|Year : 2019 | Volume
| Issue : 3 | Page : 216-227
In-vitro adventitious root production of Cichorium endivia L. and antioxidants, total phenolic, and total flavonoids assessments
Mona M Ibrahim, Mohamed K El-Bahr, Mohamed R Rady
Department of Plant Biotechnology, Genetic Engineering and Biotechnology Division, National Research Center, Giza, Egypt
|Date of Submission||19-Feb-2019|
|Date of Acceptance||23-Mar-2019|
|Date of Web Publication||26-Sep-2019|
Mona M Ibrahim
Department of Plant Biotechnology, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Giza 12311
Source of Support: None, Conflict of Interest: None
Background and objectives Chicory plant serves as a vegetable with higher nutritional value, having major antioxidant properties. The aim of this work was in-vitro production of adventitious roots from Cichorium endivia subsp., pumelum L. and exploring the capacity of adventitious roots for antioxidant activities as well as determine their contents of total phenolic and flavonoids compounds compared with different parts of C. endivia.
Materials and methods For in-vitro adventitious root induction, root segments were cultured on half-strength Murashige and Skoog solid medium supplemented with different concentrations of indole-3-butyric acid and 0.1 mg/l α-Naphthalene acetic acid. The cultures were incubated under total darkness and or 16/8 h (light/dark) photoperiod. Murashige and Skoog liquid medium with different carbon sources was used for adventitious root production. Two different solvents (aqueous ethanol and chloroform) were used for bioactive components extraction process. 2, 2′‐diphenyl 1‐Picryl-hydrazyl radical scavenging capacity (RSC) as well as total phenolic and flavonoides contents were estimated in produced adventitious roots compared with different plant parts (seeds, leaves, and roots).
Results and conclusion Medium supplemented with 0.1 mg/l α-Naphthalene acetic acid and 1.0 mg/l indole-3-butyric acid recorded maximum adventitious root induction percentage (100%) in the dark condition. High-yield production of adventitious roots (6.60±0.5 g) was found in the liquid medium that contains sucrose as the carbon source. The aqueous ethanol extracts recorded higher RSC% values than chloroform extracts in all plant parts. Aqueous ethanol extract of seeds recorded maximum RSC% (92.8%) at 2.5 mg/ml of extract. Total phenolic contents showed maximum value with aqueous ethanol extract of seeds (18.17±0.40 mg/g of extract), whereas minimum value recorded with chloroform extract of seeds (0.28±0.05 mg/g of extract). The flavonoids contents showed maximum value also with aqueous ethanol extract of seeds (94.43±1.00 mg/g of extract), followed by aqueous ethanol extract of leaves (93.68±0.1 mg/g of extract), and minimum value with chloroform extract of leaves (2.60±0.18 mg/g of extract).
Keywords: Adventitious roots, Antioxidant activity, Cichorium endivia, Phenolic and flavonoids
|How to cite this article:|
Ibrahim MM, El-Bahr MK, Rady MR. In-vitro adventitious root production of Cichorium endivia L. and antioxidants, total phenolic, and total flavonoids assessments. Egypt Pharmaceut J 2019;18:216-27
|How to cite this URL:|
Ibrahim MM, El-Bahr MK, Rady MR. In-vitro adventitious root production of Cichorium endivia L. and antioxidants, total phenolic, and total flavonoids assessments. Egypt Pharmaceut J [serial online] 2019 [cited 2021 Jul 23];18:216-27. Available from: http://www.epj.eg.net/text.asp?2019/18/3/216/267886
| Introduction|| |
Chicory (Cichorium endivia subsp., pumelum L.) is a vegetable plant that belongs to the family Asteraceae and characterized by its widespread presence in the west and south of Europe. It has achieved a communal food status owing to its nutritional value and bitter taste, eaten cooked or raw in salads . Cichorium plant has medicinal importance owing to having a number of active compounds including alkaloids, inulin, sesquiterpene lactones, coumarins, vitamins, chlorophyll pigments, unsaturated sterols, flavonoids, saponins, and tannins ,,,. Moreover, it has been used for treatment of fever, diarrhea, jaundice, and gallstones ,. Some studies on rats showed that Cichorium intybus has antihepatotoxic and anti-diabetic activities ,. Moreover, others have showed its own antibacterial properties ,, anti-inflammatory,, hyperglycemic , and anti-ulcerogenic activities .
Polyphenols of chicory plant including flavonoids act as an effective agent to promote public health, because it possesses many important influences such as antiviral, anti-carcinogenic, antibacterial, anti-inflammatory, antifungal, antimutagenic, immunostimulating, and antioxidant effects; moreover, it can conserve the alimentary tract and reduce cholesterol level in the blood ,,.
Antioxidants are essential to protect biological systems from harmful free radicals. The human organs are qualified to deal with peroxidative activities of Reactive oxygen species (ROS) owing to the existence of endogenous enzymes, including superoxide dismutase, catalase, and glutathione peroxidase . However, when the ROS level increases, the cells need external supply of antioxidant molecules, which can be obtained from plants, including phenols, flavonoids, carotenoids, and vitamins (C and E). Therefore, favorable nutrition is an important factor needed to provide effective antioxidant compounds ,.
In this respect, induction of adventitious roots by In vitro methods showed high rates of production of active secondary metabolites, and therefore cultivation of adventitious roots has been proposed as a substitution for natural compound production .
The current study aimed to improve the In vitro production of adventitious roots from C. endivia subsp., pumelum L. and exploring their capacity for antioxidant activity compared with their different parts of C. endivia as well as to determine their contents of total phenolic and flavonoid compounds.
| Materials and methods|| |
Plant material and explants preparation
Seeds of C. endivia subsp., pumelum L. were obtained from Agricultural Research Center, Ministry of Agriculture, Egypt. Seeds were immersed in 70% ethanol for 2–3 min and then rinsed three times in sterile distilled water. The seeds were then sterilized for 30 min in 20% commercial Clorox (5% NaOCl) containing 0.5% Tween 20. After rinsing three times with sterile distilled water, seeds were cultured on Murashige and Skoog (MS) medium  containing 3% (w/v) sucrose and solidified with 0.7% (w/v) agar. Culture medium was adjusted to pH 5.8. The seeds were incubated in a culture room at 24±2°C and were kept under 16-h photoperiod of fluorescent 45-µmol cool white light tubes and 8 h dark. Root segments were excised (3 cm) from 3-week-old seedlings as explants for the present study.
In-vitro adventitious root induction
Root explants were cultured on half-strength MS solid medium supplemented with different concentrations of indole-3-butyric acid (IBA) (0.25, 0.5 and 1.0 mg/l) and 0.1 mg/l α-Naphthalene acetic acid (NAA). All culture media were adjusted to pH 5.8, 0.7% (w/v) agar and 3% (w/v) sucrose were added. The cultures were incubated at 24±2°C under 16/8 h (light/dark) photoperiod with white fluorescent lights or under total darkness for 4 weeks. The responded explants that succeeded to induce adventitious roots were scored as follows:
Effect of different carbon sources on adventitious root production
This experiment aimed to evaluate the effect of different types of carbon sources in the culture media on adventitious root production. For this purpose, 1.0 g of adventitious roots was cultured on half-strength MS solid medium contained 0.1 mg/l NAA and 1.0 mg/l IBA and supplemented with 30 g of different carbon sources (glucose, fructose, sucrose, and maltose) and solidified with 0.7 g agar. Similar previous procedure and condition were done on liquid media without the gelling agent. The cultures were kept in total dark condition, and adventitious roots fresh weights (AFW) were recorded after 4 weeks of culturing.
Leaves and roots of one month seedling were separated from C. endivia, and adventitious roots were collected from the best culture medium. All samples were dried at 40°C. Moreover, seeds were taken as it is. All dried samples were grounded in the mortar. Grounded powder of the samples was weighed and macerated with 200 ml of 80% ethanol and chloroform separately and kept overnight in shaker at 110 rpm. The extracts were collected after filtration using Whatman No. 1 filter paper; this procedure was repeated once again. The extracts were collected and evaporated below 40°C. Each residue was dissolved in the same extract solvent and stored at 4°C until further use.
Percentage of the extract yield was calculated using the formula:
DPPH radical sc avenging capacity
Radical scavenging activity of plant extracts against stable 2, 2′‐diphenyl 1‐Picryl-hydrazyl (DPPH) was determined by a slightly modified method . Different concentrations of each extract (0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml) were used to evaluate antioxidant capacity. Overall, 500 μl of each extract was added to 2.5 ml of methanolic solution of DPPH (0.3 mM). After 30 min at room temperature, the absorbance values were measured at 517 nm on the spectrophotometer. Radical scavenging activity (%) was calculated by the following formula:
where As is the absorbance of plant extract and ADPPH is the absorbance of DPPH solution.
Total phenolic and total flavonoids contents
The concentration of total phenolic compounds was determined by spectrophotometric method using the Folin–Ciocalteu reagent . A calibration curve of gallic acid (20, 40, 40, 60, 80, and 100 µg/ml) was prepared, and the absorbance for tests and standard solutions was determined against the reagent blank at 550 nm with an ultraviolet/visible spectrophotometer. Total phenolic content was expressed as milligram of gallic acid equivalent/g of dry weight (DW) plant material.
The total flavonoids content was measured using a modified colorimetric . The standard curve was prepared using different concentration of quercetin. The flavonoids content was expressed as milligrams quercetin equivalents/g of plant material DW.
The experiments were carried out in triplicate, and the results were expressed as means±SE. Statistical analysis of variance was performed by analysis of variance, and least significant differences at P value less than or equal to 0.05 between the means and were determined by Duncan’s Multiple Range Test.
| Results and discussion|| |
In-vitro adventitious root induction
For adventitious root induction, root explants were cultured on MS medium supplemented with 0.1 mg/l NAA and different concentration of IBA (0.25, 0.5 and 1.0 mg/l) and incubated under light (16/8 h) photoperiod and total dark conditions. Percentages of adventitious roots were recorded after 4 weeks of cultivation ([Figure 1]).
|Figure 1 Percentage of adventitious root induction from root explant on A1 (0.1 mg/l NAA+0.25 mg/l IBA), A2 (0.1 mg/l NAA+0.5 mg/l IBA), and A3 (0.1 mg/l NAA+1.0 mg/l IBA) media cultivated under complete dark and light (16 h) conditions. IBA, indole-3-butyric acid; NAA, α-Naphthalene acetic acid.|
Click here to view
Two factors (light conditions and IBA concentrations) were taken into account to study their effect on adventitious root induction in vitro. Data presented in [Figure 1] reveal that, root segments succeed to induce adventitious roots in all different media under both dark and light (16/8 h photoperiod) conditions. The percentage of adventitious root induction was generally increased when root segments were cultivated in dark condition; they were 70, 90, and 100% on A1 (0.1 mg/l NAA+0.25 mg/l IBA), A2 (0.1 mg/l NAA+0.5 mg/l IBA), and A3 (0.1 mg/l NAA+1.0 mg/l IBA) media, respectively, versus 40, 50, and 70%, respectively, on the same three media at light condition (16/8 h photoperiod).
Concerning the effect of IBA concentration, it was noticeable that, adventitious root induction percentage increased with IBA concentration increasing, which reached the maximum value on A3 medium in both dark and light conditions, recorded at 100 and 70%, respectively. However, minimum adventitious root induction (40%) was recorded with A1 medium cultivated under light condition (16/8 h photoperiod). Therefore, it is noticeable that the two factors (light conditions and IBA concentration) have a clear effect on adventitious root induction, where it was found that, the maximum percentage of adventitious root induction was achieved when IBA was at the highest concentration (1.0 mg/l), moreover, it attained the maximum response in the dark compared with light at incubation period (16/8 h photoperiod). Such treatment (0.1 mg/l NAA+1.0 mg/l IBA) was subjected in the following experiment for adventitious root production. Induced adventitious roots on dark and light conditions with different media supplementation are displayed in [Figure 2] and [Figure 3].
|Figure 2 Adventitious root cultures of Cichorium endivia cultured 4 weeks on MS medium supplemented with A1 (0.1 mg/l NAA+0.25 mg/l IBA), A2 (0.1 mg/l NAA+0.5 mg/l IBA), and A3 (0.1 mg/l NAA+1.0 mg/l IBA) media under light (16 h) condition.|
Click here to view
|Figure 3 Adventitious root cultures of Cichorium endivia cultured 4 weeks on MS medium supplemented with A1 (0.1 mg/l NAA+0.25 mg/l IBA), A2 (0.1 mg/l NAA+0.5 mg/l IBA), and A3 (0.1 mg/l NAA+1.0 mg/l IBA) media under total dark condition.|
Click here to view
Auxin has a key role in root formation by its involvement in successive and interdependent phases . The researchers reported that, the differentiation of the roots depends upon the type and concentration of auxin, because most suitable auxins are a demand for differentiating cells to become competent to respond to the organogenic signal ,.
In this regard, adventitious roots were established cultures from C. intybus L. on MS medium with different IBA and NAA combinations. They reported that 0.5 mg/l NAA and 1.0 mg/l IBA combination was the best suited for growth promotion .
Adventitious roots were induced from Centella asiatica on MS medium containing different auxins [indole-3-acetic acid (IAA), IBA, and NAA]. They showed that, among these different auxins, IBA achieved the highest adventitious root induction .
On the contrary, adventitious roots were induced from Aloe vera. There are no or very little adventitious roots on the media containing IBA and IAA, whereas there was induction on the medium containing NAA only . Moreover, NAA is the only one that was able to induce adventitious root from Andrographis paniculata among different studied auxins (IAA and IBA) .
Recently, a study was conducted on adventitious root formation from C. intybus on MS medium containing different IAA and NAA concentrations and reached the highest induction percentage on MS medium with 1.0 mg/l NAA . Moreover, adventitious roots were induced from C. endivia on MS medium with 0.5 mg/l NAA and different concentrations of IAA and IBA . They found that, the combination of 0.5 mg/l NAA and 1.0 mg/l IBA was more responsive for root induction. Moreover, they showed the induction of adventitious roots was enhanced in total dark condition compared with the use of light condition of 16/8 h (light/dark) photoperiod. This result is perfectly consistent with our result.
In this area as well, half-strength MS medium supplemented with IBA was the most effective to promote adventitious roots of Couroupita guianensis than other auxins (IAA and NAA) .
Effect of different carbon sources on adventitious roots production
Root initiation and growth require high energy, where carbohydrates are the primary source in the metabolic substrates . In this experiment, different carbon sources (sucrose, fructose, glucose, and maltose) were used to evaluate which one increases the adventitious roots on the solid and liquid medium supplemented with 0.1 mg/l NAA+1.0 mg/l IBA and incubated under dark conditions. AFWs were recorded after 4 weeks of cultivation and are shown in [Table 1].
|Table 1 Effect of different carbon sources on adventitious roots’ fresh weights of Cichorium endivia cultured 4 weeks on solid and liquid Murashige and Skoog medium supplemented with 0.1 mg/l α-Naphthalene acetic acid+1.0 mg/l indole-3-butyric acid.|
Click here to view
As is clear from the results, AFWs were obviously increased in liquid media compared with solid media with all studied sugars; this is explained by the availability, ease of uptake of water and nutrients, and closer contact between explants and the medium if it is liquid .
Many studies were conducted to use adventitious roots to produce biomass and secondary metabolite in liquid culture, such as in Glycyrrhiza uralensis, Eurycoma longifolia, Hypericum perforatum, Prunella vulgaris L., and Eleutherococcus koreanum ,,,,.
The use of sucrose as a carbon source achieved the maximum AFW in the liquid medium recorded at 6.60±0.5 g ([Table 1] and [Figure 4]), followed by use of glucose as a carbon source (6.32±0.3 g). However, in the case of solid medium, the glucose recorded highest AFW (4.25±0.20 g) followed by sucrose (3.57±0.30 g). On the contrary, fructose and maltose recorded AFW lower than sucrose and glucose in both solid and liquid medium.
|Figure 4 Adventitious root cultures of Cichorium endivia cultured on MS liquid medium containing sucrose as the carbon source.|
Click here to view
This result is quite consistent with Hussein et al. . They reported that, among different studied carbon sources (sucrose, glucose, fructose, galactose, and sorbitol) to induce adventitious roots from the leaf explants of E. longifolia, both sucrose and glucose are the most influential.
Sucrose is the most used carbon source in tissue cultures, owing to the active absorption through the cell membrane . Sucrose is hydrolyzed to glucose and fructose by the plant cells during assimilation, and the rate of uptake varies in different plant cells . Sugars are considered as a main carbon sources and osmotic regulators .
The type of carbon source is an impact factor on adventitious root induction in many plant species . The roots’ frequency and quality can be enhanced by modify different types of carbohydrates in the culture medium . The useful effects of sucrose on rooting had been reported in apple and Gladiolus hybridus . Glucose and fructose had showed to be better carbon source in the few reports . They reported that the uptake of glucose instead of sucrose could induce higher rooting frequency.
Adventitious root production was achieved from C. intybus on half-strength MS liquid medium supplemented with 0.2 mg/l NAA and 0.5 mg/l IBA under total dark condition . Fresh weight of adventitious root reached to 5.82 g after 6 weeks of cultivation.
Adventitious roots were also produced from C. intybus L. on MS medium containing different auxins .The highest fresh weight and DW was obtained in MS liquid medium containing 0.3 mg/l NAA and 1.5 mg/l IBA.
Moreover, the highest biomass production of adventitious roots from C. endivia (4.5 g) was achieved by using root explant on the medium containing 0.5 mg/l NAA and 0.8 mg/l IBA after 6 weeks of culture . However, in the present study, the production of adventitious roots reached to 6.5 g after 4 weeks of cultivation by using sucrose as a carbon source and AFW reached to 6.3 g by using the glucose.
The highest accumulation of adventitious roots in the liquid media could be owing to the exposure of periphery tissues to sufficient nutrients and oxygen in the liquid medium; in contrast, the center of the cultures had restricted supply of nutrients and oxygen .
Extraction-yield percent estimation
Extraction step is an important for discovery of bioactive constituents from the plants. Active compounds in plants are usually found in low concentration, and an extraction technique especially type of solvent is able to obtain high yield of extract . Various studies have reported the variations in extract preparation cause difference in the biological activities, therefore, it is needful to the selection of extraction method and solvent depending on sample matrix properties, chemical properties of the analytes, matrix, efficiency, and desired properties ,.
In this study, extraction-yield percent obtained by using two extraction solvents (aqueous ethanol and chloroform) was estimated in adventitious root extracts compared with different parts of C. endivia (seeds, leaves, and roots) extracts. For aqueous ethanol used as a solvent, the extraction-yield percent of the four extracts was obviously elevated and compared with those of chloroform. Data in [Table 2] show that, in aqueous ethanol extract, the maximum value of extraction-yield (%) was found in adventitious root extract (52.5%) and root extract (51.4%), followed by leaves and seeds extracts (30.0 and 25.0%, respectively). The same order was obtained by using chloroform solvent, where adventitious root extract recorded maximum extraction-yield percent followed by root extract then leaves and seed extracts (25.0, 18.5, 12.7, and 12.5%, respectively).
|Table 2 Extraction yields percent of four different parts of Cichorium endivia extracted by aqueous ethanol and chloroform|
Click here to view
This result is consistent with Milala et al. , and also explain our results, where they showed that, different parts of C. intybus L. (root, leaves, and seeds) which subjected to the extraction by using water–ethanol resulted in the root fraction having higher fructooligosaccharides, which predominated than leave and seed fractions.
The main compounds of chicory root are carbohydrates, inclusive, saccharose, glucose, and fructose . Fructooligosaccharides and inulin represented 21% on average.
Regarding our finding, aqueous ethanol extract recorded extraction-yield percent higher than chloroform extract comparable to their counterpart to the same plant part; this has been explained by Willeman et al. . They reported that the high solubility of some compounds in alcoholic solvent may be owing to the high surface polarity especially induced by hydroxyl group.
xx2,2-diphenyl 1- picryl- hydrazyl with DPPH
In this experiment, radical scavenging capacity (RSC%) of the different C. endivia extracts was estimated, and the data are presented in [Figure 5],[Figure 6],[Figure 7],[Figure 8]. In all extracts of C. endivia, the aqueous ethanol generally recorded RSC% higher than those of chloroform extracts. This remark is entirely consistent with Montefusco et al. . They found that, hydrophilic antioxidant activity of different chicory varieties was generally higher than lipophilic extract.
|Figure 5 Radical scavenging capacity (RSC%) of Cichorium endivia seeds extracted with aqueous ethanol and chloroform.|
Click here to view
|Figure 6 Radical scavenging capacity (RSC%) of Cichorium endivia leaves extracted with aqueous ethanol and chloroform.|
Click here to view
|Figure 7 Radical scavenging capacity (RSC%) of Cichorium endivia roots extracted with aqueous ethanol and chloroform.|
Click here to view
|Figure 8 Radical scavenging capacity (%) of Cichorium endivia adventitious roots extracted with aqueous ethanol and chloroform.|
Click here to view
[Figure 5] shows RSC% of aqueous ethanol and chloroform extracts of seeds comparable with L-ascorbic acid, where it was found that, RSC% values of seeds extracted with aqueous ethanol approach to the values of L-ascorbic acid at different concentrations. The maximum RSC% reached to 92.8% at 2.5 mg/ml versus 95.3% for L-ascorbic acid at the same concentration. It is clearly observed that, aqueous ethanol extract of seeds recorded high RSC% even at the lowest concentration, where it recorded 90.2% at 0.5 mg/ml. As for chloroform extract of seeds, it reached to maximum value (60.6%) at 2.5 mg/ml.
RSC% of leaves which presented in [Figure 6] shows that, in case of aqueous ethanol extract, the high values of RSC% (89.3 and 87.7%) were recorded at 2.0 and 2.5 mg/ml, respectively, whereas the lowest extract concentration (0.5 mg/ml) showed minimum RSC% (55%). However, chloroform extract of leaves reached to maximum RSC% (48.3%) at 1.0 mg/ml, and then declined at the higher concentrations.
Regarding RSC% of roots ([Figure 7]), the values of aqueous ethanol extract approached to the values of chloroform extract especially at concentrations of 0.5 and 1.0 mg/ml, where it was found that, RSC% of aqueous ethanol recorded maximum value (59.4%) at 2.0 mg/ml and the chloroform extract (50.6%) at 2.5 mg/ml.
Aqueous ethanol extract of adventitious roots reached to maximum RSC% (63.1%) at 2.5 mg/ml, whereas chloroform extract showed lower value comparable with aqueous ethanol extract, recorded 38.8% at 2.5 mg/ml ([Figure 8]).
These results can be summarized as such, aqueous ethanol extract of seeds recorded maximum RSC%, followed by aqueous ethanol extract of leaves, and then RSC% values of aqueous ethanol extracts of adventitious roots and roots, which recorded the lowest values.
Recently, attention has increased extremely in finding naturally occurring antioxidants for use in foods or medicinal materials. Adventitious roots are considered factories for biosynthetic and production of health promoting secondary metabolites, including phenolics, flavonoids, and alkaloids. In-vitro root culture is an efficient protocol with faster growth rate and active secondary metabolites production .
The previous studies indicated that, chicory plant could be a perfect source of natural antioxidants and it is considered as remedial factor in inhibiting or slowing down oxidative stress caused by diseases. This may be attributed to the occurrence of antioxidant compounds in this plant such as phenols, flavonoids, alkaloids, tannins, coumarins, and terpenoids .
Various organic fractions of C. intybus seeds were evaluated . They reported that, seed extract showed good DPPH radical scavenging activity. This is in agreement with our results. Moreover, methanolic and ethyl acetate extracts exhibited the maximum antioxidant activity compared with other fractions (n-hexane, chloroform, and n-butanol).
Our results are also confirmed with the study of Milala et al. . They elucidated that, aqueous ethanol preparation from seeds of C. intybus was distinguished with the highest antioxidant activity compared with other plant parts (leaves, peels, and roots), whereas lowest antioxidant activity was observed in the preparation of roots.
Antioxidant activity of different parts of C. intybus L. extracted with methanol was evaluated and compared . They reported that, leaves have a distinctive ability in RSC compared with other plant parts (root, stem, and seeds), which showed lower percentage of RSC. This result was different from that we obtained, where the seeds recorded higher RSC than the other plant parts in the present study. This may be owing to the difference in the type of solvent or the variation of plant variety.
Another comparative study between leaves and root of C. intybus had been performed using different solvents (water, methanol, and chloroform)  and it showed that, maximum RSC% of leaves and roots was recorded with methanol extract (78.62 and 71.88%, respectively), followed by chloroform and water, which both recorded minimum values. This result is quite similar to ours; however, hydrophilic extract (aqueous ethanol) recorded RSC% higher than chloroform extract.
Total phenolic and flavonoids contents
Phenolic and flavonoids are a class of phytochemical compounds that have antioxidant properties and possess the ability to inhibit apoptosis, where the oxidative stress is the first step which caused apoptosis . In this part of the study, total phenolic and flavonoids contents were estimated in the different C. endivia extracts. Data presented in [Table 3] explained that, maximum phenolic and flavonoids contents were recorded with seeds extracted with aqueous ethanol (18.17±0.40 and 94.43±1.00 mg/g extract) respectively. This is perfectly consistent with DPPH antioxidant evaluation, whereas also recorded maximum RSC% value. Regarding phenolic content, there were no significant differences between leaves and adventitious root extracted with aqueous ethanol, recorded at 6.11±0.40 and 6.23±0.20, respectively; however, aqueous ethanol extract of root recorded minimum value.
|Table 3 Total phenolic and flavonoids contents of the four different Cichorium endivia extracts|
Click here to view
Regarding flavonoids contents, the aqueous ethanol extract of leaves recorded higher value than root and adventitious root (31.68±0.10, 20.87±0.30, and 21.62±0.30 mg/g extract), respectively. Chloroform extracts of the different plant parts recorded minor proportions of phenolic and flavonoids compounds compared with aqueous ethanol extracts, and there were no significant differences between the different plant parts.
In this area, the cultures of adventitious roots are an important source for the production of valuable plant secondary metabolites on a commercial scale . Moreover, ∼60% of medicinal plants used roots in the drug preparation . Therefore, the in-vitro root culture development is a highly economic source for the production of valuable plant secondary metabolites, and it used as also alternative method for clonal propagation and germplasm conservation.
Small letter express LSD significant differences (LSD0.050 value = 0.8723 for phenolic and 1.782 for flavonids. A considerable importance of C. intybus has been found owing to it containing high phenolic and flavonoid compounds. Leaves’ extract is a good source for obtaining important pharmaceutical compounds which play role in improve public health .
Moreover, C. intybus is rich in total phenolic compounds. Kaur and Singh  achieved a comparable study between leaves and roots extracted with three different solvents (methanol, chloroform, and water). Moreover, they detected that methanol extracts contain maximum values followed by chloroform extracts and then water extracts, which revealed minimum values. In three extracts, the leaves’ extracts had own phenolic compounds greater than root extracts. These results are similar to some extent with the results we obtained.
Furthermore, a comparable study was done on different parts (seeds, leaves, roots, and peels) of C. intybus, extracted with aqueous ethanol . The study concluded that the seed extract contained more than 10% of total polyphenols. The major components were dicaffeoylquinic acids (71% of total polyphenols). Seeds of chicory are considered as a good source for obtaining preparations rich in polyphenols, especially dicaffeoylquinic acids. These results are confirmed with our results.
The ethanolic and methanol fractions from seeds containing polyphenols showed higher antihepatotoxic properties than ethyl acetate and petroleum ether fractions .
| Conclusion|| |
In this study, the root explant responded to induce adventitious roots more better in the dark condition and highest IBA concentration. Adventitious root production was enhanced and attained good mass production in the liquid medium containing the sucrose as the carbon source. All studied C. endivia parts extracted with aqueous ethanol achieved considerable antioxidant activity more those extracted with chloroform; among these different extracts, aqueous ethanol extract of seeds possessed the highest antioxidant activity. This high activity can be confirmed and explained through containing the highest amount of total phenolic and flavonoids.
This research is quite compatible within the research plan of National Research Centre. The authors are greatly indebted to the NRC for the generous financing support that enabled us to achieve these results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Koudela M, Petrikova K. Nutritional composition and yield of endive cultivars − Cichorium endivia
L. Hort Science 2007; 34:6–10.
Molan AL, Duncan AJ, Barryand TN, Mc-Nabb WC. Effect of condensed tannins and sesquiterpene lactones extracted from chicory on the motility of larvae of deer lungworm and gastrointestinal nematodes. Parasitol Int 2003; 52:209–218.
Nandagopal S, Ranjitha Kumari BD. Phytochemical and antibacterial studies of chicory (Cichorium intybus
L.) − amultipurpose medicinal plant. Adv Biol Res 2007; 1:17–21.
Muthusamy VS, Anand S, Sangeetha KN, Sujatha S, Arun B, Lakshami BS. Tannins present in Cichorium intybus
enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adepocytes through PTP1B inhibition. Chem Biol Interact 2008; 174:69–78.
Atta AH, Elkoly TA, Mouneir SM, Kamel G, Alwabel NA, Zaher S. Hepatoprotective effect of methanolic extracts of Zingiber officinale
and Cichorium intybus
. Indian J Pharm Sci 2010; 72:564–570.
] [Full text]
Afzal S, Afzal N, Awan MR, Khan TS, Gilani A, Khanum R, Tariq S. Ethno-botanical studies from Nothern Pakistan. J Ayub Med Coll Abbotabad 2009; 21:52–57.
Abbasi AM, Khan MA, Ahmad M, Zafar M, Khan H, Muhammad N, Sultana S. Medicinal plants used for the treatment of jaundice and hepatitis based on socio-economic documentation. Afr J Biotechnol 2009; 8:1643–1650.
Ahmed B, Al-Howiriny TA, Siddiqui AB. Antihepatotoxic activity of seeds of Cichorium intybus
. J Ethnopharmacol 2003; 87:237–240.
Pushparaj PN, Low HK, Manikandan J, Tan BKH, Tan CH. Anti-diabetic effects of Cichorium intybus
in streptozotocin-induced diabetic rats. J Ethnopharmacol 2007; 111:430–434.
Petrovic J, Stanojkovic A, Comic LJ, Curcic S. Antibacterial activity of Cichorium intybus
; short report. Fitoterapia 2004; 75:737–739.
Cavin C, Delannoy M, Malnoe A, Debefve E, Touche A, Courtois D, Schilter B. Inhibition of the expression and activity of cyclooxygenase-2 by chicory extract. Biochem Biophys Res Commun 2005; 327:742–749.
Minaiyan M, Ghannadi A, Mahzouni P, Abed A. Preventive effect of Cichorium intybus
L. two extracts on cerulein-induced acute pancreatitis in mice. Int J Prev Med 2012; 3:351–357.
Delzenne NM, Cani PD, Daubioul C, Neyrinck AM. Impact of inulin and oligofructose on gastrointestinal peptides. Br J Nutr 2005; 93:157–161.
Rifat-uz-Zaman MS, Akhtar MS, Khan MS. In vitro antibacterial screening of Anethum graveolens L. fruit, Cichorium intybus L. leaf, Plantago ovata L. seed husk and Polygonum viviparum L. root extracts against Helicobacter pylori. Int J Pharmacol 2006; 2:674–677.
Wang M, Simon JE, Aviles JF, Zheng Q-Y, Tadmor Y. Analysis of antioxidative phenolic compounds in artichoke. J Agric Food Chem 2003; 51:601–608.
Innocenti M, Gallori S, Giaccherini C, Ieri F, Vincieri FF, Mulinacci N. Evaluation of phenolic content in the aerial parts different varieties of Cichorium intybus
L. J Agr Food Chem 2005; 53:6497–6502.
Mares D, Romagnoli C, Tosi B, Adreotti E, Chillemin G, Poli F. Chicory extracts from Cichorium intybus
L. as potential antifungials. Mycopathologia 2005; 160:85–92.
Matés JM, Pérez-Gómez C, De Castro IN. Antioxidant enzymes and human diseases. Clin Biochem 1999; 32:595–603.
Prenesti E, Berto S, Daniele PG, Toso S. Antioxidant power quantification of decoction and cold infusions of Hibiscus sabdariffa
flowers. Food Chem 2007; 100:433–438.
Jiménez AM, Martínez-Tomé M, Egea I, Romojaro F, Murcia MA. Effect of industrial processing and storage on antioxidant activity of apricot (Prunus armeniaca
). Eur Food Res Technol 2008; 227:125–134.
Zhong JJ, Pan ZW, Wang ZY, Chen F, Takag M, Yoshida T. Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis
in a centrifugal impeller bioreactor. J Biosci Bioeng 2002; 94:244–250.
Murashige T, Skoog FA. Revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 1962; 15:473–497.
Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebenson Wiss Technol 1995; 28:25–30.
Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 1999; 299:152–178.
Vabkova J, Neugebauerova J. Determination of total phenolic content, total flavonoid content and frap in culinary herbs in relation to harvest time. Acta Univ Agric Silvic Mendel Brun 2012; 20:167–172.
Bellamine J, Penel C, Greppin H, Gaspar T. Confirmation of the role of auxin and calcium in the late phases of adventitious root formation. J Plant Growth Regul 1998; 26:191–194.
Blakesley D, Chaldecott MA. The role of endogenous auxin in root initiation. J Plant Growth Regul 1997; 13:77–84.
Sabatini S, Beis D, Wolkenfelt H. An auxin dependent distal organizer of pattern and polarity in the Arabidopsis
root. Cell 1999; 99:463–472.
Ling APK, Chin MF, Hussein S. Adventitious root production of Centella asiatica
in response to plant growth regulators and sucrose concentrations. Med Aromat Plant Sci Biotech 2009; 3:36–41.
Lee YS, Yang T-J, Park S-U, Baek JH, Wu SQ, Lim K-B. Induction and proliferation of adventitious roots from Aloe vera leaf tissues for in vitro production of aloe-emodin. POJ 2011; 4:190–194.
Sharma SN, Jha Z, Sinha RK. Establishment of in vitro
adventitious root cultures and analysis of andrographolide in Andrographis paniculata
. Nat Prod Commun 2013; 8:1045–1047.
Hadizadeh H, Mohebodini M, Esmaeilpoor B. Effects of auxins on induction and establishment of adventitious and hairy roots culture of the medicinal plant chicory (Cichorium intybus
L.). Iran J Med Aromat Plants 2016; 32:389–396.
Amer A, Ibrahim M, Askr M. Establishment of in vitro
root culture of Cichorium endivia
L. − a multipurpose medicinal plant. Int J Chemtech Res 2016; 9:141–149.
Manokari M, Shekhawat MS. Implications of auxins in induction of adventitious roots from leaf explants of cannon ball tree (Couroupita guianensis
Aubl.). World Sci News 2016; 33:109–121.
Thorpe T. Carbohydrate utilization and metabolism. In Bonga JM, Durzan DJ, eds. Tissue culture in forestry. London: Martinus Nijhoff Publishers; 1982. 325–368
Pierik RLM. In vitro
culture of higher plants. Dordrecht, Netherlands: Kluwer Academic Publishers; 1997.
Yin S, Zhang Y, Gao W, Wang J, Man S, Liu H. Effects of nitrogen source and phosphate concentration on biomass and metabolites accumulation in adventitious root culture of Glycyrrhiza uralensis
Fisch. Acta Physiol Plant 2014; 36:915–921.
Lulu T, Park SY, Ibrahim R, Paek KY. Production of biomass and bioactive compound from adventitious roots by optimization of culturing conditions of Eurycoma longifolia
in balloon-type bubble bioreactor system. J Biosci Bioeng 2015; 119:712–717.
Cui X, Chakrabarty D, Lee E, Paek K. Production of adventitious roots and secondary metabolites by Hypericum perforatum
L. in a bioreactor. Bioresour Technol 2010; 101:4708–4716.
Fazal H, Abbasi BH, Akhmad N. Optimization of adventitious root culture for production of biomass and secondary metabolites in Prunella vulgaris
L. Appl Biochem Biotechnol 2014; 174:2086–2095.
Lee UJ, Park SY, Paek KY. Enhancement strategies of bioactive compound production in adventitious root culture of Eleotherococcus koreanum Nakai
subjected to methyl jasmonate and salicylic acid elicitor through airlift bioreactors. Plant Cell Tissue Org Cult 2014; 120:1–10.
Hussein S, Ling AP, Ng TH, Ibrahim R, Paek KY. Adventitious roots induction of recalcitrant tropical woody plant, Eurycoma longifolia
. Rom Biotech Lett 2012; 17:7026–7035.
Borkowska B, Szezebra J. Influence of different carbon sources on invertase-activity and growth of sour cherry (Prunus cerasus
L.) shoot cultures. J Exp Bot 1991; 42:911–915.
Srinivasan V, Pestchanker L, Moser S, Hirasuna TJ, Taticek RA, Shuler ML. Taxol production in bioreactors: kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspension of Taxus baccata
. Biotechnol Bioeng 1995; 47:666–676.
Neto VBP, Otoni WC. Carbon sources and their osmotic potential in plant tissue culture: does it matter? Sci Hortic 2003; 97:193–202.
Thompson M, Thorpe T. Metabolic and non-metabolic roles of carbohydrates. In: Bonga JM, Durzan DJ, (eds). Cell and tissue culture in forestry. Dordrecht: Martinus Nijhoff Publishers; 1987. 89–112.
Moncousin C, Ribaux MO, Rourke J, Gavillet S. Effects of type of carbohydrate during proliferation and rooting of microcuttings of Malus Jork
. Agronomie 1992; 12:775–781.
Kumar A, Sood A, Palni LMS, Gupta AK. In vitro propogation of Gladiolus hybridus Hort: synergistic effect of heat shock and sucrose on morphogenesis. Plant Cell Tiss Organ Cult 1999; 57:105–112.
Romano A, Noronha C, Martins MA. Role of carbohydrate in micropropagation of cork oak
. Plant Cell Tiss Organ Cult 1995; 40:159–167.
Min JY, Jung HY, Kang SM. Production of tropane alkaloids by small scale bubble column bioreactor cultures of Scopolia parviflora
adventitious roots. Bioresour Technol 2007; 98:1748–1753.
Quispe Candori S, Foglio MA, Rosa PTV, Meireles MAA. Obtaining b-caryophyllene from Cordia verbenacea
de Candolle by super crtitical fluid extraction. J Supercrit Fluids 2008; 46:27–32.
Hayouni EA, Abedrabba M, Bouix M, Hamdi M. The effects of solvents and extraction method on the phenolic contents and biological activities in vitro
of Tunisian Quercus coccifera
L. and Juniperus phoenica
L. fruit extracts. Food Chem 2007; 105:1126–1134.
Ishida BK, Ma J, Bock C. A simple rapid method for HPLC analysis of lycopene isomers. Phytochem Anal 2110 12:194–198.
Milala J, Grzelak K, Król B, Juśkiewicz J, Zduńczyk Z. Composition and properties of chicory extracts rich in fructans and polyphenols. Pol J Food Nutr Sci 2009; 59:35–43.
Galzka I. The composition of chicory flour of selected chicory cultivars Polanowicka and Fredonia in relation to root sizes and the date of harvest. Zywn-nauk Technol Ja 2002; 3:S37–S45.
Willeman H, Hance P, Fertin A, Voedts N, Duhal N, Goossens J, Hilbert J. A method for the simultaneous determination of chlorogenic acid and sesquiterpene lactone content in industrial chicory root food stuffs. Sci World J 2014; 2014:1–11.
Montefusco A, Semitaio G, Marrese PP, Iurlaro A, De Caroli M, Piro G et al.
Antioxidants in varieties of chicory (Cichorium intybus
L.) and wild poppy (Papaver rhoeas
L.) of southern Italy. J Chem 2015; 2015:1–8.
Khan MA, Abbasi BH, Shah NA, Yücesan B, Ali H. Analysis of metabolic variations throughout growth and development of adventitious roots in Silybum marianum L. (Milk thistle), a medicinal plant. Plant Cell Tissue Organ Cult 2015; 123:501–510.
Malik B, Pirzadah TB, Tahir I, Rehman RU. Chemo-profiling, antioxidant potential and ionomic analysis of Cichorium intybus
L. Pharmacogn J 2017; 9:917–928.
Mehmood N, Zubaır M, Rızwan K, Rasool N, Shahid M, Ahmad VU. Antimicrobial and phytochemical analysis of Cichorium intybus s
eeds extract and various organic fractions. Iran J Pharm Res 2012; 11:1145–1151.
Shad MA, Nawaz H, Rehman T, Ikram N. Determination of some biochemicals, phytochemicals and antioxidant properties of different parts of Cichorium intybus
L.: a comparative study. J Anim Plant Sci 2013; 23:1060–1066.
Kaur HP, Singh I, Singh N. Phytochemical, antioxidant and antibacterial potential of extracts of Cichorium intybus
(chicory). Eur J Pharm Med Res 2016; 3:320–326.
Hockenbery DM, Oltvai ZN, Yin X-M., Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993; 75:241–251.
Sudha CG, Seeni S. Establishment and analysis of fast-growing normal root culture of Decalepis arayalpathra, a rare endemic medicinal plant. Curr Sci 2001; 81:371–374.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]