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Identification of constituent herbs in ginseng decoctions by DNA markers.

Lo YT, Li M, Shaw PC - Chin Med (2015)

Bottom Line: All six herbal species tested in this study could be identified in decoctions.We had four main observations: (1) sample pulverization before boiling improved PCR detection; (2) prolonged boiling increased the DNA concentration but decreased the intactness of DNA fragments; (3) ginseng could be differentiated from American ginseng by multiplex PCR; (4) identification of individual herbs in multi-herb decoctions with prolonged boiling time of 180 min could be achieved.DNA could be amplified from extensively boiled ginseng decoctions, multi-herb decoctions and commercial soup although DNA degradation was critical to successful PCR.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, The Chinese University of Hong Kong, S.A.R N.T, Shatin, Hong Kong China.

ABSTRACT

Background: DNA in herbal decoctions is usually fragmented by extensive boiling and is usually regarded as unsuitable for molecular authentication. This study aims to evaluate the feasibility for molecular authentication methods by multiplex polymerase chain reaction (PCR) and DNA sequencing for decoctions.

Methods: The DNA extraction procedure, sample pulverization and boiling time were examined in (1) single herb decoction with Panax ginseng ("ginseng") or P. quinquefolius ("American ginseng"), (2) decoctions of two classical ginseng prescriptions of five herbal materials (Aconitum carmichaeli, Atractylodes macrocephala, P. ginseng, Glycyrrhiza uralensis and Zingiber officinale) and (3) a commercial ready-to-serve ginseng soup. Primers were designed from 26S-18S, ITS2 and trnH-psbA region, with DNA sequences obtained from GenBank. Multiplex PCR was also employed in ginseng or American ginseng decoctions to differentiate between these two herbs.

Results: All six herbal species tested in this study could be identified in decoctions. We had four main observations: (1) sample pulverization before boiling improved PCR detection; (2) prolonged boiling increased the DNA concentration but decreased the intactness of DNA fragments; (3) ginseng could be differentiated from American ginseng by multiplex PCR; (4) identification of individual herbs in multi-herb decoctions with prolonged boiling time of 180 min could be achieved.

Conclusions: DNA could be amplified from extensively boiled ginseng decoctions, multi-herb decoctions and commercial soup although DNA degradation was critical to successful PCR.

No MeSH data available.


Related in: MedlinePlus

Multiplex PCR in differentiation of ginseng and American ginseng. (A) Nucleotide sequences of the forward P. ginseng-specific primer (Pgi_SPF1) with comparison to the aligned reference sequences of P. ginseng (Genbank accession number: EF031239, EF031240) and P. quinquefolius (Genbank accession number: EF031236, EF031241). Open box indicates the primer sequence, dots and closed boxes indicate nucleotides identical and different from the primer sequence, respectively. (B and C) Amplification of the extracted DNA from two vouchers of pulverized (B)P. ginseng (ICM 2005–2750, ICM 2005–2779) and (C)P. quinquefolius (ICM 1713, ICM 901276) samples by multiplex PCR using primer pair 8. Samples in herbal decoction were boiled for 30 min (lanes 1–2), 60 min (lanes 3–4) and 120 min (lanes 5–6), respectively. Lane 7 is the positive control with DNA from the concerned herbal sample and lane 8 is the negative control without DNA. Lane M represents the DNA size ladder.
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Fig3: Multiplex PCR in differentiation of ginseng and American ginseng. (A) Nucleotide sequences of the forward P. ginseng-specific primer (Pgi_SPF1) with comparison to the aligned reference sequences of P. ginseng (Genbank accession number: EF031239, EF031240) and P. quinquefolius (Genbank accession number: EF031236, EF031241). Open box indicates the primer sequence, dots and closed boxes indicate nucleotides identical and different from the primer sequence, respectively. (B and C) Amplification of the extracted DNA from two vouchers of pulverized (B)P. ginseng (ICM 2005–2750, ICM 2005–2779) and (C)P. quinquefolius (ICM 1713, ICM 901276) samples by multiplex PCR using primer pair 8. Samples in herbal decoction were boiled for 30 min (lanes 1–2), 60 min (lanes 3–4) and 120 min (lanes 5–6), respectively. Lane 7 is the positive control with DNA from the concerned herbal sample and lane 8 is the negative control without DNA. Lane M represents the DNA size ladder.

Mentions: Multiplex PCR was developed to differentiate P. ginseng from P. quinquefolius. PCR master mixes involved one pair of conserved primers for P. ginseng and P. quinquefolius (Panax_F1 and Panax_R1), and a forward P. ginseng-specific primer (Pgi_SPF1) region for pairing with the same reverse conserved primer (Panax_R1) (Additional file 3). Three nucleotides at the 3′ end of the 21 nucleotide forward P. ginseng-specific primer were different from those of the DNA sequence of P. quinquefolius (Figure 3A). As a result, the 191 bp amplicon was found in P. ginseng and P. quinquefolius decoction and the 121 bp amplicon was found only in the P. ginseng decoction (Figure 3B and C). However, the 191 bp amplicon was not found in the decoction boiled for 120 min (Figure 3B and C) due to DNA degradation (Figure 2C).Figure 3


Identification of constituent herbs in ginseng decoctions by DNA markers.

Lo YT, Li M, Shaw PC - Chin Med (2015)

Multiplex PCR in differentiation of ginseng and American ginseng. (A) Nucleotide sequences of the forward P. ginseng-specific primer (Pgi_SPF1) with comparison to the aligned reference sequences of P. ginseng (Genbank accession number: EF031239, EF031240) and P. quinquefolius (Genbank accession number: EF031236, EF031241). Open box indicates the primer sequence, dots and closed boxes indicate nucleotides identical and different from the primer sequence, respectively. (B and C) Amplification of the extracted DNA from two vouchers of pulverized (B)P. ginseng (ICM 2005–2750, ICM 2005–2779) and (C)P. quinquefolius (ICM 1713, ICM 901276) samples by multiplex PCR using primer pair 8. Samples in herbal decoction were boiled for 30 min (lanes 1–2), 60 min (lanes 3–4) and 120 min (lanes 5–6), respectively. Lane 7 is the positive control with DNA from the concerned herbal sample and lane 8 is the negative control without DNA. Lane M represents the DNA size ladder.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318153&req=5

Fig3: Multiplex PCR in differentiation of ginseng and American ginseng. (A) Nucleotide sequences of the forward P. ginseng-specific primer (Pgi_SPF1) with comparison to the aligned reference sequences of P. ginseng (Genbank accession number: EF031239, EF031240) and P. quinquefolius (Genbank accession number: EF031236, EF031241). Open box indicates the primer sequence, dots and closed boxes indicate nucleotides identical and different from the primer sequence, respectively. (B and C) Amplification of the extracted DNA from two vouchers of pulverized (B)P. ginseng (ICM 2005–2750, ICM 2005–2779) and (C)P. quinquefolius (ICM 1713, ICM 901276) samples by multiplex PCR using primer pair 8. Samples in herbal decoction were boiled for 30 min (lanes 1–2), 60 min (lanes 3–4) and 120 min (lanes 5–6), respectively. Lane 7 is the positive control with DNA from the concerned herbal sample and lane 8 is the negative control without DNA. Lane M represents the DNA size ladder.
Mentions: Multiplex PCR was developed to differentiate P. ginseng from P. quinquefolius. PCR master mixes involved one pair of conserved primers for P. ginseng and P. quinquefolius (Panax_F1 and Panax_R1), and a forward P. ginseng-specific primer (Pgi_SPF1) region for pairing with the same reverse conserved primer (Panax_R1) (Additional file 3). Three nucleotides at the 3′ end of the 21 nucleotide forward P. ginseng-specific primer were different from those of the DNA sequence of P. quinquefolius (Figure 3A). As a result, the 191 bp amplicon was found in P. ginseng and P. quinquefolius decoction and the 121 bp amplicon was found only in the P. ginseng decoction (Figure 3B and C). However, the 191 bp amplicon was not found in the decoction boiled for 120 min (Figure 3B and C) due to DNA degradation (Figure 2C).Figure 3

Bottom Line: All six herbal species tested in this study could be identified in decoctions.We had four main observations: (1) sample pulverization before boiling improved PCR detection; (2) prolonged boiling increased the DNA concentration but decreased the intactness of DNA fragments; (3) ginseng could be differentiated from American ginseng by multiplex PCR; (4) identification of individual herbs in multi-herb decoctions with prolonged boiling time of 180 min could be achieved.DNA could be amplified from extensively boiled ginseng decoctions, multi-herb decoctions and commercial soup although DNA degradation was critical to successful PCR.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, The Chinese University of Hong Kong, S.A.R N.T, Shatin, Hong Kong China.

ABSTRACT

Background: DNA in herbal decoctions is usually fragmented by extensive boiling and is usually regarded as unsuitable for molecular authentication. This study aims to evaluate the feasibility for molecular authentication methods by multiplex polymerase chain reaction (PCR) and DNA sequencing for decoctions.

Methods: The DNA extraction procedure, sample pulverization and boiling time were examined in (1) single herb decoction with Panax ginseng ("ginseng") or P. quinquefolius ("American ginseng"), (2) decoctions of two classical ginseng prescriptions of five herbal materials (Aconitum carmichaeli, Atractylodes macrocephala, P. ginseng, Glycyrrhiza uralensis and Zingiber officinale) and (3) a commercial ready-to-serve ginseng soup. Primers were designed from 26S-18S, ITS2 and trnH-psbA region, with DNA sequences obtained from GenBank. Multiplex PCR was also employed in ginseng or American ginseng decoctions to differentiate between these two herbs.

Results: All six herbal species tested in this study could be identified in decoctions. We had four main observations: (1) sample pulverization before boiling improved PCR detection; (2) prolonged boiling increased the DNA concentration but decreased the intactness of DNA fragments; (3) ginseng could be differentiated from American ginseng by multiplex PCR; (4) identification of individual herbs in multi-herb decoctions with prolonged boiling time of 180 min could be achieved.

Conclusions: DNA could be amplified from extensively boiled ginseng decoctions, multi-herb decoctions and commercial soup although DNA degradation was critical to successful PCR.

No MeSH data available.


Related in: MedlinePlus