Identification of novel phytocannabinoids from Ganoderma by label-free dynamic mass redistribution assay

Located throughout the body, cannabinoid receptors (CB1 and CB2) are therapeutic targets for obesity/metabolic diseases, neurological/mental disorders, and immune modulation. Phytocannabinoids are greatly important for the development of new medicines with high efficacy and/or minor side effects. Plants and fungi are used in traditional medicine for beneficial effects to mental and immune system. The current research studied five fungi from the genus Ganoderma and five plants: Ganoderma hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang; Ganoderma capense (Lloyd) Teng, Zhong Guo De Zhen Jun; Ganoderma cochlear (Blume & T. Nees) Bres., Hedwigia; Ganoderma resinaceum Boud.; Ganoderma applanatum (Pers.) Pat.; Carthamus tinctorius L. (Compositae); Cynanchum otophyllum C. K. Schneid. (Asclepiadaceae); Coffea arabica L. (Rubiaceae); Prinsepia utilis Royle (Rosaceae); Lepidium meyenii Walp. (Brassicaceae). They show immunoregulation, promotion of longevity and maintenance of vitality, stimulant effects on the central nervous system, hormone balance and other beneficial effects. However, it remains unclear whether cannabinoid receptors are involved in these effects.

This work aimed to identify components working on CB1 and CB2 from the above plants and fungi, as novel phytocannabinoids, and to investigate mechanisms of how these compounds affected the cells. By analyzing the structure-activity relationship, we could identify the core structure for future development.

Eighty-two natural compounds were screened on stably transfected Chinese hamster ovary (CHO) cell lines, CHO-CB1 and CHO-CB2, with application of a label-free dynamic mass redistribution (DMR) technology that measured cellular responses to compounds. CP55,940 and WIN55,212-2 were agonist probe molecules, and SR141716A and SR144528 were antagonist probes. Pertussis toxin, cholera toxin, LY294002 and U73122 were signaling pathway inhibitors. The DMR data were acquired by Epic Imager software (Corning, NY), processed by Imager Beta 3.7 (Corning), and analyzed by GraphPad Prism 6 (GraphPad Software, San Diego, CA).

Transfected CHO-CB1 and CHO-CB2 cell lines were established and characterized. Seven compounds induced responses/activities in the cells. Among the seven compounds, four were purified from two Ganoderma species with potencies between 20 and 35 μM. Three antagonists: Kfb68 antagonized both receptors with a better desensitizing effect on CB2 to WIN55,212-2 over CP55,940. Kga1 and Kfb28 were antagonists selective to CB1 and CB2, respectively. Kfb77 was a special agonist and it stimulated CB1 in a mechanism different from that of CP55,940. Another three active compounds, derived from the Lepidium meyenii Walp. (Brassicaceae), were also identified but their effects were mediated through mechanisms much related to the signaling transduction pathways, especially through the stimulatory G_s protein.

We identified four natural cannabinoids that exhibited structural and functional diversities. Our work confirms the presence of active ingredients in the Ganoderma species to CB1 and CB2, and this finding establishes connections between the fungi and the cannabinoid receptors, which will serve as a starting point to connect their beneficial effects to the endocannabinoid system. This research will also enrich the inventory of cannabinoids and phytocannabinoids from fungi. Yet due to some limitations, further structure-activity relationship studies and mechanism investigation are warranted in future.