus niger, and Rhizopus sp.) were investigated. An agar dilution method was employed to determine the minimum inhibitory concentration (MIC) of essential oils and their main components. Zone inhibition tests and the inhibitory effect of the leaf sheath dip-treated with essential oils against those molds were examined. Major components of essential oils on the leaf sheath during storage were quantified by gas chromatography analysis. The MIC values of essential oils on agar and on the leaf sheath were identical. With an MIC of 50 ??g ml⁻¹, cinnamon oil had the strongest inhibitory effect. At their MICs the oils were capable of providing protection against mold growth on the leaf sheath for at least 12 weeks during storage at 25 °C and 100% RH. Scanning electron microscope examination showed that essential oils prevented spore germination. Except for menthol in peppermint oil, the main components of the essential oils, which were fairly stable over the storage period, largely contributed to the antifungal activity.     

Influence of ethephon and daminozide on growth and essential oil content of peppermint and sage

Foliar application of Daminozide at 1000 ppm reduces the growth of Salvia officinalis (sage) and decreases essential oil yield, but increases both growth and essential oil yield of Mentha piperita at the same concentration. Ethephon at 250 ppm reduces the growth and essential oil yield of peppermint and slightly increases growth and essential oil content of sage. Both growth regulators markedly reduce the level of menthone and menthol in peppermint oil and increase the level of isomenthone and neoisomenthol. Both growth regulators decrease the level of camphor and increase the level of β-pinene in sage oil. Changes in essential oil composition induced by these growth regulators are most readily explained by alterations in the levels or activities of the relevant biosynthetic enzymes.     

Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita)

(-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-)-menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone. Acid hydrolysis of the nonvolatile material released over half of the radioactivity to the steamvolatile fraction, and the major component of this fraction was identified as (+)-neomenthol by radiochromatographic analysis and by synthesis of crystalline derivatives, thus suggesting the presence of a neomenthyl glycoside. Thin layer chromatography, ion exchange chromatography, and gel permeation chromatography on Bio-Gel P-2 allowed the purification of the putative neomenthyl glycoside, and these results suggested that the glycoside contained a single, neutral sugar residue. Hydrolysis of the purified glycoside, followed by reduction of the resulting sugar moiety with NaB(3)H(4), generated a single labeled product that was subsequently identified as glucitol by radio gas-liquid chromatography of both the hexatrimethylsilyl ether and hexaacetate derivative, and by crystallization to constant specific radioactivity of both the alditol and the corresponding hexabenzoate. These results, along with studies on the hydrolysis of the glycoside by specific glycosidases, strongly suggest that (+)-neomenthyl-beta-d-glucoside is a major metabolite of (-)-menthone in flowering peppermint. This is the first report on the occurrence of a neomenthyl glycoside, and the first evidence implicating glycosylation as an early step in monoterpene catabolism.     

Feasibility study of repeated harvesting of menthol from biologically viable Mentha x piperata using ultrasonic extraction

To potentially replace the conventional destructive extraction process, we have shown the feasibility of devising a novel technique that uses ultrasound to nonlethally and repeatedly extract menthol from biologically viable peppermint plants (Mentha x piperita). Our results show that plants ultrasonicated for 1 h at 22 degrees C in a standard 40 kHz ultrasonic bath could release approximately 17.8 microg of menthol per gram of leaf tissue (2% of total product). The amount of menthol release increases with the time of treatment and is greatly affected by the temperature of the ultrasonic bath water. An increase from 2% to 12% of total product was observed when the temperature was increased from 22 degrees C to 39 degrees C. When the temperature effects were isolated, the mechanism of the product release was found to be that of cavitation. The treated plants remained viable and were ready for the subsequent ultrasound extraction after approximately 4 days of recuperation. However, the amount of product released is reduced in subsequent extractions. Scanning electron micrographs indicate that there are two mechanisms involved in extraction: (1) the diffusion of product through the cuticle of peppermint glandular trichomes and (2) the exudation of the product from broken and damaged trichomes. This study has shown the possibility of using an on-line ultrasonic, nondestructive extraction method to continuously release intracellular plant metabolites from the plants while maintaining the plant's viability.     

Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells

Menthol, a naturally occurring compound from peppermint oil, binds and activates the TRPM8 Ca(2+)-permeable channel that exhibits abnormal expression patterns in prostate cancer, suggesting that TRPM8 links Ca(2+) transport pathways to tumor biology. We thus investigated the cellular responses of prostate cancer cells to menthol. Here we found that menthol increases [Ca(2+)](i) via Ca(2+) influx mechanism(s) independent of TRPM8 in PC-3 cells. We demonstrated that menthol induces cell death at supramillimolar concentrations in PC-3 cells and the cell death is not suppressed by low extracellular Ca(2+) condition which indicates that menthol-induced cell death is not associated with Ca(2+) influx pathways. In addition, we showed that menthol increases a phosphorylated form of c-jun N-terminal kinase (JNK) in PC-3 cells through TRPM8-independent mechanisms. Thus, our data indicate that there is an apparent lack of causality between TRPM8 activation and menthol-induced cell death and that menthol can regulate TRPM8-independent Ca(2+)-transport and cellular processes.     

Plant growth regulators ameliorate the ill effect of salt stress through improved growth,photosynthesis, antioxidant system,yield and quality attributes in <Emphasis Type="Italic">Mentha piperita</Emphasis> L.

The roles of plant growth regulators (PGRs) in plants are well documented. However, there is a little information regarding their roles in alleviating salt stress in plants, particularly peppermint. This necessitated the evaluation of the efficiency of three selected PGRs in counteracting the ill effect of salt stress by conducting a pot experiment on peppermint (Lamiaceae). Three uniform size suckers were transplanted in each pot containing proper nutrients. Thirty day old plants were subjected to 4 levels of salinity, viz. 0, 50, 100 or 150 mM NaCl. Salt stress was given at 30 days after their transplantation (DAT). Plants were sprayed twice, i.e., at 60 and 75 DAT with 10^?6 M each of gibberellic acid (GA₃), salicylic acid (SA) or triacontanol (Tria). The sampling was made at 100 DAT and harvesting at 120 DAT. The graded levels of salinity decreased growth, photosynthesis, carbonic anhydrase (CA) activity, NPK content, peltate glandular trichome (PGT) density, essential oil (EO) and menthol content and herb, EO and menthol yield, but increased catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) activities and proline content linearly. Spray of PGRs particularly SA improved all parameters under both salt and salt free conditions. The maximum values for yields of herb, EO and menthol were noted with 0 mM NaCl?×?SA. However, antioxidants, proline content, PGT density and EO content were found to be maximum with 150 mM NaCl?×?SA.     

Antimicrobial activity of a traditionally used complex essential oil distillate (Olbas(?) Tropfen) in comparison to its individual essential oil ingredients

Plant extracts and essential oils have been widely studied and used as antimicrobial agents in the last decades. In our study we investigated the antimicrobial activities of Olbas(?) Tropfen (in the following named Olbas), a traditionally used complex essential oil distillate, in comparison to its individual essential oil ingredients. Olbas (10g) consists of three major components such as peppermint oil (5.3g), eucalyptus oil (2.1g), and cajuput oil (2.1g) and of two minor constituents like juniper berry oil (0.3g) and wintergreen oil (0.2g). The composition of Olbas and the five individual essential oils were characterized by GLC-MS. According to GLC-MS analysis 1,8-cineol is the main component of the complex essential oil distillate followed by menthol and menthone. The minimum inhibitory and minimum microbicidal concentrations of Olbas and each of the single essential oils were evaluated in 17 species/strains of bacteria and fungi. Time-kill assay was performed to compare the microbicidal activity of Olbas and peppermint oil during several time intervals. Olbas displayed a high antimicrobial activity against all test strains used in this study, among them antibiotic resistant MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococcus). Its antimicrobial activity was comparable to that of peppermint oil which was the most potent one of all individual essential oils tested. In the time kill assay Olbas as well as peppermint oil demonstrated similar microbicidal activities. Based on its wide antimicrobial properties Olbas can be a useful agent for the treatment of uncomplicated infections of skin and respiratory tract.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Metabolism of Monoterpenes : EVIDENCE FOR COMPARTMENTATION OF l-MENTHONE METABOLISM IN PEPPERMINT (MENTHA PIPERITA) LEAVES

Previous studies have shown that the monoterpene ketone l-[G-(3)H]-menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaf discs of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-beta-d-glucoside (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). The metabolic disposition of the epimeric reduction products of the ketone, which is a major constituent of peppermint oil, is highly specific, in that little neomenthyl acetate and little menthyl glucoside are formed. However, when l-[3-(3)H]menthol and d-[3-(3)H]neomenthol are separately administered to leaf discs, both menthyl and neomenthyl acetates and menthyl and neomenthyl glucosides are formed with nearly equal facility, suggesting that the metabolic specificity observed with the ketone precursor was not a function of the specificity of the transglucosylase or transacetylase but rather a result of compartmentation of each stereospecific dehydrogenase with the appropriate transferase. A UDP-glucose:monoterpenol glucosyltransferse, which utilized d-neomenthol or l-menthol as glucose acceptor, was demonstrated in the 105,000g supernatant of a peppermint leaf homogenate, and the enzyme was partially purified and characterized. Co-purification of the acceptor-mediated activities, and differential activation and inhibition studies, provided strong evidence that the same UDP-glucose-dependent enzyme could transfer glucose to either l-menthol or d-neomenthol. Determination of K(m) and V for the epimeric monoterpenols provided nearly identical values. The acetylcoenzyme A:monoterpenol acetyltransferase previously isolated from peppermint extracts (Croteau, Hooper 1978 Plant Physiol 61: 737-742) was re-examined using l-[3-(3)H]menthol and d-[3-(3)H]neomenthol as acetyl acceptors, and the K(m) and V for both epimers were, again, very similar. These results demonstrate that the specific in vivo conversion of l-menthone to l-menthyl acetate and d-neomenthyl-beta-d-glucoside cannot be attributed to the selectivity of the transferases, and they clearly indicate that the metabolic specificity observed is a result of compartmentation effects.     

Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.     

Durability of rubberwood (Hevea brasiliensis) treated with peppermint oil,eucalyptus oil,and their main components

Anti-fungal activities of two essential oils (peppermint oil and eucalyptus oil) and their main components (menthol and eucalyptol, respectively) against molds (Aspergillus niger, Penicillium chrysogenum, and Penicillium sp.) and a white-rot decay fungus (Trametes versicolor) identified from rubberwood surfaces were investigated. The broth dilution method and the agar diffusion technique were employed to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) using the concentration of substances between 100 and 800 μl ml^?1. Inhibitory effects of essential oils and their main components at the MICs against mold growth, fungal decay, and termite attack on rubberwood were further examined by means of the dip treatment method. It was found that MFC values against molds for all treatments examined were about 50–100 μl ml^?1 higher than MIC values. Peppermint oil and menthol exhibited high fungistatic and fungicidal activities, with MICs of 300 μl ml^?1 and 350 μl ml^?1, respectively, against the test molds and the decay fungus. Eucalyptus oil and eucalyptol were also effective against these microbes but at higher concentrations of 600 μl ml^?1 and 500 μl ml^?1, respectively. Only peppermint oil at the MIC was capable of providing a complete protection from mold growth on rubberwood for up to 12 weeks at storage conditions of 25 °C and 100% RH. Both peppermint oil and eucalyptus oil at the MICs showed moderate resistance to fungal decay and high resistance to termite attack.     

Induction of the mRNA for CYP6B2, a pyrethroid inducible cytochrome p450, in Helicoverpa armigera (Hubner) by dietary monoterpenes

A cDNA clone encoding a cytochrome P450 from H. armigera was used to examine the expression of two homologous cytochrome P450 mRNAs, one of which, CYP6B2, is probably involved in pyrethroid metabolism. The mRNA for CYP6B2 in particular can be induced up to tenfold by peppermint oil and the monoterpene α-pinene as well as to a smaller extent by menthol, butylated hydroxyanisole, and piperonyl butoxide. Both mRNAs are present in the major larval tissues, midgut, fat body, and cuticle, although only the mRNAs in the midgut and fat body are inducible by peppermint oil. The induction is a rapid process occurring within a period of 4 h with a similarly rapid rate of decrease in the absence of the inducing compounds. During normal development both mRNAs are absent from eggs and increase during the larval stage, reaching a maximum during mid fifth instar. Both mRNAs then decrease to undetectable levels during pupation and remain undetectable in the adult stage. © 1997 Wiley-Liss, Inc.     

Response of exogenous salicylic acid on cadmium induced photosynthetic damage,antioxidant metabolism and essential oil production in peppermint

The present inquest was undertaken in view of the alarming increase in the concentration of cadmium, nickel etc., in the arable soils of Uttar Pradesh, India and the little attention paid to the effect of these heavy metals on the performance of pharmaceutically imperative essential oil (EO) producing crops like peppermint (Mentha piperita L.). We devised a pot experiment to study the influence of exogenously sourced salicylic acid (SA) (10^?4 M) in the amelioration of growth, protection of photosynthesis and essential oil production against 30, 60 and 120 mg kg^?1 soil of cadmium (Cd)-accrued stress in peppermint. Plants grown with Cd showed remarkably deleterious effects on growth, photosynthesis, carbon and nitrate assimilating enzymes and yield and active constituents of EO in addition to the marked elevation in the oxidative stress. SA successfully alleviated the Cd induced toxicity in peppermint, improved photosynthesis by enhancing activity of RuBisCo and carbonic anhydrase and minimized the oxidative stress by mitigating the production of free radicals by the maintenance of free radical scavenging enzymes and reduced glutathione (GSH) pool. Furthermore, the decrease in the concentration of EO and menthol due to Cd stress was successfully alleviated by SA application which was evident from the gas chromatograms of EO of Cd stressed SA treated plants.     

Demonstration of the Intercellular Compartmentation of l-Menthone Metabolism in Peppermint (Mentha piperita) Leaves

The metabolism of l-menthone, which is synthesized in the epidermal oil glands of peppermint (Mentha piperita L. cv. Black Mitcham) leaves, is compartmented; on leaf maturity, this ketone is converted to l-menthol and l-menthyl acetate in one compartment, and to d-neomenthol and d-neomenthyl glucoside in a separate compartment. All of the enzymes involved in these reactions are soluble when prepared from whole-leaf homogenates. Mechanical separation of epidermal fragments from the mesophyll, followed by preparation of the soluble enzyme fraction from each tissue, revealed that the neomenthol dehydrogenase and the glucosyl transferase resided specifically in the mesophyll layer, whereas the menthol dehydrogenase and substantial amounts of the acetyl transferase were located in the epidermis, presumably within the epidermal oil glands. These results suggest that the compartmentation of menthone metabolism in peppermint leaves is intercellular, not intracellular.     

Metabolism of Monoterpenes: Conversion of l-Menthone to l-Menthol and d-Neomenthol by Stereospecific Dehydrogenases from Peppermint (Mentha piperita) Leaves

The monoterpene ketone l-menthone is specifically converted to l-menthol and l-menthyl acetate and to d-neomenthol and d-neomenthyl-beta-d-glucoside in mature peppermint (Mentha piperita L. cv. Black Mitcham) leaves. The selectivity of product formation results from compartmentation of the menthol dehydrogenase with the acetyl transferase and that of the neomenthol dehydrogenase with the glucosyl transferase. Soluble enzyme preparations, but not particulate preparations, from mature peppermint leaves catalyzed the NADPH-dependent reduction of l-menthone to both epimeric alcohols, and the two dehydrogenases responsible for these stereospecific transformations were resolved by affinity chromatography on Mātrex Gel Red A. Both enzymes have a molecular weight of approximately 35,000, possess a K(m) for NADPH of about 2 x 10(-5)m, are very sensitive to inhibition by thiol-directed reagents, and are not readily reversible. The menthol dehydrogenase showed a pH optimum at 7.5, exhibited a K(m) for l-menthone of about 2.5 x 10(-4)m, and also reduced d-isomenthone to d-neoisomenthol. The neomenthol dehydrogenase showed a pH optimum at 7.6, exhibited a K(m) for l-menthone of about 2.2 x 10(-5)m, and also reduced d-isomenthone to d-isomenthol. These stereochemically distinct, but otherwise similar, enzymes are of key importance in determining the metabolic fate of menthone in peppermint, and they are probably typical of the class of dehydrogenases thought to be responsible for the metabolism of monoterpene ketones during plant development.     

Influence of phosfon D and cycocel on growth and essential oil content of sage and peppermint

Foliar application of Phosfon D at 50–100 ppm stimulates the growth of Salvia officinalis (sage) and moderately retards the growth of Mentha piperita (peppermint), while increasing the essential oil yield of both species by 50–70 % Phosfon D increases the proportions of (−)-3-isothujone and (+)-3-thujone in sage oil and decreases the level of (−)-β-pinene and (+)-camphor, whereas this growth retardant increases the proportions of (+)-isomenthone and (+)-neoisomenthol in peppermint oil and decreases the level of(−)-menthone and (−)-menthoL Foliar application of Cycocel at 250–500 ppm slightly stimulates growth and essential oil formation in peppermint, and retards growth of sage with little effect on oil yield. The influence of Cycocel on sage oil composition was the opposite of that of Phosfon, with a tendency to increase the level of (−)-β-pinene and decrease the level of (−)-3-isothujone under severe stunting. The effect of Cycocel on the composition of peppermint varied with concentration. The influence of growth retardants on essential oil composition and yield are most readily explained by alterations in the levels or activities of the relevant enzymes.     

Biosynthesis of mono- and sesquiterpenes in peppermint from mevalonate-2-C

The volatile oil from peppermint (Mentha piperita L.) is composed primarily of monoterpenes with less than 2% sesquiterpenes. However, radioactivity from mevalonate-2-¹⁴C is incorporated into caryophyllene and other sesquiterpene hydrocarbons much more extensively than into monoterpenes by peppermint cuttings. Both mono- and sesquiterpenes show maximum incorporation of label after 6 hr (0·03% vs. 0·33% of the physiological isomer) and lose 75% of the incorporated label after an additional 6 hr. Caryophyllene derived from mevalonate-2-¹⁴C after 6 hr of incorporation was chemically degraded. The isoprenoid origin of caryophyllene was confirmed, and preferential labelling of the isopentenyl pyrophosphate derived portions of the molecule was noted. On the basis of such evidence it appears that separate sites may exist for the biosynthesis of mono- and sesquiterpenes and that an endogenous dimethylallyl pyrophosphate pool may participate in the biosynthesis of sesquiterpenes in peppermint.