Ontogenetic Variation of Total Phenolics and Antioxidant Activity in Roots, Leaves and Flowers of Astragalus compactus Lam. (Fabaceae)

Abstract

Introduction

The potential health risks and toxicity of synthetic antioxidants resulted in an upsurge of interest in phytochemicals as new sources of natural antioxidants. Phenolics of Astragalus L. (Fabaceae) possess antioxidant properties and have been shown to have a protective effect against several degenerative diseases. The objective of this study was to determine total phenolics and antioxidant activity of methanolic extracts from different parts of A. compactus Lam. at different phenological phases and to investigate the correlations between antioxidation and the contents of the total phenolics.

Methods

Total phenolic content (TPC) was determined using the Folin-Ciocalteau reagent and the antioxidant capacity was evaluated with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) test.

Results

Generally, the TPC in leaves was higher than that of the roots and flowers. TPC in leaves, roots and flowers of the species varied from 5.01-8.25, 4.29-7.89 and 4.19 μg GAE/mg DW, respectively. In addition, roots and leaves at fructification stage possessed higher TPC than vegetative and flowering stages. Therefore, the leaf extracts at fructification phase showed the highest TPC that accompanied with best antioxidant activity. In the root extracts, fructification stage was also characterized by the highest antioxidant activity.

Conclusion

A positive relationship between antioxidant activity and TPC showed that phenolics were the dominant antioxidant components in the species. The results obtained suggest that A. compactus methanolic extracts may serve as potential sources of natural phenolic antioxidants and that the fructification phase could be considered as the best stage for the harvesting of this plant.

Introduction

Numerous physiological and biochemical processes in the human body may produce reactive oxygen species. A vast amount of evidence implicates that free radicals are able to attack lipid membranes, proteins and DNA, and lead to some detrimental effects. The oxidative damage of biomolecules (e.g. lipids, proteins, DNA) induced by overproduction of such free radicals, eventually leads to many chronic diseases, such as atherosclerosis, cancer, diabetes, aging, and other degenerative diseases in humans (Halliwell 1994, Willcox et al 2004, Federico et al 2007).

Antioxidants are now known to play an important role in protection against disorders caused by oxidant damage. Antioxidants can delay or inhibit the initiation or propagation of oxidative chain reactions and thus prevent or repair damage done to the body’s cells by oxygen (Firuzi et al 2011). In recent years, synthetic antioxidants have been widely used by the food industry. However, because of the possible toxicities of synthetic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), the development and use of more effective antioxidants of natural origin is highly desirable (Augustyniak et al 2010, Rodil et al 2012).

Plants contain a wide variety of free radical scavenging molecules, including phenolic compounds, nitrogen compounds, vitamins, terpenoids, and some other endogenous metabolites, which are rich in antioxidant activity (Larson 1988, Cotelle et al 1996, Zheng and Wang 2001). Among the various kinds of natural antioxidants, phenolics constitute the main powerful compounds, owing to their multiple applications in food industry, cosmetics, pharmaceutical and medicinal materials (Maisuthisakul et al 2007, Dai and Mumper 2010). Structurally, phenolics comprise an aromatic ring, bearing one or more hydroxyl substituent, and range from simple phenolic molecules to highly polymerized compounds (Bravo 1998). The antioxidant activity of phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. In addition to their role as antioxidant, phenolic compounds show different medicinal properties such as antibacterial, anticarcinogenic, anti-inflammatory, anti-viral, anti-allergic, estrogenic, and immune-stimulating agents (Larson 1988, Huang et al 2010). Plant phenolics show marked qualitative and quantitative variation not only at different genetic levels (between and within species and clones) (Nichols-Orians et al 1993, Joubert et al 2008) but also between different physiological and developmental stages (Bunning et al 2010). They also vary in response to environmental factors, such as light intensity and nutrient availability (Kotilainen et al 2010, Larbat et al 2012).

Astragalus L. (Fabaceae) is generally considered as the largest genus of vascular plants with an estimated 2500-3000 species (Podlech 2008). Astragalus is widely distributed throughout the temperate regions of the world. The greatest numbers of species are found in the arid, continental regions of western North America (400 species) and central Asia (2000-2500 species). The roots and leaves of some Astragalus species are a very old and well-known drug in traditional medicine, used as antiperspirant, antihypertensive, antidiabetic, diuretic, and tonic (Tong and Eisenbrand 1992, Rios and Waterman 1997). Especially, the roots of some Astragalus species have shown interesting pharmacological properties including hepatoprotective, immunostimulant, and antiviral activities (Pistelli 2002). Regarding its medicinal properties, the interest in chemical constituents of various species of the genus Astragalus has been increased during the recent years (Fathiazad et al 2010, Movafeghi et al 2010a, 2010b). The phenolic compounds are among the most important constituent of the genus, which have been extensively investigated using modern analytical techniques in various studies (Krasteva and Nikolov 2000, Pistelli 2002). However, the information on the total phenolic compounds (TPC) and antioxidant activity of Astragalus is limited. Furthermore, there have been no reports of the variation of phenolics and antioxidant activity during ontogeny.

Therefore, the main objectives of this study were: 1) to assay antioxidant activity and to determine TPC of the Astragalus compactus Lam., as a medicinal plant; 2) to investigate the relationship between TPC and antioxidant activity; and 3) to investigate variation of the TPC during ontogeny in order to determine the optimal harvest time characterized by the highest content of bioactive compounds.

Materials and methods

Plant material

Different parts of A. compactus Lam. were harvested at three phenological phases (vegetative, flowering and fructification) from the Payam Mountains at the east Azerbaijan province of Iran, at altitudes above 2200 m. The samples were identified by Dr. S. Zare (Department of Plant Biology, University of Tehran). Voucher specimens were deposited in the herbarium of the Tabriz University of Medical Science. The samples were dried for 10 days at room temperature and then were powdered.

Extraction

A quantity of 100 g of powdered roots, leaves and flowers were extracted with methanol using a Soxhlet apparatus for 8 h. The extract was concentrated to ~1 ml under reduced pressure on a rotary evaporator. The extract was stored in sealed vials at 4°C until biological analysis.

Determination of TPC

TPC was estimated using the Folin-Ciocalteau colorimetric method described previously (Meda et al 2005) with a little modification. Briefly, 5 g of powdered samples were individually dissolved in 50 ml of acetone-water (in 4:6 ratio). After 30 min, 1 ml of these solutions were mixed with 0.2 ml of Folin-Ciocalteau reagent, and 1 ml of 2% sodium carbonate (Na₂CO₃). After incubation at 40^°C for 30 min, the absorbance of the reaction mixtures were measured at 760 nm by using a spectrophotometer (Shimadzu, Kyoto, Japan). Quantification was done on the basis of the standard curve of gallic acid. Results were expressed as μg of gallic acid equivalent (GAE) per mg of dry weight (DW).

DPPH radical-scavenging activity

The DPPH quenching ability of plant methanolic extracts was measured according to Hanato et al (1988). Five ml of the extract at different concentrations was added to 5 ml of a DPPH methanolic solution. The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark. The absorbance of the resulting solution was then measured at 517 nm. The same procedure was applied for the quercetin as a positive control. The antiradical activity was expressed as IC₅₀ (μg/ml), the antiradical concentration required to cause a 50% inhibition. A lower IC₅₀ value corresponds to a higher antioxidant activity of plant extracts. The ability to scavenge the DPPH radical was calculated using the following equation:

DPPH scavenging effect (%) = (A₀ − A₁)/A₀×100

where A₀ is the absorbance of the control at 30 min, and A₁ is the absorbance of the sample at 30 min.

Statistical analysis

The experiment was a completely randomized design with three replications. The data were subjected to ANOVA and means were separated by Duncan multiple range test at P <0.05 significant level.

Results and discussion

Total phenolic content

Based on obtained results, the TPC ranged from 5.01 to 8.28 μg GAE/mg DW for leaf extracts, and from 4.29 to7.89 μg GAE/mg DW for root extracts (Table 1). Therefore, the TPC was organ-dependent. The highest content of phenolics was detected in the leaf extracts, followed by the root and flower extracts. These findings are in agreement with previous studies on the other species of Astragalus, indicating high phenolic content in leaf compare to root and seed (Niknam and Ebrahimzadeh 2002). Similar results also have been reported for other genera such as Beta vulgaris, Petroselinum crispum and Coriandrum sativum (Pyo et al 2004, Wong and Kitts 2006). Higher amount of phenolics in leaves in comparison to that of roots may be attributed to the effect of intensity of solar radiation on the production of phenolics by plants (Mole et al 1988, Kotilainen et al 2010). Particularly when plant exposed to the UV light, the phenolics are produced as a way of reducing the photodestruction of exposed tissues.

Table 1. Total phenolic contents and DPPH scavenging ability of A. compactus leaf, root and flower extracts.

	Leaves	Roots	Flowers
	Vegetative	Flowering	Fructification	Vegetative	Flowering	Fructification
Total phenolic contents (TPC) (?g GAE/mg DW)	5.01±0.09b	5.18±0.17b	8.28±0.12a	04.29±0.15c	04.48±0.16c	7.89±0.54a	004.19±0.3c
DPPH radical scavenging activity (IC50 (?g/mL))	190.4±4.89d	176.6±4.87e	75.0±3.07f	280.5±3.15a	270.2±4.66b	78.0±2.87f	237.2±3.05c

* Values with different superscript letters are statistically different (p < 0.05).Positive control (quercetin): 0.029 μg/ml.

The results of quantitative estimation of phenolics in the roots and leaves of A. compactus during the different growth stages were shown in Fig. 1. Leaf and root extracts showed a significant increase of their TPC during ontogeny and the highest TPC indicated at fructification phase.

Fig. 1.

Ontogenic variation of total phenolic content (TPC) (μg GAE/mg DW) in A. compactus leaf, root, and flower.

Variation of TPC during the ontogeny of A. compactus confirms previous studies that indicated the influence of both phenological stages and climate factors on production and release of phenolic metabolites. It was reported that in Rhus L., Euonymus L. and Acer L. leaves, the TPC per leaf increased rapidly at the early growth stages but thereafter the content was kept rather constant (Ishikura 1976). Moreover, in some other plants such as Crithmum maritimum L. and Hypericum TPC in the aerial parts reach the highest level at flowering (Males et al 2003, Ayan et al 2007). Accordingly, the peak concentration of phenolics was observed at flowering stage in Boerhavia diffusa L. and Sida cordifolia L. (Verma and Kasera 2007).

The increase in the production of phenolic compounds at the fructification stage of A. compactus, which is characterized by the high temperature and a high exposure to solar radiation, leads us to conclude that A. compactus accumulate phenolic compounds before and during this period to protect itself against solar radiations and/or grazers. Therefore, regarding the observed ontogenic variation, the fructification is the best harvesting time for A. compactus, when phenolics are desired.

Antioxidant activity

The antioxidant activity assay was carried out to assess the ability of the medicinal extracts to scavenge free radicals in vitro. A variation in antioxidant activity of A. compactus extracts was observed with the IC₅₀ ranging from 75 to 270.2 μg/ml. Fig. 2 shows that the leaf extract again exhibited the highest antioxidant activity (corresponds with the lowest IC₅₀), whilst root and flower extracts showed the less antioxidant activity (corresponds with higher IC₅₀).

Fig. 2.

Ontogenic variation of antioxidant activity (IC₅₀ (μg/ml)) in A. compactus leaf, root, and flower.

On the other hand, the data showed that antiradical activity was significantly different in the same organ at the three developmental stages. As for TPC, the capacity to quench free radical seemed to be related to the physiological stage. The highest antioxidant activity was detected at fructification stage (IC₅₀= 75 μg/ml for leaf and 78 μg/ml for root).

A positive relationship between antioxidant activity and TPC for each organ and each developmental stage (Fig. 1, 2) indicate that the phenolic compounds have a major contribution to antioxidant activity of the investigated species. These results are consistent with the findings of many research groups who reported such positive correlation between total phenolic content and antioxidant activity (Cai et al 2004, Zheng and Wang 2001).

Conclusion

A positive relationship between antioxidant activity and TPC showed that phenolics were the dominant antioxidant components in the species. The results obtained suggest that A. compactus methanolic extracts may serve as potential sources of natural phenolic antioxidants and that the fructification phase could be considered as the best stage for the harvesting of this plant.

The findings of this work may aid further research to identify, isolate and characterize the specific compounds from the various extracts which are responsible for the higher antioxidant activity as well as to investigate the mechanisms of its antioxidant activity.

Ethical Issues

None to be declared.

Conflict of interests

The authors declare no conflict of interests.