Antioxidant properties, phenolic composition, bioactive compounds and.pdf

South African Journal of Botany 110 (2017) 240–250 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Antioxidant properties, phenolic composition, bioactive compounds and nutritive value of medicinal halophytes commonly used as herbal teas M. Qasim a,⁎, Z. Abideen a, M.Y. Adnan a, S. Gulzar a, B. Gul a, M. Rasheed b, M.A. Khan a a b Institute of Sustainable Halophyte Utilization, University of Karachi, Karachi 75270, Pakistan Centre of Excellence in Marine Biology, University of Karachi, Karachi 75270, Pakistan a r t i c l e i n f o Available online 22 October 2016 Edited by D De Beer Keywords: ABTS Coastal plants DPPH FRAP Industrial applications Secondary metabolites a b s t r a c t Halophytes, distributed from coastal regions to inland deserts have traditionally been used for medicinal and nutritional purposes. Living in sub-optimal conditions, these plants synthesize stress associated bioactive molecules, which are still remain largely unexplored. In search of natural antioxidant sources, antioxidant capacity (AC) and total phenolic content (TPC) of 100 medicinal plants (halophytes vs non-halophytes), commonly used as herbal teas, were investigated. Nutrients and phytochemical composition, especially phenolic metabolites in selected medicinal plants with higher AC were also determined. Most of the medicinal plants analysed for the first time showed considerable AC. In general, halophytes displayed higher AC and TPC than non-halophytes. High correlation indicated a major contribution of TPC in AC of these plants. Five medicinal halophytes i.e., Thespesia populneoides, Salvadora persica, Ipomoea pes-caprae, Suaeda fruticosa, and Pluchea lanceolata displayed significantly higher AC than synthetic antioxidants (BHT and BHA). Presence of bioactive phytochemicals including phenols (42.3–63.9 mg GAE g−1), flavonoids (12.3–37.1 mg QE g−1), tannins (8.7– 20 mg TAE g−1), proanthocyanidins (15.8–22.4 mg CE g−1), carotenoids (0.07–0.84 mg g−1), alkaloids (0.64– 1.1 mg g−1), and saponins (11.2–28.4 mg DAE g−1) reflected therapeutic benefits of these plants. HPLC analyses showed that the hydrolysed extracts contained chlorogenic acid, gallic acid, catechin, and quercetin as abundant phenolic metabolites which may be responsible for higher AC. These plants were also found to contain suitable amounts of proteins (8.5–17%), carbohydrates (2.6–11.4%), fibre (31.6–41.2%), and minerals (2.1–9.7%) showing their nutritional potential that has already been exploited by rural communities. The present study highlights the potential of medicinal halophytes as a source of natural antioxidants, valuable phytochemicals, and essential nutrients for pharmaceutical, nutraceutical, and chemical industries. © 2016 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction Reactive oxygen species (ROS), produced during aerobic metabolism, are essential mediators of important functions (Salganik, 2001). However, over-production of ROS results in oxidative damage of macro-molecules. Studies have demonstrated the involvement of ROS in a number of disorders including Alzheimer, atherosclerosis, diabetes, inflammation, and neurodegenerative and cardiovascular diseases. ROS also plays a key role in certain types of cancers and the ageing process. Antioxidants are molecules that neutralize harmful ROS by inhibiting oxidative chain reaction, preventing lipid peroxidation, reducing free radical concentration and chelating metal ions (Zhou and Yu, 2004). It has been recognized that consumption of vegetables and fruits reduce the risk of degenerative diseases, which may be ascribed to their antioxidant compounds (Oueslati et al., 2012). In addition, some commercial antioxidants i.e. butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which ⁎ Corresponding author. E-mail address: mqasim@uok.edu.pk (M. Qasim). http://dx.doi.org/10.1016/j.sajb.2016.10.005 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved. have been widely used in pharmaceuticals and food industry are found to be toxic (Sasaki et al., 2002). The impact of oxidative stress on human health and increasing safety concerns about synthetic antioxidants, shift the focus of the scientific community to search for new sources of safe and feasible natural antioxidants. Vegetables, cereals, fruits, and mushrooms have been screened worldwide; however, medicinal plants are more potent source of natural antioxidants (Cai et al., 2004; Li et al., 2008, 2013; Albouchi et al., 2013; Baba et al., 2015). Medicinal plants have long been used to treat infections and other human ailments. Medicinal plants share a common origin with edible plants thus it is difficult to separate medicinal plants from foods. For instance, a number of medicinal plants have been used as vegetables or salads and also for colouring, flavouring or spicing agents (Qasim et al., 2011, 2014). In this capacity, medicinal plants can provide basic nutrients and essential minerals. Studies have demonstrated a suitable composition of protein, carbohydrate, fat, fibre and minerals in some medicinal plants, comparable to or even better than common edible plants (Hussain et al., 2010). Beside nutritional importance, health benefits of medicinal plants are associated with their secondary 241 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 and eco-friendly source of natural antioxidants and other bioactive compounds. A number of coastal plants which has been used in the form of herbal tea against a range of disease conditions was reported earlier (Qasim et al., 2010, 2011, 2014). These plants thrive in harsh environments, especially hyper saline conditions which demand various adaptive mechanisms, for example redox homeostasis. Plants maintain equilibrium between ROS generation and energy consumption in enzymatic and non-enzymatic antioxidant defence to prevent cells from oxidative damage (Noctor and Foyer, 1998; Apel and Hirt, 2004). Therefore, halophytes are expected to produce bioactive compounds with high AC and hence could be better candidates for focus. The present study aimed to determine the AC and polyphenolic content of 100 medicinal plants from coastal areas of Pakistan. Nutrient, bioactive compound and phenolic metabolite contents were also determined in selected species (i.e., Thespesia populneoides, Salvadora persica, Ipomoea pescaprae, Suaeda fruticosa, and Pluchea lanceolata) showing high AC. A relationship between AC and salt resistance of medicinal plants was also determined. 2. Materials and methods 2.1. Sample collection and preparation Fig. 1. Map of study area showing site of plant collection from coastal habitats (dark flags). High Temp (oC) 72 2010 2,2′-azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation (PubChem CID: 90658258); Butylated hydroxytoluene (BHT; PubChem CID: 31404); Butylated hydroxyanisole (BHA; PubChem CID: 8456); Caffeic acid (PubChem CID: 689043), Catechin (PubChem CID: 107957), Chlorogenic acid (PubChem CID: 1794427), Coumarin (PubChem CID: Coumarin), 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical (PubChem CID: 2735032); Ferulic acid (PubChem Humidity (%) Wind (km h-1) 2011 Rainfall (cm) 72 64 56 48 48 40 40 32 32 24 24 16 16 8 8 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 56 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Values 64 Low Temp (oC) 2.2. List of chemicals used Values metabolites (Ksouri et al., 2007, 2008, 2012; Falleh et al., 2013). Medicinal plants contained a variety of secondary metabolites (e.g. phenols, flavonoids, tannins, proanthocyanidins, carotenoids, alkaloids, saponins, etc.) with a broad range of biological and pharmacological properties. Herbal remedies, usually prepared in the form of decoctions, infusions, and tonics, are commonly known as herbal teas (Qasim et al., 2011, 2014). The therapeutic effects of medicinal plants and herbal teas are associated with their antioxidant potentials (Cai et al., 2004; Li et al., 2013). Phenolic compounds were found as major contributors towards antioxidant capacity (AC) of these plants (Li et al., 2008). Interestingly, the synthesis and accumulation of polyphenols and other antioxidant metabolites are enhanced when the plant undergoes biotic/abiotic stress e.g. salinity (Navarro et al., 2006; Meot-Duros et al., 2008). Therefore it is important to characterize salt stressed plants (halophytes) for their antioxidant (Meot-Duros et al., 2008; Lee et al., 2011) and other health related effects (Trabelsi et al., 2010; Oueslati et al., 2012). Some reports are available showing halophytes as sources of polyphenolic antioxidants and other secondary metabolites of high medicinal value (e.g. Ksouri et al., 2007, 2012; Mariem et al., 2014; Stankovic et al., 2015), little is known about the phytochemical constituents and biological potential of these plants. Considering the growing demand for natural products, there is a need to search new candidates among halophytes that can serve as a safe, sustainable Medicinal plants were collected from coastal areas of Sindh and Balochistan province of Pakistan (Fig. 1). The study area represents an arid to semi-arid climate with low annual rainfall (b 250 mm) and high temperature (~ 30 °C; Fig. 2). Jafri (1966) and Ali and Qaiser (1995–2015) were used for initial identification of plants, which were further confirmed by Dr. Jahan Alam (Senior Taxonomist), Centre for Plant Conservation (CPC), University of Karachi. Voucher specimens were also deposited in the CPC for later access. At least five replicated samples per plant species were collected and dried under shade. Leaves of medicinal plants were separated and ground to fine powder using a ball mill (Retsch MM-400). Ground material (1.0 g) was extracted in 20 mL of 80% methanol using a shaking water bath (GFL-1092) at 40 °C for 3 h. After extraction, samples were centrifuged at 4000 rpm and the supernatant was recovered for further analyses (Abideen et al., 2015; Qasim et al., 2016). Fig. 2. Two years (2010 and 2011) data of study area comprising mean annual temperature, rainfall, wind velocity and humidity (Pakistan meteorological department). 242 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Table 1 Plant type, antioxidant capacity (DPPH, FRAP) and total phenolic content (TPC) of 100 medicinal plants. Values are mean (±standard error) of at least 5 replicates. Family, genus, species and author Plant type TPC DPPH FRAP (mg GAE g−1) IC50 (μg ml−1) (mMol Fe+2 g−1) Acanthaceae Peristrophe paniculata (Forssk.) Brummitt Xero 1.59 (0.29) 754.22 (56.30) 0.13 (0.02) Amaranthaceae Trianthema portulacastrum L. Achyranthes aspera L. Amaranthus viridis L. Arthrocnemum indicum (Willd.) Moq. Arthrocnemum macrostachyum (Moric.) C. Koch Atriplex stocksii Boiss. Chenopodium album L. Haloxylon stocksii (Boiss.) Benth. & Hook. Suaeda fruticosa (L.) Forssk. Aerva javanica (Brum. f.) Juss. Ex J.A. Schultes var. bovei Webb. Aerva javanica (Brum. f.) Juss. ex J.A. Schultes var. javanica Xerohal Weed Weed Hyphal Hyphal Xerohal Xerohal Xerohal Xerohal Xerohal Xerohal 3.71 (0.36) 23.45 (1.89) 10.74 (1.32) 22.29 (2.56) 13.47 (1.23) 7.64 (0.32) 32.26 (0.84) 18.52 (1.29) 46.54 (4.32) 7.91 (1.17) 21.11 (1.28) 973.26 (25.71) 421.59 (3.26) 698.35 (16.31) 193.39 (6.59) 698.12 (15.30) 701.26 (10.40) 94.23 (10.91) 311.15 (15.63) 33.31 (3.54) 798.36 (13.10) 365.35 (10.20) 0.23 (0.01) 3.85 (0.31) 2.40 (0.17) 3.57 (0.31) 3.59 (0.52) 1.19 (0.14) 3.96 (0.54) 3.12 (0.01) 5.32 (0.86) 1.34 (0.01) 3.98 (0.05) Apiaceae Ammi visnaga (L.) Lam. Weed 13.91 (1.56) 651.19 (13.60) 2.64 (0.21) Asclepiadaceae Glossonema varians Calotropis procera (Ait.) Ait. Leptadenia pyrotechnica (Forssk.) Dcne. Xero Xero Xero 5.54 (0.28) 3.73 (0.41) 2.11 (0.86) 816.24 (12.30) 725.36 (22.65) 991.62 (22.20) 0.91 (0.37) 1.86 (0.01) 1.69 (0.28) Asteraceae Artemisia scoparia Waldst. & Kit. Inula grantioides Boiss Launaea resedifolia (L.) Kuntze Pluchea lanceolata (DC.) C. B. Clarke Tridax procumbens L. Weed Xerohal Xerohal Xerohal Weed 15.13 (1.98) 7.31 (0.12) 15.53 (0.12) 42.28 (3.58) 8.54 (0.84) 516.56 (9.65) 711.89 (13.11) 512.36 (13.10) 38.76 (6.11) 621.36 (16.80) 1.57 (0.36) 1.66 (0.42) 1.75 (0.42) 5.21 (0.34) 1.86 (0.03) Avicenniaceae Avicennia marina (Forssk.) Vierh Hyphal 16.18 (1.24) 365.35 (17.78) 2.68 (0.05) Azioaceae Aizoon canariense L. Zaleya pentandara (L.) Jeffrey Xero Xerohal 1.93 (0.07) 18.52 (1.29) 712.36 (10.70) 311.15 (15.63) 1.71 (0.02) 3.12 (0.01) Boraginaceae Heliotropium currassvicum L. Trichodesma indicum (L.) R. Br. Xerohal Xerohal 7.26 (0.41) 8.53 (0.13) 796.35 (25.70) 689.21 (29.80) 1.53 (0.03) 2.87 (0.01) Brassicaceae Farsetia jacquemontii Hook.f. & Thoms. Xero 9.81 (1.23) 659.12 (19.28) 1.18 (0.05) Caesalpiniaceae Caesalpinia bonduc (L.) Roxb. Cassia holosericea Fresen Parkinsonia aculeata L. Xero Xero Xerohal 6.62 (0.69) 15.77 (1.51) 10.37 (0.32) 845.26 (15.69) 489.65 (39.71) 1003.1 (15.60) 1.39 (0.16) 1.76 (0.11) 1.31 (0.15) Capparidaceae Capparis decidua Forssk. Cleome brachycarpa Vahl ex DC. Cleome viscosa L. Xero Xero Xero 7.15 (0.85) 13.51 (1.35) 10.34 (0.11) 881.26 (18.60) 621.59 (15.28) 721.26 (41.50) 1.31 (0.18) 2.46 (0.26) 2.02 (0.36) Convolvulaceae Commicarpus boissieri (Heimerl) Cufod. Convolvulus arvensis L Convolvulus glomeratus Choisy Convolvulus prostrates Forssk. Cressa cretica L. Ipomoea pes-caprae (L.) R. Br. Xero Xero Xero Xero Hyphal Psamm 12.77 (1.20) 20.78 (2.04) 10.22 (0.84) 24.84 (1.14) 9.53 (0.27) 54.21 (2.31) 453.12 (12.45) 152.98 (22.50) 886.24 (21.70) 243.07 (12.81) 753.65 (31.70) 32.11 (3.25) 1.58 (0.03) 3.13 (0.18) 1.32 (0.29) 3.63 (0.42) 2.51 (0.03) 5.56 (0.15) Cucurbitaceae Citrullus colocynthis (Linn.) Schrad. Xero 10.61 (0.16) 825.32 (19.80) 1.21 (0.15) Cyperaceae Cyperus rotundus L. Hyphal 21.77 (2.31) 89.90 (13.27) 3.55 (0.17) Euphorbiaceae Euphorbia caducifolia Haines Euphorbia hirta L. Phyllanthus fraternus Webster Xerohal Xerohal Xero 3.11 (0.38) 3.91 (1.12) 5.21 (0.92) 825.32 (40.30) 895.12 (12.36) 789.65 (16.40) 0.91 (0.08) 1.53 (0.08) 1.58 (0.01) Xerohal 7.82 (0.72) 853.26 (18.67) 1.25 (0.01) Xero 20.34 (2.14) 197.39 (23.40) 4.03 (0.23) Gentianaceae Enicostema hyssopifolium (Willd) Verdoo Lamiaceae Leucas urticifolia (Vahl) R. Br. 243 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Table 1 (continued) Family, genus, species and author Plant type TPC DPPH FRAP (mg GAE g−1) IC50 (μg ml−1) (mMol Fe+2 g−1) Malvaceae Abutilon indicum (L.) Sweet, Hort. Brit. Digera muricata (L.) Mart. Gossypium stocksii Mast. Hibiscus micranthus L. Sida ovata Forssk. Sida spinosa L. Thespesia populneoides (Roxb.) Kostel. Xero Xero Xerohal Xero Xero Xero Hyphal 3.70 (0.15) 5.16 (0.52) 29.06 (3.29) 15.85 (0.52) 6.91 (0.10) 9.62 (1.65) 63.91 (5.28) 854.26 (10.41) 798.36 (11.30) 189.34 (10.20) 417.26 (12.81) 950.21 (53.30) 602.31 (24.80) 16.64 (1.24) 0.93 (0.02) 1.37 (0.01) 4.07 (0.14) 3.29 (0.42) 0.53 (0.07) 1.01 (0.06) 6.54 (0.56) Meliaceae Azadirachta indica Adr. Juss. Xero 19.04 (1.78) 347.23 (12.10) 3.67 (0.01) Mimosaceae Acacia nilotica (L.) Delile Acacia senegal L. Prosopis cineraria (L.) Druce Prosopis juliflora (Swartz) DC. Xerohal Xero Xerohal Xerohal 8.26 (1.89) 20.36 (2.13) 37.26 (2.73) 23.82 (2.06) 765.36 (11.11) 265.32 (15.23) 45.23 (2.21) 365.59 (11.70) 2.28 (0.03) 3.56 (0.23) 5.16 (0.11) 4.35 (0.17) Nyctaginaceae Boerhavia diffusa L. Xero 5.01 (0.32) 991.25 (20.10) 0.48 (0.08) Papilionaceae Alhaji maurorum Medic. Clitoria ternatea L. Indigofera cordifolia Heyne ex Roth Indigofera hebepetala Benth. ex Baker var. hebepetala Hook. Indigofera hochstetteri Baker Indigofera oblongifolia Forsk. Rhynchosia minima (Linn.) DC. Sesbania grandiflora (Linn.) Poir Tephrosia strigosa (Dalz.) Sant. & Maheshw. Tephrosia subtriflora Baker Tephrosia uniflora Pers. Hyphal Xero Xero Xerohal Xerohal Xerohal Xero Xerohal Weed Weed Weed 33.23 (2.89) 10.91 (0.24) 8.19 (0.13) 33.07 (2.12) 18.25 (1.39) 35.83 (4.99) 15.88 (2.20) 13.35 (1.14) 13.51 (1.25) 12.47 (1.24) 17.18 (1.24) 65.54 (7.87) 921.65 (10.90) 665.53 (29.30) 88.96 (4.19) 305.15 (15.32) 48.96 (13.41) 301.29 (22.60) 402.89 (28.04) 345.26 (18.70) 468.26 (15.36) 311.65 (21.10) 4.50 (0.27) 0.91 (0.16) 2.64 (0.47) 4.24 (0.06) 2.24 (0.12) 4.74 (0.67) 3.89 (0.01) 3.53 (0.03) 2.91 (0.02) 2.37 (0.01) 3.37 (0.11) Poaceae Aeluropus lagopoides (L.) Trin. ex Thw. Cenchrus ciliaris Rich. Cymbopogon jwarancusa (Jones) Schult. Dactyloctenium aegyptium (L.) Willd. Dactyloctenium scindicum Boiss. Desmostachya bipinnata (L.) Stapf Halopyrum mucronatum (L.) Stapf Panicum turgidum Forssk. Paspalum pasploides (Michex) Scribner Phragmites karka (Retz.) Trin. ex. Steud. Sporobolus ioclados (Nees ex Trin.) Nees Sporobolus tremulus (Willd.) Kunth. Urochondra setulosa (Trin.) C.E. Hubb. Hyphal Weed Xerohal Weed Xerohal Xerohal Psamm Xerohal Hyphal Hyphal Xerohal Hyphal Xerohal 7.58 (0.11) 8.98 (0.75) 7.85 (0.23) 3.31 (0.41) 9.41 (0.72) 7.87 (0.32) 15.84 (0.26) 16.61 (0.14) 21.29 (1.25) 10.52 (0.98) 6.81 (0.17) 7.11 (0.38) 7.32 (0.27) 771.29 (78.40) 635.26 (32.51) 782.65 (16.40) 745.32 (15.99) 721.49 (17.51) 799.36 (15.05) 214.23 (15.41) 321.26 (18.30) 289.65 (5.19) 600.12 (12.40) 775.21 (14.71) 745.21 (40.30) 702.78 (28.50) 2.38 (0.02) 1.27 (0.01) 1.23 (0.25) 0.64 (0.01) 2.22 (0.11) 2.04 (0.24) 2.18 (0.51) 2.62 (0.11) 3.35 (0.42) 2.42 (0.06) 1.54 (0.02) 2.34 (0.08) 2.46 (0.11) Portulacaceae Portulaca oleracea L. Xerohal 23.82 (2.13) 109.51 (16.66) 3.35 (0.27) Rhamnaceae Zizyphus nummularia (Burm. f.) Wight and Arn. Xero 17.65 (2.12) 325.12 (21.36) 1.53 (0.08) Salvadoraceae Salvadora oleoides Dne. Salvadora persica L. Xerohal Xerohal 25.71 (0.36) 58.23 (3.54) 313.39 (11.70) 20.92 (1.52) 2.13 (0.01) 5.96 (0.29) Solanaceae Datura fastuosa L. Solanum forskalii Dunal Solanum surattense Burm. f. Withania sominifera (L.) Dunal Xero Xero Xero Xero 24.42 (2.02) 7.21 (0.59) 22.29 (2.56) 4.79 (0.22) 289.65 (11.87) 825.32 (21.01) 305.15 (16.59) 711.45 (21.90) 3.56 (0.21) 1.85 (0.04) 3.57 (0.31) 1.72 (0.03) Sterculaceae Melhania denhamii R. Br. Xero 31.47 (3.15) 113.26 (17.17) 3.29 (0.46) Xero Xero Xero Xero Xero 23.72 (2.31) 5.27 (0.61) 9.80 (1.79) 15.31 (1.05) 7.31 (0.19) 184.29 (8.35) 811.29 (15.44) 615.32 (15.54) 322.56 (50.70) 821.36 (13.10) 4.87 (0.32) 0.97 (0.23) 2.55 (0.15) 3.83 (0.07) 1.03 (0.01) Xero Xero 8.51 (0.13) 21.72 (1.98) 689.21 (29.80) 149.29 (4.65) 2.87 (0.01) 4.76 (0.65) Tiliaceae Corchorus aestuans L. Corchorus depressus (L.) Stocks Corchorus olitorius L. Corchorus tridens L. Grewia tenax (Forsk.) Fiori Zygophyllaceae Fagonia indica ssp. schweinfurthia Hadidi Tribulus terrestris L. (continued on next page) 244 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Table 1 (continued) Family, genus, species and author Plant type Zygophyllum simplex L. BHA BHT Xerohal TPC DPPH FRAP (mg GAE g−1) IC50 (μg ml−1) (mMol Fe+2 g−1) 5.21 (0.92) 789.65 (16.40) 42.15 (2.51) 35.24 (1.65) 1.58 (0.01) 5.32 (0.42) 7.13 (0.54) Key: Hyphal — Hydrohalophyte; Psamm — Psammohalophyte; Weed — Weedy glycophyte; Xerohal — Xerohalophyte; Xero — Xerophyte. CID: 445858), Gallic acid (PubChem CID: 370), Kaempferol (PubChem CID: 5280863); Naringenin (PubChem CID: 932); Quercetin (PubChem CID: 16212154); Syringic acid (PubChem CID: 10742), 2,4,6-Tripyridyls-Triazine (TPTZ; PubChem CID: 77258). High purity HPLC grade chemicals were purchased from Sigma Aldrich especially reference standards and solvents. 2.5. High performance liquid chromatographic (HPLC) analyses 2.3. Antioxidant capacity of medicinal plants Antioxidant capacity (AC) was determined using DPPH (BrandWilliams et al., 1995) and ABTS (Re et al., 1999) radical scavenging tests. Ferric reducing antioxidant power assay (FRAP) was carried out using the method of Benzie and Strain (1996). Total antioxidant capacity (TAC) of plant samples was also evaluated by the phosphomolybdate complex method (Prieto et al., 1999). 2.4. Quantification of bioactive compounds and nutrient content of medicinal plants Total phenolic content (TPC) was determined using the Folin– Ciocalteu colorimetric method (Singleton and Rossi, 1965). Colorimetric methods were also used to quantify total flavonoids (TFC) (Chang et al., 2002), total proanthocyanidins (PC) (Sun et al., 1998) and total tannins (TTC) (Pearson, 1976). The content of total saponins (TSC) (Makkar mMol Fe+2 g-1 DPPH b a a a b b a a 4 a b 42 b b 28 a 14 2 ABTS TAC b b b b a a c b 60 b a 40 b c b b 0 40 30 20 10 TP SP IP SF PL BHT BHA TP SP IP SF PL BHT BHA 20 IC50 ( g ml-1) 80 56 IC50 (µg ml-1) a 6 100 mg GAE g-1 Dried samples (0.5 g) were extracted and phenolic glycosides hydrolysed in 40 mL methanol (62.5%) and 10 mL 6 M HCl according to the method described by Proestos et al. (2006). After purging with nitrogen for about 1 min, samples were refluxed for 2 h in a boiling water bath. Mixtures were then filtered and the final volume was adjusted to 100 mL with methanol. Mixtures were again filtered through a 0.45 μm membrane filter (Millex-HV) before injecting into a HPLC system. The HPLC system (Shimadzu LC-20AT) was equipped with LCSolution software, auto-sampler (SIL-20A), column oven (CTO-20A), and diode array detector (SPD-M20A). Analytical column, Nucleosil C18, 5 μm 100 A° (250 × 4.60 mm, Phenomenex) coupled with a guard column (Phenomenex) was used. Mobile phase was composed of (A) sodium phosphate buffer (50 mM; pH 3.3) in 10% methanol and (B) 70% methanol. The gradient program by Sakakibara et al. (2003) was used with a flow rate of 1 mL min−1. Phenolic compounds were identified by comparing retention time and UV–Vis spectra of FRAP 10 8 et al., 1995), total carotenoids (TCC) (Duxbury and Yentsch, 1956), and total alkaloids (TA) (Harborne, 1973) were also estimated. Organic content (ashing), carbohydrates (Anthrone reagent), ether extract (Soxhlet extraction), crude protein (Kjeldahl method), and crude fibre (acid base digestion) were determined using official methods described in AOAC (2005). Fig. 3. Antioxidant capacity (DPPH, ABTS, FRAP and TAC) of Thespesia populneoides (TP), Salvadora persica (SP), Ipomoea pes-caprae (IP), Suaeda fruticosa (SF) and Pluchea lanceolata (PL) in comparison with synthetic antioxidants (BHT and BHA). Similar letters are not significantly different at p b 0.05. 245 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 chromatographic peaks with that of authentic reference standards at 280 nm wavelength. y = -0.004x + 4.676 r = -0.852 R² = 0.725 DPPH (IC50 g ml-1) 1000 2.6. Statistical analysis Values are expressed as means (± standard error) of minimum 5 analytical replicates of each plant sample. Pearson's correlation coefficient (r) and the coefficient of determination (R2) were measured for the antioxidant activities and polyphenols (p b 0.05). The post-hoc LSD test was used to compare individual means. SPSS (version 16) and SigmaPlot (version 11) were used for all statistical analyses and graph preparation, respectively. 800 600 400 200 0 -200 0 1 2 3 4 FRAP (mMol Fe 5 +2 6 -1 g ) FRAP +2 -1 (mMol Fe g ) 3. Results and discussion 3.1. Antioxidant capacities of medicinal plants 3.2. Total phenolic content and its correlation with antioxidant capacity of medicinal plants TPC of 100 medicinal plants ranged from 1.59 (P. paniculata) to 63.91 mg GAE g−1 (T. populneoides), showing more than 40 fold variation. Most of the plants had high TPC (≥10 mg g−1) compared to values of some known medicinal plants such as Diplotaxis harra and Diplotaxis simplex (Falleh et al., 2013). High correlations of TPC of 100 medicinal plants with both DPPH (R2 = 0.72) and FRAP (R2 = 0.75) activities (Fig. 4) indicated the major contribution of phenolic compounds towards AC and associated therapeutic performance analogous to studies on other medicinal plants including halophytes (Cai et al., 4.5 3.0 1.5 0.0 y = -20.45x + 845.8 r = -0.848 2 R = 0.719 900 DPPH (IC50 g ml-1) Antioxidant capacities (AC) of 100 medicinal plants collected from coastal areas of Pakistan were evaluated. In traditional medicinal system, these plants are mostly used as herbal teas against a range of diseases (Qasim et al., 2010, 2011, 2014). Preparing hot or cold tea from herbs is a traditional way to extract medicinal compounds and it is a most common way or consuming herbal remedy in rural communities. In addition, it is also considered that herbs used in such preparations are either free or possess minimal toxic/side effects hence are the suitable candidates for medicinal plant research. For instance, studies have been conducted to characterize the AC and phenolic composition of plant species used in herbal teas and the focus was mostly given to their water extracts (Nagao et al., 2005; Li et al., 2013). On the other hand, several studies showed that maximum yield of phenolic antioxidants has been found in aqueous-methanolic extracts rather than pure water extracts (Li et al., 2008; Abideen et al., 2015; Qasim et al., 2016). Therefore, in this study the aqueous-methanolic extracts were prepared to determine the AC and phenolic constituents of medicinal plants. Most of the plants were analysed for the first time, which in-general, showed considerable AC (Table 1). The DPPH radical scavenging activity (IC50) of 100 medicinal plants ranged between 16.64 (Thespesia populneoides) to 1003 μg mL− 1 (Parkinsonia aculeata) indicating a 60 fold variation, where lower DPPH values represent higher AC. The AC using FRAP system showed a 50 fold variation from 0.13 (Peristrophe paniculata) to 6.54 mMol Fe+2 g−1 (T. populneoides) (Table 1). Among all plants tested, five antioxidant rich species i.e. T. populneoides, Salvadora persica, Ipomoea pes-caprae, Suaeda fruticosa, and Pluchea lanceolata, were also subjected to TAC and ABTS activity (Fig. 3). The ABTS (IC50) values ranged from 13.34 (T. populneoides) to 30.21 μg mL− 1 (S. fruticosa), while TAC from 85.43 (T. populneoides) to 58.97 mg GAE g− 1 (P. lanceolata). These species showed strong radical scavenging and reducing power capacities, which were better than synthetic antioxidants (BHT and BHA; Fig. 3). High AC found in these plants could be related to their biologically active compounds (vide infra). Such compounds make these plants superior to most of the antioxidant rich species including edible plants, herbs, medicinal plants, and some halophytes (Ksouri et al., 2008; Li et al., 2013). 6.0 y = 0.098x + 1.022 r = 0.852 R² = 0.750 600 300 0 -300 -600 0 10 20 30 40 50 60 -1 TPC (mg GAE g ) Fig. 4. Correlation of each antioxidant capacity test (DPPH and FRAP) with total phenolic contents (TPC) of 100 medicinal plants. 2004; Li et al., 2008, 2013; Oueslati et al., 2012; Stankovic et al., 2015). Strong correlation (R2 = 0.73) between DPPH and FRAP suggested radical scavenging and reducing power abilities of plant extracts, respectively (Fig. 4). Correlation (R2) was also calculated between polyphenols (TPC, TFC, TCT and PC) and AC (DPPH, ABTS, FRAP and TAC) of five selected halophytes (Table 2). The TPC and TFC were strongly correlated with each of the AC measurements (DPPH, ABTS, FRAP and TAC). Polyphenols typically possess one or more phenyl rings and hydroxyl groups and are capable of detoxifying harmful oxidants either by donating hydrogen or electron (Pereira et al., Table 2 Coefficient of determination (R2) between polyphenols (TPC, TFC, TTC and PC) and antioxidant capacity (DPPH, ABTS, FRAP and TAC) of five selected halophytes. TFC TTC PC DPPH ABTS FRAP TAC TPC TFC TTC PC DPPH ABTS FRAP 0.932 0.227 0.005 0.904 0.691 0.911 0.973 0.329 0.062 0.972 0.519 0.873 0.871 0.645 0.404 0.165 0.301 0.228 0.126 0.027 0.055 0.002 0.613 0.931 0.873 0.771 0.75 0.954 TPC — Total phenolic content; TFC- Total flavonoid content; TTC — Total tannin content; PC — Proanthocyanidin content; DPPH — DPPH radical scavenging activity; ABTS — ABTS radical scavenging activity; FRAP — Ferric reducing antioxidant power assay; TAC — Total antioxidant capacity. 246 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Table 3 Phytochemical composition of selected medicinal plants having high antioxidant capacity. Species Phenols (mg GAE g−1) Flavonoids (mg QE g−1) Tannins (mg TAE g−1) Proanthocyanidins (mg CE g−1) Carotenoids (mg g−1) Alkaloids (mg g−1) Saponins (mg DAE g−1) T. populnea S. persica I. pes-caprae S. fruticosa P. lanceolata 63.91 (5.28) 58.23 (3.54) 54.21 (2.31) 46.54 (4.32) 42.28 (3.58) 37.13 (4.28) 33.65 (3.54) 23.65 (2.31) 21.43 (4.32) 12.34 (3.58) 14.66 (3.24) 19.96 (8.43) 11.32 (1.33) 8.71 (0.76) 13.01 (2.31) 20.14 (3.54) 22.45 (2.87) 19.67 (2.54) 15.76 (1.43) 20.52 (4.31) 0.72 (0.02) 0.84 (0.02) 0.61 (0.01) 0.56 (0.01) 0.07 (0.02) 0.82 (0.03) 0.64 (0.02) 1.14 (0.06) 0.41 (0.01) 0.93 (0.07) 28.41 (2.32) 22.62 (1.12) 11.25 (1.22) 12.62 (0.87) 15.43 (1.15) GAE — Gallic acid equivalent; QE — Quercetin equivalent; TAE — Tannic acid equivalent; DAE — Diosgenin equivalent. 2013). Therefore, in most cases higher TPC have been linked with higher AC (Djeridane et al., 2006; Wong et al., 2006). With over 4000 phenolic compounds identified, any number of compounds either individually or in combination imparting synergistic effects could be responsible for higher AC and desired health benefit of medicinal plants (Williamson, 2001; Arabshahi-Delouee and Urooj, 2007; Rathore et al., 2011). 3.3. Bioactive and nutrient constituents of selected medicinal plants Efficacy of medicinal plants is a function of their bioactive ingredients. In this study, the TPC, TFC, TTC, PC, TCC, TA, and TSC content were determined in five selected plants (Table 3) indicating their therapeutic benefits. These plants contained TPC (42.28 to 63.91 mg GAE g−1), TFC (12.34 to 37.13 mg QE g−1), TTC (8.71–19.96 mg TAE g−1), and TPC (15.76 to 22.45 mg CE g−1) in high quantities. Other bioactive compounds like TCC (0.07–0.84 mg g−1), TA (0.41–1.14 mg g−1), and TSC (11.25–28.41 mg DAE g−1) were also found in considerable amounts (Table 3). These plant metabolites are reported to have several biological and pharmaceutical effects, including antimicrobial, antimalarial, antiinflammatory, antiviral, hypotensive, hypoglycemic, hepatoprotective, antioxidant and cardiovascular disease protecting activities (Niggeweg et al., 2004; Hirpara et al., 2009; Kasture et al., 2009). Besides being used as herbal remedies, medicinal plants are also consumed as food due to their nutritional components (Qasim et al., 2011, 2014; Oueslati et al., 2012). The experimental values of nutrients in five selected species of the current study ranged as follows: moisture 68.58–80.21%, dry matter 19.88–31.52%, organic content 91.31–97.94%, ash 2.14–9.69%, proteins 8.54–17.08%, carbohydrates 2.62–11.42%, ether extract 1.03–6.81%, and fibre 31.65–42.17% (Table 4). Results showed that T. populnea could be a good source of protein (17.08%), while S. persica and I. pescaprae were rich in carbohydrates (11.42%) and fibre (42.17%), respectively (Table 4). These plants also have high ash content especially in S. fruticosa (9.69%) which could be used as cheap mineral source (Table 4). Results of this study are in line with the traditional use of these plants where T. populnea, S. persica, and I. pescaprae are used as vegetables (Alzoreky and Nakahara, 2003; Burkill, 1995; Narayanan et al., 2011) and S. fruticosa as food pickle (Oueslati et al., 2012). Nutritional composition of these plants is comparable to or even higher than some of the common vegetables (Hussain et al., 2010). These plants, in sufficient quantities, can be used as a source of nutrients and essential minerals, therefore, offering a healthcare solution with conceivable dietary balance especially for the rural population of third world countries. 3.4. Phenolic profile using HPLC Phenolic composition (aglycones) of five selected halophytes were determined after hydrolysis using reference standards of gallic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, ferulic acid, coumarin, naringenin, quercetin, and kaempferol (Fig. 5). All 10 phenolic aglycones were found in S. persica and I. pes-caprae while 9, 7 and 6 phenolic aglycones were identified in P. lanceolata, T. populnea, and S. fruticosa, respectively (Table 5). Salvadora. persica was reported to contain gallic acid, caffeic acid, trans-cinnamic acid, chlorogenic acid, resorcinol, kaempferol, quercetin, and rutin (Noumi et al., 2011; Halawany, 2012), whereas derivatives of isochlorogenic acid, isocoumarin and quercetin have been reported in I. pes-caprae (Meira et al., 2012). Phenolic compounds were also reported in P. lanceolata (pluchoic acid, isorhamnetin, quercitrin, and quercetin-3rhamnoside; Srivastava and Shanker, 2012), and T. populnea (kaempferol, kaempferol 3-glucoside, quercetin, quercetin 3-glucoside, and rutin; Phanse et al., 2013). However, no report on phenolic composition of S. fruticose is available. Gallic acid, catechin, and quercetin were found in hydrolysed extracts of all test species. The dominant phenolic aglycones were chlorogenic acid and gallic acid, while catechin and quercetin were major flavonoid aglycones (Table 5). These compounds are reputed for high AC and some of these are used as standards in various antioxidant tests. Chemical structures of these compounds display antioxidant activity by allowing delocalization of electrons, neutralization of free radicals or chelation of metal ions prevent ROS damages (Zhou and Yu, 2004). Bioactive properties of phenolic compounds are known; such as chlorogenic acid displayed anticancer, antiviral, and hepatoprotective activities (Niggeweg et al., 2004), gallic acid is known for antimicrobial, antimalarial, anti-inflammatory, antitumor, and neuroprotective effects (Kasture et al., 2009), catechin bears antiplaque-forming, antiviral, hypotensive, hypoglycemic, and anticancer properties (Nagao et al., 2005)., and quercetin is used to treat hardening of arteries, cardiovascular problems, diabetes, cataracts, peptic ulcer, asthma, and prostate infections (Hirpara et al., 2009). Beside their vast application in medicinal and cosmetic industries, most of these compounds and their derivatives are also used as food additives to improve shelf life by protecting essential nutrients from oxidation and microbial deterioration (Sanches-Silva et al., 2014). Table 4 Nutrient composition of selected medicinal plants having high antioxidant capacity. Species Moisture (% FW) Dry matter (% FW) Ash (% DW) Organic matter (% DW) Ether Extract (% DW) Protein (% DW) Carbohydrate (% DW) Fibre (% DW) T. populnea S. persica I. pes-caprae S. fruticosa P. lanceolata 71.12 (4.32) 80.21 (3.21) 75.36 (4.27) 73.84 (2.05) 68.58 (3.62) 28.95 (1.71) 19.88 (1.25) 24.71 (2.15) 26.29 (0.93) 31.52 (3.11) 8.73 (0.42) 9.69 (0.26) 7.29 (0.28) 6.32 (0.15) 2.14 (0.07) 91.31 (3.24) 90.13 (4.54) 92.82 (4.42) 93.75 (4.86) 97.94 (5.22) 4.32 (0.04) 1.03 (0.02) 6.81 (0.03) 2.33 (0.02) 3.25 (0.02) 17.08 (0.15) 8.92 (0.02) 8.54 (0.01) 14.32 (0.07) 10.51 (0.02) 5.33 (0.05) 2.62 (0.01) 5.31 (0.03) 11.42 (0.02) 7.36 (0.06) 31.65 (2.31) 38.22 (3.15) 42.17 (2.41) 36.62 (1.24) 32.11 (1.35) FW — Fresh weight; DW — Dry weight. M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 247 Fig. 5. HPLC chromatograms showing phenolic profile (1 — Pyrogallol; 2 — Hydroquinone; 3 — Gallic acid; 4 — Catechin; 5 — Chlorogenic acid; 6 — Caffeic acid; 7 — Syringic acid; 8 — Ferulic acid; 9 — Coumarin; 10 — Naringenin; 11 — Quercetin and 12 — Kaempferol) of standard compounds (STD) and hydrolysed leaf extracts of Thespesia populneoides (TP), Salvadora persica (SP), Ipomoea pes-caprae (IP), Suaeda fruticosa (SF) and Pluchea lanceolata (PL). 3.5. Relationship between antioxidant capacity and salt resistance of medicinal plants Plants produce new metabolites and can also alter composition of existing chemicals to survive in different environmental stresses. For the efficient utilization of unexplored local flora, an eco-physiological approach is needed in medicinal plant research. Keeping this in mind, medicinal plants of this study were analysed on the basis of their salt resistance ability. Halophytes had 37% higher phenolic content and ~ 25% higher DPPH and FRAP activity than non-halophytes. ANOVA showed a significant effect of salt resistance on polyphenols and AC of medicinal plants (Table 6). Halophytes are naturally designed to grow 248 M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Table 5 Phenolic composition of selected medicinal plants having high antioxidant capacity. Species T. populnea S. persice I. pes-caprae S. fruticosa P. lanceolata Phenolic compounds (mg g−1) Gallic acid Catechin Chlorogenic acid Caffeic acid Syringic acid Ferulic acid Coumarin Naringenin Quercetin Kaempferol 0.391 (0.02) 0.269 (0.01) 1.419 (0.07) 0.449 (0.02) 0.652 (0.05) 0.415 (0.03) 0.890 (0.04) 0.824 (0.03) 1.667 (0.08) 1.335 (0.12) nd 0.697 (0.03) 7.369 (0.11) 1.268 (0.09) 1.888 (0.09) nd 0.373 (0.01) 0.367 (0.01) 0.383 (0.01) 0.436 (0.04) 0.373 (0.01) 0.208 (0.01) 0.618 (0.02) nd 0.571 (0.06) 0.333 (0.01) 0.210 (0.01) 0.241 (0.01) nd 0.189 (0.01) nd 0.172 (0.01) 0.289 (0.01) nd 0.251 (0.01) 0.277 (0.01) 0.175 (0.01) 0.140 (0.01) nd 0.135 (0.01) 0.338 (0.01) 0.169 (0.01) 0.191 (0.01) 0.247 (0.01) 0.164 (0.01) 0.357 (0.01) 0.190 (0.01) 0.211 (0.01) 0.176 (0.01) nd nd — not detected. but can be grown on vast degraded lands with brackish water irrigation. Sustainable development of saline/marginal lands may provide biomass of high medicinal and edible value resulting in economic gains through safe and environmental friendly measures. Conflict of interest Authors declared no conflict of interest. F = 3.25 (p < 0.05) 36 TPC (mg GAE g-1) and complete their life cycle in harsh saline environments (Alhdad et al., 2013). Under these conditions, the production of ROS, is enhanced many folds which necessitates the role of an efficient antioxidant system. As a result, tolerant plants tend to synthesize bioactive compounds including polyphenolic antioxidants in order to protect their vital metabolic functions from oxidative damage (Falleh et al., 2012, 2013). Among all plant types, TPC and AC were found higher in hydrohalophytes and xerohalophytes, whereas weedy glycophytes and xerophytes showed relatively lower TPC and AC (Fig. 6). In addition to their strong tolerance to salinity, some halophytes have also adapted to drought and waterlogged conditions. In such habitats, synergistic effects of salinity with drought or flooding amplify the magnitude of applied stress, which in turn increased the synthesis of antioxidant compounds (Alhdad et al., 2013). As a consequence, higher accumulation of polyphenols in leaves of halophytes suggest the fundamental role of these plant metabolites to protect the photosynthetic machinery from excessive light, UV and heat, and stimulates the antioxidant enzyme system (Tattini et al., 2005). For instance, mangroves and mangrove-associated halophytes such as Aegiceras corniculatum, Bruguiera parviflora, Salicornia brachiata, Suaeda maritima, and Tamarix gallica are also reported to have high AC (Ksouri et al., 2008; Alhdad et al., 2013). Halophytes 30 24 a a 18 Non-halophytes b b 12 6 750 a -1 Table 6 Comparison between halophytic and non-halophytic medicinal plants for their total phenolic content (TPC) and antioxidant capacity (DPPH and FRAP). The F values from ANOVA are given and asterisks in superscripts are showing significance level at p b 0.01. TPC (mg GAE g−1) DPPH (IC50 μg ml−1) FRAP (mMol Fe+2 g−1) 19.31 (2.16) 12.10 (0.98) 9.714** 446.46 (45.94) 585.74 (34.90) 4.502** 2.94 (0.21) 2.20 (0.16) 7.774** GAE — Gallic acid equivalent; DPPH — DPPH radical scavenging activity; FRAP — Ferric reducing antioxidant power assay. 600 a a a 450 300 150 F = 3.18 (p < 0.05) 4 3 +2 -1 FRAP (mMol Fe g ) The AC and TPC of 100 coastal medicinal plants, used to prepare herbal teas were evaluated in which most of the plants were analysed for the first time. Strong correlation between AC and TPC implied that phenolic compounds are the main contributors to AC. Five widely distributed salt tolerant species i.e. T. populneoides, S. persica, I. pescaprae, S. fruticosa, and P. lanceolata, contained considerable amount of bioactive phytochemicals as well as nutrients. Detailed HPLC analyses identified chlorogenic acid, gallic acid, catechin, quercetin and other phenolic compounds which could be responsible for high AC of these plants. Studied plants presents a promising source of natural antioxidants, bioactive compounds, and essential nutrients which can be exploited for multiple industrial and domestic applications. Higher AC and TPC in halophytes than non-halophytes suggest that applying eco-physiological approach in medicinal plant studies would help to find promising candidates with high bioactive properties. Furthermore, these plants do not require good quality soils and fresh water resources Halophytes Non-halophytes ANOVA DPPH IC50 ( g ml ) F = 1.71 (p > 0.05) 4. Conclusions a ab b b 2 1 0 alo lyco alo roh eroh dyg d e X Hy We Xe ro Fig. 6. Comparison of total phenolic content (TPC), DPPH and FRAP among different plant types. Where Hydrohalo, Xerohalo, Weedyglyco and Xero represents hydrohalophytes, xerohalophytes, weedy glycophytes and xerophytes respectively. Similar letters are not significantly different at p b 0.05. M. Qasim et al. / South African Journal of Botany 110 (2017) 240–250 Acknowledgements Khan M.A. is grateful to the Higher Education Commission (HEC) of Pakistan for providing financial support. Qasim M. and Abideen Z. are also thankful to HEC for providing Indigenous Ph.D. fellowship. We are obliged to Pakistan Meteorological Department for providing environmental data. References Abideen, Z., Qasim, M., Rasheed, A., Adnan, M.Y., Gul, B., Khan, M.A., 2015. Antioxidant activity and polyphenolic content of Phragmites karka under saline conditions. Pakistan Journal of Botany 47, 813–818. 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