Phytochemicals, Nutritional Value, Antioxidant, and Anticoagulant Activity of Lactuca sativa L. Leaves and Stems

The main ingredient in the daily salad dish is the leaves of Lactuca sativa, while the stems are usually discarded and not eaten. This study was conducted to compare the stems and leaves of two selected L. sativa varieties (L. sativa var. longifolia L. and L. sativa var. capitata L.) related to the preliminary phytochemical investigation, nutrient content, DPPH assay, and in vitro anticoagulant activity by determination of prothrombin time (PT) and activated partial thromboplastin time (aPTT). The findings revealed almost the same phytoconstituents in the leaves and stems of each variety, such as terpenoids, flavonoids, coumarins, and others. Exceptionally, tannins have only been detected in the leaves. Compared to the stems, the leaves of both varieties showed statistically significantly higher levels of raw protein and raw fat. For the DPPH assay, the leaves of both varieties have a statistically significantly higher antioxidant activity than the stems. The leaves and stem extract of the two selected varieties showed a significant prolongation of PT (P <0.05 vs. NS= 12:30 s). On the other hand, the aPTT test showed a significant increase in aPTT (P <0.05 vs. NS = 32:44 s) in the stem extract of both varieties, whereas there was no statistically significant increase in aPTT in the leaves of both varieties. Both parts of each variety have a diversity of phytochemicals and nutrients. The leaves of both varieties have a higher antioxidant activity than the stems. While the stems had higher anticoagulant activity than the leaves.


INTRODUCTION
The administration of prescribed anticoagulant medicines by patients with cardiovascular diseases is very crucial to minimize the risk of thrombosis formation and to maintain the normal blood flowability (Buttar et al., 2005). On the other hand, the serious side effects of those medicines also cause a danger of bleeding in addition to the difficulty in dose monitoring for patients been under anticoagulant therapy . Therefore, the orientation toward the plants as a source of cheap anticoagulant drugs with minimum side effects is the desired objective of anticoagulant therapy.

One of the most used up leafy vegetables under family
Asteraceae is Lactuca sativa (Alavi, 1983;Mughrbi & Auzi, 2020), which is vernacularly known as lettuce or khas in Libya. Popularly, the major consuming part in L. sativa is the leaves; whereas, the stems are usually discarded and not eaten. Most of the researches that have been conducted on L. sativa focus only on leaves while the juicy stem has a few documented studies. The leaves of L.
sativa are considered a valuable source of phenolic compounds (Ribas-Agusti et al., 2011) and sesquiterpene

Abstract
The main ingredient in the daily salad dish is the leaves of Lactuca sativa, while the stems are usually discarded and not eaten. This study was conducted to compare the stems and leaves of two selected L. sativa varieties (L. sativa var. longifolia L. and L. sativa var. capitata L.) related to the preliminary phytochemical investigation, nutrient content, DPPH assay, and in vitro anticoagulant activity by determination of prothrombin time (PT) and activated partial thromboplastin time (aPTT). The findings revealed almost the same phytoconstituents in the leaves and stems of each variety, such as terpenoids, flavonoids, coumarins, and others. Exceptionally, tannins have only been detected in the leaves. Compared to the stems, the leaves of both varieties showed statistically significantly higher levels of raw protein and raw fat. For the DPPH assay, the leaves of both varieties have a statistically significantly higher antioxidant activity than the stems. The leaves and stem extract of the two selected varieties showed a significant prolongation of PT (P <0.05 vs. NS= 12:30 s). On the other hand, the aPTT test showed a significant increase in aPTT (P <0.05 vs. NS = 32:44 s) in the stem extract of both varieties, whereas there was no statistically significant increase in aPTT in the leaves of both varieties. Both parts of each variety have a diversity of phytochemicals and nutrients. The leaves of both varieties have a higher antioxidant activity than the stems. While the stems had higher anticoagulant activity than the leaves.
From the stem lettuce (Nie et al., 2017;Nie et al., 2018) have separated a sulfated heteropolysaccharide which possesses an antioxidant, α-amylase inhibitory, and immunomodulatory activities. About twenty-one sesquiterpene lactones were isolated and characterized from the stem lettuce by (Han et al., 2010), some of these compounds have revealed a significant antioxidant activity.
Research is applied to the two parts (leaf and stem) of each variety (L. sativa var. longifolia and L. sativa var. capitata) to investigate the phytochemical of methanol extracts. Also, this study aims to determine the nutritional value, in vitro antioxidant activity, and in vitro anticoagulant activity.

Plant material collection, identification, and processing
Varieties (Romaine lettuce and iceberg lettuce) were collected from different markets in Tripoli, Libya, in November 2017. Samples were identified by Dr. The leaves and stems of each variety were separated and washed away by distilled water. The leaves had been dried in shade for two weeks, but the stems had been dried in the oven at 40°C for 16-18 hours. The dried parts were powdered using an electric grinder and stored in polyethylene sealing bags in the refrigerator at 4°C.

Extraction of phytochemicals
The powdered leaves of 350 g and the powdered stems of 300 g of both varieties were extracted by cold maceration with a powder ratio of 10 g : 100 ml of solvent (Bhat & Al-Daihan, 2014). The powders were extracted twice, using 99% methanol for four days each time, and then filtered. The filtrates were concentrated at 40°C under reduced pressure by rotary evaporator. The weights of the dry extracts were calculated and the extracts stored in the refrigerator at 4°C.

Estimation of nutritional value
The powder of the leaves and the stems of each variety have been analyzed according to the Association of Official Analytical Chemists (Association of Official Agricultural Chemists, 1990;Association of Official Agricultural Chemists, 2000). All the procedures were repeated in duplicate.

Estimation of total ash
Total ash was determined by the primary ignition of 4 g of each sample in a pre-weighed crucible until fully carbonized, and then gradually increased the temperature of the muffle furnace to 550°C overnight until carbon-free ash was obtained. The crucible has been weighed. The percentage of total ash has been calculated as follows: Total ash (%) = (Weight of ash/Weight of sample) x 100

Estimation of moisture content
The moisture content was determined by the drying method using the oven. As much as 5 g of dry powder of sample was placed in previously weighed, dry, and clean petri dish for each sample, and then the dish dried in the oven at 105°C for 3-4 hours until reaching the constant weight. The percent of moisture content was calculated using the following equation: Moisture content (%) = (Wt1 -Wt2 / Wt3) x 100 Wt1 = Weight of petri dish and sample before drying Wt2 = Weight of petri dish and sample after drying Wt3 = Weight of sample.

Estimation of crude protein
The Crude protein was determined using the Kjeldahl method. As much as 1 g of each L. sativa powder was placed in a digestion flask containing 3 g of Kjeldahl catalyst and 12 ml of concentrated H2SO4 and was digested for an hour. The flask was then transferred to the distillation unit with 25 ml of 40% NaOH for ammonia release. The flask content was titrated with 0.1 N HCl in the presence of 25 ml of 4% boric acid with two drops of bromocresol green (BCG) and methyl red 5 : 1. The protein percentage was calculated as follows: Crude protein (%) = (Sample volume -blank volume) x N x14.007 x 6.25 x 100/Sample weight (mg) N = Normality of standard HCl

Estimation of crude fat
As much as 100 ml of petroleum ether (40-60°C) was poured in the pre-weighed cup. About 3 g of each sample was transferred into extraction thimble. The extraction process has proceeded in the automated Soxhlet system by two steps, extraction step an hour followed by rinsing step for 30 minutes. Then the cup was dried in the oven at 105°C and weighed. The percentage of crude fat was calculated using the following formula: Crude fat (%) = (Weight of extracted fat/Weight of sample) x 100

In vitro analysis of antioxidant activity
The quantitative DPPH scavenging activity was determined following Kumarasamy et al. (2007) and Kumar et al. (2014)

In vitro assay of anticoagulant activity
The study pursues (Cordier et al., 2011;Chen et al., 2014) protocols for assaying the in vitro anticoagulant action of extracts with suitable amendments.

Volunteers recruitment
Four healthy volunteers of both genders (ages 25-37 years). They were asked to ensure that they had not any previous history for CVDs or medicines administration such as anticoagulant, antiplatelet, and vitamin supplement, etc. Before blood collection, informed consent has been acquired and signed via all volunteers for their participation in this study. All volunteers have subjected to twice measurements of routine PT and aPTT tests to ascertain the normality of the collected blood.

Volunteers recruitment
Four healthy volunteers of both genders (ages 25-37 years). They were asked to ensure that they had not any previous history for CVDs or medicines administration such as anticoagulant, antiplatelet, and vitamin supplement, etc. Before blood collection, informed consent has been acquired and signed via all volunteers for their participation in this study. All volunteers have subjected to twice measurements of routine PT and aPTT tests to ascertain the normality of the collected blood.

Blood collection, plasma preparation, and extracts preparation
Blood was collected by venipuncture of each volunteer and collected separately into (3.2%) sodium citrate (9 : 1 v/v, blood : anticoagulant sodium citrate) vacuum tube and mixed well. Immediately, the collected blood was centrifuged at 4000 rpm for 10 minutes to obtain PPP (platelet-poor plasma). Working solutions for each extract with concentrations (1, 2, and 4 μg/μl) were prepared in normal saline for biological assay.

Anticoagulant activity assay
Following manufacturer's information, two parameters PT and aPTT were separately tested to explore at which step of coagulation pathways were inhibited by L. sativa extracts. Commercial heparin (1 μg/μl) was used as a positive control for both parameters and regular saline was used as a negative control.

Prothrombin time assay
In a fusion tube, 25 μl of normal citrated PPP and 25 μl of extract solution was added and incubated at 37°C for 2-3 minutes. Instantly, the time for clot formation (in seconds) was recorded using a stopwatch after the addition of 100 μl of PT reagent.

Activated partial thromboplastin time assay
In a fusion tube, 25 μl of normal citrated PPP and 25 μl of extract solution was added and incubated for 2 minutes at 37°C. Then 25 μl of APTT reagent was added and incubated for 2-3 minutes. By using the stopwatch, the time for clot formation (in seconds) was recorded immediately after the addition of 50 μl of (0.025 M) CaCl2 solution.

Statistical analysis
The results were expressed as mean ± S.D with n = 2 using IBM SPSS statistics 20. The data were statistically analyzed by one-way ANOVA with Tukey's post hoc test. For the anticoagulant activity experiment, the data were statistically analyzed by one-way repeated measures RM-ANOVA with Bonferroni's post hoc test.
The P-value of less than 0.05 was considered as statistically significant.

Extraction yield
After employing a cold maceration method, the yields of L. sativa (each part of each variety) dried methanolic extracts were presented in Table I

Preliminary phytochemical investigation
The methanolic extracts of L. sativa varieties leave and stem revealed the presence of various bioactive constituents such as carbohydrates, saponins, terpenoids, steroids, flavonoids, coumarins, and mucilage as tabulated in Table II. However, there was a little dissimilarity between the leaves and the stems, i.e. the tannins have been detected only in the leaves.   (4.1%) that has been reported by Wheeler et al. (1994).

In-vitro analysis of antioxidant activity
The efficiency of the sample in scavenging the DPPH radicals is expressed by the IC50 value. A small IC50 value implies a high antioxidant activity. A significant difference (P < 0.05) in the IC50 values were found between the leaves and the stems of both varieties.
Specifically, the leaves of L. sativa var. capitata displayed the lowest IC50 (2.41 mg/ml) in comparison to the leaves of L. sativa var. longifolia (3.23 mg/ml). However, there was no significant difference in the stems of both varieties with (P > 0.05), and they displayed higher IC50 values comparing to the leaves. In comparison to the L-ascorbic acid, the resulted IC50 is (0.01 mg/ml), which is the lowest value among the tested L. sativa extracts; therefore, Lascorbic acid has a significant (P < 0.05) superior antioxidant activity more than other tested extracts   (Llorach et al., 2008;Liu et al., 2007). In this work, a higher antioxidant activity has been observed in leaves in comparison to the stems. This may be attributed to the presence of polyphenolic compounds (tannins) in the leaves. Tannins act as strong natural antioxidants and a substitute for many synthetic antioxidants (Sung et al., 2012). Under the same conditions of DPPH assay, Gan and Azrina (2016) have measured the IC50 of 70% aqueous ethanolic extracts of L.
sativa var. longifolia and L. sativa var. capitata leaves. The resulted values were (4.48 and 3.99 mg/ml), respectively, which are approaching those reported in the present work (3.23 mg/ml and 2.41 mg/ml) for the leaves methanolic extract for the same varieties. Therefore, the bit dissimilarity in the results may be due to the difference in the solvent of extraction and the phenolic content.

In-vitro analysis of anticoagulant activity
Coagulation waterfall is mediated by clotting factors, which circulate in blood plasma in an inactive form and are activated in response to any triggers. Coagulation cascade includes two correlating pathways; intrinsic and extrinsic pathways (Triplett, 2000;Palta et al., 2014).
Prothrombin time test reflects the extrinsic pathway because the PT reagent composes of calcified thromboplastin, which stimulates the clotting factors that mediate the extrinsic pathway; whereas, the intrinsic pathway can be examined by aPTT test using aPTT reagent, which is consisted of kaolin cephaeline that acts as an activator in presence of calcium ions for intrinsic clotting factors (Jesonbabu et al., 2012). In this study, the leaves and the stems extract of both selected varieties    (Rodrigues et al., 2011;Karaki et al., 2013).

CONCLUSION
From this study, the yield of stem methanol extracts was higher than the yield of leaf for both varieties. Both parts of each variety showed the presence of the same phytochemicals with a slight discrepancy between the leaves and the stems, i.e. tannins were detected only in the leaves. The content of the leaves' nutrients in both varieties showed statistically significantly higher levels of raw protein, raw fat, and fairly total ash compared to the studied stem powders. As regards the DPPH assay, the leaves of both varieties have a statistically significantly higher antioxidant activity than the stems. The stems revealed a statistically significant higher anticoagulant activity than the leaves.