[VOLUME 5 I ISSUE 4 I OCT. – DEC. 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 Carbohydrates of Moringa oleifera seeds
Sandanamudi Anudeep & Cheruppanpullil Radha Protein Chemistry and Technology Department, CSIR- Central Food Technological Research Institute, Mysore – 570 020, India
Received: Dec 11, 2018 Accepted: Des. 11, 2018
ABSTRACT Carbohydrates from defatted moringa seed flour were sequentially extracted with 70% ethanol, cold water, hot water, 0.5% EDTA and 10% alkali in order to obtain free sugars, neutral, pectic, alkali soluble and alkali insoluble polysaccharides, respectively. Ethanol extract consisted of monosaccharides such as rhamnose (4%), arabinose (6%), xylose (11.5%), mannose (9%), galactose (5%), glucose (3%) and disaccharides such as, sucrose (2.2%), maltose (1.75%) as well as raffinose family oligosaccharides such as raffinose (0.78%), stachyose (0.8%) and verbescose. The polysaccharide fractions of moringa seed flour predominantly consists of arabinogalactan (1.29%), xylan type polysaccharides (1.45%) and cellulose. Defatted moringa seed flour contained 33.5% total dietary fiber which comprises of 6.5% soluble fiber. The present investigation reports the nature of carbohydrates and dietary fiber present in moringa seeds that cab be a potential alternative source for roughage rich plant sources.
Keywords: Moringa oleifera seed; carbohydrates, non-starch polysaccharides; dietary fiber.
INTRODUCTION Carbohydrates are derived naturally from plant sources. The important carbohydrates in plants are starch, resistant starch, sucrose, raffinose, stachyose, pectin, cellulose etc. These carbohydrates are isolated and added to foods because of its functional properties like stabilizer, sweetener, thickener, emulsifying agent, gelling etc. Along with the functional advantages the carbohydrates also possess physiological functions such as dietetic functions and fermentation in the colon (Alphons, 1998). Polysaccharides or non-digestible carbohydrates (dietary fiber) that are present naturally in plant parts exert various health benefits (Requena et al., 2016). Many current applications within the food industry have been fuelled with dietary fibers, oligosaccharides, and resistant starch (Prasad et al., 2015). Moringa oleifera is the most widely cultivated species of a monogeneric family (Moringaceae) because of its nutritional and health benefits. All parts of moringa plant are used as traditional medicine in treating various ailments (Melo et al., 2013). Reports on moringa seed carbohydrates are scanty in literature. Moringa being a traditional food and its use in herbal medicine, the scientific assessment of its potential as an alternative food source is impending (Anderson et al., 2009). In addition, no reports are available on the total carbohydrate profile and polysaccharide composition of Moringa oleifera seeds. Carbohydrate profiling of moringa seeds is an essential criteria to study the functional carbohydrates present. The objective of this study is to profile moringa seed carbohydrates and `dietary fiber. An investigation was accordingly initiated to examine the type of carbohydrates and dietary fiber present in moringa seeds.
MATERIALS AND METHODS Materials Moringa oleifera seeds (PKM-1 variety) were procured from local market, Mysore, India. Termamyl, pepsin, pancreatin and dowex ion exchange resins were from Sigma Chemical Company (St Louis MO, USA). Bio-Gel P2 was from Bio Rad Laboratory, USA. HPLC columns used were analytical (4.6ID×250mm) and preparative (20ID×250mm) from Cosmosil, Kyoto, Japan. Rtx-2330 capillary column (30.0m) was used for Gas liquid chromatography. Q-Tof Ultima Global, Waters, UK, was used for ESI-MS. HPLC grade solvents were used for chromatography analysis. All other chemicals and reagents used were of analytical grade unless until mentioned. Analytical determinations Total carbohydrate and uronic acids were estimated by phenol sulphuric acid assay (Dubois et al., 1956) and MHDP methods (Blumenkrantz and Asboe-Hansen, 1973) respectively. Protein was estimated by Micro- Kjeldhal method (N×6.25) (AOAC, 2000). Dietary fiber estimation and isolation was performed by enzymatic method (Nills et al., 1983). Prebiotic effect of the soluble dietary fiber was analyzed by growth curve experiment.
Research Paper IJRAR- International Journal of Research and Analytical Reviews 헂103 [ VOLUME 5 I ISSUE 4 I OCT.– DEC. 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138 Preparation of defatted moringa seed flour Defatted moringa seed flour was prepared according to Govardhan et al., (2013) with minor modifications. Matured and dry seeds (5 kg) of Moringa oleifera was dehulled and cleaned to obtain kernels. The kernels were flaked and dried and subsequently defatted by repeated washing with hexane until the fat content was 2%. The defatted sample was air dried, milled and sieved with a 100 µm mesh stainless steel sieve and designated as defatted moringa seed flour (DMSF). The flour sample was stored in an airtight container until needed. Ethanol soluble sugars Defatted moringa seed flour was refluxed with 70% ethanol to obtain ethanol soluble free sugars (Salimath and Tharanathan, 1982). The ethanol soluble sugars were subjected to further analysis. The parameters for HPLC analysis includes - Detector: RI detector, mobile phase- acetonitrile:water (70:30) and column- 5 NH2- MS COSMOSIL with 1 ml/min flow rate). Subsequently the Electron Spray Ionization – Mass Spec (ESI-MS from Q-Tof Ultima Global, Waters, UK) was performed for the determination of molecular mass of oligosaccharides. Non Starch Polysaccharides (NSP) The alcohol insoluble residue was subjected to further extractions to obtain non starch polysaccharides (NSP) as mentioned by Rao and Muralikrishna, (2001) with minor modification as shown in Fig. 1. The isolated NSP and ethanol extracted sugars were processed to prepare their respective alditol acetates (Sawardekar et al., 1965). The component sugars were separated and identified on Rtx-2330 column using a Shimadzu gas liquid chromatography equipped with fame ionization detector (FID).
Fig 1. Isolation of oligosaccharides and non starch polysaccharides from defatted moringa seed flour. 70% ethanol was used for extraction of free sugars and oligosaccharides. The residue was further subjected to sequential extractions for isolation of non starch polysaccharides.
Isolation of dietary fiber Defatted moringa seed flour was sequentially digested with termamyl, pepsin and pancreatin as described by Nills et al., (1983). The pH of the digest mixture was adjusted to 4.5 and centrifuged at 6000 rpm for 15 min. The supernatant was decanted and allowed to precipitate for 1 hr by adding 3 volumes of warm ethanol followed by filtration and washing with 70% ethanol, acetone and dried (Daou & Zhang, 2013). The dried soluble dietary fiber was lyophilized and stored in an air tight container until further use. 헂104 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 4 I OCT. – DEC. 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 Data analysis Data presented are means of three replicate determinations ± standard deviation.
RESULTS AND DISCUSSION Proximate analysis of 100 g DMSF revealed the presence of 7.9 g moisture, 1.7 g fat content, 6.2 g crude fiber, 6.1 g ash, 48.3 g proteins and 29.6 g carbohydrates (by difference). In the present study the carbohydrate content in defatted moringa seed flour was reported to be 29.6% whereas total carbohydrates present in moringa seeds were reported to be 9-16% (Abdulkarim et al., 2005; Anhwange et al., 2004). This variability in the carbohydrate content may be due to the influence of environment, plant variety and season. Carbohydrates present in the seeds depend on the plant variety and degree of maturity at harvest (Stephen, 2000).
Profiling of ethanol soluble sugars Total carbohydrate content in ethanol extract was 42% which comprises of monosaccharides, disaccharides and oligosaccharides. Monosaccharides were identified as rhamnose (4%), arabinose (6%), xylose (11.5%), mannose (9%), galactose (5%) and glucose (3%) by gas liquid chromatography and HPLC (Fig. 2).
a 0.0003
0.0002
Charge n/C Charge 0.0001
2 4 1 3 5 6 0.0000 7
2 4 6 8 10 12 14 16 18 20 22 24 Time / min
0.0006 b
0.0005
0.0004
0.0003 Charge n/C Charge 0.0002 1
0.0001 5 2 3 4 0.0000 6 7
2 4 6 8 10 12 14 16 18 20 22 24 26 Time / min Fig 2. HPLC elution pattern for standard sugars and ethanol extract of defatted moringa seed flour. (a) Standards: 1- Fructose, 2- Glucose, 3- Galactose, 4- Sucrose, 5-Maltose, 6-Raffinose, 7-Stachyose (b) 70% ethanol extract HPLC elution pattern: 1-Glycerol, 2-Fructose, 3-Galactose, 4-Sucrose, 5- Maltose, 6-Raffinose, 7-Stachyose. The disaccharides that were identified include sucrose (2.2%) and maltose (1.75%). Among the oligosaccharide fraction; verbascose, stachyose (0.8%) and raffinose (0.78%) were identified by HPLC (Fig. 3) and characterized by ESI-MS (Fig. 4).
0.00020 a
0.00015
0.00010 Charge n/C Charge
0.00005 12.975
0.00000
2 4 6 8 10 12 14 16 Time / min
0.0005 b
0.0004
0.0003
Charge n/C Charge 0.0002
0.0001 21.467 0.0000
2 4 6 8 10 12 14 16 18 20 22 24 Time / min Fig 3. HPLC elution pattern for purified oligosaccharides isolated from defatted moringa seed flour. (a) HPLC peak at 12.9 min represents Raffinose (b) HPLC peak at 21.4 min represents Stachyose. Research Paper IJRAR- International Journal of Research and Analytical Reviews 헂105 [ VOLUME 5 I ISSUE 4 I OCT.– DEC. 2018] E ISSN 2348 –1269, PRINT ISSN 2349-5138
Intensity 100 689.4 a TOF MS ES + 152
%
0 450 500 550 600 650 700 750 800 850 900 950 1000 100 527.3 TOF MS ES + 1.00 e3 b
%
0 450 500 550 600 650 700 750
100 c 851.4 TOF MS ES + 270
%
0 700 750 800 850 900 950 m/z Fig 4. Electron spray Ionization – Mass spectrum of purified oligosaccharides from defatted moringa seed flour as obtained in the positive mode (m/z). (a) Molecular mass 689 represents stachyose (b) Molecular mass 527 represents raffinose (c) Molecular mass 851 represents verbescose with sodium adducts.
Moringa oleifera seeds have been reported for the presence of functional oligosaccharides and dietary fiber. The health benefits reported for moringa carbohydrates include cholesterol reducing and immunomodulation benefits (Soumitra et al., 2004; Anudeep et al., 2016). These functional carbohydrates open up a wide area of interest by food industries.
Profiling of Non Starch Polysaccharides (NSP) The term non-starch polysaccharide (NSP) covers a large variety of polysaccharide molecules excluding α- glucans (starch) (Choct, 1997). Non starch polysaccharides of moringa seeds are represented in Table 1. The major sugars identified in all NSP include rhamnose, arabinose, xylose and galactose. The sequential extraction of the DMSF with cold water resulted in 1.04 ± 0.03 g of cold water soluble fraction. Cold water fraction was a whitish powder with 26.8% proteins and 13.2% uronic acids. Total sugar was 64.8% which consisted of rhamnose and galactose in the ratio 1:3. The sugar composition of cold water fraction of DMSF suggests that the fraction is comprised of rhanmogalacturonans. Rhanmogalacturonans are enigmatic polysaccharide with no known function (Willats et al., 2001). Table 1: Chemical composition of non starch polysaccharides from defatted moringa seed flour and its fractions. Composition Defatted Cold Hot Pectic Hemi A Hemi B AIR moringa water Water polysaccharides seed soluble soluble Flour Yield (%) 100 1.04±0.03 1.29±0.5 0.81±0.02 6.1±0.32 1.45±0.14 27.2±0.3 Protein 48.3±0.1 26.8±0.2 28.2±0.8 30.8±0.12 55.0±0.6 9.8±0.1 11.1±0.15 Total sugar 16±0.3 64.8±0.3 45±0.22 26.8±0.18 11.4±0.32 48.1±0.52 27.8±0.34 Uronic acids -- 13.2±0.23 20.3±0.6 22.3±0.92 5.3±0.26 83.1±0.7 38.0±0.2 Sugars detected Rhamnose 12.2 8.9 7.4 6.7 2.1 1.2 - Arabinose 14.55 1.08 44.7 54.5 + 6.8 5.34 Xylose 10.2 - 2.8 2.02 16.3 11.1 - Mannose 1.02 - 2.06 - + - 1.96 Galactose 14.5 18 13.1 2.7 2.04 39.1 5.54 Glucose 1.2 3.1 9.42 17.9 4.3 10.4 4.6 -- Not determined - Not detected + Present in low amounts Values represent mean ± SD. 헂106 IJRAR- International Journal of Research and Analytical Reviews Research Paper [VOLUME 5 I ISSUE 4 I OCT. – DEC. 2018] e ISSN 2348 –1269, Print ISSN 2349-5138 http://ijrar.com/ Cosmos Impact Factor 4.236 The hot water polysaccharide fraction of the DMSF was 1.29 ± 0.5 g. The hot water polysaccharide fraction of the moringa seeds contained total sugar of 45% in which arabinose (44.7%) and galactose (13.1%) were present in the ratio of 3.4: 1. Proteins and uronic acids were estimated as 38.7% and 49.3%, respectively. The bioactive polysaccharide (with the effect on the complement system) is the pectins rich in neutral sugar side chains of (arabino) galactans containing β-(1 - 6) linkage. Based on the composition of hot water soluble fraction, we could deduce that it contained arabinogalactan proteins (AGP’s). Many researchers reported the presence of arabinogalactan proteins from various plant sources (Pellerin et al., 1995) and reported for its active role in immunomodulation (Ramberg et al., 2010). Further extractions with EDTA produced pectic polysaccharide. Propectin was destroyed by adding 50% glacial acetic acid to obtain a pH of 4.5. The pectic polysaccharide fraction consisted of 42.9% proteins and 22.3% uronic acids. The major sugars identified were rhamnose, arabinose and glucose in the ratio 1:8.13:2.67. This suggests the isolated pectic fraction is probably a polysaccharide that consists of a mixture of varied sugars namely rhamnose, arabinose, xylose, galactose, glucose and uronic acid. Many researchers isolated pectic polysaccharide comprising of varied sugars and uronic acids with protein backbone and reported health effects of pectic oligosaccharides from various other sources (Raisa et al., 2009; Belen et al., 2014). The hemicellulosic part of moringa seed flour was brown and fluffy in nature with a divergent sugar composition. Hemicellulose A fraction is mainly a proteinaceous material (77.9%) and uronic acid concentration was minimal (2.6%) in hemicellulose A in comparison with that of other non starch polysaccharide fractions. On the other hand, hemicellulose B was a cream colored fluffy material rich in carbohydrates (48.1%). Hemicelluloses B had lesser protein content (13.6%) and highest uronic acids content (83.1%) among all the other fractions. The Hemi A contained xylan type polysaccharides with galactose as major contaminant. A brownish residue in gummy nature was obtained after successive extractions of hemicelluloses with yield of 31.83%. It contains significant amount of proteins (22.3%) and uronic acids (38.0%). Among all the fractions of non starch polysaccharides, cellulose was highest in its yield (31.8%).
Dietary fiber Proximate analysis of defatted moringa seed flour revealed 6.2% of crude fiber which accords with available literature data (Compaoré et al., 2011). Dietary fiber analysis of defatted moringa seed flour showed the presence of 33.5 ± 0.5% of total dietary fiber which includes soluble dietary fiber 6.5 ± 0.25% and insoluble dietary fiber 27 ± 0.25%. In comparison, soy bean has 23% dietary fiber, legumes like Bengal gram and red gram which were considered to be important sources of dietary fiber possessed 28.3% and 22.6% dietary fiber, respectively (Gopalan et al., 2004; Katoch, 2013). This suggests that moringa seeds are potential source for dietary fiber among all the other leguminous seeds.
CONCLUSIONS This data provides information on the carbohydrate profiling of Moringa oleifera seeds. Data obtained using GLC; HPLC and ESI-MS analysis identified the nature of carbohydrates in Moringa oleifera seeds. Dietary fiber analysis of moringa seeds revealed the presence of 33.5% total dietary fiber. Polysaccharide fraction of defatted moringa seed flour (DMSF) consists of arabinogalactan, xylan type polysaccharides and cellulose. This investigation has shown that DMSF is rich in complex carbohydrates and could be a potential source of dietary fiber with varied applications. In vivo studies using these molecules can help in better understanding the mechanism of action to exploit their functional properties.
ACKNOWLEDGEMENTS The authors are grateful to the Director, CSIR–CFTRI, Mysore, for extending facilities for this study. This research was funded by Dept of Biotechnology, Government of India, New Delhi; Sanction No. BT/Bio- CARe/05/400/2010-2011.
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