Phytochemical Characterization and Solvent Fraction Depending in vitro Antioxidant Activities of Cassia absus, Gymnema sylvestre, Nigella sativa and Piper nigrum

This study was aimed to phytochemically characterize and assess the antioxidant activities of 70% methanol extract and subsequent fractions of Cassia absus (L.) seeds, Gymnema sylvestre (L.) whole plant, Nigella sativa (L.) seeds, and Piper nigrum (L.) seeds. Powdered plant materials were extracted with 70% methanol and further fractionated with ethyl-acetate, n-butanol and the residual aqueous fraction. Phytochemical analysis was performed to detect different phytocompounds. Mineral compositions were quantified, and total phenolic and flavonoid contents were determined. The antioxidant potential of methanol extracts and fractions was assessed in vitro through estimating DPPH radical and superoxide anion scavenging activities and reducing power assay. Extraction yields ranging highest of N. sativa methanol extract (30.42±1.49%) and lowest of P. nigrum ethyl-acetate fraction (4.58±0.61%) were obtained. Results revealed that methanol extracts and fractions of selected plants contain phytocompounds such as alkaloids, flavonoids, phenols, glycosides, tannins, terpenoids, saponins, carbohydrates, fats and fixed oils. The mineral analysis showed considerable quantities of calcium (C. absus methanol extract: 372.454±3.633 mg/100g), magnesium (G. sylvestre methanol extract: 131.045±1.346 mg/100g), and zinc (N. sativa methanol extract: 36.019±0.284 mg/100 g) in all fractions while minor quantities of manganese, copper and cobalt were also found. Methanol extracts showed considerably higher total phenolic (N. sativa methanol extract: 179.71±2.14 mg GAE/g) and flavonoid (N. sativa methanol extract: 189.18±3.17 mg CE/g) contents compared to other fractions, and subsequently exhibited pronounced scavenging activities on DPPH* (N. sativa methanol extract: 23.8 μg/mL) and superoxide radicals (N. sativa methanol extract: 24.9 μg/mL) and had potent reductive abilities (N. sativa methanol extract: 1.123±0.038 O.D.). Conclusively, C. absus, G. sylvestre, N. sativa and P. nigrum possess significant nutritive properties and could be used as natural antioxidant sources to prevent oxidative stress-associated diseases.


Introduction
The use of medicinal plants as therapeutic remedies is as old as human civilization and people have been continuing to rely on them for primary healthcare needs [1]. Approximately 80% population of underdeveloped or developing countries still depends on natural-sourced products to cure their acute or chronic ailments [2]. It is currently estimated that more than 0.4 million plant species have been discovered. Compounds derived from natural sources have a significant contribution to the discovery of novel chemical entities. A multi-disciplinary approach is involved in the drug discovery process and is dependent upon many disciplines such as ethnobotany, biology, phytochemistry and chemical separation techniques. At present, a considerable proportion (around 87%) of therapeutic agents is directly or indirectly derived from nature [3].
The generation of free radicals occurs throughout cellular metabolic activities. However, multiple antioxidant defense mechanisms effectively neutralize these free radicals and the body maintains the oxidation and anti-oxidation balance [4]. Oxidative stress is assumed to be the underlying cause of various human diseases. Oxidation of macromolecules like proteins, lipids and damage to DNA is

Extract preparation
Dried plant materials were coarsely powdered by a mechanical grinder to achieve maximum extraction. About 100 g of each powdered plant material was macerated in 500 mL of 70% methanol for 72 h at room temperature, with shaking at regular intervals. The extract was firstly filtered through a muslin cloth and then filtered through Whatman paper no. 1 and concentrated by using a rotary evaporator (Hei-VAP, Heidoph Rotacool ® , Germany). This process was repeated three times and the combined percentage of yield was calculated. Then, a 70% methanol sample was kept aside and the remaining concentrate of 70% methanol was mixed with water and ethyl-acetate, n-butanol and aqueous fractioning were performed [18]. Dry concentrated extracts were stored in airtight containers at -4 o C for further analysis. The percentages of extract yield were calculated by the following formula: Extraction yield (%) = [Weight (g) of dry soluble solid/Weight (g) of sample] x 10 (1)

Qualitative phytochemical screening
Extracts were subjected to phytochemical analysis for the presence of alkaloids, carbohydrates, flavonoids, fats and fixed oils, glycosides, phenols, saponins, steroids, tannins and terpenoids by following standard protocols [19,20].

Mineral composition
AOAC, (1990) method was followed to prepare dry extract samples for the detection and quantification of different elements i.e. manganese (Mn), nickel (Ni), zinc (Zn), cadmium (Cd), calcium (Ca), cobalt (Co), lead (Pb), magnesium (Mg), copper (Cu) by using atomic absorption spectrophotometer [21]. About 10 mL mixture of nitric acid: perchloric acid (7:3) added to 1 g of each extract and heated at 180-200 o C until white fumes appeared. The final volume was made up to 100 mL by adding distilled water.

Total phenolic content
The Folin-Ciocalteau's method was used for the estimation of total phenolic content of extracts [22]. In short, each extracted sample (50 mg) was diluted with 7.5 mL of double distilled water and mixed with 0.5 mL of Folin-Ciocalteau reagent. After incubating at 37 o C for 10 min, 1.5 mL of 20% w/v Na2CO3 was added to sample mixtures, further heated on the water bath for 20 min and cooled immediately in an ice bath. The absorbance of resulting blue-colored complex reaction mixtures was measured at 765 nm using a spectrophotometer (Shimadzu ® , Japan). Different concentrations of gallic acid as standard in methanol were prepared for the calibration curve (y=0.0116x+0.0927; R 2 =0.9955).

Total flavonoid content
Total flavonoid content was determined using aluminum chloride with the help of colorimetric method [22]. Briefly, a weighed quantity of each sample was mixed with 0.3 mL of 5% NaNO2 solution and 4 mL of distilled water. After 5 min, 0.3 mL of 10% AlCl3 was added and 2 mL of 1 M NaOH was mixed after incubating for 6 min and further diluted with 2.4 mL of distilled water. Absorbance was measured at 510 nm and flavonoid content was determined by using a catechin calibration curve (y=0.0027x+0.1609; R 2 =0.9938).

In vitro antioxidant activity
For the assessment of antioxidant activities, methanol extracts and fractions of selected plants were dissolved in methanol (95%) to prepare 1mg/mL concentration of samples and further reconstituted to made concentration-dependent dilutions. Ascorbic acid as a standard was used for comparison. https://doi.org/10.37358/RC.21.2.8418

DPPH* radical scavenging assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed by following a previously prescribed method [23]. Ascorbic acid as reference and extract samples of different concentrations (25 to 300 µg/mL) were dissolved in methanol. About 2 mL of reference and sample dilutions were mixed with freshly prepared 0.5 mL of 0.002% DPPH methanolic solution, incubated for 15 min at 37 o C and absorbencies were measured at 517 nm using a spectrophotometer. All values were taken in triplicate and DPPH percentage inhibition was calculated using the given formula: AControl=Absorbance of Control/Blank without extracts, ASample= Absorbance of extracts or standard. IC50 values of all extracts were calculated to determine 50% inhibition of DPPH* in comparison to standard.

Superoxide radical scavenging assay
The Beauchamp and Fridovich method [24] was adopted to determine the antioxidant activity of methanol extracts and fractions of plants through their superoxide anion radical scavenging ability. The reaction mixture comprised of 500 µL of PO4 buffer (50 mM, pH 7.6), 100 µL of nitroblue tetrazolium (0.5 mM), 250 µL of phosphor-methozine sulfate (20 mM) and 300 µL of riboflavin (50 mM) was prepared and then 1 mL of each sample was added. Sample solutions were placed under a fluorescent lamp for 20 min to trigger the reaction and absorbance was taken at 560 nm. The scavenging activity (%) was calculated as: AControl=Absorbance of Control/Blank without extracts, ASample= Absorbance of extracts or standard. IC50 values of all extracts were calculated to determine 50% inhibition of superoxide anion radical in comparison to ascorbic acid as standard.

Reducing power assay
Reducing power of a test sample is its ability to convert Fe +3 to Fe +2 . The appearance of a bluecolored complex indicates the conversion to Fe +2 and absorbance can be measured at 700 nm using a spectrophotometer [25]. In short, 2 mL of ascorbic acid (standard) and plant extracts of various concentrations were added to 2mL of 1% potassium ferricyanide and 2 mL of 0.2 M phosphate buffer (pH 6.6). The mixture was heated for 30 min at 45 o C. Then, 2 mL of trichloroacetic acid was added, centrifuged for 10 min at 3000 rpm and the upper layer was collected. Further, 2 mL of distilled water, 0.4 mL of freshly prepared ferric chloride (0.1% w/v) were mixed with 2 mL of supernatants, and after 10min, absorbance was measured at 700 nm. A higher absorbance value of the reaction mixture indicates higher reducing power of extract. The test was performed in triplicates and results were averaged.

Statistical analysis
Data were analyzed using Graphpad Prism ® (ver. 6.0) and results were presented as mean±SEM of triplicates determinations.

Extraction yields
Various parameters including solvents, sample to solvent ratio, method, temperature and time influence the efficiency and extraction yield [26]. The percentage extraction yields of methanolic extracts and fractions obtained from C. absus, G. sylvestre, N. sativa and P. nigrum are mentioned in Methanolic extract of N. sativa gave the highest yield of 30.42±1.49%, whereas the ethyl-acetate fraction of P. nigrum gave the lowest yield (4.58±0.61%). The differences in extraction yields of plants are attributed to varying concentrations of bioactive phytocompounds and their different solubility profiles [27]. Values are expressed as mean±SEM (n=3). Small letters (a-c): p<0.05 significant difference between different solvent fractions of the same plant; Capital letters (A-C): p<0.05 significant difference between same solvent type in different plants.

Estimation of phytochemical constituents
Qualitative phytochemical analysis of C. absus, G. sylvestre, N. sativa and P. nigrum methanol extract and different fractions have been shown in Table 2. Phytocompounds present in plants are responsible for pharmacological activities such as alkaloids have been known for antimalarial, antihypertensive, anticancer activities, as well as isolated pure alkaloids, have been used as antibacterial, antispasmodic and analgesic agents [28,29]. Flavonoids and phenols present in plants considerably contribute to antioxidant, anti-inflammatory and anticancer activities, owing to their reaction oxygen species (ROS) scavenging potential [30]. Saponins possess an antioxidant potential and are known to lower cancer risks, modulate blood lipids and improve blood glucose response [31]. Plants possessing tannins have been used to treat hemorrhoids and wounds and also used as diuretics, antidiarrheal, astringents and against gastric and duodenal tumors [28,32]. Terpenoids have shown anti-malaria, antiulcers, and anti-cancer activities [33]. Glycosides have been used as flavoring agents in pharmaceutical products and have anti-carcinogenic activity [34]. Phytocompounds like steroids have therapeutic effectiveness as cardiotonic, anti-microbial and insecticidal properties. Moreover, they are useful in enhancing nitrogen retention in osteoporosis [35].

Estimation of mineral contents
The mineral compositions of methanol extract and organic/aqueous fractions are shown in Table 3. Results showed the considerably high amount of manganese in GSME (13.024±0.087 mg/100 g), zinc in NSME (36.019±0.284 mg/100 g), calcium in CAME (372.454±3.633 mg/100g), magnesium in GSME (131.045±1.346 mg/100 g) and copper in GSME (15.961±0.093 mg/100 g). Results revealed that selected plants contain significant quantities of minerals including Mn, Zn, Ca, Mg and Cu. These minerals are an important part of a diet and exist in the body at low percentages. Thus, these plants could be a potential source of major and trace minerals that are required for the normal functioning of human body [36].  (n=3). Here, C. absus: CAME, CAEF, CABF, CAAF, G. sylvestre: GSME, GSEF, GSBF, GSAF, N. sativa: NSME, NSEF, NSBF, NSAF, and P. nigrum: PNME, PNEF, PNBF, PNAF are methanol extract, ethyl-acetate, n-butanol and aqueous fractions, respectively. Small letters (a-d): p<0.05 significant difference between different solvent fractions of the same plant; Capital letters (A-D): p<0.05 significant difference between same solvent type in different plants.

Estimation of total phenolic and flavonoid contents
The total phenolic and flavonoid contents of methanol extracts and solvent fractions of selected plants are shown in Figure 1. Results of total phenolic contents showed a wide variation in fractions, ranging from 13.61±1.25 mg GAE/g of CABF to 179.71±2.14 mg GAE/g of NSME, indicating higher phenolic content of NSME and PNME (179.71±2.14 and 143.91±3.21 mg GAE/g of sample). Total flavonoid content was determined through a calorimetric method, using catechin equivalent as standard.
Results of the present study demonstrated greater flavonoid content in NSME and CAME (189.18±3.71 and 127.71±2.28 mg CE/g of sample). The amount of total flavonoid content of plants studied using https://doi.org/10.37358/RC.21.2.8418 various solvents ranged from 6.79±0.68 mg CE/g of PNBF to 189.18±3.71 mg CE/g of NSME. Polyphenols are gaining increased attention from researchers due to their potential biological activities. The antioxidant capacities of polyphenols, particularly lipid peroxidation inhibition and free radicals scavenging, are pharmacologically most important [37]. Flavonoids are widely distributed phytocompounds that have been established as health-enhancing plant compounds owing to antioxidant chelating or scavenging attributes [38].

DPPH* scavenging activity
Antioxidant activity of foods and medicinal plants has been commonly determined by using DPPH* scavenging assay. The methanolic solution of DPPH* undergoes a reduction in the presence of H +donating antioxidant, appeared as a color change from purple to yellow, which is measured at 517 nm [39]. The scavenging effect of methanol extracts and fractions of C. absus, G. sylvestre, N. sativa and P. nigrum have been shown in Figure 2. Results showed the scavenging activity in the following order C. absus: CAME>CAAF>CABF>CAEF, G. sylvestre: GSME>GSAF>GSEF>GSBF, N. sativa: NSME> NSAF>NSEF>NSBF, and P. nigrum: PNME>PNAF>PNBF>PNEF, respectively. The IC50 values, as mentioned in Table 4, depicted that DPPH* scavenging activities of NSME (23.8 µg/mL) and GSAF (26.2 µg/mL) are close to ascorbic acid (17.3 µg/mL). Meanwhile, IC50 values of GSBF and PNEF were found above 300 µg/mL. It shows that N. sativa acts as a potent antioxidant while C. absus and G. sylvestre possess moderate antioxidant ability and relatively less antioxidant potential of P. nigrum was observed. Figure 3 shows the in vitro superoxide anion scavenging activity of extracts and fractions determined by the riboflavin-NBT-PMS system. Flavins reduction generates superoxide radicals in the presence of light, subsequently causes NBT reduction and forms a blue-colored formazan [40]. In this study, methanol extracts and fractions exhibited potent scavenging activity against superoxide radicals. A concentration-dependent inhibition of blue formazan formation was observed and the highest scavenging potential of NSME was found close to ascorbic acid while GSBF showed the lowest scavenging activity. The scavenging activities were in the following pattern of C. absus: CAME>CAAF>CAEF>CABF, G. sylvestre: GSAF>GSME>GSEF>GSBF, N. sativa: NSME>NSAF>NSBF>NSNSEF, and P. nigrum: PNME>PNBF>PNAF>PNEF, respectively. The IC50 values of extracts and fractions presented in Table

Reducing power activity
The reducing power activities of methanol extracts and fractions of plants in comparison to ascorbic acid, determined by measuring the conversion of Fe +3 to Fe +2 , are presented in Figure 4. Extracts containing reductants exhibit antioxidant action through donating H-atom and halt the free radical chain. In the present study, N. sativa extract and fractions showed relatively high reductive ability in following order NSME>NSAF>NSBF>NSEF than C. absus: CAME>CAAF>CABF>CAEF, G. sylvestre: GSAF> GSME>GSBF>GSEF and P. nigrum: PNME>PNBF>PNAF>PNEF, respectively. Results revealed that NSME (1.123±0.038 O.D. at 300 µg/mL) exhibit the highest reducing power while the lowest reducing power of CAEF (0.436±0.029 O.D. at 300µg/mL) in contrast to ascorbic acid (1.548±0.042 O.D. at 300 µg/mL) was observed. Antioxidant activity of extract has been believed due to peroxides breakdown, reductive capacity on metals, binding of heavy metal ion catalysts, and radical scavenging [41]. Reducing power activities of extracts and fractions of plants increased with increasing the concentrations, similarly to antioxidant activities.

Conclusions
This study revealed the presence of bioactive phytocompounds, minerals, phenolic and flavonoids in plant materials analyzed. The in vitro antioxidant activities for four solvent extracts of C. absus, G. sylvestre, N. sativa and P. nigrum were assessed and solvent effects studied in the present study demonstrated the higher phenolic and flavonoid extraction efficiency of methanol, while ethyl-acetate extraction showed the lowest polyphenol contents. Methanolic extracts exhibited the highest in vitro antioxidant activities followed by aqueous, n-butanol and ethyl-acetate fractions. Medicinal plants are a vital source of phytocompounds that have a potential impact and beneficial effects on general health. Studies like the present investigation are progressively characterizing bioactivities of herbal products and enhancing their applications in healthcare.