Velusamy Sundaresan

DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market

The past couple of decades have witnessed global resurgence of herbal-based health care. As a result, the trade of raw drugs has surged globally. Accurate and fast scientific identification of the plant(s) is the key to success for the herbal drug industry. The conventional approach is to engage an expert taxonomist, who uses a mix of traditional and modern techniques for precise plant identification. However, for bulk identification at industrial scale, the process is protracted and time-consuming. DNA barcoding, on the other hand, offers an alternative and feasible taxonomic tool box for rapid and robust species identification. For the success of DNA barcode, the barcode loci must have sufficient information to differentiate unambiguously between closely related plant species and discover new cryptic species. For herbal plant identification, matK, rbcL, trnH-psbA, ITS, trnL-F, 5S-rRNA and 18S-rRNA have been used as successful DNA barcodes. Emerging advances in DNA barcoding coupled with next-generation sequencing and high-resolution melting curve analysis have paved the way for successful species-level resolution recovered from finished herbal products. Further, development of multilocus strategy and its application has provided new vistas to the DNA barcode-based plant identification for herbal drug industry. For successful and acceptable identification of herbal ingredients and a holistic quality control of the drug, DNA barcoding needs to work harmoniously with other components of the systems biology approach. We suggest that for effectively resolving authentication challenges associated with the herbal market, DNA barcoding must be used in conjunction with metabolomics along with need-based transcriptomics and proteomics.

Chemical diversity in the genus Alpinia (Zingiberaceae): comparative composition of four Alpinia species grown in Northern India.

The essential‐oil compositions of leaves, flowers, and rhizomes of Alpinia galanga (L.) Willd., Alpinia calcarata Rosc., Alpinia speciosa K. Schum., and Alpinia allughas Rosc. were examined and compared by capillary GC and GC/MS. Monoterpenoids were the major oil constituents identified. 1,8‐Cineole, α‐terpineol, (E)‐methyl cinnamate, camphor, terpinen‐4‐ol, and α‐ and β‐pinenes were the major constituents commonly distributed in leaf and flower essential oils. The presence of endo‐fenchyl acetate, exo‐fenchyl acetate, and endo‐fenchol was the unique feature of rhizome essential oils of A. galanga, A. calcarata, and A. speciosa. On contrary, the rhizome oil of A. allughas was dominated by β‐pinene. Significant qualitative and quantitative variations were observed in essential‐oil compositions of different parts of Alpinia species growing in subtemperate and subtropical regions of Northern India. Cluster analysis was performed to find similarities and differences in essential‐oil compositions based on representative molecular skeletons. Monoterpenoids, viz., 1,8‐cineole, terpinen‐4‐ol, camphor, pinenes, (E)‐methyl cinnamate, and fenchyl derivatives, were used as chemotaxonomic markers.

Volatile terpenoid compositions of leaf and rhizome of Curcuma amada Roxb. from Northern India

The volatile constituents of leaves and rhizomes of Curcuma amada grown in the foothills of Uttarakhand, India, were studied by using capillary gas chromatography–flame ionization detector (GC–FID) and GC–mass spectrometry (GC–MS). The analysis revealed fifty-seven constituents comprising 92.69% and 98.56% of the leaf and rhizome oil compositions. The leaf essential oil of C. amada was mainly composed of furanosesquiterpenoids, namely epi-curzerenone (10.76%), curzerenone (9.53%), curzerene (3.95%) and furanogermenone (1.77%). Camphor (17.90%), isoborneol (7.30%), camphene (3.57%), borneol (1.87%) and camphene hydrate (1.25%) were the major constituents of 36.79% of the monoterpenoids fraction of leaf essential oil composition. Conversely, the rhizome essential oil of C. amada was mainly dominated by monoterpenoids (97.72%), with myrcene (88.84%) a single major constituent along with β-pinene (3.74%), (E)-β-ocimene (2.61%) and other minor constituents. Comparison of the volatile constituents of leaves and rhizome essential oil of C. amada revealed sharp qualitative and quantitative variations. Furanosesquiterpenoids, isoborneol and borneol were the exclusive constituents of leaf essential oil, and were not noticed in rhizome oil of C. amada. Myrcene, the single major constituent of rhizome oil, along with β-pinene and (E)-β-ocimene, seem to be responsible for the characteristic mango aroma of C. amada.

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

Abstract High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.

Composition and comparison of essential oils of Pogostemon cablin (Blanco) Benth. (Patchouli) and Pogostemon travancoricus Bedd. var. travancoricus.

Abstract Comparative study on the essential oil constituents of Pogostemon cablin (Blanco) Benth. (Patchouli) and P. travancoricus Bedd. var. travancoricus were investigated using GC and GC/MS analysis. Eleven compounds from P. cablin oil (Patchouli) and 13 from P. travancoricus var. travancoricus oil were identified. Both species shared compounds like α- and β-patchoulene, patchouli alcohol (patchoulol), β-caryophyllene, α-guaiene, seychellene and selinene, although quantitatively less in P. travancoricus var. travancoricus.

Chemical composition of root aroma of Decalepisarayalpathra (J. Joseph and V. Chandras.) Venter, an endemic and endangered ethnomedicinal plant from Western Ghats, India

Decalepisarayalpathra (J. Joseph and V. Chandras.) Venter, which belongs to the family Apocynaceae, is a perennial under shrub, endemic to southern Western Ghats, India. The highly aromatic tuberous roots of the D. arayalpathra are used as an effective remedy for peptic ulcer, cancer-like afflictions and as rejuvenating tonic by native tribes. The objective of this study was to characterise the root aroma of D. arayalpathra for possible industrial applications. Hydrodistilled volatile oil of the roots was analysed by gas chromatography-flame ionisation detector and gas chromatography–mass spectrometry. The volatile oil was characterised by the presence of higher amount of an industrially important flavour molecule, 2-hydroxy-4-methoxybenzaldehyde (96.8%) along with some other minor or trace constituents. Owing to characteristic vanillin-like flavour, the root oil of the D. arayalpathra can be explored as a potential substitute of vanillin-aroma in the flavour industry.

Phytochemical diversity of Murraya koenigii (L.) Spreng. from Western Himalaya.

Murraya koenigii (L.) Spreng. (Rutaceae), commonly known as ‘curry leaf tree’, is a popular spice and condiment of India. To explore the diversity of the essential‐oil yield and aroma profile of curry leaf, growing wild in foot and mid hills of north India, 58 populations were collected during spring season. M. koenigii populations were found to grow up to an altitude of 1487 m in north India. Comparative results showed considerable variations in the essential‐oil yield and composition. The essential‐oil yield varied from 0.14 to 0.80% in shade‐dried leaves of different populations of M. koenigii. Analysis of the essential oils by GC and GC/MS, and the subsequent classification by statistical analysis resulted in four clusters with significant variations in their terpenoid composition. Major components of the essential oils of investigated populations were α‐pinene (2; 4.5–71.5%), sabinene (3; <0.05–66.1%), (E)‐caryophyllene (11; 1.6–18.0%), β‐pinene (4; <0.05–13.6%), terpinen‐4‐ol (9; 0.0–8.4%), γ‐terpinene (8; 0.2–7.4%), limonene (7; 1.1–5.5%), α‐terpinene (6; 0.0–4.5%), (E)‐nerolidol (14; 0.0–4.1%), α‐humulene (12; 0.6–3.5%), α‐thujene (1; 0.0–2.5%), β‐elemene (10; 0.2–2.4%), β‐selinene (13; 0.2–2.3%), and myrcene (5; 0.5–2.1%). Comparison of the present results with those in earlier reports revealed new chemotypes of M. koenigii in investigated populations from Western Himalaya. The present study documents M. koenigii populations having higher amounts of sabinene (3; up to 66.1%) for the first time.

Essential oil composition of four Ocimum spp. from the Peninsular India

Abstract Compositional characteristics of the essential oils of four Ocimum spp., namely Ocimum adscendens Willd., Ocimum gratissimum L., Ocimum tenuiflorum L. and Ocimum americanum L. were examined using GC-FID, GC-MS, and hierarchical cluster analyses from peninsular India. The essential oil content varied from 1.0% to 2.0% in different Ocimum spp. Altogether eighty-four constituents, corresponding to 89.9−96.5% of the total oil compositions were identified. Major constituents of O. adscendens oil were eugenol (47.6%), (E)-caryophyllene (15.7%) and β-elemene (11.3%). O. gratissimum oil was characterized by the presence of higher amounts of eugenol (52.9%), caryophyllene oxide (7.2%) and (Z)-β-ocimene (3.5%). Major constituents of O. tenuiflorum oil were methyl eugenol (50.9%), caryophyllene oxide (7.5%) and (E)-caryophyllene (5.5%). Moreover, the oil of O. americanum was dominated by camphor (41.8%), limonene (7.1%), α-pinene (6.2%), β-selinene (5.6%) and camphene (5.0%). To the best of our knowledge, a detailed essential oil profile of O. adscendens is being reported for the first time.

Evaluation of single and multilocus DNA barcodes towards species delineation in complex tree genus Terminalia

DNA barcoding is used as a universal tool for delimiting species boundaries in taxonomically challenging groups, with different plastid and nuclear regions (rbcL, matK, ITS and psbA-trnH) being recommended as primary DNA barcodes for plants. We evaluated the feasibility of using these regions in the species-rich genus Terminalia, which exhibits various overlapping morphotypes with pantropical distribution, owing to its complex taxonomy. Terminalia bellerica and T. chebula are ingredients of the famous Ayurvedic Rasayana formulation Triphala, used for detoxification and rejuvenation. High demand for extracted phytochemicals as well as the high trade value of several species renders mandatory the need for the correct identification of traded plant material. Three different analytical methods with single and multilocus barcoding regions were tested to develop a DNA barcode reference library from 222 individuals representing 41 Terminalia species. All the single barcodes tested had a lower discriminatory power than the multilocus regions, and the combination of matK+ITS had the highest resolution rate (94.44%). The average intra-specific variations (0.0188±0.0019) were less than the distance to the nearest neighbour (0.106±0.009) with matK and ITS. Distance-based Neighbour Joining analysis outperformed the character-based Maximum Parsimony method in the identification of traded species such as T. arjuna, T. chebula and T. tomentosa, which are prone to adulteration. rbcL was shown to be a highly conservative region with only 3.45% variability between all of the sequences. The recommended barcode combination, rbcL+matK, failed to perform in the genus Terminalia. Considering the complexity of resolution observed with single regions, the present study proposes the combination of matK+ITS as the most successful barcode in Terminalia.

Variability in Rhizome Volatile Constituents of Acorus calamus L. from Western Himalaya

Abstract: Acorus calamus L. (Araceae), is a semi aquatic perennial herb extensively distributed in western Himalaya. To explore the variability in volatile constituents of rhizome essential oils of A. calamus growing wild in foot and mid hills of Uttarakhand, India; twenty one populations were collected during spring season from an altitude of 215 m to 1827 m. The essential oil yield was found to vary from 0.68 % to 5.80 % in the different populations of A. calamus. GC-FID and GC-MS analysis of oil samples resulted in identification of forty constituents comprising 94.0-99.9 % of oil compositions. Phenyl propanoids (81.9-96.9 %) represented by β-asarone (69.8-95.6 %), α-asarone (0.1-4.7 %), (Z)-methyl isoeugenol (<0.1-4.4 %), (E)-isoeugenol acetate (0.0-3.7 %), (E)-methyl isoeugenol (0.1-2.9 %) and γ-asarone (<0.1-1.6 %) constitute the major fraction of essential oil composition. Other major constituents identified were α-selinene (0.1-7.2 %), humulene epoxide II (<0.1-3.5 %), p-cymene (<0.1-1.9 %) and α-bisabolol (0.0-1.6 %). Comparative results and subsequent statistical analysis based on euclidean multidimensional distance scaling showed considerable variations in rhizome essential oils composition.