Immunofluorescence and Immunoelectron Microscopic Localization of  Medicinal Substance, Rb1, in Several Plant Parts of Panax ginseng

Sadaki Yokota1,*, Yuko Onohara1 and Yukihiro Shoyama

人参，即人参根的原药，是最重要的草药之一。它被用于增强体力、提高对疲劳和身体压力的应对能力，以及作为一种对抗癌症、中枢神经系统紊乱、低体温、碳水化合物和脂质代谢、免疫功能、心血管系统和放射防护的滋补品[1]。其主要活性成分是人参皂苷，包括具有大马士革骨架的原人参三醇和/或原人参二醇。众所周知，人参根和根提取物中的人参皂苷浓度因提取方法、后续处理[2]甚至采集季节[3]而异。因此，需要对质量进行标准化。为了控制中医处方中规定的草药质量，我们之前报道了一种制备抗人参皂苷-Rb1、抗人参皂苷-Rg1和抗人参皂苷Re单克隆抗体（mAbs）的方法[4, 5]，并建立了人参皂苷-Rb1的酶联免疫吸附测定（ELISA）和免疫亲和浓缩方法，实现一步分离[6]。

关于中医处方中重要草药黄芩（Scutellaria baicalensis）根和茎片中的
-葡萄糖醛酸酶（GUS）[7]，该酶是氧化爆发机制的起始酶[8]，我们使用5-溴-4-氯-3-吲哚-
beta-葡萄糖醛酸酯（X-Gluc）作为底物，对其进行了GUS活性染色。清晰的差异表明
-葡萄糖醛酸酶的分布根据组织和栽培季节而变化。最近，我们指出与大麻化合物相关的关键酶——四氢大麻酸（THCA）合酶[9]在腺毛分泌细胞中得到独特表达。表达THCA合酶与绿色荧光蛋白（GFP）融合的转基因烟草在腺毛头部显示出与贮存腔相对应的荧光。这些结果表明，腺毛的分泌细胞不仅分泌大麻化合物，还是其生物合成位点[10]。在我们正在进行的基于组织化学的研究中，我们进行了免疫荧光（IF）和免疫电镜（IEM）研究，以确定人参皂苷Rb1在人参器官（如根、叶和茎）中的积累位置。

Fig. (6). IEM of ginseng leaf and corresponding IF images. A. Parenchymal cell. Heavy gold labeling for Rb1 is observed in chloroplasts (Ch) and peroxisomes (P) which contain dense core (arrows). The vacuole (V), mitochondrion (M) and cytoplasm are weakly labeled. B. IEM control section. No gold particles are noted in the chloroplasts (Ch), peroxisomes (P), mitochondrion (M), and vacuole (V). CW: cell wall. C. Stroma of intermediate cell. It is heavily stained for Rb1. D. The corresponding image under IF. The intermediate cell is stained (arrows). E. The sieve element of the phloem. Irregular masses forming the element are strongly stained for Rb1 but the cell wall (CW) is consistently negative. F. IF image (arrow) corresponding to E. Bar=1 μm for A, B and E, 0.5 μm for C, and 10 μm for D and F.

图 6. 人参叶的免疫电镜（IEM）及相应的免疫荧光（IF）图像。A. 间质细胞。在叶绿体（Ch）和含有致密核心的泡体（P）中观察到Rb1的重金标记（箭头）。液泡（V）、线粒体（M）和细胞质弱标记。B. IEM对照切片。在叶绿体（Ch）、泡体（P）、线粒体（M）和液泡（V）中未观察到金颗粒。CW：细胞壁。C. 间质细胞基质。Rb1染色强烈。D. 对应的IF图像。间质细胞染色（箭头）。E. 韧皮部筛管元素。形成该元素的不规则块状物强烈染色为Rb1，但细胞壁（CW）始终呈阴性。 对应于E的IF图像（箭头）。比例尺=1 μm（A、B和E），0.5 μm（C），10 μm（D和F）

Fig. (8). IEM localization of Rb1 in xylem of the leaf stem and corresponding IF images. A. Perforation of the xylem. Note the disintegration of the dense primary cell wall and the presence of fine fibrous remnant (arrows). Note also the presence of gold particles in the tubular element surrounded by the secondary cell wall. The latter is disintegrated and has become very thin in the lower right area (open arrow). Inset. Corresponding IF image. B. More developed perforation. The secondary cell wall (CW) of both sides of the element is largely lost. Heavy labeling for Rb1 is noted in the fibrous remnant. Inset. Corresponding IF image. The Y-remnant is stained for Rb1. C. Perforation between two xylem cells. The primary cell wall has completely disappeared and a fine fibrous mass forms the fibrous remnant, the center of which fibers are concentrated to form a thick mass (arrows). The remnant is strongly labeled for Rb1. Upper left inset. High-power view of the fibrous remnant. Fine fibers extend from the central remnant of the primary cell wall (arrowheads). Lower right inset. Corresponding IF image. The remnant is strongly stained. D. Fibrous remnant bordering three cells. The remnant is strongly labeled for Rb1. Three fibrous remnants of primary cell wall are connected with each other at the center of the remnant (arrows). Inset. Corresponding IF image, which is stained for Rb1. Bar=1 μm for A, B, B insert and D, 2 μm for B, 5 μm for all inserts.

图 8. 人参叶柄木质部中Rb1的免疫电镜（IEM）定位及相应的免疫荧光（IF）图像。A. 木质部的穿孔。注意密实原生细胞壁的解体和微纤维残留的存在（箭头）。同时注意金颗粒存在于被次生细胞壁包围的管状元素中。后者在右下区域解体并变得非常薄（开放箭头）。插图：对应的IF图像。B. 更为发达的穿孔。元素两侧的次生细胞壁（CW）大部分消失。微纤维残留中出现Rb1的浓烈标记。插图：对应的IF图像。Y形残留物染色为Rb1。C. 两个木质部细胞之间的穿孔。原生细胞壁完全消失，微纤维形成纤维残留，其中纤维的中心聚集形成厚块（箭头）。残留物强烈标记为Rb1。左上插图：纤维残留的高倍视图。细纤维从原生细胞壁的中央残留延伸（箭头头）。右下插图：对应的IF图像。D. 界定三个细胞的纤维残留。残留物强烈标记为Rb1。三个原生细胞壁的纤维残留在残留物中心彼此连接（箭头）。插图：对应的IF图像，染有Rb1。比例尺=1 μm（A、B、B插图和D），2 μm（B），5 μm（所有插图）。