Phytochemical compositions, antioxidant properties, enzyme inhibitory effects of extracts of four endemic Lathyrus L. taxa from Türkiye and a taxonomic approach

ABSTRACT Lathyrus is an economically important genus, with different parts of some species used as foodstuff or animal feed. In this study, phytochemical compositions and bioactivities of Lathyrus brachypterus var. brachypterus, L. brachypterus var. haussknechtii, L. nivalis subsp. sahinii and L. tefennicus taxa which are endemic to Türkiye were investigated. Total phenolic and flavonoid contents (TPC, TFC) of methanolic extracts were detected. Then, phytochemical compositions, antioxidant features (radical scavenging (DPPH: 1,1-diphenyl-2-picrylhydrazyl, ABTS: 2,2′-azino-bis(3 ethylbenzothiazoline) 6 sulfonic acid), reducing power (FRAP:Ferric ion reducing antioxidant power, CUPRAC:Cupric ion reducing antioxidant capacity), metal chelating activity (MCA), and the phosphomolybdenum assays (PDA)) and enzyme inhibitory properties of the extracts were also determined. The highest values were found at L. brachypterus var. brachypterus for TPC, L. brachypterus var. haussknechtii for TFC. The highest antioxidant properties were seen in extracts of L. brachypterus var. brachypterus in DPPH, ABTS, FRAP, CUPRAC and PDA assays, while in extract of L. nivalis subsp. sahinii in MCA. The highest enzyme inhibitory activity was found in extract of L. brachypterus var. brachypterus in tyrosinase and glucosidase assays, while in extracts of L. nivalis subsp. sahinii in AChE (acetylcholinesterase), BChE (butyrylcholinesterase) and amylase. Finally, a taxonomic evaluation was made by considering the phytochemicals.


Introduction
Free radicals can be described as molecular entities or molecular fragments that capable of independent existence (hence 'free') and have unpaired electron(s) in outer atomic orbits or molecular orbits (hence 'radical') (Martemucci et al. 2022).Provenance of these radicals may be endogenous (as products of normal metabolisms of the cell organelles such as peroxisomes, mitochondria and endoplasmic reticulum, enzyme activities or phagocytosis, etc.) or exogenous (pollution, tobacco smoke, heavy or transition metals, alcohol, pesticides, etc.) (Phaniendra et al. 2015;Martemucci et al. 2022).
Many radicals are unstable and show a highly reactive property that tends to accept or donate an electron.Because of these properties, they act as oxidant or reductant (Mohammed et al. 2015).The presence of unpaired electron(s) in these radicals causes oxidative stress that can brings about the damages of the proteins, carbohydrates, enzymes, lipids and DNA and even lead to cell death through DNA fragmentation and lipid peroxidation.These outcomes of oxidative stress constitute the molecular basis of the diabetes, cancer, autoimmune diseases, cardiovascular diseases and neurodegenerative disorders (Ratnam et al. 2006).
Antioxidants, which have the potential as prophylactic and therapeutic agents in many diseases, have acquired great significance in the recent times due to the understanding of the role of free radicals in diseases and disorders mentioned above and aging (Ratnam et al. 2006).Several antioxidantbased mechanisms are used by human body to ward off effects of the oxidative stress.Antioxidants, which may be endogenous or exogenous origin, act as "free radical scavengers" in these mechanisms (Pham-Huy et al. 2008).
Since endogenously produced antioxidants are inadequate to prevent oxidative damage caused by free radicals, the antioxidants got exogenously except hige doses are beneficial in preserving against free radicals (Koçyiğit & Selek 2016).
Many plants and their derivatives have been considerably used to prohibit oxidative stress because they contain important natural antioxidants (Akbari et al. 2022).Recently, researches on the extraction methods, antioxidant and enzyme inhibitory features of the bioactive compounds, which have great importance for human health, have also increased.
Lathyrus L., which is an economically important plant genus belongs to Fabaceae family, is one of the studied plant genus on these subjects.The genus Lathyrus, which has more than 200 species naturally distributed in the world, is represented by 79 taxa at the species, subspecies and variety level in Türkiye and 25 of these taxa are endemic (Genc et al. 2022).Some species of the genus are cultivated for different purposes in different parts of the world.The seeds of some species are used as human food, while the aerial parts of some species are used as animal feed (Yildirim et al. 2023).It has been stated that L. tuberosus and L. undulatus Boiss.species have positive effects on health (Baytop 1984;Sakinoglu-Oruc et al. 2021).Within the genus, the main phenolic compounds were reported as chlorogenic acid, epicatechin, and benzoic acid for L. czeczottianus Bässler (Ceylan et al. 2021), quercetin and kaempferol for both L. cicera L. and L. digitatus (Bieb.)Fiori (Llorent-Martinez et al. 2017a) There are a number of studies testing the bioactive components, antioxidant properties and enzyme inhibitory properties of extracts obtained using the aerial parts or seeds of some taxa of the genus (Pastor-Cavada et al. 2009;Fratianni et al. 2014;Heydari et al. 2015;Llorent-Martinez et al. 2016;2017a;b;Ozbek-Yazici et al. 2020;Ceylan et al. 2021;Eyiiş & Karadeniz-Pekgöz 2021).In these studies, there are differences in the used parts of the plant, the used tests, and the way the results are expressed.In Pastor-Cavada et al. (2009), it has been stated that there are few studies on Lathyrus species, although they are seen as a source of functional compounds such as antioxidant phenolics.
The purposes of this investigation are to exhibit the phytochemical composition, total bioactive components, antioxidant capacity and enzyme inhibitory potential of the aerial parts of Lathyrus brachypterus var.brachypterus, L. brachypterus var.haussknechtii, L. nivalis subsp.sahinii and L. tefennicus taxa which are endemic for Türkiye.According to our knowledge, there is no report on these properties of these taxa.The aerial parts (as mix) of these plants were carefully separated and they were dried at the dark conditions for ten days.The dried plant materials were powdered by using a laboratory mill.The extracts were prepared using methanol through maceration.Overnight, the air-dried powdered Phytochemical compositions, antioxidant properties, enzyme inhibitory effects of extracts of four endemic Lathyrus L. taxa from Türkiye and a taxonomic approach samples (10 g) were macerated at room temperature with 200 mL of methanol.Finally, the solvents were evaporated from the mixtures.The extracts were stored at 4 °C until further analysis was required.

Phytochemical composition
In the quantitative screening of fifty-three compounds (phenolics, flavonoids, organic acid, etc.), Shimadzu brand LCMS-8040 tandem mass spectrometer and a Nexera model ultra-high performance liquid chromatograph (U-HPLC) were used.The separation system consisted of binary pumps (LC 30AD), a column oven (CTO 10 ASvp), an autosampler (SIL 30 AC), and a degasser (DGU 20 A3R) (the chromatograph).The previously developed and validated liquid chromatography-mass spectrometry/mass spectrometry method was used in the analyses (Yilmaz 2020).The analytical column used for the chromatographic seperation was a reversed phase Agilent brand Poroshell 120 EC-C18 model (150 mm×2.1 mm, 2.7 µm) column.In addition, the temperature of the column was arranged as 40°C.The mobile phases used for the gradient elution was; mobile phase A (water, 5 mM ammonium formate, 0.1% formic acid) and mobile phase B (methanol, 5 mM ammonium formate, 0.1% formic acid).Moreover, the gradient program started with 20% mobile phase B, followed by a ramp from 20% to 100% for 25 minutes.Then the mobile phase system remained constant at 100% B for 10 minutes.Finally, the initial mobile phase system (20% B) was followed for 10 minutes.Furthermore, the injection volume and the mobile phase flow rate was set to 5 µL and 0.5 mL/min, respectively.

Bioactive compounds
The total phenolic content (TPC) and total flavonoid content (TFC) were determined using previously published methods (Zengin & Aktumsek 2014).TPC and TFC values were expressed as mg gallic acid equivalents (GAE)/g extract and mg rutin equivalents (RE)/g extract, respectively.
The effects of the extracts on the activity of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, amylase, and glucosidase enzymes were also tested.In AChE and BChE assays used galanthamine as positive control, results were expressed as mg galanthamine equivalents (GALAE)/g extract.Kojic acid, a standard tyrosinase enzyme inhibitor, was used in the tyrosinase tests and the results were given as mg kojic acid equivalents (KAE)/g extract (Uysal et al. 2017).Amylase and glucosidase enzyme inhibition test results of the extracts were calculated as mmol acarbose equivalents (ACAE)/g extract.

Data analysis
All data were given as mean ± standard deviation.Statistical analysis was performed by analysis of variance (ANOVA).A post hoc test (Tukey) was done when the differences shown by data were significant (p < 0.05).Also, hierarchical clustered analysis (HCA) was achieved to assess the (dis)similarity between samples in terms of their molecules.SIMCA 14.0 statistical program was used for all analysis.

Phytochemical composition
The phytochemical structures of four taxa were revealed by LC-MS/MS analysis and the amounts of their some major compounds were calculated.The findings are given in Tab. 1.
Fifty-three compounds were considered and twentyfive of them were detected in at least one of the studied taxa.The first three compounds detected with the highest amount are Isoquercitrin, Quinic acid and Chlorogenic acid (for L. b. var.brachypterus), Quinic acid, Isoquercitrin and Chlorogenic acid (for L. b. var.haussknechtii), Quinic acid, Salicylic acid and Protocatechuic acid (for L. n. subsp.sahinii), Quinic acid, Fumaric acid and Hesperidin (for L. tefennicus).
It was stated that three of the mentioned compounds with the highest rate have favourable effects as given below: isoquercitrin against oxidative stress, cancer, cardiovascular disorders, diabetes and allergic reactions (Valentová et al. 2014), quinic acid against prostate cancer (Inbathamizh & Padmini 2013), chlorogenic acid which also has hepatoprotective and renoprotective features, against oxidative stress, inflammatory stress, cardiovascular disorders and diabetes (Maalik et al. 2016).

Total bioactive components
Phenolics, including flavonoids, have been reported to be effective on various pharmacological activities (Mondal & Rahaman 2020).There are many studies showing that flavonoids have benefical effects in many different clinical areas such as cardiovascular diseases, neurology, urology, immunology and gastroenterology (Hoensch & Oertel 2015).Various studies have been conducted to investigate the TPC and TFC values of the extracts of different Lathyrus taxa obtained by using different solvents, such as ethyl acetate, methanol, water, etc. Aerial parts of plants have been used in some of these studies (Heydari et al. 2015;Llorent-Martinez et al. 2016;2017a;b;Ceylan et al. 2021;Eyiiş & Karadeniz-Pekgöz 2021) and seeds have been used in others (Pastor-Cavada et al. 2009;Marathe et al. 2011;Fratianni et al. 2014;Ozbek-Yazici et al. 2020;Eyiiş & Karadeniz-Pekgöz 2021).It is not possible to make healthy comparisons with some of the studies mentioned above, as there are differences in solvents, used plant parts or the ways the results are expressed.

Bekir Yildirim, Mustafa Abdullah Yilmaz, Gokhan Zengin and Hasan Genc
TPC values of methanolic extracts (as mg GAE/g) have been found as 150.63, 179.69, 390.94, 397.00, 452 et al. 2017b), respectively.According to the findings of our work, TFC values of all taxa except L. n. subsp.sahinii were higher than the literature data.Although L. n. subsp.sahinii gave close results with L. pratensis, it was determined that it had higher TFC content than the other taxa given in the literature.
Even in the extracts of the same species using different solvents, the total content of bioactive components can be different.Comparisons become difficult due to differences in the methods of obtaining the extracts, the used parts of the plant, and the presentation of the results (Llorent-Martinez et al. 2016).It has been stated that differences in bioactive components are related to genetic variations, climatic conditions, soil structure, region where the plants grow, habitat properties and the other ecological factors (Llorent-Martinez et al. 2016;Ozbek-Yazici et al. 2020;Zengin et al. 2020;Ceylan et al. 2021).The total bioactive components can be different even in the extracts obtained from the same species according to differences in the solvent, the plant parts used, or region which the plants were collected.In general, the total bioactive components are closely related to the solvent polarity (Fratianni et al. 2014;Llorent-Martinez et al. 2016;Ceylan et al. 2021;Eyiiş & Karadeniz-Pekgöz 2021).

Antioxidant properties
Some commonly performed in vitro methods were used in this study: DPPH, ABTS, FRAP, CUPRAC, MCA and PDA.Tab. 3 summarizes antioxidant properties of the investigated taxa.
As seen in the Tab.3, rankings of the taxa according to the antioxidant properties are the same considering the DPPH, ABTS, CUPRAC and FRAP tests.When the MCA and PDA tests are taken into account, the rankings change.

Radical scavenging assays (DPPH • and ABTS • +)
The free radical scavenging abilities of antioxidants are used to measure their antioxidant powers.DPPH and ABTS+ are free radicals used for this purpose (Pisoschi & Negulescu 2012;Sadeer et al. 2020;Flieger et al. 2021;Muntenau & Apetrei 2021).DPPH • discovered by Goldschmidth and Renn in 1922, is one of the most commonly used stable free radicals.The DPPH scavenging test serves to reduce this radical by an antioxidant.The DPPH solution is deep violet in colour with a maximum absorbance at a wavelength of 517 nm.When it interacts with a substance (such as an

Reducing antioxidant abilities (FRAP and CUPRAC)
The Ferric Reducing Antioxidant Power (FRAP) and Cupric Reducing Antioxidant Capacity (CUPRAC) are important tests applied to determine antioxidant properties.Iron and copper ions are reduced by taking advantage of the electron-donating ability of antioxidants.The reducing power is also a measure of the antioxidant ability (Llorent-Martinez et al. 2017a).The FRAP assay is based on the reduction of the Fe(TPTZ)3+ (iron trotripyridyltriazine) complex to a strongly blue coloured Fe(TPTZ)2+ complex by antioxidants under acidic (pH 3.6) conditions.Results occur in terms of absorbance increase at 593 nm and are expressed as micromolar Fe2+ equivalent or relative to an antioxidant standard.Nevertheless, it was also found that the measured reduction capacity does not necessarily reflect antioxidant activity (Benzie & Strain 1996;Antolovich et al. 2002).In the CUPRAC test, reducing capability of the Cu(II)-neocuproine complex formed by Cu(II) and neocuproine (2,9-Dimethyl-1,10-phenanthroline) to Cu(I)-neocuproine, which gives maximum absorbance at 450 nm, by the presence of the antioxidants is utilized (Apak et al. 2004;Büyüktuncel 2013).
The reducing abilities of the investigated taxa are given in Tab. 3.According to the FRAP assay results in this study, the ferric reducing abilities of the taxa are in the form of L. b. var.brachypterus > L. b. var.haussknechtii > L. n. subsp.sahinii > L. tefennicus from strong to weak.Power of L. b. var.brachypterus to reduce Fe 3+ to Fe 2+ close to two times that of L. tefennicus.
The CUPRAC assay also gave similar results with the FRAP test.The same ranking was seen in terms of copper reduction capabilities of taxa.Nevertheless, copper reduction ability of L. b. var.brachypterus is more than two times that of L. tefennicus, and close to two times that of L. n. subsp.sahinii.
According to literature, FRAP and CUPRAC assays were performed on some Lathyrus species (Llorent-Martinez et al. 2016;2017a;b;Ceylan et al. 2021).Taking into account parameters such as solvent, plant parts used and ways of expressing the results, Llorent-Martinez et al. (2016;2017a) can be compared with current study.For methanolic extracts of the L. aureus and L. pratensis, results of the reducing power tests were 31.98 and 13.32 mg TE/g extract (FRAP), 71.22 and 35.33 mg TE/g extract (CUPRAC), respectively (Llorent-Martinez et al. 2016)

Metal chelating activity (MCA)
Chelating agents are organic compounds that bind metal ions to form a structure called a chelate.Chelators form a complex together with toxic ions.These complexes, which have lower toxicity, are more easily removed from the body by the excretory system.Metal chelation therapy is commonly used to treat metal poisoning (Flora et al. 2007).Iron and copper, which are transition metals, play an important role in the Fenton and Haber-Weiss reactions that lead to the formation of free radicals, which are very reactive and harmful in the body.Substances that can chelate these metals are important in terms of antioxidants (Llorent-Martinez et al. 2017a;b).
The metal chelating activities of the extracts obtained from the taxa (displayed in Tab. 3) are generally close to each other and vary in the range of 23.25-30.81mg EDTAE/g.It was observed that extract of the L. nivalis subsp.sahinii was more effective than the others.
The metal chelating activities of the methanolic extracts were found as 44.48, 23.78, 9.39 and 0.62 mg EDTAE/g for L. digitatus, L. cicera, L. aureus and L. pratensis, respectively (Llorent-Martinez et al. 2016;2017a).Different studies (Llorent-Martinez et al. 2017b;Ceylan et al. 2021) on the chelating abilities of extracts obtained from Lathyrus species were also carried out.However, these studies do not make it possible to compare in terms of the used solvents and the ways the results were expressed.
The results of the MCA tests showed that the taxa we investigated generally had better chelating ability than the taxa given in the literature, except L. digitatus.

Phosphomolybdenum assay (PDA)
The phosphomolybdenum assay based on the reduction of Mo(VI) to Mo(V) is a method used for the quantitative determination of antioxidant capacity.At the end of the reduction reactions, a green coloured phosphate-Mo(V) complex is formed at acidic pH.This complex has a maximum absorbance value at 695 nm (Prieto et al. 1999).
Phosphomolybdenum tests results are shown in Tab. 3. As a result of PDA, the antioxidant abilities of the taxa were found to be close to each other, as in the MCA test.L. b. var.brachypterus and L. n. subsp.sahinii have the higher values (1.21 and 1.20, respectively) while L. b. var.haussknechtii and L. tefennicus have the lower (1.08 and 1.06, respectively).
All of the taxa we studied showed less efficiency in terms of phosphomolybdate assays than the taxa given in the literature.

Enzyme inhibitory properties
A C h E ( a c e t y l c h o l i n e s t e r a s e ) a n d B C h E (butyrylcholinesterase) are enzymes that act on Alzheimer's and learning, and amylase and glucosidase on diabetes.Excess of these enzymes in the body can lead to the mentioned health problems.The excess of the tyrosinase enzyme, which catalyzes the production of melanin pigment, which helps to prevent UV light, also can casuses hyperpigmentation and neurodegenerative diseases like Parkinson's (Yırtıcı 2019).Enzyme inhibitors are secondary metabolites that bind to enzymes and reduce their activity (Rauf & Jehan 2017).Enzyme inhibition, which is an important pharmacological research area today, is popular in treatment strategies of global health problems.One of the most important characteristics of enzyme inhibitors is that they are used as drugs in many physiological conditions (Çakmak et al. 2017).For this reasons, it is necessary to search for natural compounds that do not have unfavorable effects from plants in order to gain new approaches in the treatment of the aforementioned diseases (Llorent-Martinez et al. 2016).
Extracts of four Lathyrus taxa were tested by spectrophotometric methods to determine enzyme inhibitory activities.Findings reached at the end of the tests are summarized in Tab. 4.  It is normal for extracts from different species to have different test results.Yet, even varieties of the same species are less similar to each other according to the HCA.It is understood that the morphological similarities of the taxa and their phytochemical compositions are not compatible.If these differences, seen even in varieties of L. brachypterus species, are not caused by environmental differences such as geographical and ecological conditions, it can be assumed that genetic factors may be effective.
As a result of the morphological and anatomical studies carried out on L. b. var.haussknechtii, which was first described as a species and then reduced to the variety level, it was suggested that the taxon be classified as a different species as L. haussknechtii (Çildir 2011).In other words, these two varieties (L. b. var. brachypterus and L. b. var. haussknechtii) belonging to the same species are thought to be two different species.In Güner et al. (2012), this taxon has been evaluated as a separate species as L. haussknechtii.The results of the HCA in this study also support this opinion.

Conclusions
The tests applied to determine the amount of bioactive components in plants may give different results depending on the polarity of the solvent.Antioxidant properties and enzyme inhibitory properties of the plant extracts may also vary depending on the ratio of bioactive components.The results of studies carried out on taxa of the Genus Lathyrus indicate that variations in bioactive components and their amounts are related with pedo-climatic, geographic and the other ecologic differences.In addition, genetic factors can also affect the mentioned properties of the plants.In addition, the phytochemical profiles of L. b. var.brachypterus and L. b. var.hausskenchtii are not compatible with current taxonomic affinities based on morphological similarities.It has been concluded that the phytochemical profile data in current study supports the views that accept these varieties as L. brachypterus and L. haussknechtii as separate species.Bekir Yildirim, Mustafa Abdullah Yilmaz, Gokhan Zengin and Hasan Genc Plant samples were collected from the natural distribution areas at the flowering period.Plant samples were identified by YILDIRIM and GENC.Locality informations are given below.• L. brachypterus Čel.var.brachypterus: Foothills of Erciyes mountain, 1730 m, Kayseri, Türkiye.• L. brachypterus var.haussknechtii (Širj.)P.H.Davis: Karadağ mountain, 1975 m, Karaman, Türkiye.• L. nivalis Hand.-Mazz.subsp.sahinii H.Genç: Karadağ mountain, 1680 m, Karaman, Türkiye.• L. tefennicus H.Genç&A.Şahin: Bezirgan plateau road, 1300 m, Tefenni, Burdur, Türkiye.From this point of the manuscript, the abbreviations L. b. var.brachypterus for L. brachypterus var.brachypterus, L. b. var.haussknechtii for L. brachypterus var.haussknechtii and L. n. subsp.sahinii for L. nivalis subsp.sahinii will be used.
. var. brachypterus 41.74±0.04 a 75.87±3.43a 77.32±0.23 a 114.92±2.00 a 25.28±0.36b 1.21±0.07 a L. b. var.haussknechtii 36.59±0.35b 67.61±4.37 b 56.67±1.48b 85.52±1.71b 25.29±0.16b 1.08±0.04 a L. n. subsp.sahinii 19.43±0.31c 65.23±0.98b 45.37±0.30c 62.32±0.23 c 30.81±0.31 a 1.20±0.09a L. tefennicus 18.90±0.57c 57.73±0.09c 42.33±0.25d 51.88±1.09d 23.25±0.28c 1.06±0.07 a Values are reported as mean±S.D. of three paralel experiments.Different superscripts indicate significant differences between the tested extracts.(p<0.05)antioxidant) that can donate hydrogen, the reduced form of DPPH is formed and the colour of the solution becomes pale yellow and the colour change serves as an indicator of antioxidant activity.The decrease in absorbance is linearly related to antioxidant concentration.The degree of colour change in the solution also indicates the level of antioxidant capacity.In other words, substances with strong antioxidant properties cause more lightening of the solution colour (Pisoschi & Negulescu 2012; Sadeer et al. 2020; Flieger et al. 2021).ABTS •+ assay, which was first reported in Miller et al. (1993), is also known as TEAC (Trolox Equivalent Antioxidant Capacity) (Sadeer et al. 2020; Muntenau & Apetrei 2021).ABTS •+ , a bluish-green chromophore of maximum absorption at 734 nm, is another radical used in scavenging assays, and it is formed as a result of losing an electron by the nitrogen atom of ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) (Pisoschi & Negulescu 2012).When ABTS •+ receives an electron from electron donors such as antioxidants, it forms the stable form of ABTS and changes in colour to pale blue or colourless (Pisoschi & Negulescu 2012; Sadeer et al. 2020; Flieger et al. 2021; Muntenau & Apetrei 2021).Findings of the radical scavenging assays are presented in Tab. 3. As a result of the DPPH • scavenging assay in the current study, the ordering of the taxa according to their antioxidant capacities (as trolox equivalent) is L. b. var.brachypterus > L. b. var.haussknechtii > L. n. subsp.sahinii > L. tefennicus.The antioxidant capacities of the varieties of the L. brachypterus are higher than the others.In particular, DPPH • scavenging capacity of L. b. var.brachypterus (41.74 mg TE/g) is more than twice that of L. n. subsp.sahinii and L. tefennicus.In the same assay, capacity of L. b. var.haussknechtii (36.59 mg TE/g) is also close to twice that of the mentioned taxa.Considering the ABTS •+ scavenging assay result, the ordering of the taxa in terms of their antioxidant capacities is the same as the ordering made according to the DPPH • scavenging assay result.ABTS •+ scavenging capacities of the taxa are close to each other and vary in the range of 57.73-75.87mg TE/g.ABTS •+ scavenging capacities are approximately twice the DPPH • scavenging capacities for L. b. var.brachypterus and L. b. var.haussknechtii, and approximately three times for L. n. subsp.sahinii and L. tefennicus.DPPH and ABTS radical scavenging tests were performed on Lathyrus species(Llorent-Martinez et al. 2016;2017a; b;Heydari et al. 2015;Ceylan et al. 2021;Ozbek-Yazici et al. 2020).Radical scavenging abilities were measured as 126.68 and 4.09 mg TE/g extract (for DPPH) and 67.85 and 34.37 mg TE/g extract (for ABTS) for the methanolic extracts of L. pratensis and L. aureus, respectively (Llorent-Martinez et al. 2016).For methanolic extracts of the L. digitatus and L. cicera, DPPH radical scavenging abilities were 40.28 and 25.23 mg TE/g DE, while ABTS radical scavenging abilities were 147.83 and 65.18 mg TE/g DE, respectively (Llorent-Martinez et al. 2017a).Relatively, L. b. var.brachypterus and L. b. var.haussknechtii are closer to L. digitatus; L. n. subsp.sahinii and L. tefennicus are closer to L. cicera in terms of DPPH tests.Similarly, L. b. var.brachypterus and L. b. var.haussknechtii are closer to L. pratensis, and L. n. subsp.sahinii and L. tefennicus closer to L. cicera in terms of ABTS tests.

Figure 1 .
Figure 1.Hierarchical cluster analysis for tested taxa based on their phytochemical composition (by Ward method).
Extraction yields, Total phenolic content (TPC) and Total flavonoid content (TFC) values of the extracts of the examined Lathyrus taxa were presented in Tab. 2. TPC values vary in the range of 24.91-44.31mg GAE/g in the current study.L. b. var.brachypterus possessed the highest TPC (44.31±0.48mg GAE/g), followed by L. b. var.haussknechtii (34.99±1.03mg GAE/g) and L. n. subsp.sahinii (27.25±0.62 mg GAE/g).The lowest TPC was found in L. tefennicus (24.91±0.06mg GAE/g).TFC values vary in the range of 25.78-42.98mg RE/g.TFC contents of L. b. var.haussknechtii and L. b. var.brachypterus are very close to each other, 42.98 mg RE/g and 41.84 mg RE/g, respectively.TFC content of L. tefennicus is 37.14 mg RE/g and of L. n. subsp.sahinii is 25.78 mg RE/g.Assays carried out on four Lathyrus taxa mentioned above have revealed different results on the amount of bioactive components.

Table 2 .
Extraction yields, total phenolic and flavonoid contents.

Phytochemical compositions, antioxidant properties, enzyme inhibitory effects of extracts of four endemic Lathyrus L. taxa from Türkiye and a taxonomic approach extracts
. FRAP values were determined with 55.84 and 40.99 mg TE/g DE, CUPRAC values were determined as 54.39 and 53.96 mg TE/g DE for methanolic of L. digitatus and L. cicera, respectively (Llorent-Martinez et al. 2017a).According to the FRAP test results, L. tefennicus and L. n. subsp.sahinii showed closeness to L. cicera, and L. b. var.haussknechtii to L. digitatus.Extract of the L. b. var.brachypterus has the highest FRAP value.CUPRAC test results show that L. tefennicus is closer to L. cicera and L. digitatus, while L. b. var.brachypterus and L. b. var.haussknechtii have the highest values.