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

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Abstract

Ethnopharmacological relevance

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.

Aim of the study

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.

Materials and methods

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).

Results

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 Gs protein.

Conclusions

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.

Introduction

Cannabinoid receptors (CB1 and CB2) belong to the G-protein coupled receptor (GPCR) superfamily and they are primarily coupled to inhibitory G proteins (Gi) (Howlett, 2002). They are part of the endocannabinoid system (ECS) named after the plant Cannabis sativa L. (Gaoni and Mechoulam, 1964). The ECS is a complex lipid signaling and immunomodulator system, and it is probably the most important physiologic system. Alterations of the ECS are reported to be associated with neuropsychiatric disorders and immune vulnerability (Pacher et al., 2006; Zoppi et al., 2011; Zou and Kumar, 2018). CB1 is expressed at high levels in the brain (Matsuda et al., 1990), playing a role in memory (Hebert-Chatelain et al., 2016), sleep (Murillo-Rodriguez, 2008), mood (Witkin et al., 2005), and pain management, and its agonists show neuroprotective properties (Molina-Holgado et al., 2005). CB2 receptor localization indicates a high density in immune cells (Munro et al., 1993), and multiple pathologic conditions can be mediated by CB2 activation (Atwood et al., 2012a), such as multiple sclerosis (Chiurchiu et al., 2018), HIV infection (Purohit et al., 2014), and Alzheimer's disease (Ramirez et al., 2005). CB2 stimulation is important for development of neuroprotective therapies (Lourbopoulos et al., 2011; Merighi et al., 2012). The involvement of cannabinoid receptors in health and diseases is currently a topic of interest (Pacher and Kunos, 2013).

Apart from endocannabinoids, exogenous ligands can induce effects through the receptors as well. Nature serves as a reservoir full of active ingredients with rich structural diversity. So far, more than 100 cannabinoids (also known as phytocannabinoids) have been identified from the Cannabis sativa L., among which Δ9-tetrahydrocannabinol (Δ9-THC) is the principal psychoactive component (Fig. 1) (Gaoni and Mechoulam, 1964). Naturally occurring components are an important source of novel drugs and therapies. For example, HU-210 is developed based on the structure of Δ8-THC with enhanced affinity and efficacy (Felder et al., 1995), and it has become a useful tool molecule in cannabinoid-related pharmacology studies (Devane et al., 1992). Cannabidiol (CBD, Fig. 1) is the second prevalent active ingredient in the Cannabis sativa L. but lacks detectable psychoactivity (Blessing et al., 2015). Although CBD interacts with CB1 and CB2 in vitro, it acts as an antagonist and an antidote to Δ9-THC, providing protection against certain negative effects caused by THC abuse (Hampson et al., 1998; Rong et al., 2017). Sativex (GW Pharmaceuticals, UK), an oromucosal spray with THC and CBD in a 1:1 ratio, has now received marketing authorization in Europe and Canada for the treatment for adult multiple sclerosis-related spasticity (Bifulco and Pisanti, 2015; Karschner et al., 2011; Wright et al., 2012).

Nowadays, the concept of phytocannabinoid is no longer limited to active ingredients from Cannabis sativa L., and a number of components have been identified in non-cannabis species, having important effects through the mediation of cannabinoid receptors (Gertsch et al., 2010; Sharma et al., 2015). For example, Amyrins, widely distributed in nature, are extremely potent on CB1 (Ki = 0.133 nM) over CB2 (Ki = 1,989 nM) (da Silva et al., 2011). Such naturally occurring molecules not only enrich the inventory of cannabinoids, but also offer potential leads for drug design. Cannabinoid-related drug development in return helps people understand the underlying mechanisms of how medicines act on human bodies (Hanus et al., 2016).

Multiple reports have revealed adverse health effects of synthetic cannabinoids with emphasis on psychosis-like effects. CB1 antagonism is considered as a promising therapeutic approach to treat overweight/obesity and related cardiometabolic disorders (Janero and Makriyannis, 2009). CB1-selective antagonists, SR141716A and its close analogs (MK-0364 and CP945,598), were first used for the treatment of obesity but later found to cause serious depression and anxiety (Di Marzo and Despres, 2009; Proietto et al., 2010; Traynor, 2007). Thus, clinical applications of these drugs were discontinued. How to minimize and overcome side effects is a challenge. It drives us to continuously explore the natural source to seek new active ingredients as templates to develop drugs with no or minor negative effects, for the sake of human health.

In this work, 82 compounds derived from 10 species (5 plants and 5 fungi) were screened on CB1 and CB2 receptors. These species grow in west China and have been reported to have beneficial effects to mental health and immune system (Table 1), but whether the cannabinoid receptors are engaged in their effects remains not known. A label-free dynamic mass redistribution (DMR) technology was applied that measured cellular responses to compounds by a resonant waveguide grating optical biosensor (Fang, 2011; Schroder et al., 2011). Active ingredients were found in three of the ten species. Among the 82 compounds, 4 compounds distributed in the Ganoderma species were identified to be active on the cannabinoid receptors, including three antagonists and a special agonist that may stimulate CB1 via a different mechanism. EC50 values of these compounds were between 20 and 35 μM, making them promising leads for drug design. Another three compounds were identified in the Lepidium meyenii Walp. (Brassicaceae) plant, effects of which were mediated through mechanisms much related to the signaling transduction pathways.

Section snippets

Materials

CP55,940 and U73122 were purchased from Sigma-Aldrich (St. Louis, MO), WIN55,212–2 from Absin Bioscience Inc. (Shanghai, China), and SR141716A and SR144528 from Tocris Biosciences (Ellisville, MO). Pertussis toxin was purchased from APExBio (Houston, TX), cholera toxin from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai, China), and LY294002 from Selleck Chemicals (Houston, TX). Cyclic monophosphate (cAMP)-Gi kit was purchased from cisbio (Number 62AM9PEB, Bedford, MA) for cAMP HTRF

Characterization of transfected CHO-CB1 and CHO-CB2 cell lines

DMR detections were performed with the transfected CHO-CB1 and CHO-CB2 cell lines so as to verify the transfection quality and characterize the models with specific tool molecules, and meanwhile to prove the suitability of applying the DMR technology on the cannabinoid receptors. Four high-affinity, commonly-used probe molecules were used in this work to fulfill the aims (Fig. 1).

CP55,940 is a non-selective agonist of CB1 and CB2. Reported Ki values, obtained from radiolabeling assays, are in a

Binding characteristics of CP55,940 and WIN55,212-2

During the model characterization, CP55,940 and WIN55,212–2 were used as agonist tool molecules, and some points should not be missed. First, DMR assay results showed that CP55,940 was non-selective between receptor subtypes and the transfection level of CB1 was higher than CB2. WIN55,212–2 was determined to be selective to CB2 over CB1 with EC50 differed by a factor of (CB1/CB2: 2,006/140.7 = ) 14.7. This result agrees with earlier reports (Felder et al., 1995; McPartland et al., 2007).

Second,

Conclusions

For decades, phytocannabinoids were considered as cannabinoids that occur naturally in the Cannabis sativa L. plant. Then more and more naturally occurring compounds distributed in species besides Cannabis sativa L. have been found active on the cannabinoid receptors. In this work, natural cannabinoids were identified in Lepidium meyenii Walp., and also in two species of Ganoderma (Ganoderma hainanense J.D. Zhao, L.W. Hsu & X.Q. Zhang and Ganoderma cochlear (Blume & T. Nees) Bres., Hedwigia).

Declaration of interest

None. The authors declare no competing financial interest.

Author contributions

Han Zhou and Xingrong Peng: provided equal contribution to this work. Han Zhou: conceived the present idea and designed the project; designed the models and performed the experiments; wrote the manuscript with input from all authors. Xingrong Peng: prepared the compound samples and provided information of the plants and fungi. Tao Hou: designed the models and performed the experiments. Nan Zhao: drafted the chemical structures and analyzed the data. Minghua Qiu: prepared the compound samples

Acknowledgement

This work was supported by the “State Key Program of National Natural Science of China (Grant No. U1508221)”, “Project of National Science Foundation of China (81803706)”, “Innovation Program of Science and Research” from DICP, CAS (DICP TMSR201601), and the funding for the construction of DICP-CMC Innovation Institute of Medicine. We thank Dr. Olivier Civelli at University of California, Irvine for generously providing plasmids for cell transfections.

References (91)

  • Y. Mizushina et al.

    A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases

    Biochem. Biophys. Res. Commun.

    (1998)
  • F. Molina-Holgado et al.

    Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling

    Mol. Cell. Neurosci.

    (2005)
  • E. Murillo-Rodriguez

    The role of the CB1 receptor in the regulation of sleep

    Prog. Neuro-Psychopharmacol. Biol. Psychiatry

    (2008)
  • S. Oka et al.

    GPR35 is a novel lysophosphatidic acid receptor

    Biochem. Biophys. Res. Commun.

    (2010)
  • E.B. Patay et al.

    Phytochemical overview and medicinal importance of Coffea species from the past until now

    Asian Pac. J. Trop. Med.

    (2016)
  • X.R. Peng et al.

    Protective effects of triterpenoids from Ganoderma resinaceum on H(2)O(2)-induced toxicity in HepG2 cells

    Food Chem.

    (2013)
  • X.R. Peng et al.

    Unusual prenylated phenols with antioxidant activities from Ganoderma cochlear

    Food Chem.

    (2015)
  • X.R. Peng et al.

    Lanostane triterpenoids from Ganoderma hainanense J. D. Zhao

    Phytochemistry

    (2015)
  • X.R. Peng et al.

    Racemic meroterpenoids from Ganoderma cochlear

    Fitoterapia

    (2018)
  • K. Ramalakshmi et al.

    Antioxidant potential of low-grade coffee beans

    Food Res. Int.

    (2008)
  • M. Rinaldicarmona et al.

    Sr141716a, a potent and selective antagonist of the brain cannabinoid receptor

    FEBS Lett.

    (1994)
  • C. Rong et al.

    Cannabidiol in medical marijuana: research vistas and potential opportunities

    Pharmacol. Res. : Off. J. Italian Pharmacol. Soc.

    (2017)
  • G.A. Thakur et al.

    Natural cannabinoids: templates for drug discovery

    Life Sci.

    (2005)
  • X.X. Tian et al.

    Hydantoin and thioamide analogues from Lepidium meyenii

    Phytochem. Lett.

    (2018)
  • H. Wu et al.

    Macamides and their synthetic analogs: evaluation of in vitro FAAH inhibition

    Bioorg. Med. Chem.

    (2013)
  • Y.Z. Xie et al.

    Ganoderma lucidum inhibits tumour cell proliferation and induces tumour cell death

    Enzym. Microb. Technol.

    (2006)
  • C. Zhao et al.

    Pharmacological effects of natural Ganoderma and its extracts on neurological diseases: a comprehensive review

    Int. J. Biol. Macromol.

    (2019)
  • P. Zhao et al.

    GPR55 and GPR35 and their relationship to cannabinoid and lysophospholipid receptors

    Life Sci.

    (2013)
  • X. Zhou et al.

    Towards a better understanding of medicinal uses of Carthamus tinctorius L. in traditional Chinese medicine: a phytochemical and pharmacological review

    J. Ethnopharmacol.

    (2014)
  • M. Adams et al.

    Antiplasmodial lanostanes from the Ganoderma lucidum mushroom

    J. Nat. Prod.

    (2010)
  • B.K. Atwood et al.

    Functional selectivity in CB2 cannabinoid receptor signaling and regulation: implications for the therapeutic potential of CB2 ligands

    Mol. Pharmacol.

    (2012)
  • R. Bartenschlager et al.

    The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection

    Nat. Rev. Microbiol.

    (2013)
  • M. Bifulco et al.

    Medicinal use of cannabis in Europe: the fact that more countries legalize the medicinal use of cannabis should not become an argument for unfettered and uncontrolled use

    EMBO Rep.

    (2015)
  • M.A. Bloomfield et al.

    The effects of Delta(9)-tetrahydrocannabinol on the dopamine system

    Nature

    (2016)
  • M.L. Bolognesi et al.

    Multitarget drug discovery and polypharmacology

    ChemMedChem

    (2016)
  • D. Braida et al.

    Potential anxiolytic- and antidepressant-like effects of salvinorin A, the main active ingredient of Salvia divinorum, in rodents

    Br. J. Pharmacol.

    (2009)
  • N.A. Brooks et al.

    Beneficial effects of Lepidium meyenii (Maca) on psychological symptoms and measures of sexual dysfunction in postmenopausal women are not related to estrogen or androgen content

    Menopause

    (2008)
  • E.E. Codd et al.

    Dynamic mass redistribution as a means to measure and differentiate signaling via opioid and cannabinoid receptors

    Assay Drug Dev. Technol.

    (2011)
  • K.A. da Silva et al.

    Activation of cannabinoid receptors by the pentacyclic triterpene alpha,beta-amyrin inhibits inflammatory and neuropathic persistent pain in mice

    Pain

    (2011)
  • A. Dafni

    Rituals, ceremonies and customs related to sacred trees with a special reference to the Middle East

    J. Ethnobiol. Ethnomed.

    (2007)
  • F. Degorce et al.

    HTRF: a technology tailored for drug discovery - a review of theoretical aspects and recent applications

    Curr. Chem. Genom.

    (2009)
  • E. Delshad et al.

    Medical uses of Carthamus tinctorius L. (Safflower): a comprehensive review from traditional medicine to modern medicine

    Electron. Physician

    (2018)
  • W.A. Devane et al.

    A novel probe for the cannabinoid receptor

    J. Med. Chem.

    (1992)
  • V. Deveaux et al.

    Cannabinoid CB2 receptor potentiates obesity-associated inflammation, insulin resistance and hepatic steatosis

    PLoS One

    (2009)
  • V. Di Marzo et al.

    CB1 antagonists for obesity--what lessons have we learned from rimonabant?

    Nat. Rev. Endocrinol.

    (2009)
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