Comparisons of Scutellaria baicalensis, Scutellaria lateriflora and Scutellaria racemosa: Genome Size, Antioxidant Potential and Phytochemistry

• Abstract
• Introduction
• Materials and Methods
• Plant collection and establishment in vitro
• Chromosome counts in Scutellaria racemosa
• Flow cytometry analysis
• Sample harvest and preparation
• Antioxidant potential
• Quantification of medicinal compounds
• Column and separation
• Experimental design and statistical analysis
• Results
• Discussion
• References

Abstract

The genus Scutellaria in the family Lamiaceae has over 350 species, many of which are medicinally active. One species, Scutellaria baicalensis, is one of the most widely prescribed plants in Traditional Chinese Medicine, used for neurological disorders, cancer and inflammatory diseases and has been the subject of detailed scientific study but little is known about the phytochemistry of other Scutellaria. The current study was designed to compare the medicinal phytochemistry of 3 species of Scutellaria used to treat neurological disorders. To accomplish this objective, the specific objectives were (a) to establish an in vitro collection of the South American native; S. racemosa, (b) to botanically characterize S. racemosa and (c) to compare the phytochemistry of S. racemosa with S. baicalensis and S. lateriflora. S. racemosa was established in vitro from wild populations in Florida. Botanically, S. racemosa is diploid with 18 chromosomes, and flow cytometry data indicated that S. baicalensis and S. racemosa have small nuclei with estimated small genomes (377 mbp and 411 mbp respectively). Antioxidant potential studies showed that there were no significant differences in the 3 Scutellaria species. Phytochemical analyses detected and quantified the flavonoids baicalin, baicalein, scutellarin, and wogonin as well as the human neurohormones melatonin and serotonin in leaf and stem tissues from S. baicalensis, S. lateriflora, and S. racemosa. These findings represent the first phytochemical analysis of S. racemosa and establish S. racemosa as a model system for study of medicinal plant secondary metabolism and as a potential source of important phytopharmaceuticals for treatment of human disease.

Introduction

The genus Scutellaria includes more than 350 species worldwide that range from Siberia to South, Central and North America as well as Japan, much of Europe and Asia. Scutellaria has been used by indigenous cultures across vast geographical distances to treat a variety of different ailments, including neurological disorders, chronic ailments, hepatitis, cirrhosis, jaundice, and anxiety [1]. Scutellaria species are a source of a wide range of phytochemicals with more than 200 identified to date (reviewed in [1]). Scutellaria baicalensis (Georgi.), commonly known as Huang-qin or wogon, is the most commonly prescribed plant in Traditional Chinese Medicine (TCM) and also extensively used in Japanese Kampo medicines [2]. In the scientific literature, there are more than 275 scientific reports of the effectiveness of S. baicalensis extracts in reducing prostate cancer, inhibition of proliferation of human hepatoma cell lines, inhibition of liver fibrosis, and reduction of symptoms of Type 1 allergic reactions (reviewed in [1]). Another species, S. lateriflora, also known as scullcap or mad-dog, has a long history of use by native North American peoples to stimulate blood flow, to reduce nervous tension and to treat mental or neurological illnesses (reviewed in [1]). Currently, skullcap is sold in North America as a tea, tonic or capsules made from dried aerial parts, and is used to treat epilepsy, neurological damages from bacterial infections, insomnia, anxiety, neuralgia, and withdrawal from tranquilizers or barbiturates [3]. Aqueous extracts of S. lateriflora have been shown to reduce anxiety levels of rats [4], and a recent study found that extracts bind to the serotonin 5-HT7 receptor [5]. Recently, a Central and South American Scutellaria species, Scutellaria racemosa, has been shown to have neuroprotective activity in a stressed animal model system and ethnobotanical evidence of use by the indigenous people of the Cauca Valley range of Columbia and Ecuador as a neurologically active plant [6].

The common types of uses of Scutellaria species by indigenous peoples across vast geographic distances and the similarities of reports of efficacy in animal models suggest the potential for common or conserved neurochemistry in plants of this species. In 1997, S. baicalensis was found to contain high concentrations of neurotransmitters [7]. More recently, the non-selective cation channel agonist and antidepressant hyperforin, previously thought to be characteristic of the medicinal plant St. John’s wort, was also found in the S. baicalensis leaves [8] but the vast majority of phytochemical studies of S. baicalensis have focused on flavonoid compounds with more than 60 different compounds reported in the literature [1], [9]. The flavonoids are of interest for their anti-cancer and antioxidant activity [10], but previous research has not investigated whether these medicinally active phytochemicals are common to species within the Scutellaria genus and may be linked to the medicinal activity of these other species.

The overall objective of the current study was to elucidate the phytochemical basis of the common traditional knowledge of use of the species by comparing the chemical composition of Scutellaria grown as axenic cultures under identical nutritional, growth regulator and environmental regimes. The specific objectives were (a) to collect Scutellaria racemosa from wild populations that have appeared in invasive populations in Florida [11], (b) to establish S. racemosa in in vitro cultures under the same conditions as S. lateriflora and S. baicalensis, (c) to generate botanical information about S. racemosa to supplement the limited information that was available about the species [12] and (d) to identify and quantify melatonin, serotonin, baicalin, baicalein, wogonin and scutellarin in S. racemosa and S. lateriflora for comparison with the phytochemical profiles of S. baicalensis.

Materials and Methods

Plant collection and establishment in vitro

S. baicalensis stock cultures were initiated from commercially available seed (Richter’s Greenhouses Inc) as described previously [13]. S. lateriflora cultures were established using a slightly modified procedure which included surface sterilization of seeds (obtained from Richter’s Greenhouses Inc.), germination on water-agar (0.8 %) with 4 mL/L Plant Preservative Material (PPM; Plant Cell Technology Inc) and incubation in total darkness at 24 °C [13]. The resulting etiolated seedlings were transferred to Magenta boxes containing 25 mL of culture medium (pH 5.75) consisting of MS salts [14], B5 vitamins [15], 3 % sucrose and solidified with 0.3 % gellan gum (hereafter referred to as MSO). The media were autoclaved at 121 °C, 1.4 kg/cm² and stored for at least two days to ensure sterility prior to subculture initiation. The plantlets were maintained by regular subculture of nodal segments on MSO, every three weeks. All cultures were maintained in a controlled consistent environment with a 16-h photoperiod under cool-white light (22 - 45 μmol m^-2 s^-1) at 28 °C.

S. racemosa was collected in December 2005, from three locations in North and Central Florida as listed on herbarium vouchers from the University of South Florida’s Atlas of Florida Vascular Plants database [16]. Stem segments and seeds were collected from Leon, Orange, and Nassau counties. Stem segments were collected and surface sterilized with a 20 % aqueous solution of 5.4 % sodium hypochlorite for 30 min, thoroughly rinsed with distilled water, and placed onto MSO. Surface sterilized tissues were subcultured onto fresh MSO medium in Phytotech P700 Culture Boxes (Phytotechnology Laboratories) and placed in a growth chamber with a 16-h photoperiod under cool-white light (22 - 45 μmol m^-2 s^-1) at 25 °C. Shoot apices were subcultured every three weeks. All cultures were transferred to MSO media devoid of growth regulators for 3 weeks prior to bioassays and chemical analysis.

Chromosome counts in Scutellaria racemosa

Shoot cultures of S. racemosa were transferred to MSO supplemented with 5 μM of indole-3 acetic acid (IAA) for rapid root proliferation. Small growing tips of individual roots were harvested after 4 weeks and immediately immersed into cold Carnoy′s fixative (75 % ethanol/25 % glacial acetic acid). Tissues were fixed for 24 hours and then stored for up to six weeks in a solution of 70 % ethanol. Root tips were placed onto a microscope slide and gently squashed with forceps to separate cells. Chromosomes were stained with acetocarmine (0.5 % carmine in 45 % glacial acetic acid) and observed by light microscopy.

Flow cytometry analysis

The genetic complement of the three species was determined by flow cytometry analysis using two-week-old Brassica napus cv. Topaz seedlings as an internal reference standard [17], and nuclei were isolated using the method previously optimized by Alan et al. [17]. Briefly, 50 mg of tissue from actively growing tissues were homogenized with or without an equivalent amount of leaf tissue from 2-week-old B. napus in 1 mL of ice-cold nuclei isolation buffer (NIB: 15 mM HEPES, 1 mM EDTA, 80 mM KCl, 20 mM NaCl, 300 mM sucrose, 0.2 % Triton X-100, 0.5 mM spermine, 1 % polyvinylpyrrolidone (PVP), with pH adjusted to 7.5). The homogenate was filtered through a 37 μm nylon filter into a 1.5-mL Eppendorf tube and centrifuged at 8000 × g for 5 sec. The supernatant was decanted and the nuclei pellet was resuspended in 300 μL of NIB with 25 μg/mL RNase. Nuclei were stained by addition of 10 μL of propidium iodide (1 mg/mL) per sample prior to analysis. Samples were analyzed with a Coulter EPICS Elite ESP Flow Cytometer (Beckman-Coulter, Inc.) equipped with an argon laser emitting at 488 nm using a 610 nm band pass filter. The instrument was checked for linearity with fluorescent Flow Check beads (Beckman-Coulter) and the amplification was adjusted to position the 2C peak of S. baicalensis nuclei approximately at channel 100. Five nuclei samples were prepared from each of the Scutellaria species and 5,000 to 10,000 nuclei were analyzed from each sample. Quantification was accomplished by comparison of the preparations with and without the B. napus tissues and using the formula below:[]

Sample harvest and preparation

Approximately 0.5 g of stem and leaf tissue was aseptically harvested from S. baicalensis, S. lateriflora, and S. racemosa cultures, flash frozen, dried to complete dryness under nitrogen gas, accurately weighed and transferred to a 50-mL beaker. A 10-mL aliquot of methanol: 0.45 % formic acid (40 : 60) was added to each sample and extraction was completed by sonication in an ultrasonic bath (Bransonic 1510R-MT, 42 kHz, Branson Ultrasonic Co) for 45 min. The extraction was repeated twice. The supernatant was decanted and centrifuged (Hamilton Bell Co., Inc., Model 1500) at 4500 × g for 10 min. The resulting extract was filtered through a 0.45 μm filter (Millipore). For quantification, repeated injections of 50 μL were made under each of the optimized detection conditions.

Antioxidant potential

Antioxidant potential was determined as described previously [18]. Shoot tissue (30 - 40 mg) was accurately weighed and macerated for 5 min in 0.5 mL 50 % ethanol. Particulate matter was pelleted from the extract by centrifugation for 3 min at 13,000 rpm (Eppendorf Centrifuge 5415 C, Brinkmann Instruments Inc.). A series of diluted extracts were prepared and the reaction was started by combining 0.5 mL of each test sample with 0.5 mL of 200 μmol L^-1 of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Free radical generation was monitored by colour development at 520 nm and the volume of sample extract required to cause a 50 % decrease in the absorbance at 520 nm relative to the control (100 %) was determined.

Quantification of medicinal compounds

Certified analytical standards of melatonin, serotonin and indole-3-acetic acid were purchased from Sigma Chemical Co. Certified standards of baicalin, baicalein, scutellarin, and wogonin were purchased from NIBPCP. The purity of the standards was greater than 98 % and single peaks with individual molecular weights were identified during reverse phase chromatography of each of the compounds ([Fig. 1]). The solvents used included HPLC-grade methanol and acetonitrile (VWR Scientific) and AR grade formic acid (Sigma Chemical Co.).

Column and separation

All separations were performed with an Alliance series high-performance liquid chromatographic system (Waters Inc.) coupled with a Premier series LCT MS/MS (Waters Inc.) and controlled with a MassLynx V4.0 Data Analysis System described previously [19]. Compounds were separated with an Xterra C₁₈ HPLC column (2.1 × 1000 mm, 3.5 μm; Waters Inc.) heated to 30 °C with a gradient of 0.45 % formic acid: acetonitrile (0 - 5 min, 100 : 0 % v/v; 5 - 30 min, 100 : 0 - 0 : 100 % v/v; 30 - 45 min, 0 : 100 % v/v; 45 - 46 min, 0 : 100 - 100 - 0 % v/v). The medicinal metabolites were eluted at 0.25 mL/min over a 60 min period and detected within the MS/MS in ESI positive mode using optimized parameters for each metabolite [19].

The method detection limit (LOD) of compounds was defined as the analyte concentration producing a signal of at least 2 times the height of the noise while the limit of quantification was defined as the lower limit of the linear range of quantification. A standard, high linearity (r² = 0.99) was obtained for all quantification ranges for the metabolites. The lower limits of detection of melatonin, serotonin, and IAA were 4.92 × 10 ^{- 5} ng mL^-1, 4.63 × 10 ^{- 4} ng mL^-1, and 4.82 × 10 ^{- 5} ng mL^-1, respectively with recovery at 98.4 %, 92.6 %, and 96.4 %. Lower limits of detection for the flavonoids baicalein, baicalin, wogonin, and scutellarin were 1.0 × 10 ^{- 6} ng mL^-1, 9.4 × 10 ^{- 6} ng mL^-1, 9.24 × 10 ^{- 5} ng mL^-1, 9.31 × 10 ^{- 5} ng mL^-1 respectively. Recovery was determined with authenticated standard at baicalein = 95.3 %, baicalin = 94.2 %, wogonin = 92.4 %, and scutellarin = 93.1 %.

Experimental design and statistical analysis

This experiment consisted of five replicate samples of each of three Scutellaria species injected into the analytical instrument. The whole experiment was repeated twice. Statistical differences were assessed by the Student-Newman-Keulls (SNK) means separation test based on a General Linear Model in SAS 9.0 (Statistical Analysis System Inc., 1995).

Results

The creation of a collection of axenic cultures of Scutellaria species grown in controlled environments facilitated comparative phytochemical analyses. These plant cultures were clonally propagated and grown on MSO media devoid of growth hormones prior to phytochemical analysis. S. baicalensis and S. lateriflora cultures have been described previously [8], [17], but there are no previous studies of S. racemosa. New in vitro collections of S. racemosa were established from material collected in Florida in 2005 ([Fig. 2] A). Bacterial and fungal contamination was restricted by extensive surface sterilization including 24 h soak in 1 mL/L PPM. De novo growth was visibly apparent after 20 hours in culture. Nodal cuttings cultured onto a medium devoid of growth regulators did not regenerate but supplementation of the culture medium with various combinations of IBA and BAP, ranging from 1 μmol/L to 20 μmol/L induced vigorous growth of S. racemosa. Nodal cuttings, with 2 opposite leaves, had the greatest survival, growth and continued differentiation when cultured on MSO medium supplemented with 1.0 μ μmol/L IBA and 2.5 μmol/L BAP ([Fig. 2] B). Cultures were maintained over a period of at least 6 months prior to chemical analysis and transferred to basal media devoid of growth regulators at least 3 weeks prior to chemical analysis ([Fig. 2] C).

Previous studies of S. racemosa were limited to taxonomical and descriptive studies [21], but much of the basic botanical information was unavailable. In the current studies, 18 chromosomes were visible in cytological examination of root tissue sections of S. racemosa ([Figs. 3] A and B). Flow cytometry was used to determine the size of the nucleus of S. racemosa (∼ 0.75 pg DNA/nucleus), S. baicalensis (∼ 0.84 pg DNA/nucleus) and S. lateriflora (∼ 1.95 pg DNA/nucleus) ([Table 1]). Nuclei of Scutellaria species showed sharp 2C peaks and small 4C peaks (10 - 17 % of total nuclei) which were often barely detectable above the debris ([Fig. 3] C). The simultaneous processing of internal reference standard did not change the positions of Scutellaria peaks [26]. Comparative studies of the Scutellaria results with the results from other plant samples prepared in an identical fashion showed that S. racemosa and S. baicalensis had significantly smaller nuclei and estimated genome size than other medicinal species as seen in [Table 1]. Using the measure and the equation described previously [17], the size of the genome of S. racemosa was estimated to be 377 mbp and S. baicalensis was estimated to have a genome of about 411 mbp.

Antioxidant potential was determined in clonally propagated tissues of S. baicalensis, S. lateriflora, and S. racemosa. All three species had equal capacity of detoxifying oxygen free radicals as determined by the DPPH bioassay ([Fig. 4]). Melatonin, serotonin, baicalin, baicalein, scutellarin, and wogonin were detected and quantified in shoot tissues of clonally propagated axenic cultures of S. baicalensis, S. lateriflora, and S. racemosa ([Figs. 1] and [5]). All three Scutellaria species were found to have melatonin ([Fig. 6] A) and serotonin ([Fig. 6] B). Significantly less serotonin was found in S. baicalensis than in the other two species ([Fig. 6] B) and serotonin levels were five-fold higher in S. lateriflora than S. baicalensis ([Fig. 6] B).

The flavonoids baicalin and baicalein were also detected and quantified in all three species of Scutellaria ([Figs. 7] A and B). There were no significant differences in the baicalin content but significantly more baicalein was quantified in tissues of S. lateriflora than was found in the other two species ([Fig. 7] B). Shoot tissues of S. racemosa had significantly less than baicalein than in either of the other species ([Fig. 7] B). Scutellarin was detected in the highest concentrations in S. baicalensis ([Fig. 7] C) with about 800 times the scutellarin concentration of S. racemosa and S. lateriflora ([Fig. 7] C). Wogonin was found in all three Scutellaria species and was present in significantly higher concentrations in S. racemosa than in the other two species ([Fig. 7] D).

Discussion

The establishment of an in vitro collection of plant material is the primary prerequisite for the development of high quality phytopharmaceuticals, for the investigation of the complex chemical profiles of medicinal plant species and for future studies to define the role of specific genes in plant secondary metabolism [20]. In the current study, in vitro techniques were used to establish a sterile, perpetual, axenic collection of S. baicalensis, S. lateriflora and S. racemosa. At the botanical, molecular, biochemical and genetic levels, virtually nothing is known about S. racemosa and very little is known about S. lateriflora. Chromosome counts of S. racemosa also show 18 distinct chromosomes and flow cytometry indicated that S. racemosa is diploid. The results are consistent with the published literature on S. baicalensis which also has 18 chromosomes [21]. The larger nucleus size of S. lateriflora is reflected in the literature describing this species as diploid with 88 chromosomes [22].

In this study, the antioxidant activity was determined to be statistically consistent across the 3 species of Scutellaria. Previous studies have reported the antioxidant activities of S. baicalensis [8] and S. lateriflora [23] but this is the first measure of the capacity of S. racemosa to detoxify reactive oxygen species (ROS). S. lateriflora was ranked among the most potent antioxidant species in a recent study of medicinal herbs [23] and previous researchers have hypothesized that the capacity of extracts of S. baicalensis to detoxify ROS species to reduce radical oxygen toxicity may contribute to the medicinal efficacy [24]. The indoleamines and the flavonoids measured in the phytochemical studies are potent antioxidants [24], [25].

The flavonoids baicalin, baicalein, scutellarin, and wogonin were detected and quantified in all 3 Scutellaria species. Baicalin and baicalein are the most commonly described flavonoids in S. baicalensis [26] but recently more than 20 other flavonoids have also been described [27]. Scutellarin is the main effective constituent of breviscapine, a cerebrovascular and cardiovascular drug. In our studies, the highest levels of scutellarin were found in S. baicalensis at levels of about 7 mg/g but significant quantities of scutellarin were not detected in the other species. In contrast, the highest concentrations of wogonin were found in S. racemosa. Wogonin is an antioxidant, neuroprotective, anti-inflammatory and potential anti-tumour compound with pharmaceutical potential [28].

The neurohormone indoleamines melatonin and serotonin were also detected and quantified in the 3 Scutellaria species. Prior to this study, the indoleamines had been measured only in S. baicalensis [7], [8]. The discovery that S. lateriflora and S. racemosa contain melatonin and serotonin may possibly explain earlier reports of S. lateriflora extracts to 5-HT7 receptors [5] and neuroprotective activity of S. racemosa [6]. Melatonin has been found in more than 100 other medicinal plants [29]. In human and animal model studies, melatonin was absorbed through the gut, circulated in blood and potentially affected human health (reviewed in [1]). However, there is no known role for melatonin in plant physiology (reviewed in [30]). It has been hypothesized that the role of melatonin in plant physiology may be analogous to its function in mammals as a chemical messenger of light and dark, calmodulin binding factor or an antioxidant [30] or that the high level of melatonin in medicinal plants such as S. baicalensis might be related to survival in northern latitudes, and may retard or lessen the effects of environmental stressors [30]. Melatonin may accumulate and detoxify ROS in plants exposed to heat or cold stress, drought, and some types of pollution [30].

This study is the first systematic investigation of the phytochemistry of S. racemosa, as it compares to other medicinal species within the genus Scutellaria. It is interesting to note that all of the medicinal constituents of the relatively well characterized S. baicalensis were also present in the 2 other Scutellaria species. Recent studies, including this work suggest that genera of medicinal plants may have conserved phytochemical diversity and a synergy of multiple bioactive molecules that ultimately provides the medicinal efficacy. Further studies of the complexity of medicinal plant secondary metabolism require detailed analysis of the role of specific genes in these biochemical pathways but the genetics of most medicinal plants is difficult to study because of the large genome size and complexity. Our studies with the nucleus of S. baicalensis and S. racemosa indicate that these two species maybe excellent model systems for future investigation.

The small genome size of S. racemosa and S. baicalensis coupled with the successful in vitro culture, diploid genome and extensive library of secondary metabolites [5] indicates that this species is a good potential as a model system for genomic studies. The complete sequence of the Arabidopsis thaliana and Oryza sativa genomes have created incredible resources for the study of plant physiology but for medicinal applications, the information provided in these databases may be insufficient. Medicinal plants have very unique secondary metabolism that may not be fully represented in either genome database and finding a suitable model system for study of a medicinal plant genome has been incredibly difficult. The ideal medicinal plant model genome will represent an active secondary metabolism with a wide range of products and a complex metabolome, coupled with a small genome size, short life cycle and the potential for development of phytochemical mutants.

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Susan J. Murch

Chemistry

University of British Columbia Okanagan

3333 University Way

Kelowna

British Columbia

Canada, V1V 1V7

Phone: +1-250-807-9566

Email: susan.murch@ubc.ca

References

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Susan J. Murch

Chemistry

University of British Columbia Okanagan

3333 University Way

Kelowna

British Columbia

Canada, V1V 1V7

Phone: +1-250-807-9566

Email: susan.murch@ubc.ca