Stem extract of Albizia richardiana exhibits potent antioxidant, cytotoxic, antimicrobial, anti-inflammatory and thrombolytic effects through in vitro approach

Albizia richardiana belongs Fabaceae family which different parts like fruits, flowers, barks, and roots are used medicinally. The study reports the in vitro anti-inflammatory, thrombolytic, cytotoxicity and antimicrobial activity of methanolic extract of A. richardiana stem and its different fractions. The methanolic extract of A. richardiana stem (MEAR) extracted with n-hexane (HXFAR), carbon tetrachloride (CTFAR), chloroform (CFAR), and aqueous (AQFAR) and subjected for DPPH scavenging activity and total phenol content (TPC). The cytotoxic activity evaluated by brine shrimp lethality bioassay, while the disk diffusion method used for the antimicrobial study. The anti-inflammatory and thrombolytic activities of the extracts evaluated by the hypotonic solution induced hemolysis, heat-induced hemolysis and human blood clot lysis, respectively. All the extracts exhibited excellent antioxidant activity in the DPPH scavenging assay and maximum total phenol content observed by HXFAR. Secondly, the extract showed a moderate LC50 value in brine shrimp lethality bioassay, where the CTFAR extract exhibited potential antimicrobial activities against sixteen different microorganisms. In anti-inflammatory, all the extract exhibited a significant (P < 0.0001) protection against lysis of human erythrocyte membrane induced by heat and hypotonic solution, as compared to the standard acetyl salicylic acid. An extremely significant (P < 0.0001) clot lysis was found in MEAR (16.66%) while the standard drug streptokinase (70.94%). All the fractions revealed the significant free radical scavenging activity. Moreover, CTFAR showed wide spectrum of antimicrobial activity. Thus, the results of the present study provided scientific evidence for the use of Albizia richardiana as traditional medicine.


Background
Plant is a resourceful preference to produce drug which is important to keep the world healthy [1]. Plants are used to cure, prevent the diseases whereas 75% of world population depends on herbal drugs for their primary healthcare [2,3]. Albizia richardiana King & Prain belongs Fabaceae family which is found in Asia, Africa and Australia [4]. Different parts like fruits, flowers, barks, roots etc. are used medicinally [5]. A. richardiana are used in treating depression, appetite, increase blood circulation, tightness in chest, eye problems, blurred vision and pain in back [6]. This plant reported to have several secondary metabolites in bark such as carbohydrates, saponins, glucosides, glycosides and alkaloids. It was also reported to have significant antioxidant activity while lethal concentration (LC 50 ) was extremely toxic in bark extract. The hypoglycemic and antimicrobial activity was also evaluated [7].
Secondary metabolites of plants are not necessary for survival of plant but essential for the development of plant, reproduction, growth, protection etc. [8]. Phenolic are highly distributed in plant species which are useful in physical and biological stress [9,10]. Phenolic compounds effective for human health for their various biological effects such as antioxidant, anti-microbial, anticancer, and anti-allergic effects [11].
Reactive oxygen species (ROS) incorporates both oxygen and non-radicals that are generated during usual metabolic processes [12]. Excessive production of ROS may lead to oxidative chain reactions which imbalance the antioxidants in body. This imbalance may affect in health conditions and molecular function [13][14][15]. The imbalance may cause several diseases such as neurodegenerative diseases, cancer, allergies, cardiac problem [16]. Antioxidants (present in human body or obtained by food or plants) can suppress the oxidative responses or free radicals, in this manner restricting the oxidative damage [17]. So, intake of dietary of plant derived antioxidants may reduce or prevent the oxidative stress and intake of additional medicine [18].
Our present research study design to investigate the in vitro anti-inflammatory, thrombolytic, cytotoxicity and antimicrobial activity of methanolic extract of A. richardiana and its different fractions. To our knowledge, there are no such study was design or published by using the stem of A.richardiana.

Sample collection and preparation
A. richardiana stem was collected from hilly area of Chittagong, Bangladesh which was identified by the Professor Dr. Shaikh Bokhtear Uddin, Department of Botany, University of Chittagong, Bangladesh. Stem was cut into small pieces and dried in sun-light for couple of days. For better grinding the sun-dried stem was also applied in oven. After that the dried stem was crushed with a high capacity grinding machine and the ultimate product was coarse powder of A.richardiana (0.5 kg).

Drugs and chemicals
Folin-Ciocalteu reagent (10 fold diluted), (Merck, St. Louis, MO, USA), Methanol, Chloroform, Carbon tetra chloride (Merck Darmstadt, Germany), Brine shrimp egg (SK brand, Thailand), vincristine sulphate (Sigma-Aldrich Co). The Human RBCs were collected from a 70 kg diseased free male of fair complexion. The collected RBCs were kept in a test tube with an anticoagulant EDTA under standard conditions of temperature 23 ± 2°C and relative humidity 55 ± 10%. Lyophilized Alteplase (Streptokinase) vial of 15, 00,000 I.U. was purchased from Beacon Pharmaceuticals Ltd. All other chemicals are in analytical grade.

Experimental design Extraction of plant material
The coerced powder of stem was macerated in 1.5 Liter of methanol in an amber glass bottle. The bottle was kept in a dry place with occasional shaking and stirring. After 14 days, the mixture was filtered through cotton and Whitman filter paper #1 respectively. The obtained volume of filtrate was reduced by evaporation at atmospheric temperature until 70% solvent was evaporated. Finally, a crude semi-solid methanolic extract of A. richardiana obtained (5 g).

Solvent-solvent partitioning
The crude extract is fractionated using the solventsolvent partitioning protocol, designed by Kupchan et al. and modified by Van Wagenen et al. [19]. All the fractioned (HXFAR, CTFAR, CFAR, and AFAR) were subjected to evaporate for drying to be used further analysis.

Antioxidant activity DPPH scavenging assay
To evaluate anti-oxidant potential of the plant extract, DPPH assay was used employing the method of Brand-Williams et al., 1995 [20]. Therefore, each test sample containing 2 mL of serially diluted different concentrations (500 μg/mL to 0.977 μg/mL) was mixed with 3 mL of a DPPH methanol solution (20 μg/mL). Following a 30 min reaction period (25°C) a dark place, the absorbance was measured at 517 nm by UV spectrophotometer. Finally, IC 50 values were calculated from the graph plotting concentration of the sample against percentage inhibition of free radicals. As positive control, ascorbic acid and butylated hydroxyl toluene were used.

Total phenolic component analysis
The total phenolic content of A. richardiana stem was assessed by using the previously described method of Skerget et al. (2005) [21]. Folin-Ciocalteu reagent used as an oxidizing agent whereas gallic acid was used as reference. The A. richardiana stem extract (2 mg) was dissolved in the distilled water to obtain a sample concentration of 2 mg/mL. Afterwards, a mixture consisting of 0.5 ml extract solution (conc. 2 mg/ml), 2.5 mL Folin-Ciocalteu reagent (diluted ten times with water) and 2 ml Na 2 CO 3 (7.5% w/v) solution was prepared and incubated for 20 min at room temperature. Finally, the absorbance of the mixture was determined with UVspectrophotometer at 760 nm and the total phenolic content of the sample was calculated from absorbance. The standard curve was also prepared from gallic acid solution using different concentrations. The unit of phenolic content is expressed as mg of GAE (gallic acid equivalent)/gm of the extract.

Cytotoxic activity assay
Brine shrimp lethality bioassay was performed to assess possible cytotoxic activity (Meyer et al., 1982) [22]. Brine shrimp eggs collected from pet shops were hatched in simulated seawater (prepared from 38 g sea salt dissolving in one liter of distilled water) with constant oxygen supply till they mature to nauplii. The test sample was taken in a vial and dissolved in 200 μL pure dimethyl sulfoxide (DMSO), from which each time 100 μL solution was transferred to a test tube containing 5 mL simulated sea water and 10 shrimp nauplii. Test samples of different concentrations were prepared applying the sequential dilution method. For validation of the test method and ensuring that the obtained results only reflect the activity of the test agent and nullify effects of other possible factors, positive and negative control groups were used. Vincristine sulfate was added as a positive control to DMSO to obtain different concentrations through serial dilutions, which were later added to the pre-marked test tube containing 5 mL, simulated sea water and 10 shrimp nauplii to get a positive control group. Next, a comparative analysis is done between the result of test agent and positive control group. For negative control, 100 μL DMSO is added to each of the premarked simulated sea water (5 mL), and shrimp nauplii (n = 10) filled test tubes. Rapid mortality of the brine shrimps indicates the test is invalid as the nauplii died due to reason other than the cytotoxicity of the compounds. After 24 h, visual inspection with a magnifying glass was done to count the survivors and concentration-mortality data were analyzed statistically using IBM-PC program. Median lethal concentration (LC 50 ) was used to express the effectiveness or concentration-mortality relationship of plant product. It represents the concentration of a chemical that produces death in half of the test subjects after a specific exposure period.

Antimicrobial assay
In this study, the antimicrobial property of crude extracts as well as fractions of A. richardiana was tested using the disc diffusion method [23,24]. The bacterial and fungal strains used for the experiment were obtained from the State University of Bangladesh. Standard Ciprofloxacin (30 μg/disc) and blank discs were used as positive and negative control respectively. Nutrient agar medium was used to assess the sensitivity of the organisms to the test materials. On the previously marked zones of the agar plates pre-inoculated with test bacteria and fungi; the sample discs, the standard antibiotic discs and the control discs were placed gently. The plates were then incubated at 37°C for 24 h. After incubation, the clear zone of inhibition surrounding the discs was measured which determines the antimicrobial potency of the test agents.

Anti-inflammatory activity
To evaluate anti-inflammatory activity, hypotonic solution induced hemolysis and heat-induced hemolysis techniques were adopted.

Hypotonic solution induced hemolysis
Hypotonic solution induced hemolysis was evaluated by using the previously described method [25]. The experiment included test sample consisting of stock erythrocyte (RBC) suspension (0.50 mL) with 5 mL of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffer saline (pH 7.4) containing either different methanol extracts (2 mg/mL) or Acetylsalicylic acid (0.10 mg/ mL). The acetylsalicylic acid was used as a reference standard. All these mixtures were subjected to incubation for 10 min at room temperature and then centrifuged for 10 min at 3000 g, and the absorbance (O.D.) of the supernatant was determined at 540 nm using Shimadzu UV spectrometer. The percentage inhibition of either hemolysis or membrane stabilization was calculated applying the following equation: Where, OD 1 = Optical density of hypotonic-buffered saline solution alone (control) and OD 2 = Optical density of test sample in hypotonic solution.

Heat-induced hemolysis
Heat-induced hemolysis was evaluated by using the standard protocol [25]. Aliquots (5 mL) of the isotonic buffer containing 1 mg/mL of different extractives were taken into two duplicate sets of centrifuge tubes. The vehicle, in the same amount, was added to another tube as control and erythrocyte suspension (30 μL) was added to each tube and mixed with gentle inversion. One pair of the tubes was incubated at 54°C for 20 min in a water bath, while the other pair was maintained at 0-5°C in an ice bath. The reaction mixture was centrifuged for 3 min at 1300 g, and the absorbance of the supernatant was measured at 540 nm. The percentage inhibition of hemolysis was calculated according to the equation: Where, OD 1 = test sample unheated, OD 2 = test sample heated and OD 3 = control sample heated

Thrombolytic activity assay
To investigate the thrombolytic activity of methanol extracts and different fractions of A. richardiana, 10 mg of methanol extracts along with its different fractions were added to different vials each containing 1 mL of distilled water. Aliquots (5 mL) of venous blood were drawn from healthy volunteers (without a history of use of oral contraceptive or anticoagulant therapy) and distributed in 10 different pre-weighted sterile vials (1 mL/tube). The vials were then incubated at 37°C for 45 min. In order to determine the clot weight, the vials were again weighed after clot formation. A 100 μL aqueous solution of different fractioned along with the crude extracts was added separately to each vial containing pre-weighted clot. Also, 100 μL of streptokinase (SK) and 100 μL of distilled water were separately added to the control vial as a positive and negative control respectively. All the vials were then subjected to incubation at 37°C for 90 min and observed for clot lysis. Once the incubation was over, the released fluid was removed, and vials were weighed again to express the percentage of clot lysis from the difference in weight measured before and after clot lysis.

Statistical analysis
Values are represented in Mean ± SEM (n = 3). P < 0.05 is considered as statistically significant in comparison to control group where One-way ANOVA was followed by GraphPad Prism (Ver 7).
The total phenol content in A. richardiana was evaluated by the Folin-Ciocalteu's reagent which expressed as GAE/g of dried extract. The regression equation found from standard gallic acid was: Y = 0.0162x + 0.0215; R 2 = 0.9985. The highest total phenolic content was found in n-hexane fraction of A. richardiana 54.05 mg GAE/g. The order of TPC antioxidant activity as followed: HXFAR > AQFAR > CTFAR > MEAR >CFAR.
Oxidative damage resulting an imbalance between the antioxidant defense system and free radical (FS) generation [26] which lead to several damage of protein, nucleic acid and lipids [27]. Oxidative stress initiated by tissues infection or injury by physical injury, hypertoxia, any chemical (toxin) and excessive exercise which produce increase amount of xanthine oxidase, disruption in oxidative phosphorylation and excess reactive oxygen species (ROS). Excess production of ROS, cause several complications such as cardiac disease, diabetes, aging, Parkinson's disease [28]. In biological system, phenolic and flavonoid like content are important for antioxidant 141.34 μg/mL under dark condition respectively. Mostly, the higher the lethal concentration the lower will be the LC 50 and vice versa. The value of LC 50 over 1000 μg/ml is considered to be non-toxic, ranging from 500 to 1000 μg/ml is weakly toxic, moderately toxic for 100-500 μg/ml while less than 100 μg/ml is considered as highly toxic [32]. Here, all extract are shown a moderate toxicity, Hence, the extract required cation at treatment while overdosing.

Anti-microbial activity
In anti-microbial screening performed at the dose of 400 μg/disc by disc diffusion method, the carbon tetrachloride soluble fraction (CTFAR) exhibited the highest inhibition against microbial growth with a zone of inhibition ranged from 7 to 23 mm. The HXFAR and CFAR also exhibited minimum inhibition against microbial growth while AFAR did not exhibit any. The results indicate that CTFAR possesses better antimicrobial activity against gram positive, gram negative as well as fungi and can be studied further to explore potent anti-microbial agent ( Table 4). The antimicrobial potential of plants was studied by zone of inhibition of different microorganisms of gram positive, gram negative and fungi. Different fraction of A. richardiana showed their potential activity. Except CTFAR, none of the fraction exhibits a positive inhibition of growth of microorganisms. From the result, the antimicrobial activity can be mentioned that methanol, n-hexane, carbon tetra chloride extract of A. richardiana might be a broad spectrum antibacterial agent against the tested organisms.

Anti-inflammatory activity
The anti-inflammatory activity was presented in Figs. 1 and 2. The different methanol extract and fractions of A. richardiana stem at concentration 2.0 mg/mL significantly (P < 0.0001) protected the lysis of human erythrocyte membrane induced by heat and hypotonic solution, as compared to the standard acetylsalicylic acid (0.10 mg/mL). The erythrocyte membrane resembles to lysosomal membrane and as such, the effect of drugs on the stabilization of erythrocyte could be extrapolated  to the stabilization of lysosomal membrane [33]. In both the hypotonic solution and heat-induced condition the highest membrane stabilizing activity was demonstrated by the CTFAR (62.29 ± 0.41% and 85.71 ± 0.71%, respectively). Significant membrane stabilizing activity was also exhibited in other extract in both conditions. The results clearly indicated that the extract of the stem of A. richardiana were highly effective in the membrane stabilizing activity as to prevent the lysis of erythrocyte induced by hypotonic solution and heat. Previous studies have reported that flavonoids exert profound stabilizing effects on lysosomes both in vitro and in vivo experimental animals while tannin and saponins have the ability to bind cation and other biomolecules, and are able to stabilize erythrocyte membrane [34,35].

Thrombolytic activity
In this study, the thrombolytic activity, among all extract only MEAR exhibit extremely significant (P < 0.0001) clot lysis while all other plant extracts of A. richardiana was found to be negligible (Fig. 3). The amounts of thrombolytic activity were present in Methanol soluble fraction (MEAR-16.66%), chloroform soluble fraction (CFAR-2.15%), Hexane soluble fraction (HXFAR-3.22%), carbon tetrachloride soluble fraction (CTFAR-4.68%) and AFAR have no thrombolytic activity. Therefore, it can be concluded that the extracts of A. richardiana showed very poor clot lysis activity compared to standard substance Streptokinase (SK-70.94%).
In recent years, the formation of a clot in blood has been a serious problem in blood circulation. Blocking off the blood vessel due to the formation of thrombus, which impedes the blood flow. So, the normal oxygen supply and blood flow was deprived. Plasmin is a fibrinolytic drug, blood clot formed by thrombin which lysed by plasmin. This plasmin activated by tPA. The fibrinolytic drug dissolved the thrombin in coronary arteries to reassure the blood flow [36]. In our present study, the clot lysis formation was found to be negligible while in comparison to standard drug streptokinase, whereas only MEAR found to be significant.