Isolation, characterization and biological activities of betulin from Acacia nilotica bark

Medicinal plants are in use of humankind since ancient and still they are playing an important role in effective and safer natural drug delivery systems. Acacia nilotica (native of Egypt) commonly known as babul belongs to family Fabaceae, widely spread in India, Sri Lanka and Sudan. Being a common and important plant, using in many ways from fodder (shoots and leaves to animals) to dyeing (leather coloration) to medicine (root, bark, leaves, flower, gum, pods). The present study is focused on investigating the natural chemistry and important biological activities of the plant. Employing bioassay guided fractionation coupled with TLC and column chromatography, a pure fraction named AN-10 was isolated from ethyl acetate fraction of crude methanol extract which identified as “Betulin (Lupan-3ß,28-diol)” by Liebermann-Burchard test and structure elucidation by UV–Vis, NMR and MS techniques. A battery of in vitro biological assays for antioxidant, anti-inflammatory and anticancer were performed and betulin showed excellent potential in all assays. It was found that the inhibitory potential in all assays were dose dependent manner and after a range of concentration, the activities get leveled off with no further increase in activity.

Isolation, characterization and biological activities of betulin from Acacia nilotica bark Prabhjit Kaur 1,2 , Saroj Arora 1 & Rajbir Singh 1,2* Medicinal plants are in use of humankind since ancient and still they are playing an important role in effective and safer natural drug delivery systems. Acacia nilotica (native of Egypt) commonly known as babul belongs to family Fabaceae, widely spread in India, Sri Lanka and Sudan. Being a common and important plant, using in many ways from fodder (shoots and leaves to animals) to dyeing (leather coloration) to medicine (root, bark, leaves, flower, gum, pods). The present study is focused on investigating the natural chemistry and important biological activities of the plant. Employing bioassay guided fractionation coupled with TLC and column chromatography, a pure fraction named AN-10 was isolated from ethyl acetate fraction of crude methanol extract which identified as "Betulin (Lupan-3ß, 28

-diol)" by Liebermann-Burchard test and structure elucidation by UV-Vis, NMR and MS techniques. A battery of in vitro biological assays for antioxidant, anti-inflammatory and anticancer
were performed and betulin showed excellent potential in all assays. It was found that the inhibitory potential in all assays were dose dependent manner and after a range of concentration, the activities get leveled off with no further increase in activity.
Increasing evidence from epidemiological and biological studies has shown that reactive oxygen species (ROS) are involved in variety of physiological and pathological processes 1,2 . Plant and food derived antioxidants are implicated in the prevention of cancer and aging by destroying oxidative species that initiate carcinogenesis through oxidative damage of DNA 3,4 . Previous scientific reports confirmed an inverse association between the daily consumption of fresh fruits & green vegetables and the chances of degenerative & chronic diseases 5 . The phenolic compounds of fruits and vegetable act as antioxidant through various ways, which includes complexation of redox-catalytic metal ions, scavenging of free radicals, and decomposition of peroxides. Especially in food-related systems (extracts/fractions), antioxidant activity studies using multiple experimental approaches, allow a complete screening of the putative chain-breaking capacity 6 . The phenols and polyphenols have attracted the interest of medical scientist because of their pharmacological properties 5,7 . Acacia nilotica (L.) Willd. Ex Del., (family Fabaceae) is a medicinal tree known for the versatile source of bioactive components. This plant offers a variety of compounds which are potent for their spasmogenic, vasoconstrictor, anti-hypertensive, antioxidant, antispasmodic, anti-inflammatory and anti-platelet aggregatory properties 8 . The leaves & flowers of A. nilotica, an evergreen tree are also been used as animal fodder [9][10][11] . The bark of the plant is rich with condensed tannins, catechin, epicatechin, epigallocatechin gallate and has also been used for the treatment of viral, bacterial, amoeboid, fungal, bleeding piles & leucodermal diseases 12 . The previous studies performed at Genetic Toxicology Laboratory of GNDU has shown that bark of A. nilotica enriched with kaempferol, umbelliferon, gallic acid, ellagic acid, which are responsible for their potent antioxidant, antimutagenic and cytotoxic activities 8,9,13 . The lack of detailed & systematic phenolic profiling of A. nilotica, which might be responsible for their important biological activities, led us to design the present study. In this study, HPLC based phenolic fingerprinting, bioassay guided fractionation, isolation & identification of betulin (AN-10) from ethyl acetate fraction of crude methanol extract of A. nilotica was done. The betulin was further checked for their antioxidant activities (DPPH, Deoxyribose, Chelating power, reducing power, lipid peroxidation assays), cytotoxic (SRB assay) & anti-inflammatory activities (COX-2 inhibitory assay).  (2) column (250 mm × 4.6 mm, 5 μm particle size). At a column temperature of 27 °C and a flow rate of 0.80 mL/min using solvent A (water) and solvent B (0.02% trifluroacetic acid (TFA) in acetonitrile) with a linear gradient elution: 70% A (5 min), 15-35% (7 min), 35-45% (11 min), 45-35% (16 min), 35-15% (20 min) at λ 280 nm. Stock solution containing ten analytes were prepared and diluted to appropriate concentrations for establishing calibration curves and different concentrations of theses analytes were injected thrice for the quantitative analysis and the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The selectivity of the method was determined by analyzing standards and methanol extract. The peaks of reference compounds were identified by comparing their retention times (rt in min.) with the spectrum of authentic standard (Sigma Aldrich, USA).

Bioassay guided fractionation and isolation of triterpenoids.
In the process of bioassay-guided fractionation, ethyl acetate fraction of crude methanol extract is first tested for their activities, then fractionation and separation through column and TLC and then the resulting fractions are again tested for activity. The most active fractions (#56-154) with similar spot on pre-coated TLC plates is processed further for the separation of triterpenoids by column chromatography (data shown in results section). 20 g ethyl acetate fraction of methanol extract mixed with celite '545' was suspended in methanol and subjected to column chromatography using a 75 × 3.5 cm glass column filled with acidic alumina (brockman's activity) upto 5 cm down from the top of glass column. After bedding down the silica gel, column elution stated with Identification of AN-10 fraction by NMR and MS techniques. 1 H and 13 C NMR spectra were recorded for purified "AN-10" fraction at 300 MHz, using 5-mm sample tubes on a Bruker Avance-300 spectrometer. CD 3 OD was used as solvent for measurements at 30 °C. For structure elucidation and complete spectrum analysis, other additional experiments were performed as necessary: DEPT, 13 C observation with selective 1 H decoupling, 2D H,H-COSY. 13 C chemical shifts δ are reported in ppm relative to TMS with an internal reference. With very few exceptions all NMR assignments are unequivocal. Mass spectra were recorded on QTOF-Micro of water Micromass. Melting point was determined on a Barnstead Electrothermal 9100.
Antioxidant activities testing assays. In vitro antioxidant activities (AOA) of the crude extracts/fractions of A. nilotica and "AN-10" fraction was addressed by employing DPPH scavenging assay measured in terms of hydrogen using the stable nitrogen centered radical DPPH following the method of Blois 15 . The hydroxyl radical scavenging was checked with site specific and non-site specific deoxyribose degradation method of Halliwell et al. 16 and Arouma et al. 17 . The reducing power was determined as described by Oyaizu 18 . The chelating effect on ferrous ions was determined according to the method of Dinis et al. 19 and Lipid Peroxidation (LPO) was determined according to Halliwell & Guttridge 20 . In vitro cytotoxicity assay. The Sulforhodamine B dye assay was used for In vitro cytotoxic screening of crude extracts/fractions of A. nilotica and "AN-10" fraction according to Skehan et al. 21 . For primary screening, A-549 (Lung), DU-145 & PC-3 (prostate), IGROV-1 (Ovary) and MCF-7 (Breast) cancer cell lines were used. The treatments were (OD) was recorded at 540 nm, on ELISA reader and percent growth inhibition in the presence of extract/fraction and "AN-10" was calculated.

Anti-inflammatory activity. In vitro COX-2 inhibiting activities of crude extracts/fractions of A. nilotica
and "AN-10" fraction has been evaluated using 'COX (ovine) inhibitor screening assay' kit with 96-well plates. Both ovine COX-1 and COX-2 enzymes were included 22 . This screening assay directly measures PGF 2 α produced by SnCl 2 reduction of COX-derived PGH 2 . The wells of the 96-well plate showing low absorption at 405 nm indicate the low level of prostaglandins in these wells and hence the less activity of the enzyme. Therefore, the COX inhibitory activities of the crude extracts/fractions of A. nilotica and "AN-10" fraction could be quantified from the absorption values of different wells the 96-well plate. www.nature.com/scientificreports/ Statistical analysis. All experimental analyses were performed in triplicate (n = 3) and the data was presented as mean ± SD on excel sheet. For in vitro antioxidant assays, one way ANOVA test followed by Tukey's test (P < 0.05) was used to analyze the differences among IC 50 of various AN-10 and extract/fractions for different antioxidant assays.

Results
% yield of extract, TPC and bioassay guided fractionation. The high % yield (57.69 g and 6.15% yield) and Total Phenolic Content (835 mg/g as GAE) of methanol extract than other crude extracts of A nilotica lead for the detailed bioassay-guided fractionation (Fig. 3), HPLC based phytochemical screening and biological activities. Silica-gel column chromatography was performed on ethyl acetate fraction (42.89 g) of methanol extract of A. nilotica and 692 fractions of 50 ml each were collected. All these fractions were pooled into 11 groups according to their similar spot at the same frontal ratio on thin layer chromatography profiles and biological activities (Fig. 1). 54-156 fractions (group 3) exhibited high antioxidant, anti-inflammatory & anticancer activities as compared to other fractions and group of fractions (Table 1). In order for the detailed chemical investigation and identification of active compounds, the most active fractions (group-3) were pooled, dried and fractionated through re-column chromatography (silica gel, 75 × 3.5 cm) and "AN-10" fraction was collected by solvent gradient of ethyl acetate/hexane (16:84). Other chromatography and spectroscopy techniques were used for identification and structure establishment of "AN-10" fraction.
In HPLC analysis, for the better resolution, different mobile phases were used and after several trails, mobile phase consisting of solvent A (water) and solvent B (0.02% trifluroacetic acid (TFA) in acetonitrile) as a solvent gradient was finely selected in order to achieve optimal separation & quantification, high sensitivity, and good peak shape. Table 2 shows the Retention Time (RT in minutes) and % quantification as µg/mg of 10 major polyphenols.
The presence of these polyphenols, in methanol extract of A. nilotica, was confirmed by comparison of their retention times and overlaying of UV spectra with authentic standards. The methanol extract which showed presence of these 10 polyphenols, among which catechin, epicatechin, quercetin gallic acid, umbelliferone, rutin and     (Fig. 4).
Biological activities of betulin. Figure 5 depicts the positive dose dependent DPPH radical scavenging potential of betulin. The addition of betulin led to change in colour, with a very fast reaction speed up to a concentration of 50 µg/ml. At 50 µg/ml concentration betulin exhibited 88.67% of activity (IC 50 23.75 µg/ml), and there is no change in colour and inhibition potential after this concentration. These in-vitro DPPH radical scavenging potential of Betulin revealed remarkable antioxidant potential. Previous studies reported the antioxidant activity of plant extracts has a positive correlation with percentage radical scavenging activity 25 . Therefore, an extract with high percentage radical scavenging activity ought to be a potent antioxidant in vitro and in vivo. The high percentage radical scavenging activity translates to low EC50/IC50 values 26 . Betulin also exhibited very good site (72.83%) & non-site (58.44%) specific hydroxyl radical scavenging potential at 50 µg/ml concentration and the results also showed that there is slight difference in the antioxidant potential of betulin in the site & non site specific modes of deoxyribose degradation assays.
In chelating power assay, betulin isolated from A. nilotica interfered with the formation of ferrous and ferrozine complexes, and have good chelating activity of 75.22% (IC 50 58.24 µg/ml) at 250 µg/ml concentration and are able to capture ferrous ion before ferrozine (Fig. 5). www.nature.com/scientificreports/ Figure 6 also showed the dose response ability of the betulin to reduce Fe(III) to Fe(II) at different concentrations. This reduction helps to predict the betulin ability to mimic the body's endogenous antioxidants like bilirubin and uric acid in attenuating oxidative stress 27,28 . Therefore, high ferric reducing antioxidant power is correlated with increase in absorbance values and low IC 50 values. Our results are confirmatory with previous reports which found that catechin, (epi) gallocatechin and caffeic acid present in the stem bark crude extract of S. crude have good antioxidant activities against DPPH radical scavenging and reducing power activities with low IC50 values 29 .
In lipid peroxidation assay, the betulin exhibit moderate to strong antioxidant potential i.e. 16.25-90.1567.2 ± 1.8% at 10-250 µg /ml concentration (weak-good). All the values of antioxidant activities were considered to be significant at P ≤ 0.05.
In this research work, cytotoxic activities of Betulin toward the A-549, DU-145, PC-3, IGROV-1 & MCF-7 cell lines was determined and the growth inhibition percentage by betulin is shown in Table 3     www.nature.com/scientificreports/ Betulin isolated from A. nilotica was also found to be a selective inhibitor of COX-2 (COX-2 selectivity > 10). At a concentration of 10 μM, it inhibited the COX-1 by 43.81% whereas COX-2 was inhibited by 95.03% ( Table 4). The ethyl acetate fraction of methanol extract also demonstrate strong capacity to suppress this inflammatory pathway. In the presence of betulin, the level of PGE 2 dropped too low. Flavonoids and other phenolic compounds are known to target cyclooxigenase-mediated inflammation [30][31][32] HPLC based presence of polyphenols and these polyphenols already reported to block cyclooxygenase activity induced by UVB radiation. Thus it might also be implicated in suppression of cyclooxygenase-mediated inflammatory pathway 33 .

Discussion
In the past two decades triterpenes have attracted attention because of their pharmacological potential. Among them, betulin is the most abundant and it is a representative compound of Betula platyphylla, a tree species belonging to the Betulaceae family 34 . Betulin has been demonstrated to have a selective cytotoxicity in tumor cell line [35][36][37] . It has also shown a strong reduction of hepatotoxicity 38 . Furthermore, betulin was shown to exhibit chemopreventive effects on UV induced DNA damage in congenital naevi (CMN) cells 39 . Previous studies on betulin also shown protective effects against Cd-induced cytotoxicity occur via the anti-apoptosis pathway in Hep3B cells, ethanol induced cytotoxicity in HepG2 and potent superoxide anion generation inhibitors in human neutrophils [40][41][42] . The antioxidant property of betulin was confirmed by its ability to scavenge and prevent the attack of free radicals on the membranes by increasing its negative surface charge 43 . Betulin and betulinic acid have been shown as potent phospholipase A2 inhibitors 44 . Furthermore, betulin acts as a modest TNF-α inducer by enhancing mitogen-induced TNF-α production, and betulinic acid modulates cytokine production by Th1/ Th2 cell subpopulations 45 . In the present study, we have isolated betulin from ethyl acetate fraction of methanol extract of A. nilotica and checked their different antioxidant, cytoprotective and anti-inflammatory activities employing a battery of in vitro assays. It is important to use different assays, instead of relying on a single assay to assess and compare the antioxidant capacity.
The methanol extract showed high biological potential than the other crude extracts of the A. nilotica (data not shown) and these results suggested that these high biological activities might be due to the high TPC. Many previous studies observed the direct relationship between TPC and antioxidant activity in medicinal plant extracts. The phenolic compounds may contribute directly to antioxidative action or as free radical scavengers due to their hydroxyl groups 46 . Tanaka et al. 47 , reported that 1 g phenolic compounds daily from a diet rich in fruits and vegetables have inhibitory effects on mutagenesis and carcinogenesis in humans. Currently the interests of phenolic compounds are increasing in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food 48 .
The HPLC based phenolic fingerprinting of methanol extract of A. nilotica showed the presence of many phenolic components such as gallic acid, quercetin, myricetin, rutin, kaempferol, catechin, epicatechin, ferulic acid, betulin and umbelliferone and are very significant to understand the relationship between the phenolic composition and bioactivities. The enrichment of the extract with polyphenols might responsible for the potent biological activities of the extract. Several previous studies have reported that these polyphenols exhibited strong antioxidative, anticancer, and anti-inflammatory activities [49][50][51][52] .
The methanol extract of A. nilotica showed the highest amount of TPC (835 mg/g as GAE) which lead us for the chromatographic & spectroscopic analysis of methanol extract. The chromatographic analysis on precoated Kieselgel 60 254 plate (0.2 mm thick; Merck, India), showed many spots of UV & iodine sensitive compounds. The repeated column chromatography of the fraction 54-156 (group 3) as shown in Fig. 2 24 .
In results of the present study we found that betulin was effective for reducing the stable DPPH radical to the yellow colored diphenylpicryl hydrazine, indicating their DPPH radical scavenging potential. The higher DPPH radical scavenging potential and lower IC 50 values are inverse to each other. It is pertinent to mention here that, DPPH potential may be due to the hydrogen atom donating ability of betulin, which further help in trapping free radicals. EDTA is used as the metal chelator in chelating power assay as it is a strong metal chelator. In the present study, betulin exhibits good reducing potential (Fig. 6). Natural plants/extracts having chelating potential are believed to inhibit lipid peroxidation by stabilizing transition metals 53 . In reducing power assay, the yellow colour of the test solution changes to various shades of green and blue based upon the reducing power Table 4. Cyclooxygenase enzyme mediated anti-inflammatory activities (COX-1 & COX-2) of "Betulin "isolated from bark of Acacia nilotica. *COX-2 selectivity = IC 50 (COX-1)/ IC 50 (COX-2). **Reported in literature (Kaur et al., 2009) 22 . www.nature.com/scientificreports/ of the tested compound. The reductive ability assay suggests that the betulin is able to donate electron, hence they should be able to donate electrons to free radicals in actual biological or food systems, making the radicals stable and unreactive. Reducing power is one mechanism of action of antioxidants and may serve as a significant indicator of potential antioxidant activity 54 . Previous reports also found dose dependent manner of hydroxyl radicals scavenging potential of xylose and lysine Maillard reaction products 55 . The free radical scavenging capability of phenolics are closely related with structural formation, molecular weight and presence of aromatic rings & hydroxyl groups of the phenolics 56 . Recent reports found that betulin to be active against colorectal, breast, prostate & lung cancer cell lines 57,58 . Betulin is a natural compound, which contains derivatives that have been shown to possess strong anti-tumor properties 5,59 . Recent studies also found that betulin in combination with cholesterol, is a very potent agent in killing cancer cells in vitro 60 .
Inflammation is a complex process, which involves many cell signaling pathways in addition to free radical production which are responsible for tissue degeneration and many diseases viz. rheumatoid arthritis, arteriosclerosis, myocarditis, infections, cancer, metabolic disorders [61][62][63] . COX-2 is an enzyme which is necessary for the production of pro-inflammatory prostaglandins and thus has been a target for many present anti-inflammatory and cancer-preventive drugs 64 . Several natural products of plant origin have been shown to transmit their antiinflammatory activities through suppression of COX-2 65 www.nature.com/scientificreports/