Phytochemical Recovery for Valorization of Loblolly Pine and

Mar 30, 2017 - ... 203 White Engineering Hall, Fayetteville, Arkansas 72701, United States. ‡ School of Forestry & Natural Resources, University of ...
0 downloads 11 Views 2MB Size
Research Article pubs.acs.org/journal/ascecg

Phytochemical Recovery for Valorization of Loblolly Pine and Sweetgum Bark Residues Kalavathy Rajan,† Andrew Nelson,‡,§ Joshua P. Adams,‡,⊥ and Danielle Julie Carrier*,† †

Department of Biological and Agricultural Engineering, University of Arkansas, 203 White Engineering Hall, Fayetteville, Arkansas 72701, United States ‡ School of Forestry & Natural Resources, University of Arkansas, 346 University Drive, Monticello, Arkansas 71655, United States

ABSTRACT: Bark residues from forest products production using loblolly pine (LP) and sweetgum (SG) are currently combusted in pulp and paper mills. Hot water extraction (HWE) of phytochemicals from bark prior to combustion could be an effective method to valorize these biomass streams. On average, the LP and SG-HWE contained 12% monosaccharides, 33% polysaccharides, 21% total phenolics and 8% organic acids. Major phytochemicals recorded in LP and SG-HWE were shikimic acid (15%), gallic acid (7.5%), vanillic acid (5−7%) and vanillin. Antimicrobial and antioxidant activities of LP and SG-HWE were evaluated for LP and SG bark harvested in different months (January, April and September) and from different sized trees (diameter at breast height or DBH, ranging from 2 to 33 cm). All LP and SG-HWE inhibited a Staphylococcus aureus cocktail. LPHWE harvested in April from DBH 27 and 33 cm also inhibited Listeria monocytogenes, Salmonella enterica and Escherichia coli. All SG-HWE preparations, at 12.5 μg, produced significant reduction in human low-density lipoprotein (LDL) peroxidation, as compared to the copper induced control; only LP-HWE prepared from January bark produced similar results. Knowledge of seasonal variations in bark phytochemical content would thus enhance the feasibility of phytochemical extraction. KEYWORDS: Bark phytochemicals, Loblolly pine, Sweetgum, Antioxidants, Antibiotics, Seasonal variation



INTRODUCTION

as a fuel source by the pulp mills and the rest is sold as mulch and their phytochemical content is completely disregarded.4 The overlooked phytochemical content of bark has the potential to generate a substantial amount of additional value to the forest products industry and regional economies, with technologies that could be easily integrated into existing production lines. Commercially, phytochemicals, such as shikimic acid and vanillin, can be extracted from plant biomass using ethanol or methanol, followed by complex purification pathways.7,8 Purified shikimic acid and vanillin find applications in pharmaceuticals and food industry, respectively. Aqueous extraction is an eco-friendly alternative, which is reported to recover 70% of original plant phenolic content and is comparable to organic solvents-based extraction (30 to 85%).7 Among different aqueous extraction techniques, such as ultrasound and microwave assisted extraction, hot water

Loblolly pine (Pinus taeda) (LP) and sweetgum (Liquidambar styraciflua) (SG) are tree species native across the Southern United States and LP, mixed with up to 30% of SG wood, is commonly used in the pulp and paper mills of the region.1,2 LP and SG are used to produce many forest products, including lumber, plywood, fuel and pulpwood and LP, in particular, is a strong contributor to the regional and national economy and is projected to increase in importance throughout the 21st century.1,2 Tree bark is a major low-value byproduct of the timber industry and extraction of phytochemicals from bark could increase the yield and value of timber, and as well as maximize resource utilization.3 In 2012, it was projected that bark constituted 30% of the total residues generated by primary milling operations in the U.S., about 17.5 million dry tons.4 Pine and sweetgum bark have been reported to contain 15% and 16% of ethanol-extractives, respectively, and these extractives are a major source of phytochemicals with potential health benefits.5,6 Currently, 75% of the bark residues are used © 2017 American Chemical Society

Received: January 22, 2017 Revised: March 16, 2017 Published: March 30, 2017 4258

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering extraction, at 65 to 100 °C, is a simple and effective method for extracting bioactive phytochemicals from bark.9,10 Moreover, hot water extraction has already been proposed in integrated forest biorefinery operations for hemicellulose extraction from timber wood, and therefore it can also be exploited for phytochemical extraction from bark residues.11 Hot water extraction would enable the raw material to still be used in other process-related unit operations, such as combustion. Crude hot water extracts (HWE) from pine bark have been reported to exhibit biological activities such as antiperoxidation, radical scavenging capacity, antimicrobial and anti-inflammatory activities, which have potential applications in human nutrition and therapeutics.12,13 Similarly, SG bark HWE has been reported to display antiviral, antiperoxidation and antiulcerogenic properties.14 In SG-HWE, phenolic compounds, such as shikimic acid and gallic acid, have been identified as two of its various bioactive components;14 on the other hand, it is still unclear which of the broad category of phytochemicals found in LP-HWE, such as flavonoids and hydroxycinnamates, are responsible for its bioactivity.12 Identification of principal bioactive components in bark HWE would pave the way for optimizing and improving the efficiency of hot water extraction. Moreover, the phytochemical content of bark has been established to vary according to harvest season and tree girth, irrespective of the extraction method.15,16 Oleoresin, an insect repelling phytochemical found in LP bark resin, was recorded to be the highest during winter months.17 Another study showed that the highest recovery of essential oils from LP needles was obtained in early spring.18 While screening for useful phytochemicals, Bauhinia variegata bark harvested from a girth class of 36−55 cm was shown to provide the optimum benefit.15 Similar trends in phytochemical content of LP and SG bark extracts are yet to be established. Therefore, characterization of phytochemical variations in bark HWE would prove to be useful for large-scale extractions. Due to the fact that trees are harvested year-round, it is essential to determine whether the phytochemical recovery from bark HWE is constant throughout the lifespan of the tree, irrespective of the harvest season. The objectives of this study were to (1) identify the beneficial biological activities of LP and SG bark HWE, (2) pinpoint the major phytochemicals responsible for biological activities and (3) determine the variations in biological activities according to harvest season and tree maturity. Determination of these objectives is critical for establishing time frames for phytochemical extraction operations within the forestry supply chain.



Chemicals. Reference compounds of glucose (Alfa-Aesar, Ward Hill, MA), xylose, p-coumaric acid, trans-ferulic acid, syringaldehyde, 4hydroxybenzoic acid, vanillin, salicylic acid, gallic acid (TCI chemicals, Montgomeryville, PA), vanillic acid, trans-cinnamic acid and shikimic acid (Amresco, Solon, OH) were used for general characterization of bark extracts. Sulfuric acid and formic acid, ACS grade, were purchased from EMD Millipore (Gibbstown, NJ). Folin−Ciocalteu’s phenol (F− C) reagent and phenol crystals were purchased from Sigma-Aldrich (Milwaukee, WI). Human plasma, low density lipoproteins (LDL) was purchased from EMD Millipore (Darmstadt, Germany) and dialyzed in a pH 7.4 TRIS buffer in order to remove the chelating agent. Water was prepared with a Direct-Q system (Millipore, Billerica, MA) that had a resistivity of 18.2 MΩ·cm (at 25 °C). Hot Water Extraction. All bark samples were thawed and dried in a 100 °C oven until constant weight and then ground using a Wiley Mini cutting mill (Swedesboro, NJ). The average particle size of ground bark was 0.38 mm for LP and 0.31 mm for SG. Hot water extraction of bark was performed using a one liter Parr 4525 reactor (Moline, IL), at 10% solid loading (oven dry weight). Sweetgum hot water extracts (SG-HWE) were prepared at 85 °C, for 1 h and the loblolly pine hot water extracts (LP-HWE) were prepared at 100 °C, for 1 h.10,19 After extraction, the SG-HWE or LP-HWE were separated from the bark solids using a vacuum filtration apparatus and then freeze-dried in a Labconco Freezone 18 L drier (Kansas City, MO) for 72 h, at −44 °C and 0.09 mbar vacuum. Disc Diffusion Assay. Pathogenic bacterial cocktails of Staphylococus aureus (ATCC25923, S-41), Listeria monocytogenes (Ser 1/2a, 4ab, 4b and ATCC33090), Escherechia coli O157.H7 (ATCC11775, ATCC25923, ATCC25955) and Salmonella enterica (ATCC43845, ATCC8326, ATCC8387, USDA1769NR) were obtained from the Center for Food Safety, Department of Food Science, University of Arkansas, Fayetteville, AR. Cultures of S. aureus, E. coli and S. enterica were maintained on a 3% (w/v) tryptic soy broth (Bacto, Sparks, MD) liquid media, whereas L. monocytogenes was subcultured in a 3% (w/v) tryptic soy broth liquid media, also containing 0.6% (w/v) yeast extract (Bacto, Sparks, MD). The disc diffusion assay was carried out using 3.8% (w/v) Mueller Hinton agar plates (Himedia, Mumbai, India), where the bacterial cultures were inoculated using the pour plate method. Freeze-dried SG-HWE and LP-HWE were tested at 3 and 6 mg, respectively, for their antibiotic activity. Streptomycin sulfate (Sigma-Aldrich, St. Louis, MO), a broad-spectrum reference antibiotic compound, was used as the positive control at 3 mg. The samples and positive control were dissolved in sterile water, before being transferred to 6.35 mm Taxo antibiotic detection discs (Becton Dickinson and Company, Sparks, MD) and incubated at 37 °C, for 24 h. TBARS Assay. Antioxidant potential of LP and SG-bark HWEs were examined by measuring their capacity to suppress LDL peroxidation, which was induced by Cu2+ ions (copper sulfate) over a 24 h incubation period. The control contained only TRIS buffer at pH 7.4, and the two treatments contained 12.5 μg of SG-HWE and 55 μg of LP-HWE. These quantities were chosen based on preliminary evaluations, which showed that a minimum of 12.5 μg of SG-HWE and 55 μg of LP-HWE were required to produce a significant reduction in LDL peroxidation, in at least one of the treatment levels. Thiobarbituric acid assay (TBARS) measures the amount of malondialdehyde formed as a result of lipid peroxidation20 and the method for TBARS antioxidant assay was adapted from Uppugundla et al. (2009).21 Sample absorbance at ΔOD530−600 nm was measured using a microplate reader (BioTek, Winooski, VT) and the corresponding TBARS concentration (μmol/L) was expressed in TEP (1,1,3,3-tetraethoxypropane) equivalent. CPC Fractionation. January SG-HWE from class IV material was fractionated using centrifugal partition chromatography (CPC), in an attempt to purify the principle component associated with their biological activities. A biphasic solvent system composed of ethyl acetate, ethanol and water at 2:1:2 (v/v/v) ratio was used in descending mode, in order to separate the bark polyphenols.22 A bench scale, 250 mL, CPC rotor (Armen Instrument, Saint-Avé, France) was operated by the Gilson PLC 2050 controller (Middleton,

MATERIALS AND METHODS

Bark Samples. Loblolly pine (LP) (Pinus taeda) and sweetgum (SG) (Liquidambar styraciflua) trees of varying average stem diameter at breast height (DBH; measured 1.37 m above the ground), i.e., class I- 1.7 cm, class II- 6.2 cm, class III- 27.1 cm and class IV- 32.9 cm, were chosen. These size classes represent different stages of tree development from young tree seedlings to mature trees. The selected trees were harvested during three different seasons namely, January 29th (winter), April 21st (spring) and September 10th (early autumn) of 2015, which corresponded to dormant season, early seasonal growth, and late seasonal growth, respectively. All tree samples were obtained from the School of Forestry & Natural Resources POW Camp, in Monticello, AR (33°43′51″ N, 91°43′50″ W) where the trees were subjected to identical growth conditions, such as soil and climate. Bark material was obtained via scraping the bark from the bole of the tree in the field and stored at −30 °C until further experimentation. 4259

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering WI), which was equipped with a fixed wavelength (254 nm) detector. On average, 4 g of the freeze-dried extract was fractionated per trial; elution of the mobile phase was set at 8 mL/min through the rotor. The total run time was 80 min and on an average 60 fractions, of 8 mL each, were collected. Fractionated CPC samples were analyzed by liquid chromatography and consolidated based on their λmax values and the predominant phenolic compound detected. Characterization of Bark HWE. Liquid Chromatography Analysis. All freeze-dried bark extractives were reconstituted in water and analyzed using high performance liquid chromatography (HPLC) and ultraperformance liquid chromatography (UPLC) to determine their carbohydrate and phenolic compositions, respectively. Carbohydrates were analyzed on a Waters Alliance HPLC system (Model 2695, Waters Corporation, Milford, MA) fitted with a SP-G precolumn and a SP0810 analytical column (Shodex, Kawasaki, Japan). HPLC protocol for carbohydrate analysis was obtained from NREL (National Renewable Energy Laboratory, Golden, CO) report #TP-510-42618. All reconstituted bark samples were hydrolyzed using 4% sulfuric acid to determine the polysaccharide content using HPLC.23 Phenolic acid composition was determined using a Waters Acquity UPLC system (Milford, MA), equipped with a BEH C18 (1.7 μm × 2.1 mm × 50 mm) analytical column and an Acquity VanGuard precolumn. A modified UPLC method was used, where the mobile phases were 0.1% formic acid in water and 100% methanol, mixed in a gradient of 88.5:11.5 to 30:70, during a run time of 3.5 min.24 Samples were eluted at a flow rate of 0.4 mL/min and detected using a photodiode array detector. The λmax values varied for different phenolic compounds; 230 nm for shikimic acid, vanillin and syringaldehyde, 267 nm for 4-hydroxybenzoic acid, 280 nm for gallic, cinnamic and vanillic acid and 300 nm for p-coumaric, trans-ferulic and salicylic acids. Total Phenolics Assay. A modified Folin−Ciocalteu (F−C) assay was used for the total phenolics determination of bark LP and SG HWEs.25 The samples were diluted to an approximate concentration of 0.3 g/L, and 100 μL of this aliquot was mixed with 200 μL of 0.2 N F−C reagent followed by incubation in the dark, for 5 min. Then, 800 μL of 7.5% sodium carbonate solution was added to the mixture and incubated in the dark, at room temperature, for 2 h. After the incubation period, the samples were diluted by a factor of 4 with water, and their absorbance at 765 nm was determined using a spectrophotometer (Model 517601, Beckman Coulter Inc., Indianapolis, IN). Total phenolic content was expressed in tannic acid equivalent (TAE).25 Total Carbohydrates Assay. A modified phenol-sulfuric acid method was used for total carbohydrate estimation of the bark HWE.26 Samples diluted with 80% ethanol, at 1:1 volumetric ratio, were mixed with the phenol-sulfuric acid reagent as per Buysse and Merckx (1993) and left at room temperature for 15 min, for color development.26 Sample absorbance at 490 nm was determined using a UV−vis spectrophotometer (Model 517601, Beckman Coulter Inc., Indianapolis, IN) and the total carbohydrate content was estimated in glucose equivalent. Statistical Analysis. All experiments were conducted in triplicate and the results were analyzed for statistical significance using the JMP Pro 11.0 software (Cary, NC). A two-way analysis of variance, at α = 0.05, was used to examine the compositions of LP and SG HWE, such that the effects of size class and sampling date and their interaction could be determined, with one model for each species. The technical repetitions served as the replicates (n = 3). Post-hoc analysis of the bark biological activities was done using Tukey’s HSD (honest significant difference) test in order to compare all possible pairs of means, between the control and treatments, i.e., tree size class and sampling date.



for 1 h yielded 60 g/kg (odb.) of HWE, on average. In the case of LP, tree maturity had a significant impact on the mass of HWE extracted, whereas the harvest season had no effect, even if there was an interaction between the two factors (p = 0.01). In the case of SG, both tree maturity (p = 3 × 10−05) and harvest time (p = 0.04) had a significant effect on the mass of bark HWE obtained, with a strong interaction between the two factors (p = 2 × 10−05). Materials stemming from the youngest trees (i.e., class I), at the September sampling provided the highest mass of extractives, which were 85 g/kg from LP and 90 g/kg from SG. Compositional analysis of LP-HWE is presented in Table 1. All LP-HWE, on average, were mostly composed of Table 1. Composition of Hot Water Extractives (HWE) from Loblolly Pine (LP) Bark, For Different Tree Classes and Sampling Datesa Time of harvest Components HWE yield (g/kg)

Total carbohydrates (%)c

Total phenolics (%)d,c

Total organic acids (%)c

Tree classb

January

April

September

I II III IV I II III IV I II III IV I II III IV

95.0 ± 1.64 56.8 ± 2.2 59.9 ± 6.6 48.5 ± 2.1 29.7 ± 1.7 33.5 ± 0.5 39.3 ± 0.1 37.7 ± 1.0 26.4 ± 3.8 20.0 ± 1.0 15.9 ± 0.7 16.5 ± 0.0 6.1 ± 0.3 2.9 ± 0.2 10.2 ± 0.1 1.8 ± 0.4

123.3 ± 1.1 53.6 ± 6.6 38.6 ± 0.8 62.1 ± 9.9 33.2 ± 2.4 38.3 ± 0.7 46.1 ± 1.6 42.0 ± 0.8 18.1 ± 1.0 12.9 ± 0.4 12.0 ± 0.3 10.8 ± 1.2 13.5 ± 0.1 8.9 ± 1.0 17.7 ± 0.1 5.3 ± 1.0

84.9 ± 2.0 69.7 ± 1.4 70.0 ± 4.0 73.3 ± 2.8 30.0 ± 1.1 38.3 ± 2.0 35.6 ± 0.3 32.1 ± 1.5 15.5 ± 0.0 15.8 ± 0.5 11.3 ± 0.1 13.4 ± 0.0 13.0 ± 0.1 8.0 ± 0.4 13.5 ± 0.3 7.4 ± 0.3

a

Averages and standard deviations are provided for N = 3. bDiameter at breast height of tree class I- 1.7 cm, class II- 6.2 cm, class III- 27.1 cm, class IV- 32.9 cm. c(w/w) Oven dry weight of LP-HWE. dTotal phenolics were measured in tannic acid equivalent.

polysaccharides and monosaccharides (50%) and phenolic compounds (16%). Both the tree maturity and harvest time had a significant effect on polysaccharide, monosaccharide and total phenolic content of LP-HWE, with a significant interaction between the two factors. Of the different polysaccharides, glucan and arabinan on an average accounted for 45% and 40% of all LP-HWE, respectively. A higher extraction temperature of 100 °C, likely led to hemicellulose solubilization; therefore LPHWE also contained monomeric xylose and arabinose, in addition to glucose and fructose. Monosaccharide content of LP-HWE, especially fructose, was the lowest in January, which increased during April and reached the highest during September (Table 2). Soluble carbohydrate content in tree bark changes according to the balance in nutrient demand and supply; the lowest carbohydrate content was recorded in January because the rate of nutrient consumption was probably not fulfilled by the rate of photosynthesis (during winter).27 Total phenolic content, on the other hand, was the highest for material harvested in January may be because, the secondary metabolites in bark respond positively to biotic and abiotic

RESULTS AND DISCUSSION

Compositional Changes with Maturity and Season. Hot water extraction of loblolly pine bark (LP), at 100 °C for 1 h, yielded 70 g/kg (oven dry basis or odb.) of hot water extractives (HWE) and that of sweetgum bark (SG) at 85 °C 4260

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering

Table 2. Carbohydrate Composition of Hot Water Extracts (HWE) from Loblolly Pine Bark and Sweetgum Barka Sweetgum harvest time HWE components Glucan (%)

Arabinan (%)

Xylan (%)

Glucose (%)

Fructose (%)

Sucrose (%)

Xylose (%)

Tree class I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV

b

Loblolly pine harvest time

January

April

September

January

April

September

13.4 ± 5.0 8.3 ± 2.4 11.9 ± 5.2 6.7 ± 4.4 6.8 ± 1.8 2.6 ± 0.5 3.7 ± 0.2 2.8 ± 0.2 3.1 ± 0.3 1.5 ± 0.8 1.9 ± 0.1 2.3 ± 0.2 3.5 ± 0.1 3.0 ± 0.2 2.0 ± 0.1 5.0 ± 0.3 2.7 ± 0.2 2.5 ± 0.4 1.2 ± 0.0 4.4 ± 0.1 0.5 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0

13.0 ± 5.4 10.4 ± 5.3 11.5 ± 3.7 12.5 ± 6.7 1.6 ± 0.1 3.8 ± 0.9 3.8 ± 1.5 4.3 ± 1.6 1.2 ± 0.7 2.1 ± 1.0 4.3 ± 0.2 2.3 ± 0.4 4.4 ± 0.4 5.1 ± 0.7 3.3 ± 0.3 3.4 ± 0.2 7.6 ± 0.5 7.3 ± 0.4 2.1 ± 0.3 6.1 ± 0.7 0.5 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 0.5 ± 0.0

18.9 ± 0.6 19.7 ± 0.9 13.0 ± 0.2 10.0 ± 0.7 4.5 ± 0.4 3.6 ± 0.0 2.6 ± 0.0 2.9 ± 0.1 6.4 ± 1.7 2.1 ± 0.0 2.4 ± 0.0 4.5 ± 0.1 1.7 ± 0.1 2.7 ± 0.2 2.2 ± 0.1 1.4 ± 0.1 8.2 ± 0.7 7.3 ± 0.5 6.6 ± 0.0 2.8 ± 0.3 11.4 ± 0.2 10.1 ± 0.1 7.0 ± 0.2 1.3 ± 0.2

10.9 ± 2.0 12.7 ± 0.2 15.1 ± 0.0 13.4 ± 0.1 8.3 ± 0.3 28.2 ± 1.0 31.3 ± 0.3 30.4 ± 1.3 5.5 ± 0.1 6.1 ± 0.2 8.7 ± 0.1 7.8 ± 0.1 3.4 ± 0.0 2.1 ± 0.0 2.9 ± 0.1 3.0 ± 0.0 4.8 ± 0.2 2.2 ± 0.0 2.0 ± 0.0 2.9 ± 0.1

20.3 ± 1.5 32.8 ± 1.5 20.6 ± 0.9 34.7 ± 0.1 17.6 ± 1.2 20.6 ± 2.7 17.1 ± 2.7 18.2 ± 0.4 11.5 ± 0.7 3.6 ± 0.1 10.9 ± 0.9 7.5 ± 0.7 2.6 ± 0.0 3.4 ± 0.2 0.8 ± 0.0 4.6 ± 0.0 3.5 ± 0.0 5.0 ± 0.3 5.0 ± 0.3 4.4 ± 0.1

15.1 ± 0.8 18.3 ± 0.1 18.8 ± 0.5 19.4 ± 1.1 9.6 ± 0.2 12.2 ± 0.5 15.1 ± 2.1 14.9 ± 0.4 8.9 ± 0.1 3.6 ± 0.5 7.6 ± 0.4 10.8 ± 0.1 7.6 ± 0.2 8.3 ± 0.1 7.1 ± 0.4 7.4 ± 0.0 11.9 ± 0.2 9.45 ± 0.1 7.6 ± 0.2 7.6 ± 0.3

1.7 1.3 1.9 1.5

± ± ± ±

0.0 0.0 0.0 0.0

3.1 0.7 0.4 1.2

± ± ± ±

0.1 0.0 0.0 0.0

5.6 1.6 3.2 3.9

± ± ± ±

0.2 0.0 0.0 10

a

Averages and standard deviations are provided for N = 3. Percentage calculations were based on oven dry weight of HWE. bDiameter at breast height of tree class I- 1.7 cm, class II- 6.2 cm, class III- 27.1 cm, class IV- 32.9 cm.

stresses and since winter is a stressful growth period with low sunlight and less rainfall.16 Analysis showed that LP-HWE contained among other phenolic compounds: trans-ferulic acid, p-hydroxybenzoic acid, gallic acid, vanillin and vanillic acid. Vanillic acid was detected in all LP-HWE preparations and accounted for approximately 5% of LP-HWE (Table 4). Pearl (1975) also reported the presence of phenolic compounds such as pyrocatechol, vanillin, p-hydroxybenzoic acid and vanillic acid in the hot water extracts of LP bark.28 Organic acid content of LP-HWE, especially butyric and propionic acids were the highest in April and the lowest in January. Presence of butyric and propionic acids in LP-HWE may be explained by impromptu carbonization and/or autohydrolysis of fatty acid esters and resins present in the LP bark.29 Compositional analysis of SG-HWE is presented in Table 3. All SG-HWE, on average, contained phenolic compounds (27%) and total carbohydrates (29%). The polysaccharide composition, namely glucan (54%), arabinan (20%), xylan (12%), galactan (8%) and mannan (6%), was consistent for all SG-HWE. The harvest time had a significant impact on the polysaccharide content (p = 0.03) of SG-HWE, whereas tree maturity provided no significant interaction effect. Both harvest time and tree maturity had a significant effect on the monosaccharide and organic acids content of SG-HWE, with a significant interaction between the two factors (p < 0.05).

Younger SG trees of classes I and II, especially those harvested in April and September, had significantly higher amounts of monosaccharides like fructose and sucrose (Table 2). This could once again be explained by an increase in demand for carbohydrates during active growing periods (spring and early fall), leading to an increase in soluble carbohydrate content of SG-HWE.27 Organic acids content was also higher in April and September SG-HWE (Table 3), which may be due to the degradation of monosaccharides to formic acid during the hot water extraction. On the other hand, the total phenolic content of SG-HWE was not affected by either tree maturity or harvest season and there was no interaction effect between the two factors. Minimal response to seasonal changes in total phenolic content of SG-HWE could indicate that the sweetgum tree had a better (biotic and abiotic) stress tolerance compared to loblolly pine. Major phenolic compounds identified in SGHWE were shikimic acid (15%), gallic acid (7.5%) and vanillic acid (7%) (Table 4). Vanillin, p-coumaric and ferulic acids were also detected in SG-HWE, albeit in trace quantities. Martin et al. (2010) reported the presence of shikimic acid (12%) in HWE prepared from a mixture of juvenile and mature SG bark, but did not elucidate the presence of other phenolic compounds.9 Overall, the compositional analysis of LP-HWE and SG-HWE indicated that there were differences in terms of tree maturity and time of harvest. 4261

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering

All SG-HWE contained shikimic acid and it was reported that, even at concentrations as low as 2.5 g/L, shikimic acid could inhibit the growth of S. aureus by damaging its cell membrane.31 The average concentration of shikimic acid found in the SG-HWE was 15%, but it varied significantly. SG-HWE prepared from juvenile tree bark displayed significantly higher concentrations of shikimic acid than that of mature bark preparations; however, no significant differences were observed in their inhibition of S. aureus cocktail (Figure 1A). This points to the fact that shikimic acid may not be the sole compound conferring antimicrobial activity and more than one compound present in SG-HWE may be responsible for S. aureus inhibition. Phenolic compounds other than shikimic acid found in sweetgum storax, such as hydroxycinnamates and vanillin, have also been reported to exhibit antimicrobial properties.32 LP-HWE from mature bark (i.e., classes III and IV, specifically those harvested in April) exhibited antimicrobial activity against L. monocytogenes, S. enterica and E. coli cocktails. At twice the concentration of control, LP-HWE exhibited 40%, 32% and 29% inhibition against L. monocytogenes, E. coli and S. enterica cocktails, respectively (Figure 1C). Ethanol extractives from Jack pine (Pinus banksiana) bark were reported to inhibit Listeria, where the extracts were reported to contain flavonoids and hydroxycinnamate that may have conferred their antibacterial properties.12 Analysis showed the presence of hydroxycinnamates in LP-HWE, such as trans-ferulic acid, as well as other phenolic acids, like p-hydroxybenzoic and vanillic acids, which have been reported to inhibit S. aureus, E. coli and S. typhimurium.33,34 It is more than likely that one or more of the above listed phenolic compounds acted synergistically and inhibited the growth of S. aureus, L. monocytogenes, E. coli and S. enterica (Figure 1B,C). Owing to its high procyanidins content, pine bark extractives have commonly been exploited for their antioxidant potential; this study showed that LP-HWE also displayed broad-spectrum antibiotic potential. Variations in Antiperoxidation Activity. Overall, January SG and LP-HWE preparations showed the highest antiperoxidation activities. The control, which was induced by copper for 24 h, exhibited the highest levels of lipid peroxidation, whereas SG-HWE class IV preparations, containing 12.5 μg of the mature bark, exhibited the lowest levels (Figure 2A). SG-HWE prepared from January and April class IV bark prevented 99%

Table 3. Composition of Hot Water Extractives (HWE) from Sweetgum (SG) Bark, For Different Tree Classes and Sampling Datesa Time of harvest Components HWE yield (g/kg)

Total carbohydrates (%)c

Total phenolics (%)d,c

Total organic acids (%)c

Tree classb

January

April

September

I II III IV I II III IV I II III IV I II III IV

74.0 ± 3.9 53.4 ± 1.2 44.8 ± 6.7 39.5 ± 1.6 27.5 ± 0.2 26.5 ± 1.0 27.6 ± 0.4 24.7 ± 0.8 27.6 ± 2.1 22.8 ± 0.3 22.6 ± 1.7 42.3 ± 1.7 3.4 ± 0.4 2.5 ± 0.2 6.9 ± 0.9 12.1 ± 2.1

53.6 ± 1.5 74.3 ± 7.4 31.1 ± 2.3 92.4 ± 0.4 30.9 ± 1.0 33.5 ± 2.6 30.7 ± 0.2 33.1 ± 0.4 25.6 ± 1.7 23.6 ± 0.6 16.4 ± 2.3 30.8 ± 0.9 8.9 ± 0.3 7.4 ± 0.7 2.3 ± 0.1 5.0 ± 0.6

89.6 ± 0.97 85.6 ± 15.7 44.9 ± 15.2 31.1 ± 5.9 37.7 ± 0.6 34.3 ± 0.2 32.9 ± 0.2 36.5 ± 0.3 32.1 ± 0.2 25.2 ± 0.7 28.7 ± 0.9 24.7 ± 1.0 7.3 ± 0.3 10.6 ± 1.5 7.0 ± 0.3 9.2 ± 0.4

a

Averages and standard deviations are provided for N = 3. bDiameter at breast height of tree class I- 1.7 cm, class II- 6.2 cm, class III- 27.1 cm, class IV- 32.9 cm. c(w/w) Oven dry weight of SG-HWE. dTotal phenolics were measured in tannic acid equivalent.

Variations in Antimicrobial Activity. Both LP and SGHWE exhibited antimicrobial activity against the S. aureus cocktail, averaging at 39% and 42% inhibition, respectively, compared to that of the positive control (Figure 1A,B). Interestingly, the total antimicrobial activity of LP and SGHWE remained constant irrespective of tree maturity and ̆ tree (Liquidambar orientalis) harvest season. The HWE of Sigla bark exudates was reported to produce an inhibition zone of 14 mm against S. aureus, at a concentration of 100 g/L.30 In this study, incubation of S. aureus cocktail with LP-HWE (300 g/L) and SG-HWE (150 g/L) resulted in average inhibition zones of 12 and 13 mm, respectively.

Table 4. Phenolic Acids Composition of Hot Water Extracts (HWE) from Loblolly Pine Bark and Sweetgum Barka Sweetgum harvest time

Loblolly pine harvest time

HWE components

Tree classb

January

April

September

Shikimic acid (%)

I II III IV I II III IV I II III IV

15.8 ± 0.3 14.6 ± 0.1 15.0 ± 0.1 17.6 ± 1.1 8.9 ± 0.1 7.6 ± 0.1 8.3 ± 0.1 7.0 ± 0.5 7.3 ± 0.2 6.8 ± 0.1 5.8 ± 0.0 6.4 ± 0.2

14.8 ± 0.1 14.3 ± 0.1 13.7 ± 0.1 15.2 ± 0.2 7.1 ± 1.0 6.1 ± 0.0 6.8 ± 0.2 7.0 ± 0.1 6.4 ± 0.0 5.3 ± 0.1 6.3 ± 0.1 6.4 ± 0.0

13.6 ± 0.2 13.4 ± 0.2 12.8 ± 0.2 14.3 ± 0.2 6.1 ± 0.0 5.9 ± 0.2 5.8 ± 0.2 6.3 ± 0.2 6.3 ± 0.2 6.1 ± 0.2 5.4 ± 0.1 6.4 ± 0.2

Gallic acid (%)

Vanillic acid (%)

January

5.9 4.8 5.9 5.7

± ± ± ±

0.1 0.0 0.0 0.0

April

4.3 5.4 4.1 4.2

± ± ± ±

0.2 0.2 0.0 0.1

September

5.3 4.3 3.2 4.2

± ± ± ±

0.2 0.1 0.0 0.1

a

Averages and standard deviations are provided for N = 3. Percentage calculations were based on oven dry weight of HWE. bDiameter at breast height of tree class I- 1.7 cm, class II- 6.2 cm, class III- 27.1 cm, class IV- 32.9 cm. 4262

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering

LP-HWE exhibited variable reductions in LDL oxidation (Figure 2B). HWE prepared from Monterey pine (Pinus radiata) bark have been reported to exhibit high antiperoxidation and free radical (DPPH·, O2−) scavenging capabilities, which were attributed to the presence of monomeric polyphenols, oligomeric and polymeric proanthocyanidins.36 Polymeric proanthocyanidins, especially procyanidins, found in HWE of pine bark was reported to quench the oxygen free radicals and delay the formation of the hydroperoxide ion, which is responsible for lipid peroxidation during the TBARS assay.10,36 Both the harvest season and tree maturity had a significant effect on LP-HWE’s antiperoxidation activity and there was a significant interaction between the two factors (p < 0.05). All January bark, classes I to IV, provided a significant reduction (88%) in TBARS concentration compared to the copper induced control. LP-HWE prepared from April and September bark was not as effective in reducing the LDL oxidation levels, as compared to the January material. Characterization of Bark Phytochemicals. SG-HWE prepared from class IV January material showed the highest total phenolic content, exhibited S. aureus inhibition and displayed the highest reduction in TBARS concentration. In an effort to isolate the compounds responsible for SG-HWE’s antimicrobial potential and antiperoxidation activity, SG-HWE prepared from class IV January bark material was subjected to centrifugal partition chromatography (CPC) fractionation. The CPC solvent system composed of ethyl acetate, ethanol and water, has been reported to successfully fractionate shikimic acid from SG-HWE.19 In this study, four different fractions were consolidated after CPC separation, which are highlighted in the CPC chromatogram (Figure 3A); each fraction had distinct phenolic profiles, as determined by UPLC analysis. CPC fraction #1 contained at least 30% shikimic acid, fraction #2 showed 40% vanillin, fraction #3 had 50% p-hydroxybenzoic acid, and fraction #4 held 10% salicylic acid. All CPC fractions also contained some carbohydrates; estimation using phenolsulfuric acid method showed that fractions 1, 2, 3 and 4 contained 27%, 10%, 7% and 2% of total carbohydrates, respectively. All four CPC fractions were analyzed for their respective antimicrobial activity against S. aureus cocktail at 1.5 mg each (Figure 3B). It was determined that fractions #1, 2 and 3 had antimicrobial activities similar to that displayed by the original crude SG-HWE, whereas fraction 4, containing among others salicylic acid, exhibited no antimicrobial activity. SG-HWE fractions #1, 2 and 3 exhibited at least 50% inhibition compared to that of the positive control, which was 1.5 mg of streptomycin sulfate. Shikimic and p-hydroxybenzoic acids have already been established as potential antimicrobial components of SG-HWE. Vanillin has been reported as one of the major components present in SG bark storax and has also been reported to inhibit bacterial growth by destroying their cell membrane integrity.30,37 Presence of vanillin in natural SG-HWE was demonstrated by CPC fractionation, establishing this compound as one of the antibiotic components of SGHWE. Thus, it can be concluded that the antibiotic potential of SG-HWE was imparted by, but not limited to, shikimic acid, vanillin and p-hydroxybenzoic acid. The CPC fractionated SGHWE was also tested for their antiperoxidation potential, at 1.25 μg each (Figure 3C). All SG-HWE fractions exhibited at least 75% reduction in TBARS concentration, with fraction #2 exhibiting the highest reduction of 99% (Figure 3C). CPC fraction #2 was rich in vanillin, which has been reported to

Figure 1. Disc diffusion activity of hot water extractives (HWE) from (A) sweetgum bark (SG) (3 mg) and (B, C) loblolly pine bark (LP) (6 mg) for different seasons and tree size classes (indicated by diameter at breast height or DBH). Whole bark extractives were tested against (A, B) S. aureus and (C) L. monocytogenes, S. enterica and E. coli cocktails. Positive control was 3 mg streptomycin sulfate solution. Means and standard deviations, n = 3.

of LDL peroxidation, as compared to the control. Interestingly, SG-HWE from classes I, II and III bark extractives did not exhibit significant differences in their potential to reduce TBARS concentrations (Figure 2A). SG-HWE have been previously reported to completely inhibit lipid peroxidation, at concentrations of 12.5 g/L, but the SG-HWE was not characterized for harvest season or tree maturity.19 Phenolic acids identified in SG-HWE, such as gallic and vanillic acids, may be responsible for the observed antiperoxidation activity. Gallic acid was reported to completely inhibit human LDL peroxidation, even at concentrations as low as 0.01 g/L.19 Vanillic acid was also reported to provide mild suppression of TBARS concentrations in mice blood plasma.35 Other than gallic and vanillic acids, there may be other phenolic compounds in SG-HWE that conferred the observed antiperoxidation effect. 4263

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering

Figure 2. TBARS assay to test antioxidant potential of hot water extractives (HWE) from (A) 12.5 μg sweetgum bark (SG) and (B) 55 μg loblolly pine bark (LP) for different seasons and tree size classes (indicated by DBH or diameter at breast height). Control contained only phosphate buffer at pH 7.4. Bars not connected by same letters were significantly different (n = 3, p < 0.05).

Figure 3. (A) Centrifugal partition chromatogram (CPC) of sweetgum bark extractives (Jan harvest, Class IV- 32.9 cm DBH), at 254 nm. (B) Disc diffusion activity of the CPC fractions at 1.5 mg against S. aureus cocktail, where the control was 1.5 mg streptomycin sulfate. (C) Antiperoxidation (TBARS) effect of CPC fractions, at 1.25 μg, where the control was human LDL induced by copper for 24 h. Bars not connected by same letters were significantly different (n = 3, p < 0.05).

produce significant reduction in the formation of TBARS reactive substances in mice brain and liver, thus enhancing its longevity.38

phytochemicals and, depending on season, different products can be maximized. Overall, antimicrobial and antioxidant properties of HWE did not vary significantly as a function of tree maturity, but varied as a function of seasonal development. Knowledge of seasonal variations in the composition of bark HWE is critical for establishing a forestry supply chain and for the feasible extraction of phytochemicals.



CONCLUSION Analysis of SG-HWE showed that there were no significant differences in their shikimic acid, gallic acid and vanillic acid content, across all harvested seasons and tree maturities. In the case of LP-HWE, vanillin and vanillic acid contents were significantly higher in January bark, while p-hydroxybenzoic acid was significantly higher in April bark; no significant differences were observed with respect to LP tree classes. This work demonstrated that LP and SG-HWE contained valuable



AUTHOR INFORMATION

Corresponding Author

*D. J. Carrier. Email: [email protected]. Phone: (865) 9747305. 4264

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering ORCID

ical potentials of polyphenolic bark extracts from Canadian forest species. PharmaNutrition 2013, 1 (4), 158−67. (13) Rihn, B.; Saliou, C.; Bottin, M. C.; Keith, G.; Packer, L. From ancient remedies to modern therapeutics: Pine bark uses in skin disorders revisited. Phytother. Res. 2001, 15 (1), 76−8. (14) Lingbeck, J. M.; O’Bryan, C. A.; Martin, E. M.; Adams, J. P.; Crandall, P. G. Sweetgum: An ancient source of beneficial compounds with modern benefits. Pharmacogn. Rev. 2015, 9 (17), 1−11. (15) Pandey, A. K.; Ojha, V.; Yadav, S.; Sahu, S. K. Phytochemical evaluation and radical scavenging activity of Bauhinia variegata, Saraca asoka and Terminalia arjuna barks. Res. J. Phytochem. 2011, 5 (2), 89− 97. (16) Soni, U.; Brar, S.; Gauttam, V. K. Effect of seasonal variation on secondary metabolites of medicinal plants. Int. J. Pharm. Sci. Res. 2015, 6 (9), 3654−3662. (17) Blanche, C. A.; Lorio, P. L. J.; Sommers, R. A.; Hodges, J. D.; Nebeker, T. E. Seasonal cambial growth and development of loblolly pine: Xylem formation, inner bark chemistry, resin ducts, and resin flow. For. Ecol. Manage. 1992, 49, 151−65. (18) Sharma, S.; Adams, J. P.; Sakul, R.; Martin, E. M.; Ricke, S. C.; Gibson, K. E.; Carrier, D. J. Loblolly pine (Pinus taeda L.) essential oil yields affected by environmental and physiological changes. J. Sustain. For. 2016, 35, 417−430. (19) Hurd, S. Sweetgum bark: Extraction, purification, and determination of antioxidant activity. Thesis, University of Arkansas, Fayetteville, AR, 2012. (20) Janero, D. R. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biol. Med. 1990, 9 (6), 515−40. (21) Uppugundla, N.; Engelberth, A.; Vandhana Ravindranath, S.; Clausen, E. C.; Lay, J. O.; Gidden, J.; Carrier, D. J. Switchgrass water extracts: Extraction, separation and biological activity of rutin and quercitrin. J. Agric. Food Chem. 2009, 57 (17), 7763−70. (22) Abbott, J. A.; Medina-Bolivar, F.; Martin, E. M.; Engelberth, A. S.; Villagarcia, H.; Clausen, E. C.; Carrier, D. J. Purification of resveratrol, arachidin-1, and arachidin-3 from hairy root cultures of peanut (Arachis hypogaea) and determination of their antioxidant activity and cytotoxicity. Biotechnol. Prog. 2010, 26 (5), 1344−51. (23) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of sugars, byproducts, and degradation products in liquid fraction process samples; Report NREL/TP-51042623; National Renewable Energy Laboratory: Golden, CO, 2008. (24) Spácǐ l, Z.; Nováková, L.; Solich, P. Analysis of phenolic compounds by high performance liquid chromatography and ultra performance liquid chromatography. Talanta 2008, 76 (1), 189−99. (25) Ainsworth, E. A.; Gillespie, K. M. Estimation of total phenolic content and other oxidation substrates in plant tissues using FolinCiocalteu reagent. Nat. Protoc. 2007, 2 (4), 875−7. (26) Buysse, J.; Merckx, R. An improved colorimetric method to quantify sugar content of plant tissue. J. Exp. Bot. 1993, 44 (10), 1627−9. (27) Cranswick, A. M.; Rook, D. A.; Zabkeiwicz, J. A. Seasonal changes in carbohydrate concentration and composition of different tissue types of Pinus radiata trees. New Zeal. J. For. Sci. 1987, 17 (2,3), 229−245. (28) Pearl, I. A. Water-soluble and petroleum ether-soluble extractives of loblolly and slash pine barks. Tappi 1975, 58 (7), 142−145. (29) Li, Y.-X.; Lin, H.-W. Comparative study on capillary gas chromatographic analysis of organic components of wood vinegar and water-extract of wood-tar prepared from hard wood barks. Fenxi Kexue Xuebao 2012, 28 (1), 58−62. (30) Sagdic, O.; Ö zkan, G.; Ö zcan, M.; Ö zçelik, S. A study on inhibitory effects of sigla tree (Liquidambar orientalis Mill. var. orientalis) storax against several bacteria. Phytother. Res. 2005, 19, 549−51. (31) Bai, J.; Wu, Y.; Liu, X.; Zhong, K.; Huang, Y.; Gao, H. Antibacterial activity of shikimic acid from pine needles of Cedrus

Danielle Julie Carrier: 0000-0003-3322-4660 Present Addresses §

Andrew Nelson, Department of Forest, Rangeland, and Fire Sciences, University of Idaho, 875 Perimeter Dr. MS 1133, Moscow, Idaho 83844-1133. ⊥ Joshua P. Adams, School of Forestry, Louisiana Tech University, P. O. Box 10138, Ruston, Louisiana 71272. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors are grateful for Dr. William Headlee’s contribution in providing the sweetgum bark and loblolly pine bark samples from the School of Forestry & Natural Resources, University of Arkansas, in Monticello, AR. The authors thank the Arkansas Science & Technology Authority (ASTA) for the research grant #15-B-33, which funded this study. The authors also acknowledge the Plant Powered Production (P3) Center. P3 is funded through the RII: Arkansas ASSET Initiatives (AR EPSCoR) I (EPS-0701890) and II (EPS-1003970) by the National Science Foundation and the Arkansas Science and Technology Center. The authors thank Dr. Corliss O’Bryan, Department of Food Science, University of Arkansas, Fayetteville, for graciously providing the bacterial pathogens used in this study.



REFERENCES

(1) Wear, D. N.; Prestemon, J.; Huggett, R. Southern Forest Futures Project. In The Southern Forest Futures Project Technical Report; Wear, D. N., Greis, J. G., Eds.; U.S. Department of Agriculture Forest Service, Southern Research Station: Ashville, NC, 2013; pp 183−212. (2) Smith, B. W.; Miles, P. D.; Perry, C. H.; Pugh, S. A. Forest Resources of the United States, 2007; U.S. Department of Agriculture, Forest Service, Washington Office: Washington, DC, 2009. (3) Harkin, J. M.; Rowe, J. W. Bark and its possible uses; U.S. Department of Agriculture Forest Service: Madison, WI, 1971. (4) Oswalt, S. N.; Smith, W. B.; Miles, P. D.; Pugh, S. A. Forest Resources of the United States, 2012: a technical document supporting the Forest Service 2015 update of the RPA Assessment; U.S. Department of Agriculture, Forest Service, Washington Office: Washington, DC, 2014. (5) Eberhardt, T. L. Impact of industrial source on the chemical composition of loblolly pine. For. Prod. J. 2012, 62 (7/8), 516−519. (6) Djioleu, A. C.; Martin, E. M.; Pelkki, M. H.; Carrier, D. J. Sugar yields from dilute acid pretreatment and enzymatic hydrolysis of sweetgum. J. Agric. Food Anal. Bacteriol. 2012, 2 (3), 175−186. (7) Bochkov, D. V.; Sysolyatin, S. V.; Kalashnikov, A. I.; Surmacheva, I. A. Shikimic acid: Review of its analytical, isolation, and purification techniques from plant and microbial sources. J. Chem. Biol. 2012, 5 (1), 5−17. (8) Dai, J.; Mumper, R. J. Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules 2010, 15 (10), 7313−52. (9) Martin, E.; Duke, J.; Pelkki, M.; Clausen, E. C.; Carrier, D. J. Sweetgum (Liquidambar styracif lua L.): Extraction of shikimic acid coupled to dilute acid pretreatment. Appl. Biochem. Biotechnol. 2010, 162 (6), 1660−8. (10) Ku, C.-S.; Jang, J.-P.; Mun, S.-P. Exploitation of polyphenol-rich pine barks for potent antioxidant activity. J. Wood Sci. 2007, 53 (6), 524−8. (11) Hamaguchi, M.; Cardoso, M.; Vakkilainen, E. Alternative technologies for biofuels production in Kraft pulp millsPotential and prospects. Energies 2012, 5 (7), 2288−309. (12) Royer, M.; Prado, M.; García-Pérez, M. E.; Diouf, P.-N.; Stevanovic, T. Study of nutraceutical, nutricosmetics and cosmeceut4265

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266

Research Article

ACS Sustainable Chemistry & Engineering deodara against Staphylococcus aureus through damage to cell membrane. Int. J. Mol. Sci. 2015, 16 (11), 27145−55. (32) Guenther, E. Oil of Styrax. In The Essential Oils; D. Van Nostrand Company Inc.: New York, 1952; Vol. 17. (33) Cho, J. Y.; Moon, J. H.; Seong, K. Y.; Park, K. H. Antimicrobial activity of 4-hydroxybenzoic acid and trans 4-hydroxycinnamic acid isolated and identified from rice hull. Biosci., Biotechnol., Biochem. 1998, 62 (11), 2273−6. (34) Naz, S.; Ahmad, S.; Rasool, S. A.; Sayeed, S. A.; Siddiqi, R. Antibacterial activity directed isolation of compounds from Onosma hispidum. Microbiol. Res. 2006, 161 (1), 43−48. (35) Kumar, S.; Prahalathan, P.; Raja, B. Antihypertensive and antioxidant potential of vanillic acid, a phenolic compound in LNAME-induced hypertensive rats: a dose-dependence study. Redox Rep. 2011, 16 (5), 208−15. (36) Ku, C.-S.; Mun, S.-P. Antioxidant properties of monomeric, oligomeric, and polymeric fractions in hot water extract from Pinus radiata bark. Wood Sci. Technol. 2008, 42 (1), 47−60. (37) Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Ueckert, J.; Bos, A.; Narbad, A. Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua. J. Appl. Microbiol. 2004, 97 (1), 104−13. (38) Chou, T. H.; Ding, H. Y.; Hung, W. J.; Liang, C. H. Antioxidative characteristics and inhibition of alpha-melanocytestimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Exp. Dermatol. 2010, 19 (8), 742−50.

4266

DOI: 10.1021/acssuschemeng.7b00243 ACS Sustainable Chem. Eng. 2017, 5, 4258−4266