In vitro investigation of antioxidant activities of Launea taraxacifolia and Crassocephalum rubens

Launea taraxacifolia and Crassocephalum rubens are among many wild, underutilized and under cultivated vegetables in Nigeria that are at risk of extinction. Total flavonoid contents (TFC), total phenolic contents (TPC), and antioxidant activities of different concentrations (1-5 mg ml−1) were evaluated; using in vitro assays to assess the scavenging properties of 2, 2-diphenyl-1-picryl hydrazyl (DPPHRSP), nitric oxide (NORSP) and hydroxyl (OHRSP). Phenolic profiles of the alcoholic extracts were characterized using high-performance liquid chromatography techniques. The results revealed higher TFC (mg/100g RE) in aqueous (6.06±0.02-78.79±0.01) than alcohol extracts (with methanol 0.93±0.01—12.73±0.04, and with ethanol -0.85±0.01–7.70±0.03). In a similar trend, OHRSP (%) was higher in aqueous extracts (40.83±0.10–91.74±0.19) than alcoholic extracts (with methanol 11.67±0.3–30.83±0.06; and with ethanol -14.42±0.06-40.27±0.05). TPC (mg/100g GAE) which was higher in alcoholic extracts (with methanol -21.48±0.01–133.20±0.16 and with ethanol -9.45±0.01– 59.73±0.02) than aqueous extracts (14.83±0.01–52.64±0.03) was in agreement with the trend observed for NORSP (28.24±0.05-151.76±0.08 for methanolic extracts, 21.99±0.13–49.93±0.04 for ethanolic extracts and 38.47±0.11–86.15±0.05 for aqueous extracts). DPPHRSP was also higher in alcoholic extracts (methanolic -22.81±0.01-48.41±0.05 and ethanolic–14.53±0.01-62.68±0.07) than aqueous extracts (13.66±0.13–42.86±0.03). TFC, TPC and antioxidant activities showed concentration dependent increase and strong positive correlation with TFC (r= 0.926 – 0.997and r= 0.432 – 1.000) and TPC (r= 0.825 – 0.999 and r= 0.473 0.994) for L. taraxacifolia and C. rubens respectively. Caffeic acid, chlorogenic acid, ellagic acid, quercetin and kaempferol were identified as major phenolic components in the extracts. The vegetables have high antioxidant potential for promoting good health; which could be attributed to the identified phytochemicals in them.


Introduction
The antioxidant activity of fruits and vegetables is generally positively correlated with their content of polyphenols (Wang & Lin, 2000;Koffi, Sea, Dodehe, & Soro, 2010).Free radicals generated in living organism during various pro-idants that can slow or prevent this oxidative damage of other molecules thereby reducing the risk of diseases.The most effective antioxidants seem to be flavonoids and phenolic compounds of many plant raw materials, particularly in fruits, vegetables, herbs and seeds as many studies have suggested (Kim, Tsao, Yang, & Cui, 2006;Adom & Liu, 2002).Their metal-chelating capabilities and radical-scavenging properties have enabled phenolic compounds to be thought of as effective free radical scavengers and inhibitors of lipid peroxidation (Aruoma, 1998;Aruoma et al., 1997;Liu et al., 2010;Geng, Ren, Liu, & X., 2012).Many wild plants exist in Nigeria but are underutilized probably due to inadequate knowledge of their health promoting potential and sometimes due to dietary habit.L. taraxacifolia and C. rubens are among many underutilized wild vegetables in western Nigeria.While Crassocephalum rubens is used as a vegetable by few, especially among the aged and rural dwellers, L. taraxacifolia is not yet recognized as food and cannot be found in any market in Ondo State, the part of Nigeria where this work was carried out.Launaea taraxacifolia, also known as wild lettuce belongs to the family Asteraceae.The leaf is used as a vegetable in some parts of South-Western Nigeria in West Africa where it is called efo yanrin and research has established its high nutritional and medicinal values (Adebisi, 2004).It is mostly collected from the wild and only cultivated to a limited extent for home use and for local markets; whereas, it is known only as a weed in many parts of Nigeria.L. taraxacifolia is nutritionally rich in vitamins, minerals, proteins, essential fatty acids, fibre content and flavonoids (Adinortey et al., 2012;Dickson et al., 2012;Gbadamosi & Sulaiman, 2012).Crassocephalum rubens (Juss.Exjacq.S. Moore) belongs to the plant family Asteraceae.It is a wild, economic and medicinal plant that is now threatened by genetic erosion in the Nigerian ecosystem.The scarcity of the plant is due to indiscriminate collection from the wild and the difficulty in germinating the seeds (Gbadamosi & Sulaiman, 2012).Many researchers have reported the nutritional values of L. taraxacifolia and C. rubens, with the aim of drawing attention to them, but there is a dearth of information on their antioxidant potentials.The present study was aimed at investigating phenolic composition and antioxidant activities of the different concentrations of aqueous and alcoholic extracts of the leaves of the above mentioned vegetables.

Sample collection
L. taraxacifolia was obtained from a nearby bush in Akure and C. rubens was purchased from a local market also in Akure, Ondo State, in Nigeria.Approximately 500 g was obtained for each.

Preparation of Samples and extracts
The air-dried vegetable samples were ground and sieved to give a 40 mm mesh size powder.A bioactive extract of each powdered vegetable was obtained by weighing 20 g into clean and dried reagent bottles, separately adding 400 mL of distilled water, methanol and ethanol into each bottle and subjecting them to a cold maceration process for 24 h to obtain the aqueous extract and 72 h to obtain the alcoholic extracts.Extracts were brought to dryness with a rotary evaporator at a low temperature (45 °C) and were kept at 3 °C in the refrigerator while the analyses were carried out (Iwalewa et al., 2005).

Quantification of alcohol extracts by HPLC-DAD
Chromatographic analyses were carried out under gradient conditions using a C 18 column (4.6 mm × 250 mm) in reverse phase (HPLC-DAD with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system, by Shimadzu, Kyoto, Japan), packed with 5 µm diameter particles, where the mobile phase was water containing 1% acetic acid (A) and methanol (B), and the composition gradient was: 5% of B until 10 min and changed to obtain 20%, 30% The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves.LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve (da Silva et al., 2014).

Determination of total phenol contents
The total phenol content (TPC) of the samples was determined by mixing 0.5 mL of each extract (1-5mg mL −1 ) with 2.5 mL 10 % Folin-Cioalteu's reagent (v/v) and 2.0 mL of 7.5% Na 2 CO 3 .The reaction mixture was subsequently incubated at 45 °C for 40 min, and the absorbance measured at 760 nm by spectrophotometer.All tests were performed in triplicate.Gallic acid was used as a standard phenolic compound.The amount of total phenolic compounds in the extract was determined as mg of Gallic Acid Equivalent (GAE) per g dry weight (Sin-gleton, Orthofer, & Lamuela-Raventos, 1999).

Determination of total flavonoid
A known volume (0.5 mL) of each extract (1-5 mg mL −1 ) was added to a 10 mL volumetric flask.Distilled water was added to make a volume of 5 mL.At zero time, 0.3 ml of 5 % w/v NaNO 2 was added to the flask.After 5 min, 0.6 mL of 10 % w/v AlCl 3 was added and after 6 min, 2 mL of 1M NaOH was added to the mixture followed by the addition of 2.1 mL distilled water.Absorbance was read at 510 nm against the blank (water) and flavonoid content expressed as mg Rutin equivalent/g.The test was carried out in triplicate (Zhishen, Mengcheng, & Jianming, 1999).

Determination of hydroxyl (OH) radical scavenging activity
The OH scavenging activity was determined by the method of Halliwell, Gutteridge, and Aruoma (1987).Exactly 1-5 mg mL −1 of each extract of the samples were mixed with 1 mL of reaction mixture (100 µM FeCl 3 , 104 µM Ethylene-IJFS April 2017 Volume 6 pages 82-94 diaminetetraacetic acid, 1.5 M H 2 O 2 , 2.5 M deoxyribose and 100 µM ascorbic acid in 10 mM KH 2 PO 4 -KOH, pH 7.4) and incubated for 1h at 37 °C.Thereafter, 1 mL of 0.5 % thiobarbituric acid in 0.025 M NaOH and 1 mL of 2.8 % trichloroacetic acid was added to the mixture and heated for 30 min at 80 °C before reading the absorbance at 532 nm against an appropriate blank solution.All tests were performed in triplicate.Ascorbic acid was used as a positive control.Percent inhibition of OH was calculated by the following expression: Percentage of inhibition = Ao-A1 Ao × 100, where Ao is the absorbance of the Ascorbic acid and A1 is the absorbance of the sample.The percentage inhibition was plotted against corresponding sample concentration (1-5 mg mL −1 ) and IC 50 calculated from the graph equation.

Determination of nitric oxide (NO) scavenging activity
Briefly, 5 mM sodium nitroprusside in phosphate-saline was mixed with different concentrations of the extracts: 1-5mg mL −1 , before incubation at 25 °C for 150 min.Thereafter, the reaction mixture was added to Greiss reagent (1 % sulfanilamide, 2 % H 3 PO 4 and 0.1 % naphthylethylenediamine dihydrochloride), before measuring the absorbance at 546 nm (Jagetia, Rao, Baliga, & Babu, 2004).Ascorbic acid was used as the control.All tests were performed in triplicate.The nitric oxide radicals scavenging activity of the fractions was calculated according to the following equation: Percentage of inhibition = Ao-A1 Ao × 100, where Ao is the absorbance of Ascorbic acid and A1 is the absorbance in the presence of the fractions and ascorbic acid.The percentage inhibition was plotted against the corresponding sample concentration (1-5 mg mL −1 ) and IC 50 calculated from the graph equation.

Determination of nitric oxide (NO) scavenging activity
Values are presented as the mean ± SD of three replicates.ANOVA and LSD and Pearson cor-relation analyses were performed using the commercial software SPSS 16.0 3 Results and Discussion

DPPH Radical Scavenging Property
The percentage inhibitions of DPPH in aqueous extracts were lower than in alcoholic extracts.
All extracts demonstrated relatively appreciable concentration dependent increase, compared with Trolox (Tables 5 -6).IC 50 values for free radicals are presented in Table 7.

Hydroxyl Radical Scavenging Property
Aqueous extracts exerted a stronger scavenging capacity on OH in a dosage dependent manner than the alcoholic extracts and Ascorbic acid (Tables 5 -6).The extracts showed significant in vitro antioxidant capacities.

Nitric Oxide Radical Scavenging Property
The percentage inhibition of NO was higher in methanolic extracts for both L. taraxacifolia and C. rubens than in aqueous extracts except at 1 mg mL    the ethanolic extracts.The antioxidant activities of all were appreciable, when compared with Ascorbic acid, a reference standard (Tables 5 -6).

Discussion
HPLC fingerprinting of methanolic and ethanolic extracts of L. taraxacifolia and C. rubens revealed the presence of phenolic acids; gallic acid and caffeic acid.Chlorogenic Acid which was the most abundant phenolic acid in the methanolic extract of C. rubens was absent only in the methanolic extract of L. taraxacifolia but detected in small concentrations in the ethanol extracts of both vegetables.Ellagic acid was, however, detected only in the methanolic extract of L. taraxacifolia while a caffeic acid derivative was also found only in the methanolic extracts of C. rubens.Rutin, quercetin and kaempferol are flavonoid compounds detected in methanolic and ethanolic extracts of both vegetables (Table 1).Phenolic compounds and flavonoids make up a unique category of plant phytochemicals especially in terms of their wide ranging potential health-benefial properties.In particular, phenolic compounds have been reported to modulate intracellular signaling, the activity of target enzymes and gene expression (Kumari & Jain, 2012).Flavonoids have antioxidant activity and a range of health promoting effects such as antiallergic, anti-inflammatory, anti-thrombotic, vasoprotective, tumour inhibitory and anti-viral.These effects have been associated with the influence of flavonoids on the arachidonic acid metabolism (Ajiboye, Ibukun, Edobor, Ojo, & Onikanni, 2013;Trease & Evans, 2002).Isoquercitrin was detected in the methanolic but not in the ethanolic extracts of both L. taraxacifolia and C. rubens; whilst coumarin was found in ethanolic but not the methanolic extracts.Methanolic extracts of L. taraxacifolia lacked the presence of quercitrin but contained catechins which are powerful antioxidants that have been reported to cause reduction in glycemia (blood glucose levels) either by inhibiting intestinal glucose absorption (Shimizu, Kobayashi, Suzuki, Satsu, & Miyamoto, 2000), inducing β cell regeneration (Kim et al., 2003) or enhancing insulin activity (Anderson & Polansky, 2002).Based on the findings here and in previous studies, L. taraxacifolia and C. rubens extracts may be of significant value in the management of diabetes mellitus.
TFC and TPC results revealed concentration dependent increase (Tables 2 and 3), and L. taraxacifolia for all extracts gave significantly (p < 0.05) higher values of ).The methanolic extracts of both vegetables contained the highest concentrations of TPC.This was similar to the observation of Corpuz, Osi, and Santiago (2013) where aqueous extracts of S. siliquosum presented the lower amount of TPC in mg g  4. The extracts contained a considerable amount of phenolics and possess significant in vitro antioxidant capacities (Tables 5 -6).The aqueous extract of C. ruben demonstrated the strongest inhibitory power against OH radical with an IC 50 value of 1.09 mg ml −1 , which was stronger than ascorbic acid used as a reference standard.Although, nitric oxide plays a vital role in various inflammatory processes, high levels of these radicals are toxic to tissue and contribute to the vascular collapse, various carcinoma and ulcerative colitis (Rajan et al., 2011).The results of this study showed that the percentage inhibition was significantly (p < 0.05) higher in methanolic extracts of both L. taraxacifolia and C. rubens than aqueous extracts except at 1 mg/mL concentrations.However, aqueous extracts exhibited stronger scavenging activity than the ethanolic extracts, which is also in line with the trend ob-served for TPC.All extracts of the vegetables compared favorably with vitamin C in their inhibitory effect on nitric oxide.The methanolic extract of L. taraxacifolia with an IC 50 value of 1.60 exhibited the strongest anti-radical ability against nitric oxide in this study.
The ability of L. taraxacifolia and C. rubens to effectively inhibit free radicals (OH, NO, DPPH) and their reducing properties in this study may be attributed to the presence of phenolic compounds with potent antiradical properties.These vegetables possess high antioxidant potential to boost the body antioxidants and protect humans against detrimental oxidative effects caused by free radicals (Shukla, Mehta, Bajpai, & Shukla, 2009;Corpuz et al., 2013).

Conclusions
The results of this study clearly indicate that aqueous and alcoholic extracts of L. taraxacifolia and C. rubens possess significant in vitro antioxidant capacities.Generally, strong and positive associations were observed between antioxidant activities and the total phenolic content as well as the total flavonoids' content of the vegetable extracts, although the order of antioxidant potency of each extract evaluated by different models does not follow the same pattern.These observations were in agreement with several studies which associated phenolic compounds with antioxidant activity.The reported potential health benefits of L. taraxacifolia and C. rubens may be attributed to the considerable amount of various phenolic acids and flavonoids detected in them.
L. taraxacifolia and C. rubens can therefore be viewed as potential health foods, and their cultivation and utilization should be encouraged.

Table 1 :
Phenolic compounds in the alcoholic extracts of the vegetables 3.65 ± 0.03 3.95 ± 0.01Results are expressed as mean ± standard deviations (SD) of three determinations