Abstract: Phytochemical estimation and radical scavenging activity were carried out in the barks of Bauhinia variegata, Saraca asoka and Terminalia arjuna collected from central India with the objective to determine optimum harvesting age (girth class). Bark samples were analyzed for total phenols, flavonoids and tannins. Radical scavenging activity of the barks was evaluated using DPPH assay. Significant variation was observed in total phenols and tannins in B. variegata; flavonoids and tannins in T. arjuna with respect to different girth classes. However, no significant variation was found in chemical constituents among different girth classes of S. asoka. Gallic acid was used as standard having IC50 value of 1.06±0.10 μg mL-1. Radical scavenging activity showed significant variation in the barks of different girth classes of B. variegata, S. asoka and T. arjuna. The results revealed that the optimum girth class to harvest barks of B. variegata was 36-55 cm, S. asoka 61-90 cm and T. arjuna 121-150 cm as these were found to contain maximum concentration of active ingredients and possess high radical scavenging activity. Among the studied species T. arjuna showed maximum radical scavenging activity and thus can be termed as a potent antioxidant species whose bark can be used for making various formulations containing natural antioxidants.
INTRODUCTION
Bauhinia variegata, Saraca asoka and Terminalia arjuna are important medicinal trees being used in several herbal formulations (Vileges et al., 1997; Paarakh, 2010). The therapeutic properties of herbal drugs are due to the presence of certain chemical constituents (secondary metabolites) present in plant which varies according to their age and maturity (Pandey and Kori 2009). Among different chemical constituents polyphenols (flavonoids, phenolics, condensed and hydrolysable tannins) are major bioactive compounds responsible for the prevention of chronic diseases and health care (Sati et al., 2010). They have been reported to exert antiinflammatory, antimicrobial, antioxidant, anticarcinogenic and body mass reducing activities (Arts and Hollman, 2005; Youdim et al., 2004).
Bauhinia variegata Linn. (Kachnar) belonging to family Fabaceae is a medium sized deciduous tree with a short bole and spreading crown. The various plant parts viz., stem, stem bark, leaves, seeds, roots, flower buds and flowers are used in various indigenous systems of medicine and popular among the various ethnic groups in India for the cure of variety of ailments. The bark is astringent, antileprotic, antigoitrogenic, antitumour and used in fever, skin diseases and wound healing (Kurien, 2001; Daniel, 2006; Thakur et al., 1992; Singh and Aswal, 1992). It is also reported to be useful in obesity. The leaves are used in treatment of skin diseases and stomatitis (Balajirao et al., 1995). The roots of the plant are used as an antidote for snake poisoning, in dyspepsia, flatulence and as carminative (Kurien, 2001). The stem bark is reported to contain 5,7-dihydroxy and 5,7-dimethoxy flavanone-4-O-α-L rhamnopyrosyl-β-D-glycopyranosides, kaempferol-3-glucoside, lupeol and betasitosterol (Yadava and Reddy, 2001; Zhao et al., 2005).
Saraca asoca Roxb. De Wilde. (Ashoka) belonging to family Fabaceae is a medium sized evergreen tree upto 9 m in height with numerous spreading and drooping glabrous branches. Bark is bitter and used as astringent, anthelmintic, stomachic and in various other diseases. The bark has a stimulating effect on the endometrium and ovarian tissue and is useful in menorrhagia during uterine fibroids. It also has great benefits for its uterine activity (Satyavati et al., 1970). Leaves are useful in stomachalgia and flowers are use in vitiated condition of pitta, syphilis, hyperdipsia, inflammation, dysentery, haemorrhoids and scabies in children (Nadkarni and Nadkarni, 2005). Chemical investigation showed the presence of catechols, sterols, tannins, flavonoids, glycosides, leucopelargonidin and leucocyanidin in bark (Sadhu et al., 2007).
Terminalia arjuna Roxb. Wight and Arn. (Arjuna) belonging to family Combretaceae is a large deciduous and evergreen tree, standing 20-30 m above ground level with fissured bark and numerous dropping branches. It has been considered by the Ayurvedic physicians as well as by the modern practitioners as a cardiac tonic (Dwivedi, 1996). Clinical evaluation of this botanical medicine indicates that it is beneficial in the treatment of coronary artery disease, heart failure and possibly hypercholesterolemia (Alpana et al., 1997). It has also been found to possess antibacterial, antioxidant, antimutagenic (Kaur et al., 2002) and hepatoprotective activities (Subasini et al., 2007). The pharmacology of T. arjuna have been discussed by Patnaik et al. (2007) and pharmacological activities are mainly due to the tannins present in its bark. Tannins also contribute to the hypotensive action of T. arjuna bark (Takahashi et al., 1997). It is also speculated that tannins are responsible for its astringent, wound healing and anti microbial activity (Chaudhari and Mengi, 2006; Bele et al., 2010).
Phenolic compounds are a unique category of phytochemicals especially in terms of their vast potential health-benefiting properties. They have multiple biological effects and also act as antioxidants by preventing the oxidation of Low-Density Lipoproteins (LDL), platelet aggregation and damage of red blood cells (Cheynier, 2005). These chemical constituents (secondary metabolites) present in plant vary according to their age and maturity. Root causes of numerous chronic diseases involve oxidative damage to the cellular components. The use of antioxidants to minimize the oxidative damage is one of the important approaches to the primary prevention of these health problems. Several studies have indicated that the antioxidant activities of some plants were highly correlated with their total phenolic contents (Palav and Dmello, 2006; Oboh, 2008; Gupta et al., 2010). The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides (Osawa, 1994). Further, phenolic compounds are effective hydrogen donors, which make them antioxidant (Rice-Evans et al., 1995). Flavonoids have also been reported to have multiple biological effects, including an antioxidant activity (Gil et al., 1999). Many important medicinal tree species possess antioxidant properties due to the presence of phenolic compounds.
Keeping the above into consideration a study was carried out to evaluate phytochemical constituents and radical scavenging (antioxidant) activity in the barks of B. variegata, S. asoka and T. arjuna collected from central India for optimizing the harvesting age (girth class).
MATERIALS AND METHODS
The bark samples of B. variegata and T. arjuna were collected from Jabalpur (Madhya Pradesh) and S. asoka were collected from Bolangir and Khurda (Orissa) during 2008-2010. The trees were grouped according to girth class representing different age groups. In B. variegata and S. asoka the girth class ranged from 15-90 cm as trees of higher girth classes were not found. However, in T. arjuna it ranged from 30-180 cm. The barks were collected following sustainable harvesting practices.
The harvested bark samples were brought to laboratory and dried under shade. Fresh and dry weights of the barks were recorded to determine the moisture content. The dried bark samples were ground into coarse powder and used for chemical analysis. Total phenols, flavonoids and tannins were analyzed. Total phenols in the barks were estimated by Folin-Ciocalteau method (McDonald et al., 2001), tannin by Folin-Denis method (Sadasivam and Manikam, 1996) and for flavonoids, aluminium chloride colorimetric technique was used (Chang et al., 2002). DPPH assay was used for estimation of radical scavenging activity (Nickavar et al., 2006).
Estimation of radical scavenging activity: The DPPH assay has been largely used as a quick, reliable and reproducible parameter to evaluate the in vitro radical scavenging activity of plant extracts (Koleva et al., 2002; Goncalves et al., 2005). The evaluation of the free radical scavenging activity of each of the extract was carried using the DPPH (2, 2-diphenyl-1- picrylhydrazyl) assay as described by Nickavar et al. (2006) with slight modification.
Sample preparation: Five gram of dried and powdered plant material was taken in a conical flask containing 100 mL of 2 N hydrochloric acid (HCl). The content was kept in a boiling water bath for 30 min, cooled and filtered. The filtrate was transferred to a separating funnel and extracted with 150 mL (50x3) of diethyl ether. The combined ether layer was then washed with distill water and dried over anhydrous sodium sulphate. The residue thus obtained was dissolved in 10 mL methanol and various concentrations of sample extracts were prepared in methanol. One mililiter of 0.3 mM DPPH in methanol was added to 2.5 mL solution of the extract or standard and allowed to stand at room temperature in dark for 30 min. The change in colour from deep violet to light yellow was then measured at 518 nm using spectrophotometer. Blank consisted of 1 mL methanol and 2.5 mL of each sample solution, negative control contained 1 mL DPPH solution and 2.5 mL methanol. Gallic acid was used as standard.
The decrease in absorbance by the DPPH radical with increase in concentration of the extract which manifested in the rapid discolouration of the purple DPPH, suggest that samples have antioxidant activity due to their proton donating ability (Adesegun et al., 2007). The decrease in absorbance was then converted to percentage antioxidant activity using the following formula:
Statistical analysis: Results are expressed as Mean±SD of triplicates. Using statistical package for social sciences (SPSS) version 14 did the analysis.
RESULTS AND DISCUSSION
Phytochemical analysis of B. variegata revealed that total phenols content was maximum (55.38±1.01%) in girth class 36-55 cm and minimum (22.48±7.57%) in girth class 15-35 cm. Tannins were found maximum (24.12±1.77%) in girth class 56-75 cm and minimum (15.71±0.88%) in girth class 15-35 cm. Inhibitory concentration (IC50), i.e., the amount of sample required to inhibit 50% DPPH, was found maximum (7.68±0.04 mg mL-1) in girth class 15-35 cm and minimum (5.63±0.04 mg mL-1) in girth class 36-55 cm. Total phenols, tannins and antioxidant activity (IC50) showed significant variation with respect to girth classes. However, significant variation was not observed in the moisture content and flavonoids concentration with increase in girth class. The results are depicted in Table 1.
Phytochemical analysis of S. asoka bark samples showed that the maximum concentration of total phenols (7.25±0.94%), flavonoids (0.23±0.04%) and tannins (40.15±3.55%) was found in girth class 61-90 cm and minimum concentration of total phenols (6.54±0.71%), flavonoids (0.17±0.01) and tannins (29.92±6.98%) was found in girth class 15-30 cm. IC50 was found maximum (4.82±0.04 mg mL-1) in girth class 15-30 cm and minimum (2.29±0.03 mg mL-1) in girth class 61-90 cm. The data revealed that there was not any significant variation in the moisture content and concentration of all active ingredients with respect to girth class. However, IC50 value showed a significant variation among different girth classes. The results are showed in Table 2.
Concentration of total phenols in T. arjuna bark was found maximum (17.95±2.39%) in girth class 121-150 cm and minimum (12.48±2.70%) in girth class 30-60 cm. Flavonoids content was found maximum (0.59±0.01%) in girth class 151 cm and above and minimum (0.32±0.06%) in girth class 30-60 cm. Tannins were found maximum (12.5±0.30%) in girth class 121-150 cm and minimum (10.2±0.46%) in girth class 30-60 cm. IC50 was found maximum (3.01±0.05 mg mL-1) in girth class 30-60 cm and minimum (1.21±0.03 mg mL-1) in girth class 121-150 cm. Flavonoids, tannins and IC50 showed significant variation, however, moisture content and total phenols did not show any significant variation with respect to girth classes. The results are presented in Table 3.
Radical scavenging activity of the sample extracts increased with the increase in concentration. Figure 1 represents the antioxidant activity of gallic acid having IC 50 value of 1.06±0.10 μg mL-1. Figure 2 shows the comparison between the antioxidant activities of studied species depicting that T. arjuna bark have highest DPPH percent inhibition at all the concentrations, whereas the other two species possess lower activity at all the levels tested.
| Table 1: | Phytochemical analysis and antioxidant activity of Bauhinia variegata bark |
| Values are presented as Mean±SD (n = 3). Mean value within each column followed by different letter differ significantly at p<0.05 | |
| Table 2: | Phytochemical analysis and antioxidant activity of Saraca asoka bark |
| Values are presented as Mean±SD (n = 3). Mean value within each column followed by different letter differ significantly at p<0.05 | |
| Table 3: | Phytochemical analysis and antioxidant activity of Terminalia arjuna bark |
| Values are presented as Mean±SD (n = 3). Mean value within each column followed by different letter differ significantly at p<0.05 | |
| Fig. 1: | Antioxidant activity of Gallic acid |
| Fig. 2: | Percentage inhibition of DPPH in Bauhinia variegata, Saraca asoka and Terminalia arjuna |
Total phenols have a strong negative correlation with the IC50 values in B. variegata (r = -0.706) and T. arjuna (r = -0.631); tannins have a strong negative correlation in S. asoka (r = -0.722) and T. arjuna (r = -0.671), whereas negative correlation of IC50 with flavonoids was found only in S. asoka. Higher negative suggest that the antioxidant activity is dependent upon the constituents (polyphenols), more the concentration of constituents lesser will be the IC50 value, hence higher will be the antioxidant activity. Present result for S. asoka corroborates with the study of Afolayan et al. (2008) in which they observed radical scavenging activity due to the presence of flavonoids in Malva parviflora and reported a significant positive relation between total phenols and flavonoids. Palav and Dmello (2006) and Vinson et al. (1995) also reported correlation of polyphenols with antioxidant activity. Correlation coefficients between different constituents evaluated and IC50 values in the studied species are given in Table 4.
| Table 4: | Correlation between phytochemical constituents and IC50 values in studied species |
| *Correlation is significant at 0.05 level, **Correlation is significant at 0.01 level | |
The study revealed that the concentration of phenols, flavonoids and tannins in B. variegata and T. arjuna was lower in younger trees, increased upto middle age and then decreased. However, in S. asoka, their concentration increased with age. Berrocal et al. (2004) also showed that the chemical composition of Pinus radiata trees varied significantly with the age of tree. Nadeem et al. (2002) also reported significant variations in the taxol content in bark of Taxus baccata trees of different age. Pandey and Kori (2009) also reported direct relationship of tannin and oxalic acid content with the age of T. arjuna trees. Phytochemical analysis of B. variegata bark revealed the presence of 0.02% flavonoids whereas Parekh and Chanda (2007) did not reported flavonoids in the bark collected from the semi-arid region of Rajkot Gujarat, India. In T. arjuna bark tannin content ranged between 9.6 to 12.8%. Present findings are corroborated by the findings of Pandey and Kori (2009) who reported 6.75-14.82% tannins in T. arjuna bark. Total phenols, tannins and flavonoids were found responsible for the radical scavenging activity of B. variegata, T. arjuna and S. asoka respectively. Free radical scavenging activity of different flavonoids was also demonstrated by Khlebnikov et al. (2007) and Tsimogiannis and Oreopoulou (2004).
The results of phytochemical evaluation and antioxidant activities showed that the middle aged populations of B. variegata (36-55 cm) and T. arjuna (121-150 cm) have maximum concentration of phenols and showed highest radical scavenging activity. However, in S. asoka, maximum concentration of phenols and highest radical scavenging activity was found in highest girth class (61-90 cm).
CONCLUSION
In present study significant variation was found in phytochemical constituents in B. variegata and T. arjuna barks. However, no significant variation was seen in the bark of S. asoka. The amounts of polyphenols present were directly proportional to the radical scavenging activity of the species. Among the studied species T. arjuna showed maximum radical scavenging activity and thus can be termed as a potent antioxidant species whose bark can be used for making various formulations containing natural antioxidants. The results revealed that the optimum girth class for harvesting of barks of B. variegata is 36-55 cm, S. asoka 61-90 cm and T. arjuna 121-150 cm.
ACKNOWLEDGMENT
The authors are thankful to the Director, Tropical Forest Research Institute, Jabalpur for providing facilities and encouragement. Financial assistance provided by National Medicinal Plants Board, New Delhi is also acknowledged.