104 Send Orders for Reprints to [email protected] The Natural Products Journal, 2017, 7, 104-111 RESEARCH ARTICLE ISSN: 2210-3155 eISSN: 2210-3163 Biotechnological Induction of Shikimate-based Antioxidant Accumulation in Phyla dulcis BENTHAM SCIENCE Godson O. Osuji*, Ming Gao, Laura Carson, Peter Ampim, Aruna Weerasooriya, Paul Johnson, Eustace Duffus, Sela Woldesenbet, Jeneanne Kirven, Ebonee L. Williams, Dewisha Johnson and Diadrian Clarke Plant Systems Research Unit, College of Agriculture and Human Sciences, Prairie View A&M University, P.O. Box 519; MS 2000, Prairie View, TX 77446-0519, USA Abstract: Background: Medicinal phytochemicals have been used as dietary supplements in Asia and Africa for thousands of years. Biologically active antioxidants are very diverse and low in their chemical compositions thereby limiting their efficacies. The present study focuses on the enhanced accumulation of closely related dietary antioxidants: shikimate, quinate, salicylate and tocopherol in Phyla dulcis, the Central American herb known for its anti-inflammatory medicinal properties; but its polyphenolic antioxidants had not been studied. Methods: Phyla dulcis stem cuttings were planted in the greenhouse, and in field plots and treated A R T I C L E H I S T O R Y with solutions of stoichiometric mixes of mineral salts known to double crop biomass and yield. Controls were treated with water. At maturity, P. dulcis shoots and flowers were harvested per Received: August 08, 2016 treatment, immediately frozen in liquid nitrogen, and submitted to metabolomic analyses by gas Revised: October 08, 2016 Accepted: October 12, 2016 chromatography-time-of-flight mass spectrometry. DOI: Results: Field plot P. dulcis treated with KKS-mineral salts combination induced increased shiki- 10.2174/2210315506666161017124440 mate accumulation of 1.59 g per 100 g from the 0.799 g per 100 g in the untreated control. Similarly salicylate, quinate, and tocopherols increased in accumulation by many orders of magnitude in the stoichiometric mixes of mineral salts-treated P. dulcis compared with the untreated controls. Conclusion: The accumulated polyphenolic antioxidants permitted the deduction of the unique bio- synthetic pathway of the shikimate, with a massive inhibition at the enzyme steps of dehydroquinate dehydratase and shikimate dehydrogenase. Keywords: Quinate, salicylate, shikimate dehydrogenase, stoichiometric mixes of mineral salts, time-of-flight mass spectrome- try, tocopherols. 1. INTRODUCTION Biologically active phytochemicals are very diverse in their chemical structures, the most diverse in abundance be- Currently, herbal medicine, which comprises prepara- ing those with antioxidant properties. These include poly- tions from plant parts, continue to be relied on for primary phenolics, tocophenols, polyketides, carotenoids, catechins, healthcare needs (WHO: http://www.who.int/mediacentre/ caffeine, theobromine, glutathione, flavonoids, and some factsheets/fs134/en/) by more than 80 percent of the popula- proteins. Because of this diversity, plant tissue antioxidant tion in some Asian and African countries. Medicinal plants phytochemical compositions are very low leading to their have served as the sources of phytochemicals in traditional low biological efficacies and potency. Therefore, there is a treatment of various disease conditions [1]. The Chinese need to substantially increase the antioxidant compositions Traditional Medicine derives 80 percent of its medicaments of medicinal plants. The present study focuses on the en- from higher plants, which are also popular in other Asian hanced accumulation of closely related dietary antioxidants: countries such as Hong Kong, Korea, Indonesia and Malay- shikimate, quinate, salicylate and tocopherol in P. dulcis. sia. Similarly, a high percentage of herbal drugs are used in traditional treatments of diseases in India, Pakistan, Bangla- Phyla dulcis is the Central American plant that has been desh, Sri Lanka and Nepal [2], and Africa [3]. used traditionally by the Aztec people as an herbal sweet- ener. Later, the active ingredients were identified as two ses- *Address correspondence to this author at the Plant Systems Research Unit, quiterpenes: hernandulcin and 4-β-hydroxyhernandulcin that College of Agriculture and Human Sciences, Prairie View A&M University, are 1000 times sweeter than sucrose [4]. The plant is also P.O. Box 519; MS 2000, Prairie View, TX 77446-0519, USA; used in Central America in traditional orally administered Tel: 9362615038; E-mail: [email protected] medicine to treat inflammatory conditions, cough, diarrhea, The Natural Products Journal 2210-3155/17 $58.00+.00 © 2017 Bentham Science Publishers Phyla dulcis Shikimate Herb The Natural Products Journal, 2017, Vol. 7, No. 2 105 and stomachache [1]. But the chemical compositions of the (GDH) isoenzymes [17]. A control treatment was also anti-inflammatory antioxidant activities were not studied. planted in five 10 L pots in a greenhouse using the same Also, several antioxidant flavones have been characterized in growing medium mix used for the field boxes. The experi- Phyla nodiflora [5], which serves as a herbal drink, nourish- ments were watered equally every other day. However, ing agent, immunomodulatory, and anti-inflammatory nu- whenever it rained the field plots were not watered. Weather traceutical beverage in Taiwan [6]. There is increasing inter- conditions (temperatures, rain fall, humidity, wind speed, est in plants that could be utilized as health foods and for and solar radiation) during the study period were collected their potential pharmacological activity [7]. Food quality by a USDA NCRS SCAN site a few meters away from the antioxidants are important constituents of plants because experimental plots (Table 2). The greenhouse was main- they protect against free radicals and reactive oxygen species tained at 21-24 °C and 70-80% relative humidity throughout [8] in the human body. the study; and shaded 50% during the summer months with a shade cloth. The present study was undertaken to evaluate the chemi- cal compositions and the biotechnological inducibility of the antioxidant activities of P. dulcis. Earlier studies had sug- Table 1. Mineral ion composition of treatment solutions ap- gested that the secondary metabolism of the plant was under plied to P. dulcis. the differential regulation by the primary metabolism [9]. This encouraged the search for its primary metabolomic Box Composition in 1 Liter Volume panel for the identification/quantification of antioxidants. By Treatment quantifying the antioxidant data among the P. dulcis meta- ID Applied bolic variants that are differentiated using stoichiometric mixes of mineral salts, it is envisioned that the metabolomics 1 Control (field) No nutrients approach would yield very accurate comparative and quanti- NH Cl (25 mM), Na PO (40 mM) 2 NPPK† 4 3 4 tative data concerning the environmental induction of the and KCl (4 mM) antioxidant activities and their possible metabolic pathways [10]. Biosensor quantification of the antioxidant capacity of 3 N NH4Cl (25 mM) Phyla species is novel and only determined the monopheno- 4 KKPP KCl (8 mM) and Na PO (40 mM) lic antioxidants [11]. Because of the diverse chemical classes 3 4 of antioxidants (small and large molecules, hormones, some 5 KKS KCl (8 mM) and Na2SO4 (50 mM) enzymes, and proteins), multiplicity of free radical sources, NH Cl (50 mM) and Na SO cross interference by reactive antioxidant moieties, and com- 6 NNS 4 2 4 plexity of the reaction mechanisms for neutralizing the free (50 mM) radicals [12], any single chemical or biological assay will Na PO (20 mM) and 7 PN 3 4 possibly overestimate the total antioxidants in a tissue or NH4Cl (25 mM) system [13]. The biotechnological enhancement of the anti- NH Cl (25 mM), Na PO (20 mM), oxidant induction was approached through the treatment of 8 NPK 4 3 4 the plants with stoichiometric mixes of mineral salts known and KCl (4 mM) to double the amino acids, fatty acids, carbohydrates, and 9 Control (greenhouse) No nutrients protein compositions of crops resulting to the doubling of crop yields without increasing the man-hour, and energy †NPPK represents N+P+P+K, the same applies to the other treatments with more than 1 element. inputs of the sustainable agronomic/horticultural production practice [14, 15]. 2.2. P. dulcis Harvesting Harvesting was done when the fastest growing treatment 2. EXPERIMENTAL PROCEDURE had covered the entire box (November 24th, 2015). Each 2.1. P. dulcis Cultivation treatment was harvested by cutting entire shoots and flowers with pair of scissors. The harvested biomass was immedi- Phyla dulcis (Trev.) Mold (Verbenaceae) stem cuttings ately frozen in liquid nitrogen, and stored in -80 °C freezer. were planted in 120 x 120 x 30 cm (width x length x depth) The entire harvest per treatment was ground in liquid nitro- boxes, each filled with 3 bags of professional growing mix gen to coarse powder with mortar and pestle; and 200 g was (Sungro Horticulture, Bellevue, Washington, USA) mixed freeze-dried. The dry tissues were stored in -80 °C freezer, with 2 bags of organic matter-rich top soil (Landscapers and were used for the GC/MS analyses. Pride, New Waverly, Texas, USA) in the medicinal plants garden of Prairie View A&M University Research Farm. 2.3. Chemical Analysis Metadata Each box was set up on ground level in the field on a weed blocking plastic mat. About seven cuttings were planted per Sample processing and extraction with 20