DNA analysis offers a lot of potential for botanical testing, and is incredibly reliable, but only when performed on appropriate material: A DNA test cannot be universally applied to botanical extracts because DNA must still be present after the manufacturing steps, which is not always the case. The technology dominated trade media headlines in 2015 and early 2016 after NY AG Eric Schneiderman used it to build cases against a number of retailers of herbal supplements.
“We were applying this technology to potentially problematic species years before Schneiderman came along,” Cosimo Palumbo, Indena’s Marketing Director, told NutraIngredients-USA.
Indeed, the company, working with Dr Pietro Piffanelli from the Parco Tecnologico Padano (PTP) in Lodi, northern Italy, presented a poster at the International Symposium of AOAC Europe Section in Nuremberg in 2011 which applied DNA fingerprint analysis to eight species of Echinacea found in North America*.
One of the criticisms of DNA technology in the past has been around the reference standards – or lack thereof.
While some may point to GenBank – the NIH’s database that collects all publicly available genetic sequences – as a reference library, many experts note that it is not an acceptable standard (for example, samples may be misidentified or data may be missing).
Dr Piffanelli told attendees at the recent Vitafoods education sessions in Geneva that Genbank contains potential mistakes in the attribution of DNA sequences to specific plant species. “DNA barcoding is robust and reproducible, and it makes a decisive contribution to the certification of the origin of raw materials and finished products [if DNA is still present after manufacturing],” said Dr Piffanelli. “But it is of paramount importance to have certified pure samples to derive the reference DNA sequences.”
“Herbarium vouchers are ideal and with 95 years of experience we have a unique herbarium,” noted Indena’s Palumbo.
Next Gen Sequencing
There are different types of DNA testing methods: One technique is called Sanger Sequencing, but a paper published in PLOS One by scientists from the University of Guelph concluded: “Sanger sequencing should not be used for testing herbal supplements, due to its inability to resolve mixed signal from samples containing multiple species. NGS-based approaches are far more superior, enabling reliable and effective detection of DNA in complex mixtures.”
Plants and DNA
Plants have three genomes: Chloroplast DNA and mitochondrion DNA, which are inherited from one parent, usually the female; and nuclear DNA, which is inherited from both parents.
Indena’s DNA-based technologies play an important role in quality control procedures in the dietary supplement industry when embedded in a complete testing toolbox that provides a reliable authentication platform of herbal products, explained Palumbo.
“NGS technologies are based upon high-throughput decoding of all DNA present in a given extract,” he said. “NGS technologies handle millions of small fragments of DNA on the basis of an untargeted approach that generates valuable data to assess the presence of adulterants and assign all product's ingredients at the species level. Indena is working to validate proper DNA-based technologies to include these tests also on the final extracts.
“The NGS approach is the only analytical tool enabling the precise identification of the composition of multi-ingredients products. The precision and applicability of the NGS technology relies on the presence of DNA even if only in traces and on the use of four universal barcodes representing both chloroplast and nuclear genomes. The use of multiple barcodes ensures that the identification of the ingredients, fillers and adulterants is always achievable at the species level.”
NGS methods also provide semi-quantitative data of the ratios of the DNA assigned to specific species in multi-ingredient or adulterated samples, added Palumbo. The only limitation on the use of NGS analytical tools relates to the complete degradation of DNA caused by harsh treatments of botanicals manufacturing procedures such as supercritical CO2 and solvent extraction processes.
“For dietary supplement companies aiming at the highest standards of quality, integrity and safety of their products and at enhancing consumer confidence, the integration of genomics-based NGS analyses to their testing programs is to be considered a valuable asset.”
Indena is already talking with customers about this and Indena will offer an “Indena DNA Tested” logo for its partners, which guarantees that the starting plant material used for the manufacture of the extract is also DNA tested. “Finally, someone is giving a pragmatic solution to the industry,” said Palumbo.
The system can be controlled by a mobile app, called bAPP, which provides a complete and user-friendly interface that allows the user to download the test protocol required for the desired analysis, check the reaction as it is happening, and receive immediate diagnostic information on the sample being examined.
The company is now taking the testing out of a lab and into the fields. A strategic cooperation agreement with UK-based biotech Hyris is based on unique sets of reagents for specific DNA sequences (known as bKITs) and the use of a miniaturized, portable device for the analysis of nucleic acids (known as the bCUBE).
“The bCUBE is the size of a smart phone and you don’t need to be an expert to operate it,” Palumbo told us. “You can bring it onsite in the field using a sample cartridge to test the starting material as it comes out of the ground. The technology also allows for a GPS location recorded and allows for real-time recording of quality. This geolocalization also responds to the chain of custody issue and allows us to connect the dots.”
“It’s instant ID testing on the plant starting material,” he added. “Results are obtained in approximately one hour, versus the many hours required by the traditional lab equipment. We would still redo analysis on every incoming biomass, but this shortens the timeline and it’s complimentary to other tests. And results are available anywhere in real time thanks to a data cloud.”
* “Different analytical approaches of authentication of Medicinal Plants: a case study the identification of Echinacea spp. Roots”. 2011. C. Bonardi, V. Gualdi, R. Iguera, N. Fuzzati, I. Losini, P. Piffanelli. International Symposium of AOAC Europe Section in Nuremberg.