Effects of volumetric soil water content and fertilizer rate on growth and baicalin accumulation in two species of Scutellaria

Department of Environmental Horticulture, Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Apopka, Florida, USA. Department of Plant Pathology, Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Apopka, Florida, USA. Department of Environmental Horticulture, Fort Lauderdale Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Davie, Florida, USA. Department of Entomology and Nematology, Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, Apopka, University of Florida, Florida, USA.


INTRODUCTION
Plants of the Scutellaria genus are found around the globe and are prized for their medicinal qualities.In many Asian countries, S. baicalensis or Baikal skullcap, is prescribed to patients as an anti-inflammatory, antiviral and antithrombotic medicine (Shang et al., 2010).In the United States, S. lateriflora or American skullcap, is sold for its reported anxiolytic properties.Commercial sales and marketing for medicinal applications of S. lateriflora exist despite the lack of research documenting the presence of medicinally active compounds within the plant (Shang et al., 2010).Medicinal benefits of Scutellaria spp.are attributed to flavonoids present in vegetative and root tissue of most species.Flavonoids found in many species of Scutellaria include baicalin, baicalein and wogonin (Similien et al., 2016).Baicalin, one of the main flavonoids found in many species of Scutellaria, is valued for its anxiolytic effects without any sedative or myorelaxant effects (Liao et al., 2003;Xu et al., 2006).In the United States, skullcap has not been evaluated by the Food and Drug Administration as a medicine and thus has been designated as a herb of "undefined safety" (Awad et al., 2003).Nonetheless, dried skullcap shoots are commonly found in the market as an ingredient in tea, as a vitamin, and as liquid extract.Two species of Scutellaria, native to United States, Florida, S. integrifolia and S. arenicola, possess potential for commercial production; however, neither of them have been evaluated for growth and performance or baicalin content when cultivated within a greenhouse production environment.
Scutellaria spp.containing high concentrations of baicalin had high market value (Similien et al., 2016).Production of plant secondary metabolic compounds, such as baicalin, are believed to be produced by plants to protect plant cellular tissue, especially when plant stress is experienced (Kumar and Pandey, 2013).Plant stress experienced in natural, undisturbed environments are believed to result in greater secondary compound synthesis (Shippmann et al., 2002).When cultivated in protected commercial environments, medicinal plants are thought to produce decreased concentrations of secondary compounds.Relationships between nutrient availability and synthesis of baicalin may be explained by the Carbon-Nutrient Balance Hypothesis (CNBH) (Hamilton et al., 2001).The hypothesis predicts production of secondary metabolic compounds when deficiencies in carbon and nitrogen exist (Hamilton et al., 2001).Flavonoid development within Scutellaria spp.has been reported to occur in response to water and nutrient availability.Cao et al. (2012) found that baicalin concentrations in S. baicalensis were negatively correlated with nitrogen fertilizer application.Similarly, Similien et al. (2016) observed negative correlations between baicalin concentration and nutrient availability in S. lateriflora.Yuan et al. (2012) observed increased flavonoid concentrations in root and leaf tissue of S. baicalensis when subject to mild water stress (12% soil water content) as compared to a control that received sufficient water (16% soil water content).These results are in contrast to findings by Similien et al. (2016), who observed increased baicalin concentrations in S. lateriflora when irrigation was applied to plants cultivated on open field.When cultivated under 40% shaded conditions, however, field-grown S. lateriflora flavonoid concentrations were not significantly influenced by irrigation applications.
Rising popularity and interest in herbal medicines, coupled with the unknown influence of greenhouse production practices on plant growth and synthesis of the flavonoid baicalin within two Scutellaria spp., supports the need for research on this specialty crop.The objective of this study was to investigate the influence of nutrient application and plant available water on growth and baicalin synthesis of two native North American skullcap species, S. integrifolia and S. arenicola, within a protected greenhouse production environment.Results from this experiment can be used to develop cultivation practices for these potentially valuable medicinal plant species.
Agawam, MA, United States) and placed on a bench in a 30.5 x 14.6 m gutter-connected greenhouse with 30% light reducing polycarbonate paneling located in Apopka, Florida, United States (latitude 28°38' N, longitude 81°33' W).Environmental conditions within the greenhouse were measured and recorded every 15 min by a data logger (WatchDog 2475; Spectrum Technologies, Inc., Aurora, IL, United States).Forty-eight cuttings of S. arenicola were taken on 5 July 2017 and subject to the same propagation methods previously described for S. integrifolia.Due to poor rooting of this species, only 32 cuttings of S. arenicola were transplanted on 28th July 2017.
To quantify baicalin concentrations from plants found growing in a natural, undisturbed habitat (Lake County, FL, United States), field-harvested samples of S. integrifolia and S. arenicola were collected in August 2017.Given the commercial popularity of S. latiflora as an anxiolytic herbal medicine, samples of S. latiflora leaves were purchased from a local herbal medicine market (Leaves & Roots; Orlando, FL, United States).

Volumetric water content treatment
Volumetric water content (VWC) was measured and recorded every 60 min throughout the experiment utilizing soil moisture sensors connected to a data logger (Em50; Decagon Devices, Pullman, WA, United States).Throughout WAT 1 to 3, substrate moisture was maintained among all plants at 56.2% VWC.Three weeks after transplant, half of the plants for each fertilizer treatment (n=6) for S. integrifolia were selected at random and subjected to reduced VWC of 9.9% to induce water stress.For S. arenicola, half of the plants for each fertilizer treatment (n=4) were selected at random and subjected to reduced VWC of 9.9% 4 WAT on 21st August 2017.An additional week of growth prior to implementation of reduced VWC for S. arenicola was provided to allow plants to reach sufficient size.

Measurements and data collection
Beginning from 1 WAT, plant growth indices (PGI = height × width 1 × width 2 ) were recorded weekly and continued until termination of the experiment 5 WAT. S. integrifolia were terminated 2nd August 2017 (5 WAT) when plants began flowering.Plants were destructively harvested by cutting the plant 2.5 cm above the crown and mass was recorded using a laboratory balance (Adventurer Analytical; Ohaus, Parsippany, NJ, United States).Plants were dried at room temperature until ≥ 70% moisture was lost and dry Morgan et al. 99 mass yield (DMY) was recorded.Dried plant material from the same treatment group were combined and vacuum-sealed for storage until ready for baicalin analysis using high performance liquid chromatography (HPLC).S. arenicola were also cultivated for five weeks and destructively harvested on 1 September 2017.Plant DMY was recorded and all replicates of each treatment were combined and vacuum-sealed for storage until HPLC analysis could be completed.

Preparation of leaf and stem extracts
Aliquots were prepared by randomly selecting a 0.1 g sample from the previously vacuum-sealed containers.

Standard calibration
Baicalin standard was purchased from Indofine Chemical Company (Hillsborough, NJ, United States).Solid baicalin standard was dissolved in 100% methanol to achieve a standard concentration of 250 µg mL -1 .Standard calibration was achieved by doing serial injection at rates of 1, 5, 10, and 15 µL and resulted in a calibration correlation of r 2 =0.99.

Statistical analysis
The experiment was arranged as a complete randomized design with irrigation and fertilizer application rate assigned as independent variables.Six replicates were cultivated for each treatment combination for a total of 48 experimental units for S. integrifolia.Four replicates were cultivated for each treatment combination for S. arenicola.Statistical analysis of PGI, DMY and baicalin was analyzed using mixed model analysis in JMP ® Pro 13 (SAS; Cary, NC) with post-hoc mean separation tests performed using Tukey's honest significant difference test by WAT with variance within treatment combination replicates defined as the random error term.Statistical tests were considered significant if P ≤ 0.05.

Greenhouse environmental conditions
Mean daily greenhouse temperature ranged from 24.0 to 33.6°C throughout the duration of the experiment.Mean relative humidity was 79.8% and photosynthetic active radiation varied from 59.6 to 311.6 µmol m -2 s -1 .

Plant growth index
No significant effects were observed for PGI among fertilizer or water stress treatments for S. integrifolia between 1 and 4 WAT.Plant growth was minimal between 1-2 WAT; however, beginning from 3 WAT, growth was quadratic (Figure 2).Plants subjected to the medium fertilizer application rate had the highest PGI, between 2 and 5 WAT.At termination of the experiment, 5 WAT, a water stress effect was observed (Figure 3).Least square means of PGI for plants subjected to high VWC were significantly greater (5.8 e -3 m -3 ) for plants that were maintained at a high VWC than those subjected to low VWC (2.6 e -3 m -3 ).No significant effects were observed for PGI among fertilizer or water stress treatments for S. arenicola throughout the duration of the experiment.Moreover, PGI did not increase between between 1 and 5 WAT (data not shown).Plants that received the high fertilizer rates, regardless of water stress treatment, had the highest PGI at termination of the experiment with a least square mean of 4.1 e -5 m 3 .Plants that received medium rate applications of fertilizer possessed the lowest overall PGI 5 WAT with a least square mean of 2.8 e -5 m 3 .

Plant dry mass
No significant differences among treatment combinations were observed for DMY of S. integrifolia (Figure 4).Plants that received medium fertilizer application rates and subjected to high VWC had highest dry mass (1.8 g).
Lowest DMY (0.7 g) was recorded for S. integrifolia that received no fertilizer and subjected to low VWC.Similar to results observed for S. integrifolia, no significant differences among treatment combinations were observed for S. arenicola (Figure 4).S. arenicola that received low fertilizer application rates and subjected to high VWC had the highest DMY of 0.11 g.Contrary to observations for S. integrifolia, S. arenicola that received medium fertilizer application rates and subjected to high VWC had the lowest DMY at 0.08 g.

Baicalin concentration
Baicalin concentrations ranged from 0.801 to 1.383 mg g plant shoot tissue -1 (Table 1).A fertilizer and water stress interaction was observed for baicalin concentrations of S. integrifolia.High concentrations of baicalin were observed in plants that received low and medium application rates of fertilizer, regardless of water stress treatment.S. integrifolia that received no fertilizer and subjected to low VWC possessed a high mean baicalin concentration (1.383 mg baicalin g plant shoot tissue -1 ); however, plants that received the same fertilizer treatment (none) but were maintained at high VWC, possessed significantly less mean baicalin (0.870 mg g plant shoot tissue -1 ).Based on DMY, greatest baicalin yield was observed when S. integrifolia received medium rate applications of fertilizer.When high rates of fertilizer were used during cultivation of S. integrifolia, low concentrations of baicalin were observed within the plant shoot tissue regardless of water stress treatment.Baicalin concentrations in S. arenicola ranged from 1.861 to 2.899 mg g plant shoot tissue -1 (Table 1).Baicalin concentrations resulting from each treatment combination were approximately 2-fold greater than those for similar treatment combinations for S. integrifolia; however, limited plant mass resulted in insufficient replication to perform statistical mean separation tests among imposed treatments.Greatest baicalin concentrations were observed for S. arenicola that received high fertilizer application rates and subjected to low VWC.Given poor growth, baicalin yield of S. arenicola was low (0.2 to 0.3 mg).
Field harvested S. integrifolia and S. arenicola had mean baicalin concentrations of 1.457 and 2.348 mg g plant shoot tissue -1 , respectively (Table 1).Baicalin concentrations were similar to those cultivated within the  z Volumetric water content (VWC) maintained at high (56.2%)or low (9.9%) rates for weeks after transplanting of 3 to 5 and 4 to 5 for S.
x Means (n=6) within column with the same letter are not significantly different (P ≤ 0.05; Tukey's honest significant difference test).w Means (n=4) of baicalin concentrations from plants collected from a natural, undisturbed habitat (Lake County, FL, United States) in August 2017.
v Means (n=4) of baicalin concentrations from dried plant shoot material purchased from a commercial store (Shoots & Roots; Orlando, FL, United States).
greenhouse and subjected to experimental treatments.S. lateriflora, purchased at a local medicinal herbal store, had low concentrations of baicalin 0.274 mg g plant shoot tissue -1 and were similar to concentrations observed for S. integrifolia that received high rates of fertilizer and high VWC (0.273 mg g plant shoot tissue -1 ).

DISCUSSION
S. arenicola responded poorly to clonal propagation.Unlike S. integrifolia, which displayed logarithmic growth during the five-week study, S. arenicola did not increase in plant mass.Poor growth and performance of S. arenicola suggests that commercial cultivation techniques imposed in this study were not ideal for this species.Despite poor growth, however, baicalin concentrations in this species were approximately 2-fold greater than those observed in S. integrifolia.Detection of baicalin in S. arenicola has not been reported before.Relatively high concentrations of baicalin observed in S. arenicola warrant additional investigations to better understand relationships between plant growth and baicalin synthesis.
Increased fertilizer application rate, from none (0 g) to low (3.8 g) and medium (9.2 g), resulted in increased concentration of baicalin in both S. integrifolia and S. arenicola, regardless of VWC treatment.In contradiction to this trend, high fertilizer application rates (13.8 g) resulted in low baicalin concentrations in both S. integrifolia and S. arenicola, with exception of S. arenicola subjected to the low VWC treatment.Decreased baicalin concentration in response to increased application of fertilizer rates has been observed in both S. baicalensis and S. lateriflora (Cao et al., 2012;Shiwakoti et al., 2016;Similien et al., 2016).Results observed in the current investigation largely contradict these findings, thus suggest relationships between nutrient and water availability and baicalin concentration are likely species dependent, and cannot be predicted simply by application of the CNBH.Empirical studies are therefore necessary to establish these relationships and assist in development of recommended commercial production practices.
Both S. integrifolia and S. arenicola cultivated in the current study were found to possess similar concentrations of baicalin to plants found growing in local, undisturbed natural environments.Results, therefore, do not support the assertion that medicinal plants produced within protected commercial environments will unremittingly possess lower concentrations of secondary metabolic compounds, such as baicalin, due to decreased exposure to environmental stressors.Baicalin concentrations within all cultivated plants in this study were found to be greater than concentrations observed within the limited, medicinal commercial samples of S. lateriflora analyzed in this investigation.Although, not an objective of this investigation, a more diverse sampling of commercially available Scutellaria spp.medicinal products would help define variability that exists in the marketplace.Given its relatively rapid growth rate coupled with high baicalin concentration, S. integrifolia likely possess qualities that would allow it to be successfully produced commercially for medicinal application.More specifically, production practices implemented in this investigation (medium application rates of fertilizer coupled with sufficient volumetric water content) provide a foundation for successful production of high yielding plant material that possess relatively high concentrations of baicalin.

Conclusion
Production of secondary metabolic compounds such as baicalin are believed to decrease in response to applications of fertilizer and when plants are cultivated within protected commercial greenhouse environments.Results of this investigation, however, showed that greenhouse cultivation of S. integrifolia and S. arenicola did not result in decreased production of baicalin as compared to plants obtained from local, undisturbed natural environments.Moreover, applications of fertilizer increased synthesis of baicalin in majority of the imposed treatments.S. integrifolia exhibited fast growth, reaching reproductive stage by five weeks, and responded well to commercial clonal propagation techniques.Medium fertilizer application rates and high VWC produced S. integrifolia with high baicalin yield.Although, S. arenicola exhibited poor growth trends throughout this study, high concentrations of baicalin present within this species support future studies.Empirical studies that examine the relationships between horticultural production techniques and plant growth and response, such as those presented here, are necessary for the establishment of commercial production practices for medicinally important plant species.

Figure 1 .
Figure 1.High performance liquid chromatography of baicalin illustrating retention time of 1.887 min.

Figure 3 .
Figure 3. Least square means of plant growth indices (PGI) of S. integrifolia (n=6) in response to volumetric soil water content maintained at either high or low volumetric water content (VWC) of 56.2 and 9.9%, respectively.

Figure 4 .
Figure 4. Means of shoot dry mass yield of S. integrifolia (n=6) and S. arenicola in response to application of Osmocote 15-9-12 slow release fertilizer (Everris NA Inc., Dublin, OH, United States) applied at one of four rates [0 g (none), 3.8 g (low), 9.2 g (medium), or 13.8 g (high)] and maintained at either high or low volumetric water content (VWC) of 56.2 and 9.9%, respectively.