Chemical Constituents and Biological Activity Profiles on Pleione (Orchidaceae)

Pleione (Orchidaceae) is not only famous for the ornamental value in Europe because of its special color, but also endemic in Southern Asia for its use in traditional medicine. A great deal of research about its secondary metabolites and biological activities has been done on only three of 30 species of Pleione. Up to now, 183 chemical compounds, such as phenanthrenes, bibenzyls, glucosyloxybenzyl succinate derivatives, flavonoids, lignans, terpenoids, etc., have been obtained from Pleione. These compounds have been demonstrated to play a significant role in anti-tumor, anti-neurodegenerative and anti-inflammatory biological activities and improve immunity. In order to further develop the drugs and utilize the plants, the chemical structural analysis and biological activities of Pleione are summarized in this review.


Introduction
Orchidaceae is one of the largest family of flowering plants. There are about 42 genus that are used for traditional medicine in China, but thus far, no phytochemical investigation has been conducted on 70% of them [1]. As one of unexplored medicinal orchid [2], Pleione contains about 30 species in the habitats of terrestrial, epiphytic or lithophytic, among which 12 are endangered [3]. It mainly distributes in China, Vietnam, Burma, Bangladesh and the Northeast Indian at elevation of 600-4200 m [4]. China is the central region, with 23 species distributed here and 12 of them are endemic [5][6][7][8].

Glucosyloxybenzyl Succinate Derivatives
The Pleione is also rich in glucosyloxybenzyl succinate derivatives [41]. The succinic acid is the basic structure and it often combines with saccharides to form glycosides (102-124) ( Table 7 and Figure 3). The biological studies indicated that the glucosyloxybenzyl succinate derivatives compounds were documented to exert significant activities against delaying aging and improving learning and memory ability of aging mice [42]. Cui [43] used the succinic acid derivatives as an indicator compound for High Performance Liquid Chromatography (HPLC) content determination. The result indicated that the 117 and 118 can be used to distinguish three sources of TCM shan-ci-gu. Lv [44] established the HPLC fingerprint analysis of the P. bulbocodioides via measuring the content of the indicator compound 117. The similar values in the ten producing areas were all more than 0.980 of Chinese medicine. The relative retention time (in the fingerprints was similar, but the Relative Standard Deviation (RSD) values of the relative peak areas were quite different. This was assumed to be the effects of the wild environment and growth years. On account of the difficulty to obtain high-polarity compounds 117, 118, 123 and 145, Wang [45]

Other Compounds
Other compounds consist of seven flavones (125-131), eight lignans (132-139) and 44 others (140-183) ( Table 8 & Figure 4). The flavones contained three simple flavones (125-127), two prenylated flavones (128, 129) and two biflavonoids (130, 131). The lignans consist of three simple lignans (132-134) and five tetrahydrofuran lignans (135-139). Yuan [40] isolated biflavonoids 131 from Pleione for the first time in 2012. Li [16] isolated two isomerized lignan compounds 132 and 133 from P. bulbocodioides in 1997. The pseudobulbs of P. formosana have been used as one of the substitute of Shan-ci-gu [46,47]. However no phytochemical investigation was performed on it. Thus, Shiao [24] began the chemical research in 2009 and it was the first time to isolate the cycloartane triterpenoid compound 167 from the natural product. Yang [48] analyzed the chemical compounds of the P. bulbocodiodes, P. yunnanensis and P. limprichtii from fifteen producing areas by High Performance Liquid Chromatography Diode Array Detection (HPLC-DAD). The Cluster Analysis and Principal Component Analysis were used for quality evaluation, but the compounds corresponding to the chromatographic peak were not determined.

Other Compounds
Other compounds consist of seven flavones (125-131), eight lignans (132-139) and 44 others (140-183) ( Table 8 & Figure 4). The flavones contained three simple flavones (125-127), two prenylated flavones (128, 129) and two biflavonoids (130, 131). The lignans consist of three simple lignans (132-134) and five tetrahydrofuran lignans (135-139). Yuan [40] isolated biflavonoids 131 from Pleione for the first time in 2012. Li [16] isolated two isomerized lignan compounds 132 and 133 from P. bulbocodioides in 1997. The pseudobulbs of P. formosana have been used as one of the substitute of Shan-ci-gu [46,47]. However no phytochemical investigation was performed on it. Thus, Shiao [24] began the chemical research in 2009 and it was the first time to isolate the cycloartane triterpenoid compound 167 from the natural product. Yang [48] analyzed the chemical compounds of the P. bulbocodiodes, P. yunnanensis and P. limprichtii from fifteen producing areas by High Performance Liquid Chromatography Diode Array Detection (HPLC-DAD). The Cluster Analysis and Principal Component Analysis were used for quality evaluation, but the compounds corresponding to the chromatographic peak were not determined.

Biological Activities
Previous studies showed that the compounds extracted from P. bulbocodiodes, P. yunnanensis and P. formosana exerted anti-tumor, anti-neurodegenerative, anti-inflammatory anti-oxidation activities. That is why Pleione has been gaining increasing attention. The Pleione's biological activities are tightly related to the traditional efficacy of "curing fever, detoxifying the body, mitigating the swelling and cleaning the blood stasis" in Chinese Pharmacopoeia [13]. Research on biological activities will establish a foundation for the further pharmacological researches and enlighten the drug discovery for anti-tumor usage.

Anti-Tumor Activity
The biological activities of Pleione can be attributed primarily to the phenanthrenes and bibenzyls. Among those activities, that against tumors was the most significant. Liu [37] proved that the ethyl acetate extract of P. bulbocodiodes had a certain inhibitory effect on mice cancer cells LA795, while the petroleum ether extract only had an inhibition rate of 75.58% at 800 µg·mL −1 , but no remarkable inhibition at 400 µg·mL −1 and below. However, the n-butanol extract did not exert inhibitory at all. This result laid the foundation for the later chemical compounds study, and regarded the ethyl acetate as key fraction for research. The compounds such as 26, 34, 44, 58, 60 and 153 were demonstrated certain inhibitory effects against LA795 at 100 µg·mL −1 . Liu [37] confirmed that 34 and 153 showed the cytotoxic activity against LA795 cells with IC 50 value of 66 and 12 µg·mL −1 . Compound 58 exhibited cytotoxic activity and anti-allergic activity. Wang [33] found that the bibenzyls 58 and 61 isolated from P. bulbocodiodes significantly inhibited the growth of leukemia cells K562, HL-60, liver cancer cells BEL-7402, gastric cancer cells SGC-7901, lung cancer cells A569 [50], H460 and melanoma cells M14. Wang's group [32,34] indicated that 58 isolated from P. yunnanensis, performed strong activity against the growth of LA795 cells with IC 50 value of 76.21 µM, but only moderate inhibition against A569 cells and BEL-7402 cells. Compound 40 was shown to exert moderate cytotoxic activity against A569 cells. 9 µM, respectively. It suggested that the stereochemistry of 9(10)H-phenanthren-10(9)-one is of great significance to the cytotoxic activity. Tumor cell invasion and metastasis determined the prognosis of cancer patients [51]. In a word, a number of studies confirmed that the compounds isolated from the Pleione have an optimistic effect on anti-tumor treatment.

Anti-Neurodegenerative Activity
Glycosides were found to inhibit the proliferation of tumor cells, which is meaningful for anti-tumor therapy [52]. The glucosyloxybenzyls were subjected to evaluation for learning and memory deficits of mice caused via scopolamine and D-Gal + NaNO 2 [53]. Zhang [54] discussed that the Dactylorhin B, 117, 118 and 120 isolated from Coeloglossum viride var. bractestum were demonstrated to exert activities of anti-apoptosis, promoting intelligence and delaying aging. The P. bulbocodiodes consists of the three components mentioned above, except Dactylorhin B [27]. 145 was documented to exhibit activities of neuroprotective, neurasthenia and epilepsy [55]. 29 and 58 performed certain neurotoxic activities of mice hippocampal neurons (SY-SH-5Y) at 10 −5 M [25]. In addition, 2, 32 and 39 indicated significant neurotoxic activity at 10 −5 M [45]. Han [27] reported the hepatoprotective activity of glucocopyloxybenzyl succinate derivatives for the first time in 2019. These neuroprotective effects may be related to the management of antioxidants, malondialdehyde (MDA), glutathione (GSH) levels as well as the improvement of adenosine triphosphatase (ATPase) [56]. The active metabolite of APAP was reported to deplete the glutathione and initiate mitochondrial oxidative stress. Moreover, the reactive oxygen species (ROS) produced during the latter process would destroy the normal function of the mitochondria, ultimately leading to the death of necrotic cell [57][58][59]. These studies fully clarified the pharmacodynamic basis of the Pleione and laid a material foundation for anti-dementia activity.

Anti-Inflammatory and Anti-Oxidation Activity
Some compounds of Pleione exert activities of anti-bacterial and anti-inflammatory (Table 9). Wang [25,33] illustrated that 1 significantly inhibited NO production in mice peritoneal macrophages at 10 −5 M. Compounds 1 and 3 had strong inhibition activity on NO production. Compounds 3 and 61 showed good performance in calmodulin inhibition and antifungal action. Li [28,29] suggested that the compounds of 4, 63 and 64 significantly inhibited NO production induced via LPS in BV-2 cells with IC 50 values of 5.44, 2.46 and 3.14 µM, respectively. They may be the promising compounds for the development of anti-inflammatory drugs . However 9, 25, 26, 58, 62, 73, 74, 75, 81, 82, 86 and 117 only exhibited moderate inhibition on NO production. Liu [49] suggested that 170 was documented to have strong activities of anti-cytotoxicity and anti-bacterial.

Others
In addition to the anti-tumor, anti-neurodegenerative, anti-inflammatory and anti-oxidation activities, the Pleione also exhibited activities of inhibiting antigen-induced degranulation, free radical scavenging as well as anti-oxidant. Wang [26,34] proved that 58, 69, 70, 76 and 81 were shown the activity of antigen-induced degranulation in RBL-2H3 cells [60]. The inhibition efficiency of 58, 69 and 70 was between 65.5% and 99.4%.

Conclusions
The chemical investigation of the Pleione has attracted much attention around the world and some breakthrough progress has been made. Up to now, the family of the compounds had become more and more abundant, especially 9(10)H-phenanthren-10(9). This can not only provide significant evolutionary and chemotaxonomic knowledge of the genus Pleione, but also enlighten the further development and utilization of new drugs.
The future important focal points on the Pleione researches are summarized as follows. Firstly, the research range of species of the genuns Pleione need to be widened except for the P. bulbocodioides, P. yunnanensis and P. formosana in order to seek for novel substitutes. The mechanism of the biological activity should be figured out to shine more clear therapy pattern. Secondly, water-soluble and fat-soluble extracts are necessary to be explored. Thirdly, research needs further progress for clinical application to serve for the patients. Lastly, there needs to be immediate scientific protection for Pleione plants because of their endangered status.

Conflicts of Interest:
The authors declare no conflict of interest.