Abstract: Herbal remedy is considered as one of the popular forms of alternative and complementary medicines. Plants are considered to possess a number of chemical constituents with diverse pharmacological efficacies. Bryophytes, a small group of plants, are known to contain unique secondary metabolites having pharmacological and potential therapeutic value. The primary focus of the study is to depict the role of altitude and tissue types on antioxidant capacity of the liverwort Pellia endiviifolia (Dicks.) Dumort. (Pelliaceae). In the present investigation, an attempt has been made to explore the antioxidative potential of vegetative and reproductive tissues of P. endiviifolia collected from five different altitudes of Darjeeling Himalaya, West Bengal, India. Total phenolics and flavonoids contents of the liverwort samples were also determined. Methanol extract of the thalloid liverwort was investigated for antioxidant activity by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, total phenolic and flavonoid estimation. Maximum radical scavenging activity was found to be 89.336%±4.3. Maximum total phenolics content in 1 mg of the extract was 58±0.175 μg of Gallic Acid Equivalent (GAE) per mg dry weight. Maximum flavonoids content in 1mg of the extract was 80.3±331 μg of Quercetin Equivalent (QE) per mg dry weight. The results indicate, for the first time, the antioxidative potential and possible use of the liverwort as a natural antioxidant. It also shows a variation of antioxidant capacity of the liverwort depending on their tissue type and their altitude of occurrence.
INTRODUCTION
Reactive Oxygen Species (ROS) generates oxidative stress in the body as a result of endogenous or exogenous factors (Klaunig and Kamendulis, 2004) which is associated with different ailments (Pejin et al., 2012). Beneficial effects of antioxidants against various types of diseases such as diabetes (Kaneto et al., 1999), leprosy (Vijayaraghavan et al., 2005), Alzheimer's disease (Mancuso et al., 2007), cancer (Nishino et al., 2004), AIDS (Shabert et al., 1999) etc. have been recorded. Dietary antioxidants have been used against tumor (Black and Chan, 1975) and cancer (Khan et al., 2008). Herbs, spices, fruits and vegetables have always served as a natural source of antioxidants (Nakatani, 2000; Yanishlieva et al., 2006; Peschel et al., 2006; Podsedek, 2007).
Plants are reported to possess medicinal properties (Dey and De, 2012a,b) which have been tested by pharmacological investigations (Dey et al., 2011; Mukherjee et al., 2012a). Bryophytes are known to produce a number of secondary metabolites with diverse biological functionality to resist various types of biotic and abiotic stresses (Xie and Lou, 2009). Bryophytes are reported as a natural storehouse of bioactive molecules having immense pharmacological potential. They have been reported for antibacterial (Kamory et al., 1995), antifungal (Dey and De, 2011), antioxidative (Dey and De, 2012c), nitric oxide inhibitory (Harinantenaina et al., 2006) and cytotoxic (Scher et al., 2002) properties. Antioxidative activities of bryophytes have been reported in Bryum moravicum Podp. (Bryaceae), Brachythecium rutabulum (Hedw.) Schimp. (Brachytheciaceae), Calliergonella cuspidata (Hedw.) Loeske (Hypnaceae) and Hypnum mammillatum Funck (Hypnaceae) (Pejin and Bogdanovic-Pristov, 2012; Pejin et al., 2012), Marchantia polymorpha L. (Marchantiaceae) (Gokbulut et al., 2012) and many others (Chobot et al., 2008).
Pellia endiviifolia (Dicks.) Dumort. (Pelliaceae) is a terrestrial liverwort which grows on moist rocks either in pure population or in mixed population with other bryophytes (Singh and Singh, 2009). The liverwort is reported to possess bis (bibenzyl) compounds (Hashimoto et al., 1991) which are known to exhibit prolific bioactivities. Cell suspension culture of the species has also yielded diverse types of metabolites (Ono et al., 1992) and the species is yet to be reported for its antioxidative activity. Other species of the genus Pellia such as Pellia epiphylla (L.) Corda (Pelliaceae) (Cullmann et al., 1993, 1997; Cullmann and Becker, 1998) and Pellia fabbroniana Raddi (Pelliaceae) (Matsuo et al., 1971) have been reported for phyto-constituents. The present investigation first time focuses on the antioxidative potential of the liverwort P. endiviifolia.
MATERIALS AND METHODS
Plant material: The vegetative and reproductive thalli of P. endiviifolia were collected by A. Dey and S. Mukherjee from five different altitudes of Darjeeling district of the eastern Himalayas (Tiger Hill, Lava, Lolegaon, Rishyap and Kalimpong) during the years 2010-2011 (Table 1). The voucher specimen (PE 01) was identified from the key to the specimen (Singh and Singh, 2009) and deposited at the Department of Zoology and Molecular Biology, Presidency University, Kolkata, India.
Extraction: Before extraction, the plant material was first washed with detergent Teepol® followed by Bavistin to remove microbial contamination. Final rinsing was done in autoclaved distilled water. The plant material was air dried and powdered (40 mesh size) using an electrical mixer grinder. Extraction was done by soaking 1 g of dried powder in 80% methanol for 96 h. Filtration was done using Whatman’s No.1 filter paper. The extract was concentrated using a rotary evaporator to give 0.02 g of residue (yield 0.02%). The extract was diluted in 80% methanol to make a 10 mg mL-1 solution and stored at +4°C.
Antioxidant activity and estimation of total phenolics and flavonoids
Total phenolics estimation: Folin-Ciocalteu method described by Liu
et al. (2008) with some modifications was followed to estimate the
total phenolics content. Optical density was measured at 765 nm. The obtained
result was expressed as μg GAE per mg dry weight.
| Table 1: | Places of collection of the plant materials with respective altitude and latitude |
Total flavonoids estimation: Total flavonoid was estimated following Zhishen et al. (1999) with some modification. Optical density of the solution was measured at 510 nm. Total flavonoid content was expressed as μg of QE per mg dry weight.
DPPH radical scavenging activity: A 0.5 mL of extract (1mg mL-1) was added to 3 mL of 0.1 mM solution of DPPH following the method given by Ohnishi et al. (1994) with slight modifications. The concentrations of the extract were 0.0625 to 1 g mL-1. The mixture was incubated at room temperature for 20 min. Optical density was measured at 517 nm.
Statistical analyses: All calculations were performed in triplicate with their mean values and standard error (Pagano and Gauvreau, 2000). Values are given as Mean±S.D. Student’s t-test was used to determine statistical significance. Values with p<0.05 were taken as significant. Tables were prepared using Microsoft Office Excel (2007).
RESULTS
Methanol extract of plant body of P. endiviifolia showed high DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity in a dose-dependent manner. Maximum radical scavenging activity was 89.336±2.77 (Mean±SE, n = 3) from the Tiger Hill population (Table 2). The IC50 value was found to be 0.1±0.07 mg mL-1.
| Table 2: | DPPH assay for P. endiviifolia vegetative and reproductive thalli |
| Table 3: | Phenolics content of vegetative and reproductive thalli of P. endivifolia |
| Table 4: | Flavonoids content of vegetative and reproductive thalli of P. endivifolia |
Maximum total phenolic content in 1mg of the extract was 58.3±175 μg of gallic acid equivalent from the Tiger Hill population. The flavonoid content in 1mg of the extract was 80.3±331 μg of quercetin equivalent (Table 3, 4) form the same population. Interestingly, slight differences were found regarding antioxidative capacity, total phenolic and flavonoid content of the hepatic collected from various altitudes and the vegetative thallus has shown higher activity and content than the reproductive plant body.
DISCUSSION
The results indicate considerable amount of phenolic and flavonoid content in the tested extract, which may be interpreted as the reason for the significant radical scavenging activity of the bryophyte. Results also depict altitudinal and tissue specific variation of bioactivity which may be attributed to differential exposure of different bryophyte tissues to UV radiation and temperature at various altitudes. Altitude seems to play a significant role in the secondary metabolite profile of certain plants (Spitaler et al., 2006; Ganzera et al., 2008). In another report, temperature was depicted as the key factor in such variations (Albert et al., 2009). In higher altitudes, dual role of enhanced UV-B radiation and decreased temperature seems to influence the antioxidant phenolics content in certain higher plants (Spitaler et al., 2008). In Ginkgo biloba, altitude was found to control the plant’s neutraceutical formulations by modulating secondary metabolite profiling (Kaur et al., 2012). In another eastern Himalayan hepatic, Dumortiera hirsuta, tissue type (vegetative/reproductive) and altitude are reported to influence the plants bioactivity against a number of human pathogenic bacteria (Mukherjee et al., 2012b). Antioxidant and antidiabetic potential of a Vigna sp. was also found to vary depending on altitude (Yao et al., 2012).
Plants are reported to possess antioxidative activities (Muchuweti et al., 2006; Kumbhare et al., 2012) and bryophytes are no exception. These tiny plants are also reported from the ethnic uses for their diverse healing properties (Harris, 2008). The therapeutic ability of the bryophytes may be ascribed to their diverse pharmacological properties including antioxidative potentials. The data indicate potential antioxidative properties of the liverwort of P. endiviifolia from Darjeeling Himalaya which can be exploited as a natural source of antioxidants with possible therapeutic applications. Antioxidants have been tested against aging (Pejin and Bogdanovic-Pristov, 2012) and age related disorders (Snodderly, 1995), ethanol intoxication (Xie and Lou, 2009) and in cosmetics (Darvin et al., 2006). The growing demand of antioxidants in medical science and cosmetic industry can be complemented with the newer sources of antioxidants from natural sources.
CONCLUSION
Bryophytes are subjected to face a number of stresses starting from its pioneer colonization on barren rock surface to their continuous exposure to UV, cold temperature, predation and microbial attack. A strong antioxidative response is present in bryophytes which may be exploited for medicinal and cosmetic use. Further analyses may reveal the presence of bioactive compounds with pharmacological activity which can be implicated to their potential as antioxidants.
ACKNOWLEDGMENT
We acknowledge the grant and assistance provided by University Grant Commission (Minor Research Project No. F. PSW-072/09-10 (ERO) dated: 8th October, 2009).