Comparative Phytochemical Profiling and In Vitro Antioxidant Activity of Extracts from Raw Materials, Tissue-Cultured Plants, and Callus of Oroxylum indicum (L.) Vent.

Extracts from raw materials from different plant parts, tissue-cultured plants, and callus cultures of Oroxylum indicum were analyzed for in vitro antioxidant activities determined by DPPH radical scavenging assay and evaluated for phytochemical profiles by TLC and LC-MS methods. The results were analyzed by principal component analysis (PCA) to evaluate the similarity. Stalk, pedicel, flower, seed, and whole fruit and callus extracts promoted strong antioxidant activity with high total phenolic and total flavonoid contents. The main phytochemicals found in extracts were baicalin, baicalein, and chrysin. Baicalein and baicalin promoted strong antioxidant effects and existed in most extracts while chrysin, which promoted very low antioxidant activity, was a major flavonoid in the leaves and tissue-cultured plants. From PCA analysis by total phenolic and total flavonoid contents, four main clusters including callus and tissue-cultured plant groups from different growth stages, flower group, and whole fruit and leaf group could be organized. When the results were analyzed by PCA using antioxidant activity with total phenolic or total flavonoid contents, all O. indicum samples could be grouped together except the extracts from the root of tissue-cultured plants which separated from the rest due to their low phytochemical contents and weak antioxidant activities.


Introduction
Oroxylum indicum (L.) Vent. is a medium-sized, deciduous tree of the Bignoniaceae family. The tree has very large pinnate compound leaves. The outside petals are reddish purple with the pale yellow inside. Fruits are flat capsules, broad and sword shaped [1]. The seeds are numerous, flat-like papery wings [2]. Mature fruit is acrid and sweet, which promotes antihelminthic and stomachic effects [3]. The seeds have been used as purgative while the seed paste is applied to the throat for quick relief of tonsil pain [1,2]. Our previous study reported the in vitro antibacterial and antioxidant activities of O. indicum fruit extracts [4]. Furthermore, the young fruits and young flowers of this plant are popularly consumed as vegetable in the North of Thailand while the mature seeds are one composition in a traditional multiherb drink for the treatment of aphthous ulcer and sore throat. Some phytochemicals were reported from different parts of O. indicum such as flavonoids, anthraquinones, alkaloids, saponins, and fatty acids [4][5][6][7]. Despite every part of O. indicum being useful for both nutritional and medicinal applications, however, the source of O. indicum raw materials seems to be limited. Moreover, it takes some times for this plant to fully grow as a tree for its appropriate uses. Therefore, finding the methods to increase and develop new sources of plant raw material would be beneficial in the future. Plant tissue culture technique is widely used in the conservation and utilization of rare and endangered medicinal plants due to its remarkable ability of quickly increasing their biomass [8,9].
To our knowledge, no documents have ever been published about phytochemical profiles and antioxidant activities 2 Evidence-Based Complementary and Alternative Medicine of the raw materials from different plant parts, tissue-cultured plants, and callus by chromatographic and spectrophotometric methods. This study provides the information of in vitro free radical scavenging effects tested by the DPPH assay, TLC and LC-MS fingerprints, and total phenolic and total flavonoid contents of extracts from O. indicum from different sources including flowers, leaves and fruits raw materials, tissue-cultured plants from different growth stages, and callus. The obtained results were also analyzed for similarity by principal component analysis (PCA).

Plant Material Preparations
(1) Raw Materials from Different Plant Parts. The flowers (3 growth stages), pedicels, leaves, and stalks of O. indicum were collected from Bangkok province. The fruits for preparation of fruit and callus extracts were purchased from Chiang Rai province, while the seeds for preparation of tissue-cultures plants were purchased from Pattani province, Thailand, in 2016. Plant samples were identified by Assistant Professor Dr. Pongtip Sithisarn, Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Thailand. The plant samples were separately cleaned and dried in a hot air oven at 60 ∘ C and then ground using an electric mill.
(2) Tissue-Cultured Plants (In Vitro Plants). The seeds of O. indicum were cleaned using detergent and tap water flow for 15 min. Then they were sterilized by soaking in 70% ethanol for 30 sec followed by soaking in 1% sodium hypochlorite for 7 min. The sterilized seeds were washed by shaking in deionized water for 1 min, repeated for 5 times. Then the wings of the seeds were removed and the seeds were placed on sterilized MS (Murashige and Skoog) media (pH 5.8, 3% sucrose and 0.8% agar) [10]. Each media bottle contained 3 seeds of O. indicum. The bottles were kept in the dark until week 5 to induce germination and then they were moved to store under the photoperiod of 16/8 h (light/dark) until week 8. Plant samples were randomized using True Random Number Service software (Randomness and Integrity Services Ltd., Ireland) at days 3, 5, and 7 and then weekly from week 2 to week 8. Plant samples were cleaned and dried in a hot air oven at 60 ∘ C then ground using an electric mill. Plant samples from week 5 to week 8 were separated for the aerial and root parts before they were cleaned and dried.
(3) Callus. The seeds of O. indicum were separated from the fruits from Chiang Rai province and then they were washed with tap water for 15 min before sterilization with 70% ethanol for 1 minute. After that, the seeds were further sterile with 1% sodium hypochlorite for 8 minutes and then washed with sterile distilled water (5 times) to remove the remaining sterilization reagents. After the sterilization process, the wing part of the seeds was cut with sterile scissors in a laminar cabinet and the seeds then were placed in MS media supplemented with 3% sucrose and 0.8% agar before being stored in a dark place to induce the germination. After seven days, the germinated in vitro plants were moved to store under the photoperiod of 16/8 h (light/dark).
The in vitro plants were collected after 8 weeks of cultivation and excised to explants (2-3 cm long/piece). The explants were inoculated in MS media supplemented with 3% sucrose, 0.8% agar, 2 mg/L of 2,4-dichlorophenoxy acetic acid (2,4-D), and 1 mg/l of 6-benzyladenine (BA). After 21 days, the induced callus was cut from the explants and cultured into the mentioned media and then subcultured every 21 days for three more times. After that, the callus was cultured to 28 days before being collected, dried, and then grinded to powder. The powdered callus was subjected for the further extraction process. Figure 1 shows physical characteristics of O. indicum samples.

Plant Extract Preparation.
Each O. indicum sample was separately extracted by maceration using 95% ethanol (plant : solvent ratio 1 : 20 w/v) with continuous shaking for 6 h; then it was left for 12 h [4]. After that, the solution was filtered. Each extraction was repeated three times. The extracts were then combined and dried using a water bath.

Determination of Antioxidant Activity by DPPH
Scavenging Assay. The free radical scavenging activity of extracts from O. indicum sample was investigated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method [11]. A total of 100 L of the extract or standard was added to 100 L of DPPH in a methanolic solution (152 M). After staying at room temperature for 15 min, the absorbance of each solution was determined at 515 nm using a microplate reader (Tecan, USA). The percentage of inhibition was calculated. Then the EC 50 value of the samples required for 50% scavenging of the DPPH free radical was determined. Each determination was done in triplicate, and the average EC 50 value was calculated.

Phytochemical Studies
(1) Determination of Total Phenolic Content Using Folin-Ciocalteu Method. Plant extract solutions (25 L) were oxidized with Folin-Ciocalteu reagent (25 L) in 96-well plate. 75 L of distilled water and 100 L of 20% sodium carbonate solution were added. The absorbance of the resulting blue colored solution was measured at 765 nm after 60 min using a Microplate Reader (Tecan, USA) [12]. Each sample was done in triplicate. Total phenolic content was calculated from the standard curve of gallic acid and was expressed as g gallic acid equivalent in 100 g extract (g% GAE).
(2) Determination of the Total Flavonoid Content. Plant sample solutions (100 L) were separately reacted with a 2% aluminium chloride solution in the same volume. The absorbance was measured at 415 nm after 10 min using a Microplate Reader (Tecan, USA) [13]. Flavonoid content was calculated with the standard curve of quercetin and was Evidence-Based Complementary and Alternative Medicine   [14] with Ultimate 3000 machine equipped with photodiode array and mass spectrometry detectors. A Kinetex C18 column (2.10 mm i.d. × 10 cm, 2.6 m) was used for quantitative analysis. Gradient elution was performed with 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a constant flow rate of 0.35 mL/min. The gradient program was adjusted from 30% to 90% B in 10 min and stayed at 90% B for 2 min. Then the gradient program was adjusted to 30% B and stayed at 30% B for 3 min. Column temperature was 30 ∘ C with an injection volume of 3 L. Injection concentrations of standard references and plant samples were 10 and 100 g/mL, respectively. UV detection was also performed at 280 nm.

Principal Component Analysis (PCA).
Data sets of DPPH scavenging activities (EC 50 values), total phenolic, and total flavonoid contents of extracts from different plant parts and growth stages of O. indicum were subjected to Principal Component Analysis (PCA) using Minitab5 (Minitab Pty Ltd., Australia).

Statistical Analysis.
All data are reported as means ± standard deviation of triplicate analysis. Least significant difference was used to compare means ( < 0.05). All analyses were performed using SPSS for Windows, version 16.0 (SPSS Inc., USA).    Table 1.   Table 1, extracts that promoted strong antioxidant effects including flower stage 1 (OIFL1), mature seed (OIMS), whole fruit (OIWF), pedicel (OIPC), and leaf (OIL) extracts contained high amounts of total phenolic compounds and total flavonoids (higher than 4 g% GAE and 3 g% QE, resp.). Extracts from tissue-cultured plants in early stages (OITPd5, OITPd7, OITPw2, and OTPw3) and callus cultures (OICB, OICG, OICW, and OICY) promoted the similar trends of high total phenolic contents (higher than 5 g% GAE) with moderate amounts of total flavonoids (around 1-2 g% QE). The stalk extract (OIS) promoted the highest antioxidant activity. However, the total phenolic and total flavonoid contents in this extract are in moderate level (2 g% GAE and 3 g% QE, resp.) suggesting the presence of other phytochemicals that support the antioxidant effects such as tannin and ascorbic acid. The late stages of tissue-cultured plants (OITPw4-OITPw8) promoted extracts with low to moderate phenolic and flavonoid contents (1-4 g% GAE and cannot be detected −3 g% RE, resp.) corresponding to their weak antioxidant effects. The results suggest that in O. indicum tissuecultured plants, phenolic compounds, especially flavonoids, are stored mainly in shoot, but the amounts of them are very low in the root (lower than 1 g% QE).   Baicalin and baicalein which are present in most extracts showed high antioxidant effects while chrysin which is a major flavonoid in the leaves and tissue-cultured plants promoted very low antioxidant activity. This suggests that there are other compounds responsible for the antioxidant activities of O. indicum. Some flavones and their glycosides such as scutellarein, norwogonin, acacetin, hispidulin, oroxylin A, apigenin, and tetuin were previously reported in the leaves, seeds, stem bark, and root bark of O. indicum [15][16][17][18][19][20]. Flavonols, flavonoids with a hydroxyl group at the C-3 position, were reported to promote better antioxidant effect than other flavonoids without this functional group such as flavones [21]. Some flavonols previously reported in O. indicum including kaempferol and quercetin could also support the antioxidant effect of this plant [22]. There were studies about in vitro propagation by callus and shoot induction [23][24][25][26] and protoplast isolation [27] for the production of O. indicum. Auxin and cytokinin were reported as good growth regulators for O. indicum callus induction [28]. The recent study indicated that GA3 had a significant effect on seed germination of O. indicum. Moreover, total phenolic and total flavonoid contents were reported to be maximum in in vitro developed root compared to other in vitro plant parts [29]. Another report indicated the presences of baicalein and chrysin in callus extracts of O. indicum [30].

Conclusion
From all of the results, it could be summarized that the stalk, pedicel, flowers, seeds, fruits, and callus promoted the extract with high antioxidant activity and contained high total phenolic and total flavonoid contents with baicalin, baicalein, and chrysin as main ingredients found in these extracts. The shoot extracts from tissue-cultured plants also promoted some antioxidant effects but their contents of phenolics and flavonoids are quite low. Mature seeds, buds (flowers stage 1), blossoms (flowers stage 4), stalks, and leaves are interesting plant parts which should be further investigated for other active ingredients. Biotechnological studies should be provided for more research about callus and tissue-cultured plant material, especially the callus cultures which promoted a strong antioxidant effect and a high total phenolic content.      Commission, and Mahidol University, under the National Research Universities Initiative for their financial support.