EVALUATION OF IN VITRO ANTIOXIDANT PROPERTIES OF CALLUS CULTURES OF AN ENDANGERED MEDICINAL TREE SPECIES, HILDEGARDIA POPULIFOLIA (ROXB.) SCHOTT & ENDL.HTML Full Text
Received on 01 October, 2013; received in revised form, 25 November, 2013; accepted, 10 February, 2014; published 01 March, 2013
EVALUATION OF IN VITRO ANTIOXIDANT PROPERTIES OF CALLUS CULTURESOFAN ENDANGERED MEDICINAL TREE SPECIES, HILDEGARDIA POPULIFOLIA (ROXB.) SCHOTT & ENDL.
M. Saradha 1, P. Ranjitham 2 and S. Paulsamy1*
Department of Botany, Kongunadu Arts and Science College 1, Coimbatore – 641 029, Tamil Nadu, India
Department of Botany, Sri Vasavi College 2, Erode – 638 316, Tamil Nadu, India
ABSTRACT: Protocol for efficient callogenesis in Hildegardia populifolia has been developed by using leaf, node, internode and petiole explants. Preliminary phytochemical analysis and quantitative estimation of flavonoids, phenols and tannins were made along with the evaluation of antioxidant properties by DPPH• and ABTS•+ assays in in vitro callus cultures of the same species. It is an indigenous endangered multipurpose medium sized tree being used for traditional medical practices in Tamil Nadu and Andra Pradesh. Among the explants, the efficient callogenesis was accelerated within 10-19 days in leaf explant on Murashige and Skoog (MS) medium supplemented with 2, 4-D alone at 2 mg/L. Effective calli were produced from node (BAP and IAA at 2 mg/L and 0.4 mg/L respectively in MS medium), internode (2,4-D at 2 mg/L in MS medium) and petiole (2,4-D at 2 mg/L in MS medium) explants. The phytochemical study revealed that phenolics and tannin content were higher in 60 days old leaf callus cultures. Flavonoid content was more in 60 days old internode derived callus culture. Evaluation of antioxidant property established that DPPH• radical scavenging activity was relatively higher in leaf derived callus (86.24 mg/g) and ABTS•+ activity was higher in internode derived callus (3077.98 µmol TE/g extract). This study had shown that plant tissue culture offers itself as a highly efficient method for mass propagation and conservation of H. populifolia and also helps to identify the unique in vitro culture with natural antioxidant potential to get health care benefits.
Hildegardia populifolia, Sterculiaceae, efficient callus, antioxidant properties
INTRODUCTION:Hildegardia populifolia, an endangered, medium sized tree belongs to the family Sterculiaceae is found in few tropical deciduous forests of Tamil Nadu and Andhra Pradesh in India. Various parts of this species are being prescribed by the local healers of southern India for various ailments.
It is reported to have antimicrobial activity 1-3, antioxidant activity 4 and antiinflammatory activity 5. It is used to treat malaria and dog bite also 6. The fibre extracted from the bark is used for domestic purposes and the leaf extract is prescribed in traditional medical practices of Tamil Nadu and Andhra Pradesh 7.
As per the information given in the Red data Book of Indian plants 8, this narrow endemic species is under great threat due to both extrinsic and intrinsic factors. It is an enigmatic species in that its conservation status has been variously assessed as critically endangered 9.
Therefore, it is the species of conservation priority, need modern propagation strategies for enhancing its population 10 to prevent extinction.
Vegetative propagation by means of cell and tissue culture techniques is a powerful tool for plant conservation and hence reforestation 11, 12. Exploiting callus cultures for therapeutic uses in some species is another method of conservation of wilds to some extent 13, 14. However, prior to large scale application, evaluation of therapeutic properties must be important along with the estimation of phytochemicals in the callus of respective species. Hence, in the present study, development of protocol for callus cultures was made to estimate flavonoids, phenols and tannins and to evaluate antioxidant properties to confirm the medicinal properties of callus of the species, H. populifolia.
MATERIALS AND METHODS
Collection of plant material: Plants of H. populifolia collected from their natural habitat were grown and maintained in the garden of the Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, India for the source of explants.
Surface sterilization: Different parts viz., nodes, internodes, petioles and young healthy leaves were used as explants. The explants were first thoroughly washed under running tap water for 15-20 minutes and then treated with liquid detergent (Tween – 20) for 5-10 minutes. The explants were dipped in a systemic fungicide solution of 1% Bavastin (Carbendezin 50% WP) for 2 minutes. Later, these explants were washed with double distilled water for 5 minutes. Subsequently, explants were immersed in 70% (v/v) ethanol for 2-3 minutes and washed with sterile glass double distilled water for 2-3 times. Eventually, the explants were treated with aqueous solution of 0.1% (w/v) HgCl2 for 1-2 minutes and rinsed with sterile glass double distilled water for 2-3 times to remove traces of mercuric chloride. The sterilized explants were aseptically inoculated onto MS medium with different combinations and concentrations of hormones.
Culture media and growth condition: MS medium with 3% (w/v) sucrose and pH 5.6-5.8 solidified with 0.8% agar prior to autoclave at 121˚C at 15 lbs pressure for 15-20 minutes was used. Inoculation was done under aseptic conditions in a laminar air flow cabinet, where culture bottles containing 35-40 mL medium kept under UV light for sterilization before inoculation for 30 minutes. All culture bottles were incubated in a controlled environmental in growth chamber at 25˚C±2˚C under 16-18 hrs photoperiod at 3000 lux light intensity (40w white fluorescent tubes, Philips, India) and with 55-60% relative humidity.
Preparation of Extract: Calli derived from the leaf, node, internode and petiole were collected and dried in an oven at 40±1˚C for 5 h. Dried calli were homogenized to a fine powder and stored in airtight bottles. 50g of calli powder was extracted with 250 mL of solvents viz., petroleum ether, chloroform and methanol for 24 h separately by using Soxhlet apparatus. The extracts were lyophilized after solvent removal under reduced pressure. This crude callus extract was used for the determination of phytochemicals and antioxidant activity.
Preliminary phytochemical analysis: Phyto-chemical screening of the extract was carried out to identify the secondary metabolites such as alkaloids (Mayer’s and Dragendorff’s test), flavonoids (Shinoda test), terpenoids (Salkowski test), tannins (Ferric chloride test), saponins (Frothing test), cardiac glycosides (Keller-Killiani test), steroids and triterpenoids (Liebermann-Burchard test) and phenols (Ellagic acid test) according to standard phytochemical methods as described by Trease and Evans 15, Mojab et al 16, Sofowora 17 and Harbone 18.
Estimation of Phytochemicals:
Determination of Total Flavonoids: The total flavonoid content of samples was determined by following the modified colorimetric method of Zhishen et al19. 0.5 mL extract was mixed with 2 mL of distilled water and subsequently with 0.15 mL of 5% NaNO2 solution. After 6 min, 0.15 mL of 10% AlCl3 solution was added and allowed to stand for 6 min, then 2 mL of 4% NaOH solution was added to the mixture. Immediately distilled water was added to bring the final volume to 5 mL, and then the mixture was thoroughly mixed and allowed to stand for another 15 min. Absorbance of the mixture was recorded at 510nm versus prepared water blank.
Rutin was used as standard for the quantification of total flavonoid. All the values were expressed as milligram of rutin equivalent (RE) per gram of extract.
Determination of Total Phenolics and Tannins: The total phenolic content was determined according to the method described by Siddhuraju and Becker20. Aliquots of each extract were taken in test tubes and made up to the volume of 1 mL with distilled water. Then 0.5 mL of folin-ciocalteu phenol reagent (1:1 with water) and 2.5 mL of sodium carbonate solution (20%) were added sequentially in each tube. Soon after vortexing the reaction mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725 nm against the reagent blank. The analysis was performed in triplicate and the results were expressed as gallic acid equivalents (GAE).
Using the same extract, the tannins were estimated after treatment with polyvinyl polypyrrolidone (PVPP). Hundred mg of PVPP was weighed in a 100 × 12 mm test tube and to this 1.0 mL of distilled water and then 1.0 mL of tannin containing phenolic extract were added. The content was vortexed and kept in the test tube at 4ºC for 4 h. Then the sample was centrifuged (3000 rpm for 10 min at room temperature) and the supernatant was collected. This supernatant has only simple phenolics other than tannins (the tannins would have been precipitated along with the PVPP). The phenolic content of the supernatant was measured, as monitored above and expressed as the content of non-tannin phenolics on dry matter. From the above results, the tannin content of the sample was calculated as follows:
Tannin (%) = Total phenolics (%) – Free phenolics (%).
In vitro anti-oxidant activity :
- DPPH• radical scavenging activity: The 2, 2-diphenyl-picryl-1-picryl-hydrazyl radical (DPPH) scavenging activity was measured according to the method of Blois21. Methanol extract of callus samples at various concentrations was added separately to each 5mL of 0.1mM methanolic solution of DPPH and allowed to stand for 20min. Absorbance at 517nm using spectrophotometer was measured. Rutin and Quercetin were used as standard. The corresponding blank reading was also taken and DPPH radical scavenging activity was calculated by using the following formula:
DPPH radical scavenging activity (%) = Control OD-Sample OD/Control OD×100
IC50 value is the concentration of the sample required to scavenge 50% DPPH free radical / OH+ radical which has been determined by using the software SPSS v.16.
- ABTS•+ assay: The total antioxidant activity of the samples was measured by ABTS·+ [2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation decolorization assay according to the method of Re et al22. ABTS•+ was produced by reacting 7mM ABTSaqueous solution with 2.4 mM potassium persulfate in the dark for 12–16 h at room temperature. Prior to assay, this solution was diluted in ethanol (about 1:89 v/v) and equilibrated at 30˚C to give an absorbance of 0.700 ± 0.02 at 734 nm. The stock solution of the sample extracts was diluted such that after introduction of 10 μL aliquots into the assay, which have been produced between 20% and 80% inhibition of the blank absorbance. After the addition of 1 mL of diluted ABTS solution to 10 μL of sample or trolox standards (final concentration 0–15 μM) in ethanol, absorbance was measured at 30˚C exactly 30 min after the initial mixing. Appropriate solvent blanks were also run in each assay. Triplicate determinations were made at each dilution of the standard, and the percentage inhibition was calculated from the blank absorbance at 734 nm and then it was plotted as a function of trolox concentration. The unit of total antioxidant activity (TAA) is defined as the concentration of trolox having equivalent antioxidant activity expressed as μmol/g sample extract on dry matter.
Statistical analysis: All tests were conducted in triplicate. Data were reported as means ± standard deviation (SD) and were subjected to one way analysis of variance (ANOVA) and the significance of the difference between means was determined by Duncan’s Multiple Range Test (P<0.05) using the statistical software Inc., Tulsa, OK, USA.
RESULTS AND DISCUSSION: The response of various explants for callus formation to different plant growth hormones (PGRs) in MS mediumviz., BAP, 2,4-D, NAA and IAA in various combinations and concentrations was varied widely for the study species, H. populifolia (Tables 1 and 2). Callus formation was observed to be started at the margins in explants. Further, no callus formation was observed during the culture period in the control (MS medium without growth regulators) in all explants.
In the case of solo application of PGRs, 2, 4-D and BAP in combination with 2, 4-D produced friable, embryonic and pale yellow calli. Greater number of explants viz., leaf, internode and petiole responded rapidly in MS medium fortified with 2,4-D at 2.0 mg/L for callus formation (Fig. 1a, c, d). Similar kind of higher response of leaf explant to 2,4-D at 0.5 mg/L for callus formation was noted for the medicinal herb, Ionidium suffruticosum by Arunkumar and Jayaraj 23. The time required for callus formation was also varied from 12 to 19 days across the explants used (Table 1).
Callus fresh weight produced by the leaf explant was higher (26.28 g/explant) than that of other explants used. Mohajer et al 24 investigated that generally the leaf explants of many plant species produced higher fresh weight of callus than any other explants attempted.
After 15 weeks of inoculation, it was noted that calli were turned to light brownish (Fig. 1e) due to the synthesis of phenolic compounds. The transfer of calli to fresh medium was found to be effective in reducing the browning due to phenol exudation 25. In many species viz., Ephorbia lathyris 26, Pisonia alba 27, Bambusa ventricosa 28 and Tecomella undulate 29 this method of transfer of calli to fresh medium was used to control the browning of callus. Embryoid initiation was noted during subculturing of callus on MS medium with different combinations and concentrations of PGRs. It is reported that variations in hormonal combinations may be one of the effective methods for embryoids initiation from undifferentiated callus 30-34.
FIG. 1A: CALLUS DEVELOPED FROM LEAF EXPLANTS CULTURED ON TO THE MS MEDIUM FORTIFIED WITH 2,4-D AT 2mg/L, (B) FORMATION OF NODE CALLUS ON MS MEDIUM SUPPLEMENTED WITH BAP AND IAA AT 2.0 AND 4.0 mg/L, (C AND D) INTERNODE AND PETIOLE CALLUS ON MS MEDIUM SUPPLEMENTED WITH 2,4-D AT 2mg/L RESPECTIVELY, (E) BROWNING OF CALLUS AFTER 15 WEEKS, (F) MICROSCOPIC VIEW OF THE CALLUS CELL
Results of the preliminary phytochemical studies are presented in Table 3. The data revealed the presence of alkaloids, phenols, steroids, flavonoids and terpenoids in all the calli derived from different explants. However, triterpenoid was present in higher quantity only in leaf callus and totally absent in petiole callus. Cardiac glycoside was observed to be present in leaf and internode calli only and the tannin was present with high quantity in internode callus. Saponins were not detected in any callus of various explants. The secondary metabolites investigated to be present in various calli are reported to have various medicinal properties. Addae-Mensah 35 reported that the alkaloids and tannins are used in traditional herbal preparations against various common ailments in central African countries. Phenolic compounds including flavonoids possess diverse biological properties like antiinflammatory, anticarcinogenic, antidiabetic and antioxidant activities 36-38.
TABLE 3: THE DEGREE OF PRESENCE OR ABSENCE OF SECONDARY METABOLITES IN THE CALLI DERIVED FROM DIFFERENT EXPLANTS OF HILDEGARDIA POPULIFOLIA
|S. No.||Test||Callus explants|
+, ++, +++ and – indicate the tracer, little and higher amount of respective secondary metabolite and – shows the absence of respective secondary metabolites.
Quantitative analysis of total flavonoids, phenols and tannins content showed differences among the calli derived from different explants and different days after inoculation for the study species, Hildegardia populifolia (Table 4). The results of the study exhibited that considerable amount of flavonoids were present in the calli of all explants. It was higher (13.35mg RE/g extract) in 60 days old internodal callus followed by 40 days old callus of the same explant (12.101 mg RE/g extract) and 60 days old callus of leaf explant (10.28 mg RE/g extract). Flavonoids are naturally occurring phenolic compounds which have a widespread distribution in intact plants and have been found in many in vitro cultures also 39.
TABLE 4: QUANTITATIVE ESTIMATION OF TOTAL PHENOLS, FLAVONOIDS AND TANNINS IN METHANOLIC EXTRACTS OF CALLI DERIVED FROM DIFFERENT EXPLANT OF HILDEGARDIA POPULIFOLIA
|S. No.||Explant||Age of callus (days)||Content of secondary metabolite in callus|
|Total flavonoids(mg RE/g extract)||Total Phenols(mg GAE/g extract)||Total tannins(mg GAE/g extract)|
Values are expressed as mean±SD (n=6). Values within the same column not sharing common superscript letters (a-l) differ significantly at p<0.05 by DMRT.
Phenols are very important plant constituents because of their scavenging ability on free radicals due to their hydroxyl groups. Therefore, the phenolic content of biological system may contribute directly to their antioxidant action40. The total phenolics content in the 60 days old leaf callus culture was estimated to be higher (18.20 mg GAE/g extract) than in the other calli derived from other explants (Table 4). 60 days old internodal callus with 16.38 mg GAE/g extract ranked second for phenolics contents.
Similar to phenolics, the higher content of tannins was estimated in 60 days old leaf callus of H. populifolia (25.45 mg GAE/g extract) followed by 60 days old intermodal callus (23.73 mg GAE/g extract) and 40 days old leaf and intermodal callus (20.35 and 20.22 mg GAE/g extract respectively). Tanaka et al 41reported that the presence of tannins such as gallic acid, catechins and tannin-glycosides in callus cultures of the woody species, Quercus acutissima added the value of callus of this species for antioxidant properties.
All the major secondary metabolites identified were higher in 60 days old callus cultures than that of the 40 and 20 days old cultures. It is explained that some secondary plant products were synthesized in higher quantity by differentiated tissues by accumulation in specialized tissues or organs. Therefore, it is indispensable to sustain differentiated and organized tissues for the preparation of drugs 39, 42.
DPPH scavenging activity of methanolic extracts of various age old series of callus culture of leaf, node, internode and petiole showed greater variation (Table 5). 60 days old leaf derived callus culture had relatively higher DPPH radical scavenging activity with the IC50 value, 86.24 mg/g than that of the node, internode and petiole callus cultures. However, it was not comparable to that of the standards, rutin and quercetin which were with the IC50 values 56.24 and 47.05 mg/g respectively. These results were in similar trend of that obtained by Farouk et al 43 who reported that leaf derived calluscontained relatively higher radical scavenging activity in the species, Citrullus colocynthis.
TABLE 5: DPPH• RADICAL SCAVENGING ACTIVITY AND ABTS•+ ACTIVITY IN DIFFERENT AGE OLD SERIES OF CALLI DERIVED FROM VARIOUS EXPLANTS OF HILDEGARDIA POPULIFOLIA
|S. No.||Explant||Age of callus (days)||DPPH• (IC50) µg/mL||ABTS•+(µmol TE/g extract)|
Values are expressed as mean±SD (n=6). Values within the same column not sharing common superscript letters (a-n) differ significantly at p<0.05 by DMRT.
Total antioxidant activity was measured by ABTS+ method. It was found that the 60 days old internode derived callus culture has higher antioxidant activity (3077.98 µmol TE/g extract) followed by 60 days old leaf derived callus (2109.36 µmol TE/g extract), 60 days old node derived callus (2090.90 µmol TE/g extract) and 60 days old petiole derived callus (1034.32 µmol TE/g extract).
The values were expressed as trolox equivalents. Renuka et al.44 investigated that the in vitro callus culture of the species, Ruta graveolens showed prominent antioxidant activity, which may be attributed to higher content of phenolics found in the callus of this species.
The present study concluded that the MS medium supplemented with 2,4-D produced callus from higher percentage of most of the explants used. Moreover, the leaf and intermodal callus contained greater amount of phenolic compounds and hence the antioxidant activity. As the study species, Hildegardia populifolia is a critically endangered species, priority must be given to conserve the wilds. However, to meet the demand, in vitro culture sources particularly the callus derived from leaf and internodal explants can be exploited for antioxidant property.
ACKNOWLEDGEMENT: The first author is greatly acknowledging Dr. M. Aruchami Research Foundation, Coimbatore for providing financial assistance (No. ARF/RA-2013/005 dt. 20.01.2013).
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How to cite this article:
Saradha M, Ranjitham P and Paulsamy S: Evaluation of in vitro antioxidant properties of Callus cultures of an endangered medicinal tree species, Hildegardia populifolia (Roxb.) Schott & Endl. Int J Pharm Sci Res 2014; 5(3): 839-48.doi: 10.13040/IJPSR.0975-8232.5(3).839-48
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
M. Saradha , P. Ranjitham and S. Paulsamy*
Department of Botany, Kongunadu Arts and Science College, Coimbatore – 641 029, Tamil Nadu, India
01 October, 2013
25 November, 2013
10 February, 2014