An in vitro study on anti-sickling activity of Terminalia arjuna and Terminalia bellirica

About five million people yearly in the world are affected by Sickle cell disease (SCD) and approximately 1,00,000 children in the world are born each year with SCD. It is considered as a public health problem, particularly in west and central Africa and Indian subcontinent. (Moody et al., 2003, Mpiana et al., 2007, Mandot et al., 2009). Sickle cell anemia is an autosomal genetic blood disorder where concave blood cells are changed to abnormal sickle shaped red blood cells (RBCs). In sickle hemoglobin, glutamtic acid is replaced by valine in position 6 (Mpiana et al., 2013) causes a reduction in the solubility of sickle cell hemoglobin (HbS) and its polymerization to form a mass of fibres which changes shape of erythrocytes into a sickle shape (Clarke and Pazdernik, 2013). The therapies include Journal of Herbal Drug


Introduction
About five million people yearly in the world are affected by Sickle cell disease (SCD) and approximately 1,00,000 children in the world are born each year with SCD.It is considered as a public health problem, particularly in west and central Africa and Indian subcontinent.(Moody et al., 2003, Mpiana et al., 2007, Mandot et al., 2009).Sickle cell anemia is an autosomal genetic blood disorder where concave blood cells are changed to abnormal sickle shaped red blood cells (RBCs).In sickle hemoglobin, glutamtic acid is replaced by valine in position 6 (Mpiana et al., 2013) causes a reduction in the solubility of sickle cell hemoglobin (HbS) and its polymerization to form a mass of fibres which changes shape of erythrocytes into a sickle shape (Clarke and Pazdernik, 2013).The therapies include bone marrow transplantation, gene therapy along with frequent blood transfusions, use of hydroxyurea, but are expensive (Mpiana et al., 2011, Mpiana et al., 2013, Makani et al., 2013).
The mechanism of antisickling agents includes inhibiting polymerisation of haemoglobin or modifying membrane stability and red cell hydration evaluated by measuring RBC haemolysis (Mpiana et al., 2010).The Terminalia genus belongs to the Combretaceae family with 200 species (Pietrovski et al., 2006).T. arjuna and T. bellirica are Asian species extensively used Pietrovski in traditional medicine.T. arjuna used as analgesic, antiinflammatory, antioxidant, cardiac stimulant whereas, T. bellirica as expectorant, laxative, in anaemia, and anthelmintic properties (Cock, 2015).
The therapeutic activities of traditional practice medicine are mostly unidentified.There is a need to find out the remedies which are more economical and easily available.This study is a step forward to find out a potent product from the plants which can reduce the suffering of SCD people.This work investigates the antisickling, antioxidant and erythrocyte membrane stabilizing activities of T. arjuna and T. bellirica leaf extracts which were yet not been carried out in the knowledge.

Plant material and preparation
The plants selected were used for the treatment of anemia based on the guidance of traditional herbal healers and confirmed from Ethno-medicinal plants used by tribal's in central India (Gangarde et al., 2003).Leaves of T. arjuna (Roxb.)Wight & Arn. and T. bellirica (Gaertn.)Roxb.were collected from the central India and identified from the Herbarium of the Department of Botany, Govt M.V.M., Bhopal and providing them specimen no.: Bot/08/2015.The dried leaves were pulverized, labeled and stored in airtight containers at 20°C.

Extraction
Cold extraction was conducted with powdered leaves (500 g) with 2 L of methanol in amber bottles for 48 hours, with intermittent shaking.The crude methanol extract was filtered (0.45µm Whatman filter) and concentrated using a rotary evaporator.In another set, powdered leaves were soaked in sterile; distilled water for three days at 27°C and the aqueous extract was collected by filtration and concentrated in a rotary evaporator.0.9% NaCl was used to dilute both extracts to give 10, 5, 1mg/ml concentrations for subsequent antisickling assay.

Biological material
The blood samples were taken from anemic adolescent patients attending the People's Hospital and Research Centre, Bhopal.None of the patients had been transfused recently with HbAA blood.A written, informed, witnessed consent was signed by the patients who donated blood for the study.The research project had the approval with reference no.PU/CSRD/SI/30, from Centre for Scientific Research and Development, People's University, Bhopal, India.Blood was collected in sodium EDTA bottles with gentle mixing.All experiments were performed with fresh blood.In order to confirm their sickle cell nature, the blood samples were first characterized by D-10 HPLC (Bio Rad).Packed erythrocytes were collected by washing in normal saline and were then stored at 4°C (Egunyomi et al., 2009).

Membrane stability of erythrocytes
The osmotic fragility of erythrocytes was determined by placing the cells in a graded series (0.00-0.85% with a difference of 0.10%) of buffered saline, pH 7.4 in 1 ml of each extract (10 mg/mL) to which 50 µl sickle blood was added.The mixture was incubated at 25°C for 24 h, and after incubation the mixture was centrifuged at 3000 rpm for 15 min and optical density of the supernatant was recorded at 540 nm against blank made of 0.85% buffered saline (Jaja et al., 2000).A plot of lysis (%) versus NaCl concentration was obtained to determine the concentration of saline causing 50% haemolysis of the RBC (mean corpuscular fragility).

In vitro anti-sickling activity
Bioassay of crude methanol and aqueous extract of T. arjuna and T. bellirica were carried out, viz.inhibition of sickling (antisickling) and reversal of sickled erythrocytes analyzes.The antisickling activity was carried out by following the modified protocol of (Sofowora et al., 1979).The washed erythrocytes and plant extracts were mixed at equal concentration (0.5 ml) in an uncovered test tube.Methanolic and aqueous sample extracts of different concentration were taken and incubated at 37°C for 3 h with occasional shaking.To the mixture, five drops of 2% sodium metabisulphite were added and mixed thoroughly.The test tubes were sealed with liquid paraffin and incubated at 37°C for 30 min.Samples were then taken in duplicate from the different mixtures at 0 min after which the systems were incubated at an interval of 30 min, four times.Method of (Egunyomi et al., 2009) was used to prepare smear and count sickled and unsickled cells.The positive and negative control was p-hydroxybenzoic acid (5 mg/ml) and normal saline respectively.To determine the percentage of sickling inhibition was calculated using the formula of Moody et al. (2003).
In sickling reversal activity of crude extracts, washed erythrocytes were equally (0.5 ml each) mixed with freshly prepared 2% sodium metabisulphite in a clean test tube and incubated at 37°C for 30 min and observed under the microscope (Oduola et al., 2006).To the blood-metabisulphite mixture equal volume of extracts were added and incubated at 37°C for another 30 min.Samples were observed at 0 min and at 30 min interval for up to 2 h.The smear preparation, counting of sickled and unsickled cells and microscopic images of depranocytes were examined (Egunyomi et al., 2009).

Scavenging Activity
The scavenging activity of the two methanolic and aqueous extracts on DPPH (1,1-diphenyl-2picrylhydrazyl) was determined using the method of Choi et a. (2002) with minor modifications.The changes in color were measured spectrometerically at 518 nm.In 3 mL of each diluted extract, 1 ml of 0.1 mM DPPH methanol solution was added.All the extracts were tested at final concentrations ranging from 10, 5 and 1 mg/mL.At different concentrations of extract with a final concentration of 3 mL each, one milliliter of 0.3 mM DPPH ethanol solution was added to give test solutions, while 1 mL of ethanol was added to 3 mL of sample to produce the blank solutions.The solutions were allowed to react at room temperature for 30 min in the dark.The absorbance were measured at 518 nm and converted into percentage antioxidant activity using the equation: The negative control was 1 mL of DPPH solution in 3 mL of absolute ethanol.% Inhibition = [(AB -AA)/AB] × 100, Where AB: absorption of blank sample; AA: absorption of tested samples.
Moreover the IC50 value and the kinetics of DPPH activity were determined to know the efficacy of the extract.The positive controls were Ascorbic acid and butylated hydroxytoluene (BHT).

Statistical analysis
All the experiments were repeated twice and the mean data was used for interpretation.The probability of the finding and its significance of differences were analysised.

Results and discussion
Anise Out of fifty blood sample collected, sickle cell disease was identified in twelve using D 10 HPLC (Fig. 1).

Phytochemical screening
The qualitative phytochemical screening of plant extracts provides necessary information regarding chemical constituents.The results of phytochemical screening of leaves have been shown in Table 1.The T. arjuna showed the presence of alkaloids, anthraquinones, cardiac glycosides, flavonoids, saponins, tannins while alkaloids and flavonoids were absent in T. bellirica.Alkaloids were used as nerve stimulant, muscle relaxant (Robert and Wink, 1998).Alkaloid may be useful in alleviating symptoms of pains.Anthroquinone increases peristaltic action.Flavonoids possess potential pharmacological activities such as antioxidant activity, vitamin C sparing activity (Middleton and Kandaswami, 1992).Antisickling activity of may be due to presence of antioxidant activity.The presence of cardiac glycosides may be potent in curing cardiac insufficiency, cough and circulatory problems.Tannins are phenolic derivatives, may be useful in cleansing the surface of the skin ulcers that develop as a result of sickle cell disease (Kenner and Yves, 1996).

Antisickling activity
Tables 2 and 3 are the observation of antisickling activities of both methanol and aqueous extracts of T. arjuna and T. bellirica whereas morphological changes in depranocytes showed in Fig. 2. At the tested concentrations of 5 and 10 mg/ml, both the methanol and aqueous extracts of T. arjuna exhibited antisickling activity as compared with p hydroxybenzoic acid (PHBA) (positive control).The exhibited antisickling activity was concentration dependent.The antisickling activity exhibited by the aqueous extract of T. arjuna was significantly higher (p < 0.05) at the three tested concentrations of 1, 5 and 10 mg/ml as compared to its methanol extract.In a trend that contrasts the antisickling activity of T. arjuna, T. bellirica had a weak antisickling activity.Methanolic and aqueous extract at 10 mg/ml exhibited significantly lower (p<0.05)antisickling activity than positive control.The result of sickling reversal bioassay shown in tables 2 and 3 revealed a trend similar to that of the presently reported antisickling activities of both T. arjuna and T. bellirica.The sickling reversal activities of the investigated plant were concentration dependent.The solvent used in extraction of T. arjuna had no significant effect (p > 0.05) on sickling reversal activity.
Antisickling activities of leaf of T. arjuna and T. bellirica verify their use in tribal medicine for SCD management.The antisickling activities given by positive control were maintained throughout incubation period, while of plant extracts activities was decreased after 1 h.It may take into consideration that lower antisickling activity of T. bellirica than T. arjuna may be due to the absence of alkaloids and flavonoids.This confirms that all component of therapeutic mixture are necessary for activity (Gillete et al., 2004).Osmotic fragility of erythrocytes indicates the integrity of red cell membranes in pathological and normal states (Rai et al., 2009).Result of the erythrocyte osmotic fragility showed a significant decrease of the percentage of hemolysis while increasing the concentration of salt solution (Fig. 3).All the extracts tested showed a lower hemolysis percentage compared the control.Among the four fractions tested, aqueous fraction of T. arjuna presented the most significant reduction of hemolysis compared to methanolic fraction of same plant and aqueous and methanolic fraction of T. bellirica plant.Hence, it could have good guarding effect of the erythrocyte membrane.

Antioxidant activity
In order to determine in vitro antioxidative activity of antioxidant compounds, DPPH radical is widely used.The reduction of DPPH into stable radical indicated by color change measured at 518 nm wavelength.The DPPH scavenging capacity of aqueous leaf fraction of T. arjuna (10.8 μg/mL) was the highest among the extracts tested, while methanolic leaves fraction of T. arjuna (12.3 μg/mL) had slightly less DPPH scavenging capacity (Table 4).Though, IC50 value of all the extracts recorded lowest value, methanolic leaf fraction of T. bellerica (16.2 μg/mL) demonstrated better scavenging activity than aqueous leaf fraction T. bellirica (20.6 μg/mL).Among the positive controls, ascorbic acid (1.6 μg/mL) was found to have better DPPH radical scavenging ability than BHT (1.7 μg/mL).Flavonoids are good source of antioxidant constituents of plants and along with possess radical-scavenging or chelating activities (Miliauskas et al., 2004).

Conclusion
This study evaluated the phytochemical screening and the in vitro antisickling, erythrocyte membrane stability activity and antioxidant activity of leaf extract of T. arjuna and T. bellirica.These plants displayed promising in vitro antisickling and antioxidant effects.The abilility of extract to show such pharmacological properties may give justification for the use of Termianlia plant by the traditional folklore to treat SCD.This has not been previously reported as an antisickling plant in the traditional medicine.Therefore further studies are indicated to study active compound and the toxicity profiles in transgenic mice models.

Table 1 .
Phytochemical screening of leaves of T. bellirica and T. arjuna.

Table 2 .
Antisickling effect (% inhibition of sickling) of methanol and aqueous extract of T. arjuna leaves Values in parenthesis are percentage sickling reversal of 2% metabisulphite induced sickled cells.All values are means of triplicate determinations.

Table 3 .
Antisickling effect (% inhibition of sickling) of methanol and aqueous extract of leaves of T. bellirica Values in parenthesis are percentage sickling reversal of 2% metabisulphite induced sickled cells.All values are means of triplicate determinations.

Table 4 .
Antioxidant activities of methanolic and aqueous leaf extracts of T. arjuna and T. bellirica in DPPH assay.