Cytotoxic, antioxidant, and antimicrobial activities of Celery (Apium graveolens L.)

Celery (Apium graveolens Linn, Family: Apiaceae) is a common edible herb used as a spice in the traditional medicine of several nations since time immemorial. The whole plant is extensively used in cooking as soups and salads. A. graveolens has various pharmacological properties such as anticancer, anti-obesity, anti-hepatotoxic, and antihypertensive agents. Hence, it is of interest to document the in vitro cytotoxic, antioxidant, and antimicrobial activity of A. graveolens. The plants were collected in the local market, shade dried, and different parts of the plants were extracted with 70% ethanol using a cold maceration process. Antioxidant tests were performed based on the various radical scavenging methods. Antimicrobial activity and MIC were completed using the respective cup-plate and two-fold serial dilution method. In vitro cytotoxic studies were achieved by the MTT; Sulphorhodamine B assayed total cell protein content. DLA and ESC cells determined the short-term toxicity. The leaf extract exhibited significant antioxidant properties against NO, DPPH, ABTS, LPO, and HPO methods. Thus, potential inhibition against Gram-positive, Gram-negative, and fungal strains within the MIC ranges of 250-500 µg/ml was observed. All the extracts of the plant presented in the study revealed greater cytotoxicity effects against five respective cancer cell lines, L6, Vero, BRL 3A, A-549, L929, and L-929 with the ranging of 443-168.5 µg/ml. Thus, we show that A. graveolens possess a potential cytotoxic, antioxidant, and antimicrobial activity.


Background:
Medicinal plants are used worldwide due to its antioxidant and antimicrobial effects that become most popular due to the growing ratio of drug-resistant microorganisms [1]. Nevertheless, abuse of antibiotics has become the primary cause of the development and spreading of multidrug-resistant strains of various classes of pathogens [2][3][4][5]. The antimicrobial effects of diverse medicinal plants and there by products have been extensively studied and several clinically important compounds have been validated [6,7]. The plant extracts and their derivatives have been practiced for hundreds of years in folk and alternative medicine, food preservation, pharmaceuticals, and natural remedies [8,9]. Some vegetables, fruits, spices, herbs, and various parts of the plant extracts have been described to be potential antimicrobial, anticancer, anti-inflammatory, anti-aging, and antioxidant properties [10][11][12]. These antimicrobial/antioxidant properties are mainly based on the occurrence of major bioactive compounds, including alkaloids, phenolic acids, terpenes, glycosides, and flavonoids [13,14]. Even different vegetables have also known to be antimicrobial, anti-inflammatory, anti-aging, anticancer, and antioxidant properties [15,16]. Earlier studies have also demonstrated that the antibacterial and antifungal effects of different plants against various enteric bacteria and common fungi by using their plant extracts and derivatives [17][18][19][20]. Celery (Apium graveolens L. Family: Apiaceae) is commonly consumed as a vegetable and flavoring ingredient in cooking in various nations of Asian Countries. The whole plants including, leaf, stem, root, and seed are extensively used in cooking as soups and salads. It is most popular based on its unique aroma and essential oil. The plant is a good source of carotenes, tocopherols, and vitamins with high quantities of secondary metabolites including flavonoids, alkaloids, terpenoids, and phenolic acids [21,22]. Indeed However, antioxidant and antimicrobial and cytotoxicity investigation has not been yet performed using celery in Saudi Arabia. Phytochemical contents of the plant are often varied within the species as well as nationwide. It is regulated by numerous extrinsic factors including, latitude, longitude, rainfall, physicochemical parameters of water, atmospheric temperature, moisture, soil content, and photoperiods [37]. Due to the adaptation of hydric or salt stress, plants enable to increase their osmotic tension and produce various phytochemicals including, essential oils, organic acids, alkaloids, and saponins that attract insects as well as prevent predators [38]. Thus, the same species in the plants can alter their phytochemical composition due to their extrinsic factors. A recent development toward emerging herbal drugs as complementary to synthetic medicines has elicited greater attention due to their beneficial activities. Based on the attention, herbal plants and vegetables can be characterized as bioactive ingredients, and elucidate their mode of action, therefore, creating them feasible therapeutic ingredients. Therefore, it is of interest to document the in vitro antioxidant, cytotoxic, antibacterial, and antifungal activity of A. graveolens.

Preparation of the plant extracts
The whole plant of A. graveolens L. was splashed with tap water and was shadow air-dried at 37 o C and was applied for the method of 149 ©Biomedical Informatics (2021) solvent extraction. The different parts of the plant (stem, leaf, root, whole plant) were exposed to the method of cold maceration by ethanol (70%). The suspension was exposed to evaporate and the residual crude extract was kept in the freezer for future investigation. The hydroethanolic suspension of stem, leaf, root, and the whole plant was subjected to the phytochemical screening tests as per the standard methods described earlier [39][40][41].

Antioxidant Activity: Radical scavenging method-ABTS:
The assay of ABTS•+ and DPPH has commonly used methods for the determination of the antioxidant capacity of natural products. The free radical scavenging method (ABTS) was adopted according to the earlier publications [42,43]. ABTS (54.8 mg) was dissolved in distilled water (50 ml) to be prepared as a 2 mM solution, and added along with 0.3 ml of 17 mM potassium persulphate, and kept at 37 o C overnight. Added to 0.4 ml of each concentration of the plant extracts and standards, DMSO (1.0 ml), and ABTS (0.2 ml) to make up the suspension of 1.6 ml and kept 20 minutes for incubation. Color intensity was read at 734 nm by using an ultraviolet (UV)-spectrophotometer. The value of IC50 is determined by the sample concentration, which requires to scavenge 50% radical of ABTS. The below formula was applied to estimate the inhibition (%). % Anti-radical activity = [(A0 -A1 / A0) ×100].
Where A0 -control of color intensity (blank, without extracts). A1 was the color intensity of the solvent extracts. The radical scavenging activity of vitamin C was also calculated and evaluated with the diverse solvent extracts.

Radical scavenging method-DPPH:
The DPPH radical scavenging methodology was adopted according to the previous publications [42,43]. In vitro assay was achieved in microtitre plates (96 well). Added DPPH solution (200 µl) to different plant extracts (10 µl) or the standard solution and kept the plates at room temperature for 30 minutes incubation and the color intensity was read at 490 nm using a UV-spectrophotometer. The value of IC50 is determined by the sample concentration, which requires to scavenge 50% radical of DPPH.
Where A0 -control of color intensity (blank, without extracts). A1 was the color intensity of the solvent extracts.

Radical inhibitory activity-NO:
NO radical inhibition assay was achieved based on the prior publications [44,45]. Added to sodium nitroprusside (4ml, 10 mM), PBS (1 ml), and plant extract in DMSO (1ml) and make up the final volume of 6 ml in the microtitre plates that were incubated at room temperature for 2 hours. Then, a suspension comprising nitrates (0.5 ml) was eliminated and added sulphanilic acid (1 ml), shaken well, and kept at the finishing point of diazotization for at least 5 min, and added NADD (1 ml), and kept in diffused light at 37 o C for 30 min. The color intensity of the suspension was read at 540 mm using a UV-Spectrophotometer. The value of IC50 is determined by the sample concentration, requires inhibiting 50% NO radical.

Radical scavenging method-HPO
The scavenging of HPO radical was performed based on the former publications [45,46]. Added to different parts of the plant extracts (1 ml) in methanol to HPO (2 ml; 20 mM HPO in PBS). After 15 min incubation, the color intensity was read at 230 nm using a UV-Spectrophotometer. The blank was the plant suspension in PBS without HPO.

Radical scavenging method-LPO
LPO was assessed by the TBARS method in accordance with previous literature [47 -49]. Added to100 µl of the different plant extracts to lipid mixture (1 ml) and the blank was set without the extracts of the plant. The reaction of LPO was triggered by the addition of oxidant compounds, FeCl3 (400 mM, 10 µl) and ascorbic acid (200 mM, 20 µl), and kept incubation for an hour at 37ºC. The reaction was cessation by the addition of HCl (0.25 N, 2 ml) containing TCA (15%) and TBA (0.375%) and the mixture was heated for 20 min; cooled; centrifuged, and then the color intensity of the suspension was read at 532 nm using UV-Spectrophotometer.

Antimicrobial activity:
The antibacterial and antifungal activity was performed by the method of cup-plate [13, 14, 50]. Briefly, the sterile Petri plates were prepared using Sabourd dextrose agar or sterile nutrient agar (Himedia) and kept in aseptic conditions. About 100 µl of the test organisms [Gram-positive (Bacillus aerogenes, B. coagulans, B. megatarium, B. subtilis, Lactobacillus lichmani, Staphylococcus aureus), Gram-negative (Kleibsella pheumoniae, Pseudomonas aeruginosa, Salmonella typhi, Shigella), and fungal strains (Aspergillus niger, A. flavus, Candida albicans, Cryptococcus neoformans, Trichophyton rubrum)] were spread on the defined sterile plates. Holes (diameter, 5 mm) were made by a sterile bore in the plates. The standard antibiotics and plant extracts were poured into respective holes and the plates were kept for at least an hour at 4 o C to permit the ©Biomedical Informatics (2021) diffusion into the agar medium followed by 24-48 h incubation at 37ºC.

Assay of Minimum inhibitory concentration:
MIC was performed using different parts of the plant by the method of two-fold dilution [14, 17, 19, 51-55]. Sequences of test tubes were used to contain an equal volume of medium spread with the test organisms. Reduced volume of test drugs was introduced into the tubes, typically, a succeeding two-fold dilution was adopted. In this, one tube remained without any test drug, aided as a positive control. The cultures were maintained in the incubator according to the respective strains and temperature (bacteria: 37ºC for 24 hours, Fungi: 27ºC for 48 hours) which is necessary for the multiplication of up to 15 generations of the strains. Then, the growth of the organisms was visualized based on the turbidity, however, antimicrobial agents in the tube inhibited the growth of the organism, showed transparency. Hence, MIC is the drug concentration existing in the clear tube, in which the lowest drug concentration could not generate any growth.

Cytotoxic studies: MTT assay:
In vitro cytotoxic investigation was achieved by MTT assay [56]. Briefly, the cultured cells were trypsinized and the viable cells (1.0 x 10 5 cells/ml) were inoculated in the medium containing 10 % BSA. About 0.1 ml of diluted cell suspension (10,000 cells) was added into each well of 96 well microtitre plates. A limited monolayer was formed after 24 hours and washed with the medium, added 100 µl of different drug concentrations. The plates were then kept in the CO2 incubator chamber at 37ºC in a 5% CO2; microscopic observation was performed and noted every 24 hours. The drug in the well was washed and added 50 µl of MTT in DMEM medium without phenol red for 72 hours. Then the plates were gently mixed and kept in the CO2 incubator chamber for 3 hours. The upper suspension was removed and added propanol (50 µl). The color intensity was read at a wavelength of 540 nm using a microplate reader. The % of growth inhibition was measured by the following formula: Growth inhibition (%) = 100−Mean OD of testMean OD of ControlX100

Assay of total cell protein content:
The assay of total cell protein composition was performed by SRB method [57]. The cultured cells were trypsinized and the viable cells (1.0 x 10 5 cells/ml) were inoculated in the medium containing 10 % BSA. About 0.1 ml of diluted cell suspension (10,000 cells) was added into each well of 96 well microtitre plates A limited monolayer was formed after 24 hours and washed with the medium, added 100 µl of drug concentrations. The plates were then kept in the CO2 incubator chamber at 37ºC in a 5% CO2; microscopic observation was performed and noted each 24 h. Next 72 hours, 50% TCA (50 µl) was added to the wells and observed a tinny layer over the dilutions of the drug to become 10% concentrations; incubated for at least an hour at 4ºC; then washed thrice to eliminate the turbid, and then air-dried. Then stained with SRB and maintained for 30 min under room temperature. The dye was then detached by quick washing using acetic acid (1%), air-dried, and mixed with 100 µl of Tris buffer (10 mM) to dissolve the dye. The color intensity was read at 540 nm using a microplate reader and calculated using the following calculation: Growth inhibition (%) = 100−Mean OD of testMean OD of ControlX100

Studies of short-term toxicity
A study of short-term toxicity and antitumor screening was achieved using DLA and EAC cells [58]. The cell suspension was inoculated into the mouse peritoneal cavity through injecting the dense cell volume of 1.0x 10 5 cells/ml. Then, the cells were introverted using a sterile syringe from the peritoneal cavity of the mouse while it became inflammation for about 12-14 days (This study was approved by the Committee on the Use of Live Animals in Teaching and Research of the university (Permit number: 4722-18). The cells were splashed thrice using HBBS and spinned for 3 min at 1,500 g. A known volume of HBSS used to suspend the cells and counting of the cells was maintained to 2 x 10 6 cells/ml. These cells were then treated with the drug and kept incubation for at least 3 hours. After incubation, a dye exclusion test was performed. The volume of the drug-treated cells and trypan blue (0.4%) equally were combined and loaded into a hemocytometer and the viable and nonviable cells were counted.

% Of inhibition = 100−Total Number of cells -Dead cellsTotal number of CellsX100
Results: Different plant parts (leaf, root, stem, and whole plant) used and the percentage of crude extracts of A. graveolens L. was presented in Table 1. All these extracts obtained by the cold maceration process, which were undergone to qualitative preliminary phytochemicals screening. The outcome of the study showed that A. graveolens L. contains the common secondary metabolites such as glycosides, tannins, saponins, flavonoids, steroids, terpenoids, alkaloids, carbohydrates, proteins, anthraquinones, are shown in Table 1. The whole plant of the A. graveolens L. showed higher antioxidant ©Biomedical Informatics (2021) properties, listed in Table 2. The value of IC50 is determined by the sample concentration, requires inhibiting 50% NO radical. The leaf extract of A. graveolens L exposed the higher antioxidant properties with an IC50 value of 16.23 ± 0.147 µg/ml. In addition, the leaf extract of A. graveolens L. exhibited potent antioxidant activity in all DPPH, LPO, and hydrogen peroxide method when compared to the root, stem, and whole plant extracts. All four parts of A. graveolens L were screened for their antimicrobial effects against six Gram-positive viz. , Bacillus aerogenes, B. coagulans, B. megatarium, B. subtilis, Lactobacillus lichmani, Staphylococcus aureus; four Gramnegative viz., Kleibsella pheumoniae, Pseudomonas aeruginosa, Salmonella typhi, Shigella strains and five fungal strains viz., Aspergillus niger, A. flavus, Candida albicans, Cryptococcus neoformans, Trichophyton rubrum which were performed by the cup-plate method; MIC was measured by the method of two-fold serial dilution, and both results are tabulated in Table 3. All four parts of A. graveolens L demonstrated to possess high inhibition against Gram-positive and negative strains extended between 12-21 mm and listed fungal strains had 07-21 mm. The value of MIC of A. graveolens L extended to 250-500 µg/ml and the outcomes are presented in Table 3. Cytotoxic investigations of A. graveolens L were achieved using five various cell lines namely, Vero, 3A, L-929, A-549, and L6.BRL is listed in Table 4. The cytotoxic analysis was determined using microbial growth inhibition, which was achieved by the common methods of MTT and SRB. The crude extracts of stem, leaf, root, and whole plant demonstrated moderate to high cytotoxic effects in all respective five-cell lines with the value of cytotoxicity-50 stretching between 443-168.5 µg/ml. Short-term toxicity was studied using DLA and EAC cells, listed in Table 5. The short-term antitumor investigation was done by DLA and EAC that were newly obtained from the mouse peritoneum. Both these cells were treated with the extract of the leaf with dilutions ranges from 62.5 µg/ml-1000 µg/ml and viability of the cell was noted.     The study of short term in vitro cytotoxic numbers also shows that anticancer activity of crude extracts of A. graveolens presented counter to EAC and DLA, thereby A. graveolens caused noteworthy cancer cell demise and cell growth inhibition in all respective six different cancer cell lines. Based on the present study, the constituents of any of the metabolites in the plants, especially, flavonoids and phenolic acid constituents may be recognized to the anticancer activity of the plant extract. The current study focuses on the preliminary phytochemical screening, antioxidant, and anticancer activity of A. graveolens; the underlying mechanisms of cancer-selective mechanisms and the active principles of A. graveolens accountable for the activity are underway.

Conclusion:
Data shows that crude extract of A. graveolens exhibit cytotoxic, antibacterial, antifungal, and antioxidant activity. The plant extract contains bioactive principles that are known to be free radical scavengers, cytotoxic against various tumor cell lines, and active in the inhibition of Gram-positive, Gram-negative, and fungal strains for further consideration.