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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

PCR amplification of extracted DNA in the prescriptions. (A) Two herbs decoction: Decoction of Ginseng and Prepared Aconite. The prescription was boiled for 30 min (lanes 1 and 5) and 60 min (lanes 2 and 6). Extracted DNA was amplified by primer pair 7 (lanes 1–2) for P. ginseng and primer pair 1 (lanes 5–6) for A. carmichaeli. Lanes 3 and 7 are positive controls with DNA from the concerned herbal sample and lanes 4 and 8 are negative controls without DNA. (B-E) Four herbs decoction: Ginseng and Ginger Combination. The prescription was boiled for 30 min (lane 1), 60 min (lane 2), 90 min (lane 3), 120 min (lane 4), 150 min (lane 5) and 180 min (lane 6). Primers specific for (B)A. macrocephala (Primer pair 2), (C)G. uralensis (Primer pair 3), (D)P. ginseng (Primer pair 7) and (E)Z. officinale (Primer pair 10) were used to amplify the decoction DNA. Lane 7 is positive control with DNA from the concerned herbal sample and lane 8 is negative control without DNA. Lane M represents the DNA size ladder.
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Fig4: PCR amplification of extracted DNA in the prescriptions. (A) Two herbs decoction: Decoction of Ginseng and Prepared Aconite. The prescription was boiled for 30 min (lanes 1 and 5) and 60 min (lanes 2 and 6). Extracted DNA was amplified by primer pair 7 (lanes 1–2) for P. ginseng and primer pair 1 (lanes 5–6) for A. carmichaeli. Lanes 3 and 7 are positive controls with DNA from the concerned herbal sample and lanes 4 and 8 are negative controls without DNA. (B-E) Four herbs decoction: Ginseng and Ginger Combination. The prescription was boiled for 30 min (lane 1), 60 min (lane 2), 90 min (lane 3), 120 min (lane 4), 150 min (lane 5) and 180 min (lane 6). Primers specific for (B)A. macrocephala (Primer pair 2), (C)G. uralensis (Primer pair 3), (D)P. ginseng (Primer pair 7) and (E)Z. officinale (Primer pair 10) were used to amplify the decoction DNA. Lane 7 is positive control with DNA from the concerned herbal sample and lane 8 is negative control without DNA. Lane M represents the DNA size ladder.

Mentions: In a decoction involved two herbs (ginseng and prepared aconite), A. carmichaeli (148 bp) and P. ginseng (121 bp) could be amplified in extracted DNA with samples boiled for 30 and 60 min (Figure 4A). In another decoction involved four herbs (ginseng and ginger combination), A. macrocephala (108 bp), G. uralensis (106 bp), P. ginseng (121 bp) and Z. officinale (106 bp) were amplified in extracted DNA with samples boiled for 180 min (Figure 4B-E). We could also amplify the P. ginseng component in Korean Ginseng Chicken Stew after DNA was extracted (Figure 5).Figure 4


Identification of constituent herbs in ginseng decoctions by DNA markers.

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

PCR amplification of extracted DNA in the prescriptions. (A) Two herbs decoction: Decoction of Ginseng and Prepared Aconite. The prescription was boiled for 30 min (lanes 1 and 5) and 60 min (lanes 2 and 6). Extracted DNA was amplified by primer pair 7 (lanes 1–2) for P. ginseng and primer pair 1 (lanes 5–6) for A. carmichaeli. Lanes 3 and 7 are positive controls with DNA from the concerned herbal sample and lanes 4 and 8 are negative controls without DNA. (B-E) Four herbs decoction: Ginseng and Ginger Combination. The prescription was boiled for 30 min (lane 1), 60 min (lane 2), 90 min (lane 3), 120 min (lane 4), 150 min (lane 5) and 180 min (lane 6). Primers specific for (B)A. macrocephala (Primer pair 2), (C)G. uralensis (Primer pair 3), (D)P. ginseng (Primer pair 7) and (E)Z. officinale (Primer pair 10) were used to amplify the decoction DNA. Lane 7 is positive control with DNA from the concerned herbal sample and lane 8 is 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

Fig4: PCR amplification of extracted DNA in the prescriptions. (A) Two herbs decoction: Decoction of Ginseng and Prepared Aconite. The prescription was boiled for 30 min (lanes 1 and 5) and 60 min (lanes 2 and 6). Extracted DNA was amplified by primer pair 7 (lanes 1–2) for P. ginseng and primer pair 1 (lanes 5–6) for A. carmichaeli. Lanes 3 and 7 are positive controls with DNA from the concerned herbal sample and lanes 4 and 8 are negative controls without DNA. (B-E) Four herbs decoction: Ginseng and Ginger Combination. The prescription was boiled for 30 min (lane 1), 60 min (lane 2), 90 min (lane 3), 120 min (lane 4), 150 min (lane 5) and 180 min (lane 6). Primers specific for (B)A. macrocephala (Primer pair 2), (C)G. uralensis (Primer pair 3), (D)P. ginseng (Primer pair 7) and (E)Z. officinale (Primer pair 10) were used to amplify the decoction DNA. Lane 7 is positive control with DNA from the concerned herbal sample and lane 8 is negative control without DNA. Lane M represents the DNA size ladder.
Mentions: In a decoction involved two herbs (ginseng and prepared aconite), A. carmichaeli (148 bp) and P. ginseng (121 bp) could be amplified in extracted DNA with samples boiled for 30 and 60 min (Figure 4A). In another decoction involved four herbs (ginseng and ginger combination), A. macrocephala (108 bp), G. uralensis (106 bp), P. ginseng (121 bp) and Z. officinale (106 bp) were amplified in extracted DNA with samples boiled for 180 min (Figure 4B-E). We could also amplify the P. ginseng component in Korean Ginseng Chicken Stew after DNA was extracted (Figure 5).Figure 4

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