Variation in Phenolic Profile, Antioxidant, and Anti-Inflammatory Activities of Salvadora oleoides Decene. and Salvadora persica L. Fruits and Aerial Part Extracts

(1) Background: The objective of this study was to investigate the potential of Salvadora oleoides (S. oleoides) and Salvadora persica (S. persica) polyphenols as antioxidant and anti-inflammatory agents. (2) Methods: Aerial parts and fruits of S. oleoides and S. persica were collected from the periphery of District Bhakkar, Punjab, Pakistan. Methanol extracts were prepared using the Soxhlet extraction technique. Extract yield varied from 8.15 to 19.6 g/100 g dry plant material. RP-HPLC revealed the detection of thirteen phenolic aids and five flavonoids. Gallic acid, hydroxy benzoic acid, chlorogenic acid, and cinamic acid were the major phenolic acids, whereas catechin, rutin, and myricetin were the flavonoids detected. (3) Results: Maximum total phenolic contents (TPCs) (22.2 mg/g of dry plant material) and total flavonoid contents (TFCs) (6.17 mg/g of dry plant material) were found in the fruit extract of S. persica, and the minimum TPC (11.9 mg/g) and TFC (1.72 mg/g) were found in the aerial part of S. oleoides. The fruit extract of S. persica showed the highest DPPH radical scavenging activity. In vivo anti-inflammatory activity of all the extracts was performed on albumin-induced rat paw edema that was comparable with the standard indomethacin; S. persica fruit extract showed remarkable anti-inflammatory activity. Analgesic activity of aerial part and fruit extracts of S. oleoides and S. persica was investigated using a mouse model, and the results showed that maximum possible analgesia of fruit extracts of S. persica was 53.44%, which is better than the PC group (52.98%). (4) Conclusions: The variations in the antioxidant, anti-inflammatory, and analgesic activities of methanolic extracts of S. oleoides and S. persica were found to be significant, and they have therapeutic potential as antioxidant, analgesic, and anti-inflammatory agents.


Introduction
Reactive oxygen species produced during different metabolic activities cause oxidative damages that are responsible for many diseases including, Parkinson's and Alzheimer's diseases, tumor formation, heart diseases, nervous disorders, pulmonary disorders, rheumatic premature aging, and inflammatory diseases [1,2]. Inflammation has multiple actions in the process of growth and differentiation, as well as in lymphoid and non-lymphoid cells, and purchased from Merck (Darmstadt, Germany). All chemicals used, including the solvents, were of analytical grade and used without further purification.

Preparation of Plant Extracts
Plant materials were dried in the shade and then powdered (80 mesh) using an electric grinder (BL 999SP, LG, Frankfurt, Germany). Extractions were carried out using the 500 mL Soxhlet extractor with absolute methanol, as reported previously [20]. Briefly, 50 g of powdered material was taken in a thimble and extracted with 300 mL of absolute methanol for 8 h on Soxhlet extraction apparatus. The extracts were then concentrated using a rotary evaporator (Eyela, SB-651, Rikakikai Co., Ltd., Tokyo, Japan) under reduced pressure and stored in a refrigerator at 4 • C for further studies. The percentage yield of each extract was determined using the formula given below.

Qualitative and Quantitative Analysis of Phenolic Acids and Flavonoids Simultaneously
Standard stock solutions of all the phenolic acid and flavonoids available were prepared fresh by dissolving the compounds in methanol (10 mg/mL). Working solutions (0.2-1.0 mg/mL) were prepared and standard curves were constructed by plotting concentrations against peak areas. The extracts were prepared as reported previously and filtered through a 0.45 µm non-pyrogenic filter (Minisart, Satorius Stedim Biotech GmbH, Goettingen, Germany) prior to injection [7].
The HPLC analysis was performed with the Perkin Elmer system (Perkin Elmer, Japan) equipped with gradient model binary pump systems, and a UV/Visible detector. The injection mode was manual, and the degasser (DGU-20A5) system was intact. The column oven was installed and equipped with hypersil GOLD C18 column (250 × 4.6 mm internal diameter, 5 mm particle size) (Thermo Fischer Scientific Inc., Waltham, MA, USA) supported with a guard column and a non-linear gradient consisting of solvent A (acetonitrile: methanol, 70:30) and solvent B (water with 0.5% glacial acetic acid). The quantification was based on an external standard method, whereas the analytes were identified by matching the retention times and spiking the samples with the standard.

Total Phenolic and Total Flavonoid Contents
Total phenolic contents (TPCs) and total flavonoid contents (TFCs) of the prepared extracts were measured according to the Folin-Ciocalteu phenol reagent and aluminum chloride colorimetric assays, respectively, as reported by Hussain et al. [7]; several different dilutions (10-80 ppm) of gallic acid were prepared to create a calibration curve (Y = 0.0265X − 0.1834). Similarly, different dilutions of catechin (10-160 ppm) were prepared and the calibration curve was derived (Y = 0.0063X − 0.023). The TPC and TFC of the extracts were calculated from respective curves and reported as mg per gram of dry matter, expressed as gallic acid equivalent (GAE) and catechin equivalent (CE), respectively.

DPPH Radical Scavenging Assay
DPPH (2, 2-Diphenyl-1-picrylhydrazyl) radical scavenging activity was assayed by the method reported previously [7]. Briefly, 10 µg/mL concentrations of extracts and BHT were mixed with 2 mL of 90 µM DPPH. The solution was incubated at room temperature for half an hour. The absorbance was read at 517 nm, and scavenging in terms of the percentage was calculated as follows: Adult male Wistar rats (weighing approximately 130-160 g) and mice weighing approximately 30-40 g were collected from the animal house of the Department of Physiology, Government College University, Faisalabad. Before the start of experiment, the animals were adapted for a week at the standard conditions (26 • C ± 2 • C temperature; 40-60% ambient humidity). Rats were housed in elevated wire cages with free access to food and water. The intake of food was measured on a daily basis for each animal.

Anti-Inflammatory Activity
To examine the anti-inflammatory activity of S. oleoides and S. persica aerial parts and fruit extracts in rats, thirty-six albino male Westar rats were randomly divided into the following six groups, all having six rats in each group (n = 6).
NC GROUP: Normal control group, which received no treatment. PC GROUP: Positive control group, which were supplemented with a dose of 10 mg/kg body weight (bw) of indomethacin served as a standard drug. G1 GROUP: Treatment group-1, which were supplemented with 250 mg/kg bw extract of S. oleoides aerial parts. G2 GROUP: Treatment group-2, which were supplemented with 250 mg/kg bw extract of S. persica aerial parts. G3 GROUP: Treatment group-3, which were supplemented with 250 mg/kg bw extract of S. oleoides fruits. G4 GROUP: Treatment group-4, which were supplemented with 250 mg/kg bw extract of S. persica fruits.
All the rat groups were provided water ad libitum and normal feed (approx. 20 g/rat/day). For measuring the anti-inflammatory activity, extract doses of S. oleoides and S. persica were administrated orally through gavage feeding tube (16-18 cm) for 7 days, while positive controls were orally administered indomethacin 10 mg/kg bw for 7 days before the induction of inflammation [21]. After 7 days of treatment, inflammation was induced in the right paw by injecting the 0.1 mL egg albumin in each treatment and PC group. The inflammation was measured for 4 h by screw gauge. The percentage Edema Inhibition was calculated using the NC group as a standard.

Analgesic Activity
To evaluate the analgesic activity, the mice were randomly divided into the following six groups, all having five rats in each group (n = 5).
NC GROUP: Normal Control, which received no treatment. PC GROUP: Positive Control, which were supplemented with a dose of 10 mg/kg of Diclofenac sodium served as standard. G1 GROUP: Treatment group-1, which were supplemented with 250 mg/kg bw extract of S. oleoides aerial parts G2 GROUP: Treatment group-2, which were supplemented with 250 mg/kg bw extract of S. persica aerial parts G3 GROUP: Treatment group-3, which were supplemented with 250 mg/kg bw extract of S. oleoides fruits G4 GROUP: Treatment group-4, which were supplemented with 250 mg/kg bw extract of S. persica fruits. Analgesic activity was performed as described by Hossain et al. [22]. Mice were fasted for 12 h with adequate clean water. Mice were placed on hot plates and the temperature was maintained at 55 ± 1 • C. Latency period, or the pain reaction time determined with a stopwatch, was recorded, which represented the time taken for the mice to react to the pain stimulus. The time in seconds was measured with a stopwatch by observing discomfort reactions, such as licking paws or jumping. The first reading was taken just before the administration of the drug and later at 0, 60, 120, 180 and 240 min before and after the drug administration. The cutoff time was fixed for 12 s to prevent any type of injury. This served as the control pain reaction time. The maximum possible analgesia (MPA) was calculated as follows: Reaction time f or treatment − Reaction time f or saline 12 − Reaction time f or saline × 100

Statistical Analysis
All the experiments were performed in three replicates and data are reported as the mean ± standard deviation (SD). Statistical analysis was performed by means of the statistical package, STATISTICA (Stat Sift Inc., Tulsa, OK, USA). Data from different tests were analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni/Dunnett (all mean) post hoc tests; the differences between the means were considered statistically significant at probability value p ≤ 0.05.

Yield of Extracts
The extract yields (g/100 g) from S. oleoides and S. persica aerial parts and fruits are given in Table 1. Extract yields varied from 8.15 to 19.60 g/100 g of dry plant material. The maximum extract yield (19.60 g/100 g) was obtained from the aerial part of S. persica, whereas the minimum extract yield (8.15 g/100 g) was found from S. oleoides aerial parts. The results showed a significant (p ≤ 0.05) difference in the yields among different extracts. Saleem et al. [23] reported a 12% extract yield from the aerial parts of S. oleoides. Variation in the extract yield might be due to differences in extractable components and months. The values are the mean ± SD of three independent experiments. Different alphabet letters in superscript show significant (p ≤ 0.05) differences among different extracts.

HPLC Results
Thirteen phenolic acids (gallic, hydroxy benzoic, chlorogenic, caffeic, syringic, vanillic, p-coumeric, salicylic, sinapic, ferulic, ellagic, cinamic, and benzoic acids) and five flavonoids (catechin, rutin, myricetin, quercetin, and kaempferol) were identified and quantified using Rp-HPLC from different extracts of S. oleoides and S. persica, and the data are presented in Table 2. Chlorogenic acid was the major phenolic acid from the aerial parts and fruit extracts of S. oleoides and S. persica, followed by gallic acid and hydroxyl benzoic acid. S. oleoides Life 2022, 12, 1446 6 of 15 and S. persica fruit extracts exhibited the maximum concentrations of chlorogenic acids, i.e., 1786.0 and 1473.0 mg/100 g of extract, respectively. Gallic acid was abundantly found in the S. oleoides aerial part extract (1265.0 mg/100 g), followed by the S. persica fruit extracts (942.4 mg/100 g). S. persica fruit extracts also contained 942.4 mg/100 g hydroxyl benzoic acid, whereas S. oleoides fruit extract contained 727.0 mg/100 g hydroxyl benzoic acid. Cinamic acid was found abundantly in the aerial part extracts of S. oleoides (390.6 mg/100 g) and S. persica (471.6 mg/100 g) only (Figure 1). Catechin and myricetin were the major flavonoids detected in all extracts of S. oleoides and S. persica. Salvadora persica aerial part extracts were rich in myricetin, rutin, and catechin, and the contents of these were 410.5, 254.3, and 578.5 mg/100 g, respectively. S. persica fruit extracts contained 331.8, 275.1, and 117.2 mg/100 g myricetin, rutin, and catechin, respectively. Catechin was abundant in the aerial part extracts of S. oleoides (131.5 mg/100 g) and S. persica (578.5 mg/100 g) as compared with the fruit extracts. Statistical analysis showed that the concentrations of various phenolic acids and the flavonoids were significantly (p ≤ 0.05) different among different extracts.
The phenolic and flavonoid contents were positively associated with the antioxidant activity. Both gallic acid and chlorogenic acid were the potential natural antioxidant compounds. Very few reports are available on the phenolic profile of the investigated extracts. Noumi et al. [24] also reported the separation and identification of caffeic acid, rutin trihydrate, trans-cinnamic, and gallic acids in the stems of S. persica using RP-HPLC. extracts. Noumi et al. [24] also reported the separation and identification of caffeic acid, rutin trihydrate, trans-cinnamic, and gallic acids in the stems of S. persica using RP-HPLC.

Evaluation of In Vitro Antioxidant Activity
Estimation of TPC and TFC TPC and TFC results, expressed as mg/g of dry plant material, are presented in Table  1. TPC was in the range of 11.9 to 22.2 mg TPC/g of dry plant material, measured as the GAE. The maximum TPC (22.2 mg/g of dry plant material, as the GAE) was found in fruit extracts of S. persica, and the minimum TPC (11.9 mg/g, as the GAE) was found in the aerial part of S. oleoides, followed by the aerial part extracts of S. persica (16.2 mg/g, as the GAE) and the fruit extracts of S. oleoides (19.2 mg/g, as the GAE). The TFCs in aerial parts and fruit extracts were in the range of 1.63 to 6.17 mg TFC/g of dry plant material, measured as the CE. The maximum amount of TFC (6.17 mg TFC/g of dry plant material as the CE) was exhibited by fruit extracts of S. persica, and the minimum TFC (1.72 mg TFC/g of dry plant material as the CE) was exhibited by aerial part extracts of S. oleoides. The flavonoid contents in fruit extracts of S. oleoides were found to be 5.54 mg TFC/g of

Evaluation of In Vitro Antioxidant Activity
Estimation of TPC and TFC TPC and TFC results, expressed as mg/g of dry plant material, are presented in Table 1. TPC was in the range of 11.9 to 22.2 mg TPC/g of dry plant material, measured as the GAE. The maximum TPC (22.2 mg/g of dry plant material, as the GAE) was found in fruit extracts of S. persica, and the minimum TPC (11.9 mg/g, as the GAE) was found in the aerial part of S. oleoides, followed by the aerial part extracts of S. persica (16.2 mg/g, as the GAE) and the fruit extracts of S. oleoides (19.2 mg/g, as the GAE). The TFCs in aerial parts and fruit extracts were in the range of 1.63 to 6.17 mg TFC/g of dry plant material, measured as the CE. The maximum amount of TFC (6.17 mg TFC/g of dry plant material as the CE) was exhibited by fruit extracts of S. persica, and the minimum TFC (1.72 mg TFC/g of dry plant material as the CE) was exhibited by aerial part extracts of S. oleoides. The flavonoid contents in fruit extracts of S. oleoides were found to be 5.54 mg TFC/g of dry plant material and 2.44 mg TFC/g of dry plant materials in aerial part extracts of S. persica. Significant differences (p ≤ 0.05) were observed in the TPC and TFC of the different extracts of S. oleoides and S. persica.
Phenolic compounds have good relationship and largely contribute to the antioxidant activity. The polar solvent, i.e., methanol, which was used has the capacity to extract more phenolics as compared with other solvents. Saleem et al. [23] reported total phenolic (0.4 mg QE/g) and total flavonoid contents (0.21 mg QE/g) of S. oleoides aerial parts in methanol extract which were lower than our results. Kumari et al. [25] reported the total estimated amount of phenolics in the fruit methanol extract of S. persica to be 120.38 mg/100 g DW, and flavonoids were estimated to be 77.59 mg/100 g DW; however, there have been no reported results on the TPC and TFC of S. oleoides fruit. Kaneria et al. [19] reported that S. persica showed higher total phenol contents as compared with S. oleoides. The total phenolic contents of S. oleoides in methanol extracts of leaves were 253.10 mg/g, whereas the total flavonoid contents were 43.65 mg/g. The total phenolic content of S. persica in methanol extracts of leaves was 252.770 mg/g, whereas the total flavonoid contents were estimated to be 57.94 mg/g [19], which is higher than our findings. Variation in our TFC and TPC results compared with the findings of the majority of previous studies might have been due to differences in the agro-climatic, geographical, and seasonal conditions.

DPPH Free Radical Scavenging Assay
The free radical scavenging activity of various extracts (10 µg/mL) was measured by the DPPH radical scavenging assay, and the results are presented in Table 1. The fruit extract of S. persica showed maximum radical scavenging activity (54.3%), whereas the aerial parts of S. oleoides showed minimum radical scavenging activity (46.9%) when compared with the synthetic antioxidant BHT (60.8%). Statistical analysis showed the significant (p ≤ 0.05) differences in the radical scavenging potential of S. persica fruit extracts from other extracts.
The DPPH free radical scavenging capacity increases when extract concentration increases due to increases in the concentration of phenolic compounds [5]. Saleem et al. and Noumi et al. [23,24] reported the strongest scavenging DPPH assay results (51.66%) of methanolic extracts of S. oleoides aerial parts, which were comparable to our results. Kumari et al. [25] reported the IC 50 for DPPH of the S. persica fruit (IC 50 307.06 µg crude methanol extract). Souri et al. [26] investigated the antioxidant activity and free radical scavenging activity on DPPH of 13 medicinal plants traditionally used in Iran. They reported that methanolic extracts of S. persica exhibit free radical DPPH scavenging activity with an IC 50 value of 37.19, which is contrary to our results. The variation in DPPH radical scavenging activity might be due to different plant species, geographic regions, as well as different months for sample collection.

Anti-Inflammatory Activity
The anti-inflammatory activities of aerial part and fruit extracts of S. persica and S. oleoides were evaluated, and the results are presented in Table 3. At zero hour, mean inflammation (mm ± SD) was non-significant (p > 0.05) in all groups before the start of treatment.
The inflammation was significantly reduced (p ≤ 0.05) in all treated groups, G1, G2, G3, and G4, and the positive control (PC) as compared with the negative control (NC) group one hour after the oral administration of methanolic extracts. However, reductions in inflammation were non-significant (p > 0.05) among all treated groups one hour after the oral administration of extracts. The inflammation was significantly reduced (p ≤ 0.05) in all treated groups, G1, G2, G3, and G4, and the positive control group (PC) as compared with the negative control (NC) groups 2 h after the oral administration of methanol extract. However, the anti-inflammatory response was significantly reduced in G4 as compared with NC and G1 2 h after the oral administration. Inflammation was also significantly Life 2022, 12, 1446 12 of 15 reduced in G1, G2, and G3 as compared with NC, but was non-significant between groups. The inflammation was significantly reduced (p < 0.05) in all treated groups, G1, G2, G3, and G4, and PC, as compared with the NC group 3 h after the oral administration of extracts. Inflammation was significantly reduced in G4 as compared with G1, G2, and G3, but was comparable to PC. Inflammation was also significantly reduced in G1, G2, and G3 as compared with NC, but less than G4 and PC. The inflammation was significantly reduced (p < 0.05) in all treated groups, G1, G2, G3, and G4, and PC, as compared with NC four hours after the oral administration of methanol extract. Inflammation was significantly reduced (p < 0.05) in G4 as compared with NC, G2, and G3. It could be concluded that the fruits of S. persica are more potent than the other extracts used in the study.  Polyphenols are secondary metabolites that have antioxidant capacities in addition to antiallergenic, anticancer, anti-inflammatory, anti-thrombotic, and anti-mutagenic properties [27]. The results obtained by BenSaad et al. [28] clearly indicate that ellagic acid, gallic acid, and punicalagin A & B isolated from P. granatum inhibited the production of NO, PGE2, and IL-6 in LPS-induced RAW267.4 macrophages. In the present study, gallic acid may have been the compounds responsible for the remarkable anti-inflammatory effect showed by S. presica fruit. Catechins can also inhibit the infiltration and proliferation of immune-related cells and regulate inflammation and oxidative reactions by interaction with multiple inflammation-related and oxidative-stress-related pathways [29]. Catechin was the major compound found in S. persica fruit and detected in all extracts by Rp-HPLC, which is why it could also be the reason for the anti-inflammatory effect shown by plant extracts. Ibrahim et al. [30] investigated the anti-inflammatory effect of aqueous alcoholic crude extract and the ethyl acetate extract of miswak sticks (S. persica) in carrageenan-induced rat paw edema. The inhibition percentage of inflammation was 17% for crude extract and 27% for ethyl acetate extract. Natubhai et al. [21] studied the anti-inflammatory effect of S. oleoides leaf extract. The alcoholic extracts of S. oleoides at a dose of 200 and 400 mg/kg, reduced the paw edema induced by carrageenan by 46.70 and 81.70%, respectively, whereas the water extract reduced the paw edema by 51. 16 and 83.30, respectively. No reports are available in the literature on the anti-inflammatory activity of aerial parts and fruits of S. persica and S. oleoides. Baba et al. [31] investigated the anti-inflammatory activity of ethanol, acetone, and water extract of leaves, stem bark, and fruit peels of S. persica. The anti-inflammatory activity was compared with the standard drug (Diclofenac) at a dose of 10 mg/kg. Diclofenac showed values of 87.71 and 82.85 at a dose of 300 mg/kg, respectively.

Analgesic Activity
The analgesic activity of aerial part and fruit extracts of S. oleoides and S. persica was investigated using a mouse model, and the results are presented in Table 4. Mice treated with normal saline (control) did not show any significant difference in the reaction time on the experiment throughout the 240 min observation. The longest reaction time for the treated groups with the hot plate method was 180 min. The analgesic effects of Diclofenac sodium and different plant extracts could be seen from the maximum possible analgesia (MPA) graph in Figure 2. The MPA remained elevated during the observation period, reaching its peck at 180 min. The MPA of fruit extracts of S. persica was 53.44% that is better than PC group (52.98%). Aerial part extracts of both S. oleoides and S. persica showed significantly (p ≤ 0.05) less MPA. Overall, the fruit extract of S. persica produced an excellent analgesic activity at 180 min. 3.46 ± 0.31 a 6.21 ± 0.59 b 6.11 ± 0.57 bc 6.44 ± 0.58 bc 5.98 ± 0.54 b G3 3.47 ± 0.34 a 6.33 ± 0.58 b 6.23 ± 0.58 bc 6.56 ± 0.59 bc 6.12 ± 0.56 b G4 3.51 ± 0.32 a 6.45 ± 0.58 b 6.62 ±0.59 b 6.95 ± 0.64 c 6.31 ± 0.59 b The values are presented as the mean ± SD. Different alphabet letters in superscript show significant (p ≤ 0.05) differences among normal and treatment groups.

Conclusions
The results obtained in the present study showed the comparative assessment of TPC and TFC, and antioxidant, anti-inflammatory, and analgesic activities of S. persica and S. oleoides aerial parts and fruit extracts. S. persica fruit extract showed the maximum antioxidant activities in terms of TPC, TFC, and DPPH radical scavenging. Based on the results, it is concluded that fruit extracts of S. persica have a major role in the reduction in inflammation and exhibit better analgesic activity as well. This was due to the high antioxidant results of S. persica fruit extract as compared with extracts of other plant parts that were investigated, and also due to the high presence of gallic acid and catechin, as Hooda and Pal [32] reported the analgesic activity of hydroalcoholic extract of S. persica root extract on a mouse and rat model. The analgesic activity of albino mice and albino rats was evaluated using Eddy's hot plate method. In Eddy's hot plate method, the highest analgesic activity was observed at an oral concentration of 400 mg/kg for 90 min. Our results were correlated with those of Hoor et al. [33], who suggested that extracts of