Recent Advances in Grayanane Diterpenes: Isolation, Structural Diversity, and Bioactivities from Ericaceae Family (2018–2024)

Diterpenes represent one of the most diverse and structurally complex families of natural products. Among the myriad of diterpenoids, grayanane diterpenes are particularly notable. These terpenes are characterized by their unique 5/7/6/5 tetracyclic system and are exclusive to the Ericaceae family of plants. Renowned for their complex structures and broad spectrum of bioactivities, grayanane diterpenes have become a primary focus in extensive phytochemical and pharmacological research. Recent studies, spanning from 2018 to January 2024, have reported a series of new grayanane diterpenes with unprecedented carbon skeletons. These compounds exhibit various biological properties, including analgesic, antifeedant, anti-inflammatory, and inhibition of protein tyrosine phosphatase 1B (PTP1B). This paper delves into the discovery of 193 newly identified grayanoids, representing 15 distinct carbon skeletons within the Ericaceae family. The study of grayanane diterpenes is not only a deep dive into the complexities of natural product chemistry but also an investigation into potential therapeutic applications. Their unique structures and diverse biological actions make them promising candidates for drug discovery and medicinal applications. The review encompasses their occurrence, distribution, structural features, and biological activities, providing invaluable insights for future pharmacological explorations and research.


Introduction
Diterpenes, a class of terpenoids consisting of four isoprene units, represent one of the most diverse and structurally complex families of natural products.As a prominent family of natural products, diterpenes are predominantly found in plants, where they play vital roles in various biological processes, from defense mechanisms against herbivores and pathogens to growth regulation [1].The vast structural diversity and the array of bioactivities associated with diterpenes have made diterpenes a focal point of intense scientific research.
Among the myriad of diterpenes, grayanane diterpenes stand out as particularly noteworthy.These terpenes are distinguished by their unique and intricate 5/7/6/5 tetracyclic system and are exclusive to the Ericaceae family of plants [2][3][4].The Ericaceae family, which encompasses about 4000 species spread across 126 genera, ranging from small herbs to large trees, is a rich source of terpenoids, including triterpenoids, meroterpenoids, and especially diterpenoids such as grayanane diterpenes [2,5].Grayanane diterpenes, as characteristic secondary metabolites of the Ericaceae family, are prominently found in genera like Pieris, Rhododendron, Kalmia, Craibiodendron, and Leucothoe.
Despite several reviews that have covered aspects of grayanane diterpenoids, a comprehensive and in-depth overview of the developments and discoveries in this field, especially from 2018 to January 2024, has been lacking [2,6,7,[14][15][16].This review aims to fill that gap by focusing on the recent advancements made in the isolation, structural elucidation, and bioactivity studies of these diterpenes.Through a detailed examination of various species within the Ericaceae family, the paper presents a thorough overview of their occurrence, distribution, structural features, and biological activities.This approach offers valuable insights for ongoing pharmacological research and underscores the growing significance of grayanane diterpenes in the field of natural product chemistry.
Despite several reviews that have covered aspects of grayanane diterpenoids, a comprehensive and in-depth overview of the developments and discoveries in this field, especially from 2018 to January 2024, has been lacking [2,6,7,[14][15][16].This review aims to fill that gap by focusing on the recent advancements made in the isolation, structural elucidation, and bioactivity studies of these diterpenes.Through a detailed examination of various species within the Ericaceae family, the paper presents a thorough overview of their occurrence, distribution, structural features, and biological activities.This approach offers valuable insights for ongoing pharmacological research and underscores the growing significance of grayanane diterpenes in the field of natural product chemistry.

Overview of Structural Diversity and Biological Activities of Grayanane Terpenes
After an exhaustive search of the PubMed, SciFinder, Scopus, and Google Scholar databases, utilizing the keywords "grayanane", "diterpenes", "diterpenoids", and "Ericaceae family" from 2018 to January 2024, a remarkable total of 193 novel grayanane diterpenes were isolated and identified from the Ericaceae family plants.These discoveries predominantly came from the roots, leaves, or flowers of Pieris, Rhododendron, and Craibiodendron genus.These novel grayanane diterpenes are categorized into 15 distinct carbon skeletons, including ent-kaurane [17], 4,5-seco-kaurane [18], A-home-B-nor-ent-kaurane [17], grayanane [10], 1,5-seco-grayanane [19,20], 1,10-seco-grayanane [17], 1,10:2,3-disecograyanane [17,21], mollane [20,21], kalmane [19,20,22], 1,5-seco-kalmane [23], leucothane [18,21,[23][24][25], rhomollane [23], micranthane [20,25], mollebenzylane [26], and rhodauricane [19], as illustrated in Figure 1.Most of the literature research has focused on the bioactive potential of these compounds.A significant part of the studies is dedicated to analyzing their analgesic effects in vivo, particularly in mouse models.Various models have been employed for this purpose, including the acetic acid-induced writhing test and the capsaicin-and AITC-induced writhing test model [27].Additionally, there have been studies on the antifeedant activity Most of the literature research has focused on the bioactive potential of these compounds.A significant part of the studies is dedicated to analyzing their analgesic effects in vivo, particularly in mouse models.Various models have been employed for this purpose, including the acetic acid-induced writhing test and the capsaicin-and AITC-induced writhing test model [27].Additionally, there have been studies on the antifeedant activity using Plutella xylostella [10], ion channel testing on Nav1.7 and KCNQ2 [10], anti-inflammatory properties [11], cytotoxicity [11], and PTP1B activity [11].In the subsequent sections of the study, an in-depth exploration of the phytochemistry of these compounds is conducted.For detailed compound information, including the compounds' original name, their occurrence, distribution, and publication references, please see Table 1.The bioactivities reported in the references were summarized in Table 2.         2.1.Normal Grayanane-Type Diterpenes (1-97) Normal grayanane diterpenes, a predominant class of diterpenes, have been the subject of extensive research, culminating in the discovery of 97 unique compounds.Characterized by their distinctive 5/7/6/5 tetracyclic framework, these compounds are depicted in Figures 2-4 and elaborated upon in Tables 1 and 2. This section meticulously explores the remarkable identification of these 97 novel grayanane diterpenes, each marked by a unique tetracyclic structure comprising four interconnected carbon rings.Notably, the grayanane diterpenes display a standard 5/7/6/5 configuration within their tetracyclic systems, a configuration that sets them apart from other diterpene structures.This divergence often translates into varied biological properties and potential applications, underscoring the significance of this discovery.
Five analgesic grayanane diterpene glucosides, 40 [24] and 41-44 [17], were isolated and illustrated from leaves of R. auriculatum and R. micranthum, respectively.At a dose of 1.0 mg/kg, compound 40 displayed notable analgesic activity with the acetic acidinduced writhing test.Compound 43 significantly reduced the number of writhes with an inhibition rate of over 50% at the same dosage.Compounds 45-55, isolated by Sun et al. from the leaves of R. auriculatum, and their structures were defined via extensive spectroscopic data analysis and X-ray diffraction analysis [28].Compound 45 represents the first example of a 3α,5α-dihydroxy-1-βH-grayanane diterpenoid, while 49 and 50 are the first examples of 19-hydroxygrayanane and grayan-5(6)-ene diterpenoids, respectively.Compounds 45-55 all showed significant analgesic activities at 5.0 mg/kg in an acetic acid-induced writhing test with an inhibition rate over 50%.From a leaf extract of P. japonica, twelve novel antinociceptive grayanane diterpenoids, 56-67, were isolated and determined by spectroscopic methods as well as X-ray diffraction analysis [29].Compound 56 represents the first example of a 17-hydroxygrayan-15(16)-ene diterpenoid and exhibited potent antinociceptive effects with writhe inhibition rates of 56.3% and 64.8% at doses of 0.04 and 0.2 mg/kg, respectively, with effects comparable to the positive control morphine in the HOAc-induced writhing test in mice.
Molecules 2024, 29, x FOR PEER REVIEW 12 of 26 systems, a configuration that sets them apart from other diterpene structures.This divergence often translates into varied biological properties and potential applications, underscoring the significance of this discovery.Li et al. reported six novel grayanane diterpenes (68-73) from the flowers of R. molle [23], with compound 71 inhibiting 46.0% of acetic acid-induced writhes at a dose of 2.0 mg/kg.Three 1,3-dioxolane conjugates of grayanane diterpenoids (74-76) with 5-hydroxymethylfurfural and vanillin, respectively, were isolated from the flowers of R. dauricum [19].The structures were determined by spectroscopic methods and confirmed by X-ray diffraction analysis.At a lower dose of 0.04 mg/kg, 75 and 76 exhibited more potent activity than morphine in efficacy with inhibition rates of 62.8% and 53.2%, respectively.In chemical investigation of the flowers of R. dauricum, seven highly oxygenated grayanane diterpenes (77-83) were discovered [30], with compound 79 being a notable conjugated grayan-1(5),6(7),9(10)-triene diterpenoid.Among compounds 84-86, purified from the leaves of C. yunnanense [31], 84 and 85 displayed significant anti-inflammatory activity, particularly inhibiting IL-6 release in lipopolysaccharide (LPS)-induced RAW264.7 cells.Zheng et al. identified six new diterpenes (87-92) from the flowers of R. molle as potent analgesics [32].Notably, compound 92 demonstrated remarkable activity, remaining effective even at the dose of 0.04 mg/kg in vivo pain assay screenings.Chai et al. discovered compounds 93 and 94 from the roots of R. micranthum [22], both showing strong antinociceptive effects at doses of 0.1 mg/kg and 0.8 mg/kg, respectively.More recently, three additional minor grayanane diterpenes (95-97) were isolated and elucidated from the leaves of C. yunnanense [33].
In addition, there is a category known as seco-grayanane diterpenes, of which eight varieties have been identified.These compounds are marked by a distinct feature: a structural ring opening, which results in different types, including 1,5-seco-grayanane, 1,10-seco-grayanane, and 1,10:2,3-diseco-grayanane.These are illustrated in Figure 6 and also listed in Table S1.The diversity in the structure of these diterpenes, particularly the placement and number of epoxy groups, contributes to their unique chemical properties and potential applications.The existence of both epoxy-grayanane and seco-grayanane diterpenes within the grayanane family highlights the complexity and variety inherent in natural compounds.The detailed categorization and identification of these compounds, as shown in the figures and tables, provide a valuable framework for further research and understanding of their characteristics and uses.
ing thirty-five in total, are defined by the inclusion of one or two epoxy groups in their structure.The positioning of these epoxy groups varies, occurring between different sets of carbon atoms.This variation leads to a range of configurations, such as C2-C3, C6-C10, C7-C10, C5-C9, C9-C10, C5-C20, C11-C16, and even combinations like C2-C3 with C9-C10, and C2-C3 with C11-C16.These configurations are detailed in Figures 5 and 6 and Table 1.Compound 113, the second example of a 5β,9β-epoxygrayan-1(10)-ene diterpenoid, exhibited noticeable antinociceptive activity at 5.0 mg/kg in the acetic acid-induced writhing test in mice [29].Three 6,10-epoxy grayanane diterpenes (114 [23] and 115-116 [20]) were reported from R. molle and R. micranthum, respectively.Compound 115 represents the first example of a 5αH,9αH-grayanane diterpenoid and a 6-hydroxy-6,10-epoxy grayanane diterpenoid.Compounds 117-122 with diverse epoxy groups were isolated from the flowers of R. dauricum In addition, there is a category known as seco-grayanane diterpenes, of which eight varieties have been identified.These compounds are marked by a distinct feature: a structural ring opening, which results in different types, including 1,5-seco-grayanane, 1,10seco-grayanane, and 1,10:2,3-diseco-grayanane.These are illustrated in Figure 6 and also listed in Table S1.The diversity in the structure of these diterpenes, particularly the placement and number of epoxy groups, contributes to their unique chemical properties and potential applications.The existence of both epoxy-grayanane and seco-grayanane diterpenes within the grayanane family highlights the complexity and variety inherent in natural compounds.The detailed categorization and identification of these compounds, as shown in the figures and tables, provide a valuable framework for further research and understanding of their characteristics and uses.

Grayanane Dimers-Type Diterpenes (143-149)
In the referenced scientific literature, there is a notable report detailing the discovery of seven unique grayanane dimer diterpenes.This significant finding is visually documented in Figure 7 and comprehensively listed in Table 1.These dimer compounds, which represent a unique and complex class of natural products, are characterized by their distinctive structural formation.Specifically, they are formed through the connection of two grayanane monomer units.This connection is achieved via one or two ether bonds, a type of chemical bond that involves an oxygen atom linked to two alkyl or aryl groups.Two new dimeric diterpenes (143 and 144) were characterized from the fruits of R. pumilum, representing the first examples of dimeric grayanane diterpenes with a 3-O-2′ linkage from the Ericaceae family [35].Another novel dimeric diterpene 145 [9] was identified from the leaves of R. molle but with a 13-O-2′ linkage.Compound 146 is a unique dimeric grayanoid, isolated from the flowers of R. molle [23], containing a novel 14-membered heterocyclic ring with a C2 symmetry axis.More recently, Huang et al. reported three new dimers, 147-149, also from the flowers of R. molle [27].The structures were determined by comprehensive spectroscopic data analysis, 13 C NMR calculation with DP4+ analysis, and single-crystal X-ray diffraction analysis [27].Of particular interest is compound 147, a caged dimeric grayanane diterpenoid linked through two oxygen bridges of C-2−O−C-14′ and C-14−O−C-2′, featuring a unique 1,8-dioxacyclotetradecane motif.At a dose of 5.0 mg/kg, compounds 147-149 showed significant analgesic effects, with writhe inhibition rates exceeding 50% in the acetic acid-induced writhing test.Even at a lower dose of 1.0 mg/kg, compound 148 maintained an inhibition rate of 57.3%.Furthermore, in capsaicin-and AITC-induced pain models, compound 148 effectively reduced the nociceptive responses at a dose of 5.0 mg/kg, indicating its potential as a dual antagonist of TRPV1 and TRPA1.

Leucothane-Type Diterpenes (150-163)
Leucothane-type diterpenes represent a fascinating subset within the broader category of grayanane-type diterpenes, known for their unique biosynthetic relationships.These compounds are distinguished by their distinct structural framework, which features a 6/6/6/5 fused tetracyclic ring system.Over the past five years, there has been notable progress in the identification and characterization of these compounds.Fourteen new leucothane-type diterpenes have been discovered and reported, marking a significant advancement in the study of naturally occurring diterpenes.Details are shown in Figure 8 and Tables 1 and 2. Two new dimeric diterpenes (143 and 144) were characterized from the fruits of R. pumilum, representing the first examples of dimeric grayanane diterpenes with a 3-O-2 ′ linkage from the Ericaceae family [35].Another novel dimeric diterpene 145 [9] was identified from the leaves of R. molle but with a 13-O-2 ′ linkage.Compound 146 is a unique dimeric grayanoid, isolated from the flowers of R. molle [23], containing a novel 14-membered heterocyclic ring with a C 2 symmetry axis.More recently, Huang et al. reported three new dimers, 147-149, also from the flowers of R. molle [27].The structures were determined by comprehensive spectroscopic data analysis, 13 C NMR calculation with DP4+ analysis, and single-crystal X-ray diffraction analysis [27].Of particular interest is compound 147, a caged dimeric grayanane diterpenoid linked through two oxygen bridges of C-2−O−C-14 ′ and C-14−O−C-2 ′ , featuring a unique 1,8-dioxacyclotetradecane motif.At a dose of 5.0 mg/kg, compounds 147-149 showed significant analgesic effects, with writhe inhibition rates exceeding 50% in the acetic acid-induced writhing test.Even at a lower dose of 1.0 mg/kg, compound 148 maintained an inhibition rate of 57.3%.Furthermore, in capsaicin-and AITC-induced pain models, compound 148 effectively reduced the nociceptive responses at a dose of 5.0 mg/kg, indicating its potential as a dual antagonist of TRPV1 and TRPA1.

Leucothane-Type Diterpenes (150-163)
Leucothane-type diterpenes represent a fascinating subset within the broader category of grayanane-type diterpenes, known for their unique biosynthetic relationships.These compounds are distinguished by their distinct structural framework, which features a 6/6/6/5 fused tetracyclic ring system.Over the past five years, there has been notable progress in the identification and characterization of these compounds.Fourteen new leucothane-type diterpenes have been discovered and reported, marking a significant advancement in the study of naturally occurring diterpenes.Details are shown in Figure 8 and Tables 1 and 2. Three new leucothane-type diterpenes (150-152) were isolated from the leaves and twigs of R. decorum [25].The structure of compound 150 was confirmed by X-ray crystallography.In the acetic acid-induced writhing test, compound 150 showed a significant effect at a dose of 10.0 mg/kg.Sun et al. reported five new leucothane-type terpenes (153-154 [24] and 155-157 [17]) from R. auriculatum and R. micranthum, respectively.Compounds 155-157 represent the first examples of 15α-hydroxy-leucothane diterpenoids, leucothane diterpene diglucosides, and 9β-hydroxy-leucothane diterpenoids, respectively.These compounds (153-157) all displayed potent analgesic activity in the acetic acid-induced writhing test.Four additional leucothane-type diterpenes (158-159 [23] and 160-161 [18]) were elucidated from R. molle and P. formosa, respectively.Compounds 159 and 160 demonstrated weak analgesic activity in the acetic acid-induced writhing test at Three new leucothane-type diterpenes (150-152) were isolated from the leaves and twigs of R. decorum [25].The structure of compound 150 was confirmed by X-ray crystallography.In the acetic acid-induced writhing test, compound 150 showed a significant effect at a dose of 10.0 mg/kg.Sun et al. reported five new leucothane-type terpenes (153-154 [24] and 155-157 [17]) from R. auriculatum and R. micranthum, respectively.Compounds 155-157 represent the first examples of 15α-hydroxy-leucothane diterpenoids, leucothane diterpene diglucosides, and 9β-hydroxy-leucothane diterpenoids, respectively.These compounds (153-157) all displayed potent analgesic activity in the acetic acid-induced writhing test.Four additional leucothane-type diterpenes (158-159 [23] and 160-161 [18]) were elucidated from R. molle and P. formosa, respectively.Compounds 159 and 160 demonstrated weak analgesic activity in the acetic acid-induced writhing test at 20.0 mg/kg and 5.0 mg/kg, respectively.In an antifeedant assay against Plutella xylostella larvae, compound 161 showed an inhibition effect with a ratio of 52.5% at a dose of 0.5 mg/mL.Lastly, two new leucothanetype diterpenes (162-163) were isolated and identified from P. japonica [21].The structure of 163 was definitively confirmed through X-ray diffraction analysis.Notably, compound 162 exhibited strong analgesic activity with writhe inhibition over 50% at 5.0 mg/kg (i.p.).
2.5.Ent-Kaurane (164-168)-and Seco-Ent-Kaurane (169-173)-Type Diterpenes Ent-kaurane-type diterpenes hold a crucial position in the biosynthesis of grayanane diterpenes, serving as bio-precursors in the intricate chemical pathways leading to the formation of grayanane structures.This role highlights the importance of understanding ent-kaurane-type diterpenes, not only for their inherent chemical properties but also for their contribution to the biosynthesis of other significant diterpenes.In the past five years, there has been a notable advancement in the research and identification of these compounds.Specifically, five ent-kaurane-type diterpenes and five 4,5-seco-ent-kaurane-type diterpenes have been successfully identified and reported.The 4,5-seco-ent-kaurane type represents a variation of the ent-kaurane structure, characterized by a unique opening in the ring structure, specifically between the 4th and 5th carbon atoms, which significantly alters their chemical and potentially biological properties.These discoveries are meticulously detailed in Figure 8 and Tables 1 and 2.

Conclusions
Over the past five years, the field of phytochemistry has experienced a surge of progress, particularly in the study of grayanane diterpenes from the Ericaceae family.This period has been marked by the discovery of 193 novel diterpenes, each characterized by one of fifteen distinct carbon skeletons.This remarkable diversity not only underscores the richness of natural compounds but also highlights the ongoing potential for new and groundbreaking discoveries in this area.A significant focus of these studies has been on bioassay screenings, particularly evaluating in vivo pain activity using models like the acetic acid-induced writhing test.These tests have consistently demonstrated the potent analgesic properties of grayanane diterpenes.Additionally, certain compounds within this group have shown promising activity as inhibitors of PTP1B, suggesting potential therapeutic applications.

Future Perspectives
Looking to the future, the research into grayanane diterpenoids teems with exciting possibilities and opportunities.One critical area for future research is the detailed mechanistic study of these compounds, especially regarding their therapeutic applications [7].Grayanane diterpenes are known for their potent toxicity, which is primarily attributed to their mechanism of action on the sodium channels in the nervous system, leading to a cascade of neurotoxic effects [7,[37][38][39].The limitations of using grayanane diterpenes stem from their narrow therapeutic index, the difficulty in controlling their dose-dependent toxic effects, and the potential for severe adverse reactions, including cardiac issues and central nervous system disturbances.Despite their potent bioactivity, which could be harnessed for therapeutic purposes, these limitations necessitate cautious handling and research to mitigate risks.Understanding the exact mode of action of grayanane diterpenes could revolutionize drug development and treatment strategies.This could lead to the creation of new drugs that harness the unique properties of these compounds, potentially offering more effective treatments for various conditions.
Another promising direction is the application of synthetic biology in the production of diterpenoids [40].This approach could provide a sustainable and scalable alternative to traditional extraction methods from plants.This is particularly crucial for the largescale production of these compounds, especially if they are to be used in therapeutic applications [41].Synthetic biology might not only facilitate the production of these compounds but also enable the creation of novel diterpenoid derivatives with enhanced biological activities or reduced side effects.
Furthermore, exploring grayanane diterpenoids in combination therapies presents a significant opportunity for advancing medical treatments [42,43].By combining these compounds with other drugs, there is potential to harness synergistic effects, which could lead to more effective treatments with fewer side effects.This approach aligns with the growing trend in pharmacology towards personalized medicine and treatment protocols that are more holistic and patient-specific.Moreover, exploring the broader range of biological activities of grayanane diterpenes is another avenue worth exploring.While much of the current research has focused on their analgesic and PTP1B inhibitory properties, these compounds may have other biological activities that are yet to be discovered.Investigating these potential activities could open up new therapeutic areas for these compounds.
In terms of technological advancements, the development of more sophisticated analytical techniques will play a crucial role in future research [44][45][46].Technological advances such as mass spectrometry, NMR spectroscopy, and X-ray crystallography could lead to more detailed and accurate structural elucidation of these compounds.This, in turn, would enhance our understanding of their chemical properties and biological activities.The potential for international collaboration in this field also presents an exciting opportunity.By bringing together researchers from different countries and disciplines, the study of grayanane diterpenes can benefit from a wide range of expertise and resources.Such collaborations could lead to more rapid advancements in the field and sharing knowledge and techniques across borders.
In summary, the study of grayanane diterpenes stands at a pivotal point, with numerous avenues for future research and potential applications in pharmaceuticals and therapeutics.The continued exploration of these natural compounds is poised to significantly contribute to our understanding of natural product chemistry, medicinal chemistry, and pharmacology.As research progresses, grayanane diterpenes will likely play an increasingly important role in the development of new drugs and treatment strategies, highlighting the importance of natural products in modern medicine.

Table 1 .
Compound Names, Plant Sources, Related References, and Year of Publication.

Table 2 .
Compound Names and Their Reported Activities.