Phytochemical Screening , Total Flavonoid and Total Phenolic Content and Antioxidant Activity of Different Parts of Caesalpinia bonduc ( L . )

Background: Caesalpinia bonduc (L.) Roxb are traditionally used in Indonesia to treat various diseases, but still limited study about different part of this plant. Objective: The aim of this study was to screen the phytochemicals, to evaluate the total flavonoid and total phenolic contents as well as antioxidant activity of ethanol extract of root, stem, leaves, and seed kernel of C. bonduc. Methods: Each part of plant were extracted by reflux using 70% ethanol as the solvent for 2 h and repeated 3 times. Total flavonoid content was determined by aluminium chloride colorimetric assay on 415 nm. Total phenolic content was determined with Folin-Ciocalteu 1:4 on 765 nm using microplate reader. Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenger methods. Results: Phytochemical screening showed that all of samples positively contain flavonoid and saponin. Total flavonoid content was the highest in leaf and the lowest in root whereas total phenols content was highest in leaf and the lowest in seed kernel. The crude extracts displayed DPPH free radical scavenging activity with highest value in leaf extract followed by root, stem, and seed kernel. Conclusion: The 70% ethanol leaf extract of C. bonduc showed the highest yield, total flavonoid content and total phenolic content among other parts investigated. Moreover, leaf extract has highest DPPH free radical scavenging activity (79.802 μg/ml) which could be related to its higher phenolic content.

long, 3 to 4 cm, prickled, bracts long, linear-lanceolate and deciduous.Pedicel is 0.5-1.5 cm and very short in bud. 2,7,8Each part of the plant as seen in Figure 1.Whole plant of the C. bonduc contain steroidal saponins, fatty acids, hydrocarbons, phytosterols, isoflavone, amino acids and phenolics. 2Caesalpinins H-P and norcaesalpinin F were isolated from the CH 2 Cl 2 extract of the seed kernels of Caesalpinia crista, together with 13 known diterpenes. 9n Indonesia, seed kernel of this plant traditionally used for cough, malaria, and as anthelmintic. 6,10eaves for inflammation and liver disease, and the root was used for gastric and blood disorder. 6,7Part of C. bonduc possesses several activities such as anthelmintic, antimalaria, antidiabetic, antioxidant, antibacterial, antimalarial and antidiarrhoeal. 2,11,12ntioxidants could be used for management of some pathophysiological conditions, which involve free radical, such as cardiovascular and neurodegenerative disease. 13One established in vitro method to evaluate antioxidant activity was DPPH scavenging activity.This method is furthermore rapid, not involved with many steps and reagents, and reagent and shaken for 60 sec in a flat-bottom 96-well microplate.The mixture was left for 240 secs and then 75 μL of sodium carbonate solution (100 g/L) were added and the mixture was shaken at mediumcontinuous speed for 1 min.After 2 h at room temperature, the absorbance was measured at 765 nm using the microplate reader (Versamax Absorbance Microplate Reader, USA).The absorbance of the same reaction with ethanol instead of the extract or standard was subtracted from the absorbance of the reaction with the sample.Gallic acid dilutions (10-200 mg/L) were used as standards for calibration.Total phenolic contets were expressed as mg Gallic Acid Equivalents (GAE) per g of plant extract.
Antioxidant activity DPPH scavenging ability assay was used to evaluate the antioxidant activity of each extract.Test was conducted in a 96-well plate according to Zahratunnisa et al. 21with slight modification.20 µL stock solution of extracts in different concentrations (100, 500, 1000, 1500, 2000 ppm) and 180 µL of DPPH solution 0.147 mM were added to each well.After 30 min incubation at room temperature in dark room, absorbance was read at 517 nm using micro-plate reader of Versamax Microplate Reader (USA).Methanol was used as blank.The scavenging ability (%) was calculated as follows: Ascorbic acid was used as positive standard.All tests were performed in triplicate.Concentration of samples resulting in 50% inhibition on DPPH (IC 50 value) were calculated.

Percentage Yield of Extract
The results showed the highest yield was associated with leaves (33.8%), followed by seed kernel (28.26%), root (18.52%) and then stem (10.48%) as seen on Figure 2.

Phytochemical screening
The extracts were further investigated to determine phytochemical compound in the extract.The common phytochemistry content from plant such as flavonoid, alkaloid, terpenoid, steroid, tannin, and saponin have identified (Table 1).

Total Flavonoid Content
The result of total flavonoid contents of the four crude extracts is given in Table 2. Equation of calibration curve of quercetin standard was y = 0.0291×−0.0397,R 2 = 0.9904.Among the four crude extracts, leaf contained the highest (31.05 ± 0.35 mgQE/g) amount of total flavonoid content compounds followed by stem (21.82 ± 0.46), seed kernel (13.21 ± 1.35) and then root (12.55 ± 0.08 mgQE/g).

Total Phenolic Content
The result of total phenolic content determination from 70% ethanolic extracts of different parts of C. bonduc are shown in Table 3. Calibration curve from gallic acid showed maximum absorbances at 765 nm wavelength (equation y = 0.0531x + 0.0003, R 2 = 0.9951).The total phenol contents of four crude extracts determined by Folin-Ciocalteu method were reported as gallic acid equivalents.Among the four crude extracts, leaf contained the highest (146.64 ± 3.94 mgGAE/g) amount of total phenolic content compounds followed by stem (144.42 inexpensive compare to other method. 14Flavonoid and phenolic compounds may be useful as antioxidant from natural sources.Distribution of phenolic compounds varies between different parts of plant. 15ccording to the literature search, phytochemical and antioxidant activity of different parts of C. bonduc have never been comparatively evaluated.The study was carried out to screen the phytochemicals as well as to evaluate total flavonoid contents, total phenolic contents and antioxidant activity of root, stem, leaf and seed kernel ethanolic extract.

Plant Materials
Parts of C. bonduc were collected in January 2017 from Bulukumba, South Sulawesi, Indonesia.All plants were identified by Indonesia Science Institution, Center for Plant Conservation-Bogor Botanical Garden, Indonesia.

Preparation of Samples
The plants materials were collected and cleaned, dried at room temperature, crushed into powder and stored in an air tight glass container.Fifty g of powdered samples was extracted by reflux using 70% ethanol as the solvent for 2 h and repeated 3 times.The extracts then evaporated using a vacuum rotatory evaporator, and then dried in water bath on 50°C.

Determination of Percentage Yield (%)
The percentage yield of the extract was determined using the dry weight of extract (a) and soaked samples material (b) using the formula: Percentage yield (%) = a/b × 100 The extraction yield was calculated for each extract.

Phytochemical Screening
The qualitative phytochemistry test were conducted according to Indonesian Materia Medica 16 and Harborne. 17

Determination of Total Flavonoid Content
Total flavonoid content was determined by aluminium chloride colorimetric assay adapted from Chatatikun et at 18 and Sandip et al 19 with slight modification.Standard solution of quercetin in concentration 30,40,50,60,70,80,90,100 µg/ml were prepared in 96% ethanol.50 µl of extracts (1 mg/ml) or standard solution was added to 10 µl of 10% the aluminium chloride solution and followed by 150 µl of 96% ethanol.10 µl of 1 M sodium acetate was added to the mixture in a 96 well plate.96% ethanol was used as reagent blank.All reagents were mixed and incubated for 40 min at room temperature protected from light.The absorbance was measured at 415 nm with a microplate reader (Versamax Microplate Reader, USA).Total flavonoid contents were expressed as mg Quercetin Equivalents (QE) per g of plant extract.

Determination of Total Phenolic Content
The microplate total phenolic content method was based on the 96-well microplate Folin-Ciocalteu method adapted from Ahmad et al 20

Antioxidant Activity
The ability of extract in scavenging DPPH radical are shown in Table 4.The leaf has the highest ability to scavenge free radicals than other parts.

DISCUSSION
Extraction was done using 70% ethanol as solvent.According to Jing et al, 22 this solvent is preferred to extract phenolic compounds from plants.Reflux method was chosen as extraction method to optimize phenolic and flavonoid content.Temperature was maintained not exceeding 70 o C to avoid any degradation of phenolic and flavonoid as targeted compound. 23fferent parts of plant could have different phytochemical compound, which may contribute to different pharmacological effect of each part.Phytochemical screening showed that all of extracts from root, stem, leaf and seed kernel of C. bonduc contain flavonoid and saponin, and absent in alkaloid and quinone.Among other part, only leaf contained tannin, one of polyphenol that can donate hydrogen and act as antioxidant. 15,24tal flavonoid content was determined using aluminium chloride method.Aluminium chloride will form stable complex with carbonyl group at C4 and hydroxyls at C3 (flavonols) and C5 in flavonols and flavones.It could also form labile acid complexes with hydroxyls in the ortho position in B rings of flavonoids. 25     The amount of reduced DPPH was measured in absorbance at 517 nm. 26n present study, ascorbic acid as a well known potent antioxidant, was used as positive control for DPPH scavenging activity.Leaf part showed highest ability in DPPH scavenging activity compare to other plant part which measured by the lowest IC 50 value, but it has lower antioxidant capacity compared to ascorbic acid.The phenolic content in the leaf may contribute to the antioxidative action by hydrogen donating ability.Antioxidant activity of leaf and seed kernel of C. bonduc in this study were found different from study in other country. 27,28The variation might be with some modifications.A total of 25 μL of the diluted extract of each part of C. bonduc were mixed with 100 μL of 1:4 diluted Folin-Ciocalteu ± 16.05), root (89.81 ± 3.00) and then seed kernel (70.34 ± 10.59 mgGAE/g).
Alkaloid test with Mayer, Dragendorff, and Bouchardat reagents; flavonoid test with Shinoda test; tannin test with gelatin test, gelatin-salt test, and test with ferrous (III) chloride; saponin test with froth test; quinone with Borntrager test; terpenoid/steroid test with Liebermann-Burchard reagent.

Table 4 : Antioxidant DPPH Scavenging Activity. Sample IC 50 (µg/mL)
The total phenols contents in four different crude extracts were evaluated in the present study.The highest amount of phenolics compounds was present in leaf (146.64 mg of GAE/g of crude extract) and the lowest was in seed kernel extract (70.34 mg of GAE/g).Leaf also have highest total flavonoid content (31.05 ± 0.35 mgQE/g) while the lowest was in root (12.55 ± 0.08 mgQE/g).The molecule DPPH is a free radical by the delocalisation of the spare electron over the molecule.The delocalization of electron rises to the deep violet colour.If a solution of DPPH is mixed with samples that can donate a hydrogen atom, DPPH will be converted into colourless purple.