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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

DNA degradation with time of boiling. Pulverized P. ginseng sample was boiled for (A) 30 min, (B) 60 min and (C) 120 min. The extracted DNA was amplified by PCR to produce 88 bp (lane 1) using primer pair 4, 121 bp (lane 3) using primer pair 7, 191 bp (lane 5) using primer pair 9, 249 bp (lane 7) using primer pair 5 and 311 bp (lane 9) using primer pair 6. Lanes 2, 4, 6, 8, 10 are negative controls of its previous lanes without decoction DNA. Lane M represents the DNA size ladder.
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Fig2: DNA degradation with time of boiling. Pulverized P. ginseng sample was boiled for (A) 30 min, (B) 60 min and (C) 120 min. The extracted DNA was amplified by PCR to produce 88 bp (lane 1) using primer pair 4, 121 bp (lane 3) using primer pair 7, 191 bp (lane 5) using primer pair 9, 249 bp (lane 7) using primer pair 5 and 311 bp (lane 9) using primer pair 6. Lanes 2, 4, 6, 8, 10 are negative controls of its previous lanes without decoction DNA. Lane M represents the DNA size ladder.

Mentions: We investigated the relationship between boiling time and the size of amplifiable DNA. DNA fragments with sizes (in bp) of 88, 121, 191, 249 and 311 were amplified. For pulverized samples boiled for 30 and 60 min, DNA fragments of size 88-311 bp were amplified (Figure 2A and B). However, only short PCR products with sizes ranging from 88-121 bp were amplified for samples boiled for 120 min (Figure 2C).Figure 2


Identification of constituent herbs in ginseng decoctions by DNA markers.

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

DNA degradation with time of boiling. Pulverized P. ginseng sample was boiled for (A) 30 min, (B) 60 min and (C) 120 min. The extracted DNA was amplified by PCR to produce 88 bp (lane 1) using primer pair 4, 121 bp (lane 3) using primer pair 7, 191 bp (lane 5) using primer pair 9, 249 bp (lane 7) using primer pair 5 and 311 bp (lane 9) using primer pair 6. Lanes 2, 4, 6, 8, 10 are negative controls of its previous lanes without decoction 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

Fig2: DNA degradation with time of boiling. Pulverized P. ginseng sample was boiled for (A) 30 min, (B) 60 min and (C) 120 min. The extracted DNA was amplified by PCR to produce 88 bp (lane 1) using primer pair 4, 121 bp (lane 3) using primer pair 7, 191 bp (lane 5) using primer pair 9, 249 bp (lane 7) using primer pair 5 and 311 bp (lane 9) using primer pair 6. Lanes 2, 4, 6, 8, 10 are negative controls of its previous lanes without decoction DNA. Lane M represents the DNA size ladder.
Mentions: We investigated the relationship between boiling time and the size of amplifiable DNA. DNA fragments with sizes (in bp) of 88, 121, 191, 249 and 311 were amplified. For pulverized samples boiled for 30 and 60 min, DNA fragments of size 88-311 bp were amplified (Figure 2A and B). However, only short PCR products with sizes ranging from 88-121 bp were amplified for samples boiled for 120 min (Figure 2C).Figure 2

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