Chinese Journal of Natural Chinese Journal of Natural Medicines 2013, 11(4): 03300344 Medicines

doi: 10.3724/SP.J.1009.2013.00330

Phytochemical and biological research of medicinal resources

HAO Da-Cheng1*, GU Xiao-Jie 1, XIAO Pei-Gen2*, PENG Yong2

1Biotechnology Institute, School of Environment, Dalian Jiaotong University, Dalian 116028, ; 2Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine of Ministry of Education, Insti- tute of Medicinal Development, Chinese Academy of Medical Sciences, Beijing 100193, China Available online 20 July 2013

[ABSTRACT] The genus Fritillaria is a botanical source for various pharmaceutically active components, which have been commonly used in traditional Chinese medicine for thousands of years. Increasing interest in Fritillaria medicinal resources has led to additional discoveries of steroidal alkaloids, saponins, terpenoids, glycosides and many other compounds in various Fritillaria , and to investigations on their chemotaxonomy, molecular phylogeny and pharmacology. In continuation of studies on Fritillaria pharmaco- phylogeny, the phytochemistry, chemotaxonomy, molecular biology and phylogeny of Fritillaria and their relevance to drug efficacy is reviewed. Literature searching is used to characterize the global scientific effort in the flexible technologies being applied. The interre- lationship within Chinese Bei Mu species and between Chinese species, and species distributed outside of China, is clarified by the molecular phylogenetic inferences based on nuclear and chloroplast DNA sequences. The incongruence between chemotaxonomy and molecular phylogeny is revealed and discussed. It is essential to study more species for both the sustainable utilization of Fritillaria medicinal resources and for finding novel compounds with potential clinical utility. Systems biology and omics technologies will play an increasingly important role in future pharmaceutical research involving the bioactive compounds of Fritillaria. [KEY WORDS] Fritillaria; Chemical components; Chemotaxonomy; Molecular ; Phylogeny; Medicinal resource [CLC Number] R284, R965 [Document code] A [Article ID] 1672-3651(2013)04-0330-015

Zhe Bei Mu are the most famous Chinese herbal medicines 1 Introduction obtained from Fritillaria species. The former is from F. cir- Fritillaria is a genus of about 130−165 species [1-2] rhosa, F. unibracteata, F. przewalskii, F. delavayi, F. tai- within the monocot family , and is native to temper- paiensis, and F. wabuensis, while the latter is from F. thun- ate regions of the Northern Hemisphere. Fritillaria is a bo- bergii and F. thunbergii var. chekiangensis. According to tanical source for various pharmaceutically active compo- traditional descriptions, Fritillaria is slightly cold, and affects nents which have been used in traditional Chinese medicine the lungs (to clear heat and moisten dryness, and used for for thousands of years. Many species (such as F. cirrhosa, F. hot-type bronchitis with dry cough) and the heart (to calm thunbergii and F. verticillata) are used in traditional Chinese heart fire). Fritillaria is also used for treating lumps beneath cough remedies. In the China Pharmacopoeia (http://www. the skin, such as scrofulous swellings and breast lumps. Zhe chp. org.cn/cms/home/) they are listed as Chuan Bei Mu, Zhe Bei Mu is often used for the treatment of lumps, and the Bei Mu, Yi Bei Mu, Hu Bei Bei Mu, and Ping Bei Mu, re- moistening property attributed to Chuan Bei Mu is not need- spectively, and are often found in formulations combined ed. It has been adopted into some Chinese herb formulas for with extracts of Loquat ( japonica). F. verticillata treating cancers. Different Fritillaria species possess are also traded as Bei Mu in Japan. Chuan Bei Mu and different chemical profiles, and may have different pharma- cological effects. However, it is quite difficult to authenticate

[Received on] 29-Oct.-2012 Fritillaria species only by morphology. [Research funding] This project was supported by Ministry of Sci- To date, at least 30 Fritillaria species have been charac- ence and Technology national support program (No. 2012BAI29B01) terized for their chemical components [3]. Advances in ana- [*Corresponding author] HAO Da-Cheng: Prof., E-mail: hao@djtu. edu.cn; XIAO Pei-Gen: Prof., E-mail: [email protected]. net.cn lytical chemistry and molecular biology techniques facilitate These authors have no conflict of interest to declare. the in-depth studies of Fritillaria pharmaceutical resources.

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However, it is essential to study more species for both the NMR spectroscopy and MS [5]. The new cevanine-type alka- sustainable utilization of Fritillaria medicinal resources and loids, (3β, 5α)-20-hydroxy-6-oxocevan-3-yl acetate 7 and (3β, for finding novel compounds with potential clinical utility. In 5α, 6α)-6, 20-dihydroxycevan-3-yl acetate 8, were isolated this brief review, we focus on recent progress in the phyto- from bulbs of F. hupehensis and characterized by spectral chemistry and chemotaxonomy of Fritillaria, as well as mo- analysis [6]. Bulbs of F. unibracteata in , China, lecular taxonomy and phylogeny. To date, very few studies contained the new glycoalkaloid puqiedinone-3-O-β-D- have attempted to correlate DNA based phylogeny of me- glucopyranoside 11, whose structure was elucidated by spec- dicinal with phytochemistry or with medicinal proper- troscopic and chemical methods [7]. Phytochemical investiga- [4] ties . Here the molecular phylogeny of world-wide Fritil- tion of the bulbs of F. lichuanensis yielded a new isosteroidal laria species is reconstructed and the results compared with alkaloid hupehenizioiside 13 [8]. A further investigation of those of chemotaxonomy. Phylogeny has great explanatory bulbs of F. lichuanensis resulted in the isolation of two power and offers a unique perspective to complement unique cevanine-type alkaloids, lichuanine ((20S, 25R)-5α, chemotaxonomy. 14α-cevanine-3β, 6β-diol) 14 and lichuanisinine ((20S, [9] 2 Chemical Components of the Genus Fritillaria 25S)-5α, 14α-cevanine-3β, 6β-diol-N-oxide) 15 . Extraction of the bulbs of F. puqiensis yielded three new veratra- 2.1 Steroidal alkaloids mine-type alkaloids, namely puqienine C 3, puqienine D 4 Alkaloids are a group of naturally occurring chemical and puqienine E 5 [5]. Alkaloids 9 and 10 were obtained from compounds that contain mostly basic nitrogen atoms. The F. hupehensis. Their structures were deduced to be (3β, 5α, steroidal alkaloids from Fritillaria can be classified into 13α, 23β)-7, 8, 12, 14-tetradehydro-5, 6, 12, 13-tetrahydro-3, two groups based on the carbon framework: isosteroidal 23-dihydroxyveratraman-6-one and (3β, 5α, 13α, 23β)-7, 8, alkaloids and steroidal alkaloids (Fig. 1). According to the 12, 14-tetradehydro-5, 6, 12, 13-tetrahydro-3, 13, 23- trihy- linkage patterns between rings E and F, isosteroidal alka- droxy-veratraman-6-one, respectively, on the basis of chemi- loids can be divided into cevanine (A), jervine (B) and cal shift comparisons and 1H, 1H-COSY, HMBC correlations veratramine (C) types (Figs. 1 and 2). Steroidal alkaloids [6]. Bulbs of F. ussuriensis have yielded a new alkaloid ping- can be divided into solanidine (D) and secosolanidine (E) beimunone A 16 with an aromatized D-ring, whose structure [2] types , depending on whether the nitrogen atom is incor- was elucidated on the basis of spectral analysis. Chemical porated into an indolizidine ring or a piperidine ring (Fig. 1). investigation of the bulbs of F. puqiensis afforded puqienine Steroidal alkaloids found in Fritillaria before 2005 have F 17. This novel veratramine alkaloid possessed a 12, been summarized [3]. Table 1 summarizes the steroidal al- 16-epoxy ring [10]. Peimisine-3-O-β-D-glucopyranoside 12, kaloids found since 2006. possessing the furan ring E fused onto a piperidine ring sys- Puqiedine 1 and 3α-puqiedin-7-ol 2 were isolated from F. tem forming an ether bridge between carbon atoms C17 and puqiensis, their structures being determined on the basis of C23, was isolated from F. unibracteata, which might be identical

Fig. 1 Types of steroidal alkaloids in Fritillaria. a, isosteroidal alkaloids; b, steroidal alkaloids. Refer to Table 1 for more details

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Fig. 2 Steroidal alkaloids of Fritillaria. D/E configurations of cevanine group steroidal alkaloids of 1, 2, 7, 8, 11, 13−15 are trans-form. Refer to Table 1 for more details with the known jervine-type compound peimisine on the induced contraction of guinea-pig tracheal rings [14]. The basis of NMR data [7]. Pengbeimine B 18 and pengbeimine D relaxant action of imperialine and sinpeinine A, the cis-D/E 19 from F. mo na t h a have a novel skeleton in that C13 and cevanine alkaloids, is due to their selective inhibitory effects C22 were connected through oxygen forming a furan ring. on muscarinic M2 receptors. Verticine, verticinone, imperi- Their absolute structures were established by X-ray diffrac- aline, imperialine-3beta-D-glucoside and puqietinone, i.e., tion [11]. both trans- and cis-D/E cevanine alkaloids, significantly An extract of the dried ground bulbs of F. puqiensis af- elevate the cAMP concentration in the HEK cells transfected forded the verazine-type alkaloid puqietinedione 6. Its struc- with muscarinic M(2) receptor plasmid [15]. N-Demethylpu- ture was formulated as (22R, 25S)-N-methyl-22, 26- epimi- qietinone, hupeheninoside, ebeiedinone, yibeinoside A, and nocholest-3, 6-dione by comparison of the NMR data with chuanbeinone inhibited the bioactivity of human whole blood those of puqietinone [5]. Delavidine 20, an unusual 6, 12, acetylcholinesterase and plasma butyrylcholinesterase [16]. 22-triketo verazine type steroidal alkaloid with a pyrrolidine Imperialine, chuanbeinone, verticinone, and verticine from F. side chain was isolated from the methanol extract of the bulbs cirrhosa, one of the six source plants of Chuan Bei Mu, of F. delavayi [12]. The fresh bulbs of F. anhuiensis contain the showed antitussive, expectorant, and anti-inflammatory ac- alkaloids (22S, 25S)-solanid-5-en-3β-ol 21 and (22S, 25S)- tivities [17]. The four alkaloids imperialine, imperialine-β- solanid-5, 20 (21)-dien-3β-ol 22, which are the first solanidine- N-oxide, isoverticine, and isoverticine-β-N-oxide isolated type alkaloids with a 22S configuration discovered from nature. from F. wabuensis, another source plant of Chuan Bei Mu, Their structures were elucidated by NMR spectroscopy [13]. significantly inhibited cough frequency and increased latent The alkaloids imperialine, 3β-acetylimperialine and sin- period of cough in mice induced by ammonia [18]. They may peinine A concentration-dependently relaxed carbachol- be the active ingredients of Chuan Bei Mu.

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Table 1 Steroidal alkaloids found in Fritillaria bulbs in recent years

Taxon steroidal alkaloids Type References F. puqiensis Puqiedine 1 A [5] 3α-Puqiedin-7-ol 2 A [5] Puqienine C 3 B [5] Puqienine D 4 B [5] Puqienine E 5 B [5] Puqietinedione 6 D [5] F. hupehensis (3β, 5α)-20-Hydroxy-6-oxocevan-3-yl acetate 7 A [6] (3β, 5α, 6α)-6, 20-Dihydroxycevan-3-yl acetate 8 A [6] (3β, 5α, 13α, 23β)-7, 8, 12, 14-Tetradehydro-5, 6, 12, 13-tetrahydro-3, 23-dihydroxyveratraman-6-one B [6] 9 (3β, 5α, 13α, 23β)-7, 8, 12, 14-Tetradehydro-5, 6, 12, 13-tetrahydro-3, 13, 23-trihydroxyveratraman B [6] -6-one 10 F. unibracteata Puqiedinone-3-O-β-D-glucopyranoside 11 A [7] Peimisine-3-O-β-D-glucopyranoside 12 C [7] F. lichuanensis Hupehenizioiside 13 A [8] F. lichuanensis Lichuanine ((20S, 25R)-5α,14α-cevanine-3β, 6β-diol) 14 A [9] Lichuanisinine ((20S, 25S)-5α,14α-cevanine-3β, 6β-diol-N-oxide) 15 A [9] F. ussuriensis Pingbeimunone A 16 B [82] F. puqiensis Puqienine F 17 B [10] F. monatha Pengbeimine B 18 C [11] Pengbeimine D 19 C [11] F. delavayi Delavidine 20 D [12] F. anhuiensis (22S, 25S)-Solanid-5-en-3β-ol 21 E [13] (22S, 25S)-Solanid-5, 20(21)-dien-3β-ol 22 E [13] (1) Isosteroidal alkaloids (Figs. 1 & 2): A cevanine type; B veratramine type; C jervine type; (2) Steroidal alkaloids (Figs. 1 & 2): D verazine type; E solanidine type. D/E configurations of cevanine group steroidal alkaloids of 1, 2, 7, 8, 11, 13−15 are in the trans-form

Puqienine E is an angiotensin converting enzyme inhibi- 2.2 Saponins and terpenoids tory steroidal alkaloid from F. puqiensis, which showed the Saponins are widely distributed among plants, and have anti-hypertensive effect in vitro [19]. Puqienine B and pu- a wide range of biological properties. Steroidal saponins con- qienine A of F. puqiensis also exhibited a moderate an- sist of a steroidal aglycone, a C27 spirostane skeleton, gener- ti-hypertensive effect. ally comprising of a six-ring structure (Fig. 3). Over the period The alkaloid verticinone exerted a good antinocicep- 2006-2012, eight new steroidal saponins were isolated from the tive effect on inflammatory pain and cancer-related neu- dry bulbs of F. pallidiflora [22-23] (Table 2). Compounds 1, 10, ropathic pain, probably through both peripheral and cen- 11 and 12 showed cytotoxicity against C6 and HeLa cell lines −1 [23] tral mechanisms, and it might be partly involved with with IC50 values of 5.1−75.8 μmol·L . some sedation effects [20]. The newly found peimisine- Nine terpenoid derivatives were reported during 3-O-β-D-glucopyranoside of F. unibracteata, one of the 2005−2012. Compounds 31−36, and 39 belong to the diter- six source plants of Chuan Bei Mu, showed moderate penes, compounds 37 and 38 belong to triterpenoids (Table protective effect on neurotoxicity of PC12 cell lines in- S1, Fig. S1). The new kaurane diterpene (Table S1), duced by rotenone [7]. ent-3β-butanoyloxykaur-15-en-17-ol, and four known kau- Four new steroidal penta- and hexacyclic veratraman- rane diterpenes, isolated from the bulbs of F. ebeiensis, showed and cevan-based alkaloids were obtained from F. hupe- neuroprotective effects against MPP(+)-induced neuronal cell hensis[6]. Alkaloids 1 and 2 showed significant inhibitory death in human dopaminergic neuroblastoma SH-SY5Y cells [24]. effects against HeLa and HepG2 tumor cell lines. Vertici- Two labdane diterpenes of F. ebeiensis, 6α, 7β-dihydroxy- none was shown to induce differentiation in human leu- labda-8(17), 12(E), 14-triene and 6-oxo- 2α-hydroxy-labda-7, kemia cells [21], which also induces cell cycle arrest and 12(E), 14-triene, also showed similar effects [25]. A new diterpene apoptosis in immortalized and malignant human oral isopima ra-7,15-dien-19- oic acid from F. imperialis showed keratinocytes. prolyl endopeptidase inhibitory activity [26].

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Fig. 3 Steroidal saponins of Fritillaria. Refer to Table 2 for more details

Table 2 Steroidal saponins found in Fritillaria bulbs in recent years Taxon Saponins Aglycone Oligosaccharide chain References

F. pallidiflora Pallidifloside D 23 A R = S2 [23]

Pallidifloside E 24 B R = S1 [23]

Pallidifloside G 25 C R1 = S1, R2 = S4 [23]

Pallidifloside H 26 C R1 = S1, R2 = S4 [23]

Pallidifloside I 27 C R1 = S1, R2 = S4 [23]

F. pallidiflora Pallidifloside A 28 C R1 = S1, R2 = S4 [22]

Pallidifloside B 29 C R1 = S2, R2 = S4 [22]

Pallidifloside C 30 D R = S3 [22] Aglycone (Fig. 3): A C-22 steroidal lactone type; B pregnane type; C furostanol type; D spirostanol type

S1 = Xyl-Rha-Glc-; S2 = Rha-Glc-; S3 = Glc-Gal-; S4 = Glc-. The locations of R1, R2 and R are shown in Fig. 3

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2.3 Other compounds 3 Chemotaxonomy of Fritillaria FUP-1, a water-soluble polysaccharide obtained from F. ussuriensis, exerts antioxidant activity through radi- All plants produce secondary metabolites in the cal-scavenging activity and by boosting the enzymatic and co-evolution of host and pathogen/herbivore, however, the non-enzymatic antioxidant defense system of the host [27]. structural types are often specific and restricted to taxonomi- Three nucleosides and three bases were isolated from F. pu- cally related plant groups. This observation was the basis of qiensis and identified as 2’-O-methyladenosine (I), uridine the development of chemotaxonomy. Much evidence sug- (II), adenosine (III), uracil (IV), thymine (V), and adenine (VI) gests that 5α-cevanine isosteroidal alkaloids are the charac- [39] [28]. Compounds I, II, IV-VI were isolated from Fritillaria for teristic constituents of the genus Fritillaria . In light of the the first time. Compound I is a rare, methylated ribonucleo- biogenesis of this kind of alkaloid, the C-13 and C-17 of the side. Adenosine is involved in decreasing the blood pressure, molecules may be rational positions uniting a nitrogenous slowing the heart rate, relaxing the smooth muscle and seda- group in their biosynthetic pathways that generate two kinds tive effects [29]. Adenosine, uridine, adenine, guanosine and of 5α-cevanine isosteroidal alkaloids. The two hydrogen at- thymidine were quantified with reverse phase high perform- oms of C-13 and C-17 have a trans-configuration (e.g., ver- ance liquid chromatography (RP-HPLC). The order of con- ticine and verticinone; Fig. 2) and a cis-configuration (e.g., tents in different Fritillaria bulbs was F. hupehensis > F. delavine, chuanbeinone, and songbeinine), respectively. In F. thunbergii > F. cirrhosa approximately F. ussuriensis. A sen- cirrhosa, F. cirrhosa var. ecirrhosa, F. d e l av a yi (source plants sitive and reliable HPLC-diode-array detector (DAD) method of Chuan Bei Mu) and F. pallidiflora (source plant of Yi Bei was developed to simultaneously determine nine nucleosides Mu), the trans-configuration 5α-cevanine isosteroidal alka- and nucleobases, including uracil, cytidine, guanine, uridine, loids were not detected with thin layer chromatography (TLC) thymine, inosine, guanosine, thymidine, and adenosine in and only the cis-configuration alkaloids were detected [39]. [30] thirteen Fritillaria species , which could be helpful to Only cis-configuration 5α-cevanine isosteroidal alkaloids control the quality of Fritillaria bulbs. Seven nucleosides and were obtained by ingredient separation of F. unibracteata nucleobases (uracil, cytidine, uridine, guanosine, thymidine, (source plant of Chuan Bei Mu). In contrast, the adenosine, and adenine) in F. taipaiensis, one of the six trans-configuration 5α-cevanine isosteroidal alkaloids were source plants of Chuan Bei Mu, can be rapidly and accurately detected in F. thunbergii (source plant of Zhe Bei Mu), F. [31] quantified by HPLC-DAD . Bulbs of F. delavayi, one of anhuiensis, F. ebeiensis and F. wuyangensis (source plants of the six source plants of Chuan Bei Mu, is the most commonly Anhui Bei Mu), while cis-configuration alkaloids were not used antitussive and apophlegmatic in China. It contains detected. F. hupehensis (source plant of Hu Bei Bei Mu), F. uracil, cytidine, inosine, uridine, guanosine, thymidine, ade- ussuriensis (source plant of Ping Bei Mu) and F. karelinii nosine, hypoxanthine, adenine, and 2-deoxyadenosine that (source plant of Yi Bei Mu) contained both trans- and can be quantified with HPLC, which is an efficient way to cis-configuration 5α-cevanine isosteroidal alkaloids. Fritil- evaluate the quality consistency of F. delavayi [32]. laria samples were pre-derivatized by trimethylsilylimidazole Thirty-nine volatile components were identified from F. and six isosteroidal alkaloids (ebeiedine, ebeiedinone, ver- thunbergii using gas chromatography-time-of flight ticine, isoverticine, verticinone, and imperialine) were ana- mass spectrometry (GC-TOF-MS), including the octadeca- lyzed by gas chromatography [40], which is more sensitive trienoic acid methyl esters and some aromatic aldehydes and than TLC. Sixteen species were classified into four groups. F. ketones, such as benzeneacetaldehyde and 1-(2-hydroxy-5- thunbergii, F. thunbergii var. chekiangensis, F. anhuiensis, F. methylphenyl)-ethanone [33]. The volatile component ebeiensis, F. ebeiensis var. purpurea and F. hupehensis con- 3-methyl-2-butene-1- thiol, causing the foxy odor, was iden- tained only trans-configuration 5α-cevanine isosteroidal al- tified from the headspace of F. imperialis bulbs [34]. kaloids (verticine, verticinone and ebeiedine); F. pallidiflora, Element fingerprints were deciphered for F. thunbergii from [35] F. walujewii and F. yuminensis (source plants of Yi Bei Mu) 10 major producing regions of China . Analysis by induc- tively coupled plasma optical emission spectrometry allowed contained only cis-configuration alkaloids (e.g., imperialine); the simultaneous determination of 18 elements (Al, B, Ca, Cd, F. cirrhosa, F. cirrhosa var. virdiflora, F. unibracteata, F. de- Co, Cr, Cu, Fe, K, Mg, Na, Mn, Mo, S, Ni, P, Pb and Zn) in F. lavayi and F. ussuriensis contained both trans- and thunbergii. Among the many elements of F. ussuriensis, Cu, cis-configuration alkaloids; F. meleagroides and F. maximowiczii Zn, Fe, Mg, and Mn are the most dominant [36]. In addition, a contained neither trans- nor cis-configuration alkaloids. glucosylsterol, β-sitosterol-3-O-glucopyranoside, was iso- Bei Mu is one of the most commonly used Chinese lated from F. verticillata, which inhibits sortase, a bacterial herbal medicines, and is derived from the bulbs of some surface protein anchoring transpeptidase [37]. Many other fritillaries. In recent years, many new species and compounds were also isolated from various Fritillaria spe- infraspecific taxa of the genus Fritillaria have been described cies [38]. Their functions and pharmacological effects await in China, including 80 species, 52 varieties, and six forms. further studies. The significant increase of new taxa has influenced the investigation, application, quality control, and commodity 2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 335

HAO Da-Cheng, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 330344 application, quality control, and commodity circulation of method for species identification. Previous studies based on Bei Mu. Xiao et al [2] performed a benchmark study of the morphological and histological techniques have respective morphological, geographical, phytochemical and historical limitations. To find an applicable authentication method, aspects of Chinese medicinal fritillaries, with regard to their twenty-seven steroidal alkaloids (sixteen cevanine type, six current application. The source plants of Bei Mu, from a veratramine type, one jervine type, and four secosolanidine pharmacophylogenetic point of view, were classified into six type alkaloids) in seventeen Fritillaria species and twelve groups. (l) Zhe Bei Mu is mainly derived from the cultivated Bei Mu-containing compound formulas were identified and F. thunbergii and F. thunbergii var. chekiangensis in Zhejiang characterized by a HPLC-MS method [42]. The estimated and provinces, and contains D/E trans cevanine relative composition of the steroidal alkaloids was used in a group alkaloids, predominantly verticine, isoverticine, and chemotaxonomical study of Fritillaria based on hierarchial verticinone. No D/E cis cevanine group alkaloids were de- cluster analysis. The content and composition of steroidal tected [2]. (2) Yi Bei Mu consists of F. walujewii, F. tortifolia, alkaloids are controlled more by genetic than by environ- F. yuminensis, F. karelinii and F. pallidiflora, which are dis- mental factors. F. puqiensis is basal to and far away from tributed in Xingjiang, China and central , and contains other species, suggesting its chemical uniqueness. The pres- D/E cis cevanine group alkaloids, e.g., imperialine; no D/E ence of multiple types of steroidal skeletons, especially of trans form of cevanine group alkaloids were found. (3) Ping large amounts of veratramine type (e.g., puqienine B) and Bei Mu is obtained from cultivated F. ussuriensis in north- secosolanidine type (e.g., puqietinone) alkaloids in a single eastern China and Korea, and contains both D/E trans and cis species is rare in the genus Fritillaria [5, 42], which suggests forms of cevanine group alkaloids. (4) Chuan Bei Mu is the that F. puqiensis is not a subspecies of F. thunbergii as pro- most important medicinal Bei Mu, which is derived mainly posed previously [43]. The remaining sixteen species fell into from F. cirrhosa, F. cirrhosa var. virdiflora, F. przewalskii, F. two groups. Group I, mainly containing the trans-D/E ceva- delavayi, F. wabuensis, F. taipaiensis, F. taipaiensis var. nine alkaloids, such as verticine, verticinone, isoverticine, ningxiaensis and F. unibracteata in the Hengduan mountains and ebeiedinone, includes the source plants of Zhe Bei Mu, and their adjacent regions. F. cirrhosa, F. przewalskii and F. Ping Bei Mu, Hu Bei Bei Mu, and Anhui Bei Mu, and F. unibracteata have smaller bulbs than the other species, and maximowiczii, an adulterant of Chuan Bei Mu [44]. The are the main source of the commodity Qingbei, which is re- chemical profile of Zhe Bei Mu is closer to that of Ping Bei garded as Bei Mu of the highest quality. F. cirrhosa, distrib- Mu than to those of Hu Bei Bei Mu and Anhui Bei Mu, uted in , , Sichuan, and Qinghai, shows many which is geographically closer to Zhe Bei Mu. Group II, morphological variations, and towards its northwestern limit containing the highly abundant cis-D/E cevanine alkaloids, is replaced by a very closely related species, F. taipaiensis. such as imperialine and sinpeinine A, includes the source The bulbs of F. delavayi, called Lubei, are in the group of plants of Chuan Bei Mu and Yi Bei Mu. The chemical profile Chuan Bei Mu. All kinds of Chuan Bei Mu contain both D/E of F. meleagroides, which is geographically closer to Yi Bei trans and D/E cis cevanine group alkaloids. (5) Hu Bei Bei Mu, is closer to that of Chuan Bei Mu, and renders F. me le a - Mu is mainly from bulbs of cultivated F. hupehensis [41], groides a useful medicinal resource. The above classification which is very closely related to F. monantha and F. egregia. It agrees with the early TLC studies [39], and is congruent with contains both D/E trans and cis forms of cevanine group the pharmacophylogenetic treatment of Chinese medicinal alkaloids; verticine and isoverticine are representative of the Bei Mu proposed by Xiao et al. [2]. The characteristic distri- D/E trans form, while hupehenine, hupeheninoside, hupe- bution patterns of the examined compounds were utilized to henrine and hupehenizine are representative of the D/E cis determine the botanical origin of Bei Mu-containing com- form. (6) Anhui Bei Mu is derived from F. anhuiensis, F. pound formulas. Ten out of twelve products meet their label ebeiensis, F. ebeiensis var. purpurea and F. wuyangensis and claims in terms of the Bei Mu raw material. Therefore, the contains only D/E trans cevanine group alkaloids. The six qualitative and quantitative differences in steroidal alkaloids Bei Mu groups have six “Dao-di” (genuine) production areas, were useful for the chemotaxonomy of medicinal Fritillaria respectively. This classification is in line with current clinical species, and for species identification in compound formulas. application and drug marketing. All except Anhui Bei Mu are Due to its price, most of the Chuan Bei Mu on the market is recorded in the latest edition of China Pharmacopoeia (2010). not pure; quite often the high-yield Fritillaria product Ping Xiao et al. [2] suggested that the publication and naming of new Bei Mu is mixed with it, to a greater or lesser extent [44]. This taxa of the most important economic plants, not limited to Bei kind of adulteration mitigates the therapeutic efficiency. The Mu, should be more cautious. The chemosystematic study is above-mentioned HPLC-MS method is important in the qual- very helpful for the sustainable utilization of Fritillaria me- ity control of Bei Mu-containing herbal medicine prepara- dicinal resources, and also for the conservation of critically tions, allowing for preventing Bei Mu confusion and detect- endangered Fritillaria species, such as those in western China. ing possible adulteration. Due to the complexity of the botanical origin of Bei Mu LC coupled with electrospray ionization (ESI) quadru- in the herbal markets, it is urgent to develop a reliable me- pole time-of-flight (QTOF)-MS/MS was performed to study

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HAO Da-Cheng, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 330344 the fragmentation behaviors of the steroidal alkaloids of Frit- nucleotides) are used in the PCR, which uses a large template illaria [45]. Forty-one steroidal alkaloids (twenty-nine ceva- of genomic DNA. By resolving the resulting patterns, a nine type, one jervine type, six veratramine type, and five semi-unique profile can be garnered from a RAPD reaction. secosolanidine type alkaloids) were selectively identified in RAPD has been used to discriminate Chuan Bei Mu, Hu Bei eight medicinal Fritillaria species. Twenty-six alkaloids were Bei Mu, and other source plants of Bei Mu [49-50]. RAPD was unambiguously identified by comparison with the reference also used to study the genetic diversity of four F. thunbergii compounds, and fifteen alkaloids were tentatively identified , i.e., Duozi (many ), broad-, narrow-leaf or inferred according to their MS/MS data. The and Lingxia, and five Fritillaria species (F. thunbergii, F. LC/QTOF-MS total ion chromatograms of eight species con- cirrhosa, F. anhuiensis, F. thunbergii var. chekiangensis and F. firmed the chemotaxonomy results of a previous study [42]. pallidiflora) [51]. Nine polymorphic primers were selected for For example, Ping Bei Mu shared more peaks with Zhe Bei further RAPD amplification. Fifty-seven amplicons with 48 Mu than with Chuan Bei Mu; Yi Bei Mu shared more peaks polymorphic bands, accounting for 84.21% of all the bands, with Chuan Bei Mu than with Zhe Bei Mu. Confirming the were generated. Among the four F. thunbergii cultivars, Duo- above findings, the major chemical differences between the zi differed from the other three. Lingxia was closer to the source plants of Yi Bei Mu and those of Zhe Bei Mu, Hu Bei narrow-leaf form than to the broad-leaf. F. thunbergii var. Bei Mu, and Anhui Bei Mu was revealed by the analysis of F. chekiangensis and F. thunbergii had a close relationship, pallidiflora and F. walujewii with ultra-performance LC with while F. cirrhosa was closer to Zhe Bei Mu than to F. pallidi- evaporative light scattering detection (UPLC-ELSD) [46]. flora. Screened RAPD markers can be applied to identify Peimisine and sipeimine are highly abundant in Yi Bei Mu, different F. thunbergii cultivars and Fritillaria species. while peimine and peiminine, which are highly abundant in RAPD was also used to determine whether F. wabuensis Zhe Bei Mu, Hu Bei Bei Mu and Anhui Bei Mu, are of low could be used as the source plant of Chuan Bei Mu [52]. All abundance, or not detected, in Yi Bei Mu. taxa could be divided into four groups. F. thunbergii was Besides steroidal alkaloids, chemical elements were also basal to other taxa; F. sichuanica was basal to the source analyzed to differentiate Fritillaria samples. Flame atomic plants of Chuan Bei Mu; the group consisting of F. taipaien- absorption spectrometry (FAAS) and graphite furnace atomic sis, F. delavayi, one F. cirrhosa var. ecirrhosa sample and one absorption spectrometry were used to analyze the Zn, Fe, Mn, F. cirrhosa sample is sister to the group consisting of F. Cu, Na, Mg, K, Ca, As, Pb, Cd, Co, Cr, Sr, and Al content of wabuensis, F. cirrhosa, F. cirrhosa var. ecirrhosa, F. u ni - Fritillaria samples from four production regions [47]. Zn, Mn, bracteata, and F. przewalskii. The taxonomic status of 42 Cd and Sr were highly abundant in F. thunbergii, and Cu and taxa of Fritillaria in Turkey was examined by RAPD-PCR Ca highly abundant in Xiang Bei (a kind of commercial Zhe and protein analysis [53]. F. imperialis and F. persica are Bei Mu originating in Xiangshan, Zhejiang Province), close relatives. F. acmopetala subsp. acmopetala and F. so- whereas Fe, Mg, K, Co, and Al were highly abundant in F. rorum are very close relatives according to morphological, thunbergii var. chekiangensis, and Na, As, Pb, and Cr were RAPD and protein results. Therefore these two taxa can be highly abundant in F. cirrhosa. Principal component analysis considered as synonyms. F. zagrica, F. caucasica, F. (PCA) unequivocally grouped the Fritillaria samples. baskilensis, F. armena, and F. pinardii are grouped together. Eighteen elements in F. thunbergii were analyzed with All these separate taxa could be treated as synonyms based inductively coupled plasma optical emission spectrometry to on morphological and molecular data. However, no East Asia differentiate samples from the ten major production regions [35]. species were included in this study. Despite these studies, the relationships among the Fritillaria ISSR (inter-simple sequence repeat) is a general term for species have not been totally resolved. These studies did not a genome region between microsatellite loci. The comple- include Fritillaria species outside of China, although the mentary sequences to two adjacent microsatellites are used as classification of medicinal Fritillaria should be determined in PCR primers; the variable region between them is amplified. the context of world-wide distribution. The distribution of ISSR was used to study the relationship and genetic poly- secondary metabolites and other chemical entities apparently is morphism of nineteen Fritillaria populations in Sichuan of value for taxonomy, but it has to be analyzed cautiously, as Province [54]. Eleven primers were selected from 35 ISSR for any other adaptive trait. Molecular methods have to be primers and 179 DNA fragments were amplified from nine- combined with morphology and chemotaxonomy in the teen populations. Samples were classified into four groups. elucidation of the pharmacophylogeny of Fritillaria. ISSR was also used to classify twenty-two ornamental Fritil- 4 Molecular Taxonomy, Molecular Phylogeny laria taxa, including species from both East Asia and Medi- terranean region [55]. Thirteen primers were used for ISSR and Genomics amplification and 160 polymorphic bands were generated. RAPD (random amplification of polymorphic DNA) is a Samples were classified into two major groups. In the small typw of PCR reaction, but the DNA fragments that are ampli- group, F. davidii of subgenus Davidii is closer to F. anhuien- fied are random [48]. The arbitrary and short primers (8−12 sis than to F. camtschatcensis of subgenus Liliorhiza. In the

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HAO Da-Cheng, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 330344 large group, there are three subgroups: 1. the subgroup con- sisting of the Mediterranean taxa F. acmopetala ssp. wendel- boi, F. acmopetala, F. elwesii and F. uva-vulpis; 2. the sub- group consisting of source plants of Yi Bei Mu, Zhe Bei Mu and Chuan Bei Mu, which belong to subgenus Fritillaria; 3. the subgroup consisting of the Mediterranean taxa F. imperi- alis, F. raddeana (subgenus Petillium) and F. persica (sub- genus Theresia). The subgroup 1, belonging to subgenus Fritillaria, is closer to the subgroup 2 than to the subgroup 3, which does not contradict with the RAPD results [53] and phylogeny results inferred from ITS and matK sequences (see below). Genetic diversity and differentiation of four land- races of F. thunbergii and two congeners F. cirrhosa and F. anhuiensis were evaluated using ISSR markers [56]. The ge- netic diversity of F. thunbergii was high at the specie level, but relatively low at the landrace level. F. anhuiensis was closer to F. cirrhosa than to F. thunbergii, which agrees with the previous ISSR study [57] and RAPD study [51]. Interest- ingly, in UPGMA , F. thunbergii Duozi was basal to other taxa, including other F. thunbergii cultivars, F. thun- bergii var. chekiangensis, F. anhuiensis, and F. przewalskii [57]. It is possible that Duozi is a hybrid taxon selected during long-term cultivation. Low levels of genetic diversity were observed in all seven populations of F. thunbergii from the major production area Pan’an, Zhejiang Province [57], which were lower than that of fritillary populations from other pro- duction areas. However, the high genetic diversity and het- erogeneity of F. thunbergii in Pan’an, inferred from ISSR markers, was also reported [58]. AFLP (amplified fragment length polymorphism PCR) Fig. 4 Phylogenetic relationship of Fritillaria inferred from uses restriction enzymes to cut genomic DNA, followed by nuclear ITS and chloroplast matK sequences. The evolution- ligation of adaptors to the sticky ends of the restriction frag- ary history was inferred by using the Maximum Likelihood ments. A subset of the restriction fragments are then ampli- method based on the Kimura 2-parameter model. The tree fied using primers complementary to the adaptor and part of with the highest log likelihood (-13435.1986) is shown. Initial the restriction site fragments. The amplified fragments are tree(s) for the heuristic search were obtained automatically visualized on denaturing polyacrylamide gels through auto- as follows. When the number of common sites was < 100 or radiography[59] or fluorescence[60] methodologies. AFLP was less than one fourth of the total number of sites, the maxi- mum parsimony method was used; otherwise BIONJ method used to investigate the genetic diversity of six representation- [60] with MCL distance matrix was used. A discrete Gamma dis- nal populations of F. thunbergii, including 32 individuals . tribution was used to model evolutionary rate differences High intra-population genetic diversity of cultivated F. thun- among sites (5 categories, parameter = 0.703 1). The tree is bergii was revealed, while genetic differentiation among the drawn to scale, with branch lengths measured in the number populations was not significant. AFLP was also used to study of substitutions per site. The analysis involved 56 nucleotide the genetic diversity and structure of F. cirrhosa in southwest sequences (ITS + matK) retrieved from NCBI GenBank, [69-71] China [59]. Two primer combinations produced 148 repro- which were used in the previous phylogenetic studies . There were a total of 3 082 positions in the final dataset. ducible, unambiguous and polymorphic bands, ranging from Evolutionary analyses were conducted in MEGA5 [87] 120 bp to 750 bp. Populations from Hengduan Mountains exhibited higher level of genetic diversity than those from the crossing herb, seed dispersal by wind or water plays an im- east margin of the distribution range. The majority of varia- portant role in maintaining gene flow for F. cirrhosa. For tion (72.29%) was distributed within populations, and there is such an endangered and over-harvested plant, it is unusual for a relatively high gene migration rate between populations. As F. cirrhosa to possess relatively high genetic diversity at the an important medicinal plant, wild F. cirrhosa has been har- population and species levels. It is inferred that the major vested for thousands of years, with the greatest pressure in diversification of modern Fritillaria occurred in the Hima- the past 30 years. Over-harvesting might result in loss of laya-Hengduan region during periods of the uplift of the genetic variation in wild populations. As a perennial out- Qinghai-Tibetan Plateau and the global geologic and eco-

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HAO Da-Cheng, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 330344 logical changes during the late Tertiary. Many plant groups It is inferred that these taxa are evolutionarily young and still had adaptive radiation, differentiation, and speciation after in the stage of rapid species radiation and speciation. Macro- migrating to this region, e.g., Pedicularis [61], Aconitum, scopic morphology, microscopic morphology and flower Corydalis, Primula, Silene, and Rhododendron [62] and characters have diverged considerably and therefore different Clematis [63]. Relationships among some closely related spe- species are delimited, but the molecular sequences and cies of Hengduan Mountains and adjacent regions, especially chemical profiles have not diverged significantly. The maxi- the source plants of Chuan Bei Mu and other taxa of the mum parsimony (MP) phylogenetic tree based on ITS se- subgenus Fritillaria, are not resolved [52, 54, 64], suggesting quences (ITS1 + 5.8s rDNA + ITS2) suggests that Chuan Bei that these taxa may have diverged recently. Geologic and Mu source species are closer to F. thunbergii than to F. yu- climatic changes in the late Tertiary to Quaternary (2.588 minensis, a source species of Yi Bei Mu [69]. However, only mya - now) might be important for the speciation of ten Fritillaria ITS sequences were used in this study. Rønsted Fritillaria, especially in eastern Asia. Long-distance dispersal et al. [70] performed phylogenetic analyses of 37 taxa of Frit- of the fruits by wind, water or animals, and strong illaria, fifteen species of Lilium and several outgroup taxa environmental adaptability, might explain the present from Liliaceae to explore the generic delimitation of Fritil- world-wide distribution and high species diversity. Although laria in relation to Lilium and infrageneric relationships F. cirrhosa maintains relatively high genetic diversity, the within Fritillaria. DNA sequences from the maturase-coding sharply decreasing numbers of wild populations and plastid matK gene and the trnK intron, the intron of the ribo- individuals remains a threat. Zhang et al [59] suggested a somal protein-coding rpl16 plastid gene and the nr ITS were method of sustainable collection, as protecting a proportion PCR amplified and sequenced. Fritillaria and Lilium are of mature individuals is necessary and important for sister taxa. Fritillaria subgenera Fritillaria and Liliorhiza are maintaining effective population size and evolutionary supported in part, and some enigmatic species usually in- potential. Even at the wholesale price in China, good quality cluded in Fritillaria (subgenera Petilium and Theresia and Chuan Bei Mu can cost over $200/kg. As the most valued the monotypic subgenus Korolkowia) are closely related. The traditional medicinal Bei Mu, domestication and cultivation results support the new classification of Fritillaria proposed of F. cirrhosa and other source plants of Chuan Bei Mu are by Rix [1], who suggested eight subgenera, i.e., Fritillaria, vital both to satisfying market demand and protecting the Rhinopetalum, Japonica, Theresia, Petilium, Liliorhiza, wild Withresource the [65]rapid. development of DNA sequencing tech- Davidii and Korolkowia. The independent origins of the nology, some nuclear DNA and chloroplast DNA markers underground bulbils found in F. davidii, an adulterant of have been used in Fritillaria taxonomy and phylogenetic Chuan Bei Mu [44], and the subgenus Liliorhiza was proposed study. PCR primers were designed to specifically amplify 5s [70]. Regretably, this study included few Chinese species, and rDNA sequences from two traditional source plants of Chuan the results were not cross-validated by NJ and/or other tree Bei Mu, F. prezwalskii and F. unibracteata [66], with which no reconstruction methods, while Li [71] only studied species products were amplified from the new Chuan Bei Mu source distributed in Sichuan, China. To date, there is no molecular plant F. wabuensis and other medicinal Fritillaria. The inter- phylogeny study covering both the Eurasia (including China) nal transcribed spacer 1 (ITS1) regions of the nuclear ribo- and the North America taxa. somal DNA (nrDNA) of nine species and one variety of Frit- In the present study, matK and ITS sequences of Fritil- illaria genus were sequenced [67]. A mutation site in the ITS1 laria were retrieved to reconstruct phylogenetic based region shared by F. cirrhosa and other source species of on matK, ITS and the combined dataset. NJ, MP, and maxi- Chuan Bei Mu was found, and can be recognized by the re- mum likelihood (ML) phylogenetic relationships are inferred striction endonuclease SmaI. PCR-RFLP (restriction frag- from 57 matK, 69 ITS and 56 matK + ITS sequences. On the ment length polymorphism) was used to differentiate four matK + ITS tree inferred by the ML method (Fig. 4), there Chuan Bei Mu species, recorded in the China Pharmacopoeia are two major clades, one (I) consisting of the subgenus Frit- of 2005 version, from other medicinal Fritillaria species, and illaria taxa collected from Sichuan, China [54, 71], the other (II) is a successful authentication method. This method is also comprising other taxa. Two F. cirrhosa taxa are basal to other applicable to F. wabuensis and F. taipaiensis that are recorded taxa in clade I, but other F. cirrhosa taxa intermingle with the in the China Pharmacopoeia of 2010 version as additional Sichuan species and their interrelationships are actually not source plants of Chuan Bei Mu [68]. ITS2 sequencing was resolved. F. thunbergii and F. hupehensis that Li [54, 71] used as a DNA barcode to discriminate the raw plants of claimed might not be bona fide ones, which were collected in Chuan Bei Mu and its adulterants [44]. Different Chuan Bei Sichuan and were not from the native distribution areas. The Mu plants clustered together and could be distinguished from so-called “F. thunbergii” might be a misidentification, and the their adulterants on the Neighbor-Joining (NJ) phylogenetic putative “F. hupehensis” could be a hybrid between F. c i r- tree inferred from the ITS2 sequences. Intriguingly, different rhosa and other Sichuan species, which is inferred from the samples of F. cirrhosa failed to form a single cluster on the ITS tree, matK tree (this study), and ISSR results [54]. In clade NJ tree, so did samples of other Chuan Bei Mu source species. II, the Mediterranean species of the subgenus Fritillaria are

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HAO Da-Cheng, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 330344 basal to the remaining taxa, and is supported by the NJ infer- could produce secondary metabolites (e.g. ergot alkaloids in ence, but which contradicts with the MP result [70]. F. gibbosa Convolvulaceae) [74]. Lateral gene transfer from symbiotic of the subgenus Rhinopetalum, and F. japonica of the subge- fungi is also possible. Fourth, as the rhizosphere bacteria and nus Japonica are basal to two sister subclades, one consisting the soil properties have a close relationship with the geo- of the subgenus Liliorhiza species of northeast Asia and herbalism of F. thunbergii [75], it is intriguing to investigate North America, the other comprising species of the subgenus how these biotic and abiotic factors shape the chemical pro- Dividii, Fritillaria, Theresia, Korolkowia and Petilium. F. files of various Fritillaria species. thunbergii is basal to Korolkowia and Petilium, while F. pal- The genomic and transcriptomic studies may help eluci- lidiflora and F. persica are sister to the branch containing F. date the secondary metabolism pathways in Fritillaria, and thunbergii. Interestingly, in the ITS tree, F. thunbergii is sister also facilitate taxonomic and phylogenetic investigations. to the clade of the Sichuan species, while in the matK tree it Fritillaria species have exceptionally large genomes (1C = is sister to F. pallidiflora (figures available upon request). 30 000−127 000 Mb) and a bicontinental distribution. Most The leaf epidermis of sixteen Fritillaria species was exam- North American species (subgenus Liliorhiza) differ from ined using light microscopy and scanning electron micros- Eurasian species by their discrete phylogenetic position and copy [72]. The stomatal characteristics support the origin of increased amounts of heterochromatin. Fosmid libraries are the subgenus Fritillaria in China from two floristic elements. powerful tools in dissecting the huge genome [76] and were Some species spread from Central Asia, joining the Turgai constructed from genomic DNAs of F. affinis of North Flora, along the Altai–Sayan route from the south of Siberia America and F. imperialis of Eurasia and used to explore to the Sayan Mountains and Altai Mountains. Others are dis- highly repeated sequences [77]. Repeats corresponding to tributed in the Hengduan Mountains and adjacent regions of 6.7% and 4.7 % of the F. affinis and F. imperialis genome, China and belong to the Phava Flora. It is not impossible that respectively, were identified. Chromoviruses and the Tat F. thunbergii originated from hybridization between Sichuan lineage of Ty3/gypsy group long terminal repeat retrotrans- species and F. pallidiflora, since Fritillaria hybrids are not posons were the predominant components of the highly re- rare in the Sino-Japanese floristic region [73]. Moreover, there peated fractions in the F. affinis and F. imperialis genomes, were some intriguing findings on the ITS tree. F. ussuriensis respectively. A heterogeneous, extremely AT-rich, satellite of Northeast China and North Korea is sister to F. tenella, a repeat was isolated from F. affinis. The FriSAT1 repeat local- Mediterranean species of the subgenus Fritillaria, although ized in heterochromatic bands makes up approx. 26% of the F. its twin-scaled is similar to that of Japanese endemic affinis genome, and substantial genomic fractions in several Fritillaria. F. karelinii, a source species of Yi Bei Mu [2], is other Liliorhiza species. The giant Fritillaria genomes are sister to F. gibbosa and F. stenanthera, and these three are composed of many diversified families of transposable ele- basal to the other taxa. In contrast, F. y u mi ne n si s is sister to F. ments, which are useful in phylogenetic studies. The genome pallidiflora. Since other molecular marker sequences of F. obesity may be partly determined by the failure of removal ussuriensis and F. karelini are not available or are very few, mechanisms to counterbalance effectively the retrotransposon their phylogenetic positions are still elusive. The molecular amplification [77]. In the future, it is necessary to characterize marker sequences of Hu Bei Bei Mu, Anhui Bei Mu and the the genome of Chinese species. unique F. puqiensis are not available, thus their phylogenetic The decreasing cost of sequencing makes it feasible to positions remain intangible. perform the large scale transcriptome analysis of medicinal The molecular phylogeny results are not congruent with plants [78]. Two thousand one hundred and fifty-eight those inferred from chemotaxonomy [42, 45]. Many secondary high-quality expressed sequence tags (ESTs) were generated metabolites have a taxonomically restricted distribution. from a cDNA library of F. cirrhosa bulbs, and 1 343 unique However, the same secondary metabolites (e.g., several transcripts assembled [79]. After removing ribosomal RNA groups of alkaloids and cardiac glycosides) in other plant sequences, 1 330 putative protein coding sequences were taxa that are not closely related in a phylogenetic context are obtained, among which 765 (57.5%) had at least one signifi- also found [4, 74]. The patchy distribution might be explained cant match to the Swiss-Prot protein database via a BLASTX as follows. First, the occurrence of the same set of steroidal similarity search. The 1 330 unique transcripts were further alkaloids in distantly related taxa, e.g., F. cirrhosa and F. functionally classified for gene discovery, using the gene pallidiflora, F. thunbergii, F. ussuriensis and F. maximowiczii, ontology (GO) and Kyoto Encyclopedia of Genes and Ge- might be due to rampant parallelism or convergent evolution. nomes (KEGG) databases. More than ten transcripts that are Second, the genes encoding the enzymes of steroidal alkaloid likely involved in the biosynthesis of F. cirrhosa alkaloids biosynthesis might be regulated transcriptionally, i.e., they and corresponding regulatory activities were discovered, are switched on or off in a certain context. However, little is including HMGR, FPSs, CYP450s and aminotransferases. known about the Fritillaria transcriptome and the regulation This report describes the first example of transcriptome anal- mechanism at the transcription level. Third, a patchy distri- ysis from Fritillaria and is useful for further cloning and for bution may be due to the presence of endophytic fungi, which the identification of candidate genes related to steroidal

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贝母药物资源的植物化学和生物学研究

郝大程 1*, 顾晓洁 1, 肖培根 2*, 彭 勇 2

1 大连交通大学环境学院，大连 116028； 2 中国医学科学院药用植物研究所，北京 100193

【摘 要】 贝母属药用植物含有多种药用化合物，在传统中医药中已应用上千年。近年对贝母药物资源的深入研究导致在 多种贝母中发现更多的甾体生物碱、皂苷、萜类、糖苷和许多其它化合物，由此关于贝母的化学分类，分子系统发育和药理研 究也日益增多。本文基于作者的贝母药用亲缘研究，评述近年贝母属植物化学、化学分类、分子生物和系统发育研究的进展， 及其与贝母药效的关联。通过检索文献总结了贝母研究涉及的多种技术，用基于细胞核和质体 DNA 标记的系统发育分析推断 了川贝，伊贝等复合群内部的相互关系和其与其它中国贝母物种的关系，并明确了中国贝母与国外贝母之间的关系。指出化学 分类与分子系统发育结果的不一致并讨论了原因。深入研究更多的贝母物种对于贝母药物资源的可持续利用和寻找新的治疗用 化合物十分重要。系统生物学和组学技术将对今后贝母药用化合物研究的深入起到关键作用。 【关键词】 贝母；化学成分；化学分类；分子分类；系统发育；药物资源

【基金项目】 科技部国家支撑计划 (No. 2012BAI29B01)资助

344 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期