Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L.
Abstract
1. Introduction
2. Results
2.1. MALDI-TOF MS Analysis Differentiated P. indica from P. zeylanica and P. auriculata
2.2. Sensitivity of MassArray Technique for the Identification of P. indica, P. zeylanica, and P. auriculata
2.3. Identification of P. indica in Plumbago-Mixed Samples the MassARRAY Analysis Successfully Identified Plumbago indica in the Mixed Plumbago Sample
2.4. Identification of Jettamoon Pleung Daeng Crude Drugs and the Crude Drug Composed in the Traditional Thai Medicinal Formulations
3. Discussion
4. Materials and Methods
4.1. Plant Materials, Commercial Crude Drugs, and Traditional Formulations
4.2. Primer Sets and Design
4.3. DNA Extraction, Amplification, and Nucleotide Sequencing
4.4. The iPLEX Assay on the MassARRAY System for the P. indica Identification
4.5. Sensitivity of the iPLEX Extension Assay for the Differentiation of P. indica from Its Related Species
4.6. Identification of P. indica in Plumbago-Mixed Samples
4.7. Authentication of the Commercial P. indica Crude Drugs and the Polyherbal Products
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BLAST | Basic Local Alignment Search Tool |
Da | Dalton |
ddNTPs | Dideoxynucleotides |
DEL | Deletion |
FDA | Food and Drug Administration |
GC-MS | Gas Chromatography–Mass Spectrometry |
HPLC | High-Performance Liquid Chromatography |
HPTLC | High-Performance Thin-Layer Chromatography |
INDEL | Insertion/Deletion |
ITS | Internal Transcribed Spacer |
LC-MS/MS | Liquid Chromatography–Tandem Mass Spectrometry |
LFA | Lateral-Flow Immunochromatographic Assay |
LOD | Limit of Detection |
LOH | Loss of Heterozygosity |
MALDI-TOF MS | Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry |
NGS | Next-Generation Sequencing |
PCR | Polymerase Chain Reaction |
SAP | Shrimp Alkaline Phosphatase |
SBE | Single-Base Extension |
SNP | Single Nucleotide Polymorphism |
UEP | Unextended Primer |
WHO | World Health Organization |
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Primers | Target SNP Position | Primer Sequences (UEP) | Mass (Da) | Expected Masses of the Single-Base Extension Product (Da) | ||||
---|---|---|---|---|---|---|---|---|
C | A | G | T | DEL | ||||
P1#1 | 18 | AAGGATCATTGTCGAAACCTC | 6414.2 | 6661.4 | 6685.4 | 6701.4 | 6741.3 | n.d. |
P1#2 | 112 | TTGTTCAAGCCTGGG | 4599.0 | n.d. | n.d. | 4846.2 | 4870.2 | n.d. |
P2#1 | 577 | CCGCGAAGCGTCGTGCC | 5172.4 | n.d. | n.d. | 5459.6 | 5499.5 | n.d. |
P2#2 | 623 | CCTGGGGTCGCATGG | 4625.0 | 4912.2 | n.d. | n.d. | n.d. | 4952.1 |
P2#3 | 652 | ATATGCTTAAACTCAGCGG | 5811.8 | 6059.0 | 6083.0 | 6741.3 | 6138.9 | n.d. |
Species | Target SNP Position | Expected Mass of the Single-Base Extension Products | Mass Profile of the Single-Base Extension Products from Authentic Plants | ||||||
---|---|---|---|---|---|---|---|---|---|
C | G | T | INDEL | C | G | T | INDEL | ||
P. indica | P1#1 | - | - | 6741.3 | - | - | - | 6741.3 | - |
P1#2 | - | 4846.2 | - | - | - | 4846.2 | - | - | |
P2#1 | - | 5459.6 | - | - | - | 5459.6 | - | - | |
P2#2 | - | - | - | 4952.1 | - | - | - | 4952.1 | |
P2#3 | 6059.0 | - | - | - | 6059.0 | - | - | - | |
P. zeylanica | P1#1 | - | - | 6741.3 | - | - | - | 6741.3 | - |
P1#2 | - | 4846.2 | - | - | - | 4846.2 | 4870.2 * | - | |
P2#1 | - | 5459.6 | - | - | - | 5459.6 | - | - | |
P2#2 | 4912.2 | - | - | - | 4912.2 | - | - | - | |
P2#3 | 6059.0 | - | - | - | 6059.0 | - | - | - | |
P. auriculata | P1#1 | 6661.4 | - | - | - | 6661.4 | - | - | - |
P1#2 | - | - | 4870.2 | - | - | - | 4870.2 | - | |
P2#1 | - | - | 5499.5 | - | - | - | 5499.5 | - | |
P2#2 | 4912.2 | - | - | - | 4912.2 | - | - | - | |
P2#3 | 6059.0 | - | - | - | 6059.0 | - | - | - |
Samples | DNA Concentration (ng/μL) | Extension Primers | ||||
---|---|---|---|---|---|---|
P1#1 | P1#2 | P2#1 | P2#2 | P2#3 | ||
P. indica | 10 | T | G | G | DEL | C |
1 | T | G | G | DEL | C | |
0.1 | T | G | G | DEL | C | |
0.01 | T | G | G | DEL | C | |
0.001 | T | G | G | C/DEL | C | |
0.0001 | T | G/T | G | C | C | |
0.00001 | T | G | G | C | C | |
0.000001 | T | G/T | G | C | C | |
0 | - | - | - | - | - | |
P. zeylanica | 10 | T | G | G | C | C |
1 | T | G | G | C | C | |
0.1 | T | G | G | C | C | |
0.01 | T | G | G | C | C | |
0.001 | T | T | G | C | C | |
0.0001 | T | G/T | G | C | C | |
0.00001 | T | G | G | DEL | C | |
0.000001 | T | G/T | G | C | C | |
0 | - | - | - | - | - | |
P. auriculata | 10 | C | T | T | C | C |
1 | C | T | T | C | C | |
0.1 | C | T | T | C | C | |
0.01 | C | T | T | C | C | |
0.001 | C/T | G/T | G/T | C | C | |
0.0001 | T | G | G | C | C | |
0.00001 | T | G/T | G | C | C | |
0.000001 | T | G/T | G | C | C | |
0 | - | - | - | - | - |
Samples | Extension Primers | Result | ||||
---|---|---|---|---|---|---|
P1#1 | P1#2 | P2#1 | P2#2 | P2#3 | ||
PI | T | G | G | DEL | C | P. indica |
PZ | T | T | G | C | C | P. zeylanica |
PA | C | T | T | C | C | P. auriculata |
C-1 | T | G | G | DEL | C | P. indica |
C-2 | T | G | G | DEL | C | P. indica |
C-3 | T | G | G | DEL | C | P. indica |
C-4 | T | G | G | DEL | C | P. indica |
C-5 | T | G | G | DEL | C | P. indica |
C-6 | T | G | G | DEL | C | P. indica |
C-7 | T | G | G | DEL | C | P. indica |
C-8 | T | G | G | DEL | C | P. indica |
C-9 | T | G | G | DEL | C | P. indica |
R-1 | T | G | G | DEL | C | P. indica |
R-2 | T | T | G | C | C | P. zeylanica |
R-3 | T | G | G | DEL | C | P. indica |
R-4 | T | G | G | DEL | C | P. indica |
R-5 | T | G | G | DEL | C | P. indica |
R-6 | T | T | G | C | C | P. zeylanica |
Codes | Sample Details | Locality |
---|---|---|
Authentic Plumbago species | ||
PI | P. indica L. | Maerim, Chiang Mai |
PZ | P. zeylanica L. | Mueang, Chiang Mai |
PA | P. auriculata Lam. | Mueang, Chiang Mai |
Crude drugs | ||
C-1 | Jettamoon Pleung Daeng | Mueang, Phatthalung |
C-2 | Jettamoon Pleung Daeng | Samphanthawong, Bangkok |
C-3 | Jettamoon Pleung Daeng | Samphanthawong, Bangkok |
C-4 | Jettamoon Pleung Daeng | Samphanthawong, Bangkok |
C-5 | Jettamoon Pleung Daeng | Mueang, Nakhon Pathom |
C-6 | Jettamoon Pleung Daeng | Hat Yai, Songkhla |
C-7 | Jettamoon Pleung Daeng | Mueang, Nakhon Pathom |
C-8 | Jettamoon Pleung Daeng | Samphanthawong, Bangkok |
C-9 | Jettamoon Pleung Daeng | Mueang, Chiang Mai |
Thai traditional formulations | ||
R-1 | Ya Benchakun | Faculty of Pharmacy, Chiang Mai University (In-house preparation) |
R-2 | Ya Benchakun | Company 1 |
R-3 | Ya Fai Pralaikan | Faculty of Pharmacy, Chiang Mai University (In-house preparation) |
R-4 | Ya Fai Pralaikan | Company 2 |
R-5 | Ya Fai Ha Kong | Faculty of Pharmacy, Chiang Mai University (In-house preparation) |
R-6 | Ya Fai Ha Kong | Company 3 |
Primer Set | Primer Name | Target(Region/SNP) | Direction | Primer Sequences (5′→ 3′) | Original Design |
---|---|---|---|---|---|
DNA barcode | ITS5A | ITS region | Forward | CCT TAT CAT TTA GAG GAA GGA G | [48] |
ITS4 | ITS region | Reverse | TCC TCC GCT TAT TGA TAT GC | [49] | |
Specific primer | P1F | ITS1 region | Forward | ACG TTG GAT GAA CCT GCG GAA GGA TCA TTG | This study |
P1R | ITS1 region | Reverse | ACG TTG GAT GGC GCC GTG TTT TTG TTC AAG | This study | |
P2F | ITS2 region | Forward | ACG TTG GAT GCG GTT GGC TTA AAT TCG GG | This study | |
P2R | ITS2 region | Reverse | ACG TTG GAT GCT TAT TGA TAT GCT TAA ACT | This study | |
iPLEX extension primer | Ext_P1#1 | SNP18 | Forward | AAG GAT CAT TGT CGA AAC CTC | This study |
Ext_P1#2 | SNP112 | Reverse | TTG TTC AAG CCT GGG | This study | |
Ext_P2#1 | SNP577 | Forward | CCG CGA AGC GTC GTG CC | This study | |
Ext_P2#2 | SNP623 | Reverse | CCT GGG GTC GCA TGG | This study | |
Ext_P2#3 | SNP652 | Reverse | ATA TGC TTA AAC TCA GCG G | This study |
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Thongkhao, K.; Intharuksa, A.; Phrutivorapongkul, A. Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L. Int. J. Mol. Sci. 2025, 26, 7168. https://doi.org/10.3390/ijms26157168
Thongkhao K, Intharuksa A, Phrutivorapongkul A. Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L. International Journal of Molecular Sciences. 2025; 26(15):7168. https://doi.org/10.3390/ijms26157168
Chicago/Turabian StyleThongkhao, Kannika, Aekkhaluck Intharuksa, and Ampai Phrutivorapongkul. 2025. "Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L." International Journal of Molecular Sciences 26, no. 15: 7168. https://doi.org/10.3390/ijms26157168
APA StyleThongkhao, K., Intharuksa, A., & Phrutivorapongkul, A. (2025). Unveiling Adulteration in Herbal Markets: MassARRAY iPLEX Assay for Accurate Identification of Plumbago indica L. International Journal of Molecular Sciences, 26(15), 7168. https://doi.org/10.3390/ijms26157168