Valencene is a natural product found in Xylopia sericea and Helichrysum odoratissimum, None of these plants listed by PubChem are very familiar to North Americans. Valencene is apparently found in valencia oranges. [1] María Eugenia Letelier and other scientists from the School of Chemical and Pharmaceutical Sciences, Universidad de Chile, Santiago developed a protocol for the microbial transformation of valencene from valencia orange into a compound called nootkatone. [1] Another group of scientists from Institute of Biochemistry, Heinrich-Heine University Düsseldorf produced a fusion protein of a cytochrome P450 and an alcohol dehydrogenase both of microbial origins. [2]

Microbial enzymes are used to transform valencene to the fragrance industry coveted grapefruit odorant nootkatonal.

The Cox2 binding site came from this link.

Cox2 and the histamine H1 ad potential targets [3,4]

Most of the authors of the valencene study were from various locations in Brazil with one Korean author. [3] Both countries have a substantial publication record of plant based medicines. Anti-inflammatory substances are a big interest of Big Parma and plant based medicine practitioners. Two common food ingredients were used to cause inflammation when administered by non oral routes. The nookatrone study came out of Sara Vitalini’s group that included Italian and Brazilian investigators.  The protocol was practically identical with the valencene publication with an entirely Brazilian cast of corresponding authors. [4]

Carageenan is a water-soluble extractive mixture of polysaccharides from RED ALGAE. Chief sources are the Irish moss Chondrus crispus (Carrageen), and Gigartina stellata. It is used as a stabilizer, for suspending cocoa in chocolate manufacture, and to clarify beverages. Dextran is a group of glucose polymers made by certain bacteria. Dextrans are used therapeutically as plasma volume expanders and anticoagulants. They are also commonly used in biological experimentation and in industry for a wide variety of purposes.

Materials and methods

2.2. Animals
Male and female Swiss mice 20-30g  were randomly assigned to groups.  

2.3. Evaluation of anti-inflammatory activity

  • carrageenan-induced peritonitis  systemic
  • carrageenan-induced pleurisy systemic
  • paw edema induced by carrageenan, dextran, histamine, or AA.  local
  • surgically inserted, granuloma producing, cotton pellets, chronic inflammation

2.3.1. Assessment of the anti edematogenic activity

The animals (n = 6 per group) were randomly assigned into groups
and treated orally with

  • valencene (10, 100, or 300 mg/Kg),
  • vehicle (0.9% saline, negative control)
  • positive control drugs (promethazine 10 mg/Kg or indomethacin 25 mg/Kg) 1 h before the challenge.

Each challenge was performed though the injection of 20 μL of a 1% (w/v) solution containing an inflammatory agent (carrageenan, dextran, histamine, or AA) into the right hind paw. Equal volume of saline was administered into the left hind paw. The volume of both paws was measured at different time-points using a plethysmometer and the difference between the hind and left paws was determined.

Histamine and carragean may may act by release of histamine or arachidonic acid.  Histamine and arachidonic acid were tested alone, with specific inhibitors, and with the indicated concentration of valencene.

and Nookatone

2.3.2. Carrageenan-induced peritonitis

Peritonitis is the inflammation of the peritoneum, or in other words, the abdominal cavity. The mice (n = 6) were intraperitoneal injected with 1 mL of 1% carrageenan (challenge). Four hours after the challenge, the mice were euthanized, the peritoneal cavity was washed with PBS  and assayed total leukocytes, MPO, and total proteins. (SDH-20).

Myeloperoxidase is an enzyme secreted in neutrophil granuloctes. MPO, in simple terms, produces bleach. Albumin, in this case serum albumin, is a major blood protein.

and Nookatone

2.3.3. Carrageenan-induced pleurisy

Pleurisy/Pleuritis is the inflammation of the membranes surrounding the lung and thoracic cavity. Pleurisy was induced by intrathoracic injection of carrageenan. Four hours following the carrageenan injection, the mice were anesthetized, euthanized, and the pleural cavity was washed with 1 mL of saline.   Leukocytes were collected in the pellets after a brief centrifugation.  The supernatants were used for cytokine measurement.

Interleukin 1β, or IL1β , is a pro-inflammatory cytokine that may induce cyclooxigenase 2. Cox2 is an enzyme involved in the conversion of arachidonic acid to prostaglandin E2. TNFα is also a pro=inflammatory cytokine.

and Nookatone

2.3.6. Cotton pellet-induced granuloma

The backs of anesthetized mice (n = 6) were implanted with four cotton pellets through a small incision. Twenty-four hours later, the animals were treated orally with vehicle or valencene  (10 mg/Kg) for ten consecutive days. On day 11th, the mice were killed and the pellets, as well as the surrounding tissue, were removed, dried, and weighed.

Up until this figure the authors were examining an inflammatory response to food substances in non enteric locations. Cotton is basically cellulose, another “nontoxic” substance that can cause inflammation when placed in the wrong location. Cotton absorbs fluids and solutes.

and Nookatone

2.4. In silico analysis of COX-2 inhibition and H1 receptor antagonism

Computer simulations of ligand docking to known crystal structures were performed with   protein data base entries 1PXX and 3RZE..

This post will not get into the refinements involved in these in silico experiments.

and Nookatone

The Diclofenac binding pocket

Let’s take a closer look at mouse cyclooxygenase 2 on UniProt. and the crystal structure used for molecular modeling 1PXX. This study started with the observation that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin.

  • The authors generated a series of mutants that might impact binding: Arg-120, Tyr-355, Tyr-348, and Ser-530.
  • Diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation.
  • The crystal structure of a complex of diclofenac with murine COX-2 demonstrated that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530.
  • This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120

The table on the left is from images contained in the valencene and nookagone studies. [1,2] The table on the right is from UniProt with added info (magenta) from 1PXX.

Diclofenac is a cyclooxygenase inhibitor. Other structures from PubChem are shown for comparison. In the bottom left a GUI from 1PX. is shown describing how the image in the bottom right was generated. Some of the amino acids have been labeled.


  1. Furusawa M, Hashimoto T, Noma Y, Asakawa Y. Highly efficient production of nootkatone, the grapefruit aroma from valencene, by biotransformation. Chem Pharm Bull (Tokyo). 2005 Nov;53(11):1513-4. free article
  2. Kokorin A, Urlacher VB. Artificial Fusions between P450 BM3 and an Alcohol Dehydrogenase for Efficient (+)-Nootkatone Production. Chembiochem. 2022 Jun 20;23(12):e202200065. PMC free article
  3. Dantas LBR, Alcântara IS, Júnior CPS, de Oliveira MRC, Martins AOBPB, Dantas TM, Ribeiro-Filho J, Coutinho HDM, Passos FRS, Quintans-Júnior LJ, Almeida JRGS, Cruz-Martins N, Kim B, de Menezes IRA. (2022) In vivo and in silico anti-inflammatory properties of the sesquiterpene valencene. Biomed Pharmacother. 2022 Sep;153:113478. PMC free article
  4. Bezerra Rodrigues Dantas L, Silva ALM, da Silva Júnior CP, Alcântara IS, Correia de Oliveira MR, Oliveira Brito Pereira Bezerra Martins A, Ribeiro-Filho J, Coutinho HDM, Rocha Santos Passos F, Quintans-Junior LJ, Alencar de Menezes IR, Pezzani R, Vitalini S. Nootkatone Inhibits Acute and Chronic Inflammatory Responses in Mice. Molecules. 2020 May 7;25(9):2181. PMC free article
  5. Murase T, Misawa K, Haramizu S, Minegishi Y, Hase T. Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK. Am J Physiol Endocrinol Metab. 2010 Aug;299(2):E266-75. free article

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