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Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid

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  • Traditional Chinese medicine, a cornerstone of Chinese civilization, boasts a rich history spanning thousands of years. The Pleione orchid, renowned for its medicinal properties, is a primary source of Pseudobulbus Cremastrae seu Pleiones (PCsP, 山慈菇). Given its therapeutic effects, there has been a surge in research related to Pleione in recent years, underscoring the need for a comprehensive review of this medicinal plant. Here, the latest studies on the chemical constituents, pharmacological effects, and clinical applications of Pleione are summarized, and the shortcomings of current research presented. This review encompasses advancements made over the past few decades, providing a theoretical foundation for both new drug development and the clinical application of Pleione. It also aids in the effective utilization and industrialization of medicinal and edible orchids, thereby promoting their sustainable development and societal benefits.
  • Asiatic hybrid lily (Lilium spp.) is one of the most important cultivars of Lilium, mainly distributed in East Asia, and is derived from interspecific crosses of the Sinomartagon section[1]. Asiatic hybrids are widely cultivated for landscaping, and it has high ornamental value due to its elegant floral shape and brilliant colors. The domestication of the double-flower is of great value in ornamental plants. In Lilium, the origin type of double-flower is mainly pistil or stamen petalization. Double-flower lily cultivars are favored for their layered flowers and the abortive stamens with no pollen. However, most double-flower cultivars are concentrated in Oriental hybrids. The Asiatic hybrid 'Annemarie's Dream' is a double-flowered cultivar whose stamens convert into petaloid organs in the 3rd whorl in varying degrees, and the other stamens become pollen-aborted degenerate stamens (Fig. 1). Therefore, this unique feature provides the materials for studying the stamen petaloid during the double-flower development and thus offer a basis for double-flower breeding by altering flora phenotype.

    Figure 1.  Floral phenotype of the Asiatic hybrid lily 'Annemarie's Dream'. (a) S1: bud stage. (b) S2: full-bloom stage. Bar = 1 cm.

    Owing to its important role in both plant reproduction and ornamental value, floral organ identity has been well studied in Arabidopsis and Antirrhinum. Bowman et al. proposed that flora organ identity is regulated by the ABC model[2,3]. The flower organs of angiosperms are generally composed of four classes in the whorl, the first floral whorl are the sepals, the second whorl are the petals, the third whorl are stamens and the fourth whorl are carpels[4]. Based on the functional verification of double or triple mutants of flower development[5], each floral organ development is controlled by five types of homeotic genes in the MADS-box gene family, A, B, C, D, and E, individually or in combination. The ABC model confirmed that the A-class genes including APETALA1 (AP1), APETALA2 (AP2) determines the formation of sepals in the first whorl, the combined expression of B-class genes APETALA3 (AP3), PISTILLATA (PI) and A-class genes specify the development of petals in the second whorl, with third whorl stamens being specifically controlled by B and C-class genes AGAMOUS (AG), and the C-class gene acts to form carpels in the fourth whorl[6,7]. In addition, the D-class genes SEEDSTICK (STK) and SHATTERPROOF (SHP) determine the formation of carpels[8]. Subsequent research found E-class genes SEPALLATA (SEP) can interact with other class genes and enable the normal development of four whorls of floral organs[9]. As research has advanced, a floral quartet model has been proposed, which suggests that identity of floral organs is regulated by tetrameric protein complexes of MIKC-type MADS-domain transcription factors A-, B-, C- and E-class of the MADS-box family in Arabidopsis[10]. Petal development is regulated by the interaction of genes in B-class (AP3/PI) with A-class (AP1) and E-class (SEP), while the stamen is specifically controlled by B-class genes in combination with those in E-class and C-class (AG)[11].

    Currently, the research on the regulation of the double-flower formation mechanism is mainly focused on the C-class AGAMOUS ortholog gene. Loss of function of the C-class AGAMOUS gene in Arabidopsis will change the A-class gene expression boundary, from the first and second whorls to the third whorl stamens[12,13]. In some ornamental plants, loss of function or reduced expression of the AG gene will convert stamens into petals[1416]. Ma et al. identified and isolated the homologous AG genes in single-flowered and double-flowered varieties in Kerria japonica, and ectopic expression analysis in Arabidopsis showed that the AG gene of double-flowered flowers do not display the function of the C-class[17]. In addition, François et al. identified two alleles of the A-class gene AP2 in a rose double-flowered mutant, one of the alleles contains a transposon with an insert intron that produces an AP2 mutant with miR172 resistance to regulate the expression of the AG gene to control the formation of double-flowered flowers[18]. In Lilium, an AGAMOUS-like gene was isolated from 'Elodie', which is correlated with the degree of petaloidy of the stamens[16]. Meanwhile, it has been shown that some genes involved in phytohormone signaling transduction and TFs regulation can also work with MADS-box transcription factors to control the development of plant flowers[19,20]. Transcriptome analysis during flower development revealed that MADS-box and hormone signal transduction related genes, play a vital role in the stamen petaloid in Lagerstroemia speciosa and Nelumbo nucifera[21,22].

    The above-mentioned research results indicate that the morphology of stamen petaloid may be regulated by the 'ABCE' model and phytohormone-related genes during double-flower development. However, it is unclear whether the molecular regulatory mechanism of stamen petaloid during flower development is consistent with the 'ABCE' model in Asiatic lily hybrids. In the present study, we conducted RNA-seq to comparatively analyse the transcriptomic differences during the homeotic transformation of stamen into tepal at two floral development stages (i.e., stamen, petaloid stamen and inner tepal). The enrichment analysis of key DEGs indicated that plant hormone signal transduction pathways are strongly involved in stamen petaloid. In addition, the expression patterns of these genes were analyzed in this transition. With the help of weight gene co-expression network analysis (WGCNA), our results provided insight into the molecular regulatory mechanism regulating stamen petaloid. In conclusion, we hypothesized that the significant down-regulation of CL14315.Contig2_All (LiAG), CL3014.Contig2_All (PYL) and CL5627.Contig1_All (GID2) may lead to stamen petaloid in Asiatic hybrids 'Annemarie's Dream'.

    The floral organ shape of ornamental plants is an important factor that determines the ornamental value. Normally, ornamental plants generally have four whorls of floral organs, which are sepals, petals, stamens and carpels. In lilies, sepals and petals are collectively referred to as the tepals. We divided the Asiatic hybrid lily 'Annemarie's Dream' flower development into two periods as S1 (bud stage) and S2 (full-bloom stage) (Fig. 1a & b). The double-flowered lily 'Annemarie's Dream' have three outer tepals and three inner tepals in whorls one and two, petaloid stamen and normal stamen in whorl three, and one pistil in whorl four. In the S1 stage, we discovered that stamens are converted to petaloid organs in varying degrees and the other stamens become pollen-aborted degenerate stamens. The normal tepal, staminode, petaloid stamen and pistil continue to elongate, and the flowering process is completed at the S2 stage. Therefore, 'Annemarie's Dream' provides a good model for research on the molecular mechanism of stamen petaloid formation during double-flower development.

    To further explore the molecular mechanism underlying stamen petaloid phenotype during double-flower development, 18 cDNA libraries at two double-flower development stages were sequenced using a Illumina Hiseq platform. Comparative transcriptomic analysis among bud stage stamen (Budst), bud stage petaloid stamen (Budpest), inner bud tepal (Inbud), full-bloom stage stamen (ST), full-bloom stage petaloid stamen (PEST) and full-bloom stage inner tepal (InTE) were conducted. A total of 185.94 Gb clean data were generated from 18 cDNA libraries, with an average of 10.33 Gb of clean data per sample for further analysis (Supplemental Table S1). After de novo assembly of clean reads using Trinity, 190,488 unigenes were obtained with total length, average length, N50 and GC content of 147,192,422 bp, 772 bp, 1,438 bp and 44.34%, respectively. In addition, seven databases (KEGG, GO, NR, NT, SwissProt, Pfam and KOG) were used to annotate all unigenes to provide gene function information. In total, 97,282 (51.07%) of 190,488 unigenes were annotated. Within these databases, 87,454 genes (45.91%) were annotated in Nr; 57,964 genes (30.43%) in NT; 65,424 genes (34.35%) in SwissProt; 68,882 genes (36.16%) in KOG; 67,991 genes (35.69%) in KEGG; 28,443 genes (14.93%) in GO and 60,776 (31.91%) in Pfam.

    To investigate the transcriptional differences associated with stamen petaloid in double- flowers, we conducted pairwise comparison among inner tepal, stamen and petaloid stamen at two development stages (Fig. 2). In total, 37,549 DEGs were identified at a fold change ≥ 2 and adjusted P-value ≤ 0.001, among which the highest number of DEGs was ST vs PEST with 58,887. In some comparisons (ST vs PEST, ST vs InTE), the down-regulated DEGs were detected more than up-regulated DEGs, and this trend was most notable at the full-bloom stage (Fig. 2a). We determined significant DEGs between ST vs PEST, ST vs InTE, InTE vs PEST at full-bloom stage and Budst vs Budpest, Inbud vs Budpest, Budst vs Inbud at the bud stage by estimating the gene expression level using FPKM, with a log2 fold change greater than 2 and Q-value < 0.001. We found that there were 41,683, 41,343 and 25,824 DEGs between ST vs PEST, ST vs InTE and InTE vs PEST respectively (Fig. 2a). The number of overlapping DEGs detected in ST vs PEST and ST vs InTE but not in InTE vs PEST was 24,057 (Fig. 2b), which might be related to the formation of stamen petaloid. In the comparison of bud stage, there were 24,878, 21,136 and 29,460 DEGs between Budst vs Budpest, Inbud vs Budpest and Budst vs Inbud respectively (Fig. 2c). The results were similar to those in the blooming stage, with a higher number of DEGs for the comparison of stamens vs petaloid stamens/inner bud tepal. Additionally, the largest number of DEGs in the ST vs Budst comparison was 42,059 accompanying the flowering process (Fig. 2d).

    Figure 2.  Analyses of DEGs at two development stages of stamen, petaloid stamen and inner tepal. (a) The number of up- and downregulated DEGs in 9 comparisons. (b) Venn diagram of DEGs in ST vs PEST, ST vs InTE and InTE vs PEST. (c) Venn diagram of DEGs in Budst vs Budpest, Budst vs Inbud and Inbud vs Budpest. (d) Venn diagram of DEGs in ST vs Budst, PEST vs Budpest and InTE vs Inbud. (e) KEGG pathway enrichment analysis of DEGs for ST vs PEST. (f) KEGG pathway enrichment analysis of DEGs for ST vs InTE.

    We performed GO enrichment for function classification of DEGs. The results revealed that 17 terms for biological processes, 15 terms of cellular components and 10 terms for molecular functions were concentrated in all DEGs (Supplemental Fig. S1). KEGG pathway enrichment analysis was conducted to further investigate the major regulatory pathway of DEGs. KEGG classification results indicated that DEGs were mainly enriched in cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems (Supplemental Fig. S2). We focused our research on the KEGG enrichment analysis of DEGs of ST vs PEST and ST vs InTE. The DEGs for ST vs PEST mainly enriched in plant hormone signal transduction (804), phenylpropanoid biosynthesis (808), biosynthesis of amino acids (697) and glycolysis/ gluconeogenesis (382) (Fig. 2e). The DEGs for ST vs InTE mainly enriched in hormone signal transduction (802), phenylpropanoid biosynthesis (772), biosynthesis of amino acids (654) and MAPK signaling pathway (569) (Fig. 2f).

    It has been shown that flower organ development is regulated by phytohormones such as auxin (AUX), abscisic acid (ABA), cytokinin (CK), gibberellin (GA), ethylene (ETH) and jasmonic acid (JA)[20]. In order to investigate the role of phytohormone-related genes in the regulation of stamen petaloid during double-flower development, we screened 65 DEGs involved in plant hormone biosynthesis and signal transduction pathway from all pairwise comparisons (Fig. 3). There were 34 genes involved in auxin pathways, including auxin influx carrier protein (AUX1), the auxin/indole-3-acetic acid protein (AUX/IAA), auxin-responsive protein (GH3), small auxin-up RNA (SAUR) and auxin response factor (ARF). Among them, most of the major homologs of AUX1, GH3, SAUR, ARF were downregulated in both developmental stages of stamen compared with petaloid stamen or tepal, except for Unigene22450_All (AUX1), Unigene11089_All, Unigene29394_All (SAUR), and CL6633.Contig12_All (ARF) which were upregulated. Notably, all IAA were upregulated in stamen, petaloid stamen and inner tepal phenotype, respectively. In the cytokinin signaling pathway, histidine phosphotransfer protein (AHP) and histidine kinase (AHK) were downregulated in stamen compared to petaloid stamen or tepal. A total of three genes involved in the ethylene signaling transduction pathway, among which ethylene-insensitive3 (EIN3, CL140.Contig10_All) and ethylene response factor (ERF, CL15675.Contig2_All, Unigene30251_All) were downregulated in both developmental stages of stamen compared with petaloid stamen or tepal, while ethylene receptor (ETR, CL6025.Contig6_All) and CL15628.Contig1_All (EIN3) were upregulated. Among the DEGs involved in the JA signaling pathway, the jasmonate ZIM domain-containing protein (JAZ) was expressed at a high level in both stages of stamen, whereas lipoxygenase (LOX) and MYC2 were highly expressed in petaloid stamen and tepal in contrast to the expression pattern of JAZ. In the gibberellin biosynthesis and signal pathway, the gibberellin synthesis-related gene (GA203ox, GA20ox) and GA insensitive dwarf1/2 (GID1/2) were downregulated in both stages of stamen compared with petaloid stamen or tepal. Nonetheless, the expression levels of CL452.Contig3_All (GA20ox) and Unigene10534_All (GA2ox) were upregulated. In particular, four pyrabactin resistance-like genes (PYL) involved in ABA signaling pathway were significantly downregulated in both stages of stamen compared with petaloid stamen or tepal.

    Figure 3.  Expression heat maps of DEGs involved in the biosynthesis and signaling pathways phytohormone as auxin, CK: cytokinin, ETH: ethylene, JA: jasmonic acid, GA: gibberellic acid and ABA: abscisic acid. Red and blue represent up- and downregulation in gene expression, respectively. Gene expression level (log10(FPKM+1)) are represented by color gradation.

    A large number of transcription factors (TFs) were identified as DEGs in the regulation of stamen petaloid during both development stages (S1, S2). In our study, we screened out 118 TFs with |log2FC| > 2 to be significant, including MYB family with 38 members, NAC family (20), MADS family (14), Tify family (16), bHLH family (16), mTERF family (12), and bZIP family (2). In the pairwise comparison of stamen, petaloid stamen and tepal at two stages, there were 23 significant TFs amongst 118 DEGs. Their expression profiles revealed TFs that play key roles in stamen petaloid during different stages of flower development (Fig. 4a).

    Figure 4.  Analysis of key transcription factor genes encode DEGs regulating stamen petaloid. (a) The expression heat map of key TFs in two development stages (S1, S2). (b) Phylogenetic analysis of key MADS-box family transcription factors and Arabidopsis-related proteins.

    The DEGs encoding Unigene52058_All, CL6417.Contig1_All, Unigene27099_All (MYB), CL10229.Contig2_All (NAC), CL1706.Contig4_All (bZIP), Unigene31762_All (TIFY) and Unigene489_All (ERF) were downregulated in both development stages of stamen compared with petaloid stamen or tepal. In mTERF family, Unigene774_All (mTERF) was highly expressed in petaloid stamen or tepal at S1/S2 stages. bHLH family CL11633.Contig1_All (PIF4) showed higher expression levels at S1(bud stage) than S2 (full-bloom stage) and was upregulated in stamen compared with petaloid stamen or tepal. As we know, different tetramers of MIKC-type MADS-domain TFs control the identity of floral petaloid organs[10]. Therefore, we conducted phylogenetic and expression pattern analysis of key MADS-box TFs related to stamen petaloid during the development of double-flower (Fig. 4). The phylogenetic analysis showed that MADs-box TFs of Asiatic hybrid lily 'Annemarie's Dream' are classified into six subgroups as A class (SQUA/AP1), B class (AP3/PI), C class (AG) and E class (SEP/AGL2) (Fig. 4b). In the phylogenetic tree, LiAGL6 and LiSEP are clustered into one upper major clade, indicating their close evolutionary relationship, and presumably they both perform the function of E class SEP. Among 14 MADS-box TFs, LiAP1 (CL10060.Contig3_All) and LiAG (CL14315.Contig1_All, CL14315.Contig2_All, CL815.Contig3_All, CL815.Contig4_All) were significantly downregulated in both development stages of stamen compared with petaloid stamen or tepal. Meanwhile, LiAGL6 (CL13859.Contig2_All, CL17687.Contig1_All) and LiSEP3 (CL10355.Contig2_All) have similar expression patterns being upregulated in both development stages of stamen compared with petaloid stamen or tepal. In addition, LiPI (CL4637.Contig2_All, CL4637.Contig3_All) showed higher expression levels at S1 (bud stage) than S2 (full-bloom stage).

    To illustrate further insight into the regulation of stamen petaloid throughout double-flower development stages, we performed WGCNA to gain the gene co-expression networks of key DEGs. A total of 29 gene co-expression modules were identified based on their similar expression profiles (Fig. 5a). Notably, we found that key DEGs in green modules displaying a distinctly different expression pattern which highly expressed in stamen in both stages of stamen but barely expressed in petaloid stamen and tepal. This may indicate the DEGs in this module are closely related to the regulation of stamen petaloid. Therefore, we identified the potential regulatory network for stamen petaloid in the green module (Fig. 5b). There were many genes related to plant hormones and TFs in this module. Firstly, two MADS-box family genes CL14315.Contig2_All (AG) and CL10060.Contig3_All (AP1) were identified as hubs within the potential regulatory network in the green module. Then, we explored the DEGs involved in TFs regulation and phytohormone signaling co-expressed with the hub genes. In this regulatory sub-network, the MYB family has the most members (6), followed by Tify (5), bZIP (4), bHLH (4), AP2-ERF (2) and NAC (1), they may both have an interaction with AG and AP1. Furthermore, we found that CL3014.Contig2_All (PYL) and CL5627.Contig1_All (GID2) as central components of ABA and GA signaling transduction respectively, that had a cross regulation with MADs-box TFs. Moreover, CL6583.Contig1_All (SAUR) and CL3630.Contig2_All (AHK) were also cross regulated by key co-expression TFs. All co-expressed genes (TFs and phytohormone-related DEGs) with MADs were downregulated in both development stages of stamen compared with petaloid stamen or tepal, which suggests that the downregulation of the key genes has a vital role in stamen petaloid during double-flower development.

    Figure 5.  The resolved gene regulatory network of stamen petalod in Asiatic hybrid 'Annemarie's Dream'. (a) Dendrogram showing co-expression modules identified by WGCNA across double-flower development. (b) The regulatory network of key genes involved in stamen petaloid.

    To further verify the quality of RNA-seq data, we performed quantitative real-time PCR to validate the expression patterns of MADS-box genes. As shown in Fig. 6, the expression levels of A-class AP1, B-class PI, C-class AG, E-class SEP and AGL6 are consistent with FPKM values obtained by transcription profiles. The results demonstrate the reliability of RNA-seq data and weight gene co-expression analysis.

    Figure 6.  Validation of the expression patterns of five MADS-box genes. Error bars represent standard deviation (SD) of biological replicates. Bars with different letters indicate significant differences among treatments, P ≤ 0.001, following one-way ANOVA.

    Flower type is one of the most valuable ornamental phenotypes in flowering plants. In Lilium, double-flower cultivars with petaloid stamens are of high commercial value. Based on the morphology of tepal, petaloid stamen and stamen of the Asiatic hybrid cultivar 'Annemarie's Dream', we suggest that the formation of petaloid stamen is caused by transition from stamen to tepal. To investigate the molecular mechanism underlying stamen petaloid, the comparing transcriptome analysis was conducted among stamen, petaloid stamen and tepal at two double-flower development stages. We detected 37,549 DEGs, more of which were downregulated in the comparison of stamen vs petaloid stamen and stamen vs tepal, suggesting that the down-regulation genes were significant in the transformation of stamens into petals (Fig. 2a). Moreover, we found that the number of DEGs in ST vs PEST and ST vs InTE was much larger than those in PEST vs InTE, suggesting that stamen petaloid is more like tepal than stamen. This result was further supported by the Pearson correlation coefficient analysis (Supplemental Fig. S3). Notably, the significant DEGs mainly enriched in plant hormone signal transduction (Fig. 2e), which indicates that the stamen petaloid may be regulated by a complex multilevel regulatory system. Therefore, we explored the more specific transcriptional regulatory networks containing hormone signaling gene and TFs with the help of WGCNA.

    Plant hormones are essential factors affecting flower organ development[23]. In our study, there were numerous phytohormone-related genes involved in biosynthesis and signaling of auxin, cytokinin, ethylene, jasmonic acid, gibberellic acid and abscisic acid, which were differentially expressed across stamen, petaloid stamen and tepal. Among them, biosynthesis and signaling genes of auxin and cytokinin are involved in the formation of floral organs and cell growth and proliferation of petals during flower development[2426]. In our study, the signaling related genes of auxin (AUX1, GH3, SAUR and ARF) and cytokinin (AHK, AHP) were almost downregulated in both stages of stamen compared with petaloid stamen or tepal, indicating its important role in determining the flora organ identity in Asiatic hybrids (Fig. 7). Previous studies have confirmed that auxin has significant activity during plant reproductive development, especially in anthers and filaments[27,28]. Therefore, we inferred auxin may be synthesized more in stamen than in stamen petaloid/tepal. However, we found all IAA genes showed higher expression level in petaloid stamen and tepal than stamen, indicated that these genes as auxin downstream response factors might play important roles in stamen petaloid. It has been shown that when the IAA8 protein is mutated in Arabidopsis, the transgenic plants have abnormally floral organs with short petals and stamens[29]. Furthermore, IAA8 plays its role in the development of floral organs by changes in JA levels probably via its interaction with ARF6/8 proteins, since both ARF6/8 are required for normal JA (jasmonic acid) production. Other studies have also confirmed that hydrogen peroxide dehydrogenase (AOS) and hydrolase synthesis (HPL) together facilitate the metabolism of JA and may lead to the maturation of petaloid stamens[21]. Thus, we hypothesized that that IAA and JA play an important role in regulating flora organ development and promoting the formation of petaloid stamens. Our transcriptome datasets confirmed that most of the genes related to the JA signaling pathway showed a higher expression level in petaloid stamen and tepal than stamen. In addition, PYL genes as ABA signaling receptor were downregulated in both stages of stamen compared with tepal and petaloid stamen, indicated that stamen petaloid is controlled by a complex network of hormonal regulation.

    Figure 7.  Summary of candidate DEGs involved in phytohormone signals and biosynthesis regulating stamen petaloid in the Lilium cultivar ‘Annemarie’s Dream’. The vertical up arrow represents the upregulated genes from stamen to petaloid stamen phenotype. The vertical down arrow represents the downregulated genes from stamen to petaloid stamen.

    Many TF family members, such as MYB, NAC, bZIP, TIFY, bHLH, ERF and mTERF showed significantly distinct expression patterns among stamen, petaloid stamen and tepal at two development stages, suggesting they may participate in the regulation of stamen petaloid. Previous studies have confirmed that GA can promote JA production and high levels of JA can induce MYB expression and thus promote stamen development. Furthermore, MYB has been reported to play a critical role in the development of floral organs and pollens[3032]. In this study, we found that most of the transcription factors were highly expressed in both stages of the stamen, with the largest number of MYB families, so we speculated that key MYB genes might be involved in stamen formation. In addition, the NAC transcription factor gene can be rapidly induced by ethylene and is involved in the regulation of petal size and floral boundary by activating or inhibiting the cell expansion of petals[33,34]. CL10229.Contig2_All, which was identified as NAC-like TFs, is likely involved in stamen development. It has been suggested that mTERF, Tify, MYB-related, bHLH and NAC TFs are probably involved in flower primordium differentiation such as perianth differentiation and stamen differentiation in Erythronium japonicum[35]. bHLH TFs may mediate region, organ, and floral type specific signals in L. speciosa inflorescences[36]. Meanwhile, the abundance of bHLH family TFs PIF4 was regulated by BLADE-ON-PETIOLE (BOP) genes, which have previously been proven to control leaf and flower development in Arabidopsis[37]. We found that CL11633.Contig1_All (PIF4) was upregulated in stamen compared with petaloid stamen or tepal and showed higher expression levels at S1(bud stage) than S2 (full-bloom stage), which suggests that it may play a role in promoting the flowering process and floral organ identity.

    The MADS-box transcription factor plays an important role in plant reproductive development, especially as its homologous proteins are in a pivotal position in the floral organ identity. According to the 'ABCE' model, four whorls of floral organs are regulated by tetrameric protein complexes of MIKC-type MADS-domain transcription factors A-, B-, C- and E-class[10]. Therefore, we focus on the expression patterns of MADS-box genes in different floral tissues during double-flower development. An increasing number of studies have shown that altered expression patterns of AG homologs lead to stamen petaloid in double-flower phenotypes of non-model plants. In our study, we screened 14 significantly DEGs from MADS-box TFs and classified them into four A-, B-, C-, and E-class based on their phylogenetic analysis with Arabidopsis-related genes. Among them, four homeotic LiAG genes showed highest expression levels in stamen compared to petaloid stamen and tepal. Similarly, the decreased expression of the AG ortholog PrseAG led to double-flower formation in Prunus lannesiana[38]. Ectopic expression of EjAG identified in double-flower loquat (Eriobotrya japonica) rescued the development of stamens and carpels from the double-flower phenotype in an Arabidopsis ag mutant[23]. In summary, these studies suggest the destruction of floral organ-determining gene AG may lead to the homeotic transformation of stamen into tepal. In addition, we found that LiAGL6 and E-class gene LiSEP3 were upregulated in both development stages of stamen vs petaloid stamen or tepal, in contrast to the expression pattern of LiAG. That indicates that LiAGL6 and LiSEP3 are involved in the transformation of stamen to tepal.

    Mao et al.[39] applied a strategy using in vivo fluorescence resonance energy transfer (FRET) to find complicated tepal and stamen heterotetrameric complexes in lily and verified lily (Lilium longiflorum). PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, LMADS8 and LMADS9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. In our study, B-class gene LiPI were highly expressed in bud stages and showed higher expression levels in stamen petaloid and tepal than stamen, which indicated that LiPI may form a tetramer with AP1, AP3 and SEP3 to regulate the formation of petaloid stamen.

    Through WGCNA analysis, we discovered the green gene co-expression module that may be involved in the regulation of stamen petaloid. Two MADS-box TFs (LiAG, CL14315.Contig2_All; LiAP1, CL10060.Contig3_All) were identified as hub genes in this module. Centering on these genes, we identified key co-expressed genes that determined floral organ development at a transcriptional level, specifically the stamen petaloid. These genes form a multi-level regulatory network involving auxin, GA and ABA signaling and some key TFs such as MYB. Our transcriptome data provides an insight into the molecular regulatory network underlying stamen petaloid and thereby offering a theoretical basis for double-flower breeding in Lilium.

    Asiatic Hybrids lily 'Annemarie's Dream' cultivars were grown in the nursery of Badaling Forest Farm (Beijing, China). The double-flower cultivars were cultivated in the greenhouse and three parts of the floral organ were collected from the bud stage to the full-bloom stage. Bud stage: Inner bud (Inbud), petaloid stamen (Budpest) and staminode (Budst). Full-bloom stage: Inner tepal (InTE), petaloid stamen (PEST) and staminode (ST). Each sample was obtained from nine flowers or flower buds at two development stages, three of which were used as one biological replicate, for a total of three biological replicates. Samples were immediately flash frozen in liquid nitrogen and stored at −80 °C for RNA extraction.

    Extraction of total RNA from six samples, including three biological replicates were carried out using RNAprep pure Plant Kit (TIANGEN Biotech, Beijing, China) and the RNA integrity number (RIN) of each sample needed to be > 7.3 for cDNA library construction. mRNA with polyA tail was enriched by Oligo (dT) beads and rRNA was removed using DNA hybridization probes. Subsequently the broken short mRNA fragment was used as a template to create cDNA libraries and library quality was assessed on the Agilent 2100 Bioanalyzer and ABI Step One Plus Real-Time PCR System. Illumina sequencing was performed at Illumina Hiseq platform by BGI Co. (Beijing, China).

    Clean reads were obtained by removing low-quality reads, reads containing adapters and poly N and reads with unknown base 'N' content greater than 5%. De novo assembly of clean reads using Trinity v2.0.6 was performed[40], followed by TGICL[41] to cluster the assembled transcripts and remove redundancy to obtain Unigene. Transdecoder v3.0.1 was used to identify candidate coding regions in Unigene by aligning the homologous protein sequences in the SwissProt or Pfam database[42]. For gene functional annotation, the assembled unigene were aligned and annotated using HMMER v3.0[43], BLAST v2.2.23[44], and BLAST 2GO v2.5.0 to seven functional databases as GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), NR (Non-redundant proteins), NT (Nucleotide sequence database), COG (clusters of orthologous groups), Swiss-Prot, and Pfam.

    The gene expression level of each sample was calculated by RSEM v1.2.8[45], based on Fragments Per Kilobase of transcript per Million (FPKM). Differential expression gene analysis was performed based on Poisson distribution, DEGs detection was performed according to the method described in Wang et al.[46]. In order to improve the significance of DEGs, P-values are corrected to Q-values using the strategy employed by Storey & Tibshirani[47]. In this study, we defined genes at Fold Change > 2, Q-value ≤ 0.001 and false discovery rate (FDR) < 0.05 in a comparison were recognized as significant differentially expressed genes.

    Validation of RNA-seq data related to MADS-box TFs using qRT-PCR. The qRT-PCR reactions were performed on the iQTM5 using SYBR. The primers used in this study are listed in Supplemental Table S2. The PCR protocol was initiated at 94 °C for 3 min, followed by 40 cycles of 94 °C for 20 s, 60 °C for 30 s and 72 °C for 30 s. CT values (Cycle threshold) were recorded after completing 40 cycles. The data was obtained from three biological replicates, each of which contains three technical replicates. Relative gene expression was normalized with the lily actin gene as an internal reference and was analyzed using the 2ΔΔCᴛ method[48]. The difference of the mean values for the different treatments were compared by post-hoc least significant difference tests. Values of P < 0.001 were considered to indicate significance. Origin software is used for chart drawing in Fig. 6.

    After discarding relative low expression genes (the FPKM was less than 1 in more than 18 samples), the R package WGCNA[49] was used to identify modules of highly co-correlated gene modules base on the filtered FPKM data. The co-expression modules were obtained using automatic network construction function (block wise modules) with power = 15, minModuleSize = 100, TOMtype was signed. Eigengene value was calculated for each module based on Pearson correlation. The networks were visualized by Cytoscape (v.3.8.2)[50].

    This work was supported by grants from the Opening Foundation of Beijing Engineering Research Center of Rural Landscape Planning and Design (KF2020).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Ji X, Wang S, Wu S, Lan S, Peng D, et al. 2024. Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid. Medicinal Plant Biology 3: e029 doi: 10.48130/mpb-0024-0029
    Ji X, Wang S, Wu S, Lan S, Peng D, et al. 2024. Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid. Medicinal Plant Biology 3: e029 doi: 10.48130/mpb-0024-0029

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Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid

Medicinal Plant Biology  3 Article number: e029  (2024)  |  Cite this article

Abstract: Traditional Chinese medicine, a cornerstone of Chinese civilization, boasts a rich history spanning thousands of years. The Pleione orchid, renowned for its medicinal properties, is a primary source of Pseudobulbus Cremastrae seu Pleiones (PCsP, 山慈菇). Given its therapeutic effects, there has been a surge in research related to Pleione in recent years, underscoring the need for a comprehensive review of this medicinal plant. Here, the latest studies on the chemical constituents, pharmacological effects, and clinical applications of Pleione are summarized, and the shortcomings of current research presented. This review encompasses advancements made over the past few decades, providing a theoretical foundation for both new drug development and the clinical application of Pleione. It also aids in the effective utilization and industrialization of medicinal and edible orchids, thereby promoting their sustainable development and societal benefits.

    • Medicinal plants play a pivotal role in traditional medical systems globally and they are essential for human health[1]. As traditional Chinese medicine (TCM) continues to evolve and the demand for natural remedies grows, the significance of medicinal plants has amplified. In recent years, many studies have discovered several natural active substances with anti-inflammatory, anti-cancer, and antiviral functions, with the majority of these potent ingredients being derived from medicinal plants[26].

      The orchid family is the largest and most diverse family among flowering plants, and many orchids are rare and valuable medicinal materials[7]. In China, approximately 42 genera of orchid are used in traditional medicine. Dendrobium catenatum has effects such as antioxidation, antitumor, immunity enhancement, and blood glucose lowering, among others[8]. Cremastra appendiculata has impacts including heat-clearing, detoxification, lung moistening, cough relief, blood circulation activation, pain alleviation, and swelling reduction[9]. Bletillae rhizoma has effects of swelling reduction, bleeding cessation, and lung moistening[10]. The pharmacological activities of Gymnadenia conopsea includes antioxidant, anti-allergy properties, progenitor cell proliferation promotion, and hepatitis B virus surface antigen inhibition[11]. Pholidota chinensis has the effects of nourishing yin, promoting diuresis, eliminating blood stasis, and pain relief. It is often used for diseases such as dizziness, headache, cough, and hematemesis[12]. The main chemical compositions of the genus Bulbophylum are bibenzyls, phenylpropanoids, phenanthrenes, phenolic acids, glycosides, flavonoids as anti-inflammatory, anti-bacterial, anti-microbial, anti-oxidation, anti-cholinesterase, and other activities[13]. Gastrodia elata has analgesic, anti-epileptic, sedative, memory improvement, neuroprotective, and antioxidant activities, and is widely used in the treatment of nervous system diseases, and cardiovascular and cerebrovascular diseases[14]. Furthermore, Cypripedium henryi, Paphiopedilum malipoense, Cheirostylis chinensis, Coelogyne fimbriata, Liparis distans, and many other orchids are also precious Chinese herbal materials, which have certain effects on treating many diseases.

      The Pleione genus, belonging to the orchid family, is highly valued for its medicinal properties[15]. Pleione grows on rocks or trees and possesses a pseudobulb that stores water and nutrients. The dried pseudobulb of the plant serves as the source of the TCM Pseudobulbus Cremastrae seu Pleiones (PCsP, 山慈菇)[16]. More than 1,400 years ago, in the Tang Dynasty, 'Supplements to Compendium of Materia Medica' recorded the pesticide effect of PCsP. According to the 2020 edition of the 'Pharmacopoeia of the People's Republic of China', PCsP has effects of relieving asthma, cough, inflammation, pain, and stopping bleeding[17]. Modern studies have shown that Pleione is rich in many active ingredients and exhibits pharmacological effects such as anti-tumor, anti-inflammatory, anti-oxidant effects, and reducing blood sugar levels[18]. The Pleione genus comprises approximately 33 species (including nine natural hybrids), predominantly found in China[15,19]. However, not all species are utilized for medicinal purposes. Notably, Pleione bulbocodioides (P. bulbocodioides), and Pleione yunnanensis (P. yunnanensis) have been recognized for their medicinal attributes in traditional Chinese medicine, specifically for the alleviation of asthma and anti-inflammatory effects. Furthermore, the medicinal applications of Pleione species extend beyond China to other Asian nations. In northeastern India, species such as Pleione humulis (P. humulis), Pleione praecox (P. praecox), and Pleione maculata (P. maculata) are employed for treating lacerations, wounds, colds, and liver ailments[20]. Similarly, in Nepal, both P. praecox and P. maculata serve as invigorating tonics and energy enhancers[21].

      The Pleione orchid is a rich source of chemical diversity and has been extensively studied in botanical and pharmacological research[15,20]. In this review, the clinical applications of Pleione species have been critically evaluated for the first time, which has not been done in previous reviews. This is an important step to translate laboratory findings into clinical practice. The latest research progress has also been updated and concludes with the identification of research gaps and future directions, which will provide a progressive perspective. Here, the basic research on the chemical constituents, pharmacological effects, and clinical research of Pleione are reviewed, in order to provide a theoretical basis for the new drug development and clinical application of this plant. The focused review on the clinical applications of Pleione will deepen our understanding of its therapeutic potential and thus make this review a valuable addition to the field.

    • In recent years, with the increasing application of Pleione, the chemical constituents of this plant have been extensively studied by pharmacologists. So far, researchers have isolated several types of chemical components from Pleione, including phenanthrenes, bibenzyls, glucosyloxybenzyl succinate derivatives, flavones, lignans, and other compounds[22] (Fig. 1). These studies provide a reference for the basic research of Pleione and also lay a foundation for its quality control.

      Figure 1. 

      Chemical constituents of Pleione. Phenanthrenes, bibenzyls, glucosyloxybenzyl succinate derivatives, flavones, lignans, and other compounds have been isolated from Pleione.

    • Phenanthrene is a typical compound extracted from Pleione, and 63 phenanthrene derivatives have been isolated from this genus (Table 1). Twenty-six phenanthrenes and dihydrophenanthrenes compounds were isolated from the dried pseudobulbs of P. bulbocodioides[23,24]. Compounds 13−16, and 24 were isolated from Pleione for the first time, and compounds 1−5, 7−10, 13−14, and 20−25 exhibited potent DPPH radical scavenging activity. From the pseudobulbs of P. bulbocodioides, the following compounds have been isolated: shancilin (27)[25], bletilol A-C (28−30)[26], shanciols C-H (31−37)[2730], two new phenanthro [2,3-b] furans (38, 39)[31,32], compounds 40−43[33], and four new pairs of enantiomers (44−51)[34] . Eleven phenanthrenes (52−61)[35,36] and two phenanthrenes (62, 63)[37] have been isolated from the pseudobulbs of P. yunnanensis and P. formosana, respectively. Compounds 24, 25, and 52 are simple dihydrophenanthrenes; compounds 26, 42, 53−55, 57, 58, 62, and 63 are benzyl-substituted dihydrophenanthrenes; compounds 16 and 43 are dihydrophenanthrene dimers; compounds 7, 8, 13−15, 56 are dimers of phenanthrene; compounds 9, 10, and 60 are phenanthrene and dihydrophenanthrene polymers; compounds 11, 12, 27, and 41 are dihydrophenanthrene and bibenzyl polymers; compound 33 is phenanthrene and phenylpropanoid polymer; compounds 44−51 are phenanthrene polymers, and other compounds are dihydrophenanthrene and phenylpropanoid polymers (Fig. 2).

      Table 1.  Phenanthrene compounds from Pleione.

      No. Compound Ref.
      1−11 Bulbocodioidins A−K [23]
      12 (7'S,8'R)-7-hydroxy-7-(4'-hydroxy-3',5'-dimethoxy-phenyl)-8'-hydroxymethyl-5-methoy-9,10,7',8-tetrahydro-phenanthrene-[2,3-b]furan [23]
      13 Monbarbatain A [23]
      14 2,7,2'-trihydroxy-4,4',7'-trimethoxy-1,1'-biphenanthrene [23]
      15 Blestriarene A [23]
      16 Blestrianol A [23]
      17 1-p-hydroxybenzy1-4-methoxy-9,10-dihydrophenanthrene-2,7-diol [23]
      18 1-p-hydroxybenzy1-4-methoxyphenanthrene-2,7-diol [23]
      19 Pleionesin E [23]
      20 Shanciol H [23]
      21 7-hydroxy-7'-(4'-hydroxy-3'-methoxy-phenyl)-4-methoxy-9,10,7',8'-tetrahydrophenanthrene-[2,3-b]furan-8'-yl-methyl acetate [23]
      22 Pleionesin B [23]
      23 Pleionesin D [23]
      24 Hircinol [23]
      25 Coelonin [23]
      26 7-hydroxy-2,4-dimethoxy-1-(p-hydroxybenzyl)-phenanthrene [24]
      27 Shancilin [25]
      28 Bletilol A [26]
      29 Bletilol B [26]
      30 Bletilol C [26]
      31 Shanciol [26]
      32−37 Shanciols C−H [2730]
      38 (4'-hydroxy-3'-methoxyphenyl)-10-hydroxymethyl-11-methoxy-5,6,9,10-tetrahydrophenanthrene[2,3-b]furan-3-ol [31]
      39 Hydroxy-9-(4'-hydroxy-3'-methoxyphenyl)-11-methoxy-5,6,9,10-tetrahydroohenanthrene-azaspiro[2,3-b]furan-10-yl)methylethyl [32]
      40 2,7,2'-didroxy-4,4',7'-trimethoxy-1,1'-biphenanthrene [33]
      41 Phoyunnanin A [33]
      42 (4-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol [33]
      43 4,4',7,7'-tetrahydroxy-2,2'-dimethoxy-9,9',10,10'-tetrahydro-1,1'-biphenanthrene [33]
      44 (9R) bulbocodioidin A [34]
      45 (9S) bulbocodioidin A [34]
      46 (9R) bulbocodioidin B [34]
      47 (9S) bulbocodioidin B [34]
      48 (9R) bulbocodioidin C [34]
      49 (9S) bulbocodioidin C [34]
      50 (10R) bulbocodioidin D [34]
      51 (10S) bulbocodioidin D [34]
      52 Lusianthridin [35]
      53 4,7-dihydroxy-1-(p-hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene [35]
      54 2,7-dihydroxy-4-methoxy-1-(p-hydroxybenzyl)-9,10-dihydrophenanthrene [35]
      55 2,7-dihydroxy-1-(p-Hydroxybenzyl)-4-methoxy-9,10-diphenanthrene [35]
      56 Blestriarene C [35]
      57 1-(p-hydroxybenzyl)-2,7-dihydroxy-4-methoxy-phenanthrene [35]
      58 Shancidin [35]
      59 Shancigusin G [35]
      60 Blestriarene B [36]
      61 Pleionesin A [36]
      62 Pleioanthrenin [37]
      63 (4-hydroxybenzyl)-4,7-dimethoxy-9,10-dihydrophenanthrene-2-ol [37]

      Figure 2. 

      Chemical structural formula of phenanthrene compounds from Pleione.

    • Bibenzyls are abundant in Pleione, and 43 bibenzyls have been isolated from this genus (Table 2). From the dried pseudobulbs of P. bulbocodioides, 30 bibenzyl compounds (64−93) were successfully isolated. Notably, the compound gigantol demonstrated significant DPPH radical scavenging activity[2325,27,28,38,39,41]. Also, compounds 74−77 and 90 were isolated from this genus for the first time[28,31,39]. Additionally, two new bibenzyls (96, 97), along with two known compounds (94, 95), were isolated from the pseudobulbs of P. formosana[37]. From the pseudobulbs of P. yunnanensis, nine bibenzyl compounds (98−106) were also isolated[35,40,41]. Compounds 65, 66, 70, 82−84, and 98−100 are simple bibenzyls; compounds 64, 67, 71−74, 77, 85−89, 92−97, and 101−104 are benzyl substituted bibenzyls; compounds 75, and 76 are bibenzyl and fluorene polymers; compounds 90, 91, 105, and 106 are bibenzyl and glycoside polymers; compounds 68, and 69 are bibenzyl and phenylpropanoid polymers; compounds 78−81 are bibenzylamide polymers (Fig. 3).

      Table 2.  Bibenzyl compounds from Pleione.

      No. Compound Ref.
      64 3,3'-dihydroxy-2,6-bis(p-hydroxybenzyl)-5-methoxybibenzyl [23]
      65 Gigantol [23,24]
      66 Batatasin III [23,38]
      67 Shanciguol [25]
      68 Shanciols A [27]
      69 Shanciols B [27]
      70 3'-O-methylbatatasin III [38]
      71 3,3'-dihydroxy-2-(p-hydroxybenzyl)-5-methoxybibenzyl [39]
      72 3',5-dihydroxy-2-(p-hydroxybenzyl)-3-methoxybibenzyl [39]
      73 3,3'-dihydroxy-4-(p-hydroxybenzyl)-5-methoxybibenzyl [39]
      74 Bulbocodin [39]
      75 Bulbocodin C [28]
      76 Bulbocodin D [28]
      77 Bulbocol [39]
      78 Dusuanlansins A [33]
      79 Dusuanlansins B [33]
      80 Dusuanlansins C [33]
      81 Dusuanlansins D [33]
      82 Bauhinol C [33]
      83 2,5,2',5'-tetrahydroxy-3-methoxybibenzyl [33]
      84 2,5,2',3'-tetrahydroxy-3-methoxybibenzyl [33]
      85 Arundinin [33]
      86 Isoarundinin I [33]
      87 Isoarundinin II [33]
      88 5-O-Methylshanciguol [33]
      89 Blestritin B [33]
      90 2-(4''-hydroxybenzyl)-3-(3'-hydroxyphenethyl)-5-methoxy-cyclohexa-2,5-diene-1,4-dione [31]
      91 6'-(3''-hydroxyphenethyl)-4'-methoxydiphenl-2,2',5'-triol [41]
      92 Batatsin III-3-O-glucoside [41]
      93 Gymconopin D [41]
      94 Arundin [37]
      95 2,6-bis-(4-hydroxybenzyl)-3',5-dimethoxy-3-hydroxybibenzyl [37]
      96 Pleiobibenzynin A [37]
      97 Pleiobibenzynin B [37]
      98 3,5-Dimethoxy-3'-hydroxybibenzyl [35]
      99 Hydroxy-3',5-dimethxoybibenzyl [35]
      100 3,3'-dihydroxy-5-methoxybibenzyl [35]
      101 Shancigusin A [40]
      102 Shancigusin B [40]
      103 Shancigusin C [40]
      104 Shancigusin D [40]
      105 Shancigusin E [35]
      106 Shancigusin F [35]

      Figure 3. 

      Chemical structural formula of bibenzyl compounds from Pleione.

    • Glucosyloxybenzyl succinate derivatives are abundant in Pleione (Table 3). Pleionosides A−J (107−116) were isolated from the pseudobulbs of P. bulbocodioides and P. grandiflora[43,44]. They represent four kinds of acids, (2R)-2-p-hydroxybenzylmalic acid (107−110), (2R)-2-benzylmalic acid (111), (2R, 3S)-2-benzyl tartaric acid (112), and (2R)-2-isobutylmatic (113−116). Eight other glucosyloxybenzyl compounds (117−124) were also isolated from P. bulbocodioides[43]. Shancigusins H−I were isolated from the pseudobulbs of P. yunnanensis (125, 126)[35]. The basic structure of glucosyloxybenzyl succinate derivatives is succinic acid, which often combines with saccharides to form glycosides (Fig. 4).

      Table 3.  Glucosyloxybenzyl succinate derivatives from Pleione.

      No. Compound Ref.
      107 Pleionoside A [43,44]
      108 Pleionoside B [43,44]
      109 Pleionoside C [43,44]
      110 Pleionoside D [43,44]
      111 Pleionoside E [43,44]
      112 Pleionoside F [43,44]
      113 Pleionoside G [43]
      114 Pleionoside H [43]
      115 Pleionoside I [43]
      116 Pleionoside J [43]
      117 Vandateroside II [43]
      118 Grammatophylloside A [43]
      119 Grammatophylloside B [43]
      120 Cronupapine [43]
      121 Gymnoside I [43]
      122 Militarine [43]
      123 Dactylorhin A [43]
      124 Loroglossin [43]
      125 Shancigusins H [35]
      126 Shancigusins I [35]

      Figure 4. 

      Chemical structural formula of glucosyloxybenzyl succinate derivatives from Pleione.

    • Seven flavones have been isolated from Pleione (Table 4). A new prenylated flavone (127), together with three known flavone derivatives (128−130), were isolated from the n-BuOH extract of P. bulbocodioides[45]. Amentoflavone (131), kayaflavone (132)[46], and 5,7-dihydroxy-8-methoxyflavone (133) were isolated from P. bulbocodioides[47]. Compounds 129, 130, and 133 are simple flavones; compounds 127, and 128 are prenylatedflavones; compounds 131, and 132 are bioflavonoids (Fig. 5).

      Table 4.  Flavone compounds from Pleione.

      No. Compound Ref.
      127 3,5,7,3'-tetrahydroxy-8,4'-dimethoxy-6-(3-methylbut-2-enyl)flavone [45]
      128 3,5,3'-trihydroxy-8,4'-dimethoxy-7-(3-methylbut-2-enyloxy) Flavone [45]
      129 Isorhamnetin-3,7-di-O-β-D-glucopyranoside [45]
      130 3'-O-methylquercetin-3-O-β-D-glucopyranoside [45]
      131 Amentoflavone [46]
      132 Kayaflavone [46]
      133 5,7-dihydroxy-8-methoxyflavone [47]

      Figure 5. 

      Chemical structural formula of flavone and lignan compounds from Pleione.

    • Eight lignans have been isolated from Pleione (Table 5). Two isomerized lignan compounds (134, 135), syringaresinol mono-O-β-D-glucoside, lirioresinol, phillygenin, and (E)-p-hydroxycinnamic acid (136−139) were successively isolated from P. bulbocodioides[43,45,4749]. Epipinoresinol (140) and syringaresinol (141) were isolated from the pseudobulbs of P. yunnanensis[35]. Compounds 134, 135, and 138 are simple lignans, and compounds 136, 137, and 139−141 are tetrahydrofuran lignans (Fig. 5).

      Table 5.  Lignan compounds from Pleione.

      No. Compound Ref.
      134 Sanjidin A [48]
      135 Sanjidin B [48]
      136 Syringaresinol mono-O-β-D-glucoside [43]
      137 Lirioresinol [45]
      138 Phillygenin [47]
      139 (E)-p-hydroxycinnamic acid [49]
      140 Epipinoresinol [35]
      141 Syringaresinol [35]
    • In addition to the above groups of compounds, many other compounds have been isolated from Pleione, such as aromatic, steroids, and aliphatic compounds (Table 6).

      Table 6.  Other compounds from Pleione.

      No. Compound Ref.
      142 Tetracosanol [23]
      143 Gallicacid [23]
      144 Tetacosanoic acid-2,3-dihydroxypropyl ester [23]
      145 Chrysophanol [23]
      146 Monopalmttin [23]
      147 Methy(4-OH)phenylacetate [23]
      148 Methyl3-(3-hydroxyphenyl)propionate [29]
      149 5-hydroxymethylfurfural [29]
      150 p-dihydroxy benzene [30]
      151 β-sitosterol [35]
      152 Daucostero [35]
      153 Amber acid [35]
      154 Adenosine [35]
      155 (24R)-cyclomargenyl p-coumarate [37]
      156 (24R)-cyclomargeno [37]
      157 Pleionol [39]
      158 p-hydroxybenzoic acid [41]
      159 p-hydroxybenzaldehyde [41]
      160 Ergosta-4,6,8(14),22-tetraen-3-one [24]
      161 (7S,8R)-dehydrodiconiferyl [43]
      162 Gastrodin [43]
      163 Gastrodioside [45]
      164 Phenl-β-D-glucopyranoside [45]
      165 Hydroquinone [46]
      166 Methyl4-hydroxyphenylacetate [46]
      167 Physcion [46]
      168 4,4'-dihydroxydiphenylmethane [47]
      169 Pleionin [48]
      170 3-hydroxybenzenepropanoic acid [49]
      171 Cinnamic acid [7]
      172 4-(ethoxymethyl)phenol [7]
      173 4-(methoxymethyl)phenol [7]
      174 Methyl3-(4-hydroxyphenyl)propionate [7]
      175 4-oxopentanoic [7]
      176 (E)-ferulic acid [42]
      177 (E)-ferulic acid hexacosyl ester [42]
      178 (Z)-ferulic acid hexacosyl ester [42]
      179 β-daucosterol [42]
      180 Pholidotin [42]
      181 Triphyllol [42]
      182 3-hydroxybenzoic acid [40]
      183 4-(4''-hydroxybenzyl)-3-(3'-hydroxy-phenethyl) furan [40]
      184 3-(3'-hydroxyphenethyl)furan-2(5H)-one [40]
      185 Methyl3-(3'-hydroxyphenethyl)furan-2(5H)-one [40]
    • Being rich in chemical components is an important pharmacological basis for the clinical application of Pleione[17]. With the development of science and technology, researchers have conducted in-depth studies into the pharmacological effects of Pleione in many aspects and found that this plant possesses various functions such as antitumor effect, anti-inflammatory effect, anti-dementia effect, effect on hematopoietic function, anti oxygenation, and hypolipidemic effect (Fig. 6).

      Figure 6. 

      Pharmacological effects of Pleione: antitumor effect, anti-inflammatory effect, anti-dementia effect, affecting hematopoietic function, antioxygenation, and hypolipidemic effect.

    • Numerous studies have shown that Pleione exhibits have an inhibitory effect on many types of tumors, such as colorectal cancer, breast cancer, liver cancer, thyroid cancer, gastric cancer, and so on (Fig. 7)[7,23,5054]. To study the chemical constituents of P. bulbocodioides and find their antitumor bioactive compounds, 12 compounds were obtained and identified from this plant, and the antitumor activity of these constituents was studied using the MTT assay in vitro[50]. The results showed that (8R)-4,5'dihydroxy-8-hydroxymehtyl-3'-methoxydeoxybenzoin exhibited good inhibitory activity against the SKOV-3 cell line. The compounds isolated from P. bulbocodioides have some activity in inhibiting LA795 (mouse lung adenocarcinoma cells)[7]. Compounds such as phoyunnanin A, shanciol F, batatasin III, and p-dihydroxybenzene showed inhibitory effects against LA795. Hydroxy-9-(4'-hydroxy-3'-methoxyphenyl)-11-methoxy-5,6,9,10-tetrahydroohenanthrene-azaspiro[2,3-b]furan-10-yl)methylethyl and p-dihydroxybenzene showed cytotoxic activity against LA795 cells with the IC50 value of 66 and 12 μg·mL−1, respectively. Bulbocodioidins A–D were isolated from the pseudobulbs of P. bulbocodioides[51]. The cytotoxic effects of the isolated compounds were evaluated in MCF-7 cell lines, and bulbocodioidin A, and bulbocodioidin D demonstrated cytotoxic activities. Batatasin III and gigantol inhibited the growth of gastric cancer cells SGC-7901, liver cancer cells BEL-7402, leukemia cells K562, HL-60, melanoma cells M14, and lung cancer cells A569, H460[23]. Bulbocodioidin B exerted cytotoxic activities against liver cancer cells BGC-823, colon cancer cells HepG2, and breast cancer cells MCF-7 with the IC50 values of 2.3, 8.3, and 2.5 μM, respectively. Batatasin III from P. yunnanensis showed activity against the growth of LA795 cells with the IC50 value of 76.21 μM, but only moderate inhibition against BEL-7402 cells and A569 cells. Compound 1,3',5',7-tetrahydroxy-4,7'-dimethoxy-9,9',10,10'tetrahydro-2,2'-biphenanthrene from P. maculata had good inhibitory activity against three tumor cell lines, A549, MCF-7/S, and SKOV-3[52]. Wang et al. investigated the effect of polysaccharides extracted from P. bulbocodiodes on cell proliferation and epithelial-mesenchymal transition (EMT) in ovarian cancer cells and its mechanism[53]. The results demonstrated that these polysaccharides inhibited the proliferation of ovarian cancer cells by decreasing the expression levels of β-catenin and c-myc, hindered the binding of Wnt ligands to transmembrane receptors, and downregulated the expression of downstream genes, such as CyclinD1. The extracts of the dried pseudobulb of Pleione inhibited the PI3K/Akt signaling pathway affected the expression of its downstream tumor suppressor gene Bax and the anti-apoptotic genes Bcl-2 and Caspase-3, thereby inhibiting the proliferation of breast cancer cells and inducing their apoptosis[54]. These results confirm that Pleione possesses an antitumor effect.

      Figure 7. 

      Antitumor effect of Pleione. Compounds bulbocodioidins A, B, D, batatasin III, gigantol, p-dihydroxybenzene, phoyunnanin A, shanciol F, 1,3',5',7-tetrahydroxy-4,7'-dimethoxy-9,9',10,10'tetrahydro-2,2'-biphenanthrene and (8R)-4,5'dihydroxy-8-hydroxymehtyl-3'-methoxydeoxybenzoin isolated from Pleione exhibited inhibitory activity against cancer cells.

    • Researchers studied the acute toxicity, anti-inflammatory, and antibechic tests of P. yunnanensis[18]. Using maximal dosages of the suspension of p. yunnanensis extract, it was found that the dosage was more than 8.0 g·kg−1 (equivalent to 14.16 g·kg−1 of raw pharmacognosy and 106 times the common human dosage). The results indicate that p. yunnanensis has no toxic effects. Through the anti-inflammatory and antibechic tests, p. yunnanensis was proven to exhibit anti-inflammatory and antibechic effects within certain fixed dosages. Hou induced and sampled mouse peritoneal macrophages to determine the inhibitory rate of chemical components in p. yunnanensis on cell growth[55]. The results showed that the compound shanciol D had an obvious anti-inflammatory effect at the concentration of 10 μM. Coelonin, hircinol, and gigantol isolated from P. bulbocodiodes[23], possessed the potential to inhibit the LPS-induced production of nitric oxide in murine macrophage RAW 264.7 cells, with the IC50 values ranging from 9.6 to 35.7 μM. Gigantol exhibited the potent activity toward radical-scavenging and NO production inhibition, reduced inducible nitric oxide synthase mRNA expression, thus exhibited good performance in antifungal action and calmodulin inhibition. Fifteen components exhibiting moderate inhibition of NO production were isolated from P. bulbocodiodes[33,45]. 2,5,2',5'-tetrahydroxy-3-methoxybibenzyl,4,7-dihydroxy-1-(p-hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene and 2,5,2',3'-tetrahydroxy-3-methoxybibenzyl could inhibit NO production induced by LPS in BV-2 cells with the IC50 values of 2.46 and 3.14 μM, respectively. Hydroquinone from P. bulbocodiodes had activity of anti-bacterial and anti-cytotoxicity[7]. At a concentration of 100 μg·mL−1, hydroquinone had a certain inhibitory effect on lung adenocarcinoma cells, LA795. These results show that Pleione may be a promising plant for the development of anti-inflammatory drugs.

    • The benzyl bisaccharide glycosides, which were isolated from P. bulbocodioides, have a significant improvement effect on the symptoms of learning and memory disorders induced by scopolamine in mice[56]. Pleionesin A and batatasin III from P. bulbocodioides exhibited neurotoxic activities on mice hippocampal neurons (SY-SH-5Y) at 10 μM[24]. Lusianthridin, shanciol H, pleionesin E, and gastrodin isolated from P. bulbocodioides and P. yunnanensis were also documented to exhibit activities against neurasthenia, neuroprotection, and epilepsy[57]. In summary, it is believed that Pleione possesses certain anti-dementia effects.

    • With the development of industry, the discovery rate of secondary aplastic anemia caused by exposure to various chemicals, drugs, or rays are increasing year by year. P. bulbocodioides can significantly reduce the toxicity of cyclophosphamide and toluene to bone marrow and can also stimulate bone marrow hematopoietic cells, causing myeloid cell lines to proliferate, which is conducive to the recovery of injured body functions. Hao et al. investigated the effects of the ethyl acetate (EtOAc) extract of P. bulbocodioides on the proliferation and apoptosis of human leukemia K562 and HL-60 cells, as well as the possible apoptosis pathway[58]. These results showed that the EtOAc extract of P. bulbocodioides inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through the intrinsic mitochondrial apoptosis pathway. Researchers observed the pharmacodynamic effects of the 'Shancigu compound' (a compound made from the pseudo bulbs of P. yunnanensis) on mice with aplastic anemia[59]. The results showed that the 'Shancigu compound' group had an obvious function of increasing peripheral hematocytes and strengthening the hematopoietic function of bone marrow. 'Qingduyin', a compound Chinese medicine derived from Pleione can reduce the number of leukemia cells in the liver, spleen, bone marrow, and peripheral blood of L7212 mice, prolong the survival period of the model mice, regulate immune function, and improve the activity of interleukin-2, interleukin-6, tumor necrosis factor A, and their mRNA expression[60]. Li et al. observed interleukin-2 activity and lL-2 RNA expression in L7212 leukemia mice and the influence of the recipe of 'Qingduyin' on them, and they confirmed that the recipe of 'Qingduyin' can treat leukemia in the clinic as a biological response modulator[61].

    • The antioxidant activity of extracts from P. bulbocodioides has been determined by spectrophotometry[23,36]. The results showed that some compounds had scavenging effects on DPPH free radicals and exhibited good antioxidant activity in vitro. At 10 M, monbarbatain A, 2,7,2-trihydroxy-4,4,7-trimethylhydro-1,1-polyphenanthrene, shanciol H, hircinol, coelonin, and dendrobiol showed a certain free radical scavenging ability[7]. Hircinol, batatasin III, and dendrobiol possess antioxidant activity[62]. Coelonin and hircinol exhibited DPPH free radical scavenging activity[63]. Meng studied the antioxygenation effect of polysaccharide from Pleione. 80 SPF Kunming mice (20−22 g) were randomly divided into five groups (blank control group, model group, and polysaccharide with a low, middle, and high dose group)[64]. After 22 d of continuous irrigation, CAT, GSH-PX, MDA, T-AOC, and SOD in serum, liver and kidney were measured. The results showed that compared with the blank control group, polysaccharides in each dose group significantly increased in serum, liver, and kidney of CAT, GSH-Px, and SOD activity (p < 0.01) and T-AOC decreased significantly in serum, liver, and kidney MDA levels (p < 0.01). These results indicate that Pleione has a significant antioxidant effect.

      Research indicates that PCsP, which originates from P. yunnanensis, has a good inhibitory effect on α-glucosidase, and inhibiting α-glucosidase can reduce the postprandial blood sugar peak, adjust blood sugar spike, and improve insulin sensitivity[65]. Further, it has been shown that the polysaccharide from Pleione has a hypolipidemic effect[64]. 70 SPF Kunming mice (20−22 g) were randomly divided into five groups (blank control group, model group, and polysaccharide with a low, middle, and high dose group). While the blank control group was fed with the full price of feed, other groups were fed high-fat feed. After 4 weeks, the levels of ALP, ALT, AST, CREA, DBil, Glu, LDH, LDL-C, TBil, TC, TG, HDL-C, UREA, and UA in the liver were measured. The results showed that the contents of TC and TG in the serum and liver of mice in the high-fat model group were significantly higher than those in the control group (p < 0.01), and the model was successful. Compared with the model group, mountain arrowhead polysaccharides with different dose groups can significantly reduce the serum TG, TC, and LDL-C levels (p < 0.01), and significantly increasee the content of HDL-C (p < 0.01). These studies indicate that Pleione plays an important role in the improvement of diabetes and lipid reduction.

    • In recent years, with the deepening of research into the biological activities of Pleione and its extracts, the compound prescription consisting of PCsP and other Chinese medicines is effective in the treatment of diseases affecting the respiratory system, digestive system, endocrine-metabolic system, and so on, and it holds a broad clinical application prospect.

    • In recent years, with the continuous advancement of research on TCM, it has been discovered that P. bulbocodioides, has been recognized for its therapeutic potential within the TCM framework, particularly in the treatment of respiratory conditions. Studies suggest that P. bulbocodioides is particularly effective in managing respiratory disorders, including bronchitis[66]. Additionally, contemporary research has demonstrated that P. bulbocodioides possesses anticancer properties, offering a novel perspective on its application in the treatment of respiratory system diseases. For instance, a study combined PCsP with simple targeted drug therapy in the control group to assist in the treatment of advanced non-small cell lung cancer (NSCLC)[67]. After two months of treatment, the overall effective rate and Karnofsky performance status of patients in the observation group were significantly higher than those of the control group, indicating that the adjuvant treatment with PCsP can promote improvement in patients' physical function status and has a high clinical remission rate with certain safety[67]. Furthermore, in a separate investigation involving 90 patients with advanced lung cancer, PCsP formulations demonstrated superior efficacy compared to chemotherapy[68]. The study identified qi-yin deficiency as a common syndrome in intermediate and advanced NSCLC, characterized by elements such as yin deficiency, qi deficiency, blood stasis, phlegm, and toxin, pathogenic heat, and pathogenic dampness[69]. PCsP, with the functions of promoting blood circulation, clearing heat and detoxifying, and removing blood stasis, is one of the core drug components in the treatment of NSCLC. The combination of PCsP with simple targeted drug therapy in the control group has established a solid foundation for the application of P. bulbocodioides in antitumor treatment.

    • PCsP is widely used in diseases of the digestive system and plays a key role in the treatment of nasopharyngeal cancer, liver cancer, and colon cancer. Compound Chinese medicine containing P. bulbocodioides is notably effective in reducing fever and facilitating detoxification. This offers potential supplementary therapeutic benefits in the treatment of specific cancers, including lung cancer. For example, a compound Chinese medicine primarily composed of P. bulbocodioides has been observed to significantly decrease body temperature and enhance toxin excretion, thereby benefiting treatments for lung and liver cancer treatments[70]. The study collected prescriptions prescribed for the treatment of nasopharyngeal carcinoma outpatients in the clinic[71], aiming to investigate medication rules with TCM maintenance treatment for nasopharyngeal carcinoma. The findings indicated that the medication frequency of PCsP was very high. The research amassed authenticated initial records from specialized clinical oncology practices, focusing on colorectal cancer treatment. Upon digitization of the data into an analytical framework, a comprehensive review was conducted to discern the prevalence and correlation of frequently utilized pharmaceuticals[72]. The results highlighted that PCsP is consistently utilized as an anti-cancer detoxification agent. However, due to its inherent toxicity, the use of P. bulbocodioides should be supervised by a healthcare professional.

    • PCsP is widely used in the treatment of thyroid cancer, gouty arthritis, and hyperlipidemia due to its pharmacological effects such as anti-tumor, anti-gout, and hypoglycemic properties. Clinical studies have indicated that P. bulbocodioides exhibits promising antitumor effects[30,39] and may be utilized in the treatment of diseases related to the endocrine and metabolic systems. A study analyzed the underlying patterns within TCM prescriptions to elucidate the therapeutic principles governing the treatment of acute gouty arthritis[73]. In a dataset of 732 medicinal formulas, PCsP was noted to have a medication frequency exceeding 300. Additionally, the combination of 'Yi Yi Ren' (薏苡仁) with PCsP can strengthen the spleen and eliminate dampness[73]. An analysis of 254 standardized gout treatments revealed a therapeutic approach that emphasizes the regulation of the middle burner, with the acute phase being addressed by clearing heat and dampness, as well as harmonizing the middle jiao. The analysis of 254 gout treatment protocols underscored a therapeutic focus on middle jiao regulation, especially during the acute phase, where the strategy revolves around clearing heat and dampness to address the 'excess in superficiality'. This approach often involves the substantial use of PCsP to achieve the desired therapeutic effects[74]. Research has indicated that an extract from P. bulbocodioides, also known as PCsP in TCM, can inhibit the proliferation of thyroid cancer cells, such as the SW579 cell line, and promote apoptosis by regulating the expression levels of Bcl-2 protein[75]. This finding suggests that P. bulbocodioides may serve as an adjunctive therapeutic agent in the treatment of certain cancers, including thyroid cancer. In summary, research on the application of P. bulbocodioides in diseases of the endocrine and metabolic system is currently limited, necessitating further clinical studies to verify its efficacy and safety.

    • PCsP also has therapeutic effects on ovarian cancer, breast cancer, tongue cancer, cancer metastasis pain, etc. The underlying pathogenesis of breast cancer is characterized by the interplay of phlegm, stasis, stagnation, and deficiency. Consequently, a therapeutic regimen incorporating 'Trichosanthes kirilowii-PCsP-liquorice' (瓜蒌皮-山慈菇-生甘草) has been employed to prevent the recurrence and metastasis of the disease[76]. PCsP is the main component of Louci Nodule-dissipating Decoction (LCSJ). Clinical studies have found that LCSJ can improve the disease-free survival rate and overall survival time for 1, 2, and 3 years[77]. Another study found that the application of PCsP can enhance the analgesic effect in the treatment of metastatic bone pain and reduce the dose of opiates, such as Oxycontin[78]. Additionally, a variety of traditional Chinese patent medicines have been developed using PCsP as the main ingredient, such as Cigu Xiaozhi Pills (慈菇消脂丸), Shangke Wanhua Oil (伤科万花油), Zhou's Huisheng Pills (周氏回生丸), and Ziyuan Yixiao Pills (紫元益消丸)[7982]. All of these medicines have shown promising therapeutic effects in the treatment of various diseases. It plays a significant role in improving the quality of life for survival patients.

    • As a small genus with only about 33 species, the genus Pleione is highly demanded in the medicinal market and holds great potential for development. However, the following problems persist in the development and application of Pleione (Fig. 8).

      Figure 8. 

      Shortcomings and future development prospects of Pleione. Wild resources of Pleione are seriously damaged and need to be sustainably preserved. The tiny seeds of the Pleione make it difficult for seedlings to germinate independently in the natural state, so it is necessary to establish a rapid and modern technique for the propagation of Pleione. Pleione has a wide range of pharmacological effects and is mainly used in clinical treatments. However, studies on the pharmacological effects and the action mechanism of Pleione have not been fully understood. Therefore, further studies should focus on elucidating the pharmacological effects and the action mechanism of Pleione.

    • The Pleione genus, with its beautiful flowers, is extremely popular among gardeners. The demand for wild resources by breeders and hobbyists is increasing every year, and a large number of wild resources are harvested for private sale annually. At the same time, according to incomplete statistics, about 15,000 kg of Pleione bulbs are dug up and used in medicine, resulting in a yearly reduction of wild resources year-on-year, and their sustainability therefore worrying. Wild resources are crucial for breeding, industrialized development, and application. Consequently, there is an urgent need to conduct technical research focused on key aspects of conservation, sustainable development, and utilization of Pleione. Moreover, there is a lack of in-depth exploration of the current situation regarding Pleione resources. Considering that nearly all Pleione species are assessed as Critically Endangered (CR), Endangered (EN), and Vulnerable (VU) by IUCN criteria[83], there is an imperative need for extensive conservation measures across the genus. For instance, the collection of germplasm resources, particularly from wild populations, is a crucial aspect of conservation. Furthermore, in-situ conservation is vital in protecting and managing these species within their natural habitats, thereby complementing ex-situ conservation efforts that focus on preservation outside of their indigenous environments.

    • The tiny seeds of the Pleione plant make it difficult to germinate seedlings independently in their natural state, so it is necessary to establish a rapid and modern technique for the propagation of Pleione. At present, there are few studies on the rapid propagation technology of Pleione, and researchers mostly use the traditional split method to cultivate P. bulbocodioides. However, this method has the risk of variety degradation due to the accumulation of viruses. Therefore, despite having mastered the basic cultivation technology of Pleione, there is an urgent need to establish an artificial pollution-free, and large-scale cultivation technology system to meet the demands of the pharmaceutical market and achieve an increase in added value.

    • Pleione has a wide range of pharmacological effects and is mainly used in the clinical treatment of breast cancer, liver cancer, stomach cancer, colorectal cancer, and other tumor diseases. However, studies on the pharmacological effects and the action mechanism of Pleione have not been fully understood. Although the clinical effect of Pleione has been proven, the relevant studies largely remain at the animal or cell experiment level. Therefore, further studies should focus on elucidating the pharmacological effects and the action mechanism of Pleione and it is also of great significance to carry out large-scale, randomized controlled clinical studies, and systematic evaluation for the clinical application of Pleione and its new drug development.

      • This review was supported by the National Natural Science Foundation of China (Grant No. 32400317) and the Fujian Provincial Natural Science Foundation of China (Grant No. 2024J01425).

      • The authors confirm contribution to the paper as follows: study conception and design: Liu Z, Ji X; data collection: Wang S, Wu S; analysis and interpretation of results: Zeng D, Peng D; draft manuscript preparation: Ji X, Lan S, Zeng D. All authors reviewed the results and approved the final version of the manuscript.

      • Data sharing is not applicable to this article as no new data were created or analyzed in this study.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (8)  Table (6) References (83)
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    Ji X, Wang S, Wu S, Lan S, Peng D, et al. 2024. Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid. Medicinal Plant Biology 3: e029 doi: 10.48130/mpb-0024-0029
    Ji X, Wang S, Wu S, Lan S, Peng D, et al. 2024. Review on chemical constituents, pharmacological activities, and clinical applications of Pleione orchid. Medicinal Plant Biology 3: e029 doi: 10.48130/mpb-0024-0029

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