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Table of Contents
REVIEW ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 4  |  Page : 393-407

Medicinal plant of Bletilla striata: A review of its chemical constituents, pharmacological activities, and quality control


1 TCM and Ethnomedicine Innovation and Development International Laboratory, School of Pharmacy, Innovative Materia Medica Research Institute, Hunan University of Chinese Medicine, Changsha, China
2 Research Center for New Drug Evaluation, Research Center for Modern Chinese Medicines, Tianjin Institute of Pharmaceutical Research, Tianjin, China
3 State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang, China
4 H E J Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

Date of Submission21-May-2020
Date of Acceptance22-Jun-2020
Date of Web Publication15-Oct-2020

Correspondence Address:
Prof. Wei Wang
TCM and Ethnomedicine Innovation and Development International Laboratory, School of Pharmacy, Innovative Materia Medica Research Institute, Hunan University of Chinese Medicine, Changsha
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_58_20

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  Abstract 


Bletilla striata belongs to the family Orchidaceae, and it is mainly distributed in East Asia. The tubers of B. striata have been utilized in Traditional Chinese Medicine for various ailments, such as hematemesis, tuberculosis, malignant ulcers, hemorrhoids, traumatic bleeding, and chapped skin. Phytochemical investigation on B. striata has resulted in the identification of 192 monomeric compounds, including bibenzyls, phenanthrene derivatives, triterpenoids and its saponins, steroids and its saponins, malic acid derivatives, and anthocyanins. Moreover, B. striata polysaccharide is another typical chemical constituent of this plant. Pharmacology studies have shown that the plant possesses wound healing, antimicrobial, anticancer, antioxidative, and antiviral activities. This review aims to provide the latest and comprehensive information on chemical constituents, pharmacological activities, and quality control of B. striata and to identify future research needs.

Keywords: Bletilla striata, Orchidaceae, chemical constituents, pharmacology, quality control


How to cite this article:
Jiang S, Wang MY, Yuan HW, Xie Q, Liu Y, Li BS, Jian YQ, Liu CX, Lou HY, AU, Pan WD, Wang W. Medicinal plant of Bletilla striata: A review of its chemical constituents, pharmacological activities, and quality control. World J Tradit Chin Med 2020;6:393-407

How to cite this URL:
Jiang S, Wang MY, Yuan HW, Xie Q, Liu Y, Li BS, Jian YQ, Liu CX, Lou HY, AU, Pan WD, Wang W. Medicinal plant of Bletilla striata: A review of its chemical constituents, pharmacological activities, and quality control. World J Tradit Chin Med [serial online] 2020 [cited 2021 Aug 2];6:393-407. Available from: https://www.wjtcm.net/text.asp?2020/6/4/393/303584




  Introduction Top


Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a perennial herb and mainly distributed in China, Japan, Korea, Thailand, and other Southeast Asian Nations.[1] The tubers of B. striata (“Bai-Ji ” in Chinese) have been used in China for thousands of years, which was initially recorded in ancient book, Shennong's Classic of Materia Medica (“Shen Nong Ben Cao Jing,”.[2] Bletillae Rhizoma (the dried tubers of B. striata) is bitter, pucker, and sweet in flavor and slightly cool and astringent in nature. It is used in Traditional Chinese Medicine (TCM) for the treatment of pulmonary and gastric hemorrhages, chapped skin, ulcerative carbuncle, colds, and burns.[3],[4] Besides, B. striata as a vascular embolizing agent for treating primary hepatic carcinoma has been reported to be more valid than conventional gel foam.[5],[6]

Even though B. striata was used earlier in China, the study of chemical composition was first started in Japan. At the end of the 20th century, Yamaki, Bai, and Takagi have published more than 10 papers on B. striata in the phytochemistry. Subsequently, other Asian researchers followed and deepened this subject. Today, the plant is known to produce more than 190 compounds, including bibenzyls, phenanthrene derivatives, triterpenoids, steroids, malic acid derivatives, and other compounds.[7] Furthermore, B. striata is also a rich source of polysaccharides, which was not only used in food industry but also applied in medicinal as wound dressing and drug delivery.[8] Among them, bibenzyls, phenanthrene derivatives, and polysaccharides are the typical chemical constituents of this plant. Moreover, pharmacology experiments and clinical practice have demonstrated that its active compounds possess a wide range pharmacological activities, including antibacterial, antioxidant, anti-inflammatory, anticancer, and antiaging effects.[9] Currently, a number of domestic pharmaceutical enterprises in China have been approved to produce B. striata proprietary medicines, including B. striata syrup, B. striata tablets, B. striata granule, and B. striata capsule. The clinical application is extensive.[10] The present paper aimed to review phytochemistry, pharmacological activities, and quality control of B. striata. This information might be useful in designing future studies and in developing new pharmaceuticals containing B. striata or its active ingredients.


  Chemical Constituents Top


B. striata is a rich source of bibenzyls and phenanthrene derivatives. To date, 44 bibenzyls, 73 phenanthrene derivatives, 8 triterpenoids, 16 steroids, 13 malic acid derivatives, 15 simple phenols, and a number of compounds representing a middle spectrum of constituent classes have been isolated and identified from the plant. Herein, their names, associated references, and structures are shown in [Table 1], [Table 2], [Table 3], [Table 4], [Table 5] and [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Table 1: Bibenzyls isolated from Bletilla striata

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Table 2: Phenanthrenes isolated from Bletilla striata

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Table 3: Triterpenoids and steroids isolated from Bletilla striata

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Table 4: Malic acid derivatives isolated from Bletilla striata

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Table 5: Other compounds isolated from Bletilla striata

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Figure 1: Structures of bibenzyls (1–44) isolated from Bletilla striata

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Figure 2: Structures of dihydrophenanthrenes (45–76) isolated from Bletilla striata

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Figure 3: Structures of monomeric phenanthrenes, phenanthraquinones, and triphenanthrenes (77–100) isolated from Bletilla striata

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Figure 4: Structures of biphenanthrenes (101–117) isolated from Bletilla striata

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Figure 5: Structures of triterpenoids (118–125) isolated from Bletilla striata

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Figure 6: Structures of steroids (126–141) isolated from Bletilla striata

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Figure 7: Structures of malic acid derivatives (142–154) isolated from Bletilla striata

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Figure 8: Structures of simple phenols and other compounds (155–192) isolated from Bletilla striata

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Bibenzyls

Bibenzyl is one of the representative chemotypes of B. striata. Dihydro-m-coumaroyl-CoA, with three molecules of malonyl-CoA, is used in bibenzyl biosynthesis.[11] The skeleton of bibenzyls is simple, but their various structural types are led by the amounts and the substituted positions of the methoxy group, the hydroxyl group, and the p-hydroxybenzyl group. A total of 44 bibenzyls with flexible structures have been identified. They can be arranged in Group I – simple bibenzyls (1–11), Group II – benzyl bibenzyls (12–37), and Group III – chiral bibenzyls (38–44). The Group III – chiral bibenzyls have been isolated from B. striata for the first time.

Phenanthrenes

Phenanthrenes are a rather unusual class of aromatic metabolites in nature. It is also another typical chemical constituent of B. striata. Dihydrophenanthrenes (45–76), monomeric phenanthrenes (77–96), biphenanthrenes (101–117), phenanthraquinones (97–99), and triphenanthrenes (100) make up these compounds that have been reported in B. striata. The methoxy and hydroxy groups are main substituents of dihydrophenanthrenes and monomeric phenanthrenes isolated from B. striata. These two moieties number are between 3 and 5 and can generally be found on C-2, C-3, C-4, C-7, or C-8. There structures are shown in [Figure 2], [Figure 3], [Figure 4].

Triterpenoids

Triterpenoids are extensive in many plants. Only eight triterpenoids have been isolated from B. striata. Among them, there are one saponin of oleanolic acid (125) and seven cycloartane triterpenoids, including four C-32 cycloartane triterpenoids (118–121) and two C-31 cycloartane triterpenoids (122 and 123). Their structures (118–125) are shown in [Figure 5].

Steroids

Steroids are the one of the secondary metabolites and also widely found in plants. Steroids from B. striata have diverse structures and different substituent groups. Seven steroids (126–129, 131, 133, and 135) and nine saponins of them (130, 132, 134, and 136–141) have been isolated from B. striata, as shown in [Figure 6].

Malic acid derivatives

Malic acid exists in almost all fruits, most of which are kernel fruits. Thirteen malic acid derivatives (142–154) are presented in the tubers and fibrous roots of B. striata. Militarine (146) is abundant in this plant. Their structures are shown in [Figure 7].

Others

B. striata contains so many other compounds such as simple phenolics (155–160, 163–168), anthraquinones (171, 172), lignans (174–177), glycoside compounds (178–184), and anthocyanidins (188–192), whose structures are shown in [Figure 8].


  Biological Activity Top


B. striata has been used to treat hematemesis, tuberculosis, malignant ulcers, hemorrhoids, traumatic bleeding, and chapped skin. Several extracts and isolated compounds have been evaluated for their wound-healing, antibacterial, antioxidant, anti-inflammatory, anticancer, antiaging, and cytotoxicity activities. In addition, hence, many researchers consider that B. striata polysaccharide (BSP) was the major active component responsible for the various pharmacological activities. Consequently, B. striata has enormous potential for research and exploitation.

Wound-healing activity

Wound healing, as a normal biological process in the animal, contained four main phases: hemostasis, inflammation, proliferation, and resolution.[58] At the first hemostasis stage, the tubers of B. striata have been widely used to cure hematemesis, pulmonary hemorrhage, gastrorrhagia, and traumatic bleeding because it is capable of restraining leakage of blood and stopping bleeding.[59] B. striata micron particles (350–250 μm) enable to improve blood/particle aggregation and to form rapid sealants on wound surfaces to achieve rapid hemostasis.[60] Generally, BSP has been recognized as a major compound in wound healing.[61] BSP has been serving as a wound treatment agent for fairly long period because of its inherent advantage as natural polysaccharides promoting nonspecific activation of the immune system by activating the function of macrophages to clean up the wound site after injury.[8],[62] BSP can be used as wound dress (BSP hydrogel, BSP fibers, and BSP cotton gauze) to treat bleeding.[63],[64],[65] Throughout the available literature, nonpolysaccharide components may also have hemostatic effects. It has been reported that five spirostane steroidal saponins (137–141) isolated from the roots of B. striata, all exhibited potent hemostatic activities as shown in significantly decreasing the whole blood clotting time.[9] Some studies showed that the 80% ethanol extract of B. striatahas hemostatic effect which may be related to promote the activation of platelet and coagulation–fibrinolytic system.[66] The wound-healing activities of various extracts (petroleum ether, ethyl acetate, n-butyl alcohol, and water) of B. striata were tested, and water and n-butyl alcohol fractions could significantly increase the maximum platelet aggregation rate.[67]

At the second inflammation phase, nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) have play important roles in immune and inflammatory responses. Studies reported that therapy with BSP induces coordinate changes in inducible NO synthase (iNOS), TNF-α, and IL-1β mRNA levels, enhances the expression of these cytokines, but has no effect on interferon gamma (IFN-γ) level.[68] IFN-γ is a regulator of hematopoietic stem cells (HSCs) during homeostasis and under conditions of infectious stress.[69] The effective fraction from water or organic extracts of B. striata (collected and analyzed by chromatographic system and inductively coupled plasma mass spectrometer) can reduce the inflammatory cytokine production.[70] Besides, ethyl acetate fraction from methanol extract of B. striata not only reduced NO production but also inhibited iNOS and cyclooxygenase-2 (Cox-2) production.[71] Furthermore, five spirostane steroidal saponins (137–141) isolated from the roots of B. striata displayed selective inhibition of Cox-2 (>90%).[9]

The third proliferative phase commonly follows and overlaps with the inflammatory phase, and it is characterized by epithelial proliferation and migration over the provisional matrix within the wound.[72] In this period, BSP could increase in the expression of the vascular endothelial growth factor (EGF), induce human umbilical vascular endothelial cells' proliferation and migration, and further promote healing by wound contraction.[73],[74] Furthermore, some BSP-based wound dressings such as BSP hydrogel can elevate higher expression of EGF, promote re-epithelialization and collagen deposition by means of TGF-β/Smad signal pathway activation, and then control the inflammatory responses.[73],[75]

At the last phase, B. striata may have some positive effect on remodeling. However, there is no present literature involving the further mechanism and clear explanations. Except for BSP, some simple phenolics in B. striata can improve the function of wound healing.[50]

Antibacterial activity

Due to the emergence and spread of bacterial resistance and the lack of new antimicrobial drug development, infections caused by multidrug-resistant bacteria have become an increasingly serious problem. Traditional antibiotic screening methods have not kept pace with the evolution of bacterial resistance. The mechanism of phytochemical constituents is different from that of traditional antibiotics, so it has become an international research hotspot. B. striata is one of many Chinese herbal plants with antibacterial activity. Some compounds such as bibenzyls, phenanthrenes, and biphenanthrenes isolated from B. striata are major antimicrobial constituents to against Gram-positive and Gram-negative strains such as Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, and methicillin-resistant S. aureus. Some bibenzyls such as bulbocol (16), shancigusin C (20), shanciguol (29), bletistrin G (31), bletistrin J (34), and bletistrin F (40) showed potent inhibitory activities, with MIC values ranging from 3 to 26 μg/mL against S. aureus ATCC 6538, B. cereus ATCC 6051, and methicillin-resistant S. aureus ATCC 43300.[19] Some phenanthrenes such as 2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene (46), 4,5-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (47), and 2-hydroxy-4,7-methoxy-phenanthrene (87) showed moderate inhibitory activities, with MIC of 8–64 μg/mL against S. aureus ATCC 29213, B. subtilis CGMCC 1.1470, E. faecalis ATCC 29212, and S. epidermidis CMCC 26069.[24] Some biphenanthrenes also showed strong inhibitory properties and part of which inhibited effect even more than positive control drugs. For example, 1,1'-linked biphenanthrenes, 4, 7, 4'-trimethoxy-9',10'-dihydro (1,1'-biphenanthrene)-2,2',7'-triol (105) showed strong inhibitory activities, with MIC values ranging from 2 to 4 μg/mL against S. aureus ATCC 29213, S. aureus ATCC 43300, S. epidermidis CMCC 26069, and B. subtilis CGMCC 1.1470 (all the MIC of ampicillin is 8 μg/mL).[37]The biological results are shown in [Table 6].
Table 6: The antibacterial activities of some compounds from Bletilla striata

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Apart from the secondary metabolites stated above, ethyl acetate fraction obtained from 95% ethanol extract of B. striata has higher inhibitory activity with MIC values ranging from 0.065 to 1.042 mg/mL against Gram-positive.[76] The phenanthrene fraction (EF60) isolated from the ethanol extract of the fibrous roots of B. striata showed moderate inhibitory activity with MIC values ranging from 8 to 64 μg/mL against S. aureus and B. subtilis.[77] In addition, BSP also had an obvious bacteriostatic effect against S. aureus.[78]

Anticancer activity

In 1979, Pettit et al. has found that a series of active phenanthrenes, dihydrophenanthrenes, and stilbenes isolated from Combretum caffrum which can inhibit cancer cell growth.[79],[80] Stilbenes isolated from B. striata also contain the same effect, such as 2',6'-bis (4-hydroxybenzyl)-5-methoxybibenzyl-3,3'-diol (26), 2,7-dihydroxy-l, 3-bis(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (60), and 2,7-dihydroxy-1-(p-hydroxybenzyl)-4,8-dimethoxyphenanthrene (91), with an IC50 of 3 μM, sensitized K562/breast cancer resistance protein (BCRP) cells to SN-38 (an active metabolite of CPT-11) by inhibiting BCRP function.[31] Two novel phenanthraquinones, 7-hydroxy-2-methoxyphenanthrene-3,4-dione (97) and 3',7',7-trihydroxy-2,2',4'-trimethoxy- [1,8'-biphenanthrene]-3,4-dione (98), could block the G1-to-S phase transition process and then display significant inhibitory effect against the human breast cancer cells (MCF-7), HT-29, nonsmall cell lung adenocarcinoma (A549), and HUVEC cell lines, with IC50 values ranging from 12.46 ± 2.71 to 48.35 ± 3.87 μg/mL. Some bibenzyls such as 3',4''-dihydroxy-5',3'',5''-trimethoxybibenzyl (3), batatasin III (5), and gigantol (7) showed considerable cytotoxicity against A549, ovarian cancer cells (SKOV-3), skin melanoma (SK-MEL-2), and colon cancer cells (HCT15) cells with IC50 values of 5.28–24.51 μM.[13] In addition, five spirostane steroidal saponins (137–141) displayed significant cytotoxicity against lung cancer cells (A-549), human gastric carcinoma cells (BGC-823), human hepatocellular carcinoma cells (HepG2), human myeloid leukemia (HL-60), MCF-7, hepatocellular carcinoma cells (SMMC-7721), and colon cancer cells (W480) with IC50 values <30 μM.[9] Oleanolic acid 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (125) also exhibited antiproliferative activity against the MCF-7, HT-29, and A-549 cells and could induce G0/G1 phase arrest effectively.[42]The biological results are shown in [Table 7].
Table 7: The anticancer activities of some compounds from Bletilla striata

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Furthermore, the chloroform subfraction of the fibrous root of B. striata can induce HepG2 cells apoptosis, which implied cancer therapeutic effect.[81] BSP is also illustrated to have antitumor activity.[82] BSP-1 (a backbone of 1→4-linked β-D-mannose and glucose residues) could inhibit the tumor proliferation of HepG2-bearing mice.[83] Recently, more and more studies demonstrated that BSP was a promising drug carrier due to its brilliant biocompatibility, easy availability, and low toxicity.[8] Silymarin was loaded into the nanoassembly of BSP conjugates with stearic acid (SA), which can improve cytotoxicity and cell uptake in HepG2 cell lines in vitro.[84] BSP to conjugate alendronate (ALN-BSP) demonstrated high efficiency in targeting and eliminating tumor-associated macrophages, which accordingly reduced angiogenesis, reactivated immune surveillance, and suppressed tumor growth in vivo.[85] The anticancer activity of docetaxel (DTX)-based SA-modified BSPs copolymer micelles against HepG2, HeLa, MCF-7, and SW-480 cells was superior to that of DTX injection.[86]

Antioxidative activity

Many studies have been performed to search for novel antioxidants instead of tocopherols in plant materials, such as spices, oil seeds, and polysaccharides.[87] BSP has obvious antioxidant effect which was proved by hydroxyl radical scavenging assay, superoxide anion radical-scavenging assay, DPPH-free radical scavenging activity, and chelation of ferrous ions.[88],[89] BSP film can reduce the rate of water loss in cherry tomato, inhibit its respiratory intensity, and delay the aging process of the fruit.[90] In addition, the sch of B. striata has strong DPPH radical scavenging activity, ferric-reducing antioxidant activity, and tyrosinase inhibitory activity because of the total phenolic contents.[81] It is also worth indicating that microorganism fermentation could further build the antioxidative activity of B. striata. For instance, Fusarium avenaceum and Fusarium oxysporum fermentation enhances the total phenolic contents as well as the antioxidant activities of B. striata.[91]

Antiviral activity

The influenza virus is a branch of the virus. At present, the main measures to prevent and treat influenza are vaccination and treatment drugs. Because of the rapid transmission of influenza virus and the easy occurrence of antigenic variation, the existing vaccines cannot fight against new virus infection, while the development of new specific vaccines has obvious lag, which cannot effectively prevent the outbreak and epidemic of influenza. Therefore, it is important to find new influenza antiviral drugs, especially from natural products. In Madin–Darby canine kidney models, 2-hydroxy-4,7-methoxyphenanthrene (87) and 1,1'-linked biphenanthrenes, 4, 7, 4'-trimethoxy-9',10'-dihydro (1,1'-biphenanthrene)-2,2',7'-triol (105) showed inhibitory activities against influenza virus A/Sydney/5/97 (H3N2) with IC50 values of 43.3 ± 5.3 and 28.6 ± 4.3 μM, respectively.[92] Furthermore, water and 95% ethanol extracts of B. striata play an anti-influenza virus effect by inhibiting virus HA receptor and intervening the viral RNA synthesis and neuraminidase activity.[93]


  Quality Control Top


The quality of the medicinal materials is closely related to the characteristics of regional, diversity, variability, and humanity.[94] Due to the complex situation of Chinese medicinal materials, such as multiple sources and producing areas, the quality of pharmaceutical products varies greatly, especially the quantity of active ingredients. Hence, Liu et al. proposed a new concept on Chinese medicine quality markers (Q-Marker) which can be a reflection of Chinese medicine safety and effective labeling substances for quality control. Q-Marker is a chemical substance that is inherent in or formed during the processing and preparation of TCM and its products, and it is closely related to the functional properties of TCM.[95] In China, Guizhou province has the largest output and the best quality of B striata.[17] Zhengan County in this province is accepted as the authentic region to produce the crude B. striata. This plant is as an important material of Chinese medicine; definite quality control can offer help for its effects on human health. However, the Q-Marker and potential mechanisms for producing genuine medicinal materials are not fully clear.[7] As the main method of quality control of medicinal materials, B. striata has no uniform quality standard of medicinal ingredients. The Chinese Pharmacopoeia (2015) recommends identifying the authentic properties of B. striata according to the morphologic, microscopic, and thin-layer chromatography approaches and by ensuring that the total ash points are ≤5.0%.[7],[59] Even so, there are still some studies reporting on its quality control. BSP is one of the main active components of the plant, and its content is an important quality evaluation of B. striata. Wang et al. compared the content of polysaccharides in cultivated and wild B. striata and found that the content in cultivated plant was stable, while the content in wild plant was labile.[96] There were also significant differences in the content of BSP in different production areas.[97] Zhou et al. used a HPLC method to find that the average contents of militarine (146) and gastrodin (178) of wild B. striata were higher than that of cultivated B. striata.[98] Phenols are another important active component of B. striata. Chi et al. used nine phenols as reference substances to find that the active ingredients of B. striata were correlated with the elevation.[99] Furthermore, medicinal parts also will affect the active content of B. striata. For example, the content of polysaccharides in the tubers of B. striata is higher than the fibrous roots;[100] the total phenols content in the fibrous roots of B. striata is higher than the tubers.[101] Hence, the polysaccharides and the total phenols may be used as the Q-Markers for the quality control of B. striata. In addition, cultivation style, production area, elevation, and medicinal parts all affect the active content of B. striata.[102] To sum up, its needs more experiments to verify quality control of B. striata.


  Conclusion Top


As an ancient Chinese medicine, B. striata can cure various diseases such as pulmonary and gastric hemorrhages, chapped skin, and ulcerative carbuncle. The plant contains many types of chemical constituents, including bibenzyls, phenanthrene derivatives, malic acid derivatives, steroids, and polysaccharides. These chemical constituents are the basis of substances that have a variety of biological functions such as hemostasis, anti-inflammatory, antibacterial, anticancer, antioxidant, and antiviral activities. BSP has been considered as the main active component of the plant. It not only has been exploited as useful wound dressings, but it also has been demonstrated as a promising drug carrier. However, the bioavailability of BSP in body and property evaluation for the best therapeutic function is needed for deep investigations. Thus, it is necessary to find main active compounds for wound healing with B. striata by advanced chemical and pharmacological studies. Furthermore, Bletillae Rhizoma mainly depends on wild resources, but excessive exploitation has led to the sharp shrinkage of B. striata wild resources and the loss of genetic diversity in China. Future directions of researches will be focused on cultivation, biological activities, and underlying mechanisms of B. striata. Hence, the research on breeding, cultivation and quality control of this plant is also a lot every year. This review summarized the latest chemical constituents and pharmacological activities of B. striata and tried to illuminate their connections and hope to provide further research on this medicinal plant which has great development potential.

Financial support and sponsorship

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Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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