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A new approach for the identification of a Chinese traditional medicine, “Chuanxiong” by 18S ribosomal RNA gene sequences
Phytomedicine Vol. 3 (4), pp. 387-389, 1996/97 © 1996 by Gustav Fischer Verlag
A new approach for the identification of a Chinese Traditional Medicine, "Chuanxiong" by 188 ribosomal RNA gene sequences H. FUSHIMI1, K. KOMATSU1, M. ISOBE2 and 1. NAMBA1 1
2
Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, Toyama, Japan Faculty of Engineering, Toyama University, Toyama, Japan
The medicinal plants are processed and dried to be used as crude drugs in Chinese traditional medicine. The identification of the commercial crude drugs has been a serious problem, for example, there are drugs with the same name and different botanical sources. Such problems happen with substitution of some crude drugs derived from wild plants that can not satisfy the demand, or due to the processed and dry forms of the drugs that sometimes lose the feature of the original plant. So far the determination of the botanical origin of the crude drugs and their quality control have been performed by the anatomical and chemotaxonomical studies. These methods consist in comparing the internal structures and chemical constituents of the crude drugs with those of the medicinal parts of original plant or its related species of the same genus. In order to develop a simple method of identification of crude drugs, we report a new method for the identification of a popular Chinese traditional medicine, "Chuanxiong" by gene sequences. This crude drug is described as a medicine for headache in ancient literature and now, it is frequently used for the treatment of dysmenorrhea, menopause syndrome, infertility and other diseases of the female genitals. As the materials, two samples of "Chuanxiong" (known as "Senkyu" in Japanese) were chosen that are the rhizomes of Cnidium officinale Makino, from Japan, and Ligusticum chuanxiong Hart, from China, both of family Apiaceae. Their 185 ribosomal RNA gene sequences were targetted because they can be obtained from every organs of the plant. Moreover, hundreds of their copies are contained in a genome. Before the DNA analysis of the crude drugs, a fresh rhizome of Cnidium officinale was examined. The method for extraction of the total DNA was essentially according to Murray et al. (Murray and Thompson, 1980). Rhizome tissues frozen with liquid nitrogen were ground into a fine powder. The powder was washed with PBS-EDTA solution consisting of 140 mM NaCI, 2.7 mM KCI,
8.1mM Na2HP04, 1.5mM KH 2P0 4 and 5AmM EDTA (pH 7.0). Into this precipitates were added 10 vol of extraction buffer consisting of 100 mM Tris-HCI (pH 8.0), 40 mM EDTA and 250 mM NaCI, 1 vol of 10% sodium Nlauroyl salcosinate and 1 mg of proteinase K. The mixed solution was incubated for 12 hours at 37°C with shaking. After centrifugation at 900 X g for 15 min the supernatant was extracted by 0.3 vol of PhOHICHCl 3 (1:1) and its aqueous phase was further extracted by 0.3 vol of CHCl3 after added 0.1 vol of 10% cetyl trimethylammonium bromide (CTAB) and 0.1 vol of 5 M NaCI. Then 0.1 vol of 3 M NaOAc and 2.5 vol of absolute ethanol were added into the isolated aqueous phase to get a pellet and it was resuspended in TE-buffer consisting of 10 mM Tris-HCI and 1 mM EDTA (pH 8.0). This solution was incubated for 30 min at 37°C with RNase, and the DNA was purified by treating with Geneclean kit II (BIO 101). The size of DNA was determined by electrophoresis in 1.0% agarose gel and by ethidium bromide staining (Figure 1). As a result, the total DNA of about 20 kb in average size was obtained. The DNA (10-100ng) was then amplified by the PCR using primers derived from sequence flanking 185 rRNA gene (Mitchell, 1990). Each primer had the following sequences: 185 F: 5'CAA CCT GGT TGA TCC TGC CAG T 3' and 185 R: 5' CTG ATe eTT eTG CAG GTT CAC CTA C 3'. The PCR amplification was carried out as follows: DNA amplification in 50/.11 reaction buffer consisting of 50 mM Tris-HCI (pH 9.0), 20mM (NH 4hS04, 0.7mM MgCI 2, 200 mM of each dNTP, 0.25 mM of each primer and 1.5 U of Hot Tub DNA polymerase (Amersham). Thermal cycling was carried out using a Perkin-Elmer PH 2000 machine as follows (Kainz et al., 1992): one cycle consisting of 3 min at 94°C and 8 min at 65 °C followed by 30 cycles of 40 seconds at 94°C and 8 min at 65 "C, and a final extension at 65°C for 8 min. As a result, 1.8 kb of PCR product was obtained. On the other hand, as a negative control, the PCR
388
H. Fushimi er al.
1 2
3 4
5 kb
- 3.1
- 1.0
Fig.1. Compa rison of the tot al DNA and PCR products between Cnidium officinale and a crude drug "Chuanxiong" from Japan. The total DNA and PCR products were visualized by ethidium bromide stainin g under UV. Lane 1: The total DNA extracted from a fresh rhizome of Cnidium officinale; Lane 2: The total DN A of " Chuanxiong" from Japan; Lane 3: The PCR pr oduct of 185 rRNA gene region using tot al DNA of C. officinale; Lane 4: The PCR product of 185 rRNA gene region using tot al DNA of " Chuanxiong " from Japan; Lane 5: Th e negative control (PCR with the same primers without DNA); Lane M: 1 kb ladder (GIBCO).
product with the same set of primers without DNA was loaded in 1.0 % agarose gel, which gave no fragment (Fig. 1). Therefore, it suggested that the same set of primers used in this study did not produce any product. PCR products were cloned into a TA cloning vector pCRIl (Invitrogen), and plasmid DNA was prepared on Q iagen Tip 100. Th e equa l amount of DNA from ten clones randomyl selected were mixed for sequencing. Sequencing was performed with the Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems) following the manufacturer's instructions using a set of sequencing primers described by Chaw et al., 1993. The result showed that 185 rRNA gene sequence of C. officina/e was found to be of 1808 bases. The same procedure was performed to extract the total DNA from the crude drug , " Chuanxiong " from japan (TMPW * No . 15103, depo sited in a separate box for two months after collection). Th is is a dry material previously processed with steam. Then the outer part of the drug was
peeled off before the DNA extraction to avoid the crosscontamination with another drug. The size of the tot al DNA determined by electrophoresis was less than 3.0 kb, mostly less than 1.0 kb. The DNA of crude drug was expected to be cleaved due to the physical impact such as steam and dryness and internal factor as the presence of nuclease and polyphenols such as tannins and flavonoid s. Then the 185 rRNA gene region was amplified by PCR and the PCR products (1.8 kb) obtained were cloned into pCRII vector and sequenced under the same conditions as for the fresh plant DNA. It was found that 185 rRNA gene of "Chuanxiong" from j apan was of 1808 bases and its sequence completely corresponded with that of the original plant, C. officina/e. As a result of investigation on a fresh rhizome of L. chuanxiong and the crude drug, "Chuanxiong" from China (TMPW " No . 13041, deposited for two years after collection), their 185 rRNA gene sequences were also found to be of 1808 bases, corresponding completely with each other, and also with those of C. officina/e and "Chuanxiong" from japan. In this study, we clarified that 185 rRN A gene sequences of the crud e drugs, " Chuanxiong" from japan and China completel y corresponded with tho se of their original plants, C. officina/e and L. chuanx iong, respectively. The taxonomic position of C. officinale has been ambiguous because this plant does not possess mature seeds and some morphological study suggest that it may belong to the genus Ligusticum (Masao , 1963). The tax onomic similarity between C. officina/e and L. cbuanxiong was shown in the present result. In the reported sequences on GenBank, 185 rRNA gene sequences of tomato (Lycopersicon escu/entum Mill.) and potato (Solanum tub erosum L.), both of family Solanaceae were of 1800 and 1807 bases, respectively.These sequences were different from each other and the homology between these two plants were 98.7 %, which suggest that 185 rRNA gene has sequence variati ons between different genus of the same family. However, within family Apiaceae the sequence variation was small, namely, the homolo gy between C. officinale and Angelica acutiloba Kitagawa var. sugiyamae Hikino, whose sequence was determined for reference, was 99.9 %. We also investigated on three Panax species of family Araliaceae and the corresponding Chinese medicines, three " Ginseng drugs" , (Fushimi et aI., 1996). 185 rRNA gene sequences of these plants were found to be of 1809 bases but to be different from each other. Their unique sequences were also found to be the same as the corresponding "Gin seng drugs" , respectively. The family Araliaceae is close to Apiaceae with respect to taxonomic position, belonging to the same order Apiales. So, comp aring 18S rRNA gene sequence of P. gins eng with that of C. officinale, the homology between these two species was 98.8% . Above results showed that 185 rRNA gene of different plants of the same order, family and even genus had the sequence varia-
A new approach for the identification of a Chinese Traditional Medicine tions on this gene region. As a conclusion, 185 rRNA gene sequence informations could be used for determining a plant identity, although not adapted for all genus or species. We succeeded to extract the total DNA from crude drugs which were processed and stored for a long time and to determine their 185 rRNA gene sequences that corresponded to those of original plants. This suggests that the present analytical method of gene sequencing is useful for identification of traditional medicines. This method would be applied not only in 185 rRNA gene region but also in other regions when the primers are changed. Acknowledgments This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from Science and Technology Agency, by a Grant-in Aid from the Ministry of Education, Science, Sports and Culture, and by a Grant for Research on Aging and Health from the Ministry of Health and Welfare, Japan.
References Chaw, S. M., Long, H., Wang, B.S., Zharkikh, A., Li, W. H.: The phylogenetic position of Taxaceae based on 185 rRNA sequences.}. Mol. Evol. 37: 624-630,1993. Fushimi, H., Komatsu, K., lsobe, M., Namba, T.: 185 Ribosomal RNA gene sequences of three Panax species and the corresponding Ginseng drugs. Bioi. Pharm. Bull. 19: 1530-1532, 1996.
389
Kainz, P., Schmiedlechner, A., Strack, H. B.: In vitro amplification of DNA fragments> 10 kb. Anal. Biochem. 202: 46-49,1992. Masao, K.: Notulae fractae ob floram Asiae Orienralis.j. [pn. Bot. 38: 105-111, 1963. Mitchell, L. 5.: Amplification of ribosomal RNA genes for molecular evolution studies. In: PCR Protocols. (Innis, M. A., Gelfand, O. H., Sninsky, J.J. eds., Academic Press, Inc., San Diego), 307-314,1990. Murray, M. G., Thompson, W. E: Rapid isolation of high molecular weight plant DNA. Nuc/. Acids Res. 8: 4321-4325, 1980. *The specimen number of the Museum of Materia Medica, Analytical Research Center for Ethnomedicines, Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University (TMPW). The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession number 083027 for C. officina/e, 083028 for L. chuanxiong, 083275 for P. ginseng, 084100 for P. japonicus, 085172 for P. quinquefolius, 084297 for A. acutiloba var. sugiyamae.
Address K. Komatsu, Research Institute for Wakan- Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
A new approach for the identification of a Chinese Traditional Medicine, "Chuanxiong" by 188 ribosomal RNA gene sequences H. FUSHIMI1, K. KOMATSU1, M. ISOBE2 and 1. NAMBA1 1
2
Research Institute for Wakan-Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, Toyama, Japan Faculty of Engineering, Toyama University, Toyama, Japan
The medicinal plants are processed and dried to be used as crude drugs in Chinese traditional medicine. The identification of the commercial crude drugs has been a serious problem, for example, there are drugs with the same name and different botanical sources. Such problems happen with substitution of some crude drugs derived from wild plants that can not satisfy the demand, or due to the processed and dry forms of the drugs that sometimes lose the feature of the original plant. So far the determination of the botanical origin of the crude drugs and their quality control have been performed by the anatomical and chemotaxonomical studies. These methods consist in comparing the internal structures and chemical constituents of the crude drugs with those of the medicinal parts of original plant or its related species of the same genus. In order to develop a simple method of identification of crude drugs, we report a new method for the identification of a popular Chinese traditional medicine, "Chuanxiong" by gene sequences. This crude drug is described as a medicine for headache in ancient literature and now, it is frequently used for the treatment of dysmenorrhea, menopause syndrome, infertility and other diseases of the female genitals. As the materials, two samples of "Chuanxiong" (known as "Senkyu" in Japanese) were chosen that are the rhizomes of Cnidium officinale Makino, from Japan, and Ligusticum chuanxiong Hart, from China, both of family Apiaceae. Their 185 ribosomal RNA gene sequences were targetted because they can be obtained from every organs of the plant. Moreover, hundreds of their copies are contained in a genome. Before the DNA analysis of the crude drugs, a fresh rhizome of Cnidium officinale was examined. The method for extraction of the total DNA was essentially according to Murray et al. (Murray and Thompson, 1980). Rhizome tissues frozen with liquid nitrogen were ground into a fine powder. The powder was washed with PBS-EDTA solution consisting of 140 mM NaCI, 2.7 mM KCI,
8.1mM Na2HP04, 1.5mM KH 2P0 4 and 5AmM EDTA (pH 7.0). Into this precipitates were added 10 vol of extraction buffer consisting of 100 mM Tris-HCI (pH 8.0), 40 mM EDTA and 250 mM NaCI, 1 vol of 10% sodium Nlauroyl salcosinate and 1 mg of proteinase K. The mixed solution was incubated for 12 hours at 37°C with shaking. After centrifugation at 900 X g for 15 min the supernatant was extracted by 0.3 vol of PhOHICHCl 3 (1:1) and its aqueous phase was further extracted by 0.3 vol of CHCl3 after added 0.1 vol of 10% cetyl trimethylammonium bromide (CTAB) and 0.1 vol of 5 M NaCI. Then 0.1 vol of 3 M NaOAc and 2.5 vol of absolute ethanol were added into the isolated aqueous phase to get a pellet and it was resuspended in TE-buffer consisting of 10 mM Tris-HCI and 1 mM EDTA (pH 8.0). This solution was incubated for 30 min at 37°C with RNase, and the DNA was purified by treating with Geneclean kit II (BIO 101). The size of DNA was determined by electrophoresis in 1.0% agarose gel and by ethidium bromide staining (Figure 1). As a result, the total DNA of about 20 kb in average size was obtained. The DNA (10-100ng) was then amplified by the PCR using primers derived from sequence flanking 185 rRNA gene (Mitchell, 1990). Each primer had the following sequences: 185 F: 5'CAA CCT GGT TGA TCC TGC CAG T 3' and 185 R: 5' CTG ATe eTT eTG CAG GTT CAC CTA C 3'. The PCR amplification was carried out as follows: DNA amplification in 50/.11 reaction buffer consisting of 50 mM Tris-HCI (pH 9.0), 20mM (NH 4hS04, 0.7mM MgCI 2, 200 mM of each dNTP, 0.25 mM of each primer and 1.5 U of Hot Tub DNA polymerase (Amersham). Thermal cycling was carried out using a Perkin-Elmer PH 2000 machine as follows (Kainz et al., 1992): one cycle consisting of 3 min at 94°C and 8 min at 65 °C followed by 30 cycles of 40 seconds at 94°C and 8 min at 65 "C, and a final extension at 65°C for 8 min. As a result, 1.8 kb of PCR product was obtained. On the other hand, as a negative control, the PCR
388
H. Fushimi er al.
1 2
3 4
5 kb
- 3.1
- 1.0
Fig.1. Compa rison of the tot al DNA and PCR products between Cnidium officinale and a crude drug "Chuanxiong" from Japan. The total DNA and PCR products were visualized by ethidium bromide stainin g under UV. Lane 1: The total DNA extracted from a fresh rhizome of Cnidium officinale; Lane 2: The total DN A of " Chuanxiong" from Japan; Lane 3: The PCR pr oduct of 185 rRNA gene region using tot al DNA of C. officinale; Lane 4: The PCR product of 185 rRNA gene region using tot al DNA of " Chuanxiong " from Japan; Lane 5: Th e negative control (PCR with the same primers without DNA); Lane M: 1 kb ladder (GIBCO).
product with the same set of primers without DNA was loaded in 1.0 % agarose gel, which gave no fragment (Fig. 1). Therefore, it suggested that the same set of primers used in this study did not produce any product. PCR products were cloned into a TA cloning vector pCRIl (Invitrogen), and plasmid DNA was prepared on Q iagen Tip 100. Th e equa l amount of DNA from ten clones randomyl selected were mixed for sequencing. Sequencing was performed with the Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems) following the manufacturer's instructions using a set of sequencing primers described by Chaw et al., 1993. The result showed that 185 rRNA gene sequence of C. officina/e was found to be of 1808 bases. The same procedure was performed to extract the total DNA from the crude drug , " Chuanxiong " from japan (TMPW * No . 15103, depo sited in a separate box for two months after collection). Th is is a dry material previously processed with steam. Then the outer part of the drug was
peeled off before the DNA extraction to avoid the crosscontamination with another drug. The size of the tot al DNA determined by electrophoresis was less than 3.0 kb, mostly less than 1.0 kb. The DNA of crude drug was expected to be cleaved due to the physical impact such as steam and dryness and internal factor as the presence of nuclease and polyphenols such as tannins and flavonoid s. Then the 185 rRNA gene region was amplified by PCR and the PCR products (1.8 kb) obtained were cloned into pCRII vector and sequenced under the same conditions as for the fresh plant DNA. It was found that 185 rRNA gene of "Chuanxiong" from j apan was of 1808 bases and its sequence completely corresponded with that of the original plant, C. officina/e. As a result of investigation on a fresh rhizome of L. chuanxiong and the crude drug, "Chuanxiong" from China (TMPW " No . 13041, deposited for two years after collection), their 185 rRNA gene sequences were also found to be of 1808 bases, corresponding completely with each other, and also with those of C. officina/e and "Chuanxiong" from japan. In this study, we clarified that 185 rRN A gene sequences of the crud e drugs, " Chuanxiong" from japan and China completel y corresponded with tho se of their original plants, C. officina/e and L. chuanx iong, respectively. The taxonomic position of C. officinale has been ambiguous because this plant does not possess mature seeds and some morphological study suggest that it may belong to the genus Ligusticum (Masao , 1963). The tax onomic similarity between C. officina/e and L. cbuanxiong was shown in the present result. In the reported sequences on GenBank, 185 rRNA gene sequences of tomato (Lycopersicon escu/entum Mill.) and potato (Solanum tub erosum L.), both of family Solanaceae were of 1800 and 1807 bases, respectively.These sequences were different from each other and the homology between these two plants were 98.7 %, which suggest that 185 rRNA gene has sequence variati ons between different genus of the same family. However, within family Apiaceae the sequence variation was small, namely, the homolo gy between C. officinale and Angelica acutiloba Kitagawa var. sugiyamae Hikino, whose sequence was determined for reference, was 99.9 %. We also investigated on three Panax species of family Araliaceae and the corresponding Chinese medicines, three " Ginseng drugs" , (Fushimi et aI., 1996). 185 rRNA gene sequences of these plants were found to be of 1809 bases but to be different from each other. Their unique sequences were also found to be the same as the corresponding "Gin seng drugs" , respectively. The family Araliaceae is close to Apiaceae with respect to taxonomic position, belonging to the same order Apiales. So, comp aring 18S rRNA gene sequence of P. gins eng with that of C. officinale, the homology between these two species was 98.8% . Above results showed that 185 rRNA gene of different plants of the same order, family and even genus had the sequence varia-
A new approach for the identification of a Chinese Traditional Medicine tions on this gene region. As a conclusion, 185 rRNA gene sequence informations could be used for determining a plant identity, although not adapted for all genus or species. We succeeded to extract the total DNA from crude drugs which were processed and stored for a long time and to determine their 185 rRNA gene sequences that corresponded to those of original plants. This suggests that the present analytical method of gene sequencing is useful for identification of traditional medicines. This method would be applied not only in 185 rRNA gene region but also in other regions when the primers are changed. Acknowledgments This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from Science and Technology Agency, by a Grant-in Aid from the Ministry of Education, Science, Sports and Culture, and by a Grant for Research on Aging and Health from the Ministry of Health and Welfare, Japan.
References Chaw, S. M., Long, H., Wang, B.S., Zharkikh, A., Li, W. H.: The phylogenetic position of Taxaceae based on 185 rRNA sequences.}. Mol. Evol. 37: 624-630,1993. Fushimi, H., Komatsu, K., lsobe, M., Namba, T.: 185 Ribosomal RNA gene sequences of three Panax species and the corresponding Ginseng drugs. Bioi. Pharm. Bull. 19: 1530-1532, 1996.
389
Kainz, P., Schmiedlechner, A., Strack, H. B.: In vitro amplification of DNA fragments> 10 kb. Anal. Biochem. 202: 46-49,1992. Masao, K.: Notulae fractae ob floram Asiae Orienralis.j. [pn. Bot. 38: 105-111, 1963. Mitchell, L. 5.: Amplification of ribosomal RNA genes for molecular evolution studies. In: PCR Protocols. (Innis, M. A., Gelfand, O. H., Sninsky, J.J. eds., Academic Press, Inc., San Diego), 307-314,1990. Murray, M. G., Thompson, W. E: Rapid isolation of high molecular weight plant DNA. Nuc/. Acids Res. 8: 4321-4325, 1980. *The specimen number of the Museum of Materia Medica, Analytical Research Center for Ethnomedicines, Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University (TMPW). The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession number 083027 for C. officina/e, 083028 for L. chuanxiong, 083275 for P. ginseng, 084100 for P. japonicus, 085172 for P. quinquefolius, 084297 for A. acutiloba var. sugiyamae.
Address K. Komatsu, Research Institute for Wakan- Yaku (Traditional Sino-Japanese Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan