Discrimination of Dendrobium officinale and Its Common Adulterants by Combination of Normal Light and Fluorescence Microscopy

The stems of Dendrobium officinale Kimura et Migo, named Tie-pi-shi-hu, is one of the most endangered and precious species in China. Because of its various pharmacodynamic effects, D. officinale is widely recognized as a high-quality health food in China and other countries in south and south-east Asia. With the rising interest of D. officinale, its products have a high price due to a limited supply. This high price has led to the proliferation of adulterants in the market. To ensure the safe use of D. officinale, a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. officinale from three commonly used adulterants including Zi-pi-shi-hu (D. devonianum), Shui-cao-shi-hu (D. aphyllum), Guang-jie-shi-hu (D. gratiosissimum). The result demonstrated that D. officinale could be identified by the characteristic “two hat-shaped” vascular bundle sheath observed under the fluorescence microscopy and the distribution of raphides under normal light microscopy. The other three adulterants could be discriminated by the vascular bundle differences and the distribution of raphides under normal light microscopy. This work indicated that combination of normal light and fluorescence microscopy is a fast and efficient technique to scientifically distinguish D. officinale from the commonly confused species.

of D. officinale and its commonly adulterants, including D. devonianum, D. aphyllum and D. gratiosissimum. The color figures of their microscopic characters and related descriptions are presented in detail and compared to distinguish them from each other.

Stems of D. officinale
Tie-pi-feng-dou: A commonly trade form of dried stem of D. officinale. Twist or spring-like, usually 2-6 spiral striation. When stretched, stem of 3.5-8 cm in length, 0.2-0.4 cm in diameter. Outer surface yellowish-green or golden-yellow, marked with fine longitudinal grooves. Node obvious, rudimental grayish-white leaf sheath sometimes can be found in node. Solid, breaks easily; broken surface is flat, grayish-white to grayish green, and slightly horny. Odour: faint; taste: mild; sticky when chewed ( Figure 1A1). Tie-pi-shi-hu: dried stem of D. officinale, slender and cylindrical, various in length ( Figure 1A2). Fresh Tie-pi-shi-hu: cylindrical or flat cylindrical, about 15 cm in length, 0.3-0.8 cm in diameter. Outer surface grayish-green, marked with longitudinal grooves. Texture soft and flexible, broken surface slightly fibrous. Odour: faint; taste: succulent; sticky when chewed ( Figure 1A3).

Stems of D. devonianum
Zi-pi-shi-hu (or Zi-pi-feng-dou): Trade form of dried stem of D. devonianum. Macroscopic characters are very similar to Tie-pi-feng-dou ( Figure 1B). Twist or spring-like, usually 2-7 spiral striation. Stem 0.2-0.7 cm in diameter. Outer surface dark greenish-yellow, sometimes with purple spots, marked with fine longitudinal grooves. Node obvious, rudimental grayish-white leaf sheath sometimes can be found in node. Fibrous, difficult to break, broken surface uneven. Odour: faint; taste: mild; sticky when chewed.

Stems of D. aphyllum
Shui-cao-shi-hu (or Shui-cao-feng-dou): Trade form of dried stem of D. aphyllum. Appearance is very similar to Tie-pi-feng-dou ( Figure 1C). Twist or spring-like, usually 2-5 spiral striations. Stems 0.2-1.1 cm in diameter. Outer surface dark yellowish-green, marked with fine longitudinal grooves. Node obvious, rudimental grayish-white leaf sheath sometimes can be found in node. Fibrous, difficult to break, broken surface uneven. Odour: faint; taste: slightly bitter, and not sticky when chewed.

Microscopic Characters
Characteristic microscopic differences of the four Dendrobium species in transverse section of stems are summarized in Tables 1-6 (Tables 1 and 2) (1). Epidermis: a row of cells, thin and flat, 17-45 μm in diameter, lateral walls were slightly lignified, covered with yellow to orange cuticles. A layer of pericladium consisting of parenchymatous cells and vascular bundles can be observed outside the epidermis sometimes. (2). Parenchyma: Parenchymatous cells similar in size, usually smaller near the vascular bundles.

Transverse Section of Stems (Observed Under Fluorescence Microscope)
Stems of D. officinale-Light intensity of dried samples is more strongly than fresh ones. Observed with excitation filter ex 450-500 nm and DM 505 nm emission filters, the cuticle, wall of epidermal cells and vascular bundles emitted green fluorescence. In vascular bundles, xylem vessels and fiber groups outside emitted green fluorescence. Especially, the "two hat-shaped" fiber groups can be easily observed. Some parenchymatous cells emitted green fluorescence ( Table 6). The light intensity is various in different samples. Observed with excitation filter ex 530-590 nm and DM 595 nm emission filters, the cuticle and wall of epidermal cells emitted red fluorescence. The fluorescence of vascular bundles is characteristic, sometimes xylem vessels and fiber groups outside emitted strong red fluorescence, sometimes the content of parenchymatous cell around vascular bundles emit red fluorescence, while xylem vessels and fiber groups outside emit no fluorescence. In addition, some parenchymatous cells emitted red fluorescence ( Table 6).

Stems of D. devonianum-Light intensity of dried samples is more strong than for fresh ones.
Observed with excitation filter ex 450-500 nm and DM 505 nm emission filters, only "hat-shaped" fiber groups can be easily observed. The other fluorescence characteristics of D. devonianum are similar to those of D. officinale (Table 6). Observed with excitation filter ex 530-590 nm and DM 595 nm emission filters, the fluorescence characteristics of D. devonianum are similar to those of D. officinale ( Table 6).
Stems of D. aphyllum-Light intensity of dried samples is more strongly than fresh ones. Observed with excitation filter ex 450-500 nm and DM 505 nm emission filters, the fluorescence characteristics of D. aphyllum are nearly the same as those of D. devonianum (Table 6). Observed with excitation filter ex 530-590 nm and DM 595 nm emission filters, the fluorescence characteristics of D. aphyllum are similar to those of D. officinale ( Table 6).

Stems of D. gratiosissimum-Light intensity of dried samples is more strongly than fresh ones.
Observed with excitation filter ex 450-500 nm and DM 505 nm emission filters, the fluorescence characteristics of D. gratiosissimum are nearly the same as those of D. devonianum (Table 6). Observed with excitation filter ex 530-590 nm and DM 595 nm emission filters, the fluorescence characteristics of D. gratiosissimum are similar to those of D. officinale (Table 6).

Discussion
By combining normal light and fluorescence microscopy, D. officinale and the common adulterants can be easily differentiated from each other based on the characteristics of their transverse sections. Both types of microscopy have their own merits in the authentication study. Normal light microscopy can provide the basic morphological characteristics. As a supplemental tool, fluorescence microscopy can exhibit specific auto-fluorescence from different plant tissues by virtue of their varied chemical constituents, and it excels in manifestation of the shape (as shown in Table 6), which makes the authentication work more easy and convenient. The comparison of D. officinale and its adulterants can be summarized as follows: (1) Cuticle: color and thickness of cuticle of four studied Dendrobium species are similar. We can therefore put forth the following key to identifying D. officinale and its common adulterants: (1). "Two hat-shaped" fiber groups emitted green fluorescence can be easily observed, Raphides distribute near the epidermis .. ................................................................................................ D. officinale (1). "One hat-shaped" fiber groups emitting green fluorescence can be easily observed (2). Vascular bundles similar in size, non-specific raphide distribution……………D. aphyllum (2). Vascular bundles various in size (3). Vascular bundles are much bigger in the centre than in the margin of the stem. Raphides distribute near the epidermis and vascular bundles……………………D. devonianum (3). Vascular bundles of different size distribute with no obvious rule. Raphides distribute near the vascular bundle……………………………………………D. gratiosissimum When fluorescence microscopy used, observed with excitation filter ex 450-500 nm and DM 505 nm emission filters, the cuticle, wall of epidermal cells and xylem vessels together with fiber groups outside vascular bundles emitted green fluorescence. Among them, the "two hat-shaped" fiber groups can be easily observed as the diagnostic feature of D. officinale. However, the other three Dendrobium species only possess "one hat-shaped" fiber groups. When observed with excitation filter ex 530-590 nm and DM 595 nm emission filters, though the red fluorescence can be observed in vascular bundles, it is not identical. Generally, xylem vessels in vascular bundles and fiber groups outside the vascular bundles emit red fluorescence, otherwise, the content of parenchymatous cells around the vascular bundles emit red fluorescence. Interestingly, the intensity and distribution of fluorescence are various in different samples, even of the same species, which may due to their different production areas, collection time and processing methods. Therefore, identification of the three adulterants only by fluorescence microscopy seems insufficient. The non-fluorescence microscopic features such as the distribution of rapheids, the size and amount of vascular bundles provided by the normal light microscopy were proposed to differentiate the three adulterants species.  Table 7. The voucher specimens were deposited in the Herbarium of Traditional Chinese Medicine, Zhejiang University of Technology.

Morphological Characteristics of four Dendrobium Stems
The gross exterior characters of each sample were examined by observing, measuring, touching, smelling, and tasting. The color digital photographs were taken by Canon digital camera 550D.

Transverse Section of Four Dendrobium Stems
The dried samples were moderately moistened firstly. Then, transverse section of samples (fresh sample or moistened dried sample) is made by cutting bare-handed. General procedures are as follows: three left fingers were used to fix the material at first. Then, put the blade which is held with the right hand against the material and slice smoothly from the left outward to the right inward. The tree-hand sections were investigated for possible auto fluorescence after sealing the mounted specimen along with purified water obtained from a Mili-Q water purification system (Millipore, Bedford, MA, USA).
The images observed with normal light and fluorescence microscope were recorded digitally. The transmittance spectra of two emission filters are shown in Figure 2. Excitation from HBO 100 light source through the excitation filter (EF) 450-500 nm and emission through dichroic mirror (DM) 505 nm (blue light), as well as excitation through filter BP 530-590 nm and emission through dichroic mirror DM 595 nm (green light) were used for observing fluorescence. The blue line is the excitation wavelength; the red line stands for the barrier filter wavelength; the green line stands for the dichroic mirror cut-off wavelength.

Conclusions
This is the first research combining normal light and fluorescence microscopy to thoroughly identify Dendrobium officinale and its counterfeits sold in the market through an investigation of the transverse sections of crude drug stems. Fluorescence microscopy, as a supplementary tool for routine microscopic identification, provides in vivo pictures and needs no professional researchers. According to the fluorescence characteristics, a further histochemical investigation on the chemical distribution in different tissues of D. officinale will be carried out. It is expected to be of great use in the identification and quality evaluation of Dendrobium species.