Authors: Narayan Ghorai, Sondipon Chakraborty, Shamik Gucchait, Samir Kumar Saha & Suman Biswas
Terpenes and terpenoids are primary constituents of essential oils of different type of plants and flowers. Some qualitative estimation methods of terpenoids in plant tissue have been previously described but there is no protocol of estimating the same quantitatively till date. In the present study a protocol has been attempted to estimate the total terpenoids concentration of different resin producing plants using a monoterpene, Linalool as standard reagent.
Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers. These compounds belong to the isoprenoid group. Even though isoprene itself has not been found in nature, its polymers, terpenic hydrocarbons and their oxygen derivatives are very often present in large quantities in different plant species. The resin produced by most plants is a viscous liquid, composed mainly of volatile fluid terpene3. The single isoprene unit, therefore, represents the most basic class of terpenes, the hemiterpenes. An isoprene unit bound to a second isoprene is the defining characteristic of terpene, which is also a monoterpene (C10). Sesquiterpenes contain three isoprene units (C15), while diterpenes (C20) and triterpenes (C30) contain two and three terpene units respectively. Tetraterpenes consist of four terpene units, polyterpenes more than four such units. In nature, terpenes occur predominantly in the form of hydrocarbons, alcohols and their glycosides, ethers, aldehydes, ketones, carboxylic acids and esters. Apart from broad structural diversity, terpenes also exhibit a wide array of biological actions. Essential oils have antimicrobial, antiparasitic, insecticidal and antioxidant/prooxidant activities that often represent the combined bioactivity of multiple components10. Many plant terpenoids are cytotoxic towards tumour cells and are applied as chemotherapeutic or chemo-preventive compounds1,2. Terpenes play an important role as signal compounds and growth regulators (phytohormones) of plants10. Many insects metabolize the terpenes which they may have obtained from their plant food for synthesizing growth hormones and pheromones10. Many plant and insect resins like Lac resin also have economically beneficial properties, useful for varnishes and adhesives4.
This protocol describes a rapid, small-scale, high-throughput assay for approximating the total terpenoids content in plant tissue using a monoterpene, Linalool as standard reagent with conc. sulfuric acid. In this reaction Geraniol (primary alcohol, C10H18O) may be produced from Linalool (Tertiary alcohol, C10H18O). Alpha-terpineol (C10H18O) or such monocyclic terpenoids may further be produced from geraniole3. It is very obscured to explain the exact chemical nature of the reaction in where a brick red precipitation has been formed and which is partially soluble in reaction mixture solution and chloroform but fully in methanol. All estimation has been done spectroscopically at 538 nm.
Total Terpenoid Calculation
12.Calculate a standard curve from the blank-corrected at wave length at 538nm of the Linalool standard (Fig.1). Calculate total terpenoids concentration of unknown plant sample as Linalool equivalents using the regression equation of Linalool standard curve.
Terpenoids content varies considerably among leaf and bark tissue and also with environmental and biological stress8,11. In the design of this experiment we advise the use of multiple biological and technical replicates, in order to minimize the sample errors (Table 1). Different incubation time should be tried with each plant species and tissue to optimize the assay. Automated liquid handling system may also significantly increase throughput and improve results6.
Most probably sulfuric acid rearranged the linalool and the tertiary alcoholic group reduces to primary alcohol for which the solubility of reaction product has been reduced to chloroform. It is very important to note that sulfuric acid not only reacts to terpenoids but also it may react with methanolic extract of plant tissue samples having plant phenolics and alkaloids.
Some qualitative estimation methods of terpenoids in plant tissues have been previously described and widely used9. The quantitative analytical colorimetric method of total terpenoids concentration in plants as suggested in this protocol can be used to estimate the total terpenoids of different resin producing plants.
The authors are thankful to Dr. Debaprasad Mondal, Dr. Shamee Bhattacharyya, Kartik Patra and Samarjit Jana of the Department of Zoology, and Dr. Mukut Chakraborty, Head of The Department of Chemistry, West Bengal State University, West Bengal, India for logistic assistance.
Figure 1: Standard curve
Example of a Linalool standard curve. The standard curve is used to estimate mg of terpenoides ( Linalool equivalents) in a 200-µl sample.
Table 1: Troubleshooting table.
Narayan Ghorai, Ghorai's Lab, West Bengal State University
Sondipon Chakraborty, Shamik Gucchait, Samir Kumar Saha & Suman Biswas, Unaffiliated
Correspondence to: Narayan Ghorai ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.055. Originally published online 27 November 2012.