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The Natural Products Journal, 2017, 7, 104-111

RESEARCH ARTICLE

ISSN: 2210-3155 eISSN: 2210-3163

Biotechnological Induction of Shikimate-based Antioxidant Accumulation in 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 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 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 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 () 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 mg of dry box on May 12th, 2015. There were re-plantings 4 weeks P. dulcis shoot tissue per experimental treatment; gas chro- later to replace the few cuttings that were slow to establish matography time of flight mass spectrometry; primary me- rooting. Mineral salt solutions (Table 1) were applied on tabolite identification; and quality control in GC/MS metabo- June 30th, 2015. The mineral ion compositions of these solu- lite profiling were custom performed according to the rec- tions were based on the model stoichiometric combinations ommendations of the Metabolomics Standards Initiative [16], and were formulated to mimic the binomial subunit [18], by University of California Davis Metabolomics Serv- polypeptide compositions of the glutamate dehydrogenase ice Center. Public database of chemicals and chemical infor- 106 The Natural Products Journal, 2017, Vol. 7, No. 2 Osuji et al.

Table 2. Field weather conditions† from transplanting to harvesting the P. dulcis shoots‡.

Rainfall Max. Daily Temp. Min. Daily Temp. Relative Humidity Solar Radiation Wind Speed Month (mm) (oC) (oC) (%) (lang) (km/h)

Mean ± Standard Deviation

May 11.2±18.0 26.7±2.2 18±3.9 93.4±6.9 328±158 10.5±3.5

June 5.0±10.2 ¶- - - - 6.9±3.4

July 0.3±1.0 35.2±1.1 22.9±0.9 91.3±3.6 554±36.1 8.0±2.4

August 4.3±19.8 34.9±3.02 22.2±1.6 88.7±6.1 473±113 6.4±1.8

September 1.5±4.8 31.7±2.1 20±2.3 91.4±7.8 388±85.2 6.0±1.5

October 4.8±20.1 29.3±3.2 15.5±3.5 80.5±14.5 322±120 9.1±4.8

November 4.5±13.7 22.4±5 11.6±4.9 89.2±10.9 201±104 10.1±4

†From a USDA-NRCS scan station on Prairie View A&M University farm a few meters from the P. dulcis plots. ‡Shoots included stems, leaves, and flowers (i.e. aboveground biomass). ¶Data not available. mation (PubChem) together with International Chemical maceutical application as the only precursor for the formula- Identifier key (InChl) were used as universal identifiers for tion of drugs against swine/avian flu, a pandemic which re- the accurate identification of metabolites. cently threatened primary healthcare in the world [22]. Salicylic acid accumulation increased from as low as 3. RESULTS AND DISCUSSION 0.38 mg per 100 g in the green house control plants to as high as 10.95 mg per 100 g of N-treated P. dulcis shoots in 3.1. Antioxidant Accumulation in Response to Mineral field plots (Table 3). Salicylic acid is a plant hormone that Salts Treatment of P. dulcis regulates thermogenesis, and signals the presence of micro- Among the polyphenolic-based antioxidants (shikimic bial pathogen infection [10]. For many centuries, plants (to- acid, salicylic acid, quinic acid, and tocopherols) of P. dul- mato, pepper, berries), and spices [23] containing high levels cis, shikimic acid abundance accounted for more than 97% of salicylic acid, and methyl salicylates were known to have of them (Table 3). Shikimic acid ranged from very high, 1.59 important medicinal properties [24]. Many users of aspirin, g per 100 g in the KKS-treated P. dulcis to as low as 0.34 g the chemically synthesized form of salicylic acid experi- per 100 g in the PN-treated plant, the lowest composition in enced reduced irritation of the gastro-intestinal track, relief the study. Currently, much of the world’s shikimic acid’s from pain and fever as added benefit of this non-steroidal supply comes from the seeds of Chinese star anise, a tree anti-inflammatory drug. Additional benefit of salicylate pro- which takes about 6 years to start bearing fruits [19] com- phylactic use include reduced risk of heart attack, stroke, and pared with P. dulcis which takes 8 weeks to start flowering. several cancers especially risk of colon cancer [25, 26]. The Although it is estimated that about 20% of a plant’s fixed high salicylic acid contents of P. dulcis (Table 3) may now carbon flows through the shikimate pathway [20], the shiki- begin to explain the folk medicinal usage of the plant for mate accumulated in the KKS-treated P. dulcis could be a pain relief and for treatment of inflammatory conditions and commercially important source to supplement microbial fevers [1]. fermentation production of the antioxidant. This medicinal Quinic acid similarly increased in accumulation from plant is loaded with shikimic acid because the control plant 0.45 mg per 100 g of greenhouse control P. dulcis to 11.8 mg grown in the greenhouse also had high, 1.10 g shikimic acid per 100 g of KKPP-treated P. dulcis shoot cultivated in the per 100 g of shoot tissues. The field control P. dulcis grown field (Table 3). Quinic acid, similar to shikimic acid and under the same environmental conditions as the mineral- salicylic acid is found in berries and cherries. Quinic acid is treated plants had 0.799 g shikimate per 100 g of shoot, a pro-metabolite that leads to the induction of efficacious therefore the KKS-treatment approximately doubled the levels of tryptophan and nicotinamide as antioxidants [27]. shikimate accumulation. This is the crop yield doubling bio- This together with shikimic acid and salicylic acid, make technology that stoichiometric mixes of mineral salts confer P. dulcis a loaded source of diverse antioxidants of dietary on crop plants [14, 16, 17] without the cultivation of more and health importance. Quinic acid is metabolized by the land. Shikimic acid has several human health benefits includ- intestinal microflora of the gastrointestinal tract to produce ing oral treatment of fungal infection, as an antioxidant, anti- hippuric acid (N-benzoylglycine) [28] and excreted as urine. bacterial and anti-viral medication. Shikimic acid restores The antioxidant efficacy of quinic acid has been confirmed the balance between fungi and bacteria in the intestinal tract [29], and is known to induce enhanced production of trypto- thereby preventing candidacies. Therefore, shikimic acid is phan and nicotinamide simultaneously administered orally. used in the treatment and prophylaxis, an anti-influenza drug for the A and B viruses [21]. On this account, shikimic acid Similarly, increased accumulation of the tocopherols (vi- has attracted global attention due to its characteristic phar- tamin E complex) in P. dulcis was observed in this study. Phyla dulcis Shikimate Herb The Natural Products Journal, 2017, Vol. 7, No. 2 107

Table 3. Shikimate biochemical pathway intermediates of P. dulcis.

InChl Key Stoichiometric Mineral Nutrient Treatments Shikimate Ret. Quan Pub Based Green Index t. mz Chem Field Anti-oxidants KKPP KKS N NNS NPK NPPK PN house Control Control

Shikimic Acid 611100 204 8742 JXDHGGNKMLTUBPHSUXUTPPSA-N 1103011 1589350 569082 1018352 1029740 1203331 328239 799763 1101353

Salicylic Acid 480699 267 338 YGSDEFSMJLZEOE-UHFFFAOYSA-N 1362 1508 10948 1797 6824 592 6890 1148 379

Quinic Acid 632897 345 6508 AAWZDTNXLSGCEKLNVDRNNJUSA-N 11811 2019 1212 1602 1367 2450 1417 2427 447

α Tocopherol 1067809 237 638015 NCYCYZXNIZIOKI-OVSJKPMPSA-N 4364 3428 2574 3074 1333 15328 2095 3407 855

ɣ Tocopherol 1026121 223 92729 QUEDXNHFTDJVIYDQCZWYHMSA-N 14296 3876 2320 19180 1247 15661 5569 10187 5062

Aromatic Amino Acids

Phenylalanine 537804 218 6140 COLNVLDHVKWLRTQMMMGPOBSA-N 11581 5814 12001 6687 3403 10304 46312 14454 8918

Tyrosine 671252 218 6057 OUYCCASQSFEMEQMMMGPOBSA-N 20291 6401 24765 10165 11100 21141 103926 27357 25901

Tryptophan 780482 202 6305 QIVBCDUIAJPQSVIFPVBQESA-N 78593 26507 49625 60317 19173 127955 225574 97852 107991

Related Metabolic Intermediates in the Shikimate Pathway

Pyruvic Acid 211668 174 1060 LCTONWCANYUPMLUHFFFAOYSA-N 10774 103446 66200 6661 46911 19796 31856 16841 10476

Erythritol 471922 217 222285 UNXHWFMMPAWVPIZXZARUISSA-N 4561 104265 111204 9986 93880 3014 31530 2305 1405

Benzoic Acid 339214 179 243 WPYMKLBDIGXBTPUHFFFAOYSA-N 1504 2022 5671 2952 4642 4199 5074 1727 1498

The increase was from the low µg quantities per 100 g in the 3.2. Physiological Basis of Antioxidant Accumulation control plants to 30 µg per 100 g of the NPPK stoichiometric The internal repeats in the compositions of the mineral nutrient-treated plants (Table 3). Adult human die- stoichiometric mineral salts (Table 1) induced synergistic re- tary requirement is about 15 mg per day. Wheat germ which sponses in the accumulation of the antioxidants. PN-treatment is the richest source of vitamin E has 2,500 mg per kg [30]. induced the lowest accumulation of shikimate; but NPK Tocopherols are fat-soluble antioxidants that are constituents treatment increased the accumulation up to three-fold. The of vitamin E biological machinery in fertility enhancement [31]. It is effective in preventing lipid peroxidation, radical relationship between PN treatment and N treatment was mani- fested by the relief of antagonism in the increase of salicylic driven oxidative events, and risk of cardiovascular diseases acid accumulation by the N treatment (Table 1). The push-pull [32]. The high accumulation of the antioxidants (tocopherols, regulation of antioxidant metabolism by stoichiometric min- quinate, salicylate, and shikimate) in stoichiometric mineral- eral salt mixes is the physiological basis of the biotechnology treated P. dulcis suggests that food-quality processing of this [15]. Some advantages of stoichiometric mixes of mineral medicinal plant could produce antioxidant-fortified healthful beverage drinks. salts are that they are applied in mM (miniscule) quantities, and that in addition to being mineral nutrients, their ions also Metabolite profiling is a tool for understanding the re- act as electromagnets (nucleophiles and electrophiles) as they sponses of plant metabolism to environmental stimuli, an reprogram the metabolic pathways according to the prevail- approach with substantial potential for the improvement of ing pH [16] thereby coordinating biological mechanisms for the compositional quality of crops [33]. As relevant to the increased crop performance and yield, GDH being a target P. dulcis metabolomics project, metabolite profiling has been site of mineral salts action. Agronomy is yet to decipher the applied in the description of the responses of plants to biotic mechanism and specifics of mineral nutrient antagonism- and abiotic stresses [33, 34], and investigation of the integra- synergism. Because stoichiometric mixes of mineral salts are tion of biochemical pathways [15, 16]. The reporting stan- soluble, they exert their combined signal integration-discri- dards [18, 35] formulated for metabolomics assure that com- mination (synergism-antagonism) effects on plant metabolism prehensive quality control measures for minimizing stochas- synchronously. In contrast, the mineral nutrient compositions tic variations in the GC-MS analyses are implemented of commercial fertilizers are differentially soluble, most of throughout the experimentation. Because biosensor, and them exercising delayed release to the crop; they exert their chemical assays lack comprehensive quality control meas- effects on plant metabolism non-synchronously and ineffec- ures [5, 11], they did not detect the shikimate-related poly- tively coordinated. phenolic antioxidants of Phyla species. 108 The Natural Products Journal, 2017, Vol. 7, No. 2 Osuji et al.

OH H COOH HO COOH

+ Quinic Acid O CH O O 2 CH2 OH HO OH

Pyruvate Erythrose OH

Dehydroquinate dehydratase Shikimate dehydrogenase

COOH COOH

Chorismate Shikimic O Acid OH C CH 2 OH OH COOH

NH2

COOH N NH 2 OH Trp COOH N

H Homogentisate NH Tyr CH 2 2 OH COOH COOH OH

Phe COOH Tocopherols

COOH

OH Benzoic Acid Salicylic Acid

Fig. (1). Shikimate metabolic pathway of Phyla dulcis. Note the double inhibition of the pathway at the dehydroquinate dehydratase and shikimate dehydrogenase steps.

3.3. Shikimate Pathway of P. dulcis accumulate ready to be siphoned off into lignin biosynthesis. Where shikimic acid accumulation was highest and maxi- The differential metabolomic peaks (abundances) of the mized (1.59 g per 100 g of shoot) as in the KKS-treated antioxidants, aromatic amino acids, and primary metabolites P. dulcis (Table 3), the trickle-down metabolites were very of the stoichiometric mineral salts-treated P. dulcis (Table low; and on the other hand, where the trickle-down aromatic 3), together with the molecular regulation of the metabolic amino acids (Fig. 1) were extraordinarily high (0.22 g per pathways [9], permitted the deduction of the basic biochemi- 100 g plant shoot) in accumulation as in PN-treated P. dul- cal pathways for shikimate synthesis (Fig. 1) of the medici- cis, shikimic and quinic acids accumulation were very low. nal plant. Some intrinsic control enzyme steps [9] including The extraordinary accumulation of shikimic acid, and the dehydroquinate dehydratase, and shikimate dehydrogenase enormous variations in the abundances of quinic acid, toco- in the pathway explain the extraordinary accumulation of pherols, salicylic acid, and aromatic amino acids suggested shikimic acid, and the enormous variations of up to 28 folds that dehydroquinate dehydratase and shikimate dehydro- in the abundances of quinic acid, tocopherols, and salicylic genase engaged in and coordinated the metabolomic chan- acid observed from one mineral salt-treated plant to the other neling [36]. GDH is the target site of mineral salt action; the (Table 3). Typical with the regulation of metabolic pathways stoichiometric mineral ions also acting as electromagnets, at the mRNA level by GDH-synthesized RNA [14, 15], de- induce the isomerization of GDH, and synthesis of some hydroquinate dehydratase and shikimate dehydrogenase cata- RNA by the enzyme [14]. The GDH research approach [37] lyze non-exergonic reactions in the shikimate pathway [10, has explained many hitherto inexplicable biological phe- 20]. GDH-synthesized RNA regulates metabolism by silenc- nomena including the production of arachin-free peanut [38], ing mRNAs that are homologous to them [14]. Therefore, the metabolic detoxification of xenobiotics in plants [39], dou- mRNAs encoding dehydroquinate dehydratase and shikimate bling of peanut yield [16], and enhancement of the essential dehydrogenase were silenced by GDH-synthesized RNA amino acids accumulation [15]. homologous to them. Shikimate and quinate must transiently Phyla dulcis Shikimate Herb The Natural Products Journal, 2017, Vol. 7, No. 2 109

Table 4. Comparison of time-of-flight mass spectra for the lightest fragment product ions of the shikimate-related antioxidants of P. dulcis.

Antioxidants Time-of-Flight Masses of the Lightest Fragment Product Ions

Quinic Acid 85:2409 88:587 90:40 92:677 94:756 96:319 98:778 100:594 103:1036

Shikimic Acid 85:149 89:152 90:30 92:189 94:99 96:150 98:58 100:45 104:122

Salicylic Acid 85:285 87:2 90:498 92:934 94:14 96:295 99:41 100:8 103:549

α-tocopherol 85:207 87:84 91:78 92:83 94:4 97:218 99:1 103:358 105:44

ɣ-tocopherol 85:7 86:92 88:26 91:52 94:33 97:79 99:8 104:48 105:82

In support of the suggested pathway for P. dulcis shiki- ionization of the metabolite, the standard -70 eV is so high mate metabolism was the detection of benzoic acid accumu- that the positively charged molecules suffer fragmentation to lation, the highest (5.67 µg per 100 g shoot) being in the multiple lower mass product ions, the identification of which N-treated plants, and the lowest (1.49 µg per 100 g shoot) by time-of-flight produces mass spectra at very high sensitiv- being in the greenhouse-grown control. Salicylic acid is syn- ity and speed. Therefore, the mass spectra of the lighter thesized by plants either through conversion of phenyla- product ions serve as further identifiers of the metabolites. lanine by Phenylalanine Ammonia-Lyase (PAL) to benzoic The polyphenolic antioxidants of P. dulcis (Table 3 and acid, or via the conversion of chorismate to isochorismate by Fig. 1) share several metabolic pathways in common [20, isochorismate synthase [10]. Salicylic acid accumulation was 40], the committed metabolites of which include pyruvate, lowest (0.38 µg per 100 g shoot) in the greenhouse-grown erythrose, or acetoacetate with ion molecular masses ranging control, and highest (10.9 µg per 100 g shoot) in the N- from about 85 to 105. The mass spectral properties of the treated P. dulcis (Table 3). These correlations suggested that lightest molecular ion fragments (Table 4) identified for the P. dulcis utilizes the PAL pathway to salicylic acid biosyn- shikimate-related polyphenolic antioxidants were within the thesis (Fig. 1). masses for pyruvate, acetoacetate, and erythrose. Therefore, the molecules of the antioxidants readily got fragmented 3.4. Responses of P. dulcis Metabolism to Light and Heat along the seams at which the initial metabolites were joined Metabolomic profiling of P. dulcis cultivated in the by dehydroquinate synthase [20, 40] in the same metabolic greenhouse (controlled temperature, light, humidity, water biochemical pathway. Complete mass spectral properties of etc.) compared with cultivation in field conditions (variabil- quinic acid, shikimic acid, salicylic acid, and the tocopherols ity of rainfall, temperature, winds, humidity etc.) showed of P. dulcis are provided under supplementary materials. The that field conditions dramatically increased the accumulation higher molecular ion fragments of the tocopherols, quinic of primary metabolites (pyruvic acid, erythritol), and most of acid, shikimic acid, and salicylic acid shared slightly differ- the secondary metabolites (salicylic acid, quinic acid, toco- ent mass ranges; the tocopherols displayed higher retention pherols, phenylalanine, and benzoic acid) by several orders indexes than the acidic antioxidants (Table 3). Therefore, of magnitude. Whereas, the greenhouse-grown plant was TOF-MS detection at very high sensitivity enhanced the pro- green, the field-grown plant was purple [9]. The intense and filing identification of the antioxidants. Biosensors had been variable heat, solar radiation, wind, rainfall, and relative hu- applied for the quantification of the antioxidant capacity of midity of Texas summer months (Table 2) were the envi- Phyla species, but they could determine only the monophe- ronmental conditions that switched the metabolism from nolic antioxidants [11]. Because of the diverse chemical primary to predominantly secondary resulting to great accu- classes of antioxidants (small and large molecules, hor- mulation of antioxidant polyphenolics (Table 3). In contrast, mones, some enzymes, and proteins), [12], no single chemi- the greenhouse was maintained at 21-24 oC and relative hu- cal or biological assay will accurately reflect the shikimate- midity of 70-80% throughout the study period and was related antioxidants and their biochemical pathways in a tis- shaded 50% with a shade cloth during the summer months to sue or system [13]. Metabolomics approach, with the intrin- minimize exposure of the plants to full sunlight. sic mass spectra identification of fragmented product ions at very high sensitivity yielded datasets that quantitatively sup- ported the biosynthetic pathways of the polyphenolic anti- 3.5. Time-of-Flight Mass Spectral Identifier of the Anti- oxidants. oxidants Gas chromatography and mass spectrometry combined to CONCLUSION quantitate the polyphenolic antioxidants and their metabolic pathway of P. dulcis (Table 3 and Fig. 1). This is the impor- Phytochemicals have been used in Asia and Africa in tra- tance of metabolomics as an essential technological approach ditional medicine. Biologically active antioxidants are very for profiling metabolic responses of plants [33] to the envi- diverse and low in their chemical compositions thereby lim- ronment. The ability of TOF-MS to profile metabolites has iting their efficacies. This study revealed that treating P. dul- been further enhanced by the mass spectrometers of LECO cis plants with solutions of stoichiometric mixes of mineral Corporation (Michigan, USA) in that after hard electron salts known to double crop biomass and yield enhanced the 110 The Natural Products Journal, 2017, Vol. 7, No. 2 Osuji et al. accumulation of closely related dietary antioxidants: shiki- [10] Dempsey, D.A.; Vlot, A.C.; Wildermuth, M.C.; Klessig, D.F. Sali- mate, quinate, salicylate and tocopherol in the plants. Field cylic acid biosynthesis and metabolism. The Arabidopsis Book. American Society of Plant Biologists Publisher: Rockville, 2011. plots of P. dulcis treated with KKS-salts combination ap- [11] Rodriguez-Sevilla, E.; Ramirez-Silva, M.; Romero-Romo, M.; proximately doubled (1.59 g) shikimate accumulation per Ibarra-Escutia, P.; Palomar-Pardave, M. Electrochemical quantifi- 100 g of plant material. Similarly salicylic acid, quinic acid, cation of the antioxidant capacity of medicinal plants using biosen- and tocopherols increased in accumulation by many orders sors. Sensors (Basel), 2014, 14, 14423-14439. of magnitude with the stoichiometric mixes of mineral salts- [12] Prior, R., Wu; X., Schaich, K. Standardized methods for the deter- mination of antioxidant and phenolics in foods and dietary supple- treated P. dulcis compared with the untreated controls. Me- ments. J. Agric. Food Chem., 2005, 53, 4290-4302. tabolomic profiling of the polyphenolic antioxidants revealed [13] Badarinath, A.V.; Rao, K.M.; Chetty, C.M.S.; Ramkanth, S.; Ra- the excessive accumulation of not only shikimate but also of jan, T.V.S.; Gnanaprakash, K. A review on in-vitro antioxidant benzoate, the consideration of which together with those of methods: Comparisons, correlations and considerations. Int. J. Pharm. Tech. 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