2014

Hong Kong Baptist University Library
Award for Excellence in Undergraduate Research

Comparative Authentication of Three “Snow Lotus”
Herbs by Macroscopic and Microscopic Features
Qilei Chen
with

Tao Yi, Yina Tang, Lai Lai Wong, Xiaoxuan Huang, Zhongzhen Zhao, and Hubiao Chen

This is the pre-peer reviewed version of the published article: Chen, Q.,

Yi, T., Tang, Y., Wong, L. L., Huang, X., Zhao, Z. and Chen, H. (2014),

Comparative authentication of three “snow lotus” herbs by
macroscopic and microscopic features. Microsc. Res. Tech., 77: 631–

641. doi: 10.1002/jemt.22381, which has been published in final form
at http://onlinelibrary.wiley.com/doi/10.1002/jemt.22381/abstract.

Comparative Authentication of Three “Snow Lotus” Herbs
by Macroscopic and Microscopic Features
QILEI CHEN, TAO YI,* YINA TANG, LAI LAI WONG, XIAOXUAN HUANG,
ZHONGZHEN ZHAO, AND HUBIAO CHEN*

School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special
Administrative Region, People’s Republic of China

*Correspondence to: Dr. Tao Yi or Dr. Hubiao Chen, School of Chinese Medicine, Hong Kong
Baptist University, Kowloon Tong, Hong Kong Special Administrative Region, People’s
Republic of China. E-mail: yitao@hkbu.edu.hk; hbchen@hkbu.edu.hk

1

pollen grains.-Mazz. glandular hairs. and as for microscopical identification. light microscopy. KEY WORDS Snow lotus. namely. The results indicate that. medusa Maxim. S. involucrata (Kar.-Bip. pollen grain 2 .) Sch. evolution sequence of the three species was speculated. convenient. Based on the observation of pollen grains. To differentiate three representative easily-confused snow lotus herbs.ABSTRACT “Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). simple and reliable. Scanning electron microscopy has been found to provide a number of unique characteristics of pollen grains. scanning electron microscopy. et Kir. S. and S. non-glandular hairs. as for macroscopical identification. cells of inner surface of the microdiodange.. Comprehensive table comparing the characteristics were presented in this study. microscopic features. polarized light microscopy and scanning electron microscopy. macroscopic features of the three herbs were systemically observed. and microscopic features were compared under the ordinary light microscopy. It can be widely applied to other herbal materials. laniceps Hand. and epidermal cells of the corolla can be used to authenticate the three snow lotus herbs. The authentication method was proven to be efficient. capitula situation and arrangement.

1973. has been widely used in Tibetan. S. 2010). stomachache and dysmenorrhea in Uighur folk medicine (Liu and Shawuti. 2007). e. 2007. Meyer 1983. 1999.. Xie. Moreover. 2010). Saussurea plants. Chinese Pharmacopoeia Commitee. treating rheumatoid arthritis. observing microscopic features is an efficient. In comparison. the chemical and pharmacological approaches. and S. Xiao. These latter two species are prescribed for the treatment of pain and inflammatory conditions in Tibetan folk medicine (Commission of Chinese Ethnomedicine. while accurate... snow lotus herbs are powdered. as well as expensive experimental equipment and animals. 2007). the chemical composition and pharmacological activities of these three herbs have been investigated.300 m in the Tianshan and A’er Tai areas of China (Chen et al. However. 2010.) Sch.000–4.. Xiao. Two other species of the same genus.-Bip. 1). 2007). Jiang and Li. laniceps Hand.-Mazz. For these reasons. it is necessary to distinguish the three species clearly. Xiao. et Kir. The three species have been confused because of their shared folk names. as used in that study. medusa Maxim.g. have also been used under the name “Snow Lotus”. (Fig. Xiao. involucrata (Kar. Therefore.INTRODUCTION “Snow lotus”. Since the three herbs have similar clinical uses. their chemical profiles and pharmacological effects cannot be distinguished effectively.. a famous Chinese Materia Medica (CMM) derived from the dried aerial parts of species of the genus Saussurea (Compositae). involve much time and effort. Chen et al. Powdered herbs are not only very difficult to identify by sight but also easily replaced by other materials. simple. Yi et al. this species has long been used for diminishing inflammation and facilitating blood circulation. once dried or processed for herbal use. economical. 1999. For some patent formulations. reliable. a plant growing in the mountains at heights of 4. In our previous studies (Yi et al. b. Jiang and Li. 1991. 1999. mainly distributed in the Qinghai-Tibet plateau at heights of 3500–5300 m. 1994. Yi et al. can be easily damaged or broken apart during commercial distribution. 1999. a variety of identification methods and characters are needed to accurately authenticate the three species of snow lotus herbs.. 1984. Mongolian. Yi et al. (Fig. 2009 a. 1999. One of the botanical origins of “Snow lotus” is S. thus morphological characters are usually not sufficient for their identification. and effective means for identifying CMM plant material powders (Liang et 3 . morphological appearances and medicinal uses (Yang. 1). 2012). Uighur and Chinese folk medicines (Health Bureau of Autonomous Region of Xizang.

comparing pollen grain characters under scanning electron microscope is also an effective and reliable approach for herbal identification (Wodehouse. The size. An et al. This method can be widely applied to other Saussurea herbs and even to other confused CMM. With the use of ordinary light microscope and polarized light microscope.. shape and color of the grains.. The method used is convenient and reliable. 2013). The present study represents the first attempt to systemically observe and compare the macroscopical and microscopical features of powdered materials and pollen grains of the three medicinal species of Saussurea.. Since pollen grains are single cells and spores. 1943). It has been reported that scanning electron microscopy has been widely used to observe pollen grains of plants to distinguish different species (Ceter et al. 2013). the pollen grain characters are indicative of relationships among plants (Wodehouse. CMM can be successfully identified. the number and arrangement of the germinal apertures. 4 . are of high diagnostic value (Erdtman. 1928). 2006. 1935). and the sculpturing of the exines. 2009)..al. The application and advantages of polarized light microscopy in observing CMM plant tissues are described in our previous publication (Tang et al. and can be used to authenticate the three representative snow lotus herbs. Apart from using optical microscopes.

Fig 1. laniceps and S. (C). (B). were collected from different main production areas in China. medusa Maxim. S.-Mazz.) Sch. et Kir. S. namely 5 batches each of S. MATERIALS AND METHODS Materials Fifteen batches of samples. medusa.-Bip. School of Chinese Medicine. Medicinal materials of Saussurea involucrata (Kar. (A). Table 1 summarizes the information of all the samples. The voucher specimens of standard samples were authenticated by Dr. Hong Kong Baptist University (HKBU). laniceps Hand. S. Hubiao Chen. The above materials were deposited in the Bank of China (Hong Kong) Chinese Medicines Centre of HKBU. involucrata. 5 .

odor and taste. Purified water was provided by a Millipore water purification system (Millipore. Sichuan. surface texture. passed through a 250 μm sieve. involucrata SI03 (2007) Tianshan. China SM04 (2007) Lhasa. Tibet S. Bedford. 2010). Japan). Tibet SL05 (2007) Lhasa. Xinjiang. Xinjiang.TABLE 1. Microscopic Identification of Powdered Crude Drugs. Tibet SL04 (2007) Lhasa. China S. China SI02 (2007) Tianshan. China SI04 (2006) Xinyuan. China SM02 (2007) Qinghai. laniceps SL03 (2007) Lhasa. Collection data of three “snow lotus” herbs Latin name Batch No. Each sample was measured and examined with regard to appearance. England) were used for mounting microscope slides. All herbal samples were powdered. and treated with chloral hydrate solution for observation. Chloral hydrate solution and dilute glycerin were prepared according to procedures described in Appendix XV B of the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia Commitee. Tibet. LEO1530 Field Emission Scanning Electron Microscope (Oxford Instrument) was used for observing pollen grains. Sichuan. The color photographs were taken with a digital camera (Panasonic DMC-FZ7. Leica IM50 and SmartSEM® were used. Tibet SM05 (2007) Lhasa. China SL01 (2008) Lhasa. Methods Macroscopic Identification of Crude Drugs. China S. MA) Apparatus Axioplan 2 and Axiophot 2 universal microscopes with reflector Axiophot Photo Module (Zeiss Group) were used for observing powdered samples. China SI05 (2005) Lhasa. Tibet SM01 (2007) Aba. (Collection year) Collection area SI01 (2007) Tianshan. Software Matrox inspector (Matrox Electronic Systems). Tibet SL02 (2007) Lhasa. Panasonic. images were recorded with a Leica direct current (DC) camera. medusa SM03 (2007) Aba. Xinjiang. Xinjiang. At least 6 . Tibet Reagents Chloral hydrate and glycerin (Uni-chem.

margins sharply toothed. RESULTS S. 10 mm in diameter. Palynological characters examined included equatorial and polar diameters. stigma 2-lobed. Odor specially fragrant. 23-52 μm in diameter. brownish-yellow and pubescent throughout. Bracteal leaves ovate or oval. margins serrate and ciliate. 55-70 mm long and 20-70 mm wide. limb 9 mm long. 11 mm long and 5 mm wide. Pollen grains: Subrounded. yellowish-white. surrounding capitula. Pollen grains were removed from anthers of the herbarium materials. bracts of the middle and inner layers lanceolate.10 different slides of each powder sample were examined under ordinary light microscope and polarized light microscope. anther purple. Achenes ovate. All flowers tubular. inner layer feathery and 15 mm long. Microscopic Characteristics. suspended in distilled water on coverslips glued to 12 mm specimen holders. (2) Unicellular. (3) Multicellular. lanceolate. twisted and flat. in two layers. grayish-white. same as below). yellow. in three to four layers. and air-dried (Andersen and Bertelsen. comprised of 5-6 cells as a single stalk. Non-glandular hairs: Of three kinds (numbers in accordance with respective pictures in Table 3. texture membranous. yellowish-white and pubescent only on top. Pappi grayish-white. Capitula crowded at top of stem. 1). cell walls smooth. corolla purplish-red. linear and bulgy. Optical Microscopy (Table 3): Powder yellowish-green. Distinctive microscopic features observed were digitally recorded. tips blunt or slightly acuminate. 1972). bases decurrent. (1) Unicellular. The specimens were coated with gold and observed with a field emission scanning electron microscope (SEM) operated at 20 kV. longitudinally ribbed. Microscopic Identification of Pollen Grains. outer layer coarse and 3 mm long. Leaves oblong or wide-lanceolate. bracts of the outer layer oblong. cell walls smooth. 3 mm long. with light fragrance. 15-18 mm long and 2 mm wide. 2. cell walls uneven. tube 7 mm long. taste slightly bitter (Fig. yellowish-green. 7 . shapes and ornamentation. involucrata Macroscopic Characteristics. Involucral bracts hemispherical. 3 germination ditches. warts on the outer surface. 1.

9. pale brown. Glandular hairs: Rare. 2). stomata anomocytic.or double-celled flat layers. 10. sometimes pale yellow. other cells brown. non-glandular hairs present on both upper and lower epidermal cells.3. with interlocked cells in each layer. anticlinal walls slightly curved. pit canals dense. warts coarse and widely spaced. yellow. arranged orderly. Scanning Electron Microscopy: Pollen grains subrounded. but with deeply sinuous anticlinal walls. Tips blunt. (2b) cells below the tube epidermis containing crystals that appear bright blue under polarized light. Fiber width 9-18 μm. with papillary protrusions. 5. (2a) tube cells colorless. colorless. with reticular sculpturing on the surface (Fig. anticlinal walls sinuous. 8. Cells of inner surface of microdiodange: Rectangular. irregular in shape. anticlinal walls undulate. Epidermal cells of leaf: Polygonal. 23-52 μm in diameter. 4. Epidermal cells of bracteal leaf: (1) Upper epidermis colorless. (1b) other cells of limb tightly arranged in lines. colorless or pale yellow. Stigmas: With short scale-like protrusions. Head with 5-6 single. rectangular. 8 . (2) lower epidermis similar to upper epidermis. Epidermal cells of involucral bract: Elongate spindle-shaped. Mostly broken. cell walls appearing bright blue under polarizing light. Pappi: Filamentous structure with longitudinally aligned fiber cells. arranged tightly in lines. outer peripheral walls irregularly prominent in lateral view. 6. pale yellow. Epidermal cells of corolla: (1a) Tip of limb yellow. 3 germinating ditches. stalk colorless. cell walls thickened in longitudinal stripes and appearing bright blue under polarized light. 7.

2. 9 . Characteristics of the powder of the three snow lotus herbs under optical microscope (Scale: 100 μm).Fig.

Fig. laniceps Macroscopic Characteristics. Leaves crowded. Top of plant covered with white hairs. bases cuneate. Bracteal leaves linear 10 . upper surface with arachnoid hairs. tips acute or acuminate. (Continued). margin undulate. 2. oblanceolate or spatulate. S. lower surface with dense brown tomentum.

3 germinating ditches. stigma 2-lobed. with no specific view under polarized light. 2b) or straight (2c). arranged orderly. Pappi gray. Odor light. densely covered with perforate and verrucate warts. both cell lumina and wall appeared bright blue under polarized light. (1) Comprised of 4-7 short cells with a single slender cell on top. All flowers tubular. (2a) tube cells pale yellow or brownish-yellow. Microscopic Characteristics. inner layer feathery and long. both surfaces covered with white tomentum. 3. cell walls smooth. colorless or pale yellow. 24-49 μm in diameter. Epidermal cells of corolla: (1) Limb brownish-yellow. 5. anthers light purple. 8-12 mm long. surrounded by reticulate sculpturing on the surface. Achenes cylindrical. 6. taste slightly bitter (Fig. 2. with light fragrance. 2 layers. 6 mm long and 1 mm wide. Non-glandular hairs: Abundant. 8. Pollen grains: Subrounded. Of two kinds. Glandular hairs: Rare. limbs 3 times longer than tubes. Tips blunt. 11 . covered with white or brown hairs. 7. anticlinal walls thin and undulate. anticlinal walls slightly curved. outer layer coarse and short. arranged tightly in lines. Multicellular. which can be either curly (2a. Head yellow. cell connections smooth and colorless. rectangular. in 3-4 layers. colorless or pale brown. Pappi: Filamentous structure with longitudinally aligned fiber cells. other cells of limb thin rectangular. comprised of more than 3 long cells.lanceolate. covered densely with long. outer layer lanceolate or linear lanceolate. Cells of inner surface of microdiodange: Rectangular. colorless or pale yellow. with 5-6 single-celled flat layers. inner layer lanceolate. 4. white. Epidermal cells of bracteal leaf: Irregular in shape. 9. 9 mm long and 4 mm wide. other cells dark brown. Optical Microscopy (Table 3): Powder yellowish-green. tip acuminate. pale yellow. Involucral bracts broadly campanulate. Stigmas: With short scale-like protrusions. stalk usually unicellular and colorless. 1). (2b) cells below the tube epidermis containing dense prismatic crystals that appeared bright blue or yellow under polarized light. Capitula arranged in paniculate spikes along upper stem. 3 mm long. stomata anomocytic. 15 mm in diameter. tip of limb with papillary protrusions. (2) Mostly broken. cell connections shrunken and yellow. cell walls smooth. 1. cell walls smooth. Epidermal cells of involucral bract: Elongate spindle-shaped. Fiber width 9-17 μm. dark brown hairs.

both surfaces with long white tomentum. cell connections slightly shrunken. Pollen grains: Subrounded. 1. outer layer coarse. with reticular sculpturing on the surface smoother than that of S. inner layer feathery. 3 germinating ditches. 8-9 mm long and 4-8 mm in diameter. 24-50 μm in diameter. Epidermal cells of leaf: Rectangular. stalk unicellular. anticlinal walls straight. Bracteal leaves linear lanceolate. 12 . in 3 layers. bracts of inner layer oblanceolate. pale yellow or colorless. whole hair brown. cell walls smooth. Of two kinds. All flowers tubular. Pappi white or grayish-white. pale yellow. Capitula sometimes densely crowded at top of stem. Leaves ovate or linear speculate. each layer with 1-2 rounded cells. outer surface sculptured with dense spinules. pale brown. tube purple or purplish-blue. warts more crowded than those of S. 3 germination ditches. limb as long as tube. covered with white or brown tomentum. both surfaces covered with long white tomentum. involucrata. margins whole or with laciniate. Involucral bracts narrow cylindrical. acuminate. with a single slender cell on top. 10 mm long and 4 mm wide. medusa Macroscopic Characteristics. Achenes rare. in 2 layers. bracts of middle layer oblanceolate. 2. Whole plant cylindrical. Microscopic Characteristics. (2a) yellowish-brown and (2b) pale red. Scanning Electron Microscopy: Pollen grains subrounded. taste slightly bitter (Fig. Optical Microscopy (Table 3): Powder yellowish-green. arranged in racemes. tips blunt or rounded. involucrata (Fig. 1). 30-57 μm in diameter. cell walls with longitudinal and corrugated striations. 10 mm long. arranged tightly in lines. bracts of outer layer ovate.10. 3. 5-7 mm in diameter. with light fragrance. Non-glandular hairs: Abundant. purple. cell connections shrunken and yellow. 2). anther purple. stigma 2-lobed. 11 mm long and 2 mm wide. Odor light. 11 mm long and 2 mm wide. Glandular hairs: Rare. Head with 2-4 layers. (2) Of 3-5 cells with two subcategories based on color of cell contents. covered with thick white or grayish-white long tomentum. bases cuneate. S. (1) Comprised of 4-8 or more short cells in a single row. spindle-shaped.

cell walls thickened in longitudinal stripes and appearing bright blue under polarized light. 7. 13 . other cells brown. 9. Scanning Electron Microscopy: Pollen grains subrounded. Fiber width 10-19 μm. outer peripheral walls irregularly prominent in lateral view. cell walls and lumina brown. stomata anomocytic. 3 germinating ditches. outer surface with granular sculptures (Fig. Cells of inner surface of microdiodange: Rectangular.4. 6. brownish-yellow. arranged orderly. Epidermal cells of leaf: Polygonal to rectangular. Pappi: Filamentous structure with longitudinally aligned fiber cells. 5. colorless. yellowish-brown or pale purple. (2b) cells below the tube epidermis containing a few prismatic crystals that appear bright blue under polarized light. (1b) other cells of limb thin rectangular. colorless. spinules dense and with blunt tips. slender spindle-shaped. Tips blunt. Epidermal cells of involucral bract: Spindle-shaped or rectangular. 2). 8. both cell lumina and wall appearing bright blue under polarized light. (2a) tube cells colorless. pale yellow or colorless. Epidermal cells of bracteal leaf: Irregular-shaped to rectangular. anticlinal walls smooth. with smooth anticlinal walls. Epidermal cells of corolla: (1a) Tip of limb brown or pale purple. pale brown to brown. 10. Stigmas: With short scale-like protrusions. 30-57 μm in diameter. with papillary protrusions.

Both surfaces with hairs Linear lanceolate Not observable Not observable Both surfaces with hairs Linear lanceolate Not observable Not observable Both surfaces with hairs 3-4 Broadly campanulate 6-9 mm × 1-4 mm 3 Slender cylindrical 10-11 mm × 2-4 mm Cylindrical. surrounding capitula Lanceolate 11-18 mm × 2-5 mm 9 mm long 7 mm long Purple 2-lobed Ovate. 8-9 mm long 2 2 Gray Light Slightly bitter Grayish-white Light Slightly bitter Some clump at top of stem. of layers Bract shape Bract size Capitula Flowers Achenes Shape Size Pappi No. laniceps Usually with hairs. involucrata Sparsely covered with hairs Oblong or wide lanceolate Blunt or slightly acuminate Decurrent Serrate and ciliate Sparsely covered with hairs Ovate or oval Sharply toothed Membranous Sparsely covered with hairs 3-4. longitudinally ribbed 3 mm long 2 Grayish-white Fragrant Slightly bitter 14 Some clump at top of stem. 3 mm long Spindle. paniculate spike inflorescence 6-9 mm long 2-3 mm long Light purple 2-lobed Clump at top of stem Limb Tube Anther Stigma Odor Taste S. only on top S. Comparison of macroscopic characteristics of the three snow lotus herbs Whole plant Leaves Shape Tip Base Margin Surface Bracteal leaves Shape Margin Texture Surface Involucral bracts No. medusa Entirely covered with hairs Oblanceolate or spatulate Acute or acuminate Cuneate Undulate Both surfaces with hairs Long oval or linear spatulate Blunt or rounded Cuneate Whole or with laciniate. of layers Color S.TABLE 2. hemispherical total inflorescence 5 mm long 5 mm long Purple 2-lobed .

brown Protrusion papillary. top cell slender. yellow Usually unicellular.TABLE 3. cell walls smooth 2) Unicellular. bright blue under polarized light. cell connections shrunken and yellow. sculpture reticular Pale yellow 1) Multicellular. colorless or pale yellow Stalk Cells interlocked in each layer. brownish-yellow Slender spindle-shaped Colorless A few prismatic crystals appeared bright blue under polarized light Spindle-shaped or rectangular Colorless. dark Protrusions papillary. tips blunt. brown or pale purple Anticlinal walls smooth. laniceps Subrounded 24-36-50 3 Warts dense. brown Unicellular. cell walls smooth Head 5-6 flat layers. colorless Fiber width (μm) 9-14-18 Cell wall Thickened in longitudinal stripes. cell linear and bulgy. top cell slender. outer walls unregularly prominent in lateral view Description Protrusions scale-like. linear. cell flat and twisted. tips blunt. cell walls smooth 2) Multicellular. cell connections slightly shrunken. cell walls uneven 3) Multicellular. brownish-yellow or pale purple Cell lumina and walls bright blue Irregular to rectangular Smooth Anomocytic Colorless or pale brown Rectangular Straight Pale brown Anomocytic Pale brown to brown Polygonal to rectangular Slightly curved Brown 15 . cell walls smooth 2) Multicellular. lower epidermis deeply curved Stoma Anomocytic Color Colorless Shape Polygonal Anticlinal wall Slightly curved Color Pale brown Shape Diameter (μm) Germination ditches Ornamentation Color Description S. Microscopic characteristics of the three snow lotus herbs Pollen grains Non-glandular hairs Glandular hairs Pappi Cells of inner surface of microdiodange Stigmas Epidermal cells of corolla Epidermal cells of involucral bract Epidermal cells of bracteal leaf Epidermal cells of leaf S. medusa Subrounded 30-43-57 3 Spinules dense. tips blunt. colorless 9-12-17 Smooth. no specific color under polarized microscope Protrusions scale-like. sculpture reticular Yellow 1) Unicellular. bright blue under polarized light. cell walls smooth 5-6 single-celled flat layers. brownish-yellow Rectangular Pale yellow or brownish-yellow Dense prismatic crystals appeared bright blue or yellow under polarized light Elongated spindle-shaped Colorless or pale yellow Cell lumina and walls bright blue Irregular Undulate S. pale yellow Tube: Shape Rectangular Color Colorless Prismatic crystals appeared bright blue Cell below epidermis under polarized light Shape Elongated spindle-shaped Color Colorless Under polarized light Cell walls bright blue Shape Irregular Anticlinal wall Upper epidermis sinuous. cell connections smooth and colorless. brown Limb: Tip Protrusions papillary. cell walls striated 2-4 layers with rounded cells. brownish-yellow Anticlinal walls curved. brown 10-15-19 Thickened in longitudinal stripes. cell connections shrunken and yellow. involucrata Subrounded 23-41-52 3 Warts coarse. yellow Other cells Anticlinal walls curved. sculpture granulate Pale yellow or colorless 1) Multicellular. outer walls unregularly prominent in lateral view Protrusions scale-like.

laniceps 2. Amphilaena (Stschegl.Fig 3. arranged in paniculate spikes along upper stem —S. laniceps. B 1 . usually supported or half-surrounded by involucral bracts covered with a thick layer of soft. B 2 : S.) Hook. Pollen grain from the three snow lotus herbs under scanning electron microscope (A 1 . Eriocoryne (DC. Capitula not supported or surrounded by broad. involucrata 1. A 2 : S. Capitula crowded at top of stem. membranous and colored involucral bracts. Capitula arrange in hemispherical racemes —S. Capitula abundant. C 2 : S. woolly hairs —Subgen. DISCUSSION TABLE 4. —S. supported or surrounded by broad. medusa. and colored involucral bracts —Subgen. Scale: 5 μm). f.) Lipsch. Key based on macroscopic characteristics of the three snow lotus herbs 1. membranous. some capitula crowded at top of stem. medusa 16 . involucrata. 2. C 1 .

Warts densely-arranged. outer surface with reticular sculptures 2. warts coarse and prominent. Warts widely-spaced. no evident warts. reticular sculptures relatively rough —S. outer surface with granular sculptures —S. Key based on pollen grains of the three snow lotus herbs under scanning electron microscope 1. Spinules dense and with blunt tips. laniceps 17 .TABLE 5. involucrata 2. reticular sculptures relatively smooth —S. Spinules not evident. medusa 1.

Table 5 and Figure 4.Fig. medusa. 18 . 4. laniceps. Key based on powders of the three snow lotus herbs under optical microscope (Scale: 100 μm). and S. 1. S. The most reliable microscopic characters for identification are listed in Table 4. ordinary light microscopy. involucrata. S. This is the first research that combines macroscopical identification. polarized light microscopy and scanning electron microscopy to thoroughly distinguish the three snow lotus herbs.

making authentication efficient and accurate.2. they contribute in different ways in authenticating herbal powders. we chose characters that are least affected by preparation processes and are most stable in different populations. Non-glandular hairs are frequently observed under the microscope. Polarized light microscopy reveals more information when samples are nearly identical under ordinary light. 4). sizes. involucrata have unique unicellular or multicellular non-glandular hairs. f.) Hook. 4). Fig. etc. For example. in choosing characters by which to authenticate species. polarized light microscopy and scanning electron microscopy. For the three types of microscopy. SEM gives detailed information about surfaces. 2 and Fig. involucrata in Subgen. laniceps and S. and 2) the distribution of crystals (Table 3. medusa. Because pollen grains are noteworthy for the specific ornamentation of their exine layers. while both S. S. However. Fig. For macroscopic features. we chose characteristics of non-glandular hairs.) Lipsch. and colors (Table 3. 19 . with the combined use of ordinary light microscopy. Ordinary light microscopy shows basic microscopical features. medusa share a same multicellular type. many independent characteristics of the powders can be observed. glandular hairs. SEM can give detailed specific information about pollen. medusa are in Subgen. for keys to differentiate these three snow lotus herbs. Thus. they belong to two subgenera: S. and they differ according to the two subgenera: S. as can be expected since they are all members of the genus Saussurea DC. For optical microscopy. laniceps and S. cell color can vary with the degree of permeabilization during mounting. cells of inner surface of microdiodange and crystal distribution underneath epidermal cells of corolla. 2 and Fig. this also stands for S. Eriocoryne (DC. and cell size can vary according to plant age. 3. involucrata. the three herbs are very similar. sample origin. Species from the two subgenera can be distinguished according to their capitula arrangement. laniceps and S. Therefore. and cell connections yellow and shrunken. (Fig. including 1) the cuticle properties of plant tissue cells. 3 and Table 5).. with top cell slender. such as cell morphological characters. Amphilaena (Stschegl. while S.

medusa can be identified from the three species by their dense spinules. As for exine ornamentation. and granular sculpturing on the surface. that is. 1935. 2008). SEM observation of exine ornamentations. 1979). shapes. As for size. laniceps (both with reticular sculpturing). in descending order. while the former do not. oval is considered to be more advanced than spherical (Wodehouse. pollen grains are believed to have evolved from larger to smaller (Covas et Schnack. then negative ornamentation. For scanning electron microscopy. laniceps. medusa (with granular sculpturing) is more primitive than S. protrusions (e. laniceps have. and sizes of pollen grains can reflect the evolution traits of the specific species (Wang et al.As for glandular hairs.g. involucrata and S. Mullaer. undulate or striated sculptures (Walker. laniceps share a similar type with a flat and layered head. medusa. indicating that they are relatively primitive in Saussurea genus. To further differentiate. According to this hypothesis. As shown in Figure 3 and Table 5. gradually evolving into reticular. Those from S. pollen grains from S. in contrast. distribution density and size. involucrata. are with little spinules. colpus width. the three kinds of pollen grains can be distinguished according to spinulate density. smoothness is considered most primitive. involucrata and S. granules). and S.. and spinules developed. 1944). spinulate size. 1976). the 20 . S. prominent warts and reticular sculpturing on the surface. S. S. The pollen grains of the three species are all subrounded. each with a head consisting of rounded cells. In this regard. laniceps. including exine ornamentation. laniceps. The three species can also be distinguished based on the amount of crystals underneath the epidermal cells of corolla. These cells of the latter species appear bright blue under polarized light. the two species can be further differentiated according to the walls of cells of the inner surface of the microdiodange. medusa have a unique type. interspecific differences of pollen grains. As for shape. were studied. involucrata and S. involucrata and S. while S. S. Pollen grains from S. Since glandular hairs are rare in S. involucrata have sparser warts and rougher reticular sculpturing than those from S. wart density and sculptures on the outer surface. lack of evident warts.

Zhang H. Therefore. Zhang ZF. further studies should be conducted in the future. involucrata. medusa. S. polarized light microscopy and scanning electron microscopy can be successfully applied in the authentication of the three Sausaurea species used as the medicinal herb “snow lotus”. the evolutionary trait above do not support the one in Flora Reipublicae popularis sinicae (Jiang et Li. Bertelsen F. which is. CONCLUSIONS Our study has proved that the combining characters derived from observation through macroscopical identification. from most primitive to most developed. and S. Microsc Res Tech 72: 727–736. S. ordinary light microscopy. 2009. Grana 12: 79–86.evolutionary sequence. S. 1972. medusa (43 μm). from most primitive to most developed. The method has proven to be simple. The macroscopic and microscopic characteristics presented in this article are proposed as a reference to establish authenticity of the drugs and may be used to differentiate the drugs from adulterants. convenient. Authenticating and distinguishing the eight species of traditional Tibetan medicine “Meiduoluomi” by microscopic technique. and S. Andersen TS. Wang JG. laniceps. Scanning electron microscope studies of pollen of cereals and other grasses. involucrata (average diameter 47 μm). Chen C. ACKNOWLEDGMENT This research was funded by the Faculty Research Grant of Hong Kong Baptist University (FRG/08-09/II-52) and General Research Fund of Hong Kong (HKBU-260111). As for the relationship between pollen grain characters and evolutionary sequence. 1999). REFERENCES An J. 21 . Li J. should be S. laniceps (36 μm). and reliable. We believe that this method can be widely applied to identify other CMM.

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