Pharmacognosy Identification of Hippocampus barbouri Based on the Combination of the Morphological Characteristics and DNA Barcodes
SUN Si-ya1, LAI Meng-ru1, FANG Yun1, GE Yu-qing2, ZHANG Guang-ji1, CHENG Ru-bin1*
1. College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China; 2. The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou 310006, China
Abstract：OBJECTIVE To obtain the key identification information of Hippocampus barbouri (H. barbouri), investigate the pharmacognosy of the seahorse adulteration H. barbouri, using the combination of morphological and DNA barcoding technique. METHODS Typical morphological characteristics of H. barbouri were studied based on the observations according to the previous reports and traditional pharmacognosy methods. Genomic DNAs of H. barbouri and other related seahorse species were extracted and the DNA barcoding genes of COI, 16S and ATP6 sequences were amplified and sequenced, respectively. In order to determine advantages of three barcodes for the identification of hippocampus, MEGA 7 was performed to analyze the intraspecific and interspecific distances and construct the NJ phylogenetic trees based on three different barcoding genes. RESULTS The most typical distinguishing features of H. barbouri included the streak of the nose, five radial lines in the eyelid, and the long and short intervals of the tail thorns. The lengths of COI, 16S and ATP6 sequences were 649-650, 574 and 603 bp, respectively, with the maximum K2P intraspecific distances were 0.012, 0.003 and 0.003, respectively. The intraspecific distances of the three barcodes were far smaller than the corresponding minimum K2P interspecific distances of H. barbouri and other seven related seahorse species, existing an obvious barcoding gap. The NJ phylogenetic trees based on COI, 16S and ATP6 sequences showed that H. barbouri samples formed of a group indicating that H. barbouri could be distinguished from other seahorse species through the three DNA barcoding markers. In addition, H. barbouri showed close genetic relationship with H. histrix according to the NJ trees. Furthermore, the NJ tree structures of COI and ATP6 were more stable than that of 16S. CONCLUSION The pharmacognostical investigation bases on the morphological identification and DNA barcodes obtained the key identification information of H. barbouri, providing the possibility for quick and accurate identification of this species. The COI, 16S and ATP6 barcoding genes can be used for further molecular identification markers for seahorse species. This study provides a new technical method for the development and quality control of hippocampus.
孙思雅, 来梦茹, 方昀, 葛宇清, 张光霁, 程汝滨. 基于性状鉴别与DNA条形码联用的鲍氏海马生药学研究[J]. 中国药学杂志, 2019, 54(24): 2047-2054.
SUN Si-ya, LAI Meng-ru, FANG Yun, GE Yu-qing, ZHANG Guang-ji, CHENG Ru-bin. Pharmacognosy Identification of Hippocampus barbouri Based on the Combination of the Morphological Characteristics and DNA Barcodes. Chinese Pharmaceutical Journal, 2019, 54(24): 2047-2054.
CHEN M, CHEN J Z, GE Y Q, et al. Research progress on chemical constituents and pharmacological activities of Hippocampus[J]. Chin Tradit Herb Drugs (中草药), 2017, 48(19): 4089-4099.
[3]
WEN L L, LI J D, WAN D G, et al. Market survey and identification of Hippocampus (Haima)[J]. China J Chin Mater Med (中国中药杂志), 2013, 38(7): 969-971.
[4]
LOURIE S A, POLLOM R A, FOSTER S J. A global revision of the seahorses Hippocampus rafinesque 1810 (actinopterygii: syngnathiformes): taxonomy and biogeography with recommendations for further research [J]. Zootaxa, 2016, 4146(1): 1-66.
[5]
SHAPAWI R, ANYIE A L, HUSSIEN M A, et al. Species and size composition of seahorses (genus Hippocampus, family syngnathidae) in the coastal waters and local market of Kota Kinabalu, Sabah, Malaysia [J]. Trop Life Sci Res, 2015, 26(2): 1-13.
[6]
WEN L L. Study on identification of Hippocampus and DNA barcodes[D]. Chengdu: Chengdu University of Traditional Chinese Medicine(成都中医药大学), 2014.
[7]
CHEN S L, QIAN Z Z. Study on the safety and verification of traditional Chinese medicine[J]. Chin Pharm J (中国药学杂志), 2015, 50(17): 1467-1468.
[8]
HU R, DU H, CUI L N, et al. DNA Molecular identification based on COI in Hippocampus and syngnathus[J]. Jilin J Tradit Chin Med (吉林中医药),2012, 32(3): 272-273,276.
[9]
JIANG C, LIU F Y, JIN Y, et al. Zoological origin survey of commercial Hippocampus in Chinese herbal markets by morphological and DNA sequencing identification[J]. China J Chin Mater Med (中国中药杂志), 2018, 43(23): 4553-4561.
[10]
KANG Y G, ZHU L W, YUAN D M, et al. Microscopic comparison and identification of Hippocampus powder[J]. J Chin Med Mater (中药材), 2006,29(3): 224-228.
[11]
CHEN L. Study on identification and quality standard of Chinese medicine Hippocampus[D]. Shanghai: Second Military Medical University(第二军医大学), 2015.
[12]
HOU D Y, YANG M M, LI J, et al. Molecular identification of polygonum chinensis using DNA barcoding[J]. Chin Pharm J (中国药学杂志), 2018, 53(14): 1165-1169.
ZHANG G X, LIU J X, JIA J, et al. Molecular identification of Hippocampus and its adulterants based on COI barcode[J]. Chin Pharm J (中国药学杂志), 2015, 50(17): 1469-1473.
[15]
FAN J J, SONG M, SONG C, et al. Identification of the skin of Bufo bufo gargarizans and its adulterants based on DNA barcoding[J]. Chin Pharm J (中国药学杂志), 2015, 50(15): 1292-1296.
[16]
GE Y Q, ZHU L Y, CHEN M, et al. Complete mitochondrial genome sequence for the endangered Knysna seahorse Hippocampus capensis, Boulenger 1900 [J]. Conserv Genet Resour, 2018, 10(3): 461-465.
[17]
ZHU L Y, CHEN M, CHENG R B, et al. Complete mitochondrial genome characterization of the alligator pipefish Syngnathoides biaculeatus, and phylogenetic analysis of family Syngnathidae [J]. Conserv Genet Resour, 2019,11(4):401-404.
[18]
CHENG R B, FANG Y, GE Y Q, et al. Complete mitochondrial genome sequence of the Jayakar′s seahorse Hippocampus jayakari Boulenger, 1900 (gasterosteiformes: syngnathidae) [J]. Mitochondrial DNA B, 2017, 2(2): 593-594.
[19]
CHENG R B, LIAO G H, GE Y Q, et al. Complete mitochondrial genome of the great seahorse Hippocampus kelloggi Jordan & Snyder, 1901 (gasterosteiformes: syngnathidae) [J]. Mitochondrial DNA A DNA Mapp Seq Anal, 2017, 28(2): 227-228.
[20]
XIANG M Y, ZENG L S, LI Z, et al. Analysis of 4 populations scallops and 4 species scallops based on partial 16S rRNA gene sequences[J]. Chin Fish Qual Stand (中国渔业质量与标准), 2018, 8(3): 68-74.
[21]
XIA Y D, WANG S P, LI J, et al. DNA Polymorphism in the partial mitochondrial atp6 gene among Schistosoma japonicum isolates from Hunan Province[J]. Chin J Zoonoses (中国人兽共患病学报), 2011, 27(1): 1-6.
[22]
HEBERT P D N, RATNASINGHAM S, WARD J R. Barcoding animal life: cytochrome c oxidase subunit I divergences among closely related species [J]. Proc R Soc Lond B, 2003, 270(Suppl1):96-99.
[23]
JIANG C, DAN F, YUAN Y, et al. Exploration and thoughts about zoological origin and macroscopical identification of Chinese material medica "Haima" in China Pharmacopoeia[J]. Chin Pharm J (中国药学杂志), 2017, 42(19): 3836-3842.
[24]
XI Y F, YIN F. Advances on artificial cultured technology of seahorses (Hippocampus spp.)[J]. Fish Inf Stratery (现代渔业信息), 2011, 26(10): 9-15.
[25]
GARCIA L M B, HILOMENGARCIA G V, CALIBARA R L M. Culturing seahorse (Hippocampus barbouri) in illuminated cages with supplementary acetes feeding [J]. Bamidgeh, 2010, 62(2): 122-129.
[26]
GARCIA L M B, HILOMEN-GARCIA G V, CELINO F T, et al. Diet composition and feeding periodicity of the seahorse Hippocampus barbouri reared in illuminated sea cages [J]. Aquaculture, 2012, 358-359: 1-5.
[27]
PAYNE M F. Rearing the Coral Seahorse, Hippocampus barbouri, on Live and Inert Prey[M]. CATO J C, BROWN C L. Marine Ornamental Species: Collection, Culture and Conservation. Vol:21. New Jersey: Lowa State Press, 2003: 289-296.
2019, Vol. 54 
