Pharmacognosy Identification of Xiaohuangcao (Dendrobium loddigesi Rolfe)

Miao ZHANG Li LI Jianbei TENG Hua ZHU Zhonghua DAI Long CHEN

Abstract [Objectives] Pharmacognosy identification was performed on Xiaohuangcao （Dendrobium loddigesi Rolfe）.

[Methods] The medicinal materials were identified through original plants， characters， microscopic structure and thin layer identification characteristics.

[Results] D. loddigesi has obvious plant morphology， characters， microscopic structure and thin layer identification characteristics. The stem of Xiaohuangcao is slender and cylindrical， and the surface is golden yellow; and the fiber bundles outside the vascular bundles are crescent shaped or semi circular in the stem transection. For the powder， crystal fiber can be observed; the vascular bundles are embedded with siliceous block cells; and there are more starch grains. In the thin layer chromatography， petroleum ether ethyl acetate butanone glacial acetic acid （8.5∶3.5∶1.5∶5 d） was used as a developing solvent， and 10% sulfuric acid ethanol solution was used as a color developing  agent.

[Conclusions] The research results provide reference for the application of the medicinal material and the formulation of its related quality standards.

Key words Dendrobium loddigesi Rolfe; Character identification; Microscopic identification; Thin layer chromatography

Xiaohuangcao （Dendrobium loddigesi Rolfe）， also known as Huancaoshihu， Fenghuashihu， and Xiaohuancao， belongs to Dendrobium in Orchidaceae. It is mainly produced in Guangdong， Guangxi， Guizhou and Yunnan. D. loddigesi grows on the trunk of trees in mountain forests at an altitude of 400-1 500 m or on rocks under the forests[2]. It has been recorded by the Pharmacopoeia as one of the commonly used traditional Chinese medicine Dendrobium plants. It has the effects of nourishing yin and clearing heat， promoting spleen and stomach， and moistening lung to arrest cough[3].

According to literatures[4]， the polysaccharides in Xiaohuangcao have anti tumor， immune regulating and blood sugar reducing effects， so most of the current research at home focuses on the  polysaccharides  of D. loddigesi. For example， Wu et al.[5] studied the effects of the habitat and harvesting time on polysaccharides in the medicinal material D. loddigesi. Qi et al.[6] studied the effects of different cultivation methods and growth time on polysaccharide content in D. loddigesi. Zhu et al.[7] studied the extraction process of polysaccharides from D. loddigesi. Some studies have focused on tissue culture[8]， endophytic bacteria[9]， etc. In general， there are few studies on the identification of  Xiaohuangcao.

In this study， through pharmacognosy identification， the specific characteristics of Xiaohuangcao were explored to distinguish it from other homologous varieties belonging to the same genus， so as to provide a scientific basis for future development and utilization.

Instruments， Reagents and Herbs

Optical biological microscope （DM2500， Germany Leica Microsystems）; semi automatic rotary slicer （RM2145， Germany Leica Microsystems）; slide drier （YG 280KX， Sunshine Medical Equipment Co.， Ltd.）; image analysis system （EX20， Sunny Optical Technology Group Co.， Ltd.）; ultrasonic cleaner （KQ5200B， Kunshan Ultrasonic Instruments Co.， Ltd.）; digital thermostatic water bath （HH S6， Jintan Medical Instrument Factory）; electronic balance （BP211D， Sartorius Scientific Instruments （Beijing） Co.， Ltd.）; electric blast drying oven （Shanghai YiHeng Scientific Instruments Co.，Ltd）; high speed universal pulverizer （Tianjin Taisite Instrument Co.， Ltd.）.

Anhydrous ethanol， methanol， glacial acetic acid， ethyl acetate and butanone （analytical grade， Sinopharm Chemical Reagent Co.， Ltd.）; petroleum ether （30-60℃） （analytically pure， Sinopharm Chemical Reagent Co.， Ltd.）; sliced paraffin （ Germany Leica Microsystems）; pure water; 5% phosphomolybdic acid test solution， FAA fixation solution， 0.5% saffron ethanol solution， aniline blue solution， chloral hydrate solution， dilute glycerin， etc.， provided by Guangxi Key Laboratory of Zhuang Yao Medicine.

The experimental Xiaohuangcao was collected from Guangxi， Yunnan and other places， and was identified as the whole grass of D. loddigesi Rolfe by Professor Wei from Guangxi University of Chinese Medicine. The samples were pulverized and passed through a 20 mesh sieve for use. The information of the collected samples is shown in Table 1.

Methods and Results

Experimental methods

The external characteristics of the samples were identified by traditional methods such as original plant identification and character identification. The stems of the medicinal materials were cross cut by paraffin sectioning to make permanent pieces. The characteristics of medicinal materials were observed by powder pellet method. The thin layer identification method was applied to observe the characteristic spots of the medicinal materials.

Experimental Results

Original plant identification

The plant is a perennial herb. The stem has a cylindrical shape with a height of 10-45 cm and a diameter of 2-7 mm. The base is slightly thin. The leaf is papery， long cylindrical lanceolate or tongue shaped， 4-6 cm long and  1.2-1.8  cm wide， apex acute and slightly hooked. The leaf is sessile， slightly loosely attached to the stem， with opening sheath. The flower is solitary， pale rose. The bract is small. The dorsal sepal is ovate oblong， 1.7 to 1.9 cm long， about 6 mm width，  apex  obtuse. The lateral sepal is longer than the dorsal sepal， but narrower， apex acute. The mentum is short， obtuse. The petal is oval， broad， as long as the dorsal sepal， apex obtuse， apically dentate; the lip is suborbicular， and the labial disc is depressed， shortly pubescent; and the petal has three lines thereon reaching the apex from base， and is fringed at its margin. The gynostemium is short. The flowering period is from April to May.

Character identification

The stem is slender and cylindrical， often curved. It is coiled into a mass or bundled. It is 11-40 cm long， 1-3 mm in diameter， and the internode is 0.4-2.3 cm in length. The surface is golden yellow， shiny， with fine vertical stripes. It has pliable and solid texture， and the section is flat. The stem is odorless， slightly sweet， sticky.

Microscopic identification

The characteristic of the stem transection

The stem transection is quasi circular. There is a column of epidermal cells， which are flat， and covered by brightly yellow cuticle. There is one or two columns of subcutaneous cells， which has thin wall， and the corner is slightly lignified. The basic tissue cells are similar or different in size， and there are many ectophloic type vascular bundles scattered thereamong， arranged to 4-6 circles. The fiber bundles outside the vascular bundles are crescent shaped or semi circular， and the outer parenchyma cells contain quasi circular siliceous blocks; and some of the parenchyma cells contain calcium oxalate crystals. The structure is shown in Fig. 1.

Powder characteristics

The powder is yellowish brown. The fibers are bundled or dispersive， slightly lignified， and the vascular bundles are embedded with siliceous cells， which are arranged in longitudinal rows. Calcium oxalate crystals are present in parenchyma cells， or scattered. The wood fibers are long fusiform， mostly bundled， with many pits. The vessels are spiral vessels. There are many starch granules scattered or clustered in parenchyma cells. The parenchyma cells are elliptical or quasi circular， with pits. The brown blocks are scattered. The structure is shown in Fig. 2.

Thin layer identification

A certain amount of the medicinal powders of 11 batches （1.0 g each） were added with 20 ml of methanol， and ultrasonically extracted for 30 min， respectively. Each of the extracted mixture was filtered， and the filtrate was concentrated to 1 ml. According to thin layer chromatography （appendix VI B of "Chinese Pharmacopoeia" 2015 edition）， 5 μl of each of the test solution was pipetted and spotted on the same silica gel G thin layer plate， and petroleum ether ethyl acetate butanone glacial acetic acid （8.5∶3.5∶1.5∶5 d） was used as a developing solvent to allow development to a development distance of about  8 cm.  The thin layer plate was taken out， air dried， and sprayed with 10% sulfuric acid ethanol solution， followed by heating at  105 ℃  for development until the spots were clear. The chromatographic separation of each test sample was good， and the spots were clear at the positions corresponding to the chromatogram of the reference drug. The results are shown in Fig. 3.

Results and Discussion

Xiaohuangcao is a perennial herb. The stem is cylindrical， yellow green， and the leaf is papery， sessile. The stem has quasi circular transection， and is covered by bright yellow cuticle. The fiber bundles outside the vascular bundles are crescent shaped or semi circular， and the outer parenchyma cells contain quasi circular siliceous blocks. The powder is yellowish brown; the fibers are bundled or scattered， and the vascular bundles are embedded with siliceous cells; and calcium oxalate crystals and starch granules are also observed.

In the thin layer identification， the extraction method， the extraction solvent， the spotting amount， the developing system and the color developing agent of the medicinal materials were investigated. The final development system was petroleum ether ethyl acetate butanone glacial acetic acid （8.5∶3.5∶1.5∶5 d） was used as a developing solvent to allow development to a development distance of about 8 cm. The thin layer plate was taken out， air dried， and sprayed with 10% sulfuric acid ethanol solution， followed by heating at 105 ℃ for color development. Three clear spots with good resolution were observed.

Miao ZHANG et al. Pharmacognosy Identification of Xiaohuangcao （Dendrobium loddigesi Rolfe）

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