Received: 24 October 2016 | Revised: 7 January 2017 | Accepted: 11 February 2017 DOI 10.1002/jemt.22856 RESEARCH ARTICLE Identification of “Huoshan shihu” Fengdou: Comparative authentication of the Daodi herb Dendrobium huoshanense and its related species by macroscopic and microscopic features Yujiao Zhao1 | Bangxing Han2 | Huasheng Peng1,3 | Xiao Wang4 | Shanshan Chu1 | Jun Dai2 | Daiyin Peng3,5 1College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China Abstract “ ” 2College of Biological and Pharmaceutical Huoshan shihu, derived from Dendrobium huoshanense, is a well-known, valuable, and rare Daodi Engineering, West Anhui University, herb. Because of its higher price and scarce resources, its related species D. officinale and D. monili- Synergetic Innovation Center of Dendrobia forme are usually presented as “Huoshan shihu” for sale. Fengdou, the processed form of D. Industrialization Development in Anhui huoshanense, its identification is particularly important. To effectively identify Fengdou of D. huosh- Province, Lu’an 237012, China anense and protect the Daodi herb, the morphological and microscopic characteristics of the stems 3Institute of Traditional Chinese Medicine Resources Protection and Development, of three Dendrobium species were examined. The results showed that macroscopic and micro- Anhui Academy of Chinese Medicine, Hefei scopic features helpful for identification of the three species were the total lengths and internode 230012, China numbers of stems, diameter and length of each internode, number of vascular bundles, and 4 Shandong Key Laboratory of TCM Quality presence or absence of silica masses. The key features useful for distinguishing between D. huosh- Control Technology, Shandong Analysis and Test Center, Ji’nan 250014, China anense and its related species, and also between their Fengdou, were the stem lengths and the 5Synergetic Innovation Center of Anhui trends of change in diameter in different internodes. The results of the study indicated that a com- Authentic Chinese Medicine Quality bination of macroscopic and microscopic identification techniques might conveniently and Improvement, Hefei 230012, China effectively be applied for identification of Dendrobium species. “Longtou Fengwei” is the main fea- Correspondence ture of the Daodi herb “Huoshan shihu,” (D. huoshanense Fengdou) and reflects the wisdom Hua-Sheng Peng, College of Pharmacy, behind the protection of Daodi herbs in ancient times. Anhui University of Chinese Medicine, Hefei 230012, China. Email: [email protected]; KEYWORDS Daiyin Peng, Institute of Traditional Chinese “Huoshan shihu” Fengdou, Dendrobium huoshanense, macroscopic, microscopic, identification Medicine Resources Protection and Devel- opment, Anhui Academy of Chinese Medi- cine, Hefei 230012, hina; and Institute of Traditional Chinese Medicine Resources Pro- tection and Development, Anhui Academy of Chinese Medicine, Hefei 230012, China. Email: [email protected] Funding information Revised Program for Endorsed National Forestry Certification System; Grant number: 2015-LY-152; National Agricultural Standardization Demonstration Area Pro- gram; Grant number: SEQ8-109 1 | INTRODUCTION plants (Tsi, Chen, Luo, & Zhu, 1999). According to a traditional Chinese medicine theory, the main functions of this herb are to tonify the stom- Shihu is not only an ornamental plant with beautiful flowers, but also a ach and promote body fluid, nourish yin, and clear heat (Chinese Phar- precious medicinal herb that has a wide and effective application in tra- macopoeia Committee, 2015). There are 74 species and two varieties ditional Chinese medicine. It is derived from Orchidaceae (Dendrobium) of Dendrobium in China (Tsi et al., 1999), and 50 species are used as traditional or folk medicines in different regions of China (Bao & Shun, Review Editor: Prof. Alberto Diaspro 1999; Bao, Shun, Zhang, & Jin, 2005; Ma, Xu, Xu, & Li, 1995; Zhao, Microsc Res Tech.2017;1–10 wileyonlinelibrary.com/journal/jemt VC 2017 Wiley Periodicals, Inc. | 1 2 | ZHAO ET AL. 2014). “Huoshan shihu,” produced in Huoshan, Anhui, is regarded as & Huang, 2015). Macroscopic and microscopic characteristics of some the “Daodi herb” (Tao, 1986; Zhao, 1983; Zhao, Guo, & Brand, 2012). Dendrobium species have been reported (Bao et al., 2005; Chu et al., As early as the Qing Dynasty, it was clearly documented in the “Sup- 2014; Ding, Xu, Wang, Shi, & Xu, 2001; He, Li, & Su, 1992; Li, Liu, plement to Compendium of Materia Medica” (Zhao, 1983). Shihu is Wei, & Wang, 2004; Li et al., 1986, 1989; Ma, Xu, Xu, & Wang, 1996; very expensive, with “Huoshan shihu” being the most expensive. Namba and Lin, 1981a,b; Xu, Xu, Sha, & Luo, 1980; Xu et al., 1981). Because it is more expensive than gold, “Huoshan shihu” is known as However, it is still unknown whether the macroscopic and microscopic characteristics can be used to identify them effectively when D. monili- the “soft gold” in traditional Chinese medicinal materials. forme and D. officinale are processed into Fengdou with different num- “Huoshan shihu” is derived from the fresh and dried stems of Den- bers of internodes. drobium huoshanense C. Z. Tang et S. J. Cheng (Chinese Pharmacopoeia To effectively authenticate and distinguish between D. huosha- Committee, 2015). Because it is of excellent quality and expensive, nense and its most common relatives, D. moniliforme and D. officinale, “Huoshan shihu” is in high demand and its natural resource is on the this article seeks to address 2 issues: (1) Comparison of macroscopic verge of extinction. As early as the Qing dynasty, the “Huoshan County and microscopic characteristics of D. huoshanense, D. moniliforme,and Annals” had recorded that the herb’s resource was decreasing D. officinale; and (2) Rapid identification of D. huoshanense Fengdou. (Huoshan County Annals, 2010). To obtain more benefits, the other 2 Dendrobium species growing in Huoshan, Anhui, D. moniliforme and D. 2 | MATERIALS AND METHODS officinale, were mixed with “Huoshan shihu” for a long time (Anhui Bureau of Quality and Technical Supervision, 2004; Tang and Cheng, 2.1 | Plant materials 1984). However, based on the morphological descriptions of “Huoshan shihu” in ancient herbal books, “Huoshan shihu” is only derived from D. “Shihu,” derived from fresh or dried stems of Dendrobium species, was huoshanense and not from D. moniliforme and D. officinale (Wang and usually harvested around October. The fresh aerial stems of Den- Peng, 2004; Zhao, Han, Peng, & Peng, 2016). Therefore, for the protec- drobium species that had no leaves and flowers were called “Xiantiao” tion of Daodi medicinal material, it is important to distinguish between when used as medicines. Some of the stems of the Dendrobium species D. huoshanense and its related species. had a relatively small size, thick flesh, soft texture, and were rich in Many studies have focused on the identification of Dendrobium slime, such as D. huoshanense, D. moniliforme,andD. officinale. Their species and various methods have been developed to distinguish fresh stems were heated until soft, twisted or wound into a spiral or between them. The Chinese Pharmacopoeia (2015 edition) recently spring form, and dried, also known as “Fengdou.” In the market, there provided morphological, microscopic, and thin layer chromatography are many types of Dendrobium species that can be processed into methods to differentiate between D. officinale, D. nobile, D. chrysoto- Fengdou. D. moniliforme and D. officinale were usually mixed with D. rum,andD. fimbriatum (Chinese Pharmacopoeia Committee, 2015). huoshanense. With the constant development of molecular biology techniques, Twenty-one samples derived from D. huoshanense, D. moniliforme, molecular studies on the Dendrobium genus have made great progress and D. officinale were collected from different production areas in the (Feng, Lu, Gao, Liu, & Wang, 2014; Feng et al., 2015; Hou et al., 2012; Anhui province in China. Specimens were authenticated by Professor Silva, Aceto, Liu, Yu, & Kanno, 2014; Wang, Feng, Lu, Shi, & Liu, 2009; Hua-Sheng Peng of Anhui University of Chinese Medicine. Detailed Xiang et al., 2013). However, research study by Xiang et al. (2013) information of the samples is presented in Table 1. To evaluate the revealed that D. huoshanense, D. officinale,andD. moniliforme from consistency of morphological and anatomical characteristics, more than sect. Dendrobium were in the same composite group. They used the 10 plants were examined for each sample and at least three sections internal transcribed spacers (ITS) from the plant’s nuclear ribosomal per plant piece were prepared. DNA and four plastid markers (the coding gene rbcL, matK, trnH-psbA spacers and trnL intron), and found that it was difficult to differentiate 2.2 | Identification method of Xiantiao among them on the basis of these markers. During production, the Fengdou is prepared from fresh stems. Therefore, to identify Fengdou, “ ” fresh stems of Shihu are often processed into Fengdou by sorting, the characteristics of the fresh stem should be compared first. The cleaning, frying, sectioning, baking, plastic surgery, complex fire, and changes in the macroscopic and microscopic characteristics were asso- other complex processes that generate dry goods (Anhui Bureau of ciated with the internode number in the stem of Shihu; therefore, Quality and Technical Supervision, 2015; Wei, Shun, Li, Dai, & He, internodes of all three Dendrobium species were numbered. The most 2015). After processing