Advances in Omics Studies, Biological and Clinical Potentialities of Cistanches Herba (Rou Cong Rong)

  

    
 
    
 
    
C. deserticola 
    
C. tubulosa 
    
C. salsa 
    
C. sinensis 
    
References
  
  

    
Total size (bp)
    
Plastomes
    
102,657
    
94,123
    
101,776
    
87,707
    
[6]
  
  

    
Cp genome
    
109,495
    
75,375
    
111,710
    
111,500
    
[8,9]
  
  

    
Mit genome
    
1,860,774
    
3,978,341
    
1,708,661
    
-
    
[11]
  
  

    
LSC

      (bp)
    
Plastomes
    
48,350
    
31,017
    
46,579
    
26,435
    
[6]
  
  

    
Cp genome
    
48,352
    
32,470
    
50,497
    
52,005
    
[8,9]
  
  

    
Mit genome
    
-
    
-
    
-
    
-
    
[11]
  
  

    
IRs

      (bp)
    
Plastomes
    
45,507
    
54,559
    
46,729
    
49,407
    
[6]
  
  

    
Cp genome
    
30,352
    
6,593
    
30,389
    
27,458
    
[8,9]
  
  

    
Mit genome
    
-
    
-
    
-
    
-
    
[11]
  
  

    
SSC

      (bp)
    
Plastomes
    
8,800
    
8,547
    
8,468
    
11,865
    
[6]
  
  

    
Cp genome
    
398
    
29,719
    
435
    
4,579
    
[8,9]
  
  

    
Mit genome
    
-
    
-
    
-
    
-
    
[11]
  
  

    
GC content (%)
    
Plastomes
    
37.95
    
36.53
    
37.26
    
36.95
    
[6]
  
  

    
Cp genome
    
36.27
    
34.95
    
36.11
    
36.75
    
[8,9]
  
  

    
Mit genome
    
44.59
    
44.57
    
44.52
    
-
    
[11]
  
  

    
Total number of genes
    
Plastomes
    
89
    
72
    
89
    
68
    
[6]
  
  

    
Cp genome
    
60
    
53
    
61
    
60
    
[8,9]
  
  

    
Mit genome
    
82
    
126
    
75
    
-
    
[11]
  
  

    
Total number of protein coding genes
    
Plastomes
    
27
    
22
    
20
    
23
    
[6]
  
  

    
Cp genome
    
21
    
20
    
21
    
21
    
[8,9]
  
  

    
Mit genome
    
37
    
65
    
41
    
-
    
[11]
  
  

    
tRNA
    
Plastomes
    
30
    
26
    
30
    
22
    
[6]
  
  

    
Cp genome
    
29
    
25
    
28
    
28
    
[8,9]
  
  

    
Mit genome
    
39
    
58
    
31
    
-
    
[11]
  
  

    
rRNA
    
Plastomes
    
4
    
4
    
4
    
4
    
[6]
  
  

    
Cp genome
    
4
    
4
    
4
    
4
    
[8,9]
  
  

    
Mit genome
    
5
    
4
    
3
    
-
    
[11]
  
  

    
Cp:    Chloroplast; Mit: Mitochondrial; LSC: Large Single-Copy region; SSC:    Single-Copy region; IRs: Inverted Repeats; C. deserticola: Cistanches    Deserticola; C. salsa: Cistanches salsa; C. sinensis: Cistanches sinensis; C.    tubulosa: Cistanches tubulosa; rRNA: Ribosomal RNA; tRNA: Transfer RNA 
  

Table 1: Characteristics of the four Cistanche herba organelle genomes.

Chloroplasts (Cp) are the places where plants perform photosynthesis and have attracted more attention than other plastids because of their important role in plant physiological development [7]. The chloroplasts genome of C. deserticola is 109,495 bp in length, consisting of one LSC (48,352 bp), one SSC (398 bp), and two IRs (30,352 bp) [8]. It contained 27 protein-coding genes, four rRNA genes, and 29 tRNA genes. Additionally, genes required for photosynthesis suffer from gene loss and pseudogenization, except for psbM [8]. As for the other Cistanche herba, the chloroplasts genome size was 111,710 bp in C. salsa, 111,500 bp in C. sinensis, and 75,375 bp in C. tubulosa [9]. Almost all genes related to photosynthesis were pseudogenized or lost, with the most severe loss occurring in C. tubulosa [9]. This suggests that Cistanche herba cannot photosynthesize independently, and its photosynthesis-related genes or protein products may come from the host Haloxylon ammodendron. Mitochondria (Mit) are essential for various metabolic processes, such as cellular respiration and ATP synthesis [10]. The mitochondria genome of C. tubulosa was significantly larger than other species (3,978,341 bp). The mitogenomic lengths of the C. deserticola and C. salsa were 1,860,774 bp and 1,708,661 bp, respectively [11]. It is worth mentioning that C. tubulosa has a higher number of genes than C. deserticola and C. salsa [11].

Gene Expression Profiles

For non-model organisms without reference gene sequences, transcriptome data can be used to discover gene expression profiles and biological characteristics of species on a larger scale [12]. At present, the nuclear genome data of Cistanche herba have not been published, but their gene expression profiles are crucial to elucidate its parasitic mechanism, compound synthesis pathway, and environmental adaptation.

Li Y et al. performed deep transcriptome sequencing in fleshy stem of C. deserticola using Illumina HiSeq2000 platform [13]. Using trinity assembler, they obtained 95,787 transcript sequences ranging from 200 bp to 15,698 bp, having an average length of 950 bases and the N50 length of 1,519 bases. 30098 identified actively expressed transcripts were annotated to a wide range of GO categories and KEGG pathways, such as terpenoid backbone biosynthesis, phenylpropanoid biosynthesis, and carotenoid biosynthesis, indicating that active metabolic processes were underway in the C. deserticola stem tissue [13]. A recent study by Hou et al. performed the full-length transcriptome sequencing and gene expression profiling of C. tubulosa using PacBio combined with BGISEQ-500 RNA-seq technology [14]. A total of 237,772 unique transcripts were obtained, ranging from 199 bp to 31,857 bp. Among the unique transcripts, 188,135 (79.12%) transcripts were annotated [14]. It is worth mentioning that 1080 transcripts were annotated for 22 enzymes related to PhGs biosynthesis [14].

Synthesis of Important Compounds

Traditional natural extraction or artificial chemical synthesis methods are difficult to meet the needs of scientific research and new drug development. Genomic and transcriptomic studies could be used to identify key enzyme coding genes of specific metabolic pathways and optimize these pathways by improving the expression of key enzymes encoding genes to obtain the required components [15]. Based on de novo transcriptome of C. deserticola, PhGs has two different biosynthesis pathways and 17 enzyme genes [13,14]. The possible post-caffeic acid/ferulic acid reaction process was published for the first time, in which the caffeic/ferulic acid would be first oxidized into phenylpyruvate derivate, then the carboxyl group was removed by decarboxylases, and finally, aldehyde group was converted back into alcohol group by dehydrogenase [13,14]. Another is based on phenylalanine metabolism pathway, in which the phenylalanine to phenylethanol was achieved by a known ‘Enrlich pathway’, then phenylethanol is converted to phenylethanol aglycone by monooxygenase or methyltransferase, and further phenylethyl alcohol glycoside is synthesized [13]. The complete picture of lignin biosynthesis pathways in C. deserticola was also presented by Li YL et al. [13], in which the lignin monomers are biosynthesized from phenylalanine through a series of enzymatic reactions, including hydroxylation, methylation, reduction, and oxidative polymerization process [16,17]. Phenylalanine Ammonialyas (PAL) is the first key enzyme in this pathway to convert phenylalanine to cinnamic acid by non-oxidative deamination [18,19]. 4-Coumarate-CoA Ligase (4CL) and trans-Cinnamate 4-Monooxygenase (CYP73A) subsequently convert cinnamic acid to coumaroyl-CoA. Finally, key enzymes Cinnamoyl-CoA Reductase (CCR), shikimate o-Hydroxycinnamoyl Transferase (HCT), and Ferulate-5-Hydroxylase (F5H) regulate the synthesis of different types of lignin by coumaroyl-CoA [20,21].

Biological Activities

Neuroprotection effects: AD is an irreversible and progressive neurodegenerative disorder whose pathogenesis is complex and not completely clear, including oxidative stress, mitochondrial abnormalities and neuroinflammation [22]. In vitro experiments, PhGs of Cistanche herba could reduce oxidative stress, scour free radicals, inhibit neural cell apoptosis, and promote neural cell growth and repair [23-25]. C. Tubulosa aqueous Extract (CTE) could improve memory loss in AD-like rats [24]. Specifically, Echinacoside (ECH), and Acteoside (ACT), the major components of CTE [26], could significantly improve the learning and memory ability of AD animal models, and the mechanism is related to inhibiting neural cell apoptosis and reducing oxidative stress [23,27-30]. Additionally, PhGs of Cistanche herba could regulate brain energy metabolism by regulating insulin signaling [31], and ACT is preferable to total phenylethanoid glycosides in anti-AD [32].

The typical neuropathological features of PD are degeneration of dopaminergic neurons in the substantia nigra [33], accompanied by chronic neuroinflammation [34]. Neurotoxic substances produced by activated microglia are key molecules that mediate the occurrence. ECH of Cistanche herba could significantly inhibit the activation of microglia cells induced by inhibiting the inflammatory signaling pathway NLRP3/CASP-1/IL-1β [35]. Additionally, ECH showed neuroprotective effects on PD pathological mice via regulating the IL-6/JAK2/STAT3 pathway to inhibit the activation of microglia [36]. ACT effectively alleviated rotational behavior in PD rats, and its anti-PD mechanism is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and the activation of the Nrf2/ARE signaling pathway [37-39].

PhGs from C. tubulosa could regulate neuroinflammation via microglial M1-M2 polarization and alleviate ischemic stroke caused by Middle Cerebral Artery Occlusion (MCAO) [40]. PhGs from C. deserticola could improve the stroke through enhancing endogenous neural stem cells proliferation via activating Wnt/β-catenin signaling pathway [41]. Therefore, Cistanche tubulosa and C. deserticola could be a candidate therapeutic agent or supplements for treating neuroinflammation related to ischemic stroke. Specifically, ACT could alleviate ischemic stroke by inhibiting microglial HMGB1/TLR4/NLRP3 signaling pathway in vivo [42]. Vascular Dementia (VD) is a severe cognitive disorder mainly caused by ischemic stroke. PhGs of Cistanche herba exerts its learning and memory-promoting effects in rat models of VD by weakening the accumulation of toxic proteins (Aβ and p-tau) and promoting neuronal cytoskeleton regeneration [43]. ECH could exert anti-VD activity by upregulating the expression of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) in the hippocampus of VD rats and alleviating the ischemic injury of neurons [44].

Gastrointestinal Tract Protection Effects

Cistanche herba extract could significantly improve the constipation of yang deficiency constipation rats and its mechanism may be related to the regulation of gastrointestinal hormone levels [45]. It could effectively improve the water holding capacity of feces, which is conducive to increasing the excretion of mice [46]. Furthermore, C. deserticola extract could improve defecation via SCF/C-kit signaling pathway [47]. In addition, CTE could regulate the composition of intestinal microbiota in rats [48] and restore their disordered intestinal microbiota [49]. Moreover, CTE could restore the growth of lactic acid bacteria and then regulate the structure of intestinal microbiota in mice with intestinal diseases [50]. Polysaccharide, as the main active ingredient of CTE [51], also played intestinal regulatory function mainly by maintaining the balance of intestinal microbiota composition, improving its metabolic function, or synergistic interaction with intestinal microbiota mediated drugs [52]. Additionally, alditol extract from C. Aeserticola (CDAE) attenuate functional constipation by regulating bile acid metabolism. It could increase the expression of CYP8B1, FGF15, TGR5, and FXR, thereby modulating bile acid synthesis and enterohepatic circulation [53].

Anti-Osteoporosis Effects

Osteoporosis is a chronic disease characterized by deterioration of the bone structure and low bone mineral density [54]. Many studies have demonstrated that C. deserticola extract have significant anti-osteoporosis activity [55-57]. Ethanol extract of C. Deserticola (CDHE) improves bone loss in Postmenopausal Osteoporosis (PMOP) mainly through regulating lipid metabolism pathways in vivo [58]. ECH and ACT from C. tubulosa have shown significant anti-osteoporosis activities in the rat model of osteoporosis combined with AD [59]. ECH and ACT could significantly improve the bone quality and total bone mineral density of the femur in rats and promote bone formation and inhibit bone resorption [60]. ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis [61]. In addition, Cistanoside A is able to promote bone formation and prevent bone resorption by interfering with NF-?B and PI3K/Akt pathways [62]. Moreover, total glycosides and polysaccharides from C. deserticola could promote osteogenesis formation and improve bone microstructure damage in mice by activating the Wnt/β-catenin signaling pathway [63].

Anti-Depressant Effects

The decoction of C. deserticola and C. tubulosa could improve spatial learning and memory in mice models of depression, and the mechanism may be related to the ‘gut-brain’ axis [64]. C. Deserticola Polysaccharides (CDPs) showed significant effects on improving abnormal behaviors of depressed rats by maintaining Th17/Treg balance and modulating gut immunity [65]. CTE could significantly improve depression-like behaviors in rats by regulating the structure of intestinal microbiota and restoring the levels of Hydroxytryptamine (5-HT) and BDNF through the ‘gut-brain’ axis [48]. Caffeic acid is the main degradation product of CTE in depression model rats [66], which could Produce antidepressant effects [67]. These metabolites have good intestinal absorption and bioavailability and are more likely to be absorbed into the blood [68,69]. In addition, ECH could exert antidepressant effects by activating the AMPAR-Akt/ERK-mTOR signaling pathway, in which mTOR is a key signaling target for rapid antidepressant effects [70].

Anti-Tumor Effects

Previous studies have demonstrated that PhGs of Cistanche herba exhibit antitumor effects on a variety of tumor cells through different signaling pathways. PhGs of C. tubulosa induced apoptosis in Hepatocellular Carcinoma (HCC) cells through both the mitochondria-dependent and MAPK signaling pathways in vitro [71]. In addition, PhGs of C. tubulosa could inhibit the growth of B16-F10 cells and esophageal cancer ECA-109 cells both in vitro and in vivo via mitochondria-dependent pathway [72,73]. PhGs of C. deserticola could significantly inhibit the mitochondrial respiration and glycolysis functions of HepG2 cells, increase the level of ROS, and inhibit cell proliferation [74]. ECH exerted antiproliferative and proapoptotic functions on HepG2 cells via decreasing TREM2 expression and inactivating the AKT pathway as well as miR-503-3p/TGF-β1/Smad axis [75,76]. Moreover, ECH could not only inhibit the growth of pancreatic cancer SW1990 cells by promoting ROS generation and MAPK signaling pathway [77], but also promote pyroptosis of non-small cell lung cancer cells through Raf/MEK/ERK signaling pathway [78].

Anti-Oxidant Effects

ECH could significantly attenuate ethanol-induced oxidative stress by enhancing the levels of antioxidants and reducing the level of ROS via SREBP-1c/FASN pathway [79]. ECH could also ameliorate oxidative stress and alcohol-induced liver injury by increasing the activity of erythroid 2-related factor 2 (Nrf2), which is promising for the treatment of Alcoholic Liver Disease (ALD) [80]. Additionally, Cistanoside could markedly attenuate the harmful effects of hypoxia-induced oxidative stress by affecting antioxidant enzyme activities in testes and GC-1 cells [81]. Moreover, Cistanche polysaccharide is also an effective antioxidant, and its phenolic hydroxyl structure could directly bind to free radicals and the activate antioxidant defense system [82]. The polysaccharide of C. deserticola could induce melanogenesis in melanocytes and reduce oxidative stress via activating NRF2/HO-1 pathway, which could be used as a new drug for the treatment of decolorization disease [83].

Immune Regulation Effects

PhGs of C. Tubulosa (CTPG) had an immune enhancement function, which could not only significantly increase the numbers of CD4+ and CD8+ T cells but also enhance the activation state of CD4+ T cells [71]. Moreover, CTPG could inhibit the apoptosis of splenocytes induced by cisplatin, which is characterized by the increased numbers of T cells and the decreased numbers of MDSCs and Tregs [71]. Polysaccharides of C. deserticola (WPCD) could modulate immune responses in vitro and in vivo. WPCD significantly promoted the maturation and function of murine Marrow-Derived Cendritic Cells (BM-DCs) through up-regulating the expression levels of MHC-II and allogenic T cell proliferation, as indicated by in vitro experiments [84]. Moreover, WPCD could enhance immunogenicity through a balanced Th1-/Th2-type response and an effective T-cell response, which could be used as a polysaccharide adjuvant on seasonal influenza vaccines [85].

Clinical Application

Digestive System

The clinical treatment of Cistanche herba on the digestive system is limited to constipation. Congrong tongbian decoction (Chinese herba preparation in hospital) has good therapeutic effects on yang-deficiency constipation, which refers to the patients with dry stool but difficult defecation, accompanied by cold and waist and knee soreness. After seven days of treatment, the defecation time of patients with constipation significantly shortened, and the defecation effect was strengthened with the increase in dose [86]. Cistanche herba decoction (Chinese native medicine preparation made by the hospital) could not only improve constipation caused by hemodialysis but also have long-lasting effects and no adverse reactions [87]. Moreover, Cistanche decoction (Chinese native medicine preparation made by the hospital) could effectively improve the constipation symptoms of Parkinson's patients, and the efficacy is better than that of the western medicine group [88].

Nervous System

Cistanche herba is clinically used for the treatment of mild AD. After agents treatment of C. deserticola (3222002216000), the cognitive status of mild AD patients has been significantly improved, and the mechanism is mainly related to the reduction of t-tau, TNF-a, and IL-1β in cerebrospinal fluid and delayed hippocampal atrophy [89]. Cistanche total glycoside capsules (Z20080047) also had good therapeutic effects on AD. After 12 weeks of treatment, the cognitive function of AD patients was significantly improved and the adverse reactions were less than those of the western medicine group [90]. Cistanche herba is usually combined with western medicine in the treatment of PD. Congrongjing Granules (CRJG) (Chinese native medicine preparation made by the hospital) could reduce the side effects of western medicine and improve the quality of life in patients with PD, and its efficacy and safety are better [91]. Moreover, Cistanche herba (Chinese native medicine preparation made by the hospital) combined with acupuncture and moxibustion could not only improve the motor ability of early PD patients [92], but also improve the non-motor symptoms of PD patients [93]. Additionally, the other two medicines of Cistanche could effectively improve life quality of PD patients, Cistanche Shujing granule (Chinese native medicine preparation made by the hospital) and Cistanche Yizhi (Z20194044) capsule could significantly relieve the depressive symptoms of PD patients [94,95].

Other Diseases

C. deserticola has two-way regulation of bone formation and bone resorption, and could effectively resist osteoporosis. After six months of C. deserticola treatment (Chinese native medicine preparation made by the hospital) in patients with primary osteoporosis, the achieved effects are significant, such as improved serum alkaline phosphatase, calcium, and phosphorus metabolism, increased bone density [96]. Additionally, CTE (Chinese native medicine preparation made by the hospital) is helpful to prevent motor organ dysfunction. After 12 weeks of CTE treatment, the stride length and gait speed of patients with locomotive syndrome increased, and no obvious adverse reactions were observed [97]. Moreover, CTE could effectively relieve tinnitus symptoms caused by chronic nephritis. After three months of treatment, the tinnitus symptoms of adult patients with chronic glomerulonephritis were significantly relieved [97].

Conclusions

This article summarized the progress in omics research including the organelle genome and transcriptome of Cistanche herba for the first time. We found significant differences in the genomic structure of important organelles of different Cistanche herba (Table 1), suggesting that genomic data could solve the long-controversial problem of Cistanche herba identification in the future. In addition, the synthesis pathway, biological function and clinical application of PhGs, an important compound of Cistanche herba, were reviewed. Future studies should focus on overcoming the technical problems of assembling genome with high heterozygosity, high repeat sequences and high GC content to construct high-quality nuclear genome profiles of Cistanche herba, so as to solve the difficult situation of qualitative characterization of transcriptome. Moreover, researchers should integrate the multi-omics data of Cistanche herba to elucidate the total synthesis pathways of its important compounds (taking PhGs as an example) and further explore new active compounds. In conclusion, this article provides a new insight for further pharmacological research and clinical application of Cistanche herba, and provides a high-level theoretical basis for its industrial transformation.

Author Statements

Funding

This study was supported by grants from Science and Technology Innovation Action Plan Startup Project (Sail Special) of Shanghai (grant number: 22YF1420500), the Fundamental Research Funds for the Central Universities (grant numbers: KLSB2022QN-01), Medical-Industrial Crossover Research Fund of Shanghai Jiao Tong University (grant numbers: YG2022QN070 and 19X190020005), and the National Natural Science Foundation of China (grant number: 81872274).

Authors Contributions

Huang J were responsible for study concept and design. Zou R and Huang J were responsible for drafting the manuscript. Huang WQ and Xu J participated in the review & editing. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Consent to Publish

The Author confirms that the work described has not been published before.

References

1. Song Y, Zeng K, Jiang Y, Tu P. Cistanches Herba, from an endangered species to a big brand of Chinese medicine. Med Res Rev. 2021; 41: 1539-77.
2. Jiang Y, Tu PF. Analysis of chemical constituents in Cistanche species. J Chromatogr A. 2009; 1216: 1970-9.
3. Lei H, Wang X, Zhang Y, Cheng T, Mi R, Xu X, et al. Herba Cistanche (Rou Cong Rong): A Review of Its Phytochemistry and Pharmacology. Chem Pharm Bull (Tokyo). 2020; 68: 694-712.
4. Korpelainen H. The evolutionary processes of mitochondrial and chloroplast genomes differ from those of nuclear genomes. Naturwissenschaften. 2004; 91: 505-18.
5. Sierra J, Escobar-Tovar L, Leon P. Plastids: diving into their diversity, their functions, and their role in plant development. J Exp Bot. 2023; 74: 2508-26.
6. Liu X, Fu W, Tang Y, Zhang W, Song Z, Li L, et al. Diverse trajectories of plastome degradation in holoparasitic Cistanche and genomic location of the lost plastid genes. J Exp Bot. 2020; 71: 877-92.
7. Daniell H, Lin CS, Yu M, Chang WJ. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol. 2016; 17: 134.
8. Li X, Zhang TC, Qiao Q, Ren Z, Zhao J, Yonezawa T, et al. Complete chloroplast genome sequence of holoparasite Cistanche deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from its host Haloxylon ammodendron (Chenopodiaceae). PLoS One. 2013; 8: e58747.
9. Yang QQ. Comparative and phylogenetic analysis of the complete chloroplast genomes of Cistanche [dissertation]: Peking Union Medical College. 2019.
10. Barbato A, Scandura G, Puglisi F, Cambria D, La Spina E, Palumbo GA, et al. Mitochondrial Bioenergetics at the Onset of Drug Resistance in Hematological Malignancies: An Overview. Front Oncol. 2020; 10: 604143.
11. Miao Y, Chen H, Xu W, Liu C, Huang L. Cistanche Species Mitogenomes Suggest Diversity and Complexity in Lamiales-Order Mitogenomes. Genes (Basel). 2022; 13.
12. Chen HY, Yu SL, Li KC, Yang PC. Biomarkers and transcriptome profiling of lung cancer. Respirology. 2012; 17: 620-6.
13. Li Y, Wang X, Chen T, Yao F, Li C, Tang Q, et al. RNA-Seq Based De Novo Transcriptome Assembly and Gene Discovery of Cistanche deserticola Fleshy Stem. PLoS One. 2015; 10: e0125722.
14. Hou L, Li G, Chen Q, Zhao J, Pan J, Lin R, et al. De novo full length transcriptome analysis and gene expression profiling to identify genes involved in phenylethanol glycosides biosynthesis in Cistanche tubulosa. BMC Genomics. 2022; 23: 698.
15. Li XW, Chen SL. Herbgenomics facilitates biological study of TCM. Chin J Nat Med. 2020; 18: 561-2.
16. Marita JM, Ralph J, Hatfield RD, Chapple C. NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proc Natl Acad Sci USA. 1999; 96: 12328-32.
17. Ralph J, Lapierre C, Marita JM, Kim H, Lu F, Hatfield RD, et al. Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry. 2001; 57: 993-1003.
18. Hou X, Shao F, Ma Y, Lu S. The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis. Mol Biol Rep. 2013; 40: 4301-10.
19. Bagal UR, Leebens-Mack JH, Lorenz WW, Dean JF. The phenylalanine ammonia lyase (PAL) gene family shows a gymnosperm-specific lineage. BMC Genomics. 2012; 13: S1.
20. Lacombe E, Hawkins S, Van Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, et al. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J. 1997; 11: 429-41.
21. Li L, Cheng XF, Leshkevich J, Umezawa T, Harding SA, Chiang VL. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell. 2001; 13: 1567-86.
22. Magalingam KB, Radhakrishnan A, Ping NS, Haleagrahara N. Current Concepts of Neurodegenerative Mechanisms in Alzheimer’s Disease. Biomed Res Int. 2018; 2018: 3740461.
23. Yang J, Ju B, Yan Y, Xu H, Wu S, Zhu D, et al. Neuroprotective effects of phenylethanoid glycosides in an in vitro model of Alzheimer’s disease. Exp Ther Med. 2017; 13: 2423-8.
24. Wu CR, Lin HC, Su MH. Reversal by aqueous extracts of Cistanche tubulosa from behavioral deficits in Alzheimer’s disease-like rat model: relevance for amyloid deposition and central neurotransmitter function. BMC Complement Altern Med. 2014; 14: 202.
25. Kurisu M, Miyamae Y, Murakami K, Han J, Isoda H, Irie K, et al. Inhibition of amyloid β aggregation by acteoside, a phenylethanoid glycoside. Biosci Biotechnol Biochem. 2013; 77: 1329-32.
26. Wang YM, Zhang SJ, Luo GA, Hu YN, Hu JP, Liu L, et al. Analysis of phenylethanoid glycosides in the extract of herba Cistanche by LC/ESI-MS/MS. Yao Xue Xue Bao. 2000; 35: 839-42.
27. Jia JX, Yan XS, Cai ZP, Song W, Huo DS, Zhang BF, et al. The effects of phenylethanoid glycosides, derived from Herba Cistanche, on cognitive deficits and antioxidant activities in male SAMP8 mice. J Toxicol Environ Health A. 2017; 80: 1180-6.
28. Peng XM, Gao L, Huo SX, Liu XM, Yan M. The Mechanism of Memory Enhancement of Acteoside (Verbascoside) in the Senescent Mouse Model Induced by a Combination of D-gal and AlCl3. Phytother Res. 2015; 29: 1137-44.
29. Chen J, Zhou SN, Zhang YM, Feng YL, Wang S. Glycosides of Cistanche improve learning and memory in the rat model of vascular dementia. Eur Rev Med Pharmacol Sci. 2015; 19: 1234-40.
30. Nagakura A, Shitaka Y, Yarimizu J, Matsuoka N. Characterization of cognitive deficits in a transgenic mouse model of Alzheimer’s disease and effects of donepezil and memantine. Eur J Pharmacol. 2013; 703: 53-61.
31. Revett TJ, Baker GB, Jhamandas J, Kar S. Glutamate system, amyloid Β peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology. J Psychiatry Neurosci. 2013; 38: 6-23.
32. Yang J, Ju B, Hu J. Effects of Phenylethanoid Glycosides Extracted from Herba Cistanches on the Learning and Memory of the APP/PSI Transgenic Mice with Alzheimer’s Disease. Biomed Res Int. 2021; 2021: 1291549.
33. Schneider RB, Iourinets J, Richard IH. Parkinson’s disease psychosis: presentation, diagnosis and management. Neurodegener Dis Manag. 2017; 7: 365-76.
34. Ho MS. Microglia in Parkinson’s Disease. Adv Exp Med Biol. 2019; 1175: 335-53.
35. Gao MR, Wang M, Jia YY, Tian DD, Liu A, Wang WJ, et al. Echinacoside protects dopaminergic neurons by inhibiting NLRP3/Caspase-1/IL-1β signaling pathway in MPTP-induced Parkinson’s disease model. Brain Res Bull. 2020; 164: 55-64.
36. Yang X, Yv Q, Ye F, Chen S, He Z, Li W, et al. Echinacoside Protects Dopaminergic Neurons Through Regulating IL-6/JAK2/STAT3 Pathway in Parkinson’s Disease Model. Front Pharmacol. 2022; 13: 848813.
37. Yuan J, Ren J, Wang Y, He X, Zhao Y. Acteoside Binds to Caspase-3 and Exerts Neuroprotection in the Rotenone Rat Model of Parkinson’s Disease. PLoS One. 2016; 11: e0162696.
38. Liang JQ, Wang L, He JC, Hua XD. Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. Neural Regen Res. 2016; 11: 101-6.
39. Li M, Zhou F, Xu T, Song H, Lu B. Acteoside protects against 6-OHDA-induced dopaminergic neuron damage via Nrf2-ARE signaling pathway. Food Chem Toxicol. 2018; 119: 6-13.
40. Liao YC, Wang JW, Guo C, Bai M, Ran Z, Wen LM, et al. Cistanche tubulosa alleviates ischemic stroke-induced blood-brain barrier damage by modulating microglia-mediated neuroinflammation. J Ethnopharmacol. 2023; 309: 116269.
41. Liu J, Wang Y, Li Q, Liu T, Liu X, Zhang H, et al. Phenylethanoid glycosides derived from Cistanche deserticola promote neurological functions and the proliferation of neural stem cells for improving ischemic stroke. Biomed Pharmacother. 2023; 167: 115507.
42. Liao Y, Hu J, Guo C, Wen A, Wen L, Hou Q, et al. Acteoside alleviates blood-brain barrier damage induced by ischemic stroke through inhibiting microglia HMGB1/TLR4/NLRP3 signaling. Biochem Pharmacol. 2023; 220: 115968.
43. Zhang YM, Wu W, Ma W, Wang F, Yuan J. Effect of glycosides of Cistanche on the expression of mitochondrial precursor protein and keratin type II cytoskeletal 6A in a rat model of vascular dementia. Neural Regen Res. 2017; 12: 1152-8.
44. Yang Q, Sun R. Effect of Echinacoside on learning-memory function and expression of BDNF and TrkB in hippocampal tissue of vascular dementia rats. Trational Chinese Drug Research& Clinical Pharmacology. 2017; 28: 304-9.
45. Du Q, Wu Z. Mechanism and dosage-effect relationship of Cistanche on yang deficiency constipation model. Central South Pharmacy. 2016; 14: 23-7.
46. Wang L, Sun J, Zhao B, Zhao M. Laxative function of dietary fiber from Cistanche deserticola. Journal of Food Safety and Quality. 2016; 7: 3740-4.
47. Zhang X, Zheng FJ, Zhang Z. Therapeutic effect of Cistanche deserticola on defecation in senile constipation rat model through stem cell factor/C-kit signaling pathway. World J Gastroenterol. 2021; 27: 5392-403.
48. Li Y, Peng Y, Ma P, Yang H, Xiong H, Wang M, et al. Antidepressant-Like Effects of Cistanche tubulosa Extract on Chronic Unpredictable Stress Rats Through Restoration of Gut Microbiota Homeostasis. Front Pharmacol. 2018; 9: 967.
49. Fan L, Peng Y, Wang J, Ma P, Zhao L, Li X. Total glycosides from stems of Cistanche tubulosa alleviate depression-like behaviors: bidirectional interaction of the phytochemicals and gut microbiota. Phytomedicine. 2021; 83: 153471.
50. Bao X, Bai D, Liu X, Wang Y, Zeng L, Wei C, et al. Effects of the Cistanche tubulosa Aqueous Extract on the Gut Microbiota of Mice with Intestinal Disorders. Evid Based Complement Alternat Med. 2021; 2021: 4936970.
51. Gao Y, Jiang Y. Study on laxative constituents in Cistanche deserticola Y. C. Ma. Modern Chinese Medicine. 2015; 17: 307-10.
52. Zhao WX, Wang T, Zhang YN, Chen Q, Wang Y, Xing YQ, et al. Molecular Mechanism of Polysaccharides Extracted from Chinese Medicine Targeting Gut Microbiota for Promoting Health. Chin J Integr Med. 2022.
53. Yin H, Gao X, Yang H, Xu Z, Wang X, Wang X, et al. Total alditols from Cistanche deserticola attenuate functional constipation by regulating bile acid metabolism. J Ethnopharmacol. 2024; 320: 117420.
54. Cannata-Andía JB, Rodriguez García M, Gómez Alonso C. Osteoporosis and adynamic bone in chronic kidney disease. J Nephrol. 2013; 26: 73-80.
55. Liang H, Yu F, Tong Z, Huang Z. Effect of Cistanches Herba aqueous extract on bone loss in ovariectomized rat. Int J Mol Sci. 2011; 12: 5060-9.
56. Li TM, Huang HC, Su CM, Ho TY, Wu CM, Chen WC, et al. Cistanche deserticola extract increases bone formation in osteoblasts. J Pharm Pharmacol. 2012; 64: 897-907.
57. Song D, Cao Z, Liu Z, Tickner J, Qiu H, Wang C, et al. Cistanche deserticola polysaccharide attenuates osteoclastogenesis and bone resorption via inhibiting RANKL signaling and reactive oxygen species production. J Cell Physiol. 2018; 233: 9674-84.
58. Li J, Zou Z, Su X, Xu P, Du H, Li Y, et al. Cistanche deserticola improves ovariectomized-induced osteoporosis mainly by regulating lipid metabolism: Insights from serum metabolomics using UPLC/Q-TOF-MS. J Ethnopharmacol. 2023; 322: 117570.
59. Zhang B, Yang LL, Ding SQ, Liu JJ, Dong YH, Li YT, et al. Anti-Osteoporotic Activity of an Edible Traditional Chinese Medicine Cistanche deserticola on Bone Metabolism of Ovariectomized Rats Through RANKL/RANK/TRAF6-Mediated Signaling Pathways. Front Pharmacol. 2019; 10: 1412.
60. Chen Y, Li YQ, Fang JY, Li P, Li F. Establishment of the concurrent experimental model of osteoporosis combined with Alzheimer’s disease in rat and the dual-effects of echinacoside and acteoside from Cistanche tubulosa. J Ethnopharmacol. 2020; 257: 112834.
61. Li S, Cui Y, Li M, Zhang W, Sun X, Xin Z, et al. Acteoside Derived from Cistanche Improves Glucocorticoid-Induced Osteoporosis by Activating PI3K/AKT/mTOR Pathway. J Invest Surg. 2023; 36: 2154578.
62. Xu X, Zhang Z, Wang W, Yao H, Ma X. Therapeutic Effect of Cistanoside A on Bone Metabolism of Ovariectomized Mice. Molecules. 2017; 22.
63. Chen T, Gao F, Luo D, Wang S, Zhao Y, Liu S, et al. Cistanoside A promotes osteogenesis of primary osteoblasts by alleviating apoptosis and activating autophagy through involvement of the Wnt/β-catenin signal pathway. Ann Transl Med. 2022; 10: 64.
64. Wang D, Wang H, Gu L. The Antidepressant and Cognitive Improvement Activities of the Traditional Chinese Herb Cistanche. Evid Based Complement Alternat Med. 2017; 2017: 3925903.
65. Liu X, Wu X, Wang S, Zhao Z, Jian C, Li M, et al. Microbiome and metabolome integrally reveal the anti-depression effects of Cistanche deserticola polysaccharides from the perspective of gut homeostasis. Int J Biol Macromol. 2023; 245: 125542.
66. Li Y, Peng Y, Ma P, Wang M, Peng C, Tu P, et al. In vitro and in vivo metabolism of Cistanche tubulosa extract in normal and chronic unpredictable stress-induced depressive rats. J Chromatogr B Analyt Technol Biomed Life Sci. 2019; 1125: 121728.
67. Takeda H, Tsuji M, Yamada T, Masuya J, Matsushita K, Tahara M, et al. Caffeic acid attenuates the decrease in cortical BDNF mRNA expression induced by exposure to forced swimming stress in mice. Eur J Pharmacol. 2006; 534: 115-21.
68. Xu J, Chen HB, Li SL. Understanding the Molecular Mechanisms of the Interplay Between Herbal Medicines and Gut Microbiota. Med Res Rev. 2017; 37: 1140-85.
69. Liu H, Yang J, Du F, Gao X, Ma X, Huang Y, et al. Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metab Dispos. 2009; 37: 2290-8.
70. Chuang HW, Wang TY, Huang CC, Wei IH. Echinacoside exhibits antidepressant-like effects through AMPAR-Akt/ERK-mTOR pathway stimulation and BDNF expression in mice. Chin Med. 2022; 17: 9.
71. Yuan P, Fu C, Yang Y, Adila A, Zhou F, Wei X, et al. Cistanche tubulosa Phenylethanoid Glycosides Induce Apoptosis of Hepatocellular Carcinoma Cells by Mitochondria-Dependent and MAPK Pathways and Enhance Antitumor Effect through Combination with Cisplatin. Integr Cancer Ther. 2021; 20: 15347354211013085.
72. Li J, Li J, Aipire A, Gao L, Huo S, Luo J, et al. Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10 Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway. J Cancer. 2016; 7: 1877-87.
73. Fu C, Li J, Aipire A, Xia L, Yang Y, Chen Q, et al. Cistanche tubulosa phenylethanoid glycosides induce apoptosis in Eca-109 cells via the mitochondria-dependent pathway. Oncol Lett. 2019; 17: 303-13.
74. Feng D, Zhou SQ, Zhou YX, Jiang YJ, Sun QD, Song W, et al. Effect of total glycosides of Cistanche deserticola on the energy metabolism of human HepG2 cells. Front Nutr. 2023; 10: 1117364.
75. Ye Y, Song Y, Zhuang J, Wang G, Ni J, Xia W. Anticancer effects of echinacoside in hepatocellular carcinoma mouse model and HepG2 cells. J Cell Physiol. 2019; 234: 1880-8.
76. Li W, Zhou J, Zhang Y, Zhang J, Li X, Yan Q, et al. Echinacoside exerts anti-tumor activity via the miR-503-3p/TGF-β1/Smad aixs in liver cancer. Cancer Cell Int. 2021; 21: 304.
77. Wang W, Luo J, Liang Y, Li X. Echinacoside suppresses pancreatic adenocarcinoma cell growth by inducing apoptosis via the mitogen-activated protein kinase pathway. Mol Med Rep. 2016; 13: 2613-8.
78. Shi Y, Cao H, Liu Z, Xi L, Dong C. Echinacoside Induces Mitochondria-Mediated Pyroptosis through Raf/MEK/ERK Signaling in Non-Small Cell Lung Cancer Cells. J Immunol Res. 2022; 2022: 3351268.
79. Tao Z, Zhang L, Wu T, Fang X, Zhao L. Echinacoside ameliorates alcohol-induced oxidative stress and hepatic steatosis by affecting SREBP1c/FASN pathway via PPARa. Food Chem Toxicol. 2021; 148: 111956.
80. Ding Y, Zhang Y, Wang Z, Zeng F, Zhen Q, Zhao H, et al. Echinacoside from Cistanche tubulosa ameliorates alcohol-induced liver injury and oxidative stress by targeting Nrf2. Faseb j. 2023; 37: e22792.
81. Yan F, Dou X, Zhu G, Xia M, Liu Y, Liu X, et al. Cistanoside of Cistanche Herba ameliorates hypoxia-induced male reproductive damage via suppression of oxidative stress. Am J Transl Res. 2021; 13: 4342-59.
82. Fu Z, Fan X, Wang X, Gao X. Cistanches Herba: An overview of its chemistry, pharmacology, and pharmacokinetics property. J Ethnopharmacol. 2018; 219: 233-47.
83. Hu Y, Huang J, Li Y, Jiang L, Ouyang Y, Li Y, et al. Cistanche deserticola polysaccharide induces melanogenesis in melanocytes and reduces oxidative stress via activating NRF2/HO-1 pathway. J Cell Mol Med. 2020; 24: 4023-35.
84. Zhang A, Yang X, Li Q, Yang Y, Zhao G, Wang B, et al. Immunostimulatory activity of water-extractable polysaccharides from Cistanche deserticola as a plant adjuvant in vitro and in vivo. PLoS One. 2018; 13: e0191356.
85. Zhao B, Lian J, Wang D, Li Q, Feng S, Li J, et al. Evaluation of aqueous extracts of Cistanche deserticola as a polysaccharide adjuvant for seasonal influenza vaccine in young adult mice. Immunol Lett. 2019; 213: 1-8.
86. Wang H. Clinical research on dose-effect relationship of different doses of Cistanche in compound Congrongtongbian decoction. Journal of shanxi college of traditional chinese medicine. 2018; 34: 11-3.
87. Wang L, Tian CL. Clinical observation on 87 cases of constipation caused by maintenance hemodialysis treated with Cistanche deserticola. Hebei J TCM. 2010; 32: 857-8.
88. Tian P. The clinical research of constipation symptoms of Parkinson’s disease treated by Cistanche granules [dissertation]: Nanjing University of Chinese Medicine. 2016.
89. Li N, Bai H, Lou J. Cerebral protective effect of Cistanche in Alzheimer’s patients. China Health Standard Management. 2015; 6: 115-6.
90. Wang Q. Clinical study of Cistanche deserticola total glycosides capsule in the treatment of Alzheimer’s disease. Strait Pharmaceutical Journal. 2009; 21: 103-4.
91. Chen S, Xiao S. Observation on the effects of Congrongjing granules and western medicine in the treatment of Parkinson’s disease. Chinese Journal of Intergrative Medicine on Cardio-cerebrovascular Disease. 2019; 17: 2914-7.
92. Zhang J, Zhang C. Effect of Herba Cistanche combined with acupuncture therapy on walking capacity and quality of life in patients with early Parkinson’s disease. Nursing of Integrated Traditional Chinese and Western Medicine. 2019; 5: 36-8.
93. Jiang K. The clinical research of non-motor symptoms of Parkinson’s disease treated by Cistanche deserticola [dissertation]: Nanjing University of Traditional Chinese Medicine. 2016.
94. Fan L, Cai J. Observation on the curative effect and sleep quality of patients with Parkinson’s disease depression treated with Congrong Shujing granules. World Journal of Sleep Medicine. 2021; 8(08): 1346-8.
95. Li H. Effect on compound Congrong Yizhi capsules in treatment of insomnia of liver and kidney deficiency in Parkinson’s disease. Drug Evaluation Research. 2021; 44: 390-3.
96. Wu Y, Zhang X, Zhang H, Wang XF. Clinical observation of Chinese medicine Cistanche deserticola in treating osteoporosis and regulating bone metabolomics. China Modern Doctor. 2019; 57: 120-4.
97. Inada Y, Tohda C, Yang X. Effects of Cistanche tubulosa Wight Extract on Locomotive Syndrome: A Placebo-Controlled, Randomized, Double-Blind Study. Nutrients. 2021; 13: 264.