Assessment of the Identity of Raw Material Samples Sold in Indian Markets as Asoka

 

Reviewed: Santhosh Kumar JU, Gogna N, Newmaster SG, et al. DNA barcoding and NMR spectroscopy-based assessment of species adulteration in the raw herbal trade of Saraca asoca (Roxb.) Willd, an important medicinal plant. Int J Legal Med. 2016;130(6):1457-1470.

 

Keywords: Saraca asoca, Fabaceae, asoka, DNA barcoding, proton nuclear magnetic resonance, ¹H NMR

 

This publication is a continuation of the collaboration among the University of Guelph and a number of research institutions in India^1-3 to assess the authenticity of medicinal herbs sold at local markets in India. In the study, commercial samples of asoka tree (Saraca asoca, Fabaceae) bark, which is used in traditional Indian medicine systems to treat menstrual disorders, diarrhea, pain, and as a diuretic and cardiac tonic, were evaluated using DNA barcoding and proton nuclear magnetic resonance (¹H NMR).

 

Ten authentic samples of asoka tree bark (together with leaves and flower, if available), verified independently by two taxonomists, were obtained from various regions in India for use as reference materials. Crude commercial products (n=25) were purchased at local herbal markets in the Indian states of Bihar, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Manipur, Orissa, and Tamil Nadu, using the common names for asoka tree listed in The Ayurvedic Pharmacopoeia of India and the Environmental Information System (ENVIS) database.⁴ Two loci, psbA-trnH and rbcL, were chosen for DNA barcoding. For comparison of the chemical fingerprints, ¹H NMR spectra of the reference and commercial samples were evaluated by multivariate statistics using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA).

 

Good-quality DNA was obtained from 17 (68%) and 11 (44%) of the commercial samples using the rbcL and psbA-trnH regions, respectively. Three samples matched the rbcL sequence, with only one matching the composition of the psbA-trnH region of the reference materials. Based on the DNA barcoding results, adulterating species belonged to the Fabaceae, Combretaceae, Caricaceae, Moringaceae, Meliaceae, Rhamnaceae, and Putranjivaceae families. Results from ¹H NMR also determined three commercial samples (12%) to be authentic; however, only one of these three samples matched the DNA barcode data of authentic asoka tree bark, while the other two did not match the asoka barcodes. This suggests that either the genetic or the chemical analysis provided erroneous results.

 

Comment: The use of a DNA barcoding approach with an orthogonal technique, ¹H NMR in this case, is an improvement from the experimental setup used in previous papers published by this research group from the University of Guelph.^1-3,5 Nevertheless, the sequencing success (44-68%) of these materials is disappointing, since DNA barcoding is promoted for the authentication, in particular, of crude raw plant materials. Plant part (bark material tends to be more challenging for DNA barcoding than leaves or flowers), drying and other processing methods, and interference from secondary metabolites may be some of the contributing factors. In addition, the discrepancies in results between the DNA barcoding and the ¹H NMR method raise the question of just how accurate these methods are. Based on the results, most of the tested commercial materials were not asoka tree bark. It is not clear what role, if any, the use of the common name to purchase the crude material may have been part of the issue, since “asoka” is a common name used for multiple plant species, including Saraca indica, Shorea robusta (Dipterocarpaceae), and Polyalthia longifolia (Annonaceae).⁴

 

The authors went to greater lengths to identify some of the metabolites in the asoka tree bark, using bi-dimensional NMR experiments such as ¹H-¹³C heteronuclear single quantum coherence (HSQC) or heteronuclear multiple quantum coherence (HMQC). Based on the data, they concluded that asoka tree bark contained rutin, hesperidin, naringenin, and caffeic acid. The presence of hesperidin and naringenin is somewhat surprising, since neither compound has been reported to occur in the bark, which is known mainly for its content of catechins, proanthocyanidins, lignans, and flavones. Since the HSQC cross-peak attributions for hesperidin are not in agreement with NMR data from the published literature,⁶ these findings should be considered questionable and in need of confirmation.

 

References

1.   Seethapathy GS, Ganesh D, Santhosh Kumar JU, et al. Assessing product adulteration in natural health products for laxative yielding plants, Cassia, Senna, and Chamaecrista, in Southern India using DNA barcoding. Int J Legal Med. 2015;129(4):693-700.

2.   Santhosh Kumar JU, Krishna V, Seethapathy GS, et al. DNA barcoding to assess species adulteration in raw drug trade of “Bala” (genus: Sida L.) herbal products in South India. Biochem Syst Ecol. 2015;61:501-509.

3.   Shanmughanandhan D, Ragupathy S, Newmaster SG, Mohanasundaram S, Sathishkumar R. Estimating herbal product authentication and adulteration in India using a vouchered, DNA-based biological reference material library. Drug Saf. 2016;39(12):1211-1227.

4.   ENVIS Centre on Medicinal Plants database. Bangalore, India: Foundation for Revitalisation of Local Health Traditions (FRLHT). Available at: http://envis.frlht.org/. Accessed December 13, 2016.

5.   Newmaster SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med. 2013;11:222. doi: 10.1186/1741-7015-11-222.

6.   [1H,13C] 2D NMR Spectrum (HMDB03265). The Human Metabolome Database (HMDB). Version 3.6. Available at: http://www.hmdb.ca/spectra/nmr_two_d/1877. Accessed December 13, 2016.