Introduction
Since human domestication, plants have been exploited for various reasons, such as food, feed, fiber, and medicine. Each plant and its parts (roots, leaves, fruits, bark) have unique chemicals that provide different medicinal properties-for instance, phenolic compounds contribute to antioxidant activity. Every plant owns special biochemical materials and their therapeutic effects [1]. For example, free radicals are produced due to the higher reactivity of oxygen, which is responsible for many disorders [2]. Such oxidative stress of free radicals on body tissues plays a significant role in diverse pathological conditions, such as aging and infection at the cellular stage and in medicinal and biological systems [3, 4]. Different enzymatic and non-enzymatic reactions are responsible for the production of oxygen free radicals as well as during normal physiological phenomena [5]. The pathological condition results in the overproduction of cells that cause damage [6]. Antioxidants are known as radical scavengers that defend the body over free radicals, and their abundance in nature encouraged scientists to substitute synthetic antioxidants to avoid their conflicting effects. Microorganisms are responsible for food spoilage, thus reducing its shelf life and causing various diseases. For example, Staphylococcus aureus is responsible for toxic shock, food poisoning, and osteomyelitis [4, 7, 8]. 
Cassia absus L. (Chaksu) is a vertical annual plant belonging to the Leguminosae family. C. absus is commonly found in south and tropical Asia, Africa, Australia, and India, with lengths up to 12–25 inches [9]. Chemical studies have proved that the leaves and roots of C. absus are rich in biochemicals, such as chaksine, rutin, quercetin, chrysophanol, riboflavin, aloe-emodin, and isochaksine [10, 11]. Seeds are augmented with saponins, glycosides, flavonoids, tannins, terpenoids, gums, amino acids, resins, oleic acid, steroids, and linoleic acid. C. absus can scavenge free radicals and is an essential source of antioxidants and phytochemicals. The bitter and styptic nature of leaves helps to cure dermis infections, constipation, cancer, respiratory ailment, and hemorrhoids [12, 13].
Citrus medica L. (family: Rutaceae), also recognized as Bara Nimbu, is a perennial bush or small tree with a height of about 3.6 m and found in warm moist regions in Asia and more common in Northern Punjab, Pakistan [14]. It contains glucose and citric acid in excess. The leaves contain apigenin, rutin, quercetin, isolimonene, and erucylamide. The floral part is abundant with highly valuable compounds, such as hesperetin, hesperidin, rutin, diosmin, quercetin, and apigenin. Fruit contains diosmin, hesperidin, and apigenin [15]. Phytoestrogens like resveratrol, lignans, isoflavones, and 8-prehylnarinagenin are commonly found in seeds. Its waste contains highly valuable compounds used in food additives, food supplements, energy drinks, and relieving agents in respiratory disease by reducing viral attacks and emitting SARS-CoV-2 in the nasal route, causing intestinal troubles and sea sickness [16]. 
However, no reports exist on the antimicrobial and antioxidant activities of C. absus and C. medica plants. Thus, this study was designed to assess the phenolic profiling, partition fractionation, and antimicrobial and antioxidant activity of extracts of C. absus and C. medica leaves in various polar to less polar solvents and to confine their relative action. Particular emphasis was on looking at different extract fractions with viable antimicrobial activity. 
Material and Methods
Study materials
The majority of chemicals/reagents such as Folin-Ciocalteu (FC) reagent, sodium carbonate anhydrous, gallic acid, sodium nitrite, sodium hydroxide, aluminum chloride anhydrous, 2,2-diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic acid, ferric chloride anhydrous, ascorbic acid, catechin HPLC-grade methanol, ethanol, and acetone were purchased from Sigma-Aldrich (St. Louis, MO, USA). In contrast, sulfuric acid and potassium-hexacyanoferrate were obtained from Merck (Darmstadt, Germany). Anhydrous monobasic potassium phosphate was purchased from Fisher Chemicals (Fair Lawn, NJ, USA). 
Collection and identification of plants
In a garden located in the suburb of Burewala (coordinates 30°4'4''N 72°38'19''E), southwest of Pakistan, fresh newly emerged leaves of C. absus and C. medica were harvested from September to October 2017. The plant materials were carried out to the laboratory at the Department of Botany for further analysis. Dr Zafar Ullah Zafar, a taxonomist from Bahauddin Zakariya University (BZU), Pakistan, further identified and authenticated the harvested plant materials.
Extraction and preparation of extracts fractions 
Plant samples were washed with distilled water to remove dust and foreign particles, broken down into small chunks, and cleaned specimens were air dried at room temperature under shade [17].
Plant leaves were then crushed and pulverized into a fine powder. Afterward, dried and fine powder (70 g) plant material was wrapped in a porous bag in the Soxhlet extraction procedure with the addition of 500 mL methanol solvent for about 4 h. The extract was concentrated through a rotary evaporator at 45°C (reduced pressure) (EYELIA, SB-651, and Rikakikai Co. Ltd. Tokyo, Japan), and the solvent was evaporated [2]. The suspension was filtered through a Büchner funnel, and the residue was washed twice with 100 mL of the same solvent. The filtrates were combined, and the solvent was evaporated under reduced pressure. The ethanol extract (5 g) was re-extracted with ethyl acetate (45 mL) under occasional shaking for 4 h at an ambient temperature in a conical flask. The mixture was filtered, and the solvent was evaporated under reduced pressure. Both extracts were transferred into a conical flask filled with argon and stored in a freezer at -20°C. During the extraction and sample preparation, laboratory glass was covered with aluminum foil to protect the extract against light. The percentage yield was calculated from dried extracts, kept at -40°C, and utilized for further analysis. Generally, water, methanol, and ethanol are commonly used in polar compounds, while hexane and dichloromethane are in nonpolar compound extraction. The methanol extracts from both plants was exposed to methanol fractions (Figure 1) (50%) [18].

Assessment of antioxidant activity
Determination of total phenolic contents (TPC)
The Folin-Ciocalteu procedure was used to determine the total phenolic contents of the C. absus and C. medica extracts as described previously [19], with slight modification. Briefly, 200 µL of crude extracts (1 mg/mL) was made up of 3 mL volume with deionized H2O, stirred rigorously with 0.5 mL of the Folin-Ciocalteu reagent (diluted 1:10 with deionized H2O) for about 3-5 min, followed by neutralized with 1.5 mL of sodium carbonate 20% (w/v). The reaction mixture was homogenized and incubated at room temperature for half an hour with sporadic shaking for advancement. The absorbance was measured at 755 nm wavelength using a double-beam UV/Vis Spectrophotometer-U-2001 (Hitachi Instruments Inc., Tokyo, Japan). The phenolic concentrations of C. absus and C. medica were determined by the calibration curve and the gallic acid. Also, the total phenolic content was counted within the 10-100 mg/L range (R2=0.9986). Their results were specified as g/100 g of plants’ dry matter equivalents to gallic acid (GAE), where three concordant values were captured for samples and averaged results were expressed as dry weight.
Determination of total flavonoid contents (TFC)
The total flavonoid content (TFC) of crude extracts of C. absus and C. medica was evaluated with the standard method [20], with slight alternation. Briefly, in 50 mL (1 mg/mL ethanol), methanol extracts were added to make 1 mL volume, dissolved with 4 mL of deionized H2O. Subsequently, after 5 min of incubation, 0.3 mL of 5% sodium nitrite (NaNO2) and then 10% aluminum chloride (AlCl3) solutions were added. The mixtures were made up to 10 mL with deionized H2O. The solution was mixed and kept at room temperature for 20-30 min, and absorbance was computed at λ 510 nm. The TFC was determined from a calibration curve, and the findings were demonstrated as rutin (mg) corresponding to dry weight (per g).
Estimation of reducing power
Reducing power was measured as reported previously [21]. In brief, 100 µL extract (100-500 ug/mL) was stirred with 2.5 mL of 200 m molar phosphate buffer (pH=6.6) add 2.5 mL of 1% potassium ferricyanide (C6FeK3N6) solution and incubated at 50°C for 25 min. After incubation solution was supplemented with 2.5 mL of 10% trichloroacetic acid (C2HCl3O2) and centrifuged at 10000 rpm for 10 min. Afterward, 5 mL of the supernatant was mixed with 5.0 mL deionized H2O and 1 mL of 0.1% ferric chloride (FeCl3), and the absorbance of the reaction mixtures was determined at 700 nm. 
DPPH free radical scavenging assay
Free radical inhibition activity of different concentrations of C. absus and C. medica were calculated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging protocols with minor optimization. Approximately 4 mL extract was mixed with 2 mL of 0.2 mM DPPH methanolic solution, kept in the dark at ambient temperature for 30 min, and absorbance was measured at λ 517 nm with various time zones 0, 0.5, 1, to 10 min, respectively. The following equation calculated free radical inhibition by DPPH (Equation 1):
1. Inhibition %= [(Ac-As)/Ac]×100
, where Ac refers to control reaction absorbance (with all reagents except the sample), and As refers to sample absorbance.
Determination of linoleic acid inhibition
The inhibition of peroxidation of linoleic acid was measured as reported in a study [22]. Briefly, linoleic acid (0.13 mL) and ethanol and phosphate buffer (10 mL) (pH=7) were mixed with each plant extract in a 25-mL volumetric flask and made the volume up to the mark with dH2O and incubated at 40°C for 15 days. To determine the oxidation rate, the thiocyanate method was adopted. Absorbance was measured through a spectrophotometer at 500 nm after stirring for 3 min along with the negative (without extracts) and positive (butylated hydroxytoluene) controls. For calculating the inhibition percentage of acid, the below formula was applied (Equation 2):
2. 100- [(increased sample absorbance at 360 h/ increased control absorbance at 360 h) 100]
Determination of antimicrobial activity
As reported in some studies, the antimicrobial activity of plant extracts was calculated by the disk diffusion method [23, 24]. The disks (6 mm in diameter) were soaked with 50 μL extract and placed in the inoculated agar. Rifampicin and fluconazole were used as positive references for fungi and bacteria to compare the activity with standard antibiotics such as 30 μg/disk. Both sample and standard disks were assigned separately in Petri dishes. Afterward, they were incubated at 37°C for 24 h and at 25°C for 3 days. Finally, the inhibition zones (mm) were measured to assess antimicrobial activity.
Determination of phenolic compounds by HPLC and data analysis
High-performance liquid chromatography (HPLC) was applied for the analysis of the chemical composition of plant extracts (model LC-10A, Shimadzu, Kyoto, Japan). The sample was diluted, and 20 μL was injected into analytical Supelco (Supelco Inc., Supelco Park, Bellefonte, PA, USA) reverse phase ODS (C18) column (250×4.6 mm). Two mobile phases, i.e. water with acetic acid (94:6 v/v) and 100% acetonitrile, were utilized to separate phenolic components. Isocratic elution was achieved at normal temperature with a 1.0 mL/min flow rate for separation. Wavelength was set at 280 nm for detection. The phenolics (gallic acid, vanillic acid, benzoic acid, quercetin, ferulic acid, caffeic acid, p-coumaric acid, catechin, ellagic acid, and chlorogenic acid) were identified by Sigma Chemicals Co. (St Louis, MO, USA). The calibration curve was used for quantitative determination. All assays were performed in triplicate, and the data were expressed as Mean±SD. Multiple range test analysis of variance (ANOVA) was accomplished using statistical software with P<0.05.
Results 
Percentage yield
The nature of the solvent and season affect the extractive components of plants, so the bioactive components possess a high yield in polar solvents like methanol which is considered the best fit for extraction [25]. The yields of C. absus and C. medica depict that the polar solvents, i.e. acetic acid, ethanol, and methanol, provided high fractionations, such as 10%, 11%, 14% and 12%, 14%, and 16%, respectively, followed by lower polar fractions (5%) for both in acetone (3%) and in ethyl acetate (6%) (Figure 2).

Less yield was produced by non-polar solvent, i.e. n-hexane (4% and 3%). It is revealed that compounds with sugar chains (glycosides, carbohydrates), phenolic compounds, and polar alkaloids are sufficiently reduced with polar solvents, as reported by Federica Menichini [26].
High-performance liquid chromatography 
Partitioned fractions of both C. absus and C. medica show different HPLC fingerprints of flavonoids and phenolic compounds, i.e. ellagic acid, gallic acid, coumaric acid, vanillic acid, 4-hydroxy-3-methoxy benzoic acid, kaempferol, caffeic acid, quercetin, rutin, syringic acid, myricetin and cinnamic acid with different amounts. Figure 3A shows the quantity of major phenolics of methanol fraction, i.e.

coumaric acid (172.11±0.04 µg/g), (112.21±0.01 µg/g), and (143.49±0.03 µg/g); gallic acid (192.03±0.02 µg/g), (141.51±0.03 µg/g), (175.15±0.02 µg/g); ellagic acid (159.18±0.01 µg/g), (110.31±0.04 µg/g), and (135.44±0.03 µg/g); cinnamic acid (108.27±0.02 µg/g), (97.24±0.03 µg/g), and (101.23±0.01 µg/g); and vanillic acid (133.17±0.02 µg/g), (101.24±0.03 µg/g), and (111.96±0.01 µg/g) µg/g. The quantity of minor phenolics (<100 µg/g) were caffeic acid (95.23±0.02 µg/g), (75.16±0.01 µg/g), and (83.54±0.03 µg/g); 4-hydroxy-3-methoxy benzoic acid (54.97±0.02 µg/g), (12.18±0.05 µg/g), and (36.26±0.03 µg/g) and syringic acid (76.35±0.01 µg/g), (34.34±0.02 µg/g), and (49.22±0.04 µg/g). Similarly, the major flavonoids of C. absus were quercetin (152.07±0.03 µg/g), (121.33±0.02 µg/g), and (134.23±0.01 µg/g); kaempferol (120.95±0.02 µg/g), (111.17±0.05 µg/g), and (118.87±0.01 µg/g); and rutin (134.03±0.01 µg/g), (128.12±0.04 µg/g), and (115.25±0.02 µg/g) by weight in methanol fractions. Minor flavonoids (<100 µg/g) were luteolin (75.05±0.01 µg/g), (43.44±0.06 µg/g), and (54.87±0.02 µg/g), and myricetin (98.4±0.02 µg/g), (83.13±0.03 µg/g), and (95.65±0.01 µg/g) in methanol fraction. Malpani M et al. reported that C. absus hold phenolic acid compounds, i.e. isochaksin, chaksine, and flavonoids, with various other compounds present in different quantities. Similarly, Figure 3B shows different flavonoids and phenolic compounds, such as gallic acid, coumarate, chlorogenic acid, caffeic acid, syringic acid, apigenin, ferulic acid, rutin, quercetin, kaempferol, catechin, and naringenin are present in different quantities. Gallic acid (175.02±0.01 µg/g), (161.14±0.04 µg/g), and (145.50±0.02 µg/g); ferulic acid (140.33±0.03 µg/g), (127.83±0.02 µg/g), and (107.30±0.01 µg/g) and chlorogenic acid (121.10±0.03 µg/g), (102.48±0.02 µg/g), and (81.21±0.02 µg/g) were major phenolics The minor phenolics were coumaric acid (71.18±0.01 µg/g), (57.31±0.04 µg/g), and (31.44±0.03 µg/g); caffeic acid (11.01±0.04 µg/g), (25.10±0.02 µg/g), and (10.63±0.01 µg/g); and syringic acid (34.03±0.03 µg/g), (22.12±0.04 µg/g), and (41.25±0.01 µg/g). The order of concentration was caffeic acid15, 27]. 
Total phenolic contents
Total phenolic compounds (TPC) expressed as GAE, equivalents of gallic acid (mg/g), whose total amounts in C. absus and C. medica leaves extract were 5.58-17.1/100 g and 2.28-8.17/100 g, respectively. For their quantification, the Folin-Ciocalteu procedure was opted because of reproducibility and less interference. The amount of TPC in C. absus was 17.1 g per 100 g, higher than that of C. medica extracts [28]. Figure 4A shows the TPC quantity of C.

The free radical inhibition might be declined due to polarity reduction or reduced phenolic compounds [17]. 
Inhibition of linoleic acid per oxidation
 The inhibition percentage age of linoleic acid following 360 h incubation was observed. The sample fractions illustrate the linoleic acid peroxidation of 27.21% for non-polar and 13.730%-18.930% for polar fractions of C. absus extract while 17.693% and 28.88%-49.60% for a non-polar and polar fraction of C. medica plant extract (Figure 6B). The effect of polarity (P<0.005) at the oxidation of linoleic acid was significant. The findings of this study confirmed that C. absus and C. medica plant extracts showed a higher inhibition percentage of linoleic acid in methanol fractions. This condition further proves that polarity directly affects antioxidant activity as the decline in polarity resulted in the decline of antioxidant activity that may be responsible for fewer phenolics that cause this activity [36].
Assessment of antimicrobial activity
Fractions of C. absus and C. medica extracts were exploited to access antimicrobial activity (Figure 7).