Antioxidant-phenolic content correlation of phenolics rich fractions from Dillenia suffruticosa wood bark

Dillenia suffruticosa is one of the tropical medicinal plants that has been used traditionally for treating several tropical diseases. However, phytochemical and pharmacological investigations of D. suffruticosa wood bark have not been well-investigated. This research aimed to investigate the correlation between antioxidant properties and total phenolic content of phenolics rich extract and fractions from D. suffruticosa wood bark. The phytochemical investigation revealed the existence of phenolics and alkaloids in methanolic crude wood bark extract and all fractions. Meanwhile, the presence of flavonoids was shown in chloroform and methanol fractions. The highest total phenolic content was demonstrated by methanol fraction with 254.34 ± 16.86 mg GAE/g extract. Meanwhile, the highest total flavonoid content was interestingly displayed in chloroform fraction with 15.33 ± 0.26 mg RE/g extract. The crude extract and methanol fraction had a profound antioxidant activity with IC 50 values of less than 15.63 ppm and 8.83 ppm, respectively. A strong correlation was shown between the antioxidant activity and total phenolic contents through correlation analysis. Thus, the D. suffruticosa wood bark could be considered a potential natural source of antioxidants.


INTRODUCTION
(Merck Millipore, USA) were used for Vacuum Liquid Chromatography (VLC) and Gravitational Column Chromatography (GCC), respectively. Silica gel 60 F254 on Thin Layer Chromatography (TLC) aluminum plate (Merck Millipore, USA) was employed to observe the extraction process, to recognize the type of secondary metabolites, and to conduct qualitative antioxidant activity using TLC technique. The chromatograms on TLC plate were detected using spray reagent CeSO 4 1.5% for phytochemical test and using DPPH spray reagent for the qualitative antioxidant test. All fractions were evaporated using a rotary-vacuum evaporator (Buchi, Germany). Distilled and pro-analysis solvents were employed for extraction, identification, and spectroscopic measurement.
The wood barks of D.suffruticosa collected in May 2017 at Pontianak Timur district, Pontianak, were used as plant material. The plant voucher was determined and deposited at Research Center for Biology, Cibinong, Indonesia, with letter number 2003/IPH.

Extraction and isolation
The wood barks of D.suffruticosa were air-dried and granulated. The powdered D. suffruticosa wood bark (2.5 kg) was extracted with methanol (3x24 h) to give a methanolic crude extract. The crude extract was partitioned with n-hexane and chloroform to give n-hexane, chloroform and methanol fractions. Each fraction was evaporated to yield n-hexane (3.2 g), chloroform (7.02 g), and methanol (73.8 g) fractions.

Phytochemical test
The phytochemical test was performed on all fractions using natural product reagents, including FeCl 3 5%, Shinoda, Mayer, Dragendorf, Liebermann-Burschad, and Salwkosky. Spots on TLC were detected by applying spray reagent CeSO 4 1,5% and subsequently heating the TLC plate on the hot plate (Sticher, 2008).

Antioxidant test
The antioxidant activity test was conducted using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method toward methanolic crude extract, n-hexane, chloroform, and methanol fractions as tested samples. The positive control used was Vitamin C. The DPPH method conducted was altered from the previous antioxidant protocol by using TLC-bioautograph technique ( ie a e a ) and UV-Vis spectrophotometry (Salazar-aranda et al., 2011).

Qualitative antioxidant test
A qualitative antioxidant test was conducted using TLC-bioautography technique described previously by ie a ( ie a e a ) with minor modification. 50 ml DPPH solution (20 µg/mL) was prepared as DPPH spray reagent and then stored in dark room μl of each sample (1mg/ml in methanol) were applied on TLC plate together with positive controls, Vitamin C and E, which were placed side by side with samples. After spraying the TLC plate containing spots from sampled and positive control with DPPH solution, it was directly set aside in a dark room for 30 minutes. The white or yellow spot on the purple TLC plate was spotted to indicate antioxidant property. The same protocol was applied to each sample and positive control with concentrations ranging from 125 to 1000 ppm.

Quantitative antioxidant test
A quantitative antioxidant test was carried out using the UV-Vis spectrophotometry technique protocol described by Salazar-aranda (Salazar-aranda et al., 2011) with slight modification. 10 mL stock solution in 1 mg/mL of each sample was made ready in methanol. 50 ml of vitamin C (0.5 mg/mL) as a positive control was prepared. 100 μl of each sample in a concentration of 1000 -1.95 ppm (except for crude extract, concentration ranging from 1000 -15.625 ppm) was added with 100 μl DPPH solution in methanol (0.004 %). The mixture was blended and kept aside in a dark room at ISSN: 2088 4559;e-ISSN: 24770256 Pharmaciana Vol. 11, No. 2, Page 2021 room temperature for 30 minutes. Observation on the absorbance of each fraction at wavelength 517 nm was done using a UV-Vis spectrophotometer. The absorbance value was then changed into a percentage of antioxidant activity (AA%) using formula as shown in equation (1), where A is the absorbance of control and B is the absorbance of the sample. The negative control is the mixture of DPPH and methanol, while the sample is DPPH, methanol, and sample.
A blank solution (Abs blank) used was 200 μl methanol. The control solution (Abs control) was a mixture of 100 μl DPPH (0.004 %) and 100 μl methanol. The antioxidant activity was conveyed in a percent concentration required to reduce 50% DPPH absorbance (IC 50 ). The IC 50 value was calculated with a linear regression equation between the percentages of antioxidant activity as a function of sample concentration. Each measurement was done in triplicate, and the same protocol was done with the vitamin C (positive control).

Determination of total phenolics content (TPC)
An experiment using the Folin-Ciocalteu method described by Armania (2013) with some modification was conducted to determine total phenolics content on each extract and fractions. In brief, 0.5 ml tested sample (1 mg/mL) was reacted with 2.5 mL Folin-Ciocalteu and then was added with 2 mL of Na 2 CO 3 7.5%. The mixture was incubated at 40 o C for an hour. The absorbance of the mixture was then measured using UV Spec ropho ome ry a λ 765 nm. The experiment was done in triplicate. The positive control was gallic acid, with a standard linear curve of gallic acid is Y =0.0129 X -0.0243 (R 2 = 0.9977). The total phenolics content was demonstrated as milligram gallic acid equivalent per gram extract (mgGAE/g extract)

Determination of total flavonoids content (TFC)
Determination of total flavonoid content was performed by using the aluminum chloride method described by Armania (2013) with some modifications. A μl of tested sample (1 mg/mL) was added with μl of AlCl 3 2%, and let the mixture at room temperature for 10 minutes. The absorbance of complex flavonoid-aluminum was observed using UV pec ropho ome ry a λ 4 nm and was done in triplicate. Rutin was used as a positive control. The TFC was displayed as a milligram of rutin equivalent per gram extract (mg RE/g extract).

Statistical analysis
All data were measured in triplicate. TPC and TFC values were represented as mean ± standard deviation. The mean difference between crude extract, n-hexane, chloroform, and methanol fractions was tested using a one-way analysis of variance with p<0.05. Pear on' produc -moment correlation was performed to determine the correlation coefficient.

RESULT AND DISCUSSION
The powdered D. suffruticosa wood bark (2.5 kg) was macerated with methanol solvent three times for 24h; each was given to 84.1 g of crude extract (M). Repeated partition using liquid-liquid extraction toward crude extract with n-hexane and chloroform, followed by rotary-vacuum evaporation, yielded n-hexane (KSH) concentrate (3.2 g), chloroform (KSC) (7.02 g), and methanol (KSM) (73.8 g) fractions ( Figure 1). These yields showed that the most abundant secondary metabolite from the wood bark extract of D.suffruticosa was found in methanol fraction, indicating that most of them are polar compounds.
Phytochemical screening on methanolic crude extract and all fractions were done using specific reagents. Those specific reagents were for alkaloids, flavonoids, phenolics, triterpenoids, saponin, and  (Table 1). Although the methanolic crude extract gave negative results for saponin, the chloroform and methanol fraction gave positive results for this group. Methanol fraction, which has the highest amount, gave positive results for phenolics, alkaloids, flavonoids, and saponins. The presence of alkaloids and phenolics was also shown in n-hexane and chloroform fractions, indicating that these secondary metabolites dominated the extract of D. suffruticosa wood bark. The presence of phenolics in all fractions corresponds with previous phytochemical studies on other parts of D. suffruticosa. Recently, an intensive investigation was conducted toward D. suffruticosa leaves, and it reported the existence of alkaloids from this part of the plant (Dawood Shah et al., 2020). Like an alkaloid that was detected in leaves, an alkaloid from D. suffruticosa wood bark was strongly detected. This finding is interesting because it indicated that alkaloids were also specifically deposited in the wood bark. However, there were no reports regarding alkaloids that isolated from D. suffruticosa so far. On the other side, flavonoid was likely not a major component in D. suffruticosa wood bark. Based on this data, it seems that the diversity of secondary metabolites on the wood bark of D.suffruticosa was high. Antioxidant activity screening using the TLC-bioautography technique with DPPH revealed that methanolic crude extract and methanol fraction gave the brightest spot, contrasting with the purple background Their po ' co or and hape were c o e y he ame wi h vi amin and E a po i ive controls (Figure 1). It indicated that methanol fraction was qualitatively potential as an antioxidant ie a e a . According to its spot appearance, chloroform fraction was considered to possess slightly active as antioxidant. In this study, antioxidant behaviour of different sample concentrations (125 -1000 ppm) was observed using the TLC-bioautography technique and compared to positive control with the same ranging concentrations. The chromatogram showed that the gradation spot patterns of crude extract and methanol fraction are similar to that of vitamin C; even crude extract displayed a brighter spot than vitamin C at the 125 ppm concentrations (Figure 2). The accumulation of antioxidant agents contained in crude extract might be the reason for these phenomena. Based on the spot appearance of each sample at concentration 125 ppm, it seemed that methanol and chloroform fraction would have IC 50 values lower than 125 ppm. Therefore, the lowest concentration used in a quantitative antioxidant test experiment must be below 125 ppm.

. Chromatogram of qualitative antioxidant test using TLC-based technique and DPPH assay for (a) methanol extract (M), n-hexane fraction (KSH), chloroform fraction (KSC), methanol fraction (KSM), and vitamin C (positive controls) with ranging concentration (125 -1000 ppm).; and (b) methanol fraction eluted with n-hexaneacetone (1:1)
The crude extract and all fractions were evaluated further to obtain their antioxidant IC 50 values using the UV-Vis spectrophotometry technique. The concentration range was 1000 -15.63 ppm for crude extract and 1000 -1.95 ppm for the fraction. The crude extract (M) still showed radical inhibition (82.5%) at a concentration of 15.63 ppm, indicating that the IC 50 of the crude extract was less than 15.63 ppm. This result demonstrated that the crude extract was very active as an antioxidant, compared to vitamin C (IC 50 = 4.45 ppm) as the positive control. Chloroform (KSC) and methanol (KSM) fractions exhibited moderate and pronounce antioxidant activity with IC 50 values of 58.71 and 8.83 ppm, respectively (Table 2), in comparison to vitamin C. Meanwhile, n-hexane (KSH) displayed no activity as an antioxidant. These results confirmed the corresponding active crude extract and fractions from D. suffruticosa wood bark with active fractions from other parts of D. suffruticosa, which have been reported previously. Previously, fractions reported from D. suffruticosa, which are considered as pronounce antioxidant, displayed other bioactivities, such as antibacterial (Kristiningrum et al., 2020), antiinflammatory (Abubakar et al., 2019, and cytotoxic against several cancer cell lines (Armania et al., 2013). Many studies have reported how antioxidant agent plays roles in other bioactivity mechanisms. Based on this, D. suffruticosa wood bark would also demonstrate interesting bioactivity.
The determination of total phenolic content (TPC) of crude extract and all fractions revealed that methanol fraction displayed the highest total phenolic content with 254.34 ± 16.86 mg GAE/g extract, followed by crude extract, chloroform, and n-hexane fractions (Table 2). In addition to that, TPC and TFC values between each fraction had significant differences statistically (p<0.05). These results supported phytochemical data regarding the existence of phenolics in all fractions. Therefore, it may indicate that phenolics contained in the wood bark mostly were polar compounds. Despite that the TPC value of each fraction was relatively high, which was ranging from 1.88 ± 0.00 to 254.34 ± 16.86 mg GAE/g extract, the total flavonoid content (TFC) was lower; ranging from 11.65 ± 0.17 to 15.33 ± 0.26 mg RE/g extract. These data suggested that flavonoids were not a major group in phenolics. Further, the highest total flavonoid content (TFC) was shown in the chloroform fraction, indicating that most flavonoids in D. suffruticosa wood bark were semipolar compounds.
An observation on the chromatogram profile of antioxidant-active methanol and chloroform fractions to analyze the relationship between antioxidant and phenolic content was performed using a simple TLC-bioautography for antioxidant and DPPH reagent by comparing the chromatogram profile of each fraction on TLC plate. The phenolic spots were shown by the change of the spots' co or to dark brownish yellow color on the TLC plate after being sprayed with CeSO 4 (1.5% b/v) followed by heating on the hot plate (Marliyana et al., 2019). The phenolics are usually semipolar to polar compounds. In this case, the best eluent systems chosen were n-hexane-acetone (1:1) to give semipolar property for chloroform fraction (KSC) and chloroform-methanol (10:0.2) to polar fraction, methanol (KSM). The experiment result showed that most spots that appeared in these fractions gave bright spots after being sprayed with the DPPH reagent ( Figure 3). It indicated that most compounds contained in both fractions possessed antioxidant activity ( ieś a e a ). Further, based on the TLC-bioautography experiment, the spots, which gave positive results for phenolics, displayed bright color in DPPH assay. It indicated that those phenolics possessed antioxidant capacity. Compared to the IC 50 and the chromatogram profile of the chloroform fraction, the methanol fraction was more active and possessed more antioxidant-phenolic spots. Therefore, it can be assumed that the strong antioxidant activity showed by the methanol fraction was caused by the existence of those phenolics. The relationship of phenolic content (TPC) or flavonoid content (TFC) with antioxidant activity was analyzed further u ing Pear on' produc -moment correlation. The result revealed that there was a strong negative correlation between TPC and IC 50 values with an r-value of -0.96 (p-value < 0.05) ( Table 3), indicating that IC 50 value of each fraction was inversely proportional to its TPC value. Since the antioxidant activity was stated as IC 50 value, the lower IC 50 value means a higher antioxidant activity. Thus, the result suggested that the higher TPC indicates a higher antioxidant activity. This finding was in agreement with the previous experiment using TLC bioautography. It was also relevant to previous research that mentioned the relationship between antioxidant and phenolic content (Kainama et al., 2020), which supports that antioxidant property shown by crude extract and all fractions was due to their phenolic content. Additionally, correlation between TFC and antioxidant in this study was moderate with r = -0.43 (p-value > 0.05) (Table 3). Thus, it gave another evidence that the exhibited antioxidant activity were likely caused by the phenolics rather than flavonoids. Many studies showed that most phenolics and flavonoids were known to possess antioxidant activity. Antioxidant-active phenolics were reported to be sensitive with DPPH assay (Bendary et al., 2013) Thi i becau e of he pheno ic ' abi i y o cavenge DPPH radica by i reac ive oxygen and its ability to lose the H atom. All polyphenolics that were isolated previously from D. Suffruticosa (protocatechuic acid, gallic acid, vitexin, tiliroside, kaempferide, and kaempferol) were reported to exhibit antioxidant activity. Since phenolics dominated D. suffruticosa wood bark, it can be predicted that the antioxidant-active crude extract of D. suffruticosa wood bark and methanol fraction contain similar antioxidant-active phenolics previously reported.

CONCLUSION
This study successfully revealed the existence of alkaloids, phenolics, flavonoids, terpenoids, steroids, and saponins in the methanolic crude extract of D. suffruticosa wood bark. The pronounce antioxidant activity showed by the crude extract was also demonstrated by phenolic rich containing fraction, i.e., chloroform and methanol fractions. A strong correlation between antioxidant and phenolic content from all fractions suggested that phenolics were responsible for their antioxidant activities. Moreover, the isolation and structure elucidation of the active principles on methanol fraction should be conducted in future research plans to reveal the structure of the antioxidant-active phenolics. Hence, D. suffruticosa wood bark has the potential for a natural antioxidant source.