Isolation, characterization, anticancer and antioxidant activities of 2-methoxy mucic acid from Rhizophora apiculata: An in vitro and in silico studies

In this present study, the sugar based bioactive molecule, 2-methoxy mucic acid ( 4) was isolated for the rst time from the methanolic extract from the leaves of Rhizophora apiculata . The structure of compound was well characterized by different spectroscopic analysis, including FT-IR, 1 H, 13 C NMR spectroscopy and HRMS. Anticancer activity of 2-methoxy mucic acid ( 4 ) was evaluated against HeLa and MDA-MB231 cancer cell lines and they displayed promising activity with the IC 50 values of 22.88283±0.72 µg/ml in HeLa and 2.91925±0.52 µg/ml in case of MDA-MB231, respectively. The antioxidant property of 2-methoxy mucic acid ( 4 ) was found to be (IC 50 ) 21.361±0.41 µg/ml. Apart from in vitro studies, we also performed extensive in silico studies (molecular docking and molecular dynamics simulation) on four key anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-w, Bcl-xL and Bcl-B) towards 2-methoxy mucic acid ( 4) and the results revealed that this molecule showed higher binding anity towards Bcl-B protein ( ΔG = -5.8 kcal/mol) and the structural stability of Bcl-B protein was signicantly improved upon binding of this molecule. The present study affords key insights about the importance of 2-methoxy mucic acid ( 4 ), and thus leads to open the therapeutic route for anticancer drug discovery process.


Introduction
Mangrove plants are rich sources of natural products of polyphenols, phenolic compounds, alkaloids, avonoids, and tannins which are active secondary metabolites having wide applications in pharmacy and medicine (Kathiresan and Bingham, 2001;Sachithanandam et al., 2020). However, the mangrove plants from Andaman and Nicobar Islands (ANI), one of the most ecologically diverse places in the tropical region, have not been chemically and biologically studied in detail. The marine living and nonliving resources of ANI are unique because of the volcanic and mid-oceanic characteristics (Indo-Paci c triangle), with ecologically rich marine habitats that provide a wide variety of marine goods and services.
The Rhizophora genus consists of more than 10 species widely distributed all over the world (Sachithanandam et al., 2019). The R. apiculata in the family of Rhizophoraceae, has been exploited for numerous traditional medicines by people in Indo-Paci c region for treating various diseases and ailments, such as astringent, diarrhoea, chronic typhoid fever, septic wounds, nausea, pain, diabetes, bleeding in fresh wounds and in ammation (Loo et al., 2007;Kaliamurthi et al., 2014). The tribal communities of ANI are a reservoir of vast knowledge. They have been using R. apiculate for treatment of various diseases such as Diarrhoea, Nausea, Vomiting, Typhoid, Hepatitis, Ulcers, an antiseptic, Isolation, characterization, and bioprospecting of bioactive compounds from mangrove plants have always generated interest to many researchers (Wu et al., 2008;Sachithanandam et al., 2020). In addition, many bioactive compounds exhibited various biomedical properties such as anti-oxidant (Loo et al., 2008), anticancer, antimicrobial (Prabhu and Guruvayoorappan, 2012;Ramalingam and Rajaram, 2018) anti-nociceptive, antiviral (Premanathan et al., 1999), antihypoglycemic (Sachithanandam et al., 2019), and anti-cholinesterase activities (Suganthy et al., 2009). The mangrove plant contains various natural bioactive compounds such as, alkaloids, benzoquinone, campesterol, cinnamate, diterpene, avonoid, lupeol, essential oils, polysaccharide, polyphenols, sitosterol, sterols, stigmasterol and triterpenoids (Premanathan et al., 1999;Sachithanandam et al., 2020). Novel taraxeryl triterpenoid, taraxeryl cis-phydroxycinnamate phenolic compounds (pyroligneous acid), and diterpenoid have been isolated from the leaves of R. apiculata (Kokpol et al., 1990;Loo et al., 2008;Gao et al., 2011). The nitrogen alkaloids identi ed from this plant act as agonist for PPARγ receptor as reported by Selvaraj et al., (2015). R. apiculata leaves are the bioavailable source to develop biomedical application, which comprises more bioactive products when compared to bark and seedlings (Bhakuni et al., 1992). In our search for new bioactive phyto-chemical compounds from ANI mangroves, we studied leaves of R. apiculata to explore the possibilities of isolating novel biological compounds with promising antioxidant and anticancer activities. A breakthrough result was achieved when the R. apiculata leaves were subjected to methanol extraction followed by column chromatography, a new bio-active compound, namely, 2-methoxy mucic acid (4) (Fig. 1) was obtained with high purity (Fig. 1). Moreover, we also performed molecular docking studies on 2-methoxy mucic acid (4) towards four anti-apoptotic drug-target proteins (Bcl-2, Bcl-w, Bcl-xL and Bcl-B). Herein, we present the isolation, structure characterization, biological evaluation and in silico studies of 2-methoxy mucic acid from R. apiculata. In addition, the structural stability of 2-methoxy mucic acid molecule towards Bcl-B was studied through molecular dynamics (MD) simulation.
Mucic acid (1) (Fig. 1) is a tetrahydroxylated dicarboxylic organic acid, a naturally occurring sugar acid in putre ed blood and fresh juice from sugar beet (Stark et al., 1950). It was rst isolated from full ripe peaches and pears (Anet and Reynolds, 1954). Limited research only has been carried out towards the isolation and identi cation of mucic acid and its derivatives (Thompson and Kies, 1965). Traditionally, this acid is used in skin care and cosmetic products in India, China, and Thailand (Yu and Van Scott, 1995); used as a chelator in pharma industries (Lewkowski, 2001) and as an intermediate chemical for the synthesis of polymer materials (Mehtiö et al., 2015). Earlier, the mucic acid gallate (2) and di-O-gallate (3) (Fig. 1) was isolated from Phyllanthus emblica medicinal plant and studied for antioxidant activity (Zhang et al., 2017;Olennikov et al., 2015). Naturally, the mucic acid could be produced by fungal enzyme Trichoderma reesei and Escherichia coli as biological sources and through extraction of pectin from dry citrus peel (Barth and Wiebe, 2017). The synthesis of mucic acid was made both by nitric oxidation of Dgalactose and electrolytic oxidation of D-galacturonate (Kiely and Kirk, 2010). Because of the less solubility of mucic acid in water, it could be easily precipitated as solid in the culture broth (Zhang et al. 2016). Among the large variety of carbohydrate derivatives, the mucic acid (1) and its analogues (2)(3) have evoked great interest in the industry as precursor of looking for several other derivatives. In this regard, the mucic acid has been used as a versatile intermediate for the synthesis of various natural products as well as pharmaceutical drugs such as pyrones (Leonardi et al., 2020), pectin derived galacturonic acid (Purushothaman et al., 2018), furan and its polyester (Zhao et al., 2019), salts of mucic acid (Tian et al., 2000), adipic acid (Li et al., 2014), mucic acid 1,4-lactone methyl ester 3-O-ferulate (Sengoku et al., 2012) and mucic acid polymer products (Pan et al., 2015). In addition to mucic acid, its derivatives have exhibited various biological properties such as antioxidant (Luo et

Plant material
Raw and fresh leaves of mangrove plant, R. apiculata (Fig. 2) were collected from the Sippighat mangrove area, South Andaman Island, India in September 2018. The leaves were washed thoroughly with deionized water and dried at room temperature and later pulverized into ne powder by using mixer grinder.

Chemicals, reagents, and Standards
All Chemicals, inorganic solvents, and reagents were purchased from Finar, Merck, Sigma, Himedia Pvt Ltd., India. Glass plates coated with silica gel (60-120 mesh SRL chemicals) were employed for thin layer chromatography (TLC).

Extraction
The extraction of bioactive component details described our recent study (Sachithanandam et al., 2020), obtained as ne powder (10 g) mix with high polarity organic solvent like methanol (300 ml), was done in a conical ask and kept overnight with increasing polarity at ambient temperature (27 °C). It was incubated at room temperature for 48 hours at 150 rpm in an orbital shaker (Thermo Fisher Scienti c and Floor table top). The extracts were ltered with Whatman No. 1 lter paper. Each supernatant of the extract was concentrated/dried in vacuo by using rotary evaporator (CYBER, Germany) under reduced pressure at 40 °C temperature to afford 2 g of crude components.
Puri cation of natural product molecule (4) The methanolic crude extract was dissolved in about 0.5 ml of dichloromethane (DCM) and analytical TLC (50: 50, Chloroform: Methanol) was performed on aluminium sheets precoated with silica gel G/UV-254 of 0.2 mm thickness (Merck, Germany). Plates were observed under short wavelength UV light and they showed one active intensive spot with a few low intensive multiple spots. The value of Retention factor (R f ) of active fraction was found to be 0.57 using mobile phase chloroform: methanol (50:50, v/v).
The Crude extracts (about 1g) was dissolved in 50 ml DCM solvent and mix with silica gel (100-200 mesh) which was evaporated through rotary evaporator (CYBER, Germany) under without pressure to get crude slurry. After obtained slurry, it was then subjected to column chromatography and packed with 100% chloroform solvent on a silica gel (100-200 mesh). Then crude extracts were eluted with increasing order of polarities, ranges from (10-100%) mixtures of chloroform: methanol to provide multiple fractions according to TLC. The active fraction (R f = 0.57) was collected at 60 % solvent mixture (60% methanol: 40% chloroform) and these fractions were collected through test tube and concentrated / dried in vacuo by using rotary evaporator (CYBER, Germany) under reduced pressure to get orange pure product (4). The isolated compound collected from column chromatography was further puri ed by slow evaporation method using mixture of 4.5 ml of dichloromethane solvent (90% DCM) and 0.5 ml of hexane solvent (10% Hexane) and kept in vial at room temperature for one day settlement of crude product. For further ultra-puri cation, this obtained compound was subjected to recrystallization process by using ethanol solvent and heated to 40-50 o C and cooled it to room temperature and stored in refrigerator at 4 o C for two days. Further, it gives pure crystalline form of potassium-2-methoxy mucic acid (4) (orange colour) with a yield of 10% (100 mg). The remaining low intensity fractions were unable to be characterized due to low quantity, impurity, and decomposition factors. Further, the structure of the isolated pure 2-methoxy mucic acid (4) was characterized by spectral analysis such as FT-IR, 1 H NMR, 13 C NMR spectral data and HRMS analysis. were equipped in a similar way without pure compound (sample extract). After 30 min, the absorbance of the prepared samples was measured using UV spectroscopy at a wavelength of 517 nm. This experiment was replicated in three times. The percentage of DPPH scavenging effect was calculated by the following equation given below.
% Inhibition: Absorbance of control (A0) -Absorbance of test sample (A1) / Absorbance of control X 100 Whereas A0 is the absorbance of negative control (0.004% DPPH solution) and A is the absorbance in presence of extract. The results were reported as IC 50 values and ascorbic acid equivalents (AAE, mg/g) of mangrove extracts.

Molecular docking and molecular dynamics simulation studies
Molecular docking is a computational procedure to understand the binding orientation. mode of binding, key intermolecular interactions, key interacting residues and estimated free energy of binding, estimated inhibition constant of a ligand molecule towards chosen drug target protein. To our knowledge, there is no existing reports on 2-methoxy mucic acid (4) with reference to interaction studies towards drug target proteins by either in silico or in vitro studies. We have identi ed this vital research gap and to ll this, very rst time, we have selected anti-apoptotic proteins as the suitable drug targets for this natural product. with kollman partial charges, and recorded into PDBQT (XYZ coordinates + partial charges + atom types) format. Similarly, the three-dimensional structure of 2-methoxy mucic acid (4) was also prepared with the addition of polar hydrogens, merging of non-polar hydrogens, addition of gasteiger charges and recorded into PDBQT format by the same Auto Dock Tools. Once protein and ligand preparation steps were completed, the receptor grid map was generated for four key anti-apoptotic proteins based on their binding sites or binding clefts or binding groove (Bcl-2 protein: center_x = 9.71 Å, center_y = 18. 45  . It is one of the most reliable end-point approaches for free energy calculations. The 10 ns of stable simulated trajectories were extracted, and snapshots were captured at a regular interval of 100 ps. In short, 100 structures from the 0-10 ns period were utilized for the MM/PBSA binding free energy calculations.

Results And Discussion
Characterisation of natural product 2-methoxy mucic acid (4) The 2-methoxy mucic acid (4) was isolated by TLC which was found to possess a molecular formula of Results obtained from the NMR data analyses is given in Table 1. The 1 H NMR (400 MHz) spectrum of 2-methoxy mucic acid (4) displayed a characteristic methoxy proton signal at 3.49 ppm at a singlet ( Supplementary Information, Fig. 2). The four aliphatic methine C-H protons such as C2-H, C3-H, C4-H and C5-H were appeared at 3.36-3.27 ppm as multiplet, 4.03 ppm as singlet, 3.77 ppm as doublet and 3.99 -3.73 ppm as doublet, respectively ( Supplementary Information, Fig. 2). The OH and COOH protons did not appear due to interaction between four OH and two COOH protons with potassium ions (Tian et al., 2000). The 13 C NMR (100 MHz) spectrum of 2-methoxy mucic acid (4) displayed the predicted seven signals ( Supplementary Information, Fig. 3). The methoxy carbon appeared at 57.25 ppm. The COOH carbonyl peaks were observed at δ 179.52 ppm (C-1) and 178.16 (C-6) ppm, respectively. The aliphatic CH carbons were observed at 82.52 (C-2), 69.09 (C-3), 70.60 (C-4) and 73.52 (C-5) ppm respectively. These types of carbons were further con rmed by 13 C Distortionless enhancement by polarization transfer (DEPT-135), an NMR method ( Supplementary Information, Fig. 4). The mass of the 2-methoxy mucic acid (4) was determined as C 7 H 7 K 5 O 8 with 413.2626 atomic mass units (amu) and found 413.2627 amu by Electrospray Ionisation method (ESI-HRMS) ( Supplementary Information, Fig. 5). The puri ed compound were analysed by Flame photometer spectroscopy for the total mineral content to conform the potassium salts present in the molecule (Systronics, Type 128) according to (APHA). All instrumental parameters were corrected to the best ame condition. Then calibration of the Flame photometer was done using the suitable standard solution (Perkin Elmer) for potassium. After the calibration, samples were run in triplicate to estimate the potassium concentration. Good recovery of 99% were obtained for the potassium standard solution, indicates the accuracy of the analytical methodology.  The antioxidant activity of 2-methoxy mucic acid (4) isolated from methanolic leaf extract of R. apiculata, was analysed with different doses, namely 1, 10, 25, 50, 100, 150 and 200 µg/ml, respectively. The results con rmed a dose dependent inhibitory activity of 2-methoxy mucic acid (4) and a free radical scavenging activity was found. The IC 50 values for DPPH antioxidant activity of 2-methoxy mucic (4) was 21.361±0.41 µg/ml; however, the Ascorbic acid used as positive control exhibited better radical scavenging effect with IC 50 values of 5.5486±0.81 µg/ml (Fig. 3). In the literature review on antioxidant activity of mucic acid derivatives, it was found that the fruit of Phyllanthus emblica L. yielded mucic acid derivatives with good antioxidant activity and the extracts from this fruit produced many phytochemical compounds having IC 50 values: gallic acid 18.71 ± 0.77, ellagic acid 13.81 ± 0.59, mucic acid 1,4-lactone 3-O-gallate 23.72 ± 1.05, isocorilagin 8.12 ± 0.59, chebulanin 11.27 ± 0.67, chebulagic acid 4.14 ± 0.19, and Mallotusinin 3.99 ± 0.11 µM, respectively (Luo et al., 2011). Another work also reported that mucic acid gallates isolated from P. emblica showed strong antioxidant activity with IC 50 values of 12.84 µM, by using DPPH method (Olennikov et al., 2015).

Cytotoxicity activity by MTT assay
In vitro inhibitory activity of 2-methoxy mucic acid (4), isolated from mangrove plant R. apiculata, was determined by MTT reduction assay on cervical (HeLa) and breast (MDA-MB231) cancer cell lines. The results con rmed a dose-dependent inhibitory activity of 2-methoxy mucic acid (4)  Molecular docking or in silico interaction studies was carried out between 2-methoxy mucic acid (4) and four members of anti-apoptotic proteins (Bcl-2, Bcl-w, Bcl-xL and Bcl-B) to understand the mode of binding, key interacting residues, type of intermolecular interactions, binding orientation of ligand towards binding site of the drug target proteins. The estimated free energy of binding (ΔG) and estimated inhibition constant (Ki) of 2-methoxy mucic acid (4) towards four drug target proteins was presented in Table 2. Of the four docking complexes, the 2-methoxy mucic acid (4) molecule showed higher binding a nity towards anti-apoptotic Bcl-B protein with the estimated binding free energy of -5.8 kcal/mol. The electrostatic potential surface calculation revealed that this ligand molecule mostly oriented in negatively charged portion of the drug target proteins (Fig. 5). Interaction analysis also explained that higher number intermolecular hydrogen bonding and hydrophobic interactions are observed between Bcl-B and 2methoxy mucic acid (4). The intermolecular interaction results of four protein-ligand complexes are given in Figs. 6 and 7. Interaction pattern analysis of four complexes revealed that this natural molecule strongly binds to the BH3 binding groove or binding cleft of anti-apoptotic proteins. This region is very important for anti-apoptotic function as well as this site is responsible for interaction of their pro apoptotic interacting partners. The estimated binding free energy values (large negative) were corroborated with the results obtained from interaction pattern analysis (higher number of intermolecular interactions). Based on the results obtained from molecular docking calculation of 2-methoxy mucic acid (4) with four anti-apoptotic members, we have chosen Bcl-B as a suitable drug target, since the ligand molecule showed stronger binding a nity and higher number intermolecular interactions and therefore this Bcl-B: 2-methoxy mucic acid (4) complex subsequently used for further molecular dynamics simulation in order to understand the long term structural stability, structural integrity, structural compactness and folding properties. Moreover, we also tried to understand the behavior of 2-methoxy mucic acid (4) towards the structural stability of Bcl-B protein.
The main drawback of molecular docking studies is to consider ligand as completely exible whereas protein or receptor as rigid entity (to some extent, few residues are treated as exible not as a whole protein). To validate the results obtained from molecular docking calculation and understand whether the 2-methoxy mucic acid (4) molecule enhances the structural stability, structural integrity, structural compactness, and folding properties of Bcl-B, apart from molecular docking, we subsequently performed MD simulation in two different states such as unbound Bcl-B and 2-methoxy mucic acid (4) bound Bcl-B structures. Moreover, this study was also used to validate the results obtained from molecular docking calculation which is a preliminary study to understand the intermolecular interaction between protein and ligand molecule. The number of water molecules and counter ions are used in the molecular dynamics simulation of protein and protein-ligand complexes are given in Table 3. The molecular dynamics simulation results (Figs. 7-8 and Table 3) explained that upon binding of 2-methoxy mucic acid (4) to the binding site of Bcl-B protein which will signi cantly enhance the structural stability of Bcl-B with the results obtained from essential dynamics analysis which covered the lesser region form (34.99 nm 2 ) in conformational space in comparison with unbound form (56.82 nm 2 ). Essential dynamics is very important analysis since it captures the essential motion from the global motions. The main application of Principal Component Analysis (PCA) is reduction of dimension, i.e., it can transform the high dimensional data (global dynamics or global motion) of protein dynamics or protein-ligand dynamics into low dimensional space (essential dynamics or essential motions) to derive a range of eigenvectors and eigenvalues. Generally, the rst two principal components (PC1 and PC2) are highly responsible most of the dominant motions, therefore, we have considered rst two principal components in the present study.

Conclusion
In summary, a carbohydrate based natural biomolecule, namely 2-methoxy mucic acid (4) was isolated for the rst time from the leaves of a mangrove species, R. apiculata, using methanolic extract with high purity. Remarkably, 2-methoxy mucic acid (4) has indicated a highly signi cant dual anticancer and antioxidant activity. Together the results of molecular docking and molecular dynamics simulation results suggested that 2-methoxy mucic acid (4) binds strongly to the anti-apoptotic Bcl-B protein. The in vitro and in silico results taken together indicate that the natural compound (4) is one of the most potent anticancer molecules. From the results of both in vitro anticancer and antioxidant studies, it has been concluded that these molecules possess the good apoptotic inducing activity, which may be used as promising leads for the development of more potent anti-cancer drugs. Further works on identi cation and biomedical application of more bioactive phyto-chemical products from other parts of R. apiculata as possible herbal source of medicines will be helpful in treatment of several critical diseases including cancer. Figure 1 Reported structures of mucic acid analogues (1-3) and 2-methoxy mucic acid (4, Present work). Antioxidant activity for 2-methoxy mucic acid (4) isolated from methanolic leaf extract of R. apiculata in comparison with Ascorbic acid.

Figure 4
Cytotoxicity effect of 2-methoxy mucic acid (4) on HeLa and MDA-MB231 cancer cell lines. (Doseresponse analysis of 2-methoxy mucic acid was done pertaining to inhibition of HeLa and MDA-MB231 cancer cell lines. 0.2 x 105 cells/ well were seeded in 96-well tissue culture plate, followed by treatment with indicated concentration (0.5, 5, 25, 50 and 100 µg/ml) of 2-methoxy mucic acid (4) for 24h. Cytotoxicity effect was determined by MTT assay after the speci ed incubation period).