In vitro antioxidant activity of Lippia adoensis Var. Koseret, Thymus schimperi Ronniger and Rosmarinus officinalis Leaf extracts and their effects on oxidative stability of ground raw beef meat during refrigeration storage

Rosmarinus officinalis,Thymus schimperi Ronniger, and Lippia adoensis var. koseret, locally known as Rosemary , Tosign , and Koseret respectively are widely used dietary herbs in Ethiopia. In this study, the antioxidant activity and effect of Rosemary , Tosign , and Koseret ethanol (80% v/v) extract on lipid oxidation of ground beef during storage were studied. Folin - Ciocalteu and aluminium chloride were used to determine total phenolic content (TPC) and total flavonoid content (TFC), respectively. Antioxidant activities of herbs were evaluated using 2, 2 - diphenyl - 1 - picrylhydrazyl (DPPH) scavenging, ferric reducing power and iron chelating activities. The peroxide and thiobarbituric acid reactive substances assays were used to evaluate the ability of the extracts to prevent lipid peroxidation in ground beef stored at 4°C. Tosign extract had highest TPC (70.93±1.53 mg GAE/g) and TFC (16.94±0.12 mg CE/g). Also, Tosign had the strongest effect of DPPH scavenging (IC 50 = 33.35±1.56µg/mL) and ferric reducing power (IC 50 = 175.71±1.03 µg/mL). But the strongest iron - chelating activity was observed in Rosemary extract (IC 50 = 160.24±2.55 µg/mL). Minimum peroxide value (0.185±0.09 milli equivalent peroxide/kg of beef) and the highest thiobarbituric acid reactive substances (80.20±20%) were recorded for ground beef treated with Rosemary at the end of storage days. In conclusion, these herbs are very effective antioxidants comparable to butylated hydroxytoluene. Thus, they could be a good substitution to synthetic antioxidants used in food preservation.


Introduction
Meat and meat products play an important role in providing energy, high-quality, readily digestible protein with all essential amino acids and other absorbable micronutrients which are needed for human growth, cell functioning and sound health (De Smet, 2012;Mourouti et al., 2015). It is also a rich source of lipids which are important for the flavour and aroma profile of meats and contribute to tenderness and juiciness. Lipid oxidation is one of the primary causes of deterioration in the quality of meat during storage, leading to the development of off -flavour, as well as reduced nutritional quality, shelf-life stability and acceptability. It is accelerated by the processes of grinding, chopping and cooking (Amaral et al., 2018) Synthetic and natural antioxidants have been successfully used to block or delay the oxidation process in meat. In addition to their ability to increase lipid stability, antioxidants added to foods may have the ability to reduce the risk of various diseases related to the production of free radicals (Diplock, 1994). However, the use of synthetic antioxidants like butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butyl hydroquinone (TBHQ) has been restricted because of possible health risks, toxicity and carcinogenic (Naveena et al., 2008). In this regard, there is an increasing interest in the identification and development of natural antioxidants from herbs and spices such as rosemary, oregano, broccoli, sage, black cumin, thyme or turmeric, as well as vegetables and fruits such as olives, pomegranate, grapes and berries (Naveena et al., 2008;Shahidi and Ambigaipalan, 2015).
Lippia adoensis var. Koseret, locally known as Koseret and Thymus schimperi Ronniger known as 'Tosign' are endemic aromatic herbs to Ethiopia commonly used by Ethiopian people for flavoring and eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER preserving food and also as traditional medicine (Aziz and Karboune, 2018). The dried leaves of L. adoensis var. Koseret are used as one of the ingredients in the preparation of traditional spiced butter. The special taste and flavor of the Gurage kitfo (minced meat with spiced butter) are attributed to essential oil imparted by the leaves (Nigist and Sebsebe, 2009;Engeda et al., 2019). The dried leaves of T. shimperi have been used to flavor tea, coffee, food and also boiled as a tea substitute and are believed to be good for diabetic patients (Nigist and Sebsebe, 2009). Rosemary is a popular dietary herb and evergreen plant belonging to the Lamiaceae family, and its extracts have a potential antioxidant activity (Mena et al., 2016). The antioxidant activity of Rosemary extracts has been associated with the presence of several phenolic diterpenes such as carnosic acid, carnosol, rosmanol, rosmariquinone and rosmaridiphenol (Borrás-Linares et al., 2014). According to Karre et al. (2013), it is widely used as a preservative in meat and poultry products and also has antimicrobial and antifungal effects. Different authors also reported on Rosemary extracts for the prevention of autoxidation in sunflower oil (Chen et al., 2014),pork-based products (Lara et al., 2011) and other types of foods such as sausages (Georgantelis et al., 2007) and chicken nuggets (Teruel et al., 2015). Oral toxicity of hydro-alcoholic extract of leaves R. officinalis was found to be safe at the highest dose level of 1000 mg/kg for 28 days of oral administration (Salokhe et al., 2020). Similarly, the research work by Destaw et al.
(2017) on T. schimperi leaf extracts in mice showed no significant signs of toxicity at doses of 100 mg and 200 mg/ kg body.
Many studies have been conducted on bioactive phytochemicals and their effects on human health. In particular, researchers have focused on food preservative activities of naturally occurring antioxidants. However, there are limited research efforts so far conducted to explore the evaluation of the antioxidant activity of selected endemic spices and herbs on the lipid stability of meat and meat products in Ethiopia (Engeda et al., 2015). Therefore, the main objective of the current study was to compare the potential role of natural antioxidants such as Rosemary, Tosign and Koseret on delaying lipid oxidations of ground raw beef meat.

Sample preparation and extraction
Rosemary and Koseret were collected from the Ethiopian Institute of Agriculture, Wondo Genet Research Center, in the wintertime. Whereas Tosign, was collected from the highland of Bale National Park, Bale zone, South East Ethiopia. Approximately 10 g of powdered dried herbs were mixed into 100 mL of 80% (v/v) ethanol for 8 hrs in an enclosed beaker with constant shaking (Odey et al., 2012). Thereafter, each extract was filtered and concentrated in a rotary evaporator (Buchi, 3000 series, Switzerland) under vacuum at 40°C. The extracts were sealed in a polyethylene bag container and stored at 4°C until further investigation.

Beef meat treatments
The beef was collected from Hawassa City abattoirs and transported to the laboratory using ice bags. The beef was free of bones, cartilage, exposed lymph glands, heavy connective tissue and the tendon's ends of shanks. It was cut into small pieces to grind with a diameter of 0.2 mm size by an electrical grinder (model of NIMA.NM-786). Ground beef was divided into five batches. Rosemary, Tosign and Koseret extracts (50 mL,1 mg/ mL) were added to three batches of ground beef (300 g), and the remaining batches, in the absence of the extract and in the presence of BHT, were used as negative and positive controls, respectively. Then samples were mixed and packed in low-density permeable polyethylene bags (Lloha and Mara, 2013). Then, they were stored in the refrigerator at 4 o C for fifteen days.

Total phenolic content
The total phenolic content of extracts was determined using the Folin-Ciocalteau reagent as described by Engeda et al. (2015) with slight modification. For 0.1 mL of the extracts (1 mg/mL), 1 mL of diluted Folin-Ciocalteu reagent (1:10) was added and allowed to stand for 5 mins. Then, 1 mL of sodium carbonate was added and incubated for 90 min at room temperature. The absorbance of the resulting blue color was measured at 765 nm with a UV-visible spectrophotometer (JENWAY, 96500, UK). The total phenolic content was estimated from gallic acid (1-100 µg/mL) calibration curve y = 0.015x + 0.081, R 2 = 0.991 (p < 0.001) and the results were expressed as milligram of gallic acid equivalent per gram of dried extract (mg GAE/g) using the equation, C = [ (c × V)/m].
Where C = total phenolic contents (mg GAE/g dried extract), c = concentration established from gallic acid calibration curve (µg/mL), V = volume of extract in eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER milliliter, m = the weight of dried extract in gram.

Total flavonoid content
The total flavonoid content of the extracts was determined according to Ayoola et al. (2008) with slight modification. The extracts (1 mL, 1 mg/mL) were diluted with 1.25 mL distilled water and 75 μL of 5% NaNO 2 was added. After 5 mins, 150 μL of 10% AlCl 3 was added. After 6 mins, 1 mL NaOH was added. Then immediately, the absorbance was measured at 510 nm. The TFC was determined using a standard curve (y = 0.024x + 0.112, R 2 = 0.99) of catechin and values were expressed as milligram of catechin equivalent per gram of dried extract (mg CE/g), using the equation Where C = total flavonoid contents (mg CE/g), c = concentration established from the catehin calibration curve (µg/mL), V = volume of dried extract in milliliter, m = the weight of dried extract in gram.

Antioxidant activities
2.6.1 DPPH scavenging activity DPPH free radical scavenging activity was determined as described by Jundi et al. (2021). The absorbance rates of the DPPH in the presence of herb extracts and butylated hydroxytoluene (BHT) as reference standards were measured at 520 nm. The ability to scavenge the DPPH radical was calculated as: Where Abs is the absorbance of the sample and Abscontrol is the absorbance of DPPH in the absence of the sample extract. The scavenging activity of the extracts was expressed as IC 50 . The IC 50 value was defined as the concentration (in μg/mL) of extracts that scavenges the DPPH radical by 50%.

Ferrous chelating activity
The ferrous chelating activity was determined using Ebrahimzadeh et al. (2008) . method with slight modification. The absorbance of the solution was measured at 562 nm, and ethylenediamine tetraactic acetic acid (EDTA) was used as a control. The inhibition percentage of ferrozine-Fe 2+ complex formation was calculated by using the formula: Where Abs sample is the absorbance ferrozine-Fe 2+ complex in the presence of sample extract and Abs control is the absorbance of ferrozine-Fe 2+ complex in the absence of sample extract.

Ferric reducing power
The presence of antioxidants in the extract causes the reduction of the yellow ferric/ferricyanide complex to the ferrous form, which can be monitored by measuring the formation of Perl's Prussian blue at 700 nm was determined according to the method reported by Amarowicz et al. (2004) with some modification. Different concentrations of solution of 1 mL (100 mg/ mL) in methanol were mixed with 2.5 mL potassium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. Then the mixture was incubated at 50°C for 20 min. Then 2.5 mL trichloroacetic acid of 10% was added to the mixture. Finally, 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL FeCl 3 (0.1%). Absorbance was measured to determine the amount of ferrocyanide (Perl's Prussian blue) at 700 nm against methanol as a blank using a double beam visible spectrophotometer. Higher absorbance (A 700 ) represents a stronger reducing power. IC 50 values (µg/mL) were calculated by plotting the absorbance against the corresponding sample concentration, representing the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid was used as a reference compound.

Peroxide value
The peroxide value (POV) of beef was determined by the AOAC (1999). The beef sample (3 g) was weighed in a 250 mL glass stopped Erlenmeyer flask and heated in a water bath at 60°C for 3 mins to melt the fat, then thoroughly agitated for 3 mins with 30 mL acetic acidchloroform solution (3:2 v/v) to dissolve the fat. The sample was filtered using filter paper to remove meat particles. A saturated potassium iodide solution (0.5 mL) was added to the filtrate, which was transferred into the flask. Approximately 0.5 mL of 1% starch solution (indicator) was added, and titration was run against a standard solution of sodium thiosulfate (N = 0.01) until the blue color just disappeared. Then Peroxide value (POV) was calculated by using the following equation: Where S is the volume of titration (mL), N is the normality of sodium thiosulfate solution, and W is the sample weight (g)

Thiobarbituric acid reactive substances assay
The Thiobarbituric acid reactive substances (TBARS) value was determined according to Jayathilakan et al. (2007)  Where A o is the absorbance of the control and A 1 is the absorbance of the sample extracts.

Statistical analysis
The data were subjected to ANOVA (analysis of variance) by using SAS version 9.2. The IC 50 value was determined by using origin 8. Mean separation was conducted using Duncan's multiple range tests at p < 0.05. The data were analyzed in triplicate, and the results are expressed as the mean values ± standard deviation (SD).

Total phenolic and flavonoid contents
The total phenolic content of the three ethanol extracts varied from 43.99 to 70.93 mg GAE/g (Table 1). Tosign extract had significantly (p < 0.05) the highest total phenolic content compared to Rosemary and Koseret extract. TPC of Tosign obtained from this study was higher than that of the sample collected from the central highlands of Ethiopia (Engeda et al., 2015). Similarly, the Tosign extract was the richest source of TFC (p < 0.05) and decreased in the order of Tosign > Rosemary > Koseret (Table 1). The TPC and TFC of the present study were lower than that of Tosign and Koseret reported by Engeda et al. (2015) also, the TPC of rosemary was lower than that reported by Turan. (2014). This variation may be because of geographical location or soil type (Oney-Montalvo et al., 2020).

DPPH scavenging activity
The DPPH scavenging activities of Rosemary, Tosign and Koseret leave extracts are shown in Figure 1. As the concentration of the sample increased, the percentage of inhibition of DPPH radical also increased (Labiad et al., 2017). The DPPH scavenging activity was concentration-dependent ( Figure 1). The IC 50 values of all the extracts were calculated from plotted graph of percentage scavenging activity against the concentration of the extracts ( Table 2). The lower the IC 50 value, the higher the scavenging potential. Among all extracts, Tosign extracts exhibited the strongest DPPH radical scavenging activity with IC 50 of 33.33 μg/mL followed by Rosemary (47.16 μg/mL) and Koseret (68.06 μg/mL) ( Table 2). The IC 50 values of Koseret and Rosemary were significantly different (p < 0.05). Also, these values were significantly weaker (p < 0.05) than that of Tosign extract. Whereas, Tosign has no significant scavenging difference (p > 0.05) with ascorbic acid. The DPPH scavenging activities of Tosign and Koseret in this study were weaker than that of samples collected from the central highland of Ethiopia (Engeda et al., 2015;Engeda et al., 2020). This difference may be because of geographical location, soil type and season of harvesting ( Nateqi and Mirghazanfari, 2018

Ferric reducing power
Fe (III) reducing the power of a compound is related to its ability to transfer electrons and serves as a useful indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant reaction (Rohman et al., 2010). The presence of antioxidants in the spice and herbal extracts causes the reduction of the Fe 3+/ ferricyanide complex to the ferrous form. Therefore, the concentration of Fe 2+ was monitored by measuring the formation of Perl's Prussian blue at 700 nm (Amarowicz et al., 2004). A higher absorbance value indicates a higher reduction capacity. The ferric reducing power of Rosemary, Tosign, and Koseret ethanol extracts and ascorbic shown are shown in Figure 2. Similar to DPPH scavenging activity, the ferric reducing power increased with increasing the concentration of the extracts (Hailemariam, 2013) reported that the reductive ability of Tosign extract has been found that the Fe 3+ to Fe 2+ transformation occurred mainly in the presence of phenolic compounds in the extract. In addition, Engeda et al. (2015) reported that the methanolic extract of Tosign had the strongest ferric reducing power, which has been correlated with the amount of total phenolic and flavonoid contents.
The IC 50 value of ferric reducing power was calculated from the absorbance against the concentration of the extract (Table 2). Similar to DPPH scavenging, Tosign extract had significantly (p < 0.05) the lowest IC 50 value (strongest ferric reducing power) compared to Koseret and Rosemary extracts. Ascorbic acid showed a stronger ferric reducing power activity (the smallest IC 50 ) than that of Rosemary, Tosign, and Koseret extracts. No significant difference (p > 0.05) was observed in ferric reducing power between Koseret and Rosemary extracts, but these values showed significantly weaker reducing power (p < 0.05) than that of Tosign extract. This difference might be the amount and type of phenolic compounds present in the extracts (Ayoola et al., 2008;Jemal et al., 2011).

Iron chelating activity
Transition metals have been proposed to be the catalysts for the initial formation of radicals. Chelating agents may stabilize transition metals in the living systems and inhibit radical generations, consequently reducing free radical damage. Metal chelating agents may have a dramatic effect on increasing the oxidation stability by blocking the pro-oxidant metal ions, and thus limiting the formation of chain initiators by preventing metal-assisted homolysis of hydroperoxides in lipid peroxidation (Praveen et al., 2012).
To better estimate the potential antioxidative properties of the extracts, the chelating activity of each extract was evaluated against Fe 2+ . Ferrozine can quantitatively form complexes with Fe 2+ . In the presence of chelating agents, the complex formation is disrupted, resulting in a decrease in the red colour of the complex. Measurement of the colour intensity reduction at 562 nm wavelength allows estimation of the metal chelating activity of the chelators (Yamaguchi et al., 2000). In this assay, both the extracts and reference control (EDTA) were assessed for their ability to compete with ferrozine for Fe 2+ in the solution. The percentage of iron-chelating activities of all extracts and references was concentration -dependent (from 100 to 1000 μg/mL) (Figure 3).
The IC 50 values of ferrous ion chelating were calculated from a graph of iron chelating activity against the concentration of the extracts ( Table 2). The lower the IC 50 value, the higher the antioxidant activity. The IC 50 values Rosemary and Tosign extract had no significant (p > 0.05) difference, but these values showed stronger iron -chelating scavenging activity (lower IC 50 value) than that of the Koseret extracts. This could be in the presence of more chelating agents present in Rosemary extract, and the complex formation is disrupted, resulting in a decrease in the red colour of the complex. EDTA was an excellent chelator for ferrous ions, and its chelating capacity (IC 50 = 51.17±2.59 mg/mL) was much stronger than that of the ethanol extracts of Rosemary, Tosign, and Koseret (p < 0.05). Engida et al. (2015) reported methanol extract of Tosign collected from the central highlands of Ethiopia showed weaker iron-chelating activity than that of the present study. This may be the solvent type used and the geographical location and season of harvesting. The Rosemary extract of the present study showed stronger ferrous chelating activity than that of samples collected from different countries (Nateqi and Mirghazanfari, 2018;Oney-Montalvo, 2020).

Peroxide value
The peroxide values of Rosemary, Tosign and Koseret extract-treated beef samples showed lower initial peroxide values and a lower range of increase with time compared to the control (Figure 4). A sharp increase in the level of peroxide was observed in the control sample after 3 days of storage, and its rate was exponentially increasing up to day 12 storage at 4 o C. It could be due to the presence of pro-oxidants which speeded the autooxidation process and yielded higher concentrations of oxidation product (Mohd et al., 2008). Among the antioxidants used, the beef samples treated with BHT (commercial synthetic antioxidant) showed lower initial peroxide values and lower increase ranges compared with other herbal additives. Compared with the control, the extract samples and reference (BHT) showed a lower increase in peroxidation levels over 15 days of testing. On day 12 of testing, the inhibition percentages of BHT,Rosemary,Tosign,Koseret,and control were 0.169±0.191,0.179±0.191,0.247±0.061,0.280±0.236,0.55±0.28 meq peroxide/Kg of beef, respectively. The increment of peroxide value of all treatments and control throughout the storage period of 15-day storage was due to the autocatalytic nature of the lipid oxidation reaction. In general, these results indicated that Rosemary extract was more effective in reducing the formation of peroxides in beef during storage at 4 o C than that of Koseret and Tosign extracts.

Thiobarbituric acid reactive substances assay
During lipid peroxidation, lipid peroxides are formed, with a subsequent formation of peroxyl radicals, followed by a decomposition phase to yield the aldehydes such as hexanal, malondialdehyde, and 4hydroxynonenal. This assay is based on the detection of a deep orange colour developed during the heating at 85 o C, which is formed between the reaction of malondialdehyde and thiobarbituric acid (TBA) at a late stage of lipid oxidation in the aqueous phase.
A comparison of secondary products of lipid peroxidation measured as a percentage of inhibition of TBARS was shown in Table 3. As the day of storage increased, the inhibition of the formation of malondialdehyde in control samples decreased, and the lowest inhibition was observed on day 15. Similarly, the inhibition potential of all samples and BHT decreased with increasing days of storage. Beef treated with the Rosemary extract was more effective than that of Koseret and Tosign extracts in reducing lipid oxidation by  lowering the level of malondialdehyde value. At the end of storage the percentage of inhibition of TBARS was decreased in the order BHT (83.81±5.50%) > Rosemary (80.20±2.3%) > Tosign (75.33±3.20%) > Koseret > control (46.65±1.90%) respectively. Therefore, the antioxidant activity of Rosemary extract is considered to be stronger in the inhibition of lipid oxidation than the rest extracts. Tosign and Koseret extracts showed weaker antioxidant activity (p < 0.05) than that of BHT, while the Rosemary extract showed similar activity with BHT (p < 0.05). This might be because phenolic compounds are considered to be one of the quick inhibitors of the lipid peroxidation process, abstracting hydrogen atoms from unsaturated fatty acids (Jayathilakan et al., 2007).

Conclusion
According to the results of this study, the antioxidant activities of the tested extracts were closely associated with their total phenolic contents. Based on measured results, it might be concluded that the addition of Koseret, Tosign, and Rosemary extracts exhibited inhibitory potential on lipid oxidation of beef meat during storage. However, further studies are needed to evaluate the potential of various extracting solvents, a mixture of solvents and individual bioactive compounds. Furthermore, the research revealed the bioactive compounds present in the crude extracts of these dietary herbs have the potential to be used as possible natural substitutes for controversial synthetic antioxidants currently used in food preservation.