Limits...
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

PCR amplification and DNA concentration in decoction with sliced or pulverized sample at different times of boiling. (A)P. ginseng sample was sliced (lanes 1–3, 7–9) or pulverized (lanes 4–6, 10–12) and boiled for 30 min (lanes 1, 4, 7, 10), 60 min (lanes 2, 5, 8, 11) and 120 min (lanes 3, 6, 9, 12). Crude DNA (lanes 1–6) and extracted DNA with concentration adjusted to that in the original decoction (lanes 7–12) were amplified by primer pair 9. Lane 13 is the positive control with DNA from P. ginseng and lane 14 is the negative control without DNA. Lane M represents the DNA size ladder. (B) The concentration of extracted P. ginseng DNA was adjusted to make it similar to that in the original decoction and measured by Qubit 2.0 Fluorometer. Water was used as blank and the data represent mean ± standard deviation (n = 3). Difference between boiling time was analyzed by one-way analysis of variance. Difference between sliced and pulverized samples at the same boiling time was analyzed by two sample t test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: PCR amplification and DNA concentration in decoction with sliced or pulverized sample at different times of boiling. (A)P. ginseng sample was sliced (lanes 1–3, 7–9) or pulverized (lanes 4–6, 10–12) and boiled for 30 min (lanes 1, 4, 7, 10), 60 min (lanes 2, 5, 8, 11) and 120 min (lanes 3, 6, 9, 12). Crude DNA (lanes 1–6) and extracted DNA with concentration adjusted to that in the original decoction (lanes 7–12) were amplified by primer pair 9. Lane 13 is the positive control with DNA from P. ginseng and lane 14 is the negative control without DNA. Lane M represents the DNA size ladder. (B) The concentration of extracted P. ginseng DNA was adjusted to make it similar to that in the original decoction and measured by Qubit 2.0 Fluorometer. Water was used as blank and the data represent mean ± standard deviation (n = 3). Difference between boiling time was analyzed by one-way analysis of variance. Difference between sliced and pulverized samples at the same boiling time was analyzed by two sample t test (*p < 0.05).

Mentions: We investigated if a 191 bp DNA of the P. ginseng 26S-18S intergenic spacer region could be amplified from the single herb P. ginseng decoction prepared by boiling sliced or pulverized samples for 30, 60 and 120 min. With the concentration of the crude and extracted DNA adjusted to that in the original decoction, visible PCR products were found in crude and extracted DNA with samples boiled for 30 min. More PCR product was obtained in pulverized samples boiled for 60 min (Figure 1A), and there was a significant increase in DNA released into the decoction in pulverized samples for each boiling time compared with sliced samples (30 min: P = 0.0280; 60 min: P = 0.0319; 120 min: P = 0.0479) (Figure 1B). A visible PCR product was not observed upon boiling for 120 min, although it was a time at which the highest DNA concentration was measured.Figure 1


Identification of constituent herbs in ginseng decoctions by DNA markers.

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

PCR amplification and DNA concentration in decoction with sliced or pulverized sample at different times of boiling. (A)P. ginseng sample was sliced (lanes 1–3, 7–9) or pulverized (lanes 4–6, 10–12) and boiled for 30 min (lanes 1, 4, 7, 10), 60 min (lanes 2, 5, 8, 11) and 120 min (lanes 3, 6, 9, 12). Crude DNA (lanes 1–6) and extracted DNA with concentration adjusted to that in the original decoction (lanes 7–12) were amplified by primer pair 9. Lane 13 is the positive control with DNA from P. ginseng and lane 14 is the negative control without DNA. Lane M represents the DNA size ladder. (B) The concentration of extracted P. ginseng DNA was adjusted to make it similar to that in the original decoction and measured by Qubit 2.0 Fluorometer. Water was used as blank and the data represent mean ± standard deviation (n = 3). Difference between boiling time was analyzed by one-way analysis of variance. Difference between sliced and pulverized samples at the same boiling time was analyzed by two sample t test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: PCR amplification and DNA concentration in decoction with sliced or pulverized sample at different times of boiling. (A)P. ginseng sample was sliced (lanes 1–3, 7–9) or pulverized (lanes 4–6, 10–12) and boiled for 30 min (lanes 1, 4, 7, 10), 60 min (lanes 2, 5, 8, 11) and 120 min (lanes 3, 6, 9, 12). Crude DNA (lanes 1–6) and extracted DNA with concentration adjusted to that in the original decoction (lanes 7–12) were amplified by primer pair 9. Lane 13 is the positive control with DNA from P. ginseng and lane 14 is the negative control without DNA. Lane M represents the DNA size ladder. (B) The concentration of extracted P. ginseng DNA was adjusted to make it similar to that in the original decoction and measured by Qubit 2.0 Fluorometer. Water was used as blank and the data represent mean ± standard deviation (n = 3). Difference between boiling time was analyzed by one-way analysis of variance. Difference between sliced and pulverized samples at the same boiling time was analyzed by two sample t test (*p < 0.05).
Mentions: We investigated if a 191 bp DNA of the P. ginseng 26S-18S intergenic spacer region could be amplified from the single herb P. ginseng decoction prepared by boiling sliced or pulverized samples for 30, 60 and 120 min. With the concentration of the crude and extracted DNA adjusted to that in the original decoction, visible PCR products were found in crude and extracted DNA with samples boiled for 30 min. More PCR product was obtained in pulverized samples boiled for 60 min (Figure 1A), and there was a significant increase in DNA released into the decoction in pulverized samples for each boiling time compared with sliced samples (30 min: P = 0.0280; 60 min: P = 0.0319; 120 min: P = 0.0479) (Figure 1B). A visible PCR product was not observed upon boiling for 120 min, although it was a time at which the highest DNA concentration was measured.Figure 1

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