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Caryophyllene: the Cinderella of essential oils

December 7, 2016 - 08:04 -- Pierre Lutgen

Preliminary note: In this document we will use indiscriminately the terms caryophyllene, alpha-caryophyllene, beta-caryophyllene, trans-caryopyllene, caryophyllene oxide, E-BCP 

Some recent informations on the use of catnip Nepeta cataria as mosquito repellent in Burundi alerted us to the major role the sesquiterpene caryophyllene plays in this plant. The study conducted by the University of Burundi, the Ethics Advisory Agency for International Cooperation (ACECI) in collaboration with Montreal’s Ecole Polytechnique together with the Government of Burundi showed that catnip oil reduced the number of bites from mosquitoes by 91.7%. The trial involved 60 volunteers. This had already been documented in Kenya in a well documented paper.

            Michael A. Birkett, Ahmed Hassanali, Solveig Hoglund , Jan Pettersson , John A. Pickett. Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites. Phytochemistry, 2011, 72, 109-114

that caryophyllene enhanced the repellent activity of the nepetalactones of Nepeta cataria and that this mixture was equivalent to the synthetic repellent DEET. Looking back at the extensive analysis Rosine Chougouo had made on some 15 different Artemisia annua plants in 2010 at the Laboratoire National de la Santé in Luxembourg,

             R Chougouo Kengne. Mise au point et validation des procedures analytiques pour la détermination de certains composés de la plante Artemisia anua de diverses origines. Rapport de stage. Université des Montagnes, Cameroun, 2011

we were surprised to find that in many cases caryophyllene is the second most important essential oil after camphor, but far ahead of limonene, eucalyptol, copaène, alpha-pinene. These results are similar to obtained of Artemisia annua grown in Madeira.

             Castilho PC; Gouveia, S; Artemisia annua L.: Essential oil and acetone extract composition and antioxidant capacity. Industrial Crops and Products, 2013, 45, 170- 181; DOI: /10.1016/j.indcrop.2012.12.022.

The analysis of Artemisia annua growing wild in Bulgaria shows that its essential oil contains 24.7% of caryophyllene.

              R. Tzenkova, Z. Kamenarska, A. Draganov & A. Atanassov (2010). Composition of Artemisia Annua Essential Oil Obtained from Species Growing Wild in Bulgaria, Biotechnology & Biotechnological Equipment, 24:2, 1833-1835, DOI: 10.2478/V10133-010-0030-6

A study from China shows that high concentrations of caryophyllene oxide are present in stems of their Artemisia annua and none in flowers and leaves.

              Yan LI , Hao-bin HU, Xu-dong ZHENG, Ji-hua ZHU and Li-ping LIUComposition and antimicrobial activity of essential oil from the aerial part of Artemisia annua. Journal of Medicinal Plants Research Vol. 5(16), pp. 3629-3633, 18 August, 2011

This is confirmed by a study from Iran showing that the concentration in sesquiterpenes is higher in stems. Another study from India

               Divya Goel, Richa Goel, Vijender Singh, Mohammed Ali Gopal, Rao Mallavarapu, Sushil Kumar. Composition of the essential oil from the root of Artemisia annua Journal of Natural MedicinesOctober 2007, 61:458 187-191

finds 6.05 % of caryophyllene in stems and only 2.05% in flowers and leaves. And no camphor in the stems, like in the Chinese paper. At Luxembourg we had analyzed stems from only one sample of Artemisia annua and found only two essential oils caryophyllene and camphor (not quantified). In a sample analyzed in Bangladesh caryophyllene and caryophyllene oxide were much higher during full-flowering and post-flowering.

                Zhan-nan Yang, Shi-qiong Zhu1 and Zheng-wen Yu Comparison of terpene components from flowers of Artemisia annua. J of the Bangladesh Pharm Soc 2012, 7, 114-119

The sesquiterpene caryophyllene is found in large amounts in the essential oils of many different spice and food plants , such as oregano, thyme, rosemary, cinnamon, curry and black peper. It is commercially used as food additive and in cosmetics. It is a dietary phytocannabinoid approved by US-FDA. Other medicinal plants like Melissa officinalis are also rich in caryophyllene. Curry leaves contain up to 35%. The biblical balm Commiphoara gileadensis contains 15% and is known in the Middle East for its health benefits.  The antifungal plant Orthosiphon stamineus contains 24.0 % caryophyllene in leaves and 35.1 % in stems. Cloves Syzygium aromaticum is widely used in dental care, as an antiseptic and analgesic. Its essential oil contains eugenol and caryophyllene. Peper often is very rich in caryophyllene. It contributes to the spicy smell and taste of peper and is in fact the molecule detected by dogs trained for drug detection. A Brazilian team confirmed the anti-inflammatory, antinociceptive and gastric ulcer activities of Piper aleyreanum.

                 Lima DK, Ballico LJ, Rocha Lapa F, Gonçalves HP, de Souza LM, Iacomini M, Werner MF, Baggio CH, Pereira IT, da Silva LM, Facundo VA, Santos AR. Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C.DC in rodents. Journal of Ethnopharmacology 2012;142(1): 274–282.

It improves renal functions, lowers the nephrotoxictiy of molecules like cisplatin and reduces creatinine.

                 Horváth B, Mukhopadhyay P, Kechrid M, Patel V, Tanchian G, Wink DA, Gertsch J, Pacher P. β-Caryophyllene ameliorates cisplatin-induced nephrotoxicity in a cannabinoid 2 receptor-dependent manner. Free Radic Biol Med. 2012 Apr 15;52(8):1325-33. doi: 10.1016/j.freeradbiomed.2012.01.014..

Caryophyllene is also a major component in the essential oil of Cannabis sativa, up to 35%. Alongside with the major component cannabinol. But the two bind to different receptors in the nervous system. Cannabinol binds to the CB1 receptor in the central nervous system and caryophyllene binds to the CB2 receptor in peripheral tissues. CB1 receptors lead to what is called a « high ». The non psychoactive CB2 receptors modulate immune responses, inhibit inflammation and edema formation and exhibit antinociceptive effects. They are potential targets for the treatment of atherosclerosis and osteoporosis.

                 Gertsch J, Leonti M, Raduner S, Racz I, Chen JZ, Xie XQ, Altmann KH, Karsak M, Zimmer A Beta-caryophyllene is a dietary cannabinoid.. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9099-104. doi: 10.1073/pnas.0803601105.

This research clearly showed that mice lacking the CB2 receptor did not show these effects. A Brazilian team had already shown that caryophyllene reduced acute inflammatory responses to LPS.

Medeiros R, Passos GF, Vitor CE, Koepp J, Mazzuco TL, Pianowski LF, Campos MM, Calixto JB. Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw. Br J Pharmacol. 2007 Jul;151(5):618-27.

Another Brazilian team recently showed that caryophyllene has very strong antileishmanial properties. It is the major constituent of Copaifera of the Amazonian rainforest.

                Soares DC, Portella NA, Ramos MF, Siani AC, Saraiva EM. Trans- β -Caryophyllene: An Effective Antileishmanial Compound Found in Commercial Copaiba Oil (Copaifera spp.). Evid Based Complement Alternat Med. 2013;2013:761323. doi: 10.1155/2013/761323.

It is also effective against the Chagas disease, Trypanosoma cruzi and shows in this case a strong synergy with the pentacyclic triterpene lupenone.

                 Glendy Polanco-Hernández, Fabiola Escalante-Erosa, Karlina García-Sosa, María E. Rosado,2 Eugenia Guzmán-Marín,Karla Y. Acosta-Viana,Alberto Giménez-Turba, Efraín Salamanca, and Luis M. Peña-Rodríguez. Synergistic Effect of Lupenone and Caryophyllene Oxide against Trypanosoma cruzi.Evidence-Based Complementary and Alternative Medicine. Volume 2013 (2013), Article ID 435398, doi.org/10.1155/2013/435398

There are not many papers documenting the antiplasmodial properties of caryophyllene.

                Kamaraj C, Rahuman AA, Roopan SM, Bagavan A, Elango G, Zahir AA, Rajakumar G, Jayaseelan C, Santhoshkumar T, Marimuthu S, Kirthi AV. Bioassay-guided isolation and characterization of active antiplasmodial compounds from Murraya koenigii extracts against Plasmodium falciparum and Plasmodium berghei. Parasitol Res. 2014 May;113(5):1657-72. doi: 10.1007/s00436-014-3810-

Another author recorded moderate antimalarial activities against two strains of Plasmodium falciparum.

               WE Campbelle, DW Gammon. Composition and antimalarial activities in vitro of the essential oil of Tetradonia riparia. Planta Medica, 63-3, 270-272.

The oral administration of caryophyllene attenuates acute and chronic pain in mice. Caryophyllene-induced antinociception was confirmed by Japanese teams.

                Kuwahata H, Komatsu T, Nagaoka K, Nakamura H, Bagetta G, Sakurada T, Sakurada S.Katsuyama S1, Mizoguchi H, Involvement of peripheral cannabinoid and opioid receptors in β-caryophyllene-induced antinociception. Eur J Pain. 2013 May;17(5):664-75. doi: 10.1002/j.1532-2149.2012.00242.x.

Recently it was demonstrated that caryophyllene, a known CB2 agonist, reduces anxiety and depression in mice.

                Bahi A , Al Mansouri S , Al Memari E , β-Caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice. Physiology & Behavior [2014, 135:119-124]

It also has stronger cytotoxic properties then myrcene or alpha-pinene against some cancer cells The documentation on antiparasitic activities of caryophyllene are scarce and deserve further studies Artemisia ketone and other essential oils of Artemisia plants The essential oils of Artemisia annua have been extensively studied and the major constituents are cineole, camphene, a-pinene, germacrene,, camphor and artemisia ketone. They are present at concentrations of 0.20-0.25% and have shown not only different antimicrobial activities12 but also anti-inflammatory13 and cytotoxic activities14,15. They are drastically reduced by high drying temperatures of the herb. Artemisia ketone is a major constituent of some cultivars of A. annua. Like other ketones it inhibits hemozoin crystallization in malaria infected persons. Nerolidol has an IC50 of 0.99 µmol/L and may lead to 100% growth inhibition at the schizont stage. Their concentration in Artemisia annua vary with plant development

               M L Towler and P. Weathers Industrial Crops and Products, Variations in key artemisinin and other metabolites throughout plant development in Artemisia annua L for potential therapeutic use,2015, 1 :67 ; 1885-191.

α-Pinene is a volatile constituent of essential oil present in the plant regardless of origin at levels up to 0.05% of dry weight

               Bhakuni RS, Jain DC, Sharma, RP, Kumar S (2001) Secondary metabolites of Artemisia annua and their biological activity. Curr Sci 80:35-48

               Seatholo ST (2007) The biological activity of specific essential oil constituents. Thesis for MS in Medicine, Univ Witwatersrand, So. Africa

               Song HX, Xie Y, Ye YP Advances in saponin-based adjuvants. Vaccine (2009) 27:1787-1796

In a South African study, which determined the antimalarial activity of 20 essential oils, α-pinene ranked second with an IC50 of 1.2 µM, similar to that of quinine at 0.29 µM 1,8-Cineole (eucalyptol) often comprises up to 30% of the essential oil in A. annua or 0.24-0.42 % (V/DW)

               Su V, King D, Woodrow I, McFadden G, Gleadow R (2008) Plasmodium falciparum growth is arrested by monoterpenes form eucalyptus oil. Flavour Frag J 23:315-318

               Charles DJ, JE Simon, Wood KV, Heinstein P Germplasm variation in artemisinin content of Artemisia annua using an alternative method of artemisinin analysis from crude plant extracts. J. Nat. Prod. (1990) 53:157–160.

The molecule is a strong inhibitor of the pro-inflammatory cytokines TNFα, IL-6 and IL-8.

               Juergens UR, Engelen T, Racké K, Stöber M, Gillissen A, Vetter H (2004) Inhibitory activity of 1.8cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes. Pulmon Pharmacol Therapetu 17:281-287

Growth and development of chloroquine-resistant and chloroquine-sensitive Plasmodium strains are affected and are stalled at the early trophozoite stage. This volatile terpene is rapidly absorbed into the blood when delivered either orally or as an inhalant

             Kovar K, Gropper B, D. Friess D, Ammon HPT (1987) Blood levels of 1,8-cineole and locomotor activity of mice after inhalation and oral administration of rosemary oil. Planta Medica 53:315-318.

             Stimpfl T, Nasel B, Nasel C, Binder R, Vycudilik W, Buchbauer G (1995) Concentration of 1,8-cineol in human blood during prolonged inhalation. Chem. Senses 20:349-350

reaching 15 µg mL-1 in 60 min (Kovar et al. 1987) suggesting its possible use as an antimalarial inhalant. With an IC50 of 0.02 mg mL-1 and an LD50 of ~25 mg mL-1, either inhalation or oral delivery is reasonable

            Kengne RDC (2010) Caracterisation physico-chimique de Artemisia annua (asteraceae), plante medicinale cultivee au Cameroun. MS thesis in Organic Chemistry, Univ de Dschang Republic of Cameroon

Artemisia ketone is a major constituent of A. annua often up to 60 %. The name given to artemisia ketone may be misleading. It is a small linear molecule which is not parent of heavier molecules with several rings like artemisinic acid, arteannuin B or artemisinin. A lot of research work has been devoted to other constituents like artemisinin, scopoletine, limonene, eucalyptol, borneol, luteolin, eupatin, casticin, but artemisia ketone is completely absent in the scientific literature. The molecule had been discovered in Artemisia annua in 1938

            Walter A. Jacobs and Robert C. Elderfield. The Terpenes, Saponins, and Closely Related Compounds. Annual Review of BiochemistryVol. 7: 449-472 (Volume publication date July 1938) DOI: 10.1146/annurev.bi.07.070138.002313

but then fell into oblivion. A recent paper from Serbia

           Niko Radulović, Pavle Randjelovic, Toxic essential oils. Part II: Chemical, toxicological, pharmacological and microbiological profiles of Artemisia annua L. volatiles. Food and chemical toxicology: 2013, 58, 37-49 DOI: 10.1016/j.fct.2013.04.016 ·

shows that artemisia ketone has a stronger free radical scavenging effect and a stronger antimicrobial activity than other well known monoterpenes in Artemisia annua. It is important also to remember that thujone is a ketone with strong antimalarial properties and is present in significant concentrations in most artemisia species. Our interest was further raised when we read in another recent paper ketones also can reduce pain and inflammation.

           Ruskin DN, Kawamura M, Masino SA. Reduced pain and inflammation in juvenile and adult rats fed a ketogenic diet. PLoS One. 2009 Dec 23;4(12):e8349.

It may also play a role in hemozoin formation. Plasmodium needs hemoglobin for its survival and multiplication in merozoites inside the red blood cell. Although this gives the parasite access to nitrogen, it leaves debris like heme, which is toxic. The parasite circumvents this by causing oxidation of Fe(II) in heme to Fe(III) forming hematin that polymerizes into an insoluble product called β-hematin and haemozoin, which is nontoxic to the parasite and inhibits cell-mediated immunity against the parasite. Other ketones like curcumin were implicated as inhibitors of β-hematin synthesis, so it is possible that artemisia ketone plays a similar role. Limonene is part of the so called “cineole cassette”, which include 1,8-cineole, limonene, myrcene, αpinene, β-pinene, sabinene and α-terpineol.

            Raguso RA, Levin RA, Foose SE, Holmberg MW, Mc Dade LA (2003) Fragrance chemistry, nocturnal rhythms and pollination, syndromes’ in Nicotiana. Phytochemistry 63:265–284

many of which affect particular stages of Plasmodium species. Limonene, for example, arrests isoprenoid biosynthesis in Plasmodium

            Goulart H R, Kimura E A, Peres V J, Couto A S, Aquino Duarte F A, Katzin A M Terpenes arrest parasite development and inhibit biosynthesis of isoprenoids in Plasmodium falciparum. Antimicrob Agents Chemother (2004) 48: 2502-9

and development at the ring and trophozoite stages

             Moura IC, Wunderlich G, Uhrig ML, Couto AS, Peres VJ, Katzin AM, Kimura EA (2001) Limonene arrests parasite development and inhibits isoprenylation of proteins in Plasmodium falciparum Antimicrob. Agents Chemother 45:2553-2558

while 1,8-cineole affects the trophozoite stage. Limonene also inhibits protein isoprenylation in P. falciparum, arresting parasite development within 48 h of treatment The in vitro IC50 against Plasmodium in these trials was 2.27 mM, significantly below 15.5 mM that was previously shown in vivo in patients with advanced cancer. The pharmacokinetics is favorable; limonene and its metabolites remain in the plasma for at least 48 hrs

             Miller JA, Hakim IA, Chew W, Thompson P, Chew W, Thomsen CA, Chow HHS (2010) Adipose tissue accumulation of d-limonene with the consumption of a lemonade preparation rich in d-limonene content. Nutrition Cancer 62:783-788.

This is important for the elimination of gametes and malaria transmission. A combination of essential oils may enhance the antimalarial effect of artesunate and even reverse the resistance of P. berghei against artesunate.

            Liu AR, Yu ZY, Lu LL, Sui ZY (2004) The synergistic action of guanghuoxiang volatile oil and sodium artesunate against Plasmodium berghei and reversal of SA-resistant Plasmodium berghei, Chin J Parasitol Parasitic Diseases 18:76-78

The concentration of monoterpenes is higher in the pre-flowering phase, but is drastically reduced by high drying temperatures or drying in the sun

           Khangholil S, Rezaeinodehi A (2008) Effect of drying temperature on essential oil content and composition of sweet wormwood (Artemisia annua) growing wild in Iran. Pak J Biol Sci 11:934-937

            Ferreira JFS, Luthria DL (2010) Drying affects artemisinin, dihydroartemisinic acid, artemisinic acid, and the antioxidant capacity of Artemisia annua L. leaves J. Agric. Food Chem. 2010, 58, 1691–1698

The monoterpene limonene has a very favourable toxicity profile and is easily available at low prices. Limonene is also present in A. annua at concentrations up to 7 mg kg-1

            Bhakuni RS, Jain DC, Sharma, RP, Kumar S (2001) Secondary metabolites of Artemisia annua and their biological activity. Curr Sci 80:35-48

So far, studies have concentrated on this particular monoterpene, but others like eucalyptol, present in the essential oil of Artemisia plants, might have a similar detrimental action on the apicoplast, a non-photosynthetic plastid of most apicomplexan parasites, such as Plasmodium.

The sesquiterpene nerolidol, found in Artemisia species arrests development of the intraerythrocytic stages of the parasite. It has an antiplasmodial IC50 of 0.99 µM compared to that of 533 µM for limonene

             Van Zyl, Robyn L.; Seatlholo, Sammy T.; Van Vuuren, Sandy F.; Viljoen, Alvaro M. The Biological Activities of 20 Nature Identical Essential Oil Constituents. Journal of Essential Oil Research . Jul/Aug 2006 Supplement, Vol. 18, p129-133.

              Seatholo ST, The biological activity of specific essential oils constituents. Thesis for MS in Science, Univ Witwaterstrand South Africa. 2007.

Indians of the Amazon basin in Brazil use the vapors of the leaves of Viola surinamensis to treat malaria and the sesquiterpene nerolidol was identified as the active constituent leading to 100 % growth inhibition at the schizont stage

             Lopes NP, Kato MJ, Andrade EH de A, Maia JGS, Yoshida M, Planchart AR, Katzin AM (1999) Antimalarial use of volatile oil from leaves of Virola surinamensis (Rol.) Warb. By Waipãpi Amazon Indians. J Ethnopharmacol 67:313-319

Like limonene nerolidol may affect the isoprenoid pathway in the apicoplast of Plasmodium. Nerolidol concentrations vary with the origin of A. annua. The highest value was found in A. annua from Ethiopia

             Muzemil A (2008) Determination of artemisinin and essential oil contents of Artemisia annua L. grown in Ethiopia and in vivo antimalarial activity of its crude extracts against Plasmodium berghei in mice. MS Thesis in Medicinal Chemistry, Addis Ababa University, Ethiopia.

Nerolidol was also found to be higher in the stems than in the leaves of A. annua

            Li QG, Peggins JO, Fleckenstein LL, Masonic K, Heiffer MH, Brewer TG (1998) The pharmacokinetics and bioavailability of dihydroartemisinin, arteether, artemether, artesunic acid and artelinic acid in rats. J Pharm Pharmacol 50:173-182

But the bioavailabilty of monoterpense per os is low. Moxa or inhalation is more appropriate for administration. In human volunteers for alpha-pinene absorbed by inhalation only 0.001% is eliminated by urine . The main part accumulates in adipose tissues and is released progressively into blood

            Falk AA, Hagberg MT, Löf AE, Wigaeus-Hjelm EM, Wang ZP Uptake, distribution and elimination of alpha-pinene in man after exposure by inhalation. Scand J Work Environ Health. 1990 Oct;16(5):372-8

The results of a study in Austria showed that 1,8-cineol is well absorbed from breathing air, with a peak plasma concentration after 18 min. The elimination of this fragrance compound from the blood is biphasic, with a mean distribution half-life of 6.7 min and an elimination half-life of 104.6 min.

            W. Jager, B. NaJel, C. NaSel1, R. Binder, T. Stimpfl, W. Vycudilik and G. Buchbauer. Pharmacokinetic Studies of the Fragrance Compound 1-8-Cineol in Humans during Inhalation. Chem. Senses 21: 477-480, 1996.

In human volunteers for alpha-pinene absorbed by inhalation only 0.001% is eliminated by urine .The main part accumulates in adipose tissues and is released progressively into blood.. Terpene content for milk samples is characterized by the same terpenes as those added to food. The terpenes appear to be transferred unaltered into the milk. Volatile terpenes in essential oils, which could influence milk flavor, are transferred into milk via both gastrointestinal and respiratory exposure. Most of it is probably absorbed by inhalation of pinenes emitted by plants or trees. Among all monoterpenes emitted by trees alpha-pinene has on the average the highest percentage.

             Sophie MOUKHTAR, Impact des émissions naturelles sur les épisodes de pollution photochimique. Application à la region du fossé rhénan Institut National Polytechnique de Toulouse. Thèse. 2005

Primary agents of the tree's defense are monoterpenes, aromatic molecules such as pinenes. Pinenes and limonene from Artemisia arborescens also display antiviral properties by inactivating viruses and inhibiting cell-to-cell diffusion.

             Adorjan, Barbara (2010) Biological properties of essential oils. Diplomarbeit, University of Vienna. Fakultät für Lebenswissenschaften

They increase the activity of human natural killer cells.

            Li Q, Kobayashi M, Inagaki H, Hirata Y, Li YJ, Hirata K, Shimizu T, Suzuki H, Katsumata M, Wakayama Y, Kawada T, Ohira T, Matsui N, Kagawa T. A day trip to a forest park increases human natural killer activity and the expression of anti-cancer proteins in male subjects. J Biol Regul Homeost Agents. 2010 Apr-Jun;24(2):157-65.

The Japanese call it a forest bathing trip. Pinenes have strong antimicrobial properties.

            Aristides Medeiros Leite, Edeltrudes de Oliveira Lima , Evandro Leite de Souza, Margareth de Fátima Formiga Melo Diniz, Vinícius Nogueira Trajano. Inhibitory effect of β-pinene, α-pinene and eugenol on the growth of potential infectious endocarditis causing Gram-positive bacteria. Revista Brasileira de Ciências Farmacêuticas. vol. 43, n. 1, jan./mar., 2007

These molecules also have immunostimulating, anti-inflammatory and gastroprotective properties.

             DK Lima. Evaluation of the Antinociceptive, Anti-Inflammatory and Gastric Antiulcer Activities of the Essential Oil From Piper Aleyreanum C.DC in Rodents. J Ethnopharmacol 142 (1), 274-282. 2012 May 12.