乌蕨（Stenoloma chusanum）化学成分研究及其资源利用评价
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摘要
本文概述了民间药用植物乌蕨(Stenoloma chusanum (Linn.)Ching)的形态特征和资源分布情况,综述了国内外对乌蕨的药理药效作用、化学成分的研究进展,并对乌蕨所含有的几种主要化学成分的生物活性进行了总结。作者提出应当对乌蕨生物活性的物质基础进行深入研究,还应对其引种、繁殖及栽培技术进行研究,为开发利用这一植物资源建立基础。作者的主要研究结果有以下几个方面：
1.乌蕨的化学成分研究
利用硅胶、聚酰胺、Lichroprep RP-18. Sephadex LH20等层析材料,对乌蕨的乙醇提取物进行分离,根据化合物的理化性质、波谱数据(ESI-MS、HR-MS、1H-NMR.13C-NMR、DEPT、HSQC、HMBC)、标准品对照并参考文献资料,对分离得到的化合物进行了结构鉴定,已分离并鉴定了19个化合物,它们分别为：乌蕨苷A(stenolomaside A)即[3-(9,3',5'-三甲氧基-4'-羟基-亚苄基)色满-4-酮]-5-O-β-D-葡萄糖苷[3-(9,3',5'-trimethoxy-4'-hydroxy-benzylidene) chroman-4-one]-5-O-β-D-glucoside,该化合物晶体同时存在反式(1a)和顺式(1b)两种化学结构,乌蕨素1(stenolin1)即香草酸-4-O-β-D-(6-O-龙胆酰基)-吡喃葡萄糖苷(4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid)(2),乌蕨素2(stenolin2)1即龙胆酸-2-O-β-D-(6-O-龙胆酰基)-吡喃葡萄糖苷(2-O-β-D-(6-O-gentisoyl glucopyranosyl)gentisic acid)(3),荭草苷(Orientin)(4),荭草苷-2"-O-β-D-吡喃葡萄糖苷(Orientin-2"-O-β-D-glucopyranoside)(5),牡荆素(Vitexin)(6),芹菜素(Apigenin)(7),牡荆素鼠李糖苷(Vitexin rhamnoside)(8),牡荆素吡喃葡萄糖苷(vitexin glucopyranoside)(9),香草酸(Vanillic acid)(10),丁香酸(Syringic acid)(11),龙胆酸(Vanillic acid)(12),原儿茶醛(protocatechualdehyde)(13),6,7-二羟基香豆素(6,7-dihydroxycoumarin)(14),十五烷醇(pentadecanol)(15),十六烷酸(hexadecanoic acid)(16), β-谷甾醇(β-sitosterol)(17),胡萝卜苷(daucosterol)(18)。其中化合物(1a)、(1b)、(2)和(3)为新化合物,化合物(4)、(5)、(8)、(9)、(10)、(14)、(15)和(16)为首次从该植物中分离得到的化合物。
2-乌蕨主要成分的定量分析及其资源评价
作者建立了乌蕨地上部分荭草苷和牡荆素含量的测定方法,检测不同产地乌蕨中两种黄酮苷的含量,据此对乌蕨资源进行了评价。用超声波提取法提取,RP-HPLC测定,Gemini C18色谱柱,乙腈-1%磷酸溶液(V：V=15：85)为流动相,流速1mL/min,检测波长340nm。荭草苷在0.025～0.148μg范围内有良好的线性关系,加样回收率为100.61%,相对标准偏差(RSD)2.18%；牡荆素在0.035～0.207μg范围内有良好的线性关系,加样回收率为99.77%,RSD2.37%。精密度试验测得荭草苷峰面积的相对标准偏差(RSD)为0.10%,牡荆素峰面积的RSD为0.17%。重复性试验测得荭草苷和牡荆素峰面积的RSD分别为1.10%和0.49%。稳定性试验表明样品在6h内稳定。从HPLC图谱可见,荭草苷和牡荆素为乌蕨中二种主要的黄酮类化合物。在所测定的乌蕨种源中,牡荆素含量均高于荭草苷含量,但两者的比例不是固定不变的,云南丘北所产乌蕨的荭草苷和牡荆素含量都是最高,分别达到1.166和1.457mg/g,移栽到南京地区1年后荭草苷含量下降,牡荆素含量增高。结果表明,以荭草苷和牡荆素含量为指标,云南丘北所产乌蕨是较好的种源。
用超声提取法提取不同产地乌蕨地上部分的总黄酮,以芦丁为对照品,建立标准曲线,采用NaNO2-Al(NO)3-NaOH比色法测定总黄酮含量。结果表明,云南丘北所产乌蕨总酮含量最高,达127.461mg·g-1,移栽到南京地区2年后仍保持较高水平；用孢子培育的植株总黄酮含量高达187.038mg·g-1；同一产地的乌蕨,叶片总黄酮含量显著高于叶柄。分析结果表明,以总黄酮含量为指标,云南丘北所产乌蕨是较好的种源,采集乌蕨药材应以叶片为主,通过人工栽培开发利用乌蕨资源是可行的。
3.乌蕨中荭草苷和牡荆素提取纯化方法
为了简便、安全、经济、有效地从乌蕨中得到单体化合物荭草苷和牡荆素,建立了一种从乌蕨的地上部分提取纯化荭草苷和牡荆素的方法,其特征是先从乌蕨地上部分获得粗黄酮析出物,再将粗黄酮用碱水助溶,稀释后用酸中和,经过反相硅胶柱层析,得到目标产物荭草苷和牡荆素,纯度达98%以上。
4.乌蕨中新化合物的生物活性研究
a.酚性化合物的抗真菌试验
用琼脂稀释法(试管法)测定了新化合物乌蕨素1、乌蕨素2及已知化合物香草酸、丁香酸和龙胆酸的抗真茵活性,检测菌株分别为白念珠菌Candida albicans,新生隐球菌Cryptococcus neoformans,红色毛癣茵Trichophyton rubrum,须癣毛癣菌Trichophyton mentagrophytes,犬小孢子菌Microsporum canis,絮状表皮癣菌Epidermophyton floccosum和黑曲霉Aspergillus niger。乌蕨素1、乌蕨素2、香草酸、丁香酸和龙胆酸对所检测真菌的最小抑制浓度(MIC)为25-100u g·ml-1,乌蕨素1和乌蕨素2的抗真菌活性分别低于它们糖苷化前的简单酚性酸即香草酸和龙胆酸。
b.黄酮类化合物对人肿瘤细胞的体外生长抑制作用
乌蕨苷A是从乌蕨中分离得到的新化合物,具有高异黄酮的化学结构,荭草苷和牡荆素是乌蕨中得量较多的两种黄酮苷,用MTT法测定了乌蕨苷A、荭草苷和牡荆素在四种浓度(10-4mol/L,10-5mol/L,10-6mol/L和10-7mol/L)下对5种肿瘤细胞体外生长的抑制作用。结果表明,在高浓度(10-4mol/L)时,3种黄酮类化合物对5种肿瘤细胞的抑制作用均较小或无效,抑制百分率最大值在20%左右。对于人白血病细胞HL-60,乌蕨苷A的抑制百分率为18.22%,荭草苷的抑制百分率为13.29%,牡荆素则无效。对于人白血病细胞K562,荭草苷和牡荆素的抑制百分率分别为19.29%和21.2%,乌蕨苷A的抑制百分率为9.21%。牡荆素对人肺癌细胞A549的抑制百分率为18.99%,其余样品均无效。3种黄酮类化合物对人肝癌细胞HepG2和人肝癌细胞SMMC-7721的生长基本无抑制作用。
5.乌蕨孢子萌发、配子体世代形态变化及孢子繁殖技术的研究
研究了外界条件对乌蕨孢子萌发的影响。用随机区组试验和方差分析探讨培养温度、贮藏温度、GA处理、光照强度对乌蕨孢子萌发的影响。与室温(20～25℃)相比,28℃培养的孢子最大萌发率相近而萌发速率显著较高；贮藏温度(A)极显著(P<0.01)影响孢子萌发率,-20℃贮藏降低萌发率；GA(B)对孢子萌发率无显著影响；光照强度(C)极显著(P<0.01)影响孢子萌发率,充足光照和弱光照无显著差异,黑暗处理降低萌发率；A×B,A×C,BxC及A×BxC交互效应不显著。
采用电镜、光学显微镜及解剖镜观察乌蕨从孢子到孢子叶形成阶段的形态变化,观察温度和光强对配子体生长发育的影响,确定乌蕨孢子繁殖的适宜条件。乌蕨孢子呈肾形,极轴长(48～54)51μm,赤道轴长(31～37)34μm,外壁光滑或具微小疣状纹饰。播孢后6d孢子开始萌发,细胞分裂成为丝状体,随后又分裂成心形的原叶体,播孢后45d原叶体腹面凹口下方产生精子器,播孢后55d又在腹面凹口生长点下方产生颈卵器。颈卵器形成后继续培养12d可形成幼小的孢子体。乌蕨孢子在温度23℃左右,光照强度10000Lx,培养基质为泥炭土：田园土：河沙=2：1：1,含水量处于饱和状态时能正常生长发育,形成孢子体。
This paper described the morphologic character and resource distribution of Stenoloma chusanum (Linn.)Ching, summarized the advances in pharmacological usage and chemical constituents about this plant, and discussed the biological activity of the major substance contained in this plant. The author suggested that the material basis of the biological activity of S. chusanum should be further studied and the techniques of introduction, propagation and cultivation should be investigated in order to establish the base for utilizing this plant. The main results of this research are as follows:
1. Chemical constituents of Stenoloma chusanum Ching
The ethanol extracts from whole plant of S. chusanum were separated by column chromatography with silica gel, polyamide, Lichroprep RP-18and Sephadex LH20. Nineteen compounds were obtained and their structures were elucidated by means of chemical evidence, spectral analysis (ESI-MS,1H-NMR,13C-NMR, DEPT, HSQC, HMBC), and comparing with the data of literature and standard compounds. The compounds were stenolomaside A i.e.[3-(9,3',5'-trimethox-4'-hydroxy-benzylidene) chroman-4-one]-5-O-β-D-glucoside, which exists E-(la) and Z-(lb) structures simultaneously; stenolin1i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid (2), stenolin2i.e.2-O-β-D-(6-O-gentisoylglucopyranosyl) gentisic acid (3), orientin (4), orientin-2"-O-β-D-glucopyranoside (5), vitexin (6), apigenin (7), vitexin rhamnoside (8), vitexin glucopyranoside (9), vanillic acid (10), syringic acid (11), vanillic acid (12), protocatechualdehyde(13),6,7-dihydroxycoumarin (14), pentadecanol (15), hexadecanoic acid (16),β-sitosterol (17), daucosterol (18). Among those compounds, Compounds(la),(1b),(2) and (3) were new compounds, compounds (4),(5),(8),(9),(10),(14),(15) and (16) were obtained from this plant for the first time.
2. Analysis of main components in Stenoloma chusanum Ching and evaluation of the plant resources
A method for determining orientin and vitexin from S. chusanum was established, content of the two flavonoids in different plants were measured and the plant resource were evaluated. Flavonoids were obtained by ultrasonic extraction, separation was carried out on Gemini C18column with a mixture of acetonitrile-1%phosphoric acid (V:V=15:85) as mobile phase and the flow rate was1.0mL/min, detective wavelength was340nm. Orientin showed a good linear relationship within0.025-0.148μg (R2=0.9995) and the average recovery was100.61%, RSD is2.18%. Vitexin showed a good linear relationship within0.035-0.207μg(R2=0.9999) and the average recovery was99.77%, RSD is2.37%. Stability experiment showed sample were stable within6h. Orientin and vitexin were the two main flavonoids and their ratio was variable in different plants according to the HPLC. The highest sample was from Qiubai Yunnan which contained orientin1.166mg/g and vitexin1.457mg/g, orientin decreased but vitexin increased when the plant was transplanted from Qiubai to Nanjing. Results showed that plant from Qiubai was an excellent resource based on the content of orientin and vitexin.
Total flavonoids(TF) from the aerial part of Stenoloma chusanum in different province were extracted by ultrasonic extraction, with the standard curve established with rutin using NaNO2-Al(NO)3-NaOH as chromogenic agent, the TF were determined by spectrophotometer. Results showed that the highest plant sample was from Qiubai Yunnan, which contained TF as high as127.461mg·g-1and could keep the higher level though they were transplanted to Nanjing Jiangsu; TF in leaf blade was notably higher than in leaf stalk; sample cultivated with spores had high TF. It could be concluded that the plants from Qiubai was an excellent resource and the leaf blade should be collected mainly based on the TF, and it was practicable to utilize this plant resource by cultivating.
3. Method of extraction and purification of orientin and vitexin from Stenoloma chusanum
In order to acquire pure orientin and vitexin from the aerial part of Stenoloma chusanum conveniently, safely and effectively, a method for extracting and separating the two flavonoids simultaneously was found. The essentials of this method were that:to get crude flavonoids precipitate at first, dissolve the precipitate with alkaline and dilute with water, neutralize the solution with acid, then separate the components by column chromatography with Lichroprep RP-18. The two flavonoids was obtained and their structures were elucidated by means of chemical evidence, spectral analysis (MS,1H-NMR,13C-NMR) and the data of literature. The purity of orientin and vitexin were detected with HPLC. Using the method described above; the target compounds of orientin and vitexin can be obtained with purity more than98%.
4. Bioactivities of new compounds from Stenoloma chusanum
a. Tests of antifungal activity about phenolic compounds
Two new phenolic compounds, stenolin1, i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid, stenolin2, i.e.2-O-D-D-(6-O-gentisoylglucopyranosyl) gentisic acid, together with three known compounds, vanillic acid, syringic acid and gentisic acid were isolated from Stenoloma chusanum, the antifungal activities of these compounds were evaluated using test tube dilution method against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccosum and Aspergillus niger. Results showed that the five compounds had the antifungal activity with MIC of25-100μg;·ml-1. Stenolin1and stenolin2, glucoside esters of phenolic compounds, had less antifungal activity than their simple phenolic compounds of vanillic acid and gentisic acid respectively.
b. Inhibitory effects of flavonoids on the growth of human tumour cells
Inhibitory effects of stenolomaside A, a new homoisoflavonoid extracted from Stenoloma chusanum, together with orientin and vitexin, two numerous compounds from the plant, on the growth of five kind of human tumour cells were tested at4levels of concentration of10-4ol/L,10-5mol/L,10-6mol/L and10-7mol/L with the method of MTT. Results showed that the three flavonoids all had weak or no effects on the tumour cells at high concentration. To human leukemic cell HL-60, phenolic acid had the inhibitory percentage of18.22%, orientin had less effect(13.29%) and vitexin had no effect. About human leukemic cell K562, orientin and vitexin had some inhibitory effects of19.29%and21.2%respectively, but stenolomaside A had petty effect of9.21%. Vitexin inhibited human lung adenocarcinoma cells A549with the percentage of18.99%, but stenolomaside A and orientin had no effects. All the three samples were almost ineffective on human hepatoma cells HepG2or SMMC-7721.
5. Investigation of spore germination,gametophytes morphology and breeding technique with spore about Stenoloma chusanum Ching
Spore germination of Stenoloma chusanum Ching were investigated. Effects of culture temperature, storage temperature, GA treatment and illumination intensity on spore germinating were studied using randomized blocks experiment and analysis of variance. Compared with room temperature (20-25℃), the germination velocity was higher at the culture temperature of28℃, although their maximal germination percentage was the same. Conserved temperature(A) significantly(P<0.01) affected spore germinating,-20℃reduced germination percentage. GA(B) did not affect germinating. Illumination intensity(C) significantly(P<0.01) affected germinating, there were no significant difference between full and weak illumination, but the dark reduced germination percentage. AxB, AxC, BxC and AxBxC had no significant interaction effect.
The development of gametophytes of Stenoloma chusanum Ching was observed, and the method of breeding with spores were investigated. The morphology in the developmental stage from spore to sporophyllary leaves was observed with electron microscope, optical microscope and anatomical lens. Effects of temperature and illumination intensity on the development of gametophytes were surveyed in order to understand the proper breeding condition. Spores of S. chusanum are kidney-shaped with polar axis of (48-54)51μm and equatorial axis of (31-37)34μm, and their exine were smooth or had petty verrucous ornamentation.6days after planting, spores germinated and filament emerged then split to form the heart-shaped prothallium.45days after planting, antheridium developed at the ventral side and under the notch of prothallium.55days after planting, archegonium developed at the ventral side and under the notch of prothallium.12days after the formation of archegonium, a young sporophyllary appeared. With the culture substrate composed of peaty:garden soil:sand=2:1:1, under the temperature of about23℃, illumination intensity of10000Lx and saturated moisture, spores could reproduce sporophyllary.
1.乌蕨的化学成分研究
利用硅胶、聚酰胺、Lichroprep RP-18. Sephadex LH20等层析材料,对乌蕨的乙醇提取物进行分离,根据化合物的理化性质、波谱数据(ESI-MS、HR-MS、1H-NMR.13C-NMR、DEPT、HSQC、HMBC)、标准品对照并参考文献资料,对分离得到的化合物进行了结构鉴定,已分离并鉴定了19个化合物,它们分别为：乌蕨苷A(stenolomaside A)即[3-(9,3',5'-三甲氧基-4'-羟基-亚苄基)色满-4-酮]-5-O-β-D-葡萄糖苷[3-(9,3',5'-trimethoxy-4'-hydroxy-benzylidene) chroman-4-one]-5-O-β-D-glucoside,该化合物晶体同时存在反式(1a)和顺式(1b)两种化学结构,乌蕨素1(stenolin1)即香草酸-4-O-β-D-(6-O-龙胆酰基)-吡喃葡萄糖苷(4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid)(2),乌蕨素2(stenolin2)1即龙胆酸-2-O-β-D-(6-O-龙胆酰基)-吡喃葡萄糖苷(2-O-β-D-(6-O-gentisoyl glucopyranosyl)gentisic acid)(3),荭草苷(Orientin)(4),荭草苷-2"-O-β-D-吡喃葡萄糖苷(Orientin-2"-O-β-D-glucopyranoside)(5),牡荆素(Vitexin)(6),芹菜素(Apigenin)(7),牡荆素鼠李糖苷(Vitexin rhamnoside)(8),牡荆素吡喃葡萄糖苷(vitexin glucopyranoside)(9),香草酸(Vanillic acid)(10),丁香酸(Syringic acid)(11),龙胆酸(Vanillic acid)(12),原儿茶醛(protocatechualdehyde)(13),6,7-二羟基香豆素(6,7-dihydroxycoumarin)(14),十五烷醇(pentadecanol)(15),十六烷酸(hexadecanoic acid)(16), β-谷甾醇(β-sitosterol)(17),胡萝卜苷(daucosterol)(18)。其中化合物(1a)、(1b)、(2)和(3)为新化合物,化合物(4)、(5)、(8)、(9)、(10)、(14)、(15)和(16)为首次从该植物中分离得到的化合物。
2-乌蕨主要成分的定量分析及其资源评价
作者建立了乌蕨地上部分荭草苷和牡荆素含量的测定方法,检测不同产地乌蕨中两种黄酮苷的含量,据此对乌蕨资源进行了评价。用超声波提取法提取,RP-HPLC测定,Gemini C18色谱柱,乙腈-1%磷酸溶液(V：V=15：85)为流动相,流速1mL/min,检测波长340nm。荭草苷在0.025～0.148μg范围内有良好的线性关系,加样回收率为100.61%,相对标准偏差(RSD)2.18%；牡荆素在0.035～0.207μg范围内有良好的线性关系,加样回收率为99.77%,RSD2.37%。精密度试验测得荭草苷峰面积的相对标准偏差(RSD)为0.10%,牡荆素峰面积的RSD为0.17%。重复性试验测得荭草苷和牡荆素峰面积的RSD分别为1.10%和0.49%。稳定性试验表明样品在6h内稳定。从HPLC图谱可见,荭草苷和牡荆素为乌蕨中二种主要的黄酮类化合物。在所测定的乌蕨种源中,牡荆素含量均高于荭草苷含量,但两者的比例不是固定不变的,云南丘北所产乌蕨的荭草苷和牡荆素含量都是最高,分别达到1.166和1.457mg/g,移栽到南京地区1年后荭草苷含量下降,牡荆素含量增高。结果表明,以荭草苷和牡荆素含量为指标,云南丘北所产乌蕨是较好的种源。
用超声提取法提取不同产地乌蕨地上部分的总黄酮,以芦丁为对照品,建立标准曲线,采用NaNO2-Al(NO)3-NaOH比色法测定总黄酮含量。结果表明,云南丘北所产乌蕨总酮含量最高,达127.461mg·g-1,移栽到南京地区2年后仍保持较高水平；用孢子培育的植株总黄酮含量高达187.038mg·g-1；同一产地的乌蕨,叶片总黄酮含量显著高于叶柄。分析结果表明,以总黄酮含量为指标,云南丘北所产乌蕨是较好的种源,采集乌蕨药材应以叶片为主,通过人工栽培开发利用乌蕨资源是可行的。
3.乌蕨中荭草苷和牡荆素提取纯化方法
为了简便、安全、经济、有效地从乌蕨中得到单体化合物荭草苷和牡荆素,建立了一种从乌蕨的地上部分提取纯化荭草苷和牡荆素的方法,其特征是先从乌蕨地上部分获得粗黄酮析出物,再将粗黄酮用碱水助溶,稀释后用酸中和,经过反相硅胶柱层析,得到目标产物荭草苷和牡荆素,纯度达98%以上。
4.乌蕨中新化合物的生物活性研究
a.酚性化合物的抗真菌试验
用琼脂稀释法(试管法)测定了新化合物乌蕨素1、乌蕨素2及已知化合物香草酸、丁香酸和龙胆酸的抗真茵活性,检测菌株分别为白念珠菌Candida albicans,新生隐球菌Cryptococcus neoformans,红色毛癣茵Trichophyton rubrum,须癣毛癣菌Trichophyton mentagrophytes,犬小孢子菌Microsporum canis,絮状表皮癣菌Epidermophyton floccosum和黑曲霉Aspergillus niger。乌蕨素1、乌蕨素2、香草酸、丁香酸和龙胆酸对所检测真菌的最小抑制浓度(MIC)为25-100u g·ml-1,乌蕨素1和乌蕨素2的抗真菌活性分别低于它们糖苷化前的简单酚性酸即香草酸和龙胆酸。
b.黄酮类化合物对人肿瘤细胞的体外生长抑制作用
乌蕨苷A是从乌蕨中分离得到的新化合物,具有高异黄酮的化学结构,荭草苷和牡荆素是乌蕨中得量较多的两种黄酮苷,用MTT法测定了乌蕨苷A、荭草苷和牡荆素在四种浓度(10-4mol/L,10-5mol/L,10-6mol/L和10-7mol/L)下对5种肿瘤细胞体外生长的抑制作用。结果表明,在高浓度(10-4mol/L)时,3种黄酮类化合物对5种肿瘤细胞的抑制作用均较小或无效,抑制百分率最大值在20%左右。对于人白血病细胞HL-60,乌蕨苷A的抑制百分率为18.22%,荭草苷的抑制百分率为13.29%,牡荆素则无效。对于人白血病细胞K562,荭草苷和牡荆素的抑制百分率分别为19.29%和21.2%,乌蕨苷A的抑制百分率为9.21%。牡荆素对人肺癌细胞A549的抑制百分率为18.99%,其余样品均无效。3种黄酮类化合物对人肝癌细胞HepG2和人肝癌细胞SMMC-7721的生长基本无抑制作用。
5.乌蕨孢子萌发、配子体世代形态变化及孢子繁殖技术的研究
研究了外界条件对乌蕨孢子萌发的影响。用随机区组试验和方差分析探讨培养温度、贮藏温度、GA处理、光照强度对乌蕨孢子萌发的影响。与室温(20～25℃)相比,28℃培养的孢子最大萌发率相近而萌发速率显著较高；贮藏温度(A)极显著(P<0.01)影响孢子萌发率,-20℃贮藏降低萌发率；GA(B)对孢子萌发率无显著影响；光照强度(C)极显著(P<0.01)影响孢子萌发率,充足光照和弱光照无显著差异,黑暗处理降低萌发率；A×B,A×C,BxC及A×BxC交互效应不显著。
采用电镜、光学显微镜及解剖镜观察乌蕨从孢子到孢子叶形成阶段的形态变化,观察温度和光强对配子体生长发育的影响,确定乌蕨孢子繁殖的适宜条件。乌蕨孢子呈肾形,极轴长(48～54)51μm,赤道轴长(31～37)34μm,外壁光滑或具微小疣状纹饰。播孢后6d孢子开始萌发,细胞分裂成为丝状体,随后又分裂成心形的原叶体,播孢后45d原叶体腹面凹口下方产生精子器,播孢后55d又在腹面凹口生长点下方产生颈卵器。颈卵器形成后继续培养12d可形成幼小的孢子体。乌蕨孢子在温度23℃左右,光照强度10000Lx,培养基质为泥炭土：田园土：河沙=2：1：1,含水量处于饱和状态时能正常生长发育,形成孢子体。
This paper described the morphologic character and resource distribution of Stenoloma chusanum (Linn.)Ching, summarized the advances in pharmacological usage and chemical constituents about this plant, and discussed the biological activity of the major substance contained in this plant. The author suggested that the material basis of the biological activity of S. chusanum should be further studied and the techniques of introduction, propagation and cultivation should be investigated in order to establish the base for utilizing this plant. The main results of this research are as follows:
1. Chemical constituents of Stenoloma chusanum Ching
The ethanol extracts from whole plant of S. chusanum were separated by column chromatography with silica gel, polyamide, Lichroprep RP-18and Sephadex LH20. Nineteen compounds were obtained and their structures were elucidated by means of chemical evidence, spectral analysis (ESI-MS,1H-NMR,13C-NMR, DEPT, HSQC, HMBC), and comparing with the data of literature and standard compounds. The compounds were stenolomaside A i.e.[3-(9,3',5'-trimethox-4'-hydroxy-benzylidene) chroman-4-one]-5-O-β-D-glucoside, which exists E-(la) and Z-(lb) structures simultaneously; stenolin1i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid (2), stenolin2i.e.2-O-β-D-(6-O-gentisoylglucopyranosyl) gentisic acid (3), orientin (4), orientin-2"-O-β-D-glucopyranoside (5), vitexin (6), apigenin (7), vitexin rhamnoside (8), vitexin glucopyranoside (9), vanillic acid (10), syringic acid (11), vanillic acid (12), protocatechualdehyde(13),6,7-dihydroxycoumarin (14), pentadecanol (15), hexadecanoic acid (16),β-sitosterol (17), daucosterol (18). Among those compounds, Compounds(la),(1b),(2) and (3) were new compounds, compounds (4),(5),(8),(9),(10),(14),(15) and (16) were obtained from this plant for the first time.
2. Analysis of main components in Stenoloma chusanum Ching and evaluation of the plant resources
A method for determining orientin and vitexin from S. chusanum was established, content of the two flavonoids in different plants were measured and the plant resource were evaluated. Flavonoids were obtained by ultrasonic extraction, separation was carried out on Gemini C18column with a mixture of acetonitrile-1%phosphoric acid (V:V=15:85) as mobile phase and the flow rate was1.0mL/min, detective wavelength was340nm. Orientin showed a good linear relationship within0.025-0.148μg (R2=0.9995) and the average recovery was100.61%, RSD is2.18%. Vitexin showed a good linear relationship within0.035-0.207μg(R2=0.9999) and the average recovery was99.77%, RSD is2.37%. Stability experiment showed sample were stable within6h. Orientin and vitexin were the two main flavonoids and their ratio was variable in different plants according to the HPLC. The highest sample was from Qiubai Yunnan which contained orientin1.166mg/g and vitexin1.457mg/g, orientin decreased but vitexin increased when the plant was transplanted from Qiubai to Nanjing. Results showed that plant from Qiubai was an excellent resource based on the content of orientin and vitexin.
Total flavonoids(TF) from the aerial part of Stenoloma chusanum in different province were extracted by ultrasonic extraction, with the standard curve established with rutin using NaNO2-Al(NO)3-NaOH as chromogenic agent, the TF were determined by spectrophotometer. Results showed that the highest plant sample was from Qiubai Yunnan, which contained TF as high as127.461mg·g-1and could keep the higher level though they were transplanted to Nanjing Jiangsu; TF in leaf blade was notably higher than in leaf stalk; sample cultivated with spores had high TF. It could be concluded that the plants from Qiubai was an excellent resource and the leaf blade should be collected mainly based on the TF, and it was practicable to utilize this plant resource by cultivating.
3. Method of extraction and purification of orientin and vitexin from Stenoloma chusanum
In order to acquire pure orientin and vitexin from the aerial part of Stenoloma chusanum conveniently, safely and effectively, a method for extracting and separating the two flavonoids simultaneously was found. The essentials of this method were that:to get crude flavonoids precipitate at first, dissolve the precipitate with alkaline and dilute with water, neutralize the solution with acid, then separate the components by column chromatography with Lichroprep RP-18. The two flavonoids was obtained and their structures were elucidated by means of chemical evidence, spectral analysis (MS,1H-NMR,13C-NMR) and the data of literature. The purity of orientin and vitexin were detected with HPLC. Using the method described above; the target compounds of orientin and vitexin can be obtained with purity more than98%.
4. Bioactivities of new compounds from Stenoloma chusanum
a. Tests of antifungal activity about phenolic compounds
Two new phenolic compounds, stenolin1, i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid, stenolin2, i.e.2-O-D-D-(6-O-gentisoylglucopyranosyl) gentisic acid, together with three known compounds, vanillic acid, syringic acid and gentisic acid were isolated from Stenoloma chusanum, the antifungal activities of these compounds were evaluated using test tube dilution method against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccosum and Aspergillus niger. Results showed that the five compounds had the antifungal activity with MIC of25-100μg;·ml-1. Stenolin1and stenolin2, glucoside esters of phenolic compounds, had less antifungal activity than their simple phenolic compounds of vanillic acid and gentisic acid respectively.
b. Inhibitory effects of flavonoids on the growth of human tumour cells
Inhibitory effects of stenolomaside A, a new homoisoflavonoid extracted from Stenoloma chusanum, together with orientin and vitexin, two numerous compounds from the plant, on the growth of five kind of human tumour cells were tested at4levels of concentration of10-4ol/L,10-5mol/L,10-6mol/L and10-7mol/L with the method of MTT. Results showed that the three flavonoids all had weak or no effects on the tumour cells at high concentration. To human leukemic cell HL-60, phenolic acid had the inhibitory percentage of18.22%, orientin had less effect(13.29%) and vitexin had no effect. About human leukemic cell K562, orientin and vitexin had some inhibitory effects of19.29%and21.2%respectively, but stenolomaside A had petty effect of9.21%. Vitexin inhibited human lung adenocarcinoma cells A549with the percentage of18.99%, but stenolomaside A and orientin had no effects. All the three samples were almost ineffective on human hepatoma cells HepG2or SMMC-7721.
5. Investigation of spore germination,gametophytes morphology and breeding technique with spore about Stenoloma chusanum Ching
Spore germination of Stenoloma chusanum Ching were investigated. Effects of culture temperature, storage temperature, GA treatment and illumination intensity on spore germinating were studied using randomized blocks experiment and analysis of variance. Compared with room temperature (20-25℃), the germination velocity was higher at the culture temperature of28℃, although their maximal germination percentage was the same. Conserved temperature(A) significantly(P<0.01) affected spore germinating,-20℃reduced germination percentage. GA(B) did not affect germinating. Illumination intensity(C) significantly(P<0.01) affected germinating, there were no significant difference between full and weak illumination, but the dark reduced germination percentage. AxB, AxC, BxC and AxBxC had no significant interaction effect.
The development of gametophytes of Stenoloma chusanum Ching was observed, and the method of breeding with spores were investigated. The morphology in the developmental stage from spore to sporophyllary leaves was observed with electron microscope, optical microscope and anatomical lens. Effects of temperature and illumination intensity on the development of gametophytes were surveyed in order to understand the proper breeding condition. Spores of S. chusanum are kidney-shaped with polar axis of (48-54)51μm and equatorial axis of (31-37)34μm, and their exine were smooth or had petty verrucous ornamentation.6days after planting, spores germinated and filament emerged then split to form the heart-shaped prothallium.45days after planting, antheridium developed at the ventral side and under the notch of prothallium.55days after planting, archegonium developed at the ventral side and under the notch of prothallium.12days after the formation of archegonium, a young sporophyllary appeared. With the culture substrate composed of peaty:garden soil:sand=2:1:1, under the temperature of about23℃, illumination intensity of10000Lx and saturated moisture, spores could reproduce sporophyllary.
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