Protective Effect of Various Extracts of Allium hirtifolium and Satureja khuzestanica Plants on AAPH-Induced Oxidative Hemolysis

Background: Allium hirtifolium and Satureja khuzestanica are the Iranian endemic plants and proper candidates for antioxidant studies. This study investigated the antioxidant, anti-hemolytic properties, and phytochemical composition in different extracts of A. hirtifolium and S. khuzestanica. Methods: Hydroalcoholic, methanolic, and n-hexane extracts of A. hirtifolium and S. khuzestanica plants were prepared using soaking and ultrasonic methods. Different plant extracts were evaluated for the presence of secondary metabolites using standard methods based on colorimetric analysis. The antioxidant properties of the compounds were measured by the ferric reducing ability of power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Finally, the anti-hemolytic effects of the extracts were investigated using the 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) methods. Results: Phytochemical analysis indicated the presence of flavonoids, phenolic acids, glycosides, steroids, and terpenoids in S. khuzestanica, as well as amino acid compounds, flavonoids, saponins, glycosides, and phenols in A. hirtifolium. FRAP assay showed that the methanolic extract of S. khuzestanica and the n-hexane extract of A. hirtifolium have the highest total antioxidant activity. The results of the DPPH assay also demonstrated that the minimum IC50 was related to the hydroalcoholic extract of S. khuzestanica (18.58 μg/mL) and the n-hexane extract of A. hirtifolium (87.95 μg/mL). In general, the extracts of both plants could reduce the percentage of AAPH-induced hemolysis while being significant only in some concentrations (P < 0.001). Conclusion: Overall, A. hirtifolium and S. khuzestanica can be used as a herbal supplement in the human diet due to their anti-hemolytic effects.

Due to phenolic and flavonoid compounds of S. khuzestanica and A. hirtifolium, it seems that they may be the proper supplement for oxidative damages. This study aimed to investigate the antioxidant and anti-hemolytic properties of different extracts of S. khuzestanica and A. hirtifolium.

Ethical Considerations
The animal experiments were approved by the Ethics

Plant Materials: Collection and Extraction Procedure
The S. khuzestanica and A. hirtifolium plants were collected from Lorestan and Hamadan provinces, Iran, respectively. These plants were identified by the herbarium section of HUMS, Hamadan, Iran. Hydroalcoholic, methanolic, and n-hexane extracts were prepared by soaking, ultrasonic, and heating methods. Briefly, 25 g of the shade-dried plants were crushed, extracted with methanol-water (50/50 v/v %), methanol, and n-hexane, and then separately shaken (72 hours at 37°C) and filtered through a Whatman No.1 filter paper. Finally, the filtered solutions were concentrated and kept at 4°C.

Phytochemical Screening
The phytochemical analyses were conducted on different extracts using the standard techniques to identify secondary metabolites such as alkaloids, flavonoids, saponins, tannins, steroids, terpenoids, proteins, amino acids, and glycosides.

DPPH Radical Scavenging Assay
Overall, 0.25 mL of the extract was mixed with 0.75 mL of the methanol solution of DPPH (0.1 mM) and incubated at room temperature for 30 minutes in darkness. Then, its optimum absorbance was recorded at 517 nm using a microplate reader (Synergy HTX, BioTek, USA). The percentage of free radical scavenging capacity (RSC) was calculated as follows: It should be noted that the IC 50 index was computed against different concentrations of ascorbic acid (vitamin C) as the standard.

Measurement of the Total Antioxidant Capacity
Ferric reducing antioxidant power (FRAP) is based on the ability of the extracts to reduce Fe (III) to Fe (II) in the presence of TPTZ. The complex from Fe (II) and TPTZ reactions has a maximum absorbance at 593 nm. The aqueous solution of known Fe II concentration was used for calibration in the range of 7-125 µg/mL. Different concentrations of the extract were added to 0.25 mL of the FRAP solution (1 volume of 20 mM FeCl3, 10 volumes of 300 mM acetate buffer, and 1 volume of 10 mM TPTZ in 40 mM HCL) and allowed to react for 40 minutes at room temperature, and eventually, the absorbance was measured at 593 nm. The results were reported as nmol Fe 2+ /mg of the sample using the ferric chloride standard curve: y = 1.6723x+ 0.1261, r2 = 0.998.

Measurement of Anti-hemolytic Activity
The blood samples were obtained from male Wistar rats and centrifuged at 3000 rpm for 10 minutes. Subsequently, 10% v/v erythrocyte suspension was prepared by red blood cell (RBCs) pellets in phosphate buffer (pH = 7.4). The 0.1% Triton X-100 solution was used for complete hemolysis, and phosphate buffer and vitamin C were considered as negative and positive controls, respectively. For the assessment of the hemolysis ability of S. khuzestanica and A. hirtifolium, RBC suspension was incubated with different concentrations of the extracts and AAPH (50 mM) at 37°C for 6 hours. The optimum absorbance was measured at 540 nm. Ultimately, the hemolysis percentage was calculated as follows:

Statistical Analysis
Data were presented as the mean ± SD and analyzed using SPSS software (version 16) by the analysis of variance (ANOVA) and Tukey's post hoc test. The value of P < 0.05 was considered to be statistically significant.

Phytochemical Screening
Based on the results (Table 1), amino acids, flavonoids, saponins, glycosides, and phenols were found in the A. hirtifolium extract. Further, flavonoids, phenolic acid, glycosides, steroids, and terpenoids were observed in the S. khuzestanica extract. These results showed that the hydroalcoholic extract of S. khuzestanica and A. hirtifolium has more phenolic, flavonoid, and terpenoid compounds compared to other extracts.

Measurement of Total Antioxidant Capacity
As shown in Figure 1, the n-hexane extract of A. hirtifolium has the most antioxidant power while its hydroalcoholic extract has the lowest value compared to other extracts. Based on these results, the antioxidant potential of the n-hexane extract of A. hirtifolium is 32.6% and 61.5% higher than that of methanolic and hydroalcoholic extracts respectively. In addition, the methanolic and n-hexane extracts of S. khuzestanica demonstrated the highest and lowest antioxidant activity, respectively.

DPPH Radical Scavenging Assay
The IC 50 index for methanolic, hexane, and hydroalcoholic extracts of S. khuzestanica was determined as 22.674, 89.70, and 19.537, respectively. Furthermore, this index was calculated 279.95, 100.7857, and 114.35 for the methanolic, hexane, and hydroalcoholic extracts of A. hirtifolium, respectively. The reported IC 50 levels for hydroalcoholic and methanolic extracts were 27.53% and 15.86% less than vitamin C, respectively. In contrast, the amount of the IC 50 for the n-hexane extract of S. khuzestanica was 330.63% higher than vitamin C ( Table 2).

Measurement of Anti-hemolytic Activity
AAPH (Figure 2) could increase RBC hemolysis compared to the negative control group (PBS, P < 0.01). On the other hand, vitamin C (100 μg/mL) could significantly reduce the AAPH hemolytic effects (75.78%). The minimum and maximum anti-hemolytic effects of hydroalcoholic, methanolic, and n-hexane of the S. Khuzestanica extract were estimated to be 54.4-69.8, 48.4-74.7, and 29.2-58.9%, respectively. Regarding the minimum applied concentration for each extract, it seems that the antihemolytic activity of the hydroalcoholic extract is higher   than that of the other two extracts. On the other hand, the anti-hemolytic activity of the A. hirtifolium hydroalcoholic extract in the concentration range of 62.5-1000 µg/mL was estimated to be equivalent to 163-216.2% of vitamin C.

Discussion
In biological systems, the production of free radicals and ROS is unavoidable, and the body can partially neutralize their harmful effects through intrinsic antioxidant defense mechanisms. Antioxidants protect tissues against the destructive effects of free radicals and can inhibit or delay the oxidation process. These compounds are divided into synthetic and natural categories (23). Among these, natural antioxidants are often secondary metabolites such as flavonoid and phenolic acid compounds, which are responsible for plant sustenance under adverse environments (24). In this study, the phytochemical screening of secondary  metabolites proved the presence of flavonoids, phenolic acids, glycosides, steroids, and terpenoids in various extracts of S. khuzestanica (more properly in the methanolic extract), which is in line with previous studies (9,25). Previously, Golparvar et al (9) reported that the major components of the S. khuzestanica essence were carvacrol (69.62%), γ-terpinene (9.25%), and p-cymene (8.36%). Likewise, Mahboubi et al indicated that the S. khuzestanica essential oil has more phenolic content while the ethanol extract had higher flavonoid contents (25). Additionally, the presence of amino acids, flavonoids, saponins, glycosides, and phenols was confirmed in different extracts of A. hirtifolium, especially in the hydroalcoholic extract (26). However, it was shown that the collected A. hirtifolium species from different regions of Iran have different total phenolic contents, and the highest amount belongs to the Sahneh region (8.4 mg GAE/g sample). It seems that the phytochemical properties of these plants may be related to the geographical location (27). In this study, two different biochemical assays, including FRAP and DPPH experiments, were used to determine the antioxidant properties of the extracts. Our DPPH findings revealed that the hydroalcoholic extract of the S. khuzestanica and n-hexane of A. hirtifolium has the lowest IC 50 (18.58 and 93.47 μg/mL, respectively), indicating their remarkable antioxidant properties. In addition, our result indicated that the lowest anti-radical property is related to the hydroalcoholic extract of A. hirtifolium and the n-hexane extract of S. khuzestanica (IC 50 ; 270.33 and 85.03 μg/mL, respectively). Additionally, the n-hexane extract of A. hirtifolium and the hydroalcoholic extract of S. khuzestanica had the highest total antioxidant capacity, which may be related to their phenolic and polyphenolic constituents. In this regard, Mahboubi et al (28) showed that the methanolic extract of S. khuzestanica has the highest antioxidant activity with the lowest IC 50 (40 μg/ mL) for its aqueous extract (80 μg/mL) and the essential oil (95 μg/mL). This discrepancy could be related to the accumulation of secondary metabolites such as phenol and flavonoid in this plant. Phenolic compounds are good electron donors via their hydroxyl groups and exhibit free radical inhibition (29). Therefore, due to the existence of phenol and flavonoids in the hydroalcoholic and methanolic extracts of S. khuzestanica and the hydroalcoholic extracts of A. hirtifolium, their antioxidant properties are extremely higher compared to the n-hexane extract. Previously, the antioxidant activity of the methanolic extract of the Sahneh (Iran) population of A. hirtifolium was reported to be higher in comparison with other similar species (IC 50 : 60.9 ± 4.7 mg/mL). However, A. hirtifolium belonging to Nahavand (Iran) had the lowest level of antioxidant activity (IC 50 267.2 ± 21 mg/mL), which was determined by DPPH and FRAP. In this respect, Pirbalouti et al (30) reported that the ethanolic extract of A. hirtifolium from Dasht-e Laleh areas has the highest phenolic compounds and thus the highest antioxidant activity compared to the population of Khaki and Samsami (IC 50 : 1.90 ± 0.31 mg/ mL). The different antioxidant activity of S. khuzestanica and A. hirtifolium may be related to different kinds of extraction and their methods, and/or their geographical locations.
AAPH, as a water-soluble radical, decreases the level of glutathione. Glutathione depletion can induce the oxidation of cellular membranes and subsequently, the hemolysis of the cells. The pretreatment of RBCs with antioxidant compounds can reduce these destructive effects. Therefore, this in-vitro model can be considered as a suitable laboratory method for evaluating the ability of new protective compounds through free radical scavenging. In this study, S. khuzestanica and A. hirtifolium extracts can reduce hemolysis caused by AAPH. The protective effect of these plants may be due to phenolic and flavonoid components such as kaempferol and carvacrol which is moderate AAPH-induced intracellular glutathione depletion (31)(32)(33). It was previously shown that flavonoids have free radical scavenger and antioxidant properties (34). Flavonoids as polyphenolic compounds have anti-hemolytic properties and prevent the lysis of RBCs in a dose-dependent manner (35). For instance, Cacciatore et al concluded that kaempferol, as a flavonoid of allium species, can inhibit RBC hemolysis (26%) at the dose of 10 µg/mL (36).

Conclusion
Generally, the S. khuzestanica and A. hirtifolium extract could prevent AAPH-induced oxidative hemolysis. This feature could be due to the phenol and flavonoid content of these plants and their inhibitory effects on free radical generation. Therefore, our findings can offer a new application for S. khuzestanica and A. hirtifolium plants as herbal supplements in the human diet.