Antioxidant Properties and Structure-Antioxidant Activity Relationship of Allium Species Leaves

Allium is a genus that is widely consumed and used as traditional medicine in several countries. This genus has two major species, namely cultivated species and wild species. Cultivated species consist of A. cepa L., A. sativum L., A. fistulosum L. and A. schoenoprasum L. and wild species consist of A. ursinum L., A. flavum L., A. scorodoprasum L., A. vineale L. and A. atroviolaceum Boiss. Several studies report that the Allium species contain secondary metabolites such as polyphenols, flavonoids and tannins and have bioactivity such as antioxidants, antibacterial, antifungal, anti-inflammatory, pancreatic α-amylase, glucoamylase enzyme inhibitors and antiplatelets. This review summarizes some information regarding the types of Allium species (ethnobotany and ethnopharmacology), the content of compounds of Allium species leaves with various isolation methods, bioactivities, antioxidant properties and the structure-antioxidant activity relationship (SAR) of Allium compounds.


Introduction
Antioxidants play important roles in health. They are also used to reduce disease risk and have the ability to protect the body against oxidative damages, which cause several diseases (diabetes, cancer, and neurodegenerative disorders, etc.). Antioxidants can control oxidative processes, leading to food quality descent caused by reactive oxygen species (ROS) and free radical reactions in the body [1,2].
ROS and free radicals are the main causes of oxidative stress, which can trigger several degenerative diseases such as cancer, coronary heart disease and vascular disease [3]. Based on the dangers posed by ROS and free radical reactions, it is necessary to have natural antioxidants that can prevent oxidative stress. Several studies reported that the leaves of the Allium species have good antioxidant activity [4][5][6].

Allium cepa
Several studies report the isolation of A. cepa with many methods. Samples were extracted using the solvent extraction/conventional maceration method with 70% ethanol solvent in a ratio 5:1 to the sample [7]. In another study conducted by Amabye et al., the ethanol extract was characterized by HPLC to determine the amount of phenolics [40].

Allium ursinum
Studies concerning A. ursinum also use several isolation methods. In a study conducted by Barla et al., a sample was extracted with an aqueous-ethanol solvent for 2 h at 80 • C to obtain a powdered extract [69]. In another study, a sample was extracted using two maceration methods, namely by water infusion and water decoction. Then, the determination of the amount of phenolic content was conducted by the colorimetric assays [70].

Allium flavum
Rezgui et al. also conducted research concerning A. flavum. The study conducted refluxing samples two times for 1 h with MeOH-H 2 O (7:3) [11]. The solvent was evaporated and then VLC (silica gel RP-18, MeOH-H 2 O 50:50) was conducted to produce several fractions to separate using CC (Sephadex LH-20, MeOH). The last step was the MPLC method (RP-18 silica gel, MeOH/H 2 O gradient 40-100%) to obtain pure compounds [11]. In addition, in another study, samples were suspended in 5 mL 1 mol/l K 2 HPO 4 with pH 7.0 and centrifuged for 10 min to obtain an aliquot of the supernatant, which will be used in the SOD activity test [27].

Allium scorodoprasum
Prior to the antioxidant activity test, Tasci et al. first performed a sample extraction with 80% using an ultrasonic bath [12].

Allium vineale
Several methods were used to isolate compounds from A. vineale. One of the methods used was a sample hydrodistilled first for 4 h and extracted with CH 2 Cl 2 . The filtrate was obtained and then evaporated to remove the solvent to obtain essential oils that have a sharp odor [45]. In other research, a sample A. vineale was first boiled using distilled water before being extracted with ethyl acetate and a concentrated organic layer. The extract obtained was then partitioned using silica gel chromatography with hexane, ethyl acetate and methanol as mobile phases to obtain twenty-five fractions [16].
Based on the previous studies using different isolation methods of this species, it

Allium Scorodoprasum
Prior to the antioxidant activity test, Tasci et al. first performed a sample extraction with 80% using an ultrasonic bath [12].

Allium Vineale
Several methods were used to isolate compounds from A. vineale. One of the methods used was a sample hydrodistilled first for 4 h and extracted with CH2Cl2. The filtrate was obtained and then evaporated to remove the solvent to obtain essential oils that have a sharp odor [45]. In other research, a sample A. vineale was first boiled using distilled water before being extracted with ethyl acetate and a concentrated organic layer. The extract obtained was then partitioned using silica gel chromatography with hexane, ethyl acetate and methanol as mobile phases to obtain twenty-five fractions [16].

Allium Atroviolaceum
Based on the research method conducted by Sebtosheikh et al., samples were cut into small pieces and extracted by hydrodistillation using a Clevenger-a type of apparatus for 4 h. The extract obtained was then evaporated using anhydrous sodium sulfate and stored at 4°C. Then, the dry extract was analyzed using GC-MS [73,74]. In another research, dry samples were extracted by maceration with H2O and 70% ethanol for 48 h. The extract was filtered and evaporated to remove the solvent to obtain water and ethanol extract [46].

Allium atroviolaceum
Based on the research method conducted by Sebtosheikh et al., samples were cut into small pieces and extracted by hydrodistillation using a Clevenger-a type of apparatus for 4 h. The extract obtained was then evaporated using anhydrous sodium sulfate and stored at 4 • C. Then, the dry extract was analyzed using GC-MS [73,74]. In another research, dry samples were extracted by maceration with H 2 O and 70% ethanol for 48 h. The extract was filtered and evaporated to remove the solvent to obtain water and ethanol extract [46].

Antimicrobial
One of the activities found in several species Allium is as an antimicrobial. This activity serves as a source of antibiotics against microorganisms such as pathogens and microorganisms that can cause defects in food [75]. Amabye et al. reported that the antimicrobial activity of the ethanol extract of A. cepa leaves can inhibit the pathogen Streptococcus pneumoniae [40]. S. pneumoniae is a pathogen that has an important role in causing invasive diseases such as pneumonia, septicemia, meningitis and some types of eye infections [76][77][78]. An antimicrobial activity assay was carried out using the agar well diffusion with sterile dimethyl sulfoxide (DMSO) as a negative control and gentamicin as a positive control to determine the sensitivity of each bacterial species to be tested. The study showed that there was antimicrobial activity in the ethanol extract of A. cepa leaves, which managed to inhibit S. pneumoniae with an inhibition zone between 11.87-19.23 mm at 20 mg/mL and the minimum inhibitory concentrations (MIC) value at 10 mg/mL.
In other studies also using the agar cup method, A. cepa leaf extract showed good antimicrobial activity in inhibiting the growth of bacteria and fungi such as Aspergillus sp., Botrytis sp. and Penicillium sp., each with an inhibition zone of 817 sq.mm, 817 sq.mm and 377 sq.mm, respectively [79]. Antimicrobial activity was also found in other Allium species such as A. ursinum [69], A. sativum to Listeria monocytogenes [78,79] and A. atroviolaceum [46]. Krivokapic et al. reported that antimicrobial activity was also present in A. ursinum leave. The test was carried out by the microdilution plate method to determine the MIC and minimum microbial concentration (MMC) value. The result indicated that the leaf extract had antimicrobial activity inhibiting the growth of 20 bacteria and fungi [10]. Other studies also state that A. ursinum leaf extracts contain organosulfur compounds such as propylene sulfide, (E) methyl-2-propenyl disulfide and (Z) methyl-2-propenyl disulfide and several other compounds that have antimicrobial activity [72].

Antibacterial
Research conducted by Solomon et al. reported the presence of antibacterial activity in A. cepa leaves with different types of extracts, namely ethanol extract, hot extract and cold extract. The three extracts were compared to see the difference in their compound content and the efficiency of their antibacterial activity. The hot extract contains more flavonoids and saponins than the other two extracts. Hot extract also showed the best antibacterial activity among ethanol extract and cold extract in inhibiting the growth of Escherichia coli, Streptococcus and Stephylus [80].

Antifungal
Parvu et al. reported that the ethanol extract of A. ursinum leaves has antifungal activity. This study uses the agar dilution assay by determining the MIC value. Although the content of allicin compounds in the leaves is not as much as in the flowers, the antifungal activity in the ethanol extract of A. ursinum leaves is able to fight several types of fungi with the MIC value of 120 µL/mL (A. niger), 80 µL/mL (B. cinerea), 100 µL/mL (B. paeoniae), 160 µL/mL (F. oxysporum f. sp. tulipae),120 µL/mL (P. gladioli) and 80 µL/mL (S. sclerotiorum) [21].

Antioxidant
Research on antioxidant activity in Allium species has been widely reported. Antioxidant activity is found in many Allium species with different test methods. Dominguez et al. reported the antioxidant activity of A. schoenoprasum based on the total iron reduction's potential technique [62]. In several studies, the antioxidant activity test of A. schoenoprasum and A. ursinum used different methods such as DPPH radical-scavenging ability, ferric reducing antioxidant power (FRAP) assay and ABTS radical scavenging assays [17,52,54,55]. The use of some of these methods is intended to compare the results of activities between one method and another and to determine which method is more appropriate to be used in certain species. Parvu et al. did the same, which uses two methods, namely DPPH bleaching method and the trolox equivalent antioxidant capacity (TEAC) assay, where the antioxidant activity of the leaf extract was shown to be higher by the TEAC method than DPPH bleaching method [64].

Anti-Inflammatory
Inflammation is a condition in which catabolism is more dominant or faster than anabolism [81]. Inflammation can also be defined as a reaction to defend the body in eliminating factors that can cause damage and the formation of homeostasis in the body. This causes increased blood flow due to the increased permeability of capillaries and white blood cells to the site of inflammation, resulting in inflammatory symptoms such as redness, swelling and pain [82]. Several previous studies reported that the Allium species has several bioactivities, one of which is anti-inflammatory [83][84][85][86][87]. Parvu et al. reported that A. schoenoprasum leaf extract with three different concentrations (25, 50 and 100%) had anti-inflammatory activity. The research was conducted by the method of testing in vivo using a turpentine oil-induced inflammation model, while in terms of in vitro, the three extracts were able to inhibit phagocytosis by reducing nitro-oxidative stress [64].
Pan et al. reported that anti-inflammatory activity was also present in A. sativum aqueous leaf extract. The extract was previously screened for phytochemicals. The results showed that the extract contained carbohydrates, reducing sugars, lipids, flavonoids, ketones, alkaloids, steroids and triterpenes. The study used two different anti-inflammatory activity testing methods, namely carrageenan-induced paw edema and histamine-induced paw edema. Both methods showed that A. sativum aqueous leaf extract was able to lower the paw edema significantly [88]. The same activity is also found in A. fistulosum aqueous leaf extract or welsh onions. However, this study only used a carrageenan-induced paw edema method. The results showed that the aqueous extract was able to inhibit the development of paw edema by reducing the activity of the catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes found in paw edema mice by 43, 74 and 50%, respectively [87].

Antitumor
Tumors are pathological cells that can interfere with cell growth to be abnormal. Tumors, often called neoplasms, are divided into benign tumors and malignant tumors. Malignant tumors are often referred to as cancer [89]. The prevention and cure of tumors can be obtained from natural ingredients that contain compounds that have antitumor activity. A number of Allium species are known to have antitumor activity, such as A. cepa, A. sativum, A. fistulosum and A. schoenoprasum [90]. Shirshova et al. reported that the aqueous and EtOH-H 2 O extract of A. schoenoprasum leaves have antitumor activity. Several compounds such as sitosterol, stigmasterol, campesterol, cholesterol, deltonin, saponin A and mono-, di-and triacylglycerin were isolated and tested for antitumor activity in 40 male BDF mice. EtOH-H 2 O extract of A. schoenoprasum leaves was given to mice that have been divided into four groups and given previous treatment and injected with tumor strains Ehrlich carcinoma (EC). The results showed that the extract of A. schoenoprasum leaves can inhibit tumor growth. The results can be seen by comparing tumor volume and mass between the experimental group and the control group [67].

Antiplatelet
Platelets are small cell fragments that clump in the area of the injured blood vessel [91]. Platelets have an important role in hemostasis, which functions in stopping bleeding [92].
Dysfunctional or abnormal platelets can cause cardiovascular damage, such as myocardial infarctions and strokes. Several studies have developed antiplatelet drugs such as aspirin to overcome the problem of abnormal platelets [93]. Several studies have reported that some Allium species have antiplatelet activity. Saplonţai-Pop et al. reported that A. cepa bulbs have antiplatelet activity. The activity test used platelet-rich plasma (PRP), which is based on the kinetic curve of decreasing plasma OD [94], while the research conducted by Ko et al. used the platelet aggregation turbidometric assay [95]. Hiyasat et al. reported that A. ursinum leaf extract has antiplatelet activity. Testing of antiplatelet activity in vitro was conducted using light transmission aggregometry which has been induced with adenosine diphosphate (ADP), collagen, A23187, epinephrine and arachidonic acid (ARA) [17]. In other species such as A. fistulosum and A. schoenoprasum was also found the presence of antiplatelet activity using the electrical impedance aggregator method. Although the activities possessed by these two species are not as good as in other species, such as A. sativum and A. ascalonicum [65]. While A. atroviolaceum extract has excellent antiplatelet activity and is able to inhibit platelet aggregation in vitro induced by ARA and ADP with each IC 50 value of 0.4881 (0.4826-0.4937) and 0.4945 (0.4137-0.5911) mg/mL [74].

Pancreatic α-Amylase and Glucoamylase Enzyme Inhibitor
The reduction of carbohydrate digestion can be carried out by controlling the activity of hydrolysis enzymes, α-amylase and glucoamylase. Controlling this activity can affect postprandial hyperglycemia, which is considered to have a prophylactic healing effect in patients with type 2 diabetes mellitus [96]. The inhibition of hydrolytic enzyme activity is one of the right efforts to suppress carbohydrate digestion and monosaccharide absorption [97]. In human physiology, pancreatic α-amylase is a type of α-amylase which is found in plants, fungi and bacteria [98]. The amount of pancreatic α-amylase synthesized in the rough endoplasmic reticulum is regulated by the amount of food substrates [99], whereas glucoamylase is an enzyme that can be produced from a number of organisms such as Aspergillus niger and Aspergillus awamori. This enzyme plays a role in producing a certain amount of glucose [100] (Table 1).
Meshram and Khamkar succeeded in isolating oleanolic acid compounds from the chloroform fraction of A. sativum leaves using an enzyme activity inhibition test of pancreatic α-amylase and glucoamylase carried out by the Miller method [101], which was modified. The study reported that oleanolic acid showed excellent inhibition of both enzymes. The highest inhibition value occurred at a concentration of 100 µg/mL was 57.50% with IC 50 83.56 µg/mL for the glucoamylase enzyme, while it was 62.43% for pancreatic α-amylase with IC 50 55.51 µg/mL [53].

Antioxidant Properties
Antioxidants are a system to protect our bodies from osulfide, allyl methyl tetrasulfide, allyl (methylthio)methyl trisulfide, 4-mexidative stress caused by free radical and reactive oxygen species (ROS) [48]. Oxidative stress can occur due to the formation of ROS and the detoxification of increased levels of ROS in balance, causing impaired cellular function [106]. Oxidative stress due to ROS can cause several chronic diseases such as cancer, coronary heart disease and osteoporosis. Free radical reactions can attack biomolecules, especially the polyunsaturated fatty acids of cell membranes. ROS which are included as free radicals include superoxide anion (O 2 •− ), perhydroxyl radicals (HO 2 • ), hydroxyl radicals ( • OH) and nitric oxide and other species such as hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), hypochloric acid (HOCl) and peroxynitrite (ONOO − ) [107,108]. The formation of ROS starts from the uptake of O 2 , then activates NADPH oxidase and produces superoxide anion radicals and continues with the conversion of O 2 that becomes H 2 O 2 by SOD [109]. Antioxidants break the chain of free radical reactions by donating their own electrons to free radicals without becoming free radicals [106,110].
Based on their activity, antioxidants are classified into two types, which are enzymatic and non-enzymatic. Enzymatic antioxidants are antioxidants that involve several enzymes such as GPx, CAT and SOD in catalyzing free radical and ROS neutralization reactions, while non-enzymatic antioxidants can come from natural materials such as fruits, onions and others. These natural materials contain several compounds that have antioxidant activity such as flavonoids, alkaloids, carotenoids and phenolic groups [111]. Testing of antioxidant activity can be carried out by some test methods such as DPPH free radical scavenging assay, oxygen radical absorbance capacity (ORAC) assay, trolox equivalent antioxidant capacity (TEAC) assay, ferric reducing antioxidant power (FRAP) assay, cupric reducing antioxidant capacity (CUPRAC) assay, reducing power assay and other methods [112][113][114].
Antioxidant compounds can also be obtained from some Allium species such as A. fistulosum, A. ursinum, A. schoenoprasum, A. flavum, A. cepa, A. scorodoprasum, A. sativum, A. cepa and A. vineale [69,70,102]. These compounds can be isolated from all parts of the plant such as bulbs, leaves, roots, flowers and bark [109]. This study will discuss the antioxidant activity of the compounds contained in Allium species leaves.
Testing of antioxidant activity in the species A. sativum was carried out using the DPPH and FRAP assay. Some studies reported that the antioxidant activity of the A. sativum leaves is very high, with IC 50 7.21 ± 0.39 mg/mL in the DPPH assay and 7.99 mol/g in the FRAP assay [114]. The four compounds were tested for their antioxidant activity using the DPPH method, hydroxyl radical-scavenging activity and the ferric thiocyanate method [57]. Singh and Kumar also reported the presence of antioxidant activity in A. sativum leaves using the phosphomolybdenum reduction assay. The method is based on the reduction of Mo (IV) to Mo (V) in the methanol extract by formatting a green phosphate complex subsequence or Mo (V) [102].
El Hadidy et al. reported that there were three major compounds isolated from A. fistulosum leaf extract. They are myricetin, quercetin and rutin. From the three compounds, myricetin is the most abundant compound in the Giza 6 and photon varieties, among other compounds, which is 38.75%. The antioxidant activity test using the DPPH method showed that the activity decreased after three months of storage based on the percentage of antioxidants [61]. The ethanol extract of A. ursinum leaves, which also uses the DPPH radical scavenging assay, showed antioxidant activity of 77% with an EC 50 value 322 g/mL. The activity was influenced by the presence of phenolic compounds in the extract [69]. Research on antioxidant activity was also carried out on A. schoenoprasum leaves which used two methods, DPPH bleaching assay and TEAC. The results using DPPH showed weak antioxidant activity with an EC 50 value (6.72 ± 0.44 g/mg), whereas the TEAC method used to determine the total oxidant scavenging activity showed a value of 132.8 ± 23 g Trolox eq./g [64].

Structure-Antioxidant Activity Relationship Compounds in Allium
The structure-activity relationship (SAR) is an approach used to determine the relationship between the structure of a compound and its bioactivity [115]. The presence of certain substituents can affect the strength of compound activity; for example, the different number and position of a hydroxyl group will provide different antioxidant activities [116]. The following are some of the compound structures that have been isolated from Allium leaf extract: (Figure 10).  186) [34]. Li et al. reported that apigenin has low antioxidant activity, which is due to the absence of a single hydroxyl group in ring A and a single hydroxyl group in ring B with an activity value (1.5 mM) [122].

Naringenin
Naringenin 4 ,5,7-trihydroxyflavanon is a flavanone compound of the flavonoid group with a molecular weight of 272.26 (C 15 H 12 O 5 ) [124][125][126][127]. This compound had been isolated from A. fistulosum [61]. It has a saturated heterocyclic ring C with hydroxyl substituent at positions 4 ,5,7. The presence of a hydroxyl group in ring A and a single hydroxyl group in ring B affect the naringenin value in the TEAC test (1.5 ± 0.05 mM), so that its antioxidant activity is lower than that of quercetin which has two hydroxyl substituents in ring B [122,124].

Kaempferol
Kaempferol can be found in fruits and vegetables [102,115]. This compound is also easily found in some Allium species [119]. In recent years, several studies had reported the presence of kaempferol in A. fistulosum, A. ursinum, A. schoenoprasum, A. sativum and other species [40,103]. Kaempferol (3,4 ,5,7-tetrahydroxyflavone) is a yellow tetrahydroxyflavone compound that belongs to the flavonoid group with hydroxyl groups at positions 3, 4 , 5, and 7 [115,122,127]. Kaempferol has a wavelength band (367 nm) which is longer than compounds that only have three hydroxyl groups such as apigenin (337 nm). The presence of a reduction of the 2,3-unsaturated bond in ring C did not affect its antioxidant activity, whereas the presence of a single hydroxyl group in ring B, which is conjugated with a conjugated double bond, has little effect on increasing antioxidant activity [124]. Farkas et al. reported that kaempferol has antioxidant activity in inhibiting heat-induced oxidation in a β-carotene-linoleic acid-model-system (65.3%) [128]. In testing using the DPPH radical scavenging activity method, kaempferol has an IC 50 value 28.05 ± 0.28 µM and 1.3 ± 0.08 mM in tests using the TEAC (Trolox equivalent antioxidant activity) [129,130].

Catechin
Catechin (3,3 ,4 ,5,7-pentahydroxyflavan) is a compound commonly found in several types of green tea, cocoa, red grapes and onions [49,120,[131][132][133]. It also can be found and isolated from A. schoenoprasum [62]. This compound belongs to flavanol compound groups which have five hydroxyl substituents at positions 3, 3 , 4 , 5 and 7 [133]. In recent years, this compound had been reported to have antioxidant activity [125,133]. Silva et al. reported that catechin had antioxidant activity of 1.9 ± 0.1 µmol in the DPPH radical scavenging assay and 1.4 ± 0.3 µM trolox equivalents/µM flavonoids in the ORAC ROO assay. o-catechol group in ring B showed a good effect on antioxidant activity [134]. In addition, the high planarity due to the intramolecular hydrogen bonding between the 3-OH and 6 -H substituents in flavanol compounds such as catechin can also provide a good antioxidant activity [135,136].

Quercetin
Quercetin (3,5,7,3 ,4 -pentahydroxyflavon) is one of the flavonoid compounds found in plants such as onions, apples, berries and others. Its presence can be easily found in several Allium species such as A. cepa, A. sativum, A. ursinum and A. fistulosum [4,56]. This flavonol compound consists of three rings and five hydroxyl groups [123,137,138]. Several studies reported that quercetin has the ability as an antioxidant in reducing the formation of ROS [139][140][141][142][143][144][145]. The strength of antioxidant activity depends on the number of hydroxyl groups possessed, such as quercetin, which will provide stronger activity than naringenin and apigenin with three hydroxyl groups [116,117,121]. This is indicated by the value of IC 50 that is smaller and TEAC values that are larger compared to the two compounds with each IC 50 (10.89 ± 0.03; >1000; 463.40 ± 22.28 µM) and TEAC (4.7, 1.53, 1.45 mM) . At the same time, the formation of a resonance-stabilized quinone structure due to the hydroxyl group adjacent to the ring C [125]. Antioxidant activity will decrease when there is glycolation of the hydroxyl group at position 3 on ring C [121].

Conclusions
Allium species such as A. cepa, A. sativum, A. fistulosum, A. schoenoprasum, A. ursinum, A. flavum, A. scorodoprasum, A. vineale and A. atroviolaceum have a great role in the health field. Those contain secondary metabolites that have several bioactivities such as antioxidant, antimicrobial, antibacterial, antifungal, anti-inflammatory and others. Their bioactivities are influenced by certain structure and functional groups.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The study did not report any data.