New Steroidal Glycosides from Rhizomes of Clintonia udensis

A total of 10 steroidal glycosides, together with three new spirostanol glycosides (6–8), a new furostanol glycoside (9), and a new cholestane glycoside (10), were isolated from the rhizomes of Clintonia udensis (Liliaceae). The structures of the new compounds were determined on the basis of extensive spectroscopic analyses, including 2-D nuclear magnetic resonance (NMR) data, and of hydrolytic cleavage followed by chromatographic or spectroscopic analyses. The isolated glycosides were evaluated for their cytotoxic activity against HL-60 leukemia cells. Spirostanol glycosides 1 and 2, and furostanol glycoside 4 showed cytotoxic activity with IC₅₀ values of 3.2±0.02, 2.2±0.12, and 2.2±0.06 μg/ml, respectively. Neither the spirostanol and furostanol saponins with a hydroxy group at C-1 (6 and 9) and C-12 (7 and 8) nor cholestane glycosides (5 and 10) exhibited apparent cytotoxic activity at a sample concentration of 10 μg/ml.     

Reproductive biology of Clintonia udensis

To clarify the life history of Clintonia udensis, we investigated its reproductive systems and spatio‐temporal population structure. Pollination experiments and the observation of floral visitors revealed that C. udensis was compatible with both self‐ and outcross‐pollen, and it potentially produces seeds by insect‐mediated outcrossing in natural conditions. In addition, propagation by clonal reproduction from rhizomes was evident. In this study, it was clarified that C. udensis potentially propagates by sexual and asexual reproduction and maintains its population through a stable frequency of flowering. The differences in the dependence on each reproduction mode could be one of the contributing factors for creating a variety of population sizes and distribution patterns of ramets in populations.     

New steroidal saponins from rhizomes of Costus spiralis

Two new steroidal saponins were isolated from the rhizomes of Costus spiralis Rosc. Their structures were established as (3beta,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->4)]-beta-D-glucopyranoside (1) and (3beta,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl O-D-apio-beta-D-furanosyl-(1-->4)-O-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-glucopyranoside (2). Their structural identifications were performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (DEPT, COSY, HETCOR and COLOC) and chemical conversions. The steroidal saponins were evaluated for anti-inflammatory activity.     

七筋菇自然居群的遗传结构分析

采用ISSR分子标记,对七筋菇(Clintonia udensis)17个居群的遗传多样性与遗传结构进行了研究。结果表明:七筋菇不同居群的多态位点百分率PPB为11.90%~59.52%,总的多态位点百分率PPB为98.8%,具有高的遗传多样性。Shannon多样性指数(0.6903)和基因分化系数(GST=0.6944)均揭示出七筋菇居群间存在明显的遗传差异,AMOVA分析结果也显示遗传变异主要发生在居群之间(81.47%),而居群内部的遗传变异仅为18.53%。七筋菇居群间的遗传距离从0.1871~0.6632,平均为0.3838,大于同一物种居群间的平均遗传距离值(0.05),同样表明七筋菇居群间的遗传多样性存在较大差异。七筋菇居群间的基因流Nm=0.2200,远远低于一般广布种植物的基因流(Nm=1.881)。Mantel检测显示居群间的遗传距离与地理距离之间没有显著相关性(r=0.029,P=0.3196)。七筋菇分布范围广以及其进化历史是其具有高遗传多样性的原因;居群间存在较高遗传变异可能是由于七筋菇本身的生物学特性、有限的基因流以及遗传漂变等原因造成的。     

东亚七筋姑多倍体ITS区的序列测定与分析

通过对13个东亚七筋姑二倍体居群和5个四倍体居群的ITS区(包括ITS1、5.8SrDNA和ITS2)进行克隆测序。ITS序列长度显示出比较高的多态性,ITS1长度为171~270bp,ITS2长度为205~238bp,5.8S序列的长度在162~164bp之间,序列中G C含量为45.83%~54.13%,当空位(gap)作缺失处理时,东亚七筋姑ITS区全序列排序后的长度为682bp,其中简约性位点289个;最大简约法分析获得一致性指数(CI)为0.630,保留系数(RI)为0.684,四倍体居群没有单独聚为一支,而是和相邻地理单元的二倍体居群聚为一支,本研究结果支持东亚七筋姑异地多起源的推断。     

A Cytogeographical Study on Clintonia udensis (Liliaceae)

The genus Clintonia has four species in North America and one in eastern Asia (Fig．1)．In this paper,the karyotypes and the intraspecific morphological and cytogeographic differentiations of ten populations of C．udensis from China are analysed,and the probable origin area of the genus is also discussed．The conclusions are as follows： (1)Based On the chromosome numbers 2n=28 from the North American species,the Japanese and Himalayan groups,the earlier investigators established x=14 as the basic chromosome number of Clintonia,and they thought that there was polyploid in this genus except for aneuploid only in C．borealis (Utech,1975;Utech and Suda,1975),but a few authors(Sen,1975;Wang et al,1993)pointed out the x=7 basic number of this genus based on 2n=14 in C．udensis from Primorskiy Kray of Russia(Skolovskaya,1966)and Yunlong,Yunnan Province of China(Wang et al,1993)respectively．Our result along with there reports by previous authors(see table 1)shows that at least two ploid levels exist in C．udensis,i.e．2n＝14 and 2n＝28．The diploids are widely distributed from northwest Yunnan of China to Primorskiy Kray of Russia,while the tetraploids are located in northwest Yunnan,Himalayas,Japan,and a narrow area in Mt．Hualongshan of southern Shaanxi(07 population)(Fig．2)．Therefore,the basic chromosome number of Clintonia certainly is x=7 ratherthan x=14,while 2n=28 in North America,Japan and Himalayan area are tetraploid, not diploid, According to the previous data,so far no diploid taxa of Clintonia has been found in North America and Himalayas．So we consider that the primitive type of Clintonia is in eastern Asia,and the secondary diversity center and the present distribution center of this genus are in North America． (2)C．udensis is widely distributed in eastern Asia(Fig．2);it has two cytotypes．The karyotypes for all the diploid populations are remarkablely similar. Taken together, they can be roughly repersented by the formula:K(2n)=14=2V+6J+2v+4j(2NOR+2j). The chromosomes range in length 25.55～12.78 μm, with the ratio of the longest to the shortest 2.0. The karyotype belongs to Stebbins (1971)2A(→2B). For the tetraploid taxa, except for 07 population, karyotypes are also identical and may be roughly symbolized as: K (2n)=28=4V + 12J + 4v+8j ( 2NOR + 6j ). The length of chomosomes is from 27.87 to 13.93 μm, with the ratio of the longest to the shortest 2.0, and thus the karyotype belongs to 2A(→2B). The karyotype of 07 population is similar to those of above tetraploid taxa but also has some differences, especially in the position of satellites and the morphology of 10th and 14th pairs of chromosomes. Its formula is K(2n)=28=4V+12J(2NOR+10J)+4v+ 8j(2n=28=10m+16sm(2SAT)+2st). The ratio of the longest (23.72μm)to the shortest(12.97μm) chromosomes is 1.83. The Karyotype belongs to 2A. And the distribution range of this population is very narrow. We think that it is probably a recent evolutionary event in C. udensis. (3)Wether Clintonia in eastern Asia has 1 sp. or 2 spp. or 1 spp. and 1 var. has been debated for a long period. According to our observation, within C. udensis, only the size of seeds is related to its ploidy level, i.e. diploid individuals have smaller seeds and tetraploid ones have larger ones; the colour of seeds is related to its geographic distribution, i.e. the materials from the Himalayas through Yunnan, Sichuan to eastern Qinling Range have pale brown seeds, while those from Mt. Lüliang, Shanxi Province via Hebei, Liaoning, Jilin to Siberia and Japan have dark brown seeds. Some other morphological characters, such as the size of leaves and fruits, inflorescence type and flower numbers between individuals in one locality, even within one populaion have evident variation. Therefore, we consider that evidence(see Table 4 )for separating C. alpina or C. udensis var. alpina from C. udensis is notsufficient.     

东亚七筋姑的传粉生物学研究

以陕西镇坪化龙山地区的东亚七筋姑自然居群为对象,通过重力玻片法、套袋法和人工授粉对其进行连续的传粉生物学观察和研究,以明确其散粉规律和繁育系统.结果表明,东亚七筋姑的花期在五月中下旬,单花花期2～3 d,整个花序的花期随花序大小的不同而异,一般为3～7 d;东亚七筋姑为虫媒传粉,其访花昆虫大多为小型昆虫,主要种类有小型甲虫、蚁类和蝇类等;东亚七筋姑自花传粉是可育的,存在异花传粉,不存在无融合生殖,自然条件下结籽率为58%,人工异株异花授粉结籽率为82%.可见,东亚七筋姑的繁育系统为兼性异交类型,需要传粉者.     

New steroidal saponins with l-arabinose moiety from the rhizomes of Smilax scobinicaulis

Phytochemical investigations of the rhizomes of Smilax scobinicaulis led to the isolation of seven steroidal saponins (1–7) of which four (1, 3, 4 and 6), nameed, Smilscobinosides C-F, respectively) are new. Five of these steroidal saponins with l-arabinose moiety are reported here for the first time in the genus Smilax. The structures were elucidated by spectroscopic analysis of the isolates and their hydrolysis products. The isolated compounds were evaluated for their cytotoxicity against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116 and Lovo). Compounds 3 and 4 exhibited significant inhibition on HCT-116 (with IC₅₀ values of 10.5 and 7.8 μM) together with inhibition on SGC-7901 (with IC₅₀ values of 21.4 and 15.8 μM), respectively.     

Genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udensis

Polyploidy is an important factor shaping the geographic range of a species. Clintonia udensis (Clintonia) is a primary perennial herb widely distributed in China with two karyotypic characteristics—diploid and tetraploid and thereby used to understand the ploidy and distribution. This study unraveled the patterns of genetic variation and spatiotemporal history among the cytotypes of C. udensis using simple sequence repeat or microsatellites. The results showed that the diploids and tetraploids showed the medium level of genetic differentiation; tetraploid was slightly lower than diploid in genetic diversity; recurrent polyploidization seems to have opened new possibilities for the local genotype; the spatiotemporal history of C. udensis allows tracing the interplay of polyploidy evolution; isolated and different ecological surroundings could act as evolutionary capacitors, preserve distinct karyological, and genetic diversity. The approaches of integrating genetic differentiation and spatiotemporal history of diploidy and tetraploidy of Clintonia udens would possibly provide a powerful way to understand the ploidy and plant distribution and undertaken in similar studies in other plant species simultaneously contained the diploid and tetraploid.     

New glycosides of the campesterol derivative from the rhizomes of Tacca chantrieri

Seven new glycosides of the campesterol derivative (24R,25S)-ergost-5-ene-3beta,26-diol (1-7) were isolated from the rhizomes of Tacca chantrieri (Taccaceae). Their structures were determined by extensive spectroscopic analysis, including 2D NMR data, and a few chemical transformations.     

Steroidal glycosides from the rhizomes of Ruscus hypophyllum

Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structures of the compounds to be assigned as (23S,25R)-23-hydroxyspirost-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (1), 1beta-hydroxyspirosta-5,25(27)-dien-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (2), (22S)-16beta,22-dihydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (3), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl O-alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranoside (4), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3beta-yl beta-d-glucopyranoside (5), (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-(3,4-di-O-acetyl-beta-d-xylopyranoside) (6), and (22S)-16beta-[(beta-d-glucopyranosyl)oxy]-3beta,22-dihydroxycholest-5-en-1beta-yl O-alpha-l-rhamnopyranosyl-(1-->2)-O-[beta-d-xylopyranosyl-(1-->3)]-beta-d-xylopyranoside (7), respectively. This is the first isolation of a series of cholestane glycosides from a Ruscus species.     

Cycloartane glycosides from the rhizomes of Curculigo orchioides

Cycloartane glycosides (1-9) were isolated from rhizomes of Curculigo orchioides (Hypoxidaceae), and this structures were determined by spectroscopic analysis and a few chemical transformations. Cytotoxic activity of glycosides (1-9) and their common aglycone (1a) against HL-60 human promyelocytic leukemia cells was also examined.     

C21 steroidal glycosides from Hemidesmus indicus

Two novel pregnane glycosides, denicunine (1) and heminine (4), have been isolated from the dried stem of Hemidesmus indicus R.Br. (family: Asclepiadaceae). Chemical transformations and spectroscopic evidence viz: 1H and 13C NMR spectroscopy and FABMS are consistent with the structures calogenin 3-O-3-O-methyl-beta-D-fucopyranosyl-(1-->4)-O-beta-D-oleandropyranosi de and calogenin 3-O-beta-D-cymaropyranosyl-(1-->4)-O-beta-D-digitoxopyranoside+ ++, respectively.     

Polyhydroxylated steroidal constituents from the fresh rhizomes of Tupistra yunnanensis

Six new polyhydroxylated steroidal saponins, tupistrosides A-F (1-6), together with nine known steroids, were isolated from the fresh rhizomes of Tupistra yunnanensis. Their structures were elucidated to be (25S)-1beta,4beta,5beta-trihydroxy-spirostane-3beta-yl O-alpha-l-arabinopyranoside (1), 1beta,24beta-dihydroxy-spirost-5,25(27)-dien-3alpha-yl O-beta-d-glucopyranoside (2), (22S,25S)-1alpha,2beta,3alpha,5alpha-tetrahydroxy-furo-spirostane-26-yl O-beta-d-glucopyranoside (3), 1beta,3alpha,22 xi-trihydroxy-furost-5,25(27)-dien-26-yl O-beta-d-glucopyranoside (4), 26-O-beta-d-glucopyranosyl-1beta,22-dihydroxy-furost-5-en-3alpha-yl O-beta-d-glucopyranoside (5) and 22-methoxy-1beta,2beta,3beta,4beta,5beta,7alpha-hexahydroxy-furost-25(27)-en-6-one-26-yl O-beta-d-glucopyranoside (6), respectively, by means of spectroscopic analysis and the results of acid hydrolysis.     

基于叶绿体DNA序列分析东亚七筋姑的遗传进化

通过对东亚七筋姑(Clintoniaudensis Trautv.etMey.)19个居群的cpDNA基因间隔区(cpDNA intergenic regions)rpl20-rps12和trnL-F进行序列分析,结果表明,当空位(Gap)作缺失处理时,trnL-F全序列排序后的长度为754个位点,G C含量为35.2%～35.8%,rpl20-rps12序列排序后长度为814bp,G C含量为34.1%～34.3%.聚类结果显示四倍体居群没有单独聚为一支,其多倍体可能是同源多倍体,起源方式属于异地多起源.     

Isoflavonoids and flavone glycosides from rhizomes of Iris carthaliniae

Two new isoflavonoid biosides, tectorigenin 4′-glucosyl (1→6)glucoside and iristectorigenin B 7-glucosyl (1→6)glucoside, a new isoflavonoid monoside, 4′-methyltectorigenin 7-glucoside and a new flavone glucoside, 6,4′-dimethoxy-5-hydroxyflavone 7-glucoside, together with tectoridin and tectorigenin 4′-glucoside were isolated from rhizomes of Iris carthaliniae. The structures of the isolated compounds were determined by NMR spectral analysis.     

Bioactive steroidal alkaloid glycosides from Solanum aculeastrum.

Solanum aculeastrum Dunal was investigated for the presence of molluscicidal compounds. This led to the isolation of solaculine A, from the root bark in addition to known steroidal alkaloids; solamargine and beta-solamarine from the berries. The structures were elucidated by spectroscopic techniques. Molluscicidal activity of the aqueous extracts of the berries and root bark, and the isolated compounds were investigated.     

Seco-pregnane steroidal glycosides from the roots of Cynanchum stauntonii

By analyzing the steroidal content of the roots of Cynanchum stauntonii, we isolated three new C₂₁ steroidal glycosides named cynastauosides A-C (1–3), all of which shared the 14,15-secopregnane-type skeleton. Their structures were determined on the basis of detailed spectroscopic analysis, including 2D NMR and qualitative chemical tests. Anti-inflammatory activities of compounds 1–3 were evaluated in vitro, with 2 and 3 inhibiting nitric oxide production in C57bl/6j mouse peritoneal macrophages by 17.0% and 6.9% of inhibition rate at a concentration of 10 μM, respectively.     

Cholestane and spirostane glycosides from the rhizomes of Dioscorea septemloba

Cholestane glycosides, dioseptemlosides A (1) and B (2), together with six spirostane glycosides, dioseptemlosides C-H (3-8), were isolated from the rhizomes of Dioscorea septemloba. Their structures were established on the basis of physical data, spectroscopic analysis (HRESIMS, 1D and 2D NMR), and chemical methods. Spirostane aglcones containing hydroxyl group at C-7, as found in compounds 4-7, were reported in the family Dioscoreaceae for the first time. These compounds did not show considerable inhibitory anti-tumor activities at a concentration of 10 microM.     

Three new phthalide glycosides from the rhizomes of Ligusticum chuanxiong

Three new phthalide glycosides, named chuanxiongoside A (1), chuanxiongoside B (2) and ligusticoside B (3) together with two known glycosides, celephthalide A (4) and icariside F₂ (5), were isolated from the rhizomes of Ligusticum chuanxiong. This is the first report of compounds 4⿿5 from the title plant. The structures of compounds 1⿿3 were identified using various spectroscopic methods.