To the best of our knowledge, the amounts of the target phenolic compounds, total bioactive components and bioactivities of AEs, EEs and WEs of
L. annuus,
L. hierosolymitanus and
L. hirsutus were revealed for the first time in this research.
Phenolic compounds
Phenolic compounds found everywhere in plants have a scavenger role against reactive oxygen/nitrogen species and some of them have various helpful effects on healthcare. It has been reported that more than 8000 phenolic compounds are naturally found in plants, half of which are flavonoids
(Papuc et al., 2020). Many phenolic compounds, such as gallic acid, quercetin, caffeic acid, protocatechuic acid, p-coumaric acid, luteolin and chlorogenic acid, have biological and pharmaceutical effects against many diseases and disorders
(Kumar et al., 2022).
In the current study, some phenolic compounds of the studied species were determined by HPLC analysis. Fig 1 reflects the standard chromatogram and retention times (Rt, min.). HPLC chromatograms of the studied species are also given in Fig 2.
In the quantitative screening, twelve compounds were assessed and the majority of them were defined in all species (Table 1). The highest gallic acid (1334.7), p-hydroxybenzoic acid (362.1), quercetin (64.0), caffeic acid (47.0) and p-coumaric acid (22.8) values (µg/g) were detected in
L. hirsutus, protocatechuic acid (42.0), vanillin (20.0), luteolin (19.1), rutin (256.0) and epicatechin (178.0) values (µg/g) in
L. hierosolymitanus, hesperidin (16.7) and chlorogenic acid (6.7) values (µg/g) in
L. annuus. Compounds epicatechin in
L. annuus, chlorogenic acid in
L. hierosolymitanus, rutin, hesperidin and chlorogenic acid in
L. hirsutus could not be detected.
In
Ceylan et al., (2021), chlorogenic acid 2510 and 12090, epicatechin 5320 and 5710, p-hydroxybenzoic acid 430 and 960, gallic acid 40 and 650, quercetin 190 and 400, caffeic acid nd and 360, luteolin 250 and 290, protocatechuic acid 30 and 180 and hesperidin 30 and 30 µg/g were indicated in the ethyl acetate extracts and WEs of
L. czeczottianus (aerial parts), respectively (nd: not determined). Vanillin, p-coumaric acid and rutin were not determined. In the extracts of aerial parts of
L. brachypterus var.
brachypterus,
L. brachypterus var.
haussknechtii,
L. nivalis subsp.
sahinii and
L. tefennicus, respectively, the amounts of some phenolic compounds (µg/g) were specified as given in parentheses. Chlorogenic acid (3582, 2408, 15, 27), luteolin (412, 32, 11, 39), p-coumaric acid (121, 84, 28, 30), quercetin (1158, 230, nd, 8), protocatechuic acid (283, 93, 79, nd), hesperidin (139, 323, nd, 259), rutin (109, 282, nd, 240), vanillin (nd, 75, nd, 80), caffeic acid (27, nd, nd, nd) and gallic acid (10, nd, nd, nd). Epicatechin and p-hydroxybenzoic acid were not detected in any of the extracts
(Yildirim et al., 2023).
As seen in Table 1, the amounts of gallic acid and p-hydroxybenzoic acid in
L. hirsutus and rutin in
L. hierosolymitanus are remarkable. These plants have the potential to be used as a source of the aforementioned phenolic compounds, as they contain relatively more of these compounds than the taxa reported in the literature.
TPCs, TFCs and antioxidant properties
The highest TPC and TFC values were determined in the EEs of
L. hirsutus and
L. hierosolymitanus, respectively, while the lowest values were determined in the WE of
L. hierosolymitanus and the AE of
L. annuus, respectively. Table 2 demonstrates the findings. Considering all species and extracts, calculated TPC and TFC values varied between 1.65-3.33 mgGAE/g dw and 1.37-3.82 mgQE/g dw, respectively.
The phenolic contents of widely used legumes vary from 0.325 to 6.378 mgGAE/g
(Marathe et al., 2011). The findings of this study regarding the TPC values are consistent with the mentioned study. Other summarized literature data are shown in Table 3. TPCs of the species investigated in our work were lower compared to the literature given in Table 3. Even extracts of the same species prepared using various solvents may give different findings. The reason why the TPC values are lower compared to the literature may be related to the species or solvents. TFC results have been expressed as mgRE/g in the literature. As in here, differences in the expression of results may not always allow for comparison with the other works.
It has been guessed that a cell has fallen into free radical harm thousands of times in 24 hours. Although most of these damages are restored, some are not and accumulate
(Pruteanu et al., 2023). Antioxidants can exert a protective effect by eliminating the adverse impacts of these radicals, which are toxic by-products of the normal metabolic processes of cells
(Sen et al., 2010). The physiological function of antioxidants is to inhibit harm to cells due to free radical-induced chemical reactions (
Young and Woodside, 2001).
In this study, some commonly used tests (DPPH, ABTS and RP) were applied for the determination of antioxidant activities. As seen in Table 2, the highest DPPH scavenging activities were found in extracts of
L. hierosolymitanus,
L. hirsutus and
L. annuus for acetone, ethanol and water, respectively. The EE of
L. hirsutus had the best DPPH scavenging activity of all extracts. The lowest DPPH scavenging activities were shown by WEs for all species. Especially WE of
L. hirsutus was the weakest. Considering the ABTS tests, the highest radical scavenging properties were found in extracts of
L. hierosolymitanus (for AE and WE) and
L. annuus (for EE). The weakest ABTS scavenging activities were shown by
L. annuus (for WE) and
L. hirsutus (for EE and AE). Another assay applied to find the antioxidant properties of the species was the RP test. In the RP tests, the most powerful effects were found in extracts of
L. hierosolymitanus (for WE and EE) and
L. hirsutus (for AE). AE of
L. annuus had the lowest RP in all extracts. In the current study, the results of antioxidant tests were calculated as IC
50 values (mg dw/ml). DPPH, ABTS and RP values of extracts of all studied species were in the range of 3.74-2.09, 2.09-1.81 and 5.21-1.87 mg dw/ml as IC
50, respectively.
As seen in Table 3, the results have been generally expressed as mgTE/g in the literature. Comparisons are impossible due to differences in the expression of the results. According to the literature, DPPH and ABTS scavenging activity values are 3.19 and >5 for
L. nissolia and 1.42 and 1.80 (mg/ml) for
L. czeczottianus, respectively. While AEs and EEs of all species analyzed in this study showed better DPPH radical scavenging activity than
L. nissolia, ABTS radical scavenging activity of all extracts of all three species was also found to be more successful than
L. nissolia. The DPPH and ABTS radical scavenging activities of all extracts of the studied species were found to be close to or lower than those of
L. czeczottianus. These results revealed that radical scavenging activities may vary depending on the species studied or the solvent used.
Enzyme inhibitory effects
Type 2 diabetes mellitus (T2DM) is a well-known risk factor for Alzheimer’s disease (AD). T2DM and AD are significant health problems that have diverse symptoms but share a complex and linked mechanism. It is widely accepted that diabetic patients, especially those with T2DM, have a much greater risk of getting AD compared to normal people
(Song et al., 2017). It has been stated that amylase inhibitors can keep postprandial hyperglycemia under control and reduce the risk of developing diabetes by delaying the increase of blood glucose (
Poovitha and Parani, 2016). Obesity, which is also associated with diabetes and is closely linked to the appearance of many chronic diseases, including cardiovascular diseases and even cancer, is caused by excessive intake and accumulation of fat in the body. Pancreatic lipase is an enzyme that breaks down triacylglycerols found in ingested fats and ensures their absorption in the intestine. Some clinical studies have shown that lipase inhibitors, which are important in the treatment of clinical obesity, can reduce obesity caused by a high-fat diet
(Liu et al., 2020).
It is known that the occurrence of some diseases and disorders is associated with the overactivity of various enzymes. Reducing the activity of these enzymes to normal levels or partially inhibiting them may prevent or delay the appearance of diseases. Therefore, inhibition tests were carried out to determine the enzyme inhibitory potentials of the extracts. Amylase and lipase were chosen as target enzymes. Table 4 reflects the findings of inhibition tests.
It was exhibited that AEs gave better results in the amylase inhibition test, while EEs were more successful in lipase inhibition tests. The WEs gave the most unsuccessful results in all enzyme inhibition tests except the lipase test of
L. hirsutus. When all extracts and species were considered, the strongest and weakest amylase inhibitory effects were observed in the AE and WE of
L. hierosolymitanus, respectively. The strongest and weakest lipase inhibitory effects were also monitored in the EE of
L. annuus and in the AE of
L. hirsutus, respectively.
Amylase inhibitory activities of the water, methanol and ethyl acetate extracts of
L. aureus and
L. pratensis are 0.17, 0.39, 0.55 and 0.13, 0.37, 0.39 mmol ACAE/g, respectively (
Llorent-Martinez et al., 2016). These values for the water and ethyl acetate extracts of
L. czeczottianus are 0.14 and 0.34 mmol ACAE/g, respectively
(Ceylan et al., 2021). Amylase inhibitory activities of the methanolic extracts of
L. cicera, L. digitatus, L. tefennicus,
L. brachypterus var.
haussknechtii,
L. brachypterus var.
brachypterus and
L. nivalis subsp.
sahinii have been found as 0.53, 0.56, 0.50, 0.53, 0.55 and 0.57 mmol ACAE/g, respectively (
Llorent-Martinez et al., 2017a;
Yildirim et al., 2023). In the studies above, enzyme inhibitory activity values have been determined as ACAE (Acarbose equivalent).
In another work (
Llorent-Martinez et al., 2017b), amylase inhibitory activities of the methanolic extracts of
L. nissolia and
L. czeczottianus have been calculated and the results expressed as 3.66 and 3.87 mg/ml (IC
50), respectively. Amylase inhibitory activities of extracts prepared using acetone, ethanol and water in the current work are relatively compatible with the values specified in the above-mentioned work. The results, especially with the AEs of the species (4.46, 4.50 and 4.86 for
L. hierosolymitanus,
L. hirsutus and
L. annuus, respectively), are close to the literature data. The high amylase inhibitory activities of the AEs may be related to their high TPCs.
As far as we know, studies investigating the lipase inhibitory activities of
Lathyrus species are not common. The lipase inhibitory activities (IC
50) of the WE and organic extract of
L. hierosolymitanus collected from the ecological conditions of Palestine are 17.48 and 125.83 mg/ml, respectively
(Jaradat et al., 2017). In the current research, more promising results were obtained compared to the above-mentioned work. EEs of all species showed high inhibitory activity in lipase inhibition assays. Lipase inhibitory activity values were 3.29, 4.80 and 5.19 mg/ml for EEs of
L. annuus,
L. hierosolymitanus and
L. hirsutus, respectively. The most effective extract, EE of
L. annuus, showed a much better inhibitory effect on lipase activity than those reported in the literature.
