|Year : 2019 | Volume
| Issue : 2 | Page : 124-127
Analysis of phenolics in Calligonum polygonoides in vitro cultured roots
Asmaa I Owis1, Nada S Abdelwahab2, Adel A Abul-Soad3
1 Department of Pharmacognosy, Beni-Suef University, Beni-Suef, Egypt
2 Department of Pharmacognosy and Analytical Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt
3 Department of Pharmacognosy and Plant Tissue Culture Laboratory, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt
|Date of Web Publication||30-Oct-2019|
Prof. Asmaa I Owis
Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, 62111, Beni-Suef.
Source of Support: None, Conflict of Interest: None
Background: Calligonum polygonoides L. subsp. Comosum (L’Hér.) Sosk. is an endangered plant species belonging to family Polygonaceae. Although the plant is rich in phytoconstituents and has multipurpose medicinal applications, but in vitro root culture studies and phytochemical investigations of these cultures are rare. Objectives: To establish in vitro root, callus and cell suspension cultures from in vitro germinated fruits of C. polygonoides to investigate the production of phenolics through root, callus and cell suspension cultures and attempt to enhance cell capacity to accumulate phenolics. Materials and Methods: Modified Murashige and Skoog medium supplemented with 1 mg l-1 indole-3-butyric acid was used to establish the root culture. Elicitation of cell suspension culture was performed using salicylic acid and yeast extract. The phenolic compounds in root, callus and cell suspension cultures were evaluated using reversed phase high performance liquid chromatography technique. Results: The unorganized cell suspension culture contained fewer amounts of phenolic compounds than the differentiated roots tissue. Elicitation produced quantitative reprogramming of phenolic content. Conclusion: The present study provides a chance to improve secondary metabolite yield from this valuable natural plant.
Keywords: Analysis, Calligonum polygonoides, high-performance liquid chromatography, phenolics
|How to cite this article:|
Owis AI, Abdelwahab NS, Abul-Soad AA. Analysis of phenolics in Calligonum polygonoides in vitro cultured roots. J Rep Pharma Sci 2019;8:124-7
| Introduction|| |
Calligonum polygonoides L. subsp. comosum (L’Hér.) Sosk. (Polygonaceae) is a small shrub, which has reputation in folklore medicine as a stimulant and astringent, under the local names “ghardaq,” “rusah,” “arta,” or “Wargat Al-shamas.”, Leaves and stems are chewed to treat gummosis, whereas young shoot infusion is used as tonic. It was reported that C. polygonoides possesses hypoglycemic, cytotoxic, antioxidant, antimicrobial, anti-ulcer, anti-inflammatory, antifungal, mosquitocidal, and estrogenic activities. Chemical analysis from previous studies revealed the presence of (+)-catechin, dehydrodicatechin A, kaempferol-3-O-rhamnopyranoside, quercitrin, β-sitosterol-3-O-glucoside, isoquercitrin, kaempferol-3-O-glucuronide, and miquelianin in the aerial parts of the plant. Campesterol, stigmasterol, (3β,5α,24S)-stigmastan-3-ol, and stigmast-4-en-3-one were isolated from the roots, whereas β-sitosterol, kaempferol, quercetin, taxifolin, gallic acid, and astragalin were isolated from leaves.
C. polygonoides has been quoted in the Red Data Book of the International Union for Conservation of Nature and Natural Resources as an endangered plant species. Plant tissue culture is found to be an attractive alternative approach to traditional methods of propagation as it offers a controlled supply of biochemical, independent of plant availability. Apart from open field cultivation hindrances, cell suspension culture systems could be used for large-scale culturing of plant cells from which secondary metabolites could be extracted, so it can ultimately provide a continuous, reliable source of natural products. Furthermore, elicitation is one of the most important approaches to enhance the yield of secondary metabolites produced by in vitro cultures. Previous attempts for studying phenolic production from tissue culture of C. polygonoides shoot were fruitful; however, no report was available concerning the accumulation of phenolic constituents by root culture. Therefore, achieving a protocol for in vitro establishment of root culture and subsequent cell cultures from the fruit explants will definitely assist investigations of phytoconstituents of such valuable plant species. Thus, the objectives of this study were to establish in vitro root culture, callus to be used in cell suspension cultures from in vitro germinated plantlets using the fruit as an explant, investigate the production of phenolics through in vitro root and cell suspension cultures, and attempt to enhance cell capacity to accumulate phenolics using salicylic acid and yeast extract.
| Materials and Methods|| |
C. polygonoides fruits were collected from western desert, Egypt, on April 2012 during flowering season and kindly authenticated by Dr. Abdelhalim Mohamed (Plant Taxonomy Department, Agricultural Research Center, Cairo, Egypt) for whom we are thankful. Voucher specimen was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University under the registration number BUPD-40. The fruits were surface sterilized by dipping in a solution of commercial Clorox (The Clorox Co., Oakland, California, USA) (5%) for 5min followed by H2O2 for 15min. The sterilized fruits were germinated aseptically on quarter-strength Murashige and Skoog (MS) basal liquid medium, supplemented with 30g/L sucrose and 7g/L agar. The pH of the medium was adjusted to 5.7. The fruits were kept at 27°C ± 2°C in the dark.
Establishment of root, callus, and cell suspension cultures
C. polygonoides roots obtained from 14-day-old in vitro germinated plantlets were cultured in liquid MS media supplemented with 1 mg/L indole-3-butyric acid (IBA) in the dark at 25°C on 100rpm rotary shaker then subcultured regularly every 3 weeks under the same conditions. Segments (1cm long) obtained from 14-day-old root were cultured on the same liquid MS media to obtain callus. Cell suspension culture was established from 30-day-old callus. About 1g callus was added to 100-mL Erlenmeyer flasks containing 30mL full strength of the same liquid MS medium and incubated at 27°C ± 2°C in the dark on a rotary shaker at 100rpm. Cells were subcultured on the same media under the aforementioned conditions every 3 weeks.
Growth time course for cell suspension culture
Growth ratio of the cultured cells was assessed by harvesting cells at specified times (7, 14, 21, and 28 days) after inoculation. Final fresh weight of the harvested cells was determined, and the growth ratio was calculated according to the following equation:
Growth ratio = final fresh weight/initial fresh weight
Treatment of cell suspension cultures with elicitors
Two grams fresh weight cells were inoculated into 100-mL Erlenmeyer flasks containing 30-mL liquid MS medium. The 7-day-old cultured cells were separately treated for 48h with filter sterilized aqueous solution of crude yeast extract (Sigma-Aldrich, St. Louis, Missouri, United States) (50 and 100 μg/mL) or salicylic acid (100 and 200 μg/mL). Control received equivalent volumes of solvent.
Phenolic extraction and high-performance liquid chromatography analysis
In vitro root and cell suspension cultures were separately harvested. The cells of suspension cultures were separated from the medium by filtration. All cultures were separately extracted with 75% aqueous methanol (3 × 10mL) at room temperature and filtered. Each filtrate was separately pooled and evaporated to dryness. One gram of each dried extract was separately dissolved in 1mL methanol (high-performance liquid chromatography [HPLC] grade, Sigma-Aldrich), clarified using Millipore filters (0.22 μm) and subjected to HPLC analysis to quantify their phenolic content. The used HPLC (Agilent 1260 Infinity) instrument was equipped with an Agilent 1260 Infinity Preparative Pump (G1361A), Agilent 1260 Diode Array Detector VL (G1315D), Agilent 1260 Infinity Thermostatted Column Compartment (G1361A), and Agilent 1260 Infinity Autosampler (G2260A). Separation and quantitation were performed on a ZORBAX Eclipse Plus C8 Analytical Column (Agilent, Santa Clara, USA) (250 × 4.6mm ID, 5 µm particle size). An aliquot of 50 µL was injected. Phenolics (Sigma-Aldrich) were quantified at 275 nm using peak area by comparing to a calibration curve derived from commercially available standards (taxifolin, isoquercitrin, astragalin, quercetin, and kaempferol). Ambient temperature was used. Elution was carried out at a flow rate of 1mL/min using water:formic acid (99.97:0.03, v/v) as solvent A and methanol:formic acid (99.97:0.03, v/v) as solvent B in a gradient mode as following: 0–5min with 80% A, 5–20min with 80%–50% A, 20–25min with 50%–20% A, and 25–30min with 20%–0% A.
| Results|| |
Establishment of root, callus, and cell suspension cultures
Fruits of C. polygonoides showed an excellent ratio of germination (100%) on quarter strength MS media after treatment with Clorox (5%) for 5min followed by H2O2 for 15min [Figure 1]A. The roots of the in vitro germinated plantlets [Figure 1B] showed fast growth when cultured in MS liquid media supplemented with 1 mg/L IBA [Figure 1C]. Callus produced from root segments was very friable, white to off-white in color [Figure 1D]. Growth parameters of cell cultures in liquid MS medium are shown in [Figure 2]. It showed high growth ratios with an exponential phase between 7th and 14th day of culture. The growth ratios were almost constant after 21 days of inoculation.
|Figure 1: (A) In vitro germinated Calligonum polygonoides L. fruits. (B) Root from the in vitro germinated seedling. (C) Root culture. (D) Callus induced from the in vitro root segments|
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|Figure 2: Time growth course of Calligonum polygonoides L. cultured cells cultivated in Murashige and Skoog (MS) liquid medium supplemented with 1 mg/L indole-3-butyric acid. Mean values ± standard error|
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Production of phenolics and high-performance liquid chromatography analysis
The capacity of root and cell suspension cultures of C. polygonoides to produce secondary metabolites was investigated by determining their phenolic content using HPLC. Levels and profiles of phenolics varied significantly with respect to differentiation state of the cultures [Table 1]. The unorganized callus and cell suspension cultures contained less amounts of all phenolic compounds than differentiated tissues of in vitro roots. Astragalin and kaempferol were the major compounds detected in roots, whereas astragalin, kaempferol, and taxifolin were the majors in case of callus. Quercetin was not detected in both callus and cell suspension cultures, whereas the latter did not accumulate isoquercetin.
|Table 1: High-performance liquid chromatography determination of phenolic content in different in vitro cultures of Calligonum polygonoides L.|
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Effect of elicitors on the production of phenolics
Cell suspension cultures of C. polygonoides undergo quantitative reprogramming in response to elicitation with salicylic acid (100 and 200 µg/mL) and yeast extracts (50 and 100 µg/mL), added to 7-day-old cultures for 48h [Table 1]. Salicylic acid and yeast extracts induced the accumulation of isoquercitrin and quercetin, which was not detected primarily in the untreated control. Incubation of the cells with salicylic acid, at a concentration of 100 µg/mL, produced an increase in taxifolin, astragalin, and kaempferol levels by approximately 2.7-, 1.1-, and 1.5-fold, respectively, whereas an increase of approximately 4.2-, 1.3-, and 2.4-fold, respectively, was observed when 200 µg/mL salicylic acid was used in comparison to that of control cells. On the contrary, incubation of the cells with crude yeast extract, at a concentration of 50 µg/mL, increased taxifolin, astragalin, and kaempferol levels by approximately 1.0-, 1.0-, and 1.4-fold, respectively, whereas an increase of approximately 3.0-, 1.6-, and 2.2-fold, respectively, was observed when 100 µg/mL crude yeast extract was used in comparison to that of control cells. Extensive browning and considerable loss of viability were detected in all yeast extract cultures after 48h. This browning of cells as well as the culture medium is a common feature observed after treating cell suspension cultures with fungal elicitors.
| Discussion and Conclusion|| |
This study provides a strategy to get benefit from one of the valuable medicinal plant species, C. polygonoides, through the application of in vitro culture techniques using the fruit as an initial explant. Root was successfully established from in vitro germinated plantlets. Callus when cultivated in MS liquid medium produced cell suspension culture, which showed stable growth and accumulated phenolic constituents. This phenolic content was higher when the cells were organized as roots. Our results were in agreement with previous studies where the production of secondary metabolites was higher in differentiated structures than in non-differentiated cells.,, The expression of secondary metabolic pathways in redifferentiated cultures is actually not surprising because it mimics exactly what the plant does. However, the inconvenience of manipulating plants or parts of them and their relatively slow growth remain the main disadvantages of this plant tissue culture system. To overcome these problems, a great effort has been directed toward increasing secondary metabolite production from cell culture using various techniques such as elicitation.
Elicitation is an effective strategy to enhance the production of secondary metabolites such as alkaloids, terpenoids, flavonoids, and phenolic compounds. Cell suspension cultures are preferred due to its rapid growth cycles. They have been used for generating large amounts of cells for quantitative or qualitative analysis of growth responses and metabolism of novel chemicals. It can ultimately provide a continuous, reliable source of natural products. This was in agreement with our results where the capacity of cell suspension culture to accumulate phenolics was enhanced after application of salicylic acid and yeast extract, thus providing a chance to improve yield.
In conclusion, this study with C. polygonoides is another example for the power of tissue culture technique to offer an alternative and renewable source for secondary metabolites from a valuable medicinal plant and provide a good chance to improve such secondary metabolite yield, which is mandatory for using biotechnological methods instead of field crops as a basic source of this important pharmaceutical raw material. Our results are reported for the first time.
The authors acknowledge the Scientific Research Development Unit, Beni-Suef University, Beni-Suef, Egypt, for funding and following up the project.
Financial support and sponsorship
This work was funded by the Scientific Research Development Unit, Beni-Suef University, Beni-Suef, Egypt.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]