Antioxidant, cytotoxic, antibacterial and thrombolytic activities of Centella asiatica L.: possible role of phenolics and flavonoids

Centella asiatica L. (Apiaceae), a green leafy vegetable, has been used as a popular folk medicine in Bangladesh in the treatment of various ailments. The plant is reported to contain phenolics and flavonoids, but their bioactive potentials are not yet completely known. The present study was designed to investigate the role of the phenolic and flavonoids in the antioxidant, cytotoxicity, antibacterial and thrombolytic activities. Four solvent fractions viz. methanol (MSF), ethyl acetate (ESF), chloroform (CSF) and petroleum ether (PSF) were prepared from the dried powder of the whole plant by the modified Kupchan method. Total phenolic content and flavonoid content were determined by Folin Ciocalteu method and aluminum chloride colorimetric method, respectively. The antioxidant activity was assessed by the DPPH radical scavenging and total antioxidant capacity assays. The antibacterial activity was determined by the disc diffusion method and cytotoxicity was evaluated by the brine shrimp lethality bioassay. Thrombolytic activity was assayed using streptokinase as standard. Qualitative analysis of phytochemical revealed the presence of phenolics and flavonoids along with other bioactive constituents. Among the extractives, CSF contained the highest content of phenolics (155.46 ± 0.52 mg GAE/g) and flavonoids (345.17 ± 1.12 mg QE/g) and exhibited the most potent antioxidant activity in terms of total antioxidant capacity (179.01 ± 0.89 mg AAE/g) and DPPH scavenging ability (IC50; 15.31 ± 0.32 µg/mL). Similarly, CSF showed the highest cytotoxicity with LC50 values of 13.80 ± 0.23 µg/mL, and thrombolytic activity with 43.94 ± 0.62% clot lysis. The fraction also exhibited broad spectrum antibacterial activity. A significant correlation was observed between the flavonoid content and total antioxidant activity (r2 = 0.894, p < 0.05), while high correlation was seen between phenolic and flavonoid content and DPPH radical scavenging, total antioxidant capacity and cytotoxicity (r2 = 0.612–0.928). Similarly, a positive correlation was found between phenolic and flavonoid content with thrombolytic and antibacterial activities. These results revealed that C. asiatica is a rich source of phenolics and flavonoids and correlated with antioxidant, cytotoxicity, antibacterial and thrombolytic activities. Hence isolation of phenolics and flavonoids from this plant may offer potential candidates which may be effective in the prevention of many chronic diseases.


Introduction
Fruits and vegetables rich in nutrition and nutraceuticals have received a great deal of attention due to their potential health benefits. Epidemiologic studies suggest that consumption of fruits and vegetables reduce the risk of cancer, cardiovascular disorder and many other chronic diseases [1][2][3]. They contain different classes of phytochemicals such as phenolics and flavonoids, alkaloids and saponins, steroids and glycosides which are attributable to the bioactivities. Among the phytoconstituents, phenolics and flavonoids have attracted much interest as they contain hydroxyl groups and able to neutralize the free radicals, quenching singlet and triplet oxygen, or decomposing peroxides by donating an electron or a hydrogen atom. They are commonly known as natural antioxidant and found effective in the prevention and repair of oxidative damage [4][5][6]. It is well known that free radicals and reactive oxygen species (ROS) liberated from the cell are capable of destroying cellular constituents and lead to the development of numerous chronic diseases, such as cancer, Alzheimer's disease, Parkinson's disease, diabetes and coronary heart diseases [7]. A large number of studies have shown that malignant cells have greater levels of ROS which can promote carcinogenesis by causing oxidative damage to DNA and macromolecules within cells, altering signal transduction pathways, and promoting a malignant phenotype [8]. Dietary antioxidants such as flavonoids have been shown to prevent or slow down malignancy by reducing oxidative damage and promoting DNA repair [9].
Thrombosis is the pathophysiological process of the formation of a blood clot within the blood vessel obstructing the flow of blood through the circulatory system. The consequence of thrombosis is embolism, ischemia, heart attack, stroke and so forth [10]. It is one of major cause of mortality and morbidity. A handful of drugs including tissue plasminogen activator, urokinase, streptokinase is currently in use [11], thus demands for development of new or alternative drugs for its treatment. Plants with high content of polyphenols have been reported to exhibit good thrombolytic activity [12,13].
The burden of bacterial infection is high in the globe and the condition has been exacerbated due to increasing resistance to antimicrobial agents [14]. Indiscriminate use of antimicrobial agents and mutational capacity of bacteria results in the development of antimicrobial resistance strain. Edible plants rich in antioxidants have shown therapeutic potential in the treatment of bacterial infection [15]. Hence search of plant for high content of phenolic and flavonoid with potential bioactivity is important to develop alternative medicine or functional food for prevention and management of the diseases.
Centella asiatica L., locally known as Thankuni is a member of the Family Apiaceae and found in marshy areas all over Bangladesh. The plant is widely considered as a health food due to high content of nutrients and health promoting properties [16]. Its leaves are eaten as raw or mixed with other green vegetable. In traditional medicine, the leaves are commonly used in the treatment of dysentery, cholera, tuberculosis, common cold, urinary tract infection, leprosy, psoriasis and eczema, bronchitis and asthma, wound healing and gastrointestinal problem [17,18]. The plant has been described in Ayurvedic medicine to improve cognitive function and to treat mental and neurological disturbances [19]. Biological investigations of this plant have shown a multitude of bioactivities including antioxidant, antimicrobial, neuroprotective, thrombolytic, and cytotoxic activities [20][21][22][23][24][25]. Phytochemical analysis revealed the presence of triterpenoids, steroids, alkaloids, saponins, phenolics and flavonoids [26,27]. Much work has been done on the neuroprotective properties and found the triterpenes as the major active constituents for cognitive activity [28,29]. Although the plant has been reported to contain the phenolics and flavonoids [30,31], their biological activities are not yet clearly known.
In the present work, we have prepared four solvent fractions of different polarity from the dried powder of Centella asiatica, determined their phenolics and flavonoids content, and evaluated the antioxidant, cytotoxicity, antibacterial, and thrombolytic activities. Finally, Pearson's correlation studies were performed to analyze the relationship between phenolics and flavonoids with the bioactivities.

Collection of plant materials
The whole plant thankuni (Centella asiatica, Family: Apiaceae) was collected from rural areas of Narayanganja district, Bangladesh when it became mature during April-May, 2018. The plant was taxonomically identified by the expert of Bangladesh National Herbarium (BNH), Dhaka, Bangladesh where a voucher specimen has been deposited (DACB 41,559) for future reference. The plant materials were cleaned, cut into pieces, sun-dried and finally dried in an oven at 45 °C and crushed.

Preparation of plant extract
The 250 g powdered materials were soaked in 1.5 L of methanol in an amber-colored glass bottle at room temperature for 7 days with occasional shaking. The extracts were filtered to a clear solution and concentrated with a rotary evaporator under reduced pressure at 50 °C and evaporated to dryness (MSF: 18.0 gm, 7.2%w/w). An aliquot (10.0 g) of the methanol extract (MSF) was partitioned with petroleum ether, chloroform, and ethyl acetate according to the modified Kupchan protocol [32]. Yield was calculated based on the starting material. The resultant fractions were evaporated to dryness to yield pet-ether (PSF, 2.13 g, 1.534%), chloroform (CSF, 2.2 g, 1.584%), and ethyl acetate (ESF, 3.12 g, 2.26%) soluble fractions. All these fractions were then stored in a refrigerator until further use.

Organisms and chemicals
Cultures of bacteria and shrimp eggs were collected from the Institute of Nutrition and Food, University of Dhaka, and International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). Kanamycin disc (K-30) from BBL and Cookeville (TN, USA); nutrient agar media was purchased from Becton, Dickinson and Company (Sparks, MD, USA). Dimethyl sulphoxide (DMSO), DPPH, gallic acid, quercetin and Folin-Ciocalteau (FC) reagent were from BDH chemicals Ltd., Poole, England. The standard drugs streptokinase, ascorbic acid, and vincristine sulfate (VC) used in this study were obtained as kind gifts from Beacon Pharmaceuticals Ltd., Bangladesh. All other chemicals and organic solvents were of the highest grade.

Phytochemical analysis
Qualitative tests were performed on the crude methanol extract (MSF) to detect the presence of different phytochemicals including phenolics, flavonoids, alkaloids, saponins, and phytosterols by using the standard phytochemical methods as described earlier [33].

Determination of total phenolic content
The total phenolic contents of the fractions of C. asiatica were assayed by Folin-Ciocalteu method as described earlier [34]. To a mixture of 2.5 ml of 10% (v/v) Folin-Ciocalteu reagent and 2.5 mL of 7.5% (w/v) sodium carbonate solution, 0.5 ml sample was added and left in the dark for 20 min at 25 °C. The absorbance of the reaction mixture was recorded by a spectrophotometer (Shimadzu, Japan) at 760 nm. A standard curve was obtained for gallic acid and the phenolic content was determined from extrapolation of this curve.

Determination of total flavonoid content
The total flavonoid contents of the fractions of C. asiatica were estimated by aluminum chloride colorimetric method as described [35]. To a mixture of methanol (3.0 ml), 10% w/v AlCl 3 (0.2 mL), 1 M potassium acetate (0.2 ml) and 5.6 mL of distilled water, individual fraction (1.0 mL) was added and left at room temperature for 30 min. The absorbance of the reaction mixture was recorded by a spectrophotometer at 420 nm. A standard curve was obtained for quercetin and the flavonoid content was determined from extrapolation of this curve.

Antioxidant activity Total antioxidant capacity
The antioxidant capacity of the fractions of C. asiatica was assessed by the method as described earlier [36]. To a mixture of sulphuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM), the individual fraction was added and heated in a water bath at 95 °C for 90 min. After cooling to room temperature, the absorbance of the mixture was recorded at 695 nm against blank. A standard calibration curve was prepared using ascorbic acid and the total antioxidant capacity was determined from extrapolation of this curve.

DPPH free radical scavenging assay
The ability of the fractions of C. asiatica to scavenge DPPH radical was determined by the method as described by Brand-Williams et al., [37]. Ascorbic acid was used as positive control. Methanolic solution of fraction or standard compound was mixed with 0.135 mM of methanolic DPPH and left in dark for 30 min. The absorbance of the reaction mixture was recorded at 517 nm. The percent scavenging was calculated using the equation: where, A control is the absorbance of control and A sample is the absorbance of extract or standard compound. IC 50 values were determined from the straight-line equation obtained from the plot of % inhibition against log of sample concentration.

Cytotoxicity
The cytotoxic activity of the fractions of C. asiatica was determined by the Brine shrimp (Artemia salina Lech) lethality bioassay [38]. Briefly, shrimp eggs were allowed for 36 h in simulated seawater to hatch and mature as nauplii (Larvae). Five milligrams of each fraction was dissolved in 1.0 ml DMSO. Test solutions 5, 10, 20, 40, and 80 μL were taken in separate vials and 5 mL of the seawater was added to each vial containing 20 nauplii. A control group was used containing 80 μL of DMSO and 20 nauplii in 5 mL of seawater. After 24 h, the number of survivors in each vial was counted. The LC 50 (median lethal concentration) values were determined from the straight-line equation obtained from the plot of % mortality against log of sample concentration.

Antibacterial activity
In-vitro antibacterial activity of the fractions of C. asiatica was performed against five gram-positive (Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Sarcina sarcinaceae, and Bacillus cereus) and six gram-negative (E. coli, Shigella dysenteriae, Shigella shiga, Pseudomonas aeruginosa, Vibrio mimicus, and Salmonella typhi) bacteria by disc diffusion method [39]. The fractions MSF, PSF, CSF, and ESF were dissolved in a sufficient amount of methanol individually, so that 10 μL solution contains 100 μg of crude extract. The inhibitory activity was performed with 400 μg of extractives per disc. A disc with 40 µL methanol was used as the negative control. The test bacteria were inoculated in sterilized nutrient agar media, mixed thoroughly and transferred to an aseptic condition immediately. The disc containing test fraction and standard drug were placed in the petri dish and were incubated at 37 °C for 24 h. Clear zones of inhibition were measured in millimeter. The antibacterial activity of the fractions was compared with the standard kanamycin disc (K-30).

Thrombolytic activity
The thrombolytic activity of the fractions of C. asiatica was evaluated by the method as described earlier [40]. To prepare the sample, each fraction (100 mg) was suspended in 10 ml of sterile isotonic solution and shaken vigorously. The suspension was kept overnight and the supernatant was collected by filtration through a filter paper for in vitro evaluation of clot lysis activity. Streptokinase (SK) was used as positive control and isotonic solutions as negative control. Venous blood from volunteer was distributed in pre-weighted sterile microcentrifuge tubes and then incubated at 37 °C for 45 min. Serum was removed completely after clot formation and the weight of clot was measured. To each tube containing clot, sample or standard drug or isotonic solution were added separately. After incubation at 37 °C for 90 min, the released fluid was removed and tubes were again weighed. The differences in weights were used to calculate the percent of clot lysis according to the following equation:

Statistical analysis
All the assays were done in three replicates of each sample and values were taken as mean ± SD by statistical analysis. Microsoft excel 2010 were used to calculate the experimental results. Significant differences (p-value < 0.05) between the means were determined using the t-test. Correlation studies were performed using Pearson's correlation test.
Percent of clot lysis = weight of released clot ∕ clot weight × 100%

Phytochemical analyses Qualitative phytochemical analysis
Qualitative analyses of the crude methanol extract (MSF) of C. asiatica indicated the presence of phenolics, flavonoids, alkaloids, tannins, steroids, and glycosides (Supplementary Table S1).

Total phenolic content (TPC)
The total phenolic content of the four solvent fractions of C. asiatica was estimated by Folin-Ciocalteu method and the result is shown in the

Total flavonoid contents (TFC)
The flavonoid content of the different fractions of C. asiatica was determined by aluminium chloride colorimetric method and the result is presented in the Table 1. The total flavonoid content of MSF, PSF, CSF and ESF were 211.34 ± 0.98, 90.52 ± 0.52, 345.17 ± 1.12, and 193.68 ± 0.99 mg of QE/g of extract, respectively, suggesting that CSF exhibited the highest content of flavonoid followed by MSF, ESF, and PSF which is similar to the phenolic content.

Antioxidant activity Total antioxidant capacity (TAC)
The total antioxidant capacity of the different fractions of C. asiatica was determined based on their ability to reduce Mo (VI) to Mo (V) and the result is given in the Table 2. The total antioxidant capacities of MSF, PSF, CSF, and ESF of C. asiatica were 102.32 ± 0.58, 68.97 ± 0.48, 179.01 ± 0.89, and 80.11 ± 0.53 mg of AAE/g of extract, respectively. Among the four fractions, CSF showed the highest capacity and PSF, the lowest (Fig. 1A). Using Pearson's correlation analysis, a significant correlation was observed between TFC and total antioxidant capacity (r 2 = 0.894, p < 0.05), whereas a high correlation was seen between TPC and total antioxidant capacity (r 2 = 0.649) ( Table 3).

DPPH radical scavenging assay
The free radical scavenging potential of the fractions of C. asiatica was evaluated by spectrophotometric method using the synthetic DPPH free radicals. The percent scavenging of DPPH radical at various concentrations of the fraction was calculated and the results have been shown in the Fig. 1B. Our results demonstrated the ability of the fractions to scavenge the free radical. For comparison of potency, the IC 50 values of each of the fractions were calculated and shown in the Table 2. Among the fractions, CSF showed highest activity with the IC 50 value of 15.31 ± 0.32 µg/mL followed by MSF, ESF and PSF. Ascorbic acid was used as standard whose IC 50 was found to be 9.94 ± 0.42 µg/ml. From correlation analysis, we found high correlation of TPC and TFC with DPPH scavenging activity (r 2 = 0.728 & 0.612) ( Table 3).

Cytotoxicity
The in-vivo cytotoxicity of the fractions of C. asiatica was determined against brine shrimp by brine shrimp lethality bioassay. The cytotoxicity of the fractions was expressed in terms of LC 50 values which were estimated from the straight line obtained from the plot of % mortality versus the log of sample concentrations ( Fig. 2A & 2B). The LC 50 values of MSF, CSF, and ESF were found to be 25.47 ± 0.45 µg/mL, 13.80 ± 0.23 µg/ mL and 17.82 ± 0.34 µg/mL, respectively. These results indicated the highest cytotoxicity in the CSF followed by ESF and MSF. Under the same experimental condition, the standard cytotoxic agent vincristine showed an LC 50 of 1.85 ± 0.23 µg/mL. Correlation studies showed a significant correlation between total phenolic content and cytotoxicity (r 2 = 0.928).

Antibacterial screening
The antibacterial activity of the fractions of C. asiatica was screened against eleven Gram-positive and Gramnegative bacteria by disc diffusion method. The results of antibacterial activity have been shown in the Table 4. All the fractions exhibited inhibition against test bacteria having the zone of inhibition ranging from 5 to

Thrombolytic effect
The different fractions of C. asiatica were assayed for thrombolytic activities by determining the ability to clot lysis. Streptokinase (SK) was used as a positive control that showed 63.34 ± 0.58% lysis as compared to the isotonic solution that had 7.0 ± 0.23% lysis activities. In this study, the different fractions of C. asiatica exhibited varying degrees of thrombolytic activities ranging from 17.58 ± 0.23% to 43.94 ± 0.62% (Table 5). Highest clot lysis activity (43.94 ± 0.62%) was found in CSF followed by PSF, ESF and MSF.

Discussion
Natural foods or food derived nutraceuticals are considered to be the promising approach at present for the prevention and treatment of various chronic diseases.
Interest in foods has increased so much in recent times because they are safe and exhibit multiple biological and therapeutic activities due to the presence of high content of diverse secondary metabolites [41,42]. Many food    [17][18][19]. Phenolics and flavonoids are important classes of phytochemicals that exhibit antioxidant and other biological activities [4][5][6]. Although the occurrence of these phytochemicals is evident in this plant [30,31], little is known about their roles in the bioactivities. Herein we report for the first time that C. asiatica is a rich source of phenolics and flavonoids and correlated with antioxidant, cytotoxicity, thrombolytic and antibacterial activities.
The methanol extract (MSF) of C. asiatica were fractionated into three different fractions PSF, CSF and ESF for phytochemical and biological investigation. Preliminary analysis of MSF revealed the presence of steroids, glycosides, tannins, alkaloids, phenolics and flavonoids as the potential bioactive constituents (Supplementary Table S-1). Quantitative analysis of phenolic and flavonoid content revealed that the CSF contained the highest content of phenolics (155.46 ± 0.52 mg GAE/g dried extract), flavonoids (345.17 ± 1.12 mg QE/g dried extract) followed by ESF (116.59 ± 0.57 mg GAE/g dried extract and 193.68 ± 0.99 mg QE/g dried extract), MSF (88.62 ± 0.41 mg GAE/g dried extract and 211.34 ± 0.98 mg QE/g dried extract), and PSF (34.76 ± 0.21 mg GAE/g dried extract and 90.52 ± 0.52 mg QE/g dried extract) ( Table 1). The phenolic and flavonoid content in the methanol extract were reported earlier by Zainol et al. [20] and Mustafa et al. [21] which is consistent with our result. Numerous phenolic compounds such as ferulic acid, chlorogenic acid, isochlorogenic acid, 3,5-di-O-caffeoyl quinic acid, 1,5-di-O-caffeoyl quinic acid, and 3,4-di-O-caffeoyl quinic acid have been isolated from the leaves of C. asiatica [30]. Similarly, two new flavonoids castilliferol and castillicetin has been identified from the plant in addition to the common flavonoids quercetin, catechin, kaempferol, kaempferol-3-O-b-D-glucoside, and quercetin-3-Ob-D-glucoside [31]. Our results revealed that C. asiatica is a rich source of ployphenolics. The presence of large amount of polyphenolics in the CSF suggests that it might play a role in biological activity.
Natural antioxidants are capable of ameliorating the oxidative damage induced by free radicals and thus considered to be the potential candidates for treating many chronic disorders [4][5][6]. In this study, we have assessed the antioxidant activity of the fractions using the two widely accepted antioxidant assays such as DPPH radical scavenging and total antioxidant activity assays. In both assays, CSF showed the highest activity followed by MSF and ESF (Fig. 1). The IC 50 values of the fractions for DPPH radical scavenging was 15.31 ± 0.32, 24.19 ± 0.52 and 28.83 ± 0.61 µg/mL, respectively, while the same were 179.01 ± 0.89, 102.32 ± 0.58, and 80.11 ± 0.53 mg of AAE/g of fraction for total antioxidant activity. The antioxidant activity of the methanol extract of C. asiatica has been reported earlier by Zainol et al., [20] and Mustafa et al., [21] which is consistent with our result. In many plants, the antioxidant activity has been found to be correlated with the phenolics and flavonoids content [6][7][8]. We also found a strong correlation of TPC and TFC with DPPH scavenging and total antioxidant capacity (Table 3). Our results suggested that CSF possesses strong antioxidant activity and that might be able to prevent or reduce the oxidative damage in oxidative stress induced diseases.
Brine shrimp lethality bioassay is a simple method used widely for evaluating the possible cytotoxicity of the plant extract/compound. This bioassay has shown a good correlation with the human tumor solid cell lines [43]. Antioxidants from dietary sources was shown to slow down the pathogenesis of cancer [9]. In this study we found the highest cytotoxicity in the CSF with LC 50 of 13.80 μg/mL followed by ESF and MSF (Fig. 2). Earlier Soyingbe et al., [44] reported that methanol and ethyl acetate extract of C. asiatica have significant anti-proliferative effect in several human cancer cell lines including human breast adenocarcinoma (MCF-7) cells, human colorectal carcinoma cells (Caco), human cervical cancer cells (HeLa) [36]. From Pearson,s correlation studies, we found a high correlation between phenolic content and cytotoxicity (r 2 = 0.928) and a moderate correlation between flavonoid content with cytotoxicity (r 2 = 0.473) ( Table 3). A similar relationship between flavonoid content and anti-tumor activity has been shown earlier by Pittella et al., [22] in human and mouse cancer cell lines. Our results suggest that CSF has high cytotoxicity which require investigation against these cell lines.
Plant has enormous potential to cure bacterial infection due to presence of antibacterial constituents. Antioxidants from plants have been reported to exhibit potential antibacterial activity [45]. In this study we found the antibacterial activity in all the test fractions (Table 4). A comparison of activity between the fractions revealed that CSF is the most active against both the Gram-positive and Gram-negative bacteria followed by ESF, MSF and PSF. It is worth noting that CSF displayed the highest zone of inhibition (30 mm) against S. dysentriae which support its traditional use in dysentery. ESF and MSF also showed good activity against the test organisms. PSF was the least active. In a previous study, Panda et al., [15] reported the antibacterial activity in the methanol and aqueous extract of C. asiatica, which is in good agreement with our result. Our results revealed that CSF exhibited the highest antibacterial activity among the test fractions and the activity showed a positive correlation with phenolics and flavonoids.
Thrombus formation is the key to the development of many vascular complexities [10]. Blood clotting is a complex process that involves a series of events. Majority of the thrombolytic agents activate plasminogen that breaks down the cross-linked fibrin mesh work which is further proteolysed by other enzymes [11]. In this study, we found the highest thrombolytic activity in the CSF followed by PSF and ESF. The thrombolytic activity of the methanol extract and ethyl acetate soluble phase was shown first by Satake et al., [30] which is in accordance with our result. Similar to antibacterial activity, a positive correlation was apparent between phenolics and flavonoids and thrombolytic activity. Our data showed the highest thrombolytic activity of CSF and the activity is associated with the polyphenolic content.

Conclusion
In conclusion, this study showed that C. asiatica is a rich source of phenolics and flavonoids and correlated with antioxidant, cytotoxicity, antibacterial and thrombolytic activities. These findings suggest a role of phenolics and flavonoids in the bioactivities. Further exploration of this plant may lead to the development of bioactive phenolic and flavonoid compounds which may be effective in the management of many chronic diseases.