Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species Brajesh N

Journal of Medicinally Active Plants Volume 2 Issue 3 Vol 2 Issues 3-4

January 2014 Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species Brajesh N. Vaidya Fort Valley State University, [email protected]

Terri A. Brearley Fort Valley State University, [email protected]

Nirmal Joshee Fort Valle State University, [email protected]

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Recommended Citation Vaidya, Brajesh N.; Terri A. Brearley; and Nirmal Joshee. 2014. "Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species." Journal of Medicinally Active Plants 2, (3):42-49. DOI: 10.7275/R5J9649K https://scholarworks.umass.edu/jmap/vol2/iss3/4

This Article is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Journal of Medicinally Active Plants by an authorized editor of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

Journal of Medicinally Active Plants Volume 2 Issue 3 Vol 2 Issues 3-4

October 2014 Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species Brajesh N. Vaidya Fort Valley State University, [email protected]

Terri A. Brearley Fort Valley State University, [email protected]

Nirmal Joshee Fort Valle State University, [email protected]

Follow this and additional works at: http://scholarworks.umass.edu/jmap

Recommended Citation Vaidya, Brajesh N., Terri A. Brearley, Nirmal Joshee. 2014. "Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species," Journal of Medicinally Active Plants 2(Vol 2 Issues 3-4):42-49. DOI: https://doi.org/10.7275/R5J9649K Available at: http://scholarworks.umass.edu/jmap/vol2/iss3/4

This Article is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Journal of Medicinally Active Plants by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Scutellaria Species Brajesh N. Vaidya, Terri A. Brearley, and Nirmal Joshee* College of Agriculture, Family Sciences and Technology, Fort Valley State University, Fort Valley, GA 31030 *Corresponding author: [email protected]

Date received: November 15, 2013

Keywords: Bioactive compounds, flavonoid, free radical, polyphenol.

ABSTRACT native to China, and S. barbata, from Korea, have The antioxidant capacity of 16 Scutellaria been extensively employed in traditional Chinese species was examined using Trolox equivalent medicine (TCM) and in Japanese Kampo medicine antioxidant capacity (TEAC) assay. Total poly- (JKM) (Watanabe et al., 2002; Murch et al., 2004). phenol, antioxidant capacity estimation and Leaf extract from S. ocmulgee has demonstrated flavonoid content measurements were conducted inhibitory properties against malignant gliomas on fresh and air dried leaf extracts. The highest (Parajuli et al., 2009, 2011). total polyphenol content was obtained in dry leaf Plants, such as basil (Ocimum spp.), mint extracts of S. ocmulgee at 732.41 67 mg/g of gallic (Mentha spp.), rosemary (Rosmarinus officinalis), acid equivalent. Dry leaf extracts of S. ocmulgee lavender (Lavandula spp.), and Baikal skullcap registered 2480.93 μmol/g of Trolox equivalent (Scutellaria baicalensis), are known to contain antioxidant capacity and the highest total relatively high levels of phenolics and have flavonoid content with 200.63 μg/mL from fresh demonstrated antioxidant activity (Zheng and Wang, leaf extracts of S. alpina. Rosemary (Rosmarinus 2001; Shao et al., 2004; Waisundara, 2010; officinalis), a common herb with known high Atanassova and Georgieva, 2010). Leaves from antioxidant potential, was used as a standard for peppermint (M. x piperita), rosemary, sage (Salvia comparison with the Scutellaria species. spp., spearmint (M. spicata), and thyme (Thymus spp. growing in a greenhouse exhibit total polyphenol INTRODUCTION (TPP) and Trolox equivalent antioxidant capacity The genus Scutellaria, commonly known as (TEAC) as plants grown under field conditions, plus skullcap (scullcap) belongs to the family Lamiaceae anti-tumorigenic activity against colon cancer cells (Mint family). Of the 400 known Scutellaria species, (Yi and Wetzstein, 2010; 2011). over 90 of these plants have been recorded as Free radicals can cause disorders, such as growing in North America. In the current study, 20 atherosclerosis, central nervous system injury, and Scutellaria spp. that grow in and adjacent to the state gastritis in the human body (Kumpulainen and of Georgia were used (Joshee et al., 2002). Salonen, 1999; Pourmorad et al., 2006). By boosting Scutellaria ocmulgee and Scutellaria montana, two the human immune system, plant-based antioxidants species that grow in Georgia, are relatively rare and block free radicals produced through oxidation protected by the state and federal government (Schuler, 1990), thus inhibiting chain reactions that (Chafin, 2007). could lead to degradation and death of cells (Pratt, Scutellaria species are known for their potential 1992; Velioglu et al., 1998; Hu and Willett, 2002). pharmaceutical/therapeutic properties and have been The antioxidant activity delays and inhibits oxidation used in the traditional medicine of many countries for of cellular components and molecules (Nijveldt et al., their anti-inflammatory, antioxidant, and anti-viral 2001). Thus, the level of activity of various phyto- properties useful in treating human ailments (Huang chemicals is important for the evaluation of their et al., 2005b; Shang et al., 2010). S. baicalensis, potential health benefit to humans.

Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

The consumption of a diet rich in natural anti- Constituent extraction. For extraction, the tissue oxidants has been associated with a reduced risk of samples were homogenized in a 50 mL capacity human illnesses, such as cardiovascular problems, chilled mortar using a pestle kept at -20 oC (by inflammation, neurodegenerative diseases, and some submerging in liquid nitrogen for 2 h) to facilitate cancers (Kong et al., 2003; Beretta et al., 2009). A grinding and minimize any degradation of bioactive recent study suggests that presence of wogonin in constituents. The homogenized leaf powder was Scutellaria extract plays a key role in the biochemical transferred to a 125 mL Erlenmeyer flask containing pathways leading to anti-cancer properties (Patel et 50 mL of HPLC grade 100% methanol (Burdick and al., 2013). Jackson, USA). The flasks were left overnight (18 h Of the several classes of plant secondary at 28.5 oC) in the dark on an orbital shaker at 200 metabolites known as antioxidants, phenolics, the RPM (Benchmark Mini Incu-Shaker, Edison, NJ, major antioxidants are classified into six groups: USA). The suspension was then transferred to 50 mL simple phenolics and phenylpropanoids, coumarins, Falcon tubes (BD, Franklin Lakes, NJ, USA) to be lignans, quinones, tannins, and flavonoids. The centrifuged at 2057 x g (Eppendorf centrifuge 5810 flavonoids consist of five sub-groups: antho- R, Brentwood, NH) at 25 oC for 40 min. The cyanidins, flavones and flavonols, flavanones, supernatant was collected and the remaining pellet catechins, and leucoanthocyanidins and proantho- was extracted again for 1 h, with 25 mL of methanol. cyanidins based on their chemical structures (Jedinak Table1. Natural distribution of Scutellaria species used in the et al., 2004). More than 295 flavonoid compounds present study. have been isolated from 35 Scutellaria species Dis Distribution Species (Shang et al., 2010). S. drummondii & S. elliptica, Major flavonoids in Scutellaria species are North America S. incana & S. integrifolia, apigenin, luteolin, scutapins, and diterpenoids S. montana & S. ocmulgee, (scutalpin C), neo-clerodanes, chrysin, iridoids, S. ovata & S. suffrutescens isoscutellarein, wogonin, baicalin, and baicalein. Central & South America S .costaricana & S. racemosa These polyphenolic compounds are known to Europe S. albida, S. alpine, & scavenge free radicals, inhibit hydrolytic and S. altissima oxidative enzymes, and act in anti-inflammatory East Asia S. baicalensis & S. barbata pathways (Frankel, 1995). In this study, the South Asia S. angulosa & S. scandens polyphenol content and antioxidant capacity of leaf After the second extraction, the two extracts extracts of Scutellaria species were analyzed and were combined and the pellets were discarded. The compared. combined extract was filtered through a double layer of Whatman filter paper No. 2 (GE Healthcare Life MATERIALS AND METHODS Sciences, Piscataway, NJ, USA ) and stored in air Plant material. A total of 16 Scutellaria tight 50 mL Falcon tubes at 4 oC in the dark, until species, grown and maintained in a greenhouse at analysis of the extracts, following the procedures Fort Valley State University, Fort Valley, Georgia, optimized by Vaidya (2013) for various Scutellaria were used in this study (Table 1). Rosemary species. (Rosmarinus officinalis) leaves collected locally were Extract analysis. Total polyphenol content used fresh and dried as comparison standards for was determined by the Folin-Ciocalteu reagent antioxidant capacity. For each species of the method (Lowry et al., 1951) as modified by Yi and Scutellaria and for the rosemary, two samples (2 g Wetzstein (2010) for the herbs in the Lamiaceae each) of leaf tissue were randomly collected from family. Gallic acid (3, 4, 5-trihydroxybenzoic acid) multiple plants, cleaned, and weighed. One sample (Sigma Life Science, USA) was used to develop was extracted fresh while the second sample was o standard reference points as outlined by Singleton dried at room temperature (25 C) in the dark for and Rossi, 1965. A spectrophotometer (Nanodrop seven days, weighed, and then extracted. 2000c, Thermo Scientific, USA) was used to read the 43

Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

solution absorbance at 765 nm. For each sample, five 0.02 was achieved, 2970 µL of ABTS was re- replicates were measured at 20 sec intervals. Total measured. To the same measured ABTS, 30 µL of a phenolic content was expressed as mg gallic acid Scutellaria species extract was added and then equivalent/g dry or fresh extract (GAE mg/g). remeasured after 6 min. The measurements from all Total flavonoid content was evaluated using the extracts were plotted against Trolox standards for aluminum chloride (AlCl3) colorimetric method percent inhibition at 6 min. Calculation of anti- (Chang et al., 2002). A standard solution (1 mg oxidant capacity was expressed as percent inhibition AlCl3/mL) was prepared by dissolving HPLC grade according to the equation: quercetin dihydrate (Alfa Aesar, UK) in 80% ethanol. This solution was further diluted with 80% ethanol to % Inhibition = [(AbsControl – AbsSample)/AbsControl] × 100 provide concentrations of 10, 25, 50, 80, 100 and 125 Where AbsControl is the absorbance of the control reaction μg/mL concentrations in distilled water. 0.5 mL of (containing all reagents except the test compound), diluted solutions were mixed with 1.5 mL of 95% AbsSample is the absorbance of the test compound, and % inhibition is the inhibition of ABTS absorbance by TROLOX. EtOH, 0.1 mL of 10% AlCl3, 0.1 mL of 1 M potassium acetate (CH3CO2K) and 2.8 mL of distilled Rosemary, a plant known to have a high water in 50 mL beakers separately and incubated at antioxidant capacity (Yi and Wetzstein, 2011), was 25 ºC for 30 min. Absorbance was read at 415 nm used as a standard to establish a comparative measure using spectrophotometer to generate the standard of the medicinal potential of the tested Scutellaria curve where regression equation is determined by species on the basis of TPP content and TEAC assays. y=128x + 4.5545. Test solutions included a mix of Total polyphenol measurements. The TEAC 0.5 mL of plant extracts with 1.5 mL of 95% EtOH, assay (Re et al., 1999; Yi and Wetzstein, 2010) was 0.1 mL of 10%, 0.1 mL of 1 M CH3CO2K and 2.8 used to measure the antioxidant capacity of the mL distilled water. These solutions with fresh and dry polyphenols, as compared with the standard Trolox. extract samples were also incubated at 25 ºC for 30 Measurements of TEAC utilized the ABTS decolori- min and measurements were taken at 415 nm. The zation assay where the change in color was directly samples were run in triplicate and blank was prepared proportional to the concentration of antioxidant present without the addition of AlCl3. The absorbance data in the liquid (Huang et al., 2005a). The intensity of was then converted to total flavonoid content using color in these assays were measured with a standard curve generated by quercetin dihydrate. spectrophotometer to provide a numerical value of total Antioxidant capacity measurement. Trolox equi- polyphenol and antioxidant capacity of fresh and dry valent antioxidant capacity (TEAC) assay of a sample extracts. Redox properties of polyphenol prevent was calculated based on the inhibition of radical peroxide decomposition to free radicals (Li et al. 2009). cation absorption exerted by the standard TROLOX Statistical analysis. All data are presented as solution (6-hydroxy-2, 5, 7, 8-tetramethy-chroman-2- means ± SE for at least three replications for each carboxylic acid) (Sigma-Aldrich, USA), a vitamin E sample. Statistical analysis was based on two way analogue (Davies et al., 1988). A 7 mM ABTS analysis of variance (ANOVA) with results at P≤ solution [2, 2’-azinobis (3-ethylbenzothiazo-line-6- 0.05 level and means were separated using Tukey’s sulfonic acid) diammonium salt] (Sigma-Aldrich, post-hoc mean separation test. USA) was mixed with 6.6 mg of potassium persulfate (Fisher Scientific, USA) to make final concentration RESULTS of 2.45 mM. This ABTS radical solu-tion was Fresh and dry leaf extracts of the Scutellaria incubated in the dark at 25 ºC for 16 h, and then species and rosemary exhibited large variations in diluted with ethanol (EtOH) to get an optical density antioxidant capacity (Table 2). In fresh extracts, TPP (OD) A734 =0.70 ± 0.02 with spectrophoto-meter, results ranged from 52.11 to 281.93 mg/g gallic acid Nanodrop 2000c using a disposable cuvette equivalent (GAE). In dry extracts, two species, S. (Plasibrand, NJ, USA). Once the OD A734 =0.70 ± drummondii and S. incana, failed to register any TPP

Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc

values. In the other species, TPP values ranged between both fresh and dry extracts. Total polyphenol content 50.56 and 281.93 mg/g GAE in S. albida to 732.41 mg/g in the fresh leaf sample of S. ocmulgee was 281.93 GAE in S. ocmulgee. Both S. ocmulgee and S. mg/g GAE whereas dry leaf sample contained montana, exhibited a relatively high TPP content in 732.41.

mg/g GAE. In S. montana, fresh leaf and dry leaf Table 2. Polyphenol content, antioxidant activity, and flavonoid content of Scutellaria species. extract had 250.24 and 630.67 mg/g GAE, respectively. Rosemary fresh and dry leaf extracts used as a standard for comparison had 175.51 and 325.28 mg/g GAE.

Means ± standard error; those means followed by the same letter within a column are not significantly different, P ≤ 0.05; NA*= no sample.

Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc Flavonoid content. Total flavonoid content of These values were among the highest of all the leaf extracts ranged between 73.93 µg/mL for S. samples tested in the current study. On the contrary, ovate to 200.63 µg/mL for S. alpine in fresh leaf S. drummondii, registered the lowest values for both extracts. In dry leaf extracts, flavonoid content fresh and dry leaf extracts, 16.99% and 9.38%, ranged from 21.29 µg/mL in S. drummondii to 170.80 respectively. µg/mL in S. ocmulgee. S. angulosa, which grows at The TEAC values obtained ranged from 234.88 2000 m above sea level in the Central Himalayas, had µmol/g (dried leaf extract of S. drummondii) to a high flavonoid content in both fresh and dry leaf 2,480.93 µmol/g (dried leaf extract of S. ocmulgee). extracts with 167.87 µg/mL for dry and 122.75 Next to S. ocmulgee, S. montana along with R. µg/mL for fresh leaf extracts. Similarly, S. baicalensis officinalis had the highest measured TEAC values in also exhibited high flavonoid content of 157.87 the dried extract. In the fresh tissue extracts, S. µg/mL in fresh leaf and 135.55 µg/mL in dry leaf ocmulgee with 1701.64 µmol/g and S. montana with extract. 1630.04 µmol/g extracts were the Scutellaria species Antioxidant capacity measurement. The with the highest TEAC values. percent inhibition exhibited against Trolox by Of the three test assays, the TPP concentration Scutellaria leaf extracts ranged from 9.38% to in fresh tissue and TEAC value for antioxidant 99.24%. In case of S. ocmulgee fresh and dry leaf capacity in fresh tissue exhibited a strong correlation extracts, average inhibition values were 68.07% and (Table 3). Significant correlation was also observed 99.24%, respectively, and for S. montana 65.20% for for TPP fresh, TPP dry, and TEAC fresh with the fresh and 76.14% for dry leaf extract, respectively. flavonoid levels in dry tissue.

Table 3. Pearson’s correlation coefficients for antioxidant capacity of Scutellaria species.* Species* TPP Fresh TPP Dry TEAC Fresh TEAC Dry FLAV Fresh FLAV Dry

Species 1.00000 0.24459 TPP Fresh 1.00000 0.3441 0.16498 0.65721 TPP Dry 1.00000 0.5568 0.0078 0.04772 0.86689 0.62131 1.00000 TEAC Fresh 0.8557 <.0001 0.0134 0.10240 -0.10473 -0.18025 -0.22532 TEAC Dry 1.00000 0.6957 0.6891 0.5203 0.3846 -0.35783 0.49601 0.13553 0.63366 -0.26573 FLAV Fresh 1.00000 0.1585 0.0429 0.6301 0.0063 0.3026 -0.00190 0.64458 0.79487 0.73609 -0.02040 0.53054 FLAV Dry 1.00000 0.9942 0.0052 0.0004 0.0008 0.9381 0.0285 *Includes the R. officinalis sample and significance level

DISCUSSION S. montana (Chafin, 2007). Plants are endowed with complex antioxidant S. ocmulgee, which registered one of the highest activity that protects them against oxidative damage. antioxidant values, is the most effective Scutellaria This study demonstrates that among Scutellaria species against glioma cells (Parajuli et al., 2009, species the antioxidant capacity can vary con- 2011, Dandawate et al., 2012). S. lateriflora has siderably. Differences in total polyphenol content, anxiolytic properties (Awad et al., 2003) and thus has Trolox equivalent antioxidant capacity, and total been used as sedative and for treating nervous flavonoid content in the fresh and dried leaf extracts disorders (Islam et al., 2011). Specific flavonoids affected antioxidant levels. The relatively high levels isolated from the leaf extracts of Scutellaria species of TPP, TEAC, and flavonoid content detected in S. are known to have specific medicinal properties ocmulgee and S. montana emphasize the need for (Shang et al., 2010). conservation of the lesser known, rare, and threaten- Other species, such as lemon balm (Melissa ed species of Scutellaria, such as S. ocmulgee and officinalis, oregano (Origanum vulgare), marjoram 46

Vaidya et al.: Antioxidant Capacity of Fresh and Dry Leaf Extracts of Sixteen Sc (Origanum majorana) and rosemary, are used in whereas, higher TPP values were measured in dry Traditional Chinese Medicine (TCM) and Ayurveda leaf extract from S. montana and S. ocmulgee. medicine practices because of noted beneficial effects These differences between fresh and dry tissues on human health (Lutomski, 2001; Capecka et al., could due to differences in the quantity and type of 2005). The polyphenolic compounds produced by flavonoid groups within the various Scutellaria these and other plants exhibit high levels of species and/or the thermostability of flavonoid groups antioxidant activity (Rohman et al., 2010). Similarly, during leaf drying and extract preparation. Under- extensive work with S. baicalensis and S. lateriflora standing the relationship among antioxidant activity, in relation to their chemical constituents and anti- flavonoids, plant species, and extraction may help oxidant properties, have been published (Li, et al., clarify differences in biological activity. A 2012; Shao et al., 2004). Previous studies conducted relationship between high antioxidant activity and by our group using S. ocmulgee leaf extract have therapeutic properties has been established for some shown specific anti-tumor activity against gliomas plant materials (Kong et al., 2003; Beretta et al., through inhibition of Akt, GSK-3 αβ, and NF-kB 2009). Understanding the anti-oxidative mechanisms phosphorylation in rat tumors (Parajuli et al., 2009, within plants and the effects of post-harvest 2011). environments and extraction methodologies could Hence, a positive correlation between higher lead to the development and improvement of amounts of TPP, TEAC, and flavonoid content can be medicinal plant materials for human health. associated with potential medicinal property of a plant extract. The current study with Scutellaria ACKNOWLEDGMENT supports the concept that determining the quantitative The authors acknowledge with thanks to Dr. and qualitative elucidation of polyphenol levels in Hazel Y. Wetzstein, and her lab members at the plant extracts will most likely enable connection of University of Georgia. This research was done with plants to various medicinal properties. Our study financial support from a USDA-NIFA Capacity with several Scutellaria species demonstrated a Building Grant (CSREES Award No. 2011-38821- positive correlation between TPP and TEAC values. 30928) (PI: Dr. N. Joshee). Extracts with the highest antioxidant activity showed the highest flavonoid content, indicating an important REFERENCES relationship found among these variables. Atanassova, M. and S. Georgieva. 2010. Comparative Qualitative and quantitative analyses of major polyphenol composition and antioxidant individual phenolics in plant extracts could be helpful capacity of the Bulgarian plants (Dry herbs). in explaining the relationship between phenolic content Electronic J. Environ. Agri. Food Chem. and antioxidant capacity within a species. In a variety 9:1514-1523. of medicinal plants, Djeridane et al., (2006) and Awad, R., J. T. Arnason, V. Trudeau, C. Bergeron, J. Katalinic et al., (2006) have demonstrated a linear W. Budzinski, B. C. Foster, and Z. Merali. correlation between the content of total phenolic 2003. Phytochemical and biological analysis of compounds and antioxidant capacity. Others, skullcap (Scutellaria lateriflora L.): a (Capecka et al., 2005; Wong et al., 2006), however, medicinal plant with anxiolytic properties. have indicated a poor linear correlation between Phytomedicine 10(8):640-649. antioxidant activity and phenolic content. Beretta, G., G. Rossoni, N. Alfredo Santagati, and R. Observed differences among the presence of Maffei Facino. 2009. Anti-ischemic activity and polyphenolics and antioxidant activity in our study endothelium-dependent vasorelaxant effect of with Scutellaria appeared to be due to the use of fresh hydrolysable tannins from the leaves of or dry tissue. In fresh tissue extracts, TPP content Rhuscoriaria (Sumac) in isolated rabbit heart and positively correlated with TEAC activity, but in dry thoracic aorta. Planta Med. 75(14):1482-1488. tissue extracts, TPP did not correlate with TEAC of Capecka, E., A. Mareczek, and M. Leja. 2005. the Scutellaria species. Although some variability Antioxidant activity of fresh and dry herbs of was apparent among species as fresh extract had a some Lamiaceae species. Food Chem. 93:223- higher value of TPP in S. alpine and S. costaricana, 226.

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