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 Table of Contents  
DPU: INTERDISCIPLINARY CONFERENCE
Year : 2020  |  Volume : 7  |  Issue : 5  |  Page : 41-48

Purification and characterization of pectins from Abelmoschus esculentus (okra pods) and Citrus limetta (citrus peels) and in silico binding study of pectin and pectic polysaccharides with galectin-1


1 Protein Biochemistry Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth (Deemed to be University), Pune, Maharashtra, India
2 Bioinformatics Research Laboratory, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth (Deemed to be University), Pune, Maharashtra, India
3 Department of Physiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth (Deemed to be University), Pune, Maharashtra, India

Date of Web Publication26-Feb-2020

Correspondence Address:
Rajesh Kumar Gupta
Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth (Deemed to be University), Tathawade, Pune - 411 033, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdrr.jdrr_62_19

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  Abstract 


Galectins are a class of animal lectins that bind to β-galactoside residues through a conserved carbohydrate recognition domain of about 130 amino acids. The carbohydrate-binding specificity of mammalian galectins revealed its affinity toward lactose, related beta-galactosides, and any glycoconjugates with a nonreducing galactoside terminus. Recent studies have indicated that galectin-1 upregulation impacts progression of tumor through its pleiotropic roles such as cell transformation and proliferation, angiogenesis, adhesion, and invasiveness and immunosuppression. Several galectin-1 inhibitors such as thiodigalactoside, Anginex GM-CT-01 and GR-MD-02 have been designed for applications in cancer therapy. GM-CT-01 (DAVANAT), a modified polysaccharide, composed of mannose and galactose (galactomannan) and a variant of DAVANAT, GR-MD-02, which is a polysaccharide with a backbone of rhamnogalacturonate and branches that terminate with galactose and arabinose residues are produced from chemically processed and modified industrial-grade apple pectin are also found to be beneficial in therapy toward cancer as well as nonalcoholic steatohepatitis. Various reports have also presented evidence that galectin-1-targeted therapy will play a major role to reduce the distribution of tumor cells, inhibit angiogenesis, and restrict tumor growth, thus seeing the importance of pectins and pectin polysaccharides in cancer therapy, we purified and characterized pectins from okra pods (Abelmoschusesculentus) and citrus peels (Citruslimetta) and studied the residual protein content, total carbohydrate content, characteristic properties using Fourier-transform infrared, galacturonic acid content, equivalent weight, and methoxyl content and methyl esterification of extracted okra and citrus pectins. We further performed insilico binding study of pectin and pectic polysaccharides with Galectin-1.

Keywords: Cancer, galectin, lectin, pectin, tumor


How to cite this article:
Gupta RD, Parekh K, Warrier VU, Lokhande KB, Swamy K V, Sood RS, Gupta RK. Purification and characterization of pectins from Abelmoschus esculentus (okra pods) and Citrus limetta (citrus peels) and in silico binding study of pectin and pectic polysaccharides with galectin-1. J Dent Res Rev 2020;7, Suppl S2:41-8

How to cite this URL:
Gupta RD, Parekh K, Warrier VU, Lokhande KB, Swamy K V, Sood RS, Gupta RK. Purification and characterization of pectins from Abelmoschus esculentus (okra pods) and Citrus limetta (citrus peels) and in silico binding study of pectin and pectic polysaccharides with galectin-1. J Dent Res Rev [serial online] 2020 [cited 2020 Apr 8];7, Suppl S2:41-8. Available from: http://www.jdrr.org/text.asp?2020/7/5/41/278902

Editor: Dr. Sarika Chaturvedi





  Introduction Top


Galectin-1 is a monomer of 14 kDa or a noncovalent homodimer and is composed of one carbohydrate-recognition domain (CRD) per subunit. Compelling indications show that galectin-1 upregulation can stimulate tumor advancement through its pleiotropic role in cell transformation, proliferation, angiogenesis, adhesion, invasiveness, and immunosuppression. An increase in expression of galectin-1 is perceived in a number of neoplasms, which includes colorectal, lung, breast, pancreas, liver, thyroid, and hematological malignancies. Several galectin-1 inhibitors have already been designed such as thiodigalactoside, anginex (β pep-25), 6DBF7, DB16, DB21, OTX008 (0018), F8.G7, GM-CT-01 (DAVANAT®), and oraz GR-MD-02 that may have a prospective clinical application toward curing cancer. Of these, GM-CT-01 (DAVANAT®) is an improved polysaccharide, composed of mannose and galactose (galactomannan) extracted from Guar seeds, is of particular interest, as it shows an affinity to the dimer interface instead of the CRDs in galectin-1 and galectin-3. Another variant of DAVANAT®, GR-MD-02, a polysaccharide having a rhamnogalacturonate backbone that has its branches terminate with galactose and arabinose, can be derived from chemical processing and modification from industrial grade apple pectin. Certainly, Gal-1-targeted therapy will play a major role to weaken the distribution of tumor and constrain angiogenesis.[1] Thus, in view of the above and seeing the importance of various pectins in inhibition of galectins, we decided to (i) purify and characterize pectins from Abelmoschus esculentus (okra pods) and Citrus limetta (citrus peels) and (ii) in silico binding study of pectin and pectic polysaccharides with Galectin-1.


  Materials and Methods Top


Materials

Ethyl alcohol, chloroform, butanol, sodium hydroxide (NaOH), HCl, H2 SO4, phenol, phenol red indicator, carbazole, pure pectin, Na2 CO3, sodium potassium tartrate, CuSO4.5H2O, Folin–Ciocalteu reagent, galactose, arabinose, and rhamnose were purchased from Fisher Scientific, HIMEDIA, MERCK, SD Fine chemicals, SRL chemicals, etc., All chemicals used were of highest analytical grade available. All experiments were carried out in Ultrapure (Type 1) and Pure (Type 3) water from Direct-Q3 water purification system from Millipore.

Methods

Processing of okra pods

Freshly bought okra pods (A. esculentus) were purchased local market. All okra pods were disease-free and washed thoroughly with tap water and then oven-dried, set at 60°C for 2–3 days until it is completely dried. Dried okra pods were finely ground to powder with grinder, then filtered through 50 mesh sieves to obtain fine powder and weighed.

Processing of sweet lime peels

Fresh sweet lime (C. limetta) peels were obtained from local fruit market. Peels were fresh without contamination, washed and dried in a hot air oven at 60°C for 2–3 days until it dried completely. Dried peels were grounded into powder with the help of Blender and then filtered through 50 mesh sieves to obtain fine powder and weighed.

Extraction of pectin from okra pods

Five grams of okra pod powder was stirred in 75% ethanol (liquid: solid ratio = 20:1) for 4–5 h at room temperature followed by separation of alcohol-insoluble substance (AIS) from alcohol mixture using muslin cloth. AIS obtained was then stirred in 100 ml of ultrapure (Type 1) water (1:20 ratio, i.e., 1 g of okra AIS and 20 ml of water) maintained at 50°C–80°C for 90 min. After the solution reaches room temperature, it was centrifuged at 10,000 rpm (9055 rcf) for 10 min at 20°C. Pellet was again resuspended in water (1:15 ratio, pellet-ultrapure water) and centrifuged again at 10,000 rpm (9055 rcf) for 10 min at 20°C. Total 160 ml of solution was obtained, in which Sevag reagent was added in 1:1 ratio (1 part of supernatant and 1 part of Sevag reagent) (4:1 ratio of chloroform: butanol) to denature the protein completely and to obtain pure carbohydrate. While addition and mixing, the whole process was carried out in the dark condition. Further, the solution was centrifuged at 2500 rpm (503 rcf) for 10 min at 4°C. Three layers were obtained after centrifugation, and it was expected that first layer as desired polysaccharide (Pectin), second layer pellet, and third clear white solution. The obtained supernatant was added with alcohol in 1:2 ratio (Supernatant [1 part] and alcohol [2 part]) and kept overnight at 4 °C). Further, the solution was centrifuged at 10,000 rpm (9055 rcf) at 4°C for 30 min. Supernatant was further discarded and pellet (pectin) was further dialyzed against water for 30 h at room temperature against ultrapure water. After the dialysis, the pellet was transferred into 2 ml microcentrifuge tubes and centrifuged at 10,000 rpm (9055 rcf) for 30 min. The obtained pectin pellet was resuspended in minimum volume of ultrapure water and transferred into 2 ml microcentrifuge tubes. Pectin-containing microcentrifuge tubes were then lyophilized for 4–5 h until they completely dried and converted into powder state. The remaining material after freeze-drying is referred to as okra pectin.[2]

Extraction of citrus pectin from Citrus limetta

Eighty grams of citrus powder were taken and digested in 400 ml of 0.01 N HCl for 1.5 h on a stirrer set at 150 rpm at 80°C–90°C. After 1.5 h, the heated solution was allowed to be cooled and filtered through muslin cloth and Whatman filter paper. 500 ml filtrate was obtained. For the further processing, 100 ml filtrate was taken and precipitated with 95% ethanol in a ratio of 1:2 (filtrate: ethanol) and kept overnight at 4°C. Further, it was centrifuged at 10,000 rpm for 30 min at 4°C. Supernatant and pellet were collected separately for further processing. The pellet was resuspended in 20 ml ultrapure water and dialyzed against pure water for 30 h. Further, it was centrifuged at 10,000 rpm for 30 min at 4°C and pellet was recovered. Pellet was resuspended in a minimal volume of ultrapure water and aliquoted in 2 ml microcentrifuge tubes. Tubes were lyophilized for 2–3 h until fine powder of pectin was obtained. The remaining material after freeze-drying was referred to as dried pure citrus pectin and weighed to calculate the total yield.[3]

Characterization of okra and citrus pectin

Residual protein estimation in extracted okra and citrus pectin

The total residual protein content in extracted purified okra and citrus pectin was estimated as Lowry et al., 1951,[4] using bovine serum albumin calibration curve.

Total carbohydrate content estimation of extracted okra and citrus pectin

Phenol–sulfuric acid method was used to estimate total carbohydrate (Dubois et al., 1951).[5] Dubois test in microplate format was essentially performed as described by Masuko et al., 2005.[6] In a reaction set, 50 μl of various sugars reported to be present in Okra and citrus pectin, i.e., rhamnose, galactose, arabinose, fucose, and mannose (0.5–4 μg in ultrapure water) was taken in 96 well microplate for preparation of calibration curve of these sugars. Thirty microliter of 5% phenol was added straight in 96 well plate. To this, 150 μl of concentrated H2 SO4 was added directly into the solution quickly. Reaction mixture was incubated for 30 min at room temperature. Blank was also prepared in the same way substituting ultrapure water for the sugar solution. Readings were taken at 490 nm against a blank. A microplate reader (Epoch biotek) was used for the absorbance measurement at 490 nm. For the estimation of sugars present in okra and citrus pectin, stock solution of okra and citrus pectin was made using 1 mg of pectin (solubilized after heating at 60°C in 1 ml of ultrapure Type 1 water) and further diluted by pipetting out 100 μl in 900 μl of water (total 1000 μl). From this stock solution of pectin, 50 μl of was taken for the sugar estimation.

Fourier-transform infrared spectra of extracted okra and citrus pectin

Fourier-transform infrared (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas. An FTIR spectrometer same time collects high spectral resolution data over a large spectral range. This confirms a major advantage over a dispersive spectrometer. The extracted okra pectin was analyzed spectrophotometrically. Each of the pectin sample obtained was ground and mixed with dry potassium bromide in mortar-pestle and compressed into disc in hydraulic pellet press applying 100 g/cm pressure. The spectra of pure pectin (from SRL chemicals containing galacturonic acid (GA) minimum 80%; methoxyl content minimum 7%), extracted okra and citrus pectin were obtained between 4500 and 400 cm−1 in attenuated total reflection mode at a resolution of 4 cm−1 using FTIR-8400s (SHIMADZU).

Galacturonic acid content of extracted okra and citrus pectin

The GA content was estimated according to the method of Dische[7] with some modifications. Briefly, 1 ml of pectin (10 mg/100 ml ultrapure water) solution was mixed carefully with 6 ml of concentrated H2 SO4 and allowed to stand for 20 min. After that, 200 μL of 0.1% carbazole solution (prepared in absolute ethanol) was added and kept for 2 h to develop pink color. Absorbance was recorded at 520 nm using a double-beam spectrophotometer (UV-1800 SHIMADZU). The GA content was calculated from a standard curve of pure pectin (0–200 μg/ml) obtained from SRL chemicals (containing galacturonic content min 80% and methoxyl content min 7%) and concentration was expressed as mg/g of pectin.

Equivalent weight of extracted okra and citrus pectin

The gel-forming ability of pectin is determined by its equivalent weight.[8] The equivalent weight is inversely related to the free acid content and is linked to the gelling power of the pectin extracted. Equivalent weight (Eq. W.) of okra pectin was estimated by the method of Ranganna 1986).[9] Briefly, 10 mg of okra pectin was dissolved in 10 ml of ultrapure water at 60°C and stirred for 2 h until it get completely dissolved. One gram of sodium chloride (NaCl) was added in okra pectin solution, and further, the okra pectin solution was titrated with 0.1 M sodium hydroxide (NaOH) using 2 drops of phenol red as an indicator. For citrus pectin, 50 mg of pectin was dissolved in 10 ml of distilled water at 60°C and stirred for 2 h until completely dissolved. 0.1 g of NaCl was added and titrated with 0.1 M of NaOH using 5 drops of phenol red as an indicator. Eq. W. was calculated using the following equation:

Equivalent weight



Methoxyl content of extracted okra and citrus pectin

Methoxyl content is an important measure in determining the settling time of pectin. It was calculated by saponification of extracted pectin and titration of liberated carboxyl group. Methoxyl content was estimated according to method of Ranganna (1995).[9] The methoxyl content was determined using the neutralized solution collected from the determination of Eq. W. The neutralized solution was mixed with 2.5 ml of 0.25 M NaOH and allowed to stand at room temperature for 30 min, after which 2.5 ml of 0.25 M HCl was added and titrated against 0.1 M NaOH. The methoxyl content was calculated using the following equation:

Methoxyl content =



Degree of methyl esterification of extracted okra pectin

Degree of methyl esterification (DM) was calculated from the GA and methoxyl content values. DM esterification was calculated according to the equation[10] where 176 and 31 are the molecular weight of anhydrous GA and methoxyl, respectively.



Galectin-1 and ligands structure preparation

The crystal structure of human Galectin-1 was retrieved from the Protein Data Bank (PDB) database (PDB ID: 4Q27) having 136 amino acid length and 1.2 Š resolution. The crystal structure of Galectin-1 was prepared using protein preparation wizard[11] module of Schrodinger suite. Hydrogen atoms were added to the crystal structure and the ionization state of amino acids was done at pH 7.5, at which Galectin-1 crystalize. To relieve steric clashes, hydrogen bonds optimized using the OPLS-2005 force field. The structures of pectin, pectin-disaccharides, D-Arabinose, tetragalacturonic acid, rhamnogalacturonan (RG)-I, and RG-II were retrieved from PubChem and ChEMBL database and subjected to energy minimization using energy minimization to obtain energetically stable confirmations using OPLS-2005 force filed in Maestro software.[12]

Molecular docking calculations

To appraise binding mode ligands with Galectin-1n, molecular-docking calculations was performed on pectin, pectin-disaccharides, D-Arabinose, tetragalacturonic acid, RG-I, and RG-II, using FlexX software.[13] The crystal structure of Galectin-1 protein (PDB ID: 4Q27) was retrieved from the PDB database kept as rigid molecule during the docking. The binding site of the Galectin-1 was defined using receptor preparation wizard in FlexX, considering all amino acids are located within 6.5 Š from bound crystal ligand (prop-2-en-1-yl 2-(acetylamino) 2-deoxy 4-O-[3-O-(prop-2-yn-1-yl)-beta-D-galactopyranosyl] beta-D-glucopyranoside) of Galectin-1 (PDB ID: 4Q27). All compounds are then docked into the binding cavity of Galectin-1 to predict binding conformations for the ligands, using incremental construction algorithm.[13] The docking parameters are kept at its default value and numbers of possible binding conformations within binding cavity of Galectin-1 are generated. The best conformations of docked compounds were considered by the binding energy and its binding interaction.


  Results and Discussion Top


Extraction of okra pectin

One kilogram of okra pods was taken and dried at 60°C. Dried okra pods were obtained after 2–3 days of incubation at 60°C. After grinding, 70 g of okra powder was obtained from 1 kg of okra. Five grams of okra pod powder were taken and stirred in 75% ethanol for 4–5 h and AIS was separated. A total of 2.96 mg of AIS was obtained from 5 g of okra powder. 2.96 g AIS extract was dissolved in 100 ml of ultrapure water and kept on stirring for 4–5 h at 50°C–80°C for 90 min, followed by centrifugation and further pellet was resuspended again in 60 ml of ultrapure water, and centrifuged at 10,000 rpm (9055 rcf) for 10 min at 20°C. Both the supernatants were pooled (100 ml + 60 ml = 160 ml). A total of 160 ml of combine supernatant was added with 160 ml Sevag reagent (1:1 ratio), precaution was taken, while addition of Sevag reagent, dark condition was always maintained. Solution was further centrifuged at 2500 rpm (565 rcf) for 10 min at 4°C. Three layers were obtained after centrifugation. The first supernatant layer (desired pectin) was added with alcohol in 1:2 ratio and kept overnight at 4°C followed by centrifugation at 10,000 rpm (9055 rcf), 4°C, 30 min. The pellet was resuspended in minimum volume of water and dialyzed against ultrapure water for 30 h with several changes of ultrapure water. After centrifugation, the pellet was centrifuged at 10,000 rpm (9055 rcf), 30 min at 4°C. Pellet was reconstituted in minimum volume of ultrapure okra pectin was freeze-dried to get powdered pectin. A total of 2 mg of purified okra pectin was obtained from 5 g of okra pod powder.

Extraction of citrus pectin

Two kilograms of C. limetta peels were taken and dried at 60°C for 2–3 days. After grinding, 455 g of citrus powder was obtained from 1.5 kg of citrus peels. Eight grams of citrus powder was taken and was digested in 400 ml of 0.01 N HCl for 1.5 h on magnetic stirrer at 80°C–90°C. After 1.5 h, the heated solution was allowed to be cooled and filtered through muslin cloth and Whatman No. 3 filter paper. The 100 ml filtrate was taken and precipitated with 95% ethanol in a ratio of 1:2 and kept overnight at 4°C. Further, it was centrifuged at 10,000 rpm (9055rcf) for 30 min at 4°C. The pellet was re-suspended in ultrapure water and dialyzed for 30 h. Further, it was centrifuged at 10,000 rpm (9055rcf) for 30 min at 4°C. Pellet was resuspended in minimal volume of ultrapure water and freeze-dried to get powdered pectin. A total of 1.3 g of pure citrus pectin was obtained from 80 g of citrus powder.

Characterization of okra and citrus pectin

Residual protein content estimation in purified okra and citrus pectin

No residual protein content was found in purified okra and citrus pectin which confirms that extracted pectin was homogenous and free from any residual protein. This concludes that the purified okra pectin is pure.

Carbohydrate estimation of okra and citrus pectin

Standard calibration curves of galactose, rhamnose, arabinose, mannose, and fucose were prepared using phenol-sulfuric acid method as described in materials and methods. The concentration of these sugars in extracted okra pectin was estimated using standard curve (0.5–4 μg) and the concentration of galactose, rhamnose, and arabinose in 50 μg of extracted okra pectin was found to be 3.2 μg, 2.62 μg, and 0.67 μg/50 μg, respectively. Similarly, concentration of various sugars in citrus pectin was calculated using standard calibration curve of galactose, rhamnose, arabinose, mannose, and fucose and found to be 2.13 μg, 1.71 μg, 0.17 μg, 1.83 μg, 0.17 μg, and 50 μg of citrus pectin, respectively.

Fourier-transform infrared spectroscopy okra and citrus pectin

The FTIR spectra of purified pectin from okra [Figure 1] and citrus [Figure 2] were obtained using SHIMADZU (FTIR-8400). The spectra of okra and citrus pectin are given below:
Figure 1: Fourier-transform infrared spectra of extracted okra pectin. 503.44 540.09 553.59 650.03 788.91 937.44 1043.52 1261.49 1356.00 1402.30 1653.05 1847.87 1979.03 2127.55 2181.56 2341.66 2364.81 2542.26 2698.50 2785.30 2989.76 3203.87 3215.44 3352.39 3464.27 3607.01 3751.67 3853.90 3981.214139.38 4197.24 4420.99

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Figure 2: Fourier-transform infrared spectra of extracted citrus pectin. 526.58 613.38 786.98 1070.53 1296.21 1435.09 1523.82 1629.90 1726.35 1847.87 1982.89 2077.40 2332.02 2445.82 2553.84 2928.04 3076.56 3271.38 3404.47 3481.63 3992.78 4073.79

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The IR spectra showed a broad peak at 3352.39 cm−1 due to stretching of O–H group (range 3500–3000 cm−1); peak at 2989.76 cm−1 is due to C–H stretching. It showed typical peaks at 1653.05 (range 1750–1350 cm−1) representing ester carbonyl C = O group and also a peak at 1402.30 and 1435.09 cm−1 corresponds to carboxylate, COO group stretching (range 1600–1650 cm−1, antisymmetric stretch and 1400–1450 cm−1, symmetric stretch). An absorption peak at 1043.52 cm−1 indicates the presence of ether linkage or (RO-R), glycosidic bond in pectin. The extracted product showed the presence of all the characteristic bands. Thus, from the IR spectra, it is confirmed that our extract is pectin.

The IR spectra showed a broad peak at 3404.47 cm−1 due to stretching of O–H group (range 3500–3000 cm−1); peak at 2928.04 cm−1 is due to C–H stretching. It showed typical peaks at 1726.35 (range 1750–1350 cm−1) representing ester carbonyl C = O group and also a peak at 1629.90 and 1435.09 cm−1 corresponds to carboxylate, COO group stretching (range 1600–1650 cm−1, antisymmetric stretch and 1400–1450 cm−1, symmetric stretch). An absorption peak at 1070 cm−1 indicates the presence of ether linkage or (RO-R), glycosidic bond in pectin. The extracted product showed the presence of all the characteristic bands. Thus, from the IR spectra, it is confirmed that extracted product is pectin.

Galacturonic acid content estimation of extracted okra and citrus pectin pectin

The GA content of okra pectin was estimated using the calibration curve of GA from pure pectin (Pure pectin from SRL chemicals containing GA min 80% and methoxyl content min 7%). The GA acid content estimation was done according to Dische with some modifications. The GA content in the okra and citrus pectins was found to be 95.92 μg/ml and 83.36 μg/ml, respectively.

Equivalent weight of extracted okra and citrus pectin

The Eq. W. of extracted okra pectin was calculated as per the equation mentioned in materials and methods. The pectin 10 mg (0.01 g) was added in the formula. The NaOH concentration was used for equivalent weight determination is 0.1 N and the volume of NaOH used was 0.1 ml for getting pink color. The Eq. W. of okra and citrus pectin was found to be 1000 g/equivalence and 2500 g/equivalence.

Methoxyl content of extracted okra and citrus pectin

The methoxyl content in purified okra pectin was measured as reported by Ranganna (1995). The neutralized solution obtained from equivalent weight was mixed with 2.5 ml of 0.25 M NaOH and left at room temperature for 30 min, followed by addition of 2.5 N HCl. The methoxyl content of purified okra and citrus pectin was found to be 3.1% and 12.4%, respectively.

Degree of methyl esterification of extracted okra and citrus pectin

The degree of methoxyl esterification was calculated as per the equation mentioned in materials and methods with value obtained from GA content and methoxyl content. DM esterification of the extracted okra and citrus was found to be 18.34% and 84.4% in okra and citrus pectin, respectively.

Molecular docking studies

The docked compounds obtained from the docking calculations were subjected to molecular interaction analysis using Maestro. Out of six compounds, RG-I shows high binding affinity toward Galcetin-1 than others [Table 1]. The docking result of pectin gives binding energy of −17.763 kcal/mol with Galectin-1. It shows four hydrogen bonds and one aromatics hydrogen bond interaction with Galectin-1. The amino acid Arg48, Asn61, and Glu71 form hydrogen bonding with pectin and one aromatics bond found between His44 and pectin [Figure 3]a.
Table 1: Binding energies of docked compounds with Galectin-1 protein and their interaction studies

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Figure 3: (a) Binding pose of pectin with galectin-1 protein. (b) Binding pose of pectin-Disaccharides with galectin-1 protein. (c) Binding pose of D-Arabinose with galectin-1 protein. Galectin-1 shown in ribbon, docked compounds represent in ball and st

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In case of pectin-disaccharides, hydrogen bonds, and salt bridge formation occurred with Galectin-1 with-13.38 kcal/mol binding energy. [Figure 3]b shows, binding mode and binding pattern of pectin-disaccharides with Galectin-1. The amino acid residues, Arg48, Asn61, and Arg73 was fond to be involved in hydrogen binding and the same residues Arg48 and Arg73 forms salt bridge with pectin-disaccharides. The compound D-Arabinose gives −15.976 kcal/mol binding energy with only hydrogen binding. The amino acid residues Arg48, Asn61, and Glu71 were fond to be involved in hydrogen bonding with D-Arabinose [Figure 3]c.

On the other hand, tetragalacturonic acid was found to be unstable within the Galectin-1-binding cavity, because the binging energy of tetragalacturonic acid with Galectin-1 is very high (9.047 kcal/mol), as compared to others. The molecular docking studies suggest the conformation of tetragalacturonic acid obtained from docking calculation us entropically unfavorable [Figure 4]a. In case of RG-I, binding conformation is very stable and entropically favored within the binging cavity of Galectin-1 having the more negative binding energy, i.e., −23.120 kcal/mol and higher binding affinity toward Galectin-1, as compared to other compounds. It forms seven hydrogen bonds and one salt bridge with Galectin-1. The amino acids residues involved in binding with RG-I are Arg48, Asn61, and Glu71 [Figure 4]b. The compound RG-II gives −14.854 binding energy, and it forms four hydrogen bonds and two salt bridges with Galectin-1. The amino acid residues Asn61 and Arg48 were involved in hydrogen binding, Arg48 and Arg73 forms salt bridge with RG-II [Figure 4]c.
Figure 4: (a) Binding pose of tetragalacturonic acid with galectin-1 protein. (b) Binding pose of rhamnogalacturonan-II with galectin-1 protein. (c) Binding pose of rhamnogalacturonan-II with galectin-1 protein. Galectin-1 shown in ribbon, docked compound

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  Conclusion Top


In the present study, we extracted, purified, and characterized pectins from Indian okra and sweet lime. Several standard tests were performed for the confirmation of extracted pectins in order to determine its biochemical and biophysical properties. Extraction of pectin from raw peels of C. limetta (sweet lime) was also carried out to assess their potential to serve as commercial source of pectin. It was compared with standard pectin purchased from SRL. Indian okra and sweet lime are a cheap and viable source for extracting pectin in comparison to the available sources. Pectin has these three major structural units of polysaccharide: homogalactouronan, RG-I, and RG II. Out of which RG-II is rarely found and explored but okra pectin, isolated in this study, has this polysaccharide which when present in pectin acts as an efficient and effective inhibitor of cancer cell when bound to galectin.

Further, molecular-docking simulations studies were also performed to reveal the binding mode of pectin and pectic polysaccharides with Galectin-1. From the docking calculations, the RG-I found to be energetically more favorable and having high binding affinity toward Galectin-1 than others. Furthermore, the critical role of amino acid residues i.e., Arg48, Asn61, Glu71 and Arg73 in the binding interaction revealed from docking studies.

Thus, in these preliminary studies, we extracted and partially characterized okra and citrus pectins and studied the in silico binding of pectin and pectic polysaccharides with Galectin-1. Further binding studies of pectin and pectic polysaccharides with purified Galectin-1 are under progress.

Generation of interdisciplinary work

Rohit D. Gupta (RDG), Krish Parekh (KP), Vaishnavi U. Warrier (VUW), and Kiran Bharat Lokhande (KBL) performed the experiment and wrote the experimental part. K. Venkateswara Swamy (KVS), Rajkumar S. Sood (RSS), and Rajesh Kumar Gupta (RKG) discussed the idea. RKG designed the experiments and wrote the manuscript.

Financial support and sponsorship

This work was supported by the Department of Science & Technology-Science and Engineering Research Board (DST-SERB), Govt. of India, Project Grant (File No. ECR/2016/001187) and by the Dr. D. Y. Patil Vidyapeeth (Deemed to be University) Project Grants (DPU/106(18)/2015 and DPU/17/2016).

Conflicts of interest

There are no conflicts of interest.



 
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