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ORIGINAL ARTICLE
Year : 2014  |  Volume : 3  |  Issue : 1  |  Page : 33-39

Morphological, physico-chemical and structural characterization of mucilage isolated from the seeds of Buchanania lanzan Spreng


1 Department of Pharmaceutics, H.S.B.P.V.T.'s Group of Institutions, College of Pharmacy, Kashti, Ahmednagar, Maharashtra, India
2 Department of Pharmacology, Shree G. M. Bilakhia College of Pharmacy Rofel, Vapi, Gujarat, India

Date of Web Publication15-Apr-2014

Correspondence Address:
Sudarshan Singh
H.S.B.P.V.T's Group of Institutions, College of Pharmacy, Kashti, Ahmednagar 414 701, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.130609

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  Abstract 

Context: Mucilage isolated from the seeds of Buchanania lanzan a wild Indian plant. This mucilage can be commercially exploited, by evaluating physico-chemical properties of this mucilage. Materials and Methods: Various physico-chemical parameter using scanning electron microscopy (SEM), molecular weight, X-ray diffraction (XRD) spectrometry, zeta potential (ZP), Fourier transform infrared (FT-IR) spectroscopy and 1D ( 1 H and 13 C) nuclear magnetic resonance (NMR) have been employed to characterize mucilage in the present study. Results: SEM analysis suggests that the mucilage has irregular particle size. Thermogravimetry analysis suggested that mucilage had good thermal stability with two stage decomposition. The weight-average molecular weight of mucilage was determined to be 4883, by gel permeation chromatography. The XRD pattern of the mucilage indicated a completely amorphous structure. The ZP was obtained 1.56 and −9.49 mV in water and 0.1 N NaCl respectively. The major functional groups identified from FT-IR spectrum include 3459/cm (-OH), 1667/cm (Alkenyl C-H and C = C stretch), 1407/cm (-COO-) and 1321/cm (-CH 3 CO). Analysis of mucilage by paper chromatography and 1D NMR indicated the presence of rhamnose, arabinose and fructose. Conclusion: The spectral and chromatography analysis of mucilage indicate that the mucilage is composed of basic sugar moiety such as (arabinose, rhamnose, fructose and mannose) as complex carbohydrates.

Keywords: 1D nuclear magnetic resonance spectroscopy, Buchanania lanzan, thermal analysis, X-ray diffraction spectrometry


How to cite this article:
Singh S, Bothara SB. Morphological, physico-chemical and structural characterization of mucilage isolated from the seeds of Buchanania lanzan Spreng. Int J Health Allied Sci 2014;3:33-9

How to cite this URL:
Singh S, Bothara SB. Morphological, physico-chemical and structural characterization of mucilage isolated from the seeds of Buchanania lanzan Spreng. Int J Health Allied Sci [serial online] 2014 [cited 2021 Jan 16];3:33-9. Available from: https://www.ijhas.in/text.asp?2014/3/1/33/130609


  Introduction Top


Natural gums are obtained as exudates from different species, which exhibit unique and diverse physico-chemical properties and have a wide variety of applications. [1] Commercially important tree gums include gum arabic, gum karaya and gum tragacanth. [2]

Buchanania lanzan spreng is a tree of 12-15 m height, with straight trunk, belong to the family Anacardiacea. Earlier, toxicological evaluation of the seed mucilage of B. lanzan spreng was carried out to use as a food additive. [3] The oil extracted from the kernels of the fruit is used as a substitute for almond oil in native medicinal preparation and confectionery. It is also applied to glandular swellings to the neck. [4],[5],[6]

The present paper, reports morphological, physicochemical and structural aspects of mucilage from the seeds of B. lanzan spreng in order to validate this tree gum. The methods employed for the analysis include (i) scanning electron microscopy (SEM), (ii) gel permeation chromatography (GPC), (iii) thermo-gravimetric analysis (TGA), (iv) zeta potential (ZP), (v) X-ray diffraction spectrometry (XRD), (vi) Fourier transform infrared spectroscopy (FT-IR), (vii) 1D nuclear magnetic resonance (NMR) and (viii) paper chromatography analysis.


  Materials and Methods Top


Materials

Fruits of B. lanzan spreng was collected from the forest of Rajgamar, District Korba, Chhattisgarh in the month of April-June and authenticated by Prof. H. B. Singh, NISCAIR, New Delhi, India. Mucilage was isolated from the seeds using maceration techniques [7] and stored in airtight polypropylene jars in desiccated condition. De-ionized water was used for all experiments. All other chemicals used were of analytical grade.

SEM

The SEM photograph of mucilage was recorded by using SEM (XL-30, Philips, Lancashire, UK). The processed specimen was subjected to SEM analysis. [8]

Molecular weight by GPC

GPC was carried out to estimate molecular weight of the mucilage relative to dextran polysaccharide as standard, using Waters Alliance model (Waters 2695 separation module with auto injector) coupled with Waters 2414 refractive index detector. Mobile phase was 0.2 M NaNO 3 in water, flow rate was 1.0 mL/min, Ultrahydrogel 500 and Ultrahydrogel 120 (7.8 mm × 30 cm × 9 um) was in series. Detector and column was operated at 30°C, which was started from Mw: 5200; 48,600; 203,000; 668,000; 1,400,000 spectra was processed using  Empower software.

TGA

The analysis of mucilage was carried out using thermal analyzer (Linseis, Germany, Stapt-1600), accurately weighed (15 mg) sample was placed into platinum cups and sealed. The temperature range was from 0°C to 900°C at a heating rate of 15°C/min. [9]

Electro kinetic studies: ZP

ZP analysis of mucilage was carried out using Zetatrac (Microtrac, NPA152-31A) by measuring the response of charged particles to an electric field. ZP was calculated for mucilage from the modulated power spectrum signal using formula [10] in water and pH dependence of the ZP was investigated with the background electrolyte of 0.1 N NaCl.

ζ = μη/ε (1)

where ζ = ZP, μ = mobility, η = viscosity, ε = dielectric constant, for water at 25°C, ZP (mV), ~12.8 × Mobility (μ/s/V/cm).

Powder XRD pattern

Powder XRD (PXRD) patterns of mucilage was recorded using X-ray diffractometer (Goniometer, BI-200SM). The experiment was carried out at 25°C under the following conditions: Voltage 40 kV, current 30 mA, 2θ angle with scan step time of 10.33 s with specific length of 10 mm. [11]

Fourier transform analysis

Fourier transform analysis (Thermo Scientific, Nicolet IS 10) spectra of mucilage was recorded on a FT-IR spectrometer (Thermo Scientific, Nicolet IS 10). The dry powder was mixed with KBr and pressed into pellets under mechanical pressure. The FT-IR spectra was obtained by scanning between 4000 and 400/cm.

1D NMR

NMR spectra of 1 H and 13 C of mucilage was recorded using NMR (400 MHz) spectrometer (Bruker Avance II 400 NMR Spectrophotometer). Mucilage (100 mg) was dissolved in D 2 O and chemical shifts were reported in ppm relative to an internal standard TSP (3-trimethylsilylpropionic-2, 2, 3, 3,-d4 acid, sodium salt, 98% D) for 1 H NMR and 1,4-dioxane (d 66.67 ppm) for 13 C spectra. Proton NMR spectra was obtained at a base frequency of 400 MHz, with 16 transitions and delay time 1.5 s and for 13 C, the base frequency was 100 MHz, with 3000 scans and delay time 2 s. [12]

Sugar composition by paper chromatography

Carbohydrate or sugars occupy a central position in plant metabolism and hence the method of their detection and estimation are very important. Sugars are conveniently classified into three groups, on the basis of molecular size: The simple monosaccharide's (e.g., glucose, fructose) and their derivatives; the oligosaccharides, from by condensation of two or more monosaccharide's units (e.g., sucrose); and the polysaccharides which consist of long chains of monosaccharide's units, joined head to tail, either as straight chains or without branching. [13] The sample was hydrolyzed and then prepared for paper chromatography. The paper chromatography analysis was performed using n-butanol: acetic acid: water (4:1:1) as solvent system using arabinose, fructose, mannose, rhamnose and xylose as reference standard.


  Results Top


SEM

SEM of mucilage obtained is represented in [Figure 1] at different magnifications. The microphotographs of mucilage indicate amorphous nature of the sample. The particles are mostly seen as aggregates of irregular shapes and dimensions which were fibrous in nature.
Figure 1: Scanning electron microscopy of mucilage at different magnification (×35, ×100, ×270 and ×500) using scanning electron microscopy

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Molecular weight by GPC

The molecular weight of mucilage was determined by GPC and expressed as the "Dextran polysaccharide equivalent" molecular weight. The computed average molecular weights (Mw) was obtained as 4883, number average molecular weight (Mn) was obtained as 3628 and polydispersity (Mw/Mn) was obtained as 1.345836.

TGA

TGA is a simple and accurate method for studying the decomposition pattern and the thermal stability of polymers. [Table 1] and [Table 2] give the details of thermal behavior according to the primary thermograms and derivative thermograms of mucilage's. The details of thermal behavior and thermal stability data according to the primary thermograms and derivative thermograms for the mucilage showed that heating at a rate of 10°C/min from 0°C to a maximum of 900°C resulted in two mass loss events.
Table 1: Thermogravimetry analysis of mucilage


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Table 2: Thermal stability characterization


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Electro kinetic studies (ZP)

The ZP measurement was performed to collect information on the stability and charge behavior of the polymer. The ZP of mucilage in aqueous medium (water) was recorded to be 1.56 mV and in 0.1 N NaCl was recorded to be –9.49 mV respectively.

PXRD pattern

PXRD analysis of mucilage is represented in [Figure 2]. Absence of characteristic peaks in the spectrum was observed, indicating that the mucilage was completely amorphous in nature.
Figure 2: Powder X-ray diffraction spectra of mucilage obtained from seeds of Buchanania lanzan using goyenimeter

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Fourier transform analysis

The interpretation of IR spectral [Figure 3] analysis was carried out using "CHEMIX School software". Spectra exhibited the typical bands and peak characteristic of polysaccharides. The spectra of mucilage showed band occurring at 3541.46-3295.52/cm, results from the presence of hydroxyl (-OH and -CO) groups. The peak obtained at 3075.63-2838.37/cm results from stretching modes of the C-H bonds of methyl groups (-CH 3 ) and carboxylic acid C = O and O-H. The peak obtained at 1689.72-1667.53/cm results from stretching mode of the alkynyl C-H, C = C, amide N-H and C = O. The peak obtained at 841.96-772.52/cm results from stretching and bending modes of the amine N-H.
Figure 3: Fourier transform infrared (FT-IR) spectral characterization of mucilage using thermo scientific FT-IR spectrophotometer

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1D NMR

The interpretation of 1 H and C 13 NMR spectra analysis was carried out using "CHEMIX School software" and represented in [Figure 4] and [Figure 5]. Spectra exhibit the typical bands and peak characteristic of polysaccharides of 1 H NMR spectra between d 5.41 and d 3.40 ppm can be attributed to CH and OH group of rhamnose. The signals between d 3.65 and d 3.38 ppm can be attributed to OH and CH group of mannose. The signals between d 3.65 and d 3.38 ppm can be attributed to OH and CH group of arabinose. The signal at d 72.2 ppm of C 13 NMR spectra can be attributed to CH group of rhamnose and mannose. The signal at d 70.3 ppm of C 13 NMR spectra can be attributed to CH group of arabinose. The signals between d 3.0 and d 3.8 ppm have also been assigned to -O-CH 3 . Further-CH 3 indicates the presence of methylated sugar (rhamnose). The overall analysis reveals that mucilage may contain mannose, rhamnose, arabinose, fructose and related sugar moiety.
Figure 4: 1D 1H nuclear magnetic resonance (NMR) spectral characterization of mucilage using Bruker Avance II 400 NMR spectrophotometer

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Figure 5: 1D 13C nuclear magnetic resonance (NMR) spectral characterization of mucilage using Bruker Advance II 400 NMR spectrophotometer

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Sugar composition by paper chromatography

The Rf value of standards arabinose, fructose and rhamnose matched with the obtained Rf values of test sample (mucilage).


  Discussion Top


SEM

Microscopy is a major tool for the characterization of the ultra-structure of polymer material ultra-structure. In case of drug delivery system, microscopical examination enables an understanding of a number of variables that govern delivery. Such imaging technique is used to examine the detail shape, size and distribution of polymeric micro and nanoparticles, as well as interaction with biological environment. [14] The SEM results of the present study suggest that hydration capacity of mucilage depends on the surface property as presented in powder XRD studies. The shape and structure or surface topography of the mucilage may have been affected by the method of extraction and purification or preparation of mucilage. [15]

Molecular weight by GPC

Polymer molecular weight determination is important because it determines many physical properties such as the temperatures for transitions from liquids to waxes to rubbers to solids and mechanical properties such as stiffness, strength, viscoelasticity, toughness and viscosity. The polydispersity of isolated mucilage was obtained as 1.3. The polydispersity index (Mw/Mn) is used as a convenient measure of the range of molecular weight present in a distribution and is in the range of 1.5-7.0 for natural polysaccharide gums. [14] If molecular weight is too low, the transition temperatures and the mechanical properties will generally be too low for the polymer material to have any useful commercial applications. For a polymer to be useful it must have transition temperatures to waxes or liquids that are above room temperatures and it must have mechanical properties sufficient to bear design loads.

TGA

For a polymer research, the TGA is especially suited to determine residual solvent, residual monomer and the released components in the earliest stage of a thermal degradation process. [16] Thermogravimetry analysis of mucilage showed two stages of decomposition. The early minor weight loss in samples is attributed to the loss of adsorbed and structural water of biopolymers as related by other authors [17] or due to desorption of moisture as hydrogen bound water to the saccharide structure. The second weight loss event may be attributed to the polysaccharide decomposition [9] and is described by a weight loss. The weight loss onset (representing the onset of oxidation or decomposition) of polymer suggests that mucilage's have good thermal stability.

Electro kinetic studies

Polarity of mucilage may be due to the presence of -OH group, which are readily available for the formation of hydrogen bonds on the interface of reinforced systems. However, plant mucilages are covered with pectin and waxy substances, [18] thus hindering the hydroxyl groups (-OH) from reacting with polar matrices and deteriorate adhesion, but polar structure is only responsible for hydrophilic behavior of mucilage. Further this indicates mucilage's swell strongly in an aqueous environment. For high adhesion forces it is necessary to have polar functional groups, but swelling processes should be prevented. The conductivity and dielectric constant often increases with thermal and electrical stress. These changes therefore are indicative of decomposition of material to yield some smaller molecules. Furthermore difference in electrical potential across an interface indicates that there must be particular distribution of charges near to the interface.

PXRD pattern

PXRD analysis of mucilage sample showed no characteristic sharp peaks in the spectrum, indicated that the mucilage is completely amorphous in nature. Natural gums, such as Arabic, guar gum and karaya gum, also show amorphous nature. [19]

Fourier transform analysis

The FT-IR spectra exhibit the typical bands and peak characteristic for mucilage. The broad band occurring at 3500-3200/cm, results from the presence of hydroxyl (-OH) groups. The peak obtained at 2885-2705/cm results from stretching modes of the C-H bonds of methyl groups (-CH 3 ). Absorption bands around 1618 and 1430/cm are typical of carboxylate groups of the galacturonic acid residues as reported by Okafor [20] and Figueiro. [21] The region between 1500 and 1800/cm is typically used to detect presence of carboxylic groups. Furthermore, absorption peaks at 1740 and 1258/cm are typical of acetyl groups. [22] The wave numbers between 800 and 1200/cm represents the finger print region for carbohydrates. [23]

1D NMR

The 1 H NMR spectrum for mucilage showed few singlet's at high field (d 1.1785 ppm [s], d 1.9559 ppm [s]), which is related to the environments of methyl groups of rhamnose and the protons linked to C-6 (d 3.65, d 3.70 ppm) and C-4 of galactose (d 3.98, 4.28 ppm), respectively and this suggests the existence of different galactose derivatives. [24] The anomeric protons have been assigned to β-sugar residues (d 4.92-4.96 ppm) and the α-sugar residue (d 5.1-5.3 ppm), as reported earlier by Agrawal [25] and by Vogt. [24] The two closely neighbored signals observed in the 1 H NMR spectrum of mucilage (d 4.02 and d 3.84 ppm) were assigned to H-1 of a-glucose. [26] The 1 H NMR spectrum showed that crowded narrow region between 3 and 5 ppm typical of polysaccharides and confirms the presence of many similar sugar residues. [27] The signals between 3.1 and 4.3 ppm can be assigned to non-anomeric protons (H 2 -H 6 ) while signals between 4.3 and 4.8 and 4.9-5.5 ppm arise from α-anomeric and β-anomeric protons respectively. [28] 13 C NMR spectra of mucilages gave line widths which are typical of an amorphous natural polymer with broad band signals. There are resonance spectra due to the methyl group of rhamnose (d 16.67 ppm) as reported earlier by Martinez. [28]

Sugar composition by paper chromatography

The paper chromatography analysis was performed in order to check the available carbohydrates in mucilage. The result obtained from 1D NMR, FT-IR and paper chromatography studies indicated the presence of arabinose, fructose, rhamnose, xylose and glucose. The result obtained from the study indicated the similar Rf values to the standard available data for sugars. According to Lee arabinose is reported to be a free but minor sugar component of onions, grapes, strawberries, commercial beer, corn and alfalfa. [27]


  Conclusions Top


Mucilage of the seeds of B. lanzan is a stable polysaccharide gum which is a natural, non-toxic and cost-effective material. The PXRD and SEM revealed that mucilage is amorphous in nature which indicated that wet granulation technology will be more suitable for dosage formulation. The thermal stability study by TGA showed that mucilage had good thermal stability. FT-IR, solid 1D 1 H and C 13 NMR and paper chromatography confirmed presence of non-reducing sugars. The overall findings of the study indicated that mucilage isolated from the seeds of B. lanzan have an inherent property which can be used in various dosage as pharmaceutical aid with suitable physiochemical property or with a form on modification.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]


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