Lipoxygenase Inhibition by Plant Extracts

Lipoxygenases are widespread enzymes that catalyze oxidation of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic acid) to produce hydroperoxides. Lipoxygenase reactions can be desirable, but also lipoxygenases can react in undesirable ways. Most of the products of lipoxygenase reactions are aromatic compounds that can affect food properties, especially during long-term storage. Lipoxygenase action on unsaturated fatty acids could result in off-flavor/off-odor development, causing food spoilage. In addition, lipoxygenases are present in the human body and play an important role in stimulation of inflammatory reactions. Inflammation is linked to many diseases, such as cancer, stroke, and cardiovascular and neurodegenerative diseases. This review summarized recent research on plant families and species that can inhibit lipoxygenase activity.


Introduction
Lipoxygenase (LOX) was discovered as an enzyme that catalyzes oxidation of carotene destruction, as first reported by Bohn and Haas. The enzyme was named carotene oxidase. Throughout the years, LOX has been identified in a variety of ways. From 1932 to 1940, names such as unsaturated lipid oxidase, lipoxidase, fat oxidase, and lipid oxidase were proposed [1]. In 1940, it was recognized as the same enzyme. All previously used names were consolidated and the enzyme was officially named lipoxygenase [2].
Lipoxygenases are present in the human body and play an important role in the stimulation of inflammatory reactions. Excessive amounts of reactive oxygen species can cause inflammation that stimulates a release of cytokines and subsequent activation of LOXs. Inflammation is linked to many diseases, such as cancer, stroke, and cardiovascular and neurodegenerative diseases. LOXs are involved in the synthesis of prostaglandins and leukotrienes. They are associated with disease development and their inhibition is considered as a crucial step in disease prevention [6].
Water Aerial parts 29.08 1 35.00 Roots 1 results are expressed as mg gallic acid equivalent (GAE)/mL of extract. 2 results are expressed as mg gallic acid equivalent (GAE)/g of extract.
Inhibition of 15-lipoxygenase with seed extracts of Apium graveolens and Petroselinum crispum was performed by Danciu et al. [17]. It was found that 86.15 mg of Apium graveolens and 69.45 mg of Petroselinum crispum extract is necessary to inhibit 50% of 15-lipoxygenase activity. Phytochemical compositions of obtained methanolic extracts were estimated and nine components were detected. Apigenin and apigenin glucoside were the most prevalent compounds in both extracts. Aerial parts of Bupleurum marginatum were extracted in two different solvents, methanol and dichloromethane [18]. Both extracts showed 5-lipoxygenase inhibitory activity at concentration of 25 µg/mL. Methanolic extract showed higher IC 50 value (45.28 µg/mL) than the extract obtained in dichloromethane (25.92 µg/mL). Phytochemical studies showed that B. marginatum extracts are rich in lignans, flavonoids, saikosaponins, and other terpenoids. Methanol extract possessed more flavonoids, while dichloromethane extract had more lignans and terpenoids. The potential ability of soybean lipoxygenase inhibition was also tested by the methanolic extract of Scandix pectenveneris [19]. Enzyme inhibition was measured on five different concentrations of plant extracts, of 100, 200, 300, 600, and 900 µg/mL, and obtained results showed 3. 50, 16.42, 34.62, 44.56, and 77.49% of LOX inhibition, respectively. Total phenolic content of examined extract was 377.94 mg equivalent (GAE)/g extract. It was reported that coumarin umbelliprenin found in the plant family Apiaceae strongly inhibits lipoxygenase [24]. Water extract of Pituranthos chloranthus showed strong lipoxygenase inhibition with IC 50 value of 0.02 mg/mL [20]. Total polyphenol content of the extract was 1.36 mg GAE/g. Paun et al. extracted aerial parts of Eryngium planum in 50% ethanol with water (v/v) [21]. The obtained extract was subjected to lipoxygenase inhibition testing and showed high inhibitory activity with an IC 50 value of 31.30 µg/mL. The polyphenol profile of E. planum showed a high concentration of flavonoids, especially isoquercetin (36.11 µg/mL) and rutin (290.52 µg/mL).
To increase polyphenol production, Złotek and co-workers used jasmonic acid (1, 10, 100 µM) and yeast extract (0.01, 0.1, 1%) as elicitors [22]. The best results were observed in Levisticum officinale leaves treated with 10 µM jasmonic acid, where total polyphenol content increased by 55% in comparison with the control sample. All extract showed the ability to inhibit lipoxygenase, but inhibition rate of elicited leaves did not differ significantly from the control. Similar research was conducted by Majdoub et al., who monitored the effect on 2 mM Zn on polyphenol amount in Anethum graveolens (dill), Pimpinella anisum (anise), and Foeniculum vulgare (fennel) [23]. Total polyphenol content increased in dill and anise plants, while in fennel, a decrease of phenol content was observed. Treated and non-treated samples had the ability to inhibit 5-lipoxygenase. Anise showed strong lipoxygenase inhibitory activity, with IC 50 of 0.035 and 0.015 mg/mL for Zn non-treated and treated plants, respectively. In the case of dill, no significant difference was observed in LOX inhibition before (IC 50 = 0.043 mg/mL) and after (IC 50 = 0.041 mg/mL) treatment with Zn. Fennel showed higher IC 50 value in the sample treated (IC 50 = 0.062 mg/mL) with Zn, compared to non-treated (IC 50 = 0.052 mg/mL) one.

Lipoxygenase Inhibition by Plant Extracts of Family Asteraceae
Asteraceae is the biggest family of flowering plants and counts about 1100 genera and 20,000 species. Many plants from the Asteraceae family are used as traditional medicine due to the possession of numerous phytochemical components, such as polyphenols and diterpenoids. Antifungal, antibacterial, insecticide, anti-inflammatory, and antitumor activities were investigated in the Asteraceae species [26]. Many plants from this family were assayed on the inhibitory activities against the lipoxygenase enzyme (Table 2). 1 results are expressed as mg gallic acid equivalent (GAE)/mL extract; 2 results are expressed as mg gallic acid equivalent (GAE)/g extract; 3 results are expressed as g gallic acid equivalent (GAE)/100 g dry extract.
Bhat et al. extracted leaves of Artemisia nilagirica in three different solvents (hexane, methanol, and ethanol) [27]. All extracted inhibited lipoxygenase, but the most effective was the methanolic extract, with an IC 50 value of 128 µg/mL, followed by hexane extract (196 µg/mL). Ethanolic extract showed poor inhibition of 21%. Burlec et al. extracted two plants from the Asteraceae family (Chrysanthemum indicum and Tagetes erecta), in four different organic solvents (chloroform, dichloromethane, hexane, and methanol) in order to investigate extract inhibition ability of 15-lipoxygenase [28]. The strongest enzyme inhibition of Chrysanthemum indicum was in chloroform extract with EC 50 value of 26.06 µg/mL while Tagetes erecta methanol extract showed similar inhibition (EC 50 = 25.85 µg/mL). The highest phenolic concentration was obtained in T. erecta chloroform extract, while dichloromethane extract of C. indicum showed the highest polyphenol content. Five different organic solvents (methanol, dichloromethane, ethyl acetate, n-hexane, and n-butanol) were employed in the extraction of Vernonia oligocephala roots [29]. All extracts were investigated for their lipoxygenase inhibition potential. Among all extracts, only butanol extract showed considerable LOX inhibition (IC 50 = 132.20 µg/mL). Phytochemical composition analysis of the roots showed the presence of saponins, terpenoids, flavonoids, glycosides, phenolic compounds, and steroids. Methanol extract had the highest flavonoid (97.35 mg quercetin equivalent (QE)/g of extract) and phenolic (113.11 mg GAE/g of extract) content. Yildirim et al. extracted different parts of Tanacetum cilicicum (capitula, leaves, and stem) in different solvents (ethanol, n-hexane, chloroform, and ethyl acetate) [30]. Extract of capitula in ethyl acetate showed significant LOX inhibition, with an IC 50 value of 9.44 µg/mL, which is even greater than the standard indomethacin (IC 50 = 22.39 µg/mL). Ethyl acetate was also proven to be the best choice for the leaves extraction, in respect to LOX inhibition (IC 50 = 85.20 µg/mL), while the best inhibition among stem extracts was observed for chloroform extracts (IC 50 = 112.40 µg/mL). Total polyphenol content in extracts varied 6.10-175.60 mg GAE/g of dry weight, while total flavonoid content was in the range of 6.13 to 30.48 mg QE/g of dry weight. Phenolic contents were the highest in all ethyl acetate extracts. Water extract of Artemisia vulgaris investigated by Nasr et al. had a high ability of lipoxygenase inhibition (IC 50 = 40.00 µg/mL) [20]. Total phenolic content of aqueous extract was 0.91 mg GAE/g of extract.
Ethanolic extract of Cnicus benedictus investigated by Paun et al. also showed very good ability of lipoxygenase inhibition (IC 50 = 52.70 µg/mL) [21]. Total flavonoid content in extract was 0.47 mg QE/mL and total phenolic content was 2.65 mg GAE/mL. High concentrations of phenolic acids were determined (especially sinapic acid and chlorogenic acid). Further, methanolic extract of Matricaria chamomilla showed good inhibition of lipoxygenase with EC 50 value of 166.32 mg/mL [17]. Total polyphenol content (458.10 µg GAE/mL of extract) and total flavonoids (342.40 µg/mL of extract) of extract were determined. Özek et al. investigated LOX inhibition ability of essential oils and methanolic extracts from Galatella villosa and Galatella tatarica [31]. The 5-LOX was inhibited with both extracts and essential oils. The oils proved to be better inhibitors with percentages of inhibition of 45.0 and 57.0% for G. tatarica and G. villosa, respectively. Both methanolic extracts showed poor 5-LOX inhibition of 29.0% and 19.0%, respectively. Content of total phenolic compounds did not differ significantly in the extracts. Similar research was conducted with the oil and extract of Cota fulvida and similar results were obtained [32]. The essential oil inhibited 5-LOX for 53.7% in comparison to poor inhibition of 23.9% of methanol extract.
The highest capacity of LOX inhibition was achieved with ethyl acetate extract of T. cilicicum capitula. Among all extracts, this one showed high phenolic content (174.10 mg GAE/g of extract) as well as flavonoid concentration (26.94 mg QE/g of extract).

Lipoxygenase Inhibition by Plant Extracts of Family Clusiaceae
The Clusiaceae family has approximately 400 species widespread in Africa, Asia, Polynesia, and South America. Garcinia is the most numerous genus of Clusiaceae, and its plant parts, such as fruit, leaves, stems, and flowers, are commonly used for the treatment of inflammation and oxidative stress. Some plants also possess biological activities, such as antioxidant, anti-inflammatory, and antibacterial [33]. Potential lipoxygenase inhibition was investigated for extract of Garcinia plants (Table 3). Sari and Elya performed LOX inhibition assay on the stem bark extracts of Garcinia porrecta Laness [34]. Extraction was conducted in different organic solvents (methanol, n-hexane, and ethyl acetate). LOX inhibition activity of extracts with IC 50 values were 0.23, 0.52, and 4.87 µg/mL for methanol, ethyl acetate, and n-hexane extracts, respectively. Total flavonoids of methanolic extract were equal to 5.66 mg QE/g. Extraction of Garcinia hombroniana Pierre leaves in n-hexane, methanol, and ethyl acetate was performed by Marlin and Elya [35]. It was proven that all leaf extracts are excellent LOX inhibitors with IC 50 values of 0.13, 1.31, and 2.05 µg/mL for ethyl acetate, methanol, and n-hexane extracts, respectively. Determined flavonoid content of the most active ethyl acetate extract was 42.00 mg QE/g sample. LOX inhibition potential of Garcinia lateriflora Blume leaf extracts was investigated [36]. IC 50 values of the LOX inhibition were 0.69, 0.79, and 1.32 µg/mL for methanol, ethyl acetate and n-hexane extracts, respectively. The most active extract was the methanolic one, with total flavonoid content of 6.29 mg QE/g of extract. Further, Garcinia kydia Roxburgh leaves were extracted with three previously mentioned solvents [37]. Obtained results on LOX inhibition had IC 50 values of 0.21, 0.55, and 3.57 µg/mL ethyl acetate, methanol, and n-hexane extracts, respectively. Total flavonoid content of 30.65 mg QE/g of extract was determined in the most active ethyl acetate extract.
Four mentioned Garcinia plants from the Clusiaceae family were extracted in the same solvents, and all showed strong inhibition of the LOX enzyme. It could be noticed that the best results are obtained with methanol and ethyl acetate extracts, while n-hexane extracts showed a slightly lower inhibition activity. The strongest LOX inhibitor was the ethyl acetate extract of G. hombroniana with the highest amount of total flavonoids among all investigated extracts.

Lipoxygenase Inhibition by Plant Extracts of Family Fabaceae
The Fabaceae family, also commonly known as Leguminosae, is the third largest plant family after Orchidaceae and Asteraceae, with 730 genera and more than 19,400 species. It includes shrubs, trees and herbaceous plants, which are easily recognized by their fruits (legume) and stipulated leaves. This plant family is mostly found in dry forests and in tropical rainforests [38]. Table 4 presents current knowledge on the extracts of plants from the family Fabaceae and their effect on LOX inhibition.
Souleymane et al. investigated 12-LOX inhibition by dichloromethane and ethyl acetate extracts of Erythrina senegalensis roots and stem bark [39]. Ethyl acetate extracts showed higher enzyme inhibition with IC 50 values of 4.95 and 7.21 µg/mL for the stem bark and roots, respectively. IC 50 values, concerning dichloromethane extracts, were 21.46 µg/mL for stem bark and 22.03 µg/mL for roots. Root extracts contained more phenolic acids, while stem bark contained more flavonoids, but the inhibition was found to be strongly dependent on the solvent used.

Lipoxygenase Inhibition by Plant Extracts of Family Lamiaceae
Lamiaceae family is known as a mint family of flowering plants. It contains about 236 genera and 7200 species. The planta are usually aromatic, similar to some culinary herbs (mint, basil, sage, rosemary, oregano, lavender) [46].
Ajuga reptans and Ajuga genevensis are used as traditional medicine for their diuretic, tonic, and astringent activity [48]. Their ethanolic extracts were investigated as potential LOX inhibitors. Obtained results showed that extract of Ajuga genevensis is a better 15-LOX inhibitor, but both extracts were found as weak LOX inhibitors with inhibition rates lower than 30.0% at a concentration of 5 mg/mL. A. genevensis extract had a higher phenolic and flavonoid content than A. reptans extract. Six individual phenolic components (caffeic acid, coumaric acid, rosmarinic acid, luteolin-O-glucoside, luteolin, and apigenin) were identified in both extracts. The most abundant compound was luteolin-O-glucoside in concentrations of 83.25 and 63.21 µg/g of dry weight for A. reptans and A. genevensis, respectively.
Clerodendrum leaves are used for the treatment of diseases, such as asthma and rheumatism. Phosrithong and Nuchtavorn extracted Clerodendrum disparifolium and Clerodendrum laevifolium leaves in ethanol and hexane in order to investigate their ability to inhibit 5-LOX activity [49]. Both plant extracts showed great potential in the inhibition of LOX activity. IC 50 values were more promising for ethanol extract, 24.39 and 14.12 µg/mL, while IC 50 values in hexane extract were slightly higher, 30.56 and 30.70 µg/mL for C. disparifolium and C. laevifolium, respectively (Table 5). Total phenolic and flavonoid content were determined in all extracts. Total polyphenols were in the range of 1167.20 to 3344.50 mg GAE/g of dry weight, while flavonoid amount was in the range 9.05-59.91 mg QE/g of dry extract. Ethanol extract with the highest polyphenol content also showed the strongest LOX inhibition.

Lipoxygenase Inhibition with Plant Extracts of Family Melastomataceae
Plants Memecylon talbotianum, Memecylon umbellatum, and Memecylon malabaricum belong to the family Melastomataceae. Some of the Memecylon species are traditionally used as a cure for skin problems [50].
Leaves of mentioned plants are dried and extracted in methanol in order to examine the 15-LOX inhibition [51]. Obtained IC 50 values were 29.87, 39.19, and 54.60 µg/mL for M. malabaricum, M. umbellatum, and M. talbotianum, respectively (Table 6). Estimated amount of phenolic content were 1.12, 1.08, and 1.96 g GAE/100 g dry weight for M. talbotianum, M. umbellatum, and M. malabaricum, respectively. Among the tested extracts, the one with the best LOX inhibitory activity also showed the highest polyphenol content. Afagnigni et al. extracted leaves of Dissotis multiflora from the family Melastomataceae in ethanol [52]. Inhibition of 15-LOX was examined at different concentrations of the extract. Obtained result showed that inhibition of 15-LOX was lower than 50% at the concentration of 100 µg/mL. 1 results are expressed as g gallic acid equivalent (GAE)/100 g dry weight.

Lipoxygenase Inhibition by Plant Extracts of Family Ericaceae
Gaultheria plants belong to the Ericaceae family and there is more than 1700 species of this plants. Gaultheria trichophylla has blue berries and pink to red flowers. It also known as Himalayan Snowberry. It is commonly used as a folk medicine for the treatment of arthritis. Methanolic and chloroform extracts of Gaultheria trichophylla were found to inhibit 5-LOX with 90.5% and 66.9% inhibition at 0.5 mg/mL and IC 50 values of 277.30 µg/mL and 379.50 µg/mL, respectively (Table 7). n-Hexane extract showed lower inhibition of 57.0%. The highest flavonoids (41.30 mg QE equivalent/g) and phenolic (17.50 mg GAE equivalent/g) concentrations were found in methanolic extract [53]. Leaves of Gaultheria procumbens L. were extracted in petroleum ether and chloroform [54]. Lipophilic extracts of the wintergreen leaves showed inhibitory effects on LOX with IC 50 values of 738.50 µg/mL and 899.97 µg/mL in the case of petroleum ether and chloroform extract, respectively. The performed GC-MS analysis of petroleum ether extracts revealed the presence of four major compound groups (aliphatic hydrocarbons, alcohols, carboxylic acids, and terpenoids). The docanose and octacosane were present in the highest amounts. Simple phenols and terpenoids were also present in significant percentage (methyl benzoate, ursolic acid, α-amirin, β-sitosterol, methyl salicylate, and oleanolic acid). Chloroform extract contained a lower amount of alcohol, simple aliphatic hydrocarbons, and carboxylic acid compared with petroleum ether extract. Dominant compounds in chloroform extract were ursolic acid, methyl benzoate, methyl salicylate, and oleanolic acid.
It could be noticed that all examined extracts from the family Ericaceae had poor LOX inhibition with high IC 50 values.

Lipoxygenase Inhibition by Plant Extracts of Family Menispermaceae
Plant Cyclea Barbarata belong to the plant family of Menispermaceae. Leaves of this plant contain many secondary metabolites, such as flavonoids, steroids, tannins, and saponins. It was reported that flavonoids have many benefits, such as antibacterial, antiinflammatory, anticancer, antioxidant, hepatoprotective, and antiviral properties [55,56]. Handayani et al. reported inhibition of LOX with leaf extracts of C. Barbarata [57]. Three various solvents (ethyl acetate, n-hexane, and methanol) were employed for extraction. Among the investigated extracts, only ethyl acetate extract showed significant inhibition of LOX with an IC 50 value of 0.26 µg/mL (Table 8). Total flavonoids content of ethyl acetate extracts was 21.62 mg QE/g. LOX inhibition activity of ethanolic extracts of Sphenocentrum jollyanum was investigated by Sinbad et al. [58]. S. jollyanum possesses many biological and pharmaceutical properties, including antimalarial, antioxidant, anti-inflammatory, antidiabetic, and antiangiogenic. Roots and stems were extracted and both extracts showed poor inhibition of LOX enzyme with IC 50 values of 541.00 and 426.00 µg/mL, respectively.

Lipoxygenase Inhibition by Plant Extracts of Family Rosacea
Cydonia oblonga is a plant from the Rosacea family and it is a rich source of antioxidants. Phytochemical screening revealed the presence of cardiac glycosides, alkaloids, terpenoids, tannins, flavonoids, saponins, and steroids [59]. Berkoz determined lipoxygenase inhibition and total phenolic content in the crude extract, aqueous and organic fractions (chloroform, ethyl acetate, butanol) of dry fruit of Cydonia oblonga [60]. The best LOX inhibition was achieved by methanol extract (IC 50 = 99.30 µg/mL), which showed the highest phenolic content of 367.00 mg GAE/g of extract. Other extracts also appeared to be the prominent inhibitors of LOX with IC 50 values in the range of 101.80 to 227.30 µg/mL (Table 9). Similar research was conducted by Marchelak and co-workers [61]. Prunus Spinosa flowers are known as traditional medicine for treatment of inflammation, urinary tract disorders, and cardiovascular diseases. Flowers of P. spinosa were subjected to the extraction in five different solvents. Up to 57 constituents, mostly phenolic acids, flavonoids, and proantocyanidins were identified in these extracts. Ethyl acetate extract showed the strongest inhibition of LOX with IC 50 value of 135.36 µg/mL and had high polyphenol content of 584.07 mg GAE/g of dry weight. Total flavonoid amounts of extracts were in the range of 1.88 to 490.63 mg/g of dry weight.

Lipoxygenase Inhibition by Plant Extracts of Family Sapindaceae
Paullinia pinnata is a plant from family Sapindaceae, whose phytochemical profile showed the presence of numerous secondary metabolites [52]. Different plant parts showed various activities, such as antioxidant, antibacterial, antidiarrheal, anti-inflammatory, antityphoid, and anxiolytic proprties. Ethanolic extracts (50% ethanol) of P. pinnata showed poor 15-lipoxygenase inhibitory potential.
Ferreres et al. extracted leaves and stem bark of Allophylus africanus with water [62]. Both extracts showed good LOX inhibitory activity with IC 50 values of 41.28 and 107.77 µg/mL for leaves and stem bark, respectively (Table 10). In aqueous leaf extracts, 30 flavones were identified, with the dominant ones being apigenin derivatives and luteolin derivatives. Stem bark was characterized by mono-C-glycosides-O-glycosylated and apigenin di-C-glycosides.

Lipoxygenase Inhibition by Plant Extracts of Other Plant Families
Many authors reported inhibition of lipoxygenase enzymes by plant extracts from various plant families, as listed in Table 11. Plant extracts varied from poor to excellent LOX inhibitors.
LOX inhibition of water extract of Lavatera cretica L. (Malvaceae) should also be mentioned [82]. Leaf and flower extracts showed very strong inhibition of LOX, with IC 50 values of 0.01 µg/mL. Extracts had high polyphenolic content of 254.62 and 191.38 mg GAE/g dry weight.

Conclusions
This paper gives an overview of the effect of various plant extracts of various plant families on LOX inhibition. It turned out that soybean lipoxygenases, both 5-LOX and 15-LOX (found in mammalians), were successfully inhibited by plant extracts. Ethanol extract of Cnicus benedictus showed great inhibition of 15-LOX with an IC 50 value of 52.7 µg/mL. The best inhibitor of 5-LOX was the ethanolic extract of Sophora tokinensis with an IC 50 value of 1.61 µg/mL. Soybean lipoxygenase was successfully inhibited by Lavatera cretica leaf and flower water extracts with an IC50 value of 0.01 µg/mL.
Based on the abovementioned, it becomes clear that the selection of appropriate solvents for phytochemical extractions, as well as the corresponding phytochemical profiles, play important roles in LOX inhibition. While some of the extracts show excellent inhibitory activities, others seem poorly effective in LOX inhibition. Moreover, several reports shows greater LOX inhibitory activity of isolated compounds themselves, than extracts on the whole. Therefore, it seems that there is lot of space to fill in the gaps in regards to finding the most novel, prominent LOX inhibitors among the multitude of plant phytochemicals.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.