Growth and Nutritional Quality Comparison Between Two Common Purslanes, Portulaca granulatostellulata and P. edulis

Portulaca granulatostellulata and P. edulisare two subspecies ofcommon purslane. The biomass of both plants was similar; however, with long upright stems and a long vegetative period, P. edulis production was easier. Both subspecies were rich in vitamin C and flavonoids, main organic nutrients’ contents in 25-d-old plants were higher than those in15-d-old plants, and were higher in P. granulatostellulata. Both purslanes were rich in minerals, with the K content being the highest, and was higher in P. edulis, whereas the Ca content was higher in P. granulatostellulata. A similarly high content of polyunsaturated fatty acid, particularly α-linolenic acid, in both purslanes showed their high value in balancing the ω-6/ω-3 intake ratio. Furthermore, all the contents of nitrate and heavy metals in this study were within the safe range, and the oxalic acid content in mature plants decreased to the high level of oxalic acid in vegetables. In brief, both purslanes are excellent reserve crops for use as vegetables and medicines. P. edulis has advantages over P. granulatostellulata regarding plant appearance and the K content. P. granulatostellulata has advantages regarding Vc, flavonoid and other mineral contents; Both purslanes are similar in fatty acid composition and are suitable for picking at a mature stage.


Introduction
Purslane is an annual succulent herb which belonging to Portulaca L., Portulacease. As a C4 and facultative CAM plant, it is widely distributed in temperate and tropical regions all over the world, and has been ranked as the eighth most common plant in the world [1]. Purslane is rich in total flavonoids and K, contains high levels of vitamin C and beta-carotene, and also contains norepinephrine, melatonin etc [2]. In addition, purslane has been reported to be the richest vegetable source of omega-3 (ω-3) fatty acids (FA) yet examined [3]. The abundance of high levels of these nutrients in purslane lead to its multiple effects as improving blood circulation and human immunity, prevention and treatment of cardiovascular disease and inhibition of microbial growth and so on [2,4]. Although purslane is still considered as a wild weed in most countries, it is now known as one of the most promising green foods, the most used medicinal plants and the "power food for the future" [5,6], and is eaten fresh, cooked, or dried and the interest of cultivating it as a food crop and herd-medicine has increased all over the world in recent years [5,7].
There are various types of purslane available in different locations of the world [6]. Those variations are not only phenotypic or physiological but also genetic [6]. Although they may be different in their tolerance to stresses [8], or different in their nutrient contents [9], according to their cultivation purpose, purslane nowadays can be divided into two types, the common and the ornamental type. Common purslane usually has small yellow flowers and produce large amount of seeds, while ornamental purslane usually has big and colorful flowers and does not produce seeds [5]. Chinese people only accept common purslane as vegetables and medicines. Currently, only two types of common purslane could be found in China, the wild type and the imported type which is called as "Dutch purslane". The wild common purslane grows prostrate with small leaves. Analysis with GC-MS and FT-IR spectroscopy method showed that wild common purslane samples from different places of China were consistent to great extent [10]. The "Dutch purslane" grows erect with large leaves, which also has names as "summer purslane" or "gardern pueslane" in European and American markets, and is the most common cultivated common purslane in the world. According to the seed size and seed coat micro-structure, purslanes from Sudan, Mediterranean etc. were divided into 19 subspecies by Danin and Raus [11]. And according to Danin and Raus [11], the wild common purslane from China was determined as P. granulatostellulata Poelln. C. Ricceri et P.V. Arrigoni subspecies (4N=36), and the "Dutch purslane" was determined as P. edulis Danin and Bagella subspecies (6N=54) [12].
After thousands of years of usage on food and medicine, as well as its widely recognized beneficial nutrients, purslane' santi-nutrients such as oxalic acid and possible heavy metal accumulations has gradually attracted people's attention [13,14]. Looking for subspecies with high yield, high nutritive value, low anti-nutrients is essential for common purslane usage. Up to now, there are a few literatures on the nutrition quality comparison among different sources [5,15,16,17], or different stages [13,18,19,20], or different parts of purslane [9,17]. In these literatures, some materials were directly from the field or market, their germplasms were uncertain and their growing environments were unknown [15]; some comparisons were among ornamental and common purslanes [16], while the ornamental purslaneis rarely eaten; orjust scant indices were compared, such as antioxidant activity [5], or FA content [15,20], or mineral contents [5] etc. All these literatures showed that variation in many traits, such as tatal flavoroids, mineral elements, FA etc. from different sources of purslane do exist, and it is possible to select for types with multiple desirable traits.
P. granulatostellulata has been used in foods and medicines in China for thousand years and P. edulis has being planted in China for over 10 years. From the perspective of production and comprehensive quality, which one is more nutritive and which one is more suitable for deep development, no data has been published. Therefore, the objective of this study was to compare the differences in growth and nutritional quality between these two common purslanes which are grown under the same condition, and to select the more suitable for cultivation as a vegetable and medicinal crop.

Chemicals
Coomassie Brilliant Blue G250 was obtained from Sigma Chemical Co., USA. Research grade bovine albumin (BSA, >99%) was purchased from Serva, Germany. Methylated fatty acid ester standards were supplied by AccuStandard Inc, USA. All other chemicals were of analytical grade obtained from Sinopharm Chemical Reagent Co., Ltd., China.

Plant Material and Plant Rearing
P. granulatostellulata seeds were collected from the Botanical Garden in Xianlin Campus of Nanjing Normal University (118°95′E ， 32°15′N). P. edulis seeds were purchased from Shangdong Hezhiyuan Seed Company (China). After the seeds of P. granulatostellulata being soaked in 35°C water for 12h, both seeds were sowed onto the surface of pre-soaked 41mm-peats (Jiffy, Norway), and placed in an Adaptis A350 incubator (Conviron, Canada). The culture conditions were as follow: 14h/10h (light/dark), 32°C/28°C (light/dark), humidity 70%, and light intensity 500µmol·m -2 ·s -1 . The peats were watered every other day with water in the first week, and with 0.5‰ Peters Professional 1 (Scotts, USA) one week later. Growth indexes were measuredon20 th day after the seeds being sowed, contents of mineral elements and fatty acids were detected on25 th day, and other indexes were detected on15 th and 25 th day.

Growth Measurement
Plant height was measured on the main stem with a ruler. Stem diameter was measured 1cm above the ground with a vernier caliper. The area of the 2nd fully expanded leaf was measured with a Multi-purpose Leaf Area Analyzer (WinFOLIA, Regent Instruments Inc., Canada). Biomasses of aboveground parts (stem and leaf) and roots were measured after the samples being oven-dried first at 105°C for 15 min, and then at 75°C for 48 hours. The branch being calculated in the branch number was at least with leaves not less than 4.

Contents Determination of Main Organic Nutrients, Nitrate and Oxalic Acid
On 15 th day and 25 th day after sowing, stems and leaves were sampled and washed with distilled water through a fine sieve separately. The samples were fresh used, or oven-dried as above and powered, or frozen in liquid nitrogen and stored at -80°C until used for subsequent analysis.
Soluble protein content was determined with fresh samples according to Bradford1976 using Bovine Serum Albumin (BSA) as a standard [21]. Soluble sugar was extracted by boiling the fresh sample in water and content was estimated by the anthrone method [22]. A standard curve was prepared using D-glucose, and the soluble sugar was calculated and expressed as mg.g -1 fresh weight (FW). For measurement of Vc content, 0.2 g fresh plant leaves were homogenized with ice-cold 5% (w/v) trichloroacetic acid using a cold mortar and pestle. The supernatant was removed after centrifugation at 14,000 g for 10 min at 48°C. Vc content was determined according to previous methods [23]. Anthocyanin was extracted by 0.1 M HCl from frozen samples and determined with the method described by Kong et al 2003 [24]. One unit of anthocyanin was defined as 0.1 increases in the absorbance at 530 nm per g fresh weight. Caroteoids were extracted by 96% ethanol from fresh samples and its content was analyzed according to the method of Porra et al 1989 [25]. Flavonoids were extracted from 3 grams of powdered samples with methanol on a Soxhlet extractor according to the method of Alothman et al 2009 [26]. The filtrate was separated by a filter paper (Whatman number 1) and concentrated under reduced pressure using a rotator evaporator (Buchi Rotavapor R-210, Switzerland). Then the total flavonoid content was determined using a colorimetric method described by Alam et al 2014 [5], and results were expressed as milligrams of Rutin equivalent in 1 g of sample (mg RE.g −1 DW).
Nitrate was extracted from fresh samples by boiling water bath and contents were assayed according to the method of salicylic acid-sulphuric acid with KNO 3 as a standard [27]. The oxalic acid was extracted also from fresh samples by water, clarified with 0.5mol/L HCl in 75°C water bath for 30 min, and contents were determined using the method of sulfonic acid as described by Ilarslan et al. with oxalic acid as a standard [28].

Determination of Mineral Contents
Samples of leaves and stems from 25 th day plants were taken for estimation of mineral contents. Fresh samples of plant material (0.5 g) were dried and powdered as above, and digested in 35% (v/v) HNO 3 at 100°C for 30 min; and mineral content (K, Ca, Mg, Fe, Cu, Zn, Pb As and Cd) was analyzed by Inductively Coupled Plasma Atomic Emission Spectrum (ICP)(Prodigy, Leeman Labs Inc. USA). Mineral content was normalized to the dry weight.

Fatty Acid Extraction, Methylation and Detection
Dried powdered samples of stems and leaves were obtained as above. FA were extracted on a Soxhlet extractor and methylated on a Rotary evaporator referring to the method of Liu et al2000 [15]. Absorbent filter bag with 2 g powders in it were placed in a cylinder in a Soxhlet extractor. With ether as the solvent, the mixture was first in 50°C water bath for 18h, and then be filtered and evaporated to nearly dry in a round flask on a Rotary evaporator. Then, 3mL of 0.5mol/l NaOH-methanol solution was added to the flask and refluxed in 60 °C water bath for 20m, and then 3mL 13% boron trifluoride-methanol solution was added and refluxed for another 20m, and 1 mL hexane was added for oscillating extraction. Finally, the saturated NaCl solution was added to the flask neck, and the supernatant liquidwas taken after the static setting. After drying with anhydrous sodium sulfate, the supernatant liquid (1 mL) were injected via an autosampler onto a fused-silica capillary column (VARIAN company; SP TM -2560; 30 m×0.25 mm I.D., 20µm filmthickness) in a Shimadzu Model GC-2010 Plus gas chromatography (Shimadzu, Japan) system fitted with a flame ionisation detector (FID) and eluted with H 2 at 30±1 mL /min with a split ratio of 1:17. The injector and detector were heated to 285°C. The column was temperature programmed from 150°C (hold 2 min) to 225°C (hold 46 min)at 5°C/min. FA methyl esters were identified by comparing GC retention time with those of a mixture of methylated FA ester standards. FA relative contents were quantified using peak areas. The unsaturated fatty acid index (IUFA) was calculated with the formula: IUFA(%)= 1×monoenoic acid content(%)+2×diolefinic acid content(%)+3×triolefinic acid content(%)

Statistics
The data are presented as means in all tables and analyzed by one-way analysis of variance (ANOVA) using the statistical software Origin 7.0. Values of the same index followed by different capital letters denote significant difference at p<0.01, and those followed by different small letters denote significant difference at p<0.05.

Comparison of Growth and Biomass
Both P. granulatostellulata and P. edulis grew well under 32°C/28°C (light/dark) in the incubator. Their flower buds appeared onthe 40th and 50th day, respectively, suggesting a longer picking period of P. edulis. On the 20thday, the plant height, stem diameter and leaf area of P. edulis were 60.56%, 76.6%and 54.22% higher than those of P. granulatostellulata, respectively (Table 1). P. edulis was tall and sturdy, but because P. granulatostellulata had more branches, no obvious difference was observed in their biomass (Table 1). P. granulatostellulata grows prostrate with small leaves, whereas P. edulis grows vertically with large leaves. This implies that P. edulis seedlings are more easily picked and bundled. All these observations show that P. edulis has more advantages in production.

Contents Comparison of General Nutrients and Anti-nutrients
In the reproduction stage, stem elongation of both purslanes stopped, and their bases began to become woody, which decreased their texture and quality. Therefore, this study selected the stems and leaves of 15-d-old (young stage) and 25-d-old (mature stage) plants as materials for detecting the contents of six types of conventional organic nutrients and twotypes of conventional anti-nutrients ( Table 2).
Several studies have reported that the most important bioactive components of purslane are carotenoid, flavonoids, and vitamins etc [6,10,18]. Table 2 showed that the contents of soluble sugar, soluble protein, and anthocyanin in both purslanes were not higher than those in conventional vegetables. Moreover, the contents of carotenoid, Vc, and total flavonoids, which have many functions, such as free radical scavenging, cardiovascular system regulation, anticancer functions, antiinflammatory functions and immune improvement [3],were extremely high. The contents of Vc and total flavonoids in the 25-d-old P. granulatostellulata plants reached 0.31 mg g -1 FW and 45.4 mg g -1 DW, respectively, which are much higher than those in conventional vegetables [29]. Contents of anthocyanin, Vc and total flavonoids in 15d-old and 25d-old P. granulatostellulata plants were significantly higher than those in P. edulis, and contents of soluble protein and carotenoid were lower than those in P. edulis. Contents of five kinds of organic nutrients except for soluble protein in 25d-old plants of both purslanes were significantly higher than those in their15d-old plants. All these suggested that the nutritive, healthy and drug effective values of P. granulatostellulata were higher than those of P. edulis, and purslane picking should be at the mature stage.
Due to the appliance of large amount of nitrogen fertilizer, nitrate in vegetables has become the main source of nitrate in the human food; and the potential carcinogenic characteristic of nitrate has made it become the most important anti-nutrient in vegetables [30]. Nitrate content in P. granulatostellulata was slightly lower than that in P. edulis. It increased by 115% and 212% from 15d to 25d in P. granulatostellulata and P. edulis, and the increasing range in P. edulis was much higher. This suggested that although the nitrate contents in this experiment had not exceeded the food standards [31], if large amount of nitrogen fertilizer was added, the mature P. edulis would be the first one to exceed the standard.
Although no study has reported the standard content of oxalic acid in vegetables or foods worldwide and people from Europe and America consider purslane to be flavorful, the main obstacle inhibiting the widespread use of common purslane is its high oxalic acid content, which can reduce the effectiveness of mineral nutrition and increase the risk of lithiasis in humans [13,32] Palaniswamy et al 2004 [13] had found that soluble oxalic acid contents in 16-true leaf purslane seedlings was about 36-45% lower than that in 8-true leaf purslane seedling. In this paper, soluble oxalic acid contents in both 15d-oldcommon purslane plants were over 15mgg -1 FW, and in 25d-old plants, they dropped by near 60% to less than 8 mgg -1 FW, the level of the high oxalic acid vegetable spinach [32]. This finding suggested that picking purslane at the mature period cannot only yield purslane with a high organic nutrient content but also avoid the shortcoming of high oxalic acid content.
The optimal picking period of purslane is at its mature period just as indicated by the contents of FA and α-linolenic acid (ALA) [19], the follow-up mineral content and FA composition were detected in 25-d-old plants.

Comparison of Mineral Contents
In addition to providing organic nutrients, vegetables provide a large amount of dietary minerals which is good for the human body, and accumulate heavy metals which can harm the human body [14]. As a promising green food and medicine, purslane is noted for its high contents of K, Ca and Mg, which play crucial roles in improving blood circulation and human immunity and reducing depression [2,4]. Table 3 shows that dietary mineral contents in both common purslanes were higher than that in conventional vegetables; in particular, the K content was the highest [6,20]. Regarding the K supply capacity, P. edulis, whose K content was 13% higher than that of P. granulatostellulata, had a greater advantage. By contrast, P. granulatostellulata had obvious advantages in the mineral content, including not only a high K content but also relatively high contents of minerals such as Ca, Mg, Fe, and Cu. Purslane has a capacity for heavy metal enrichment [14], and the dietary intake of heavy metals may lead to various diseases, including cancer, emphysema, bronchiolitis, and alveolitis [13]. However, the contents of Pb, Cd, and As in both common purslanes in this study did not exceed the food standards [34] (Table 3). Furthermore, the contents of Pb and Cd were lower in P. granulatostellulata than in P. edulis; the As content was higher in P. granulatostellulata than in P. edulis. These observations revealed that different purslanes may have different abilities of heavy metal enrichment; purslane growing in non polluted areas may not have the heavy metal accumulation problem. However, if purslane is to be planted in heavy metal-contaminated soil for food or medicinal purposes, appropriate purslane subspecies should be selected.

Comparison of Fatty Acid Composition
In addition to providing energy, different types of FA have different functions in human growth and development and in preventing diseases [35]. A balanced intake of FA has become one of the important factors affecting modern health. According to the ω-6/ω-3 polyunsaturated fatty acid (PUFA) ratio in human milk and human erythrocyte membrane and because ω-6 and ω-3are not interconvertible in the human body, the recommended intake ratio of ω-6/ω-3 PUFA indiets is 1/1 to 2/1 [35]. However, because of the abundance of ω-6PUFA and scarcity of ω-3 PUFA, the actual intake ratio ofω-6/ω-3PUFA was only 20 to 25/1(Western countries) to 38 to 50/1(India) [35]. This results in a high incidence of cardiovascular disease, diabetes, and other diseases [7] and the growing interest in introducing P. oleracea as a new cultivable vegetable [5,36].
Although Yan et al 2009 reported significant differences in the composition and content of FA among different P. oleracea accessions [17], Table 4 shows a similar total lipid content and FA composition in both common purslanes. Fifteen types of FA were detected in P. granulatostellulata, accounting for 99.745% of total FA, and 17 types of FA were detected in P. edulis, accounting for 99.141% of total FA. The FA composition of both purslanes was similar to that reported by Siriamornpun and Suttajit 2010 [9]. The most abundant FA wasα-linolenic acid (ALA)(c18:3), followed by linoleic acid (c18:2) and palmitic acid (c16:0). The relative contents of total unsaturated fatty acids (UFAs) and PUFA and index of UFA of both purslanes were higher than those of conventional vegetables, as reported by Siriamornpun and Suttajit 2010 [9], whereas the UFA content in P. granulatostellulata was higher than that in P. edulis. The total saturated fatty acid (SFA) content in both purslanes was relatively low and that in P. granulatostellulata was lower. Furthermore, the ALA content in both stems and leaves was more than 51%, which is higher than that previously reported [9,36]. The ω-6/ω-3 PUFA ratios of P. granulatostellulata and P. edulis were 0.363/1 and 0.372/1, respectively, which are lower than the recommended ω-6/ω-3 ratio and much lower than the actual ω-6/ω-3 intake ratio [35]. These results prove that unlike fish oils with high contents of cholesterol and calories, purslane can provide an excellent source of ω-3 UFA to balance the ω-6:ω-3 PUFA intake; by contrast, the slightly higher content of UFA and lower content of SFA in P. granulatostellulata showed that it has a slightly higher FA nutritive value than P. edulis does. Notes: "-" indicates undetected. Means followed by the same capital letter within indexes are notsignificantly different at ≤0.01 (LSD), and followed by the same small letter are not significantly different at ≤0.05 (LSD). 1 FA: fatty acid; 2 SFA: saturated fatty acid; 3 UFA: unsaturated fatty acid; 4 MUFA: monounsaturated fatty acid; 5 PUFA: polyunsaturated fatty acid; 6 IUFA: unsaturated fatty acid index.

Conclusions
Both P. granulatostellulata and P. edulis have high contents of Vc, total flavonoids, K, and ALA; P. granulatostellulata has advantages over P. edulis regarding Vc, flavonoid, and mineral contents, except for the K content. P. edulishas advantages over P. granulatostellulata in plant appearance, the K content, and producibility. Both subspecies are similar in FA composition and are suitable for picking at the mature stage (25d in this study) because they are more nutritious with a high content of main organic nutrients and low content of oxalic acid during this period.