Development of a Genus-Universal Nucleotide Signature for the Identification and Supervision of Ephedra-Containing Products
Abstract
:1. Introduction
2. Materials and Methods
2.1. Collection of Materials
2.2. DNA Extraction
2.3. Primer Design, PCR Amplification and Sequencing
2.4. Sequence Analysis
3. Results
3.1. Development of the Genus-Universal Nucleotide Signature for Ephedra L.
3.2. Verification of the Nucleotide Signature in the Species and Varieties within Ephedra L.
3.3. Application of the Nucleotide Signature on the Detection of Ephedra Herb in Compound Preparations
4. Discussion
4.1. Significance of the Development of a Nucleotide Signature for Ephedra
4.2. Supervision of Ephedra-Containing Products with the Genus-Universal Nucleotide Signature
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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No. | Species | Voucher No. | Collection Site | GenBank Accession |
---|---|---|---|---|
1 | E. equisetina | IMD0001954 | Hebei | OL456774 |
2 | E. equisetina | IMD0001953 | Shaanxi | OL456770 |
3 | E. equisetina | IMD0001955 | Shanxi | OL456771 |
4 | E. equisetina | IMD0001952 | Hebei | OL456772 |
5 | E. equisetina | IMD0001951 | Xinjiang | OL456773 |
6 | E. equisetina | IMD0001950 | Xinjiang | OL456775 |
7 | E. equisetina | IMD0001949 | Xinjiang | OL456776 |
8 | E. equisetina | IMD0001946 | Xinjiang | OL456777 |
9 | E. equisetina | IMD0001964 | Beijing | OL456763 |
10 | E. equisetina | IMD0001963 | Beijing | OL456764 |
11 | E. equisetina | IMD0001966 | Xinjiang | OL456765 |
12 | E. equisetina | IMD0001961 | Hebei | OL456766 |
13 | E. equisetina | IMD0001958 | Jiangsu | OL456767 |
14 | E. equisetina | IMD0001957 | Beijing | OL456768 |
15 | E. equisetina | IMD0001956 | Beijing | OL456769 |
16 | E. fedtschenkoae | IMD0001967 | Qinghai | OL456778 |
17 | E. gerardiana | IMD0001970 | Xizang | OL456779 |
18 | E. gerardiana | IMD0001969 | Xizang | OL456780 |
19 | E. gerardiana | IMD0001968 | Xizang | OL456781 |
20 | E. gerardiana | IMD0001971 | Xizang | OL456782 |
21 | E. gerardiana | IMD0001972 | Xizang | OL456783 |
22 | E. intermedia | IMD0001980 | Qinghai | OL456784 |
23 | E. intermedia | IMD0001978 | Qinghai | OL456785 |
24 | E. intermedia | IMD0001976 | Xinjiang | OL456786 |
25 | E. intermedia | IMD0001975 | Qinghai | OL456787 |
26 | E. intermedia | IMD0001974 | Xinjiang | OL456788 |
27 | E. intermedia | IMD0001995 | Sichuan | OL456789 |
28 | E. intermedia | IMD0001994 | Neimenggu | OL456790 |
29 | E. intermedia | IMD0001992 | Xinjiang | OL456791 |
30 | E. intermedia | IMD0001991 | Xinjiang | OL456792 |
31 | E. intermedia | IMD0001989 | Xinjiang | OL456793 |
32 | E. intermedia | IMD0001988 | Hebei | OL456794 |
33 | E. intermedia | IMD0001996 | Neimenggu | OL456795 |
34 | E. intermedia | IMD0001986 | Xinjiang | OL456796 |
35 | E. intermedia | IMD0001984 | Gansu | OL456797 |
36 | E. intermedia | IMD0001983 | Xinjiang | OL456798 |
37 | E. intermedia | IMD0001981 | Gansu | OL456799 |
38 | E. intermedia | IMD0001985 | Sichuan | OL456800 |
39 | E. intermedia | IMD0001987 | Xinjiang | OL456801 |
40 | E. intermedia var. tibetica | IMD0001982 | Xizang | OL456802 |
41 | E. intermedia var. tibetica | IMD0002003 | Xizang | OL456803 |
42 | E. intermedia var. tibetica | IMD0002002 | Xizang | OL456804 |
43 | E. intermedia var. tibetica | IMD0002001 | Xizang | OL456805 |
44 | E. intermedia var. tibetica | IMD0002000 | Xizang | OL456806 |
45 | E. intermedia var. tibetica | IMD0001999 | Xizang | OL456807 |
46 | E. intermedia var. tibetica | IMD0001998 | Xizang | OL456808 |
47 | E. intermedia var. tibetica | IMD0001997 | Xizang | OL456809 |
48 | E. intermedia var. tibetica | IMD0002016 | Xizang | OL456810 |
49 | E. intermedia var. tibetica | IMD0002014 | Xizang | OL456811 |
50 | E. intermedia var. tibetica | IMD0002013 | Xizang | OL456812 |
51 | E. intermedia var. tibetica | IMD0002012 | Xizang | OL456813 |
52 | E. intermedia var. tibetica | IMD0002011 | Xizang | OL456814 |
53 | E. intermedia var. tibetica | IMD0002010 | Xizang | OL456815 |
54 | E. intermedia var. tibetica | IMD0002009 | Xizang | OL456816 |
55 | E. intermedia var. tibetica | IMD0002007 | Xizang | OL456817 |
56 | E. intermedia var. tibetica | IMD0002006 | Xizang | OL456818 |
57 | E. intermedia var. tibetica | IMD0002005 | Xizang | OL456819 |
58 | E. intermedia var. tibetica | IMD0002008 | Xizang | OL456820 |
59 | E. intermedia var. tibetica | IMD0002021 | Xizang | OL456821 |
60 | E. intermedia var. tibetica | IMD0002017 | Xizang | OL456822 |
61 | E. intermedia var. tibetica | IMD0002019 | Xizang | OL456823 |
62 | E. intermedia var. tibetica | IMD0002020 | Xizang | OL456824 |
63 | E. intermedia var. tibetica | IMD0002018 | Xizang | OL456825 |
64 | E. intermedia var. tibetica | IMD0002024 | Xizang | OL456826 |
65 | E. intermedia var. tibetica | IMD0002023 | Xizang | OL456827 |
66 | E. intermedia var. tibetica | IMD0002022 | Xizang | OL456828 |
67 | E. likiangensis | IMD0002027 | Yunnan | OL456829 |
68 | E. likiangensis | IMD0002026 | Sichuan | OL456830 |
69 | E. likiangensis | IMD0002025 | Yunnan | OL456831 |
70 | E. minuta | IMD0002029 | Xizang | OL456832 |
71 | E. minuta | IMD0002030 | Xizang | OL456833 |
72 | E. minuta | IMD0002028 | Sichuan | OL456834 |
73 | E. monosperma | IMD0002037 | Qinghai | OL456835 |
74 | E. monosperma | IMD0002043 | Xizang | OL456836 |
75 | E. monosperma | IMD0002044 | Xizang | OL456837 |
76 | E. przewalskii | IMD0002060 | Gansu | OL456841 |
77 | E. przewalskii | IMD0002059 | Qinghai | OL456842 |
78 | E. przewalskii | IMD0002057 | Gansu | OL456843 |
79 | E. przewalskii | IMD0002056 | Xinjiang | OL456844 |
80 | E. przewalskii | IMD0002055 | Qinghai | OL456845 |
81 | E. przewalskii | IMD0002063 | Gansu | OL456846 |
82 | E. przewalskii | IMD0002062 | Gansu | OL456838 |
83 | E. przewalskii | IMD0002061 | Gansu | OL456839 |
84 | E. przewalskii | IMD0002051 | Xinjiang | OL456840 |
85 | E. regeliana | IMD0002066 | Qinghai | OL456850 |
86 | E. regeliana | IMD0002064 | Xinjiang | OL456847 |
87 | E. regeliana | IMD0002067 | Qinghai | OL456848 |
88 | E. regeliana | IMD0002070 | Xinjiang | OL456849 |
89 | E. saxatilis | IMD0002071 | Xizang | OL456851 |
90 | E. saxatilis | IMD0002078 | Xizang | OL456852 |
91 | E. saxatilis | IMD0002076 | Xizang | OL456853 |
92 | E. saxatilis | IMD0002075 | Xizang | OL456854 |
93 | E. saxatilis | IMD0002077 | Sichuan | OL456855 |
94 | E. saxatilis | IMD0002081 | Xizang | OL456856 |
95 | E. saxatilis | IMD0002080 | Xizang | OL456857 |
96 | E. saxatilis | IMD0002079 | Xizang | OL456858 |
97 | E. saxatilis | IMD0002090 | Xizang | OL456859 |
98 | E. saxatilis | IMD0002089 | Xizang | OL456860 |
99 | E. saxatilis | IMD0002088 | Xizang | OL456861 |
100 | E. saxatilis | IMD0002087 | Xizang | OL456862 |
101 | E. sinica | IMD0002098 | Hebei | OL456887 |
102 | E. sinica | IMD0002099 | Hebei | OL456863 |
103 | E. sinica | IMD0002097 | Hebei | OL456864 |
104 | E. sinica | IMD0002095 | Shanxi | OL456865 |
105 | E. sinica | IMD0002106 | Hebei | OL456866 |
106 | E. sinica | IMD0002105 | Neimenggu | OL456867 |
107 | E. sinica | IMD0002104 | Hebei | OL456868 |
108 | E. sinica | IMD0002103 | Hebei | OL456869 |
109 | E. sinica | IMD0002101 | Shanxi | OL456870 |
110 | E. sinica | IMD0002109 | Neimenggu | OL456871 |
111 | E. sinica | IMD0002108 | Hebei | OL456872 |
112 | E. sinica | IMD0002118 | Neimenggu | OL456873 |
113 | E. sinica | IMD0002117 | Neimenggu | OL456874 |
114 | E. sinica | IMD0002115 | Neimenggu | OL456875 |
115 | E. sinica | IMD0002112 | Neimenggu | OL456876 |
116 | E. sinica | IMD0002111 | Jilin | OL456877 |
117 | E. sinica | IMD0002125 | Neimenggu | OL456878 |
118 | E. sinica | IMD0002122 | Neimenggu | OL456879 |
119 | E. sinica | IMD0002123 | Neimenggu | OL456880 |
120 | E. sinica | IMD0002121 | Neimenggu | OL456881 |
121 | E. sinica | IMD0002119 | Hebei | OL456882 |
122 | E. sinica | IMD0002136 | Neimenggu | OL456883 |
123 | E. sinica | IMD0002135 | Neimenggu | OL456884 |
124 | E. sinica | IMD0002134 | Neimenggu | OL456885 |
125 | E. sinica | IMD0002132 | Neimenggu | OL456886 |
No. | Sample Name | Type | Collection Site | Number of Ingredients | Ingredients on Label |
---|---|---|---|---|---|
ZCY-01 | Mahuang Fuzi Xixin Soup | Concentrated pills | Guangdong | 3 | Ephedrae Herba, Aconiti Lateralis Radix Praeparata, Asari Radix Et Rhizoma |
ZCY-02 | Maxing Zhike Tablets | Tablets | Jilin | 4 | Ephedrae Herba, Armeniacae Semen Amarum, Gypsum Fibrosum, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle |
ZCY-03 | Mahuang Zhisou Pills | Watered pills | Shandong | 7 | Citri Exocarpium Rubrum, Ephedrae Herba, Platycodonis Radix, Fritillariae Cirrhosae Bulbus, Schisandrae Chinensis Fructus, Poria, Asari Radix Et Rhizoma |
ZCY-04 | Tongxuan Lifei Pills | Honeyed pills | Beijing | 11 | Perillae Folium, Peucedani Radix, Platycodonis Radix, Armeniacae Semen Amarum, Ephedrae Herba, Glycyrrhizae Radix Et Rhizoma, Citri Reticulatae Pericarpium, Pinelliae Rhizoma, Poria, Aurantii Fructus, Scutellariae Radix |
ZCY-05 | Xiaoer Feire Kechuan Granules | Granules | Heilongjiang | 11 | Ephedrae Herba, Armeniacae Semen Amarum, Gypsum Fibrosum, Glycyrrhizae Radix Et Rhizoma, Lonicerae Japonicae Flos, Forsythiae Fructus, Anemarrhenae Rhizoma, Scutellariae Radix, Isatidis Radix, Ophiopogonis Radix, Houttuyniae Herba |
ZCY-06 | Zhengtian Pills | Watered pills | Guangdong | 15 | Uncariae Ramulus Cum Uncis, Paeoniae Radix Alba, Chuanxiong Rhizoma, Angelicae Sinensis Radix, Rehmanniae Radix, Angelicae Dahuricae Radix, Saposhnikoviae Radix, Notopterygii Rhizoma Et Radix, Persicae Semen, Carthami Flos, Asari Radix Et Rhizoma, Angelicae Pubescentis Radix, Ephedrae Herba, Aconiti Lateralis Radix Praeparata, Spatholobi Caulis |
ZCY-07 | Qiguanyan Pills | Watered pills | Beijing | 31 | Ephedrae Herba, Armeniacae Semen Amarum, Gypsum Fibrosum, Glycyrrhizae Radix Et Rhizoma, Peucedani Radix, Cynanchi Stauntonii Rhizoma Et Radix, Stemonae Radix, Asteris Radix Et Rhizoma, Farfarae Flos, Meretricis Concha Cyclinae Concha, Descurainiae Semen Lepidii Semen, Citri Grandis Exocarpium, Platycodonis Radix, Poria, Pinelliae Rhizoma, Polygalae Radix, Inulae Flos, Pumice, Perillae Fructus, Codonopsis Radix, Jujubae Fructus, Schisandrae Chinensis Fructus, Cinnamomi Ramulus, Allii Macrostemonis Bulbus, Paeoniae Radix Alba, Mori Folium, Belamcandae Rhizoma, Scutellariae Radix, Indigo Naturalis, Taraxaci Herba, Eriobotryae Folium |
No. | Sequences (5′-3′) | Length/bp | Intergeneric Specificity | Intragenus Conservation |
---|---|---|---|---|
S1 | TCGGGGGGACGGCCTTGACCGTCCGGTCCGCCTCGGCGGTGCGGTCGGTTGAAAT | 55 | √ | × |
S2 | GGGGGACGGCCTTGACCGTCCGGTCCGCCTCGGCGGTGCGGTCGG | 45 | √ | × |
S3 | GCCTTGACCGTCCGGTCCGCCTCGGCGGTGCGGTC | 35 | √ | × |
S4 | GACCGTCCGGTCCGCCTCGGCGGTGCGGTC | 30 | √ | × |
S5 | CCGTCCGGTCCGCCTCGGCGGTGCGGT | 27 | √ | × |
S6 | GTCCGGTCCGCCTCGGCGGTGCG | 23 | √ | √ |
S7 | CCGGTCCGCCTCGGCGGTGC | 20 | × | √ |
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Wang, G.; Bai, X.; Chen, X.; Ren, Y.; Han, J. Development of a Genus-Universal Nucleotide Signature for the Identification and Supervision of Ephedra-Containing Products. Molecules 2022, 27, 2342. https://doi.org/10.3390/molecules27072342
Wang G, Bai X, Chen X, Ren Y, Han J. Development of a Genus-Universal Nucleotide Signature for the Identification and Supervision of Ephedra-Containing Products. Molecules. 2022; 27(7):2342. https://doi.org/10.3390/molecules27072342
Chicago/Turabian StyleWang, Gang, Xuanjiao Bai, Xiaochen Chen, Ying Ren, and Jianping Han. 2022. "Development of a Genus-Universal Nucleotide Signature for the Identification and Supervision of Ephedra-Containing Products" Molecules 27, no. 7: 2342. https://doi.org/10.3390/molecules27072342