| 摘要: | 槲蕨 (Drynaria fortunei (KUNZE) J. SMITH) 為傳統中藥『骨碎補』的基原植物。近來由於森林開發及過度採摘造成槲蕨自然棲地的破壞，此物種已面臨消失的危機，然而，據文獻所知目前並沒有人工栽種槲蕨的記錄，更沒有相關的研究報告，故建立槲蕨組織培養之大量繁殖系統可提供大量種源及物種開發。本研究先期探討不同因子(基礎鹽類、蔗糖濃度、酸鹼值、醣類、光譜性質)對無菌播種下孢子發芽、原葉體生長及生殖器官形成的影響，並研究在溫室栽植下原葉體形成孢子體的最佳條件，此系統將有助於進行此物種GAP(Good agriculture practice)栽培及避免野外族群的消失，所得結果列述如下：
1. 槲蕨孢子可在7天內發芽，以不對稱的方式進行細胞分裂，形成假根及原絲體細胞，約2個月後原葉體成熟。
2. 以半量之MS鹽類為基礎，添加2 % sucrose、酸鹼值7.7之培養基，於白光照射培養下可獲得最佳的發芽率(74.7 %)。
3. 在不同酸鹼值下藏卵器的形成數目與孢子發芽率趨勢相同，平均每一原葉體在酸鹼值為7.7時形成最多藏卵器(27.6個)。
4. 光譜會影響原葉體形成藏卵器與藏精器，以白光下可獲得較高比率(23.3 %)的雙性原葉體及50.0 %的雌性原葉體。
5. 瓶內原葉體可經由體細胞分化出二次原葉體而增殖。
6. 移植到溫室生長的原葉體，以培養在Peat moss介質的穴洞(直徑6 cm)可獲得最多孢子體幼苗(167株)。孢子體在溫室生長五個月後根莖明顯，10個月後長出收集葉。整個幼苗量化可在一年完成。
六種台灣產藥用蕨類植物包括：水龍骨科(Polypodiaceae)的槲蕨(Drynaria fortunei)和崖薑蕨(Pseudodrynaria coronans)及骨碎補科(Davalliaceae)的大葉骨碎補(Davallia divaricata)、海州骨碎補(Davallia mariesii)、闊葉骨碎補(Davallia solida)和杯蓋陰石蕨(Humata griffithiana) ，這些物種粗大根莖被稱為“骨碎補” 或 “碎補” 藥材並使用，宣稱可以治療酸痛、抗發炎、抗癌、抗老化、活血化瘀及骨損傷。本研究乃進行綜合評估及比較這六種台灣產骨碎補類藥材之水粗萃物與乙醇粗萃物的特性，希望能提供開發台灣產藥用資源及提供進一步研究之科學依據。茲將所得結果列述如下：
1. 海州骨碎補的粗萃率最高(29 %)。
2. 總抗氧活性以海州骨碎補(Davallia mariesii)的水粗萃物最大(TEAC =1.27 mM)；闊葉骨碎補(Davallia solida)的乙醇粗萃物有極高的自由基清除能力 (EC50=26.89 µg extract ml-1)；闊葉骨碎補(Davallia solida)的水粗萃物有較高的還原能力 (EC50=26.58 µg extract ml-1)。
3. 海州骨碎補(Davallia mariesii) 的水粗萃物有最多的總多酚類含量(1635.16 μg catechin equivalent/mg dry weight)、總類黃酮醇化合物(295.70 μg)、縮合單寧類化合物(1422.35 μg)及原花青素類化合物(467.85 μg)；總類黃酮化合物，則以闊葉骨碎補(Davallia solida)的乙醇粗萃物(122.44 μg)與水粗萃物(123.98 μg)含量最多，而乙醇粗萃物的崖薑蕨(Pseudodrynaria coronans)、海州骨碎補(Davallia mariesii) 及闊葉骨碎補(Davallia solida)不含類黃酮化合物。
4. 台灣產骨碎補類藥材之抗氧化能力與總多酚類化合物含量具有高度正相關。
骨碎補類藥材的基原物種間差異達「科」之層級，因此，骨碎補藥材基原鑑定尤其重要。透過外部形態鑑定，內部顯微組織的鑑定及HPLC指紋圖譜的鑑定綜合性的比較分析，建立各項彩色圖譜，並提出一鑑定上的重點為杯狀蓋陰石蕨內皮層外側之薄壁組織緊鄰內皮層面，明顯增厚於其他五種約3-4倍。並對分析台灣市售骨碎補類藥材鮮品、乾品及濃縮科學中藥，進行綜合性的比較分析，所的結果如下：
1. 市售品之三種鮮品為大葉骨碎補、崖薑蕨及杯狀蓋陰石蕨。
2. 乾品生藥飲片可從外觀及組織判定為大葉骨碎補。
3. 濃縮科學中藥，均非水龍骨科槲蕨，分析出明顯標示不符的情形。
Drynaria fortunei (KUNZE) J. SMITH (Polypodiaceae), a large epiphytic or petrophilous fern is a source of traditional Chinese medicine known as Gusuibu. The medicine for long time has been used in treatment of bone injuries and has proved very effective in the treatment of inflammation, hyperlipemia and arteriosclerosis. Studies on this valuable medicinal plant have not been carried out.
Present report describes results on spore germination, early gametophyte development and change of reproductive phase influenced by pH and light spectra in D. fortunei, a medicinal fern. In contrast to the previous reports on other fern species, germination of D. fortunei spores occurred on a wide range of pH from 3.7 to 9.7 in varying percentages. The highest germination (74.7 %) occurred on 1/2 strength of Murashige and Skoog’s basal medium supplemented with 2 % sucrose, at pH 7.7 and incubation under white light. Different pH values had significant effects on germination of spores, development of gametophytes and reproductive organs in this fern species. Contrary to earlier reports on several other ferns that blue light inhibits spore germination, 52.7 % Drynaria spores showed germination. Among the different light spectra, red, far-red (FR), blue and white light resulted in the 71.3 %, 42.3 %, 52.7 % and 71.0 % spore germination, respectively. There were no morphological differences among gametophytes grown under white or blue light. Elongated or filamentous but multi-seriate gametophytes developed under red light, while under FR, gametophytes grew as uniseriate filaments consisting of mostly elongated cells. Different light spectra influenced development of antheridia and archegonia in the gametophytes. On transfer to culture flasks, gametophytes gave rise new gametophytes and developed antheridia and archegonia. Development of the maximum number of sporophytes occurred on transfer of these gametophytes to plastic tray cells with Peat moss used as potting mix. Sporophytes grown in pots developed rhizome.
Rhizomes of Drynaria fortunei (KUNZE) J. SMITH, Pseudodrynaria coronans (WALLICH) CHING (both from Polypodiaceae), Davallia divaricata BLUME, Davallia mariesii MOORE ex BAKER, Davallia solida (FORST) SWARTZ, and Humata griffithiana (HOOKER) C. CHRISTENSEN (from Davalliaceae) are used or called as “Gusuibu” or “Shibu” in Taiwan. These have been claimed to cure physique ache, inflammation, cancer, ageing, blood stasis and bone injuries. However, no systematic investigation has been carried out so far, to evaluate comparative values of these different sources.
In the present report, ethanol and aqueous extracts of these six sources were characterized for their antioxidant, scavenging activities, reducing power, total polyphenols, flavonols, flavonoids, condensed tannins and proanthocyanidin contents. Results showed wide variation among the six sources. Most samples in aqueous extracts had higher antioxidant potencies and polyphenol contents, than the ethanol extracts indicating that aqueous preparation of “Gusuibu” was more potent than the ethanol. EC50 values of reducing capacities, and scavenging activities against DPPH radicals showed significant variation among the six sources within ethanol or aqueous extracts and between the two solvents. The maximum (1.27mM) Trolox Equivalent Antioxidant Capacity (TEAC) was recorded in aqueous extract of fern Davallia mariesii. The correlation coefficients (R2) values of TEAC and total polyphenol contents showed higher correlation (aqueous extract, R2 = 0.971; ethanol extract, R2 = 0.981).
Based on these results, there are six kinds of rhizome used or called as Gusuibu, and differences were observed in the "family" level. It is particularly important in identification of the original botanicals of Gusuibu with other species. Hence, external morphological characterization, internal microstructure studies and HPLC fingerprinting and their comparative analysis of these six genotypes through various color maps and various fresh crude drugs, dry crude drugs and concentrated herbal extracts were also analyzed. The results are as follows :
1. Three kinds of fresh Gusuibu rhizome viz., Davallia divaricata, Pseudodrynaria coronans and Humata griffithiana are available in the market.
2. The origin of dry crude drug of Gusuibu in Taiwan is from Davallia divaricata.
3. HPLC fingerprint clearly established, the concentrated herbal extracts which are available in the market, are not Drynaria fortunei. |
