Introduction

Tree leaves provide strong protective effects against diseases associated with oxidative damage (Podsędek, 2007) for antioxidant property (Uusiku et al., 2010). Antioxidants of tree leaves also play pivotal role in prevention of chronic and degenerative diseases of animals. The tree leaves are the main components of roughage feeding to the dairy animals in Mizoram, particularly during lean season i.e. winter months of the year for scarcity of grasses and forages in the state. A few studies had been conducted on nutritional assessment of tree foliages fed to dairy animals in Mizoram (Das et al., 2006, Sarma et al., 2007 and Samanta et al., 2009); however, no information is available about their anti-oxidant properties. In the present study, therefore, an attempt was made for estimation of antioxidant properties of the commonly fed tree foliages to dairy animals in Mizoram.

Materials and Methods

Location of the Study

The study was conducted in three Rural Development Blocks (RDB) namely, Tlangnuam, Thingsulthiah and Aibawk of Aizawl district, Mizoram.  Fifteen farmers were randomly selected from five villages of each RDB for the study.

Survey and Preparation of Sample for Analysis

Information about utilization of tree leaves were collected from the selected farmers. Based on the ‘Mizo’ names of the tree species and available literatures of their english names, botanical names were confirmed in collaboration with the Department of Forestry, Mizoram University, Aizawl, Mizoram and referring to literatures of Botanical Survey of India, Kolkata, India. The healthy leaves of each species were collected separately from the selected farmers, pooled together and six representative samples were drawn for analysis. The samples of leaves were air dried under the shade and made in mesh with powdery consistency for preparation of aqueous extracts using soxhlet extraction system.

Methods for Evaluation of Anti-Oxidant Activity

Iron (III) to Iron (II) Reducing Activity

In vitro antioxidant activities of the tree leaves extracts were evaluated by iron (III) to iron (II) reducing activity following methods of Oyaizu (1986). One ml of plant extract was dissolved in PBS (0.2M, pH 6.6) followed by addition of 2.5 ml of 1% aqueous potassium hexacyanoferrate [K₃Fe (CN)₆] solution. After 30 minutes incubation at 50ºC, 2.5 ml of 10% trichloroacetic acid (TCA) was added and the mixture was centrifuged at 2000 rpm for 10 min. To 2.5 ml aliquot of the upper layer, 2.5 ml of distilled water and 0.5 ml of 0.1% aqueous FeCl₃ were added and the absorbance was recorded at 700 nm. The data were presented as ascorbic acid equivalents (Asc AE) in mmol ascorbic acid per ml of plant extract.

Ascorbate Iron (III) Catalyzed Phospholipid Peroxidation

The ability of the selected tree leaves to scavenge hydroxyl radicals was determined by the method as Aruoma et al. (1997). Mouse liver sample was mixed (1:10) with 10 mM phosphate buffered saline (PBS, pH 7.4) and sonicated in an ice bath for preparation of homogenate. To 0.1 ml of plant extract 0.2 ml liposomes, 0.5 ml PBS and 0.1 ml of 1 mM FeCl₃ solution were added. Peroxidation was initiated by adding 0.1 ml of 1 mM ascorbic acid. The mixture was incubated at 37⁰ C for 60 minutes. The reaction was stopped by adding 1 ml of 10% trichloroacetic acid (TCA) and was centrifuged at 2000 rpm for 10 minutes. After centrifugation, 1 ml of 0.67% 2-thiobarbituric acid (TBA) in 0.05 M NaOH was added to the supernatant. The mixture then vortexed and heated in a water bath at 100⁰ C for 20 minutes. After cooling, 1 ml distilled water was added and absorbance was taken at 532 nm using UV-Visible spectrophotometer. The percentage inhibition was calculated where the control contained all the reaction reagents except sample.

Results and Discussion

Tree Fodder Species Utilised for feeding Dairy Animals

The tree foliages species identified were An-ku/ An-ku-hawng-tial (Celtis tetrandra Roxb.), Bel-phuar [Trema orientalis (L.) Blume], Bung (Ficus benghalensis Linn.), Ba-tling (Wendlandia budleioides Wall. Ex Wight & Arn.), Bil [Protium serratum (Wall. ex Colebr.) Engl.], Chawm-zil [Ligustrum robustum (Roxb.) Blume], Chhawn-tual  [Aporosa octandra (Buch.-Ham. ex D. Don) Vickery], Hnum (Engelhardtia spicata Lechen ex Blume), Hnah-thap [Colona floribunda (Kurz) Craib], Hnahkiah (Callicarpa arborea Roxb.), Hriang (Betula alnoides Buch.-Ham. ex D. Don.), Hmui-pui/ Len-hmui [Syzygium cumini (L.) Skeels], Khiang (Schima wallichii Choisy), Leh-ngo (Sarcochlamys pulcherrima Gaudich), Lam-khuang (Artocarpus heterophyllus Lam.), Muk-fang (Cordia dichotoma G. Forst), Ngiau [Magnolia oblonga (Wall. ex Hook.f. & Thomson) Figlar], Nau-thak [Litsea monopetala (Roxb.) Pers.], Phan (Ulmus lanceifolia Roxb. Ex Wall.), Ri-hnim (Ficus retusa L.), Si-hneh [Eurya cerasifolia (D.Don) Kobuski], Sa-zu-thei-pui (Ficus hirta Vahl.), Thing-kha [Derris robusta (DC.) Benth.], Thel-ret [Ficus elastica Roxb. Ex Hoemen.], Thing-lung [Homalium ceylanicum (Gardner) Benth. Subsp. minutiflorum (Kurz.) Mitra], Tlai-zawng/ Pai-vun [Cerasus cerasoides (Buch. Ham.exD.Don) S.Y.Sokolov], Thing-hmar-cha[Celtis timorensis Span.], Thei-tat (Artocarpus lakoocha Roxb.), Thingsia [Castanopsis tribuloides (Sm.) A.DC.], Thing-ri [Albizia odoratissima (L.f.) Benth.], Thei-ria [Carallia brachiata (Lour.) Merr.], Tiar (Saurauia punduana Wall.), Thlan-vawng [Gmelina arborea Roxb.], Thing-vawk-pui [Balakata baccata (Roxb.) Esser], Thei-tit [Ficus prostrate (Wall. ex Miq.) Buch.-Ham. ex Miq.], Vau-be [Bauhinia variegate L], Vang [Albizia chinensis (Osb) Merr.], Zih-nghal [Stereospermum chelonoides (L.f.) DC.], Zai-rum [Anogeissus acuminate (Roxb. Ex DC) Gull.] and Khawmhma (Rhus chinensis Mill).

Das et al. (2006) reported about utilization of 12 tree species for feeding dairy animals; whereas, Sarma et al. (2007) described 14 different forage species utilised for feeding dairy animals by the Mizo communities in Mizoram. However, in the present study, the personal interview with the dairy farmers revealed that they utilised tree leaves of forty one (41) different species throughout the year, out of which 21 tree species were evergreen and the rest were either large or small deciduous trees.

Anti-Oxidant Properties of Tree Foliages

Antioxidant inhibits the oxidation involving the loss of electrons or an increase in oxidation state. Oxidation reactions produce ROS (free radicals) which cause death or damage of body cells.

Table 1: Iron (III) to iron (II) reducing activity and ascorbate iron (III) catalyzed phosphor-lipid peroxidation (% inhibition) of tree foliages fed to dairy animals in Mizoram

* Not detected

Antioxidants neutralise chain reactions caused by free radicals by removing the free radical intermediates and inhibiting other oxidation reactions. The plants are known to possess antioxidant activity. In the present study, antioxidant activity of aqueous extracts of leaves of plant species identified were carried out in terms of Iron (III) to iron (II) reducing activity and ascorbate iron (III) catalyzed phosphor-lipid peroxidation (Table 1).

The ascorbic acid equivalent values for different tree species were varied from 0.1324±0.00 Asc (mg/g) to 0.2227±0.00 Asc (mg/g). However, for most of the tree species values were similar in nature indicating similar reducing power and hence antioxidant activity. Highest reducing power was estimated in Protium serratum (Wall. ex Colebr.) Engl. i.e. Bill followed by Rhus chinensis Mill i.e. Khawmhma, Syzygium cumini (L.) Skeels i.e. Hmui-pui/ Len-hmui and Ficus retusa L. i.e. Ri-hnim. Other tree species were found to have similar reducing power i.e. antioxidant activity. The reducing power assay is generally used to assess the ability of an antioxidant to donate an electron. Fe³⁺ reduction is very often used as indicator of electron-donating activity. The reducing power of the extract increases as the concentration increases suggesting that some compounds in the extract may be able to terminate the radical chain reaction. Brighente et al. (2007) also mentioned that the levels of antioxidant activity of plants are variable and phenolic content provides substantial antioxidant activity. Similar findings are also reported by Gholivand and Piryaei (2014), Ardekani et al. (2011), Vanisha et al. (2013) and Nambiar et al. (2013). The % inhibition was calculated as shown by various plant aqueous extracts under the ascorbate iron (III) catalyzed phosphor-lipid peroxidation. The percentage inhibitions were variable ranging from 33.94±1.14% to 93.05±0.36%. This indicated that the plant species might contain some compounds which were having antioxidant activity responsible for higher % of inhibition.

Conclusion

The results of the present study, thus, indicated that the tree species utilised as fodders for dairy animals in Mizoram have noticeable anti-oxidant activity and can be regarded as promising candidates for plant-derived anti-oxidant compounds and for utilizing them as feeds as well as herbs for the dairy animals in Mizoram.

Acknowledgement

 The author is thankful to the Vice-Chancellor, Central Agricultural University, Imphal, Manipur, India for financial support and providing the facilities to carry out the study in the form of research project and the Department of Forestry, Mizoram University, Aizawl, Mizoram for rendering help in identification of the species.

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