Indian Journal of Chemistry Vol. 428, February 2003, pp. 437-442

Note

Isolation and characterization of pectic polysaccharides from the fruits of

Naringi crenulata

Saroj K Mondal, Bimalendu Ray*, Swapnadip Thakur & Pradyot K Ghosal

Department of Chemistry, Burdwan University, Burdwan, 71 3 104, India.

Email: konica2@dte.vsn l.net.in; Tel: +91 3425608 10. Fax: +91 342 564452

Received 20 March 2001; accepted (revised) 13 March 2002

Pectic polysaccharides have been isolated rrom the fruits o r Naringi crenu!ata by extraction with water. The water extract contains large amount of protein . The polymers present in the water extract are fractionated by graded precipitation with ethanol, anion exchange chromatography, and size exclusion chromatography. Characterization or the subrractions obtained thererrom by various chemical and physico-chemical methods of analys is reveal s that the water extract contain pectic polymers substituted to various degrees with side chains comprising mainly or terminal , 1,4-, 1,6-, I ,3,6-linked galac tose, together with lesser amounts or 1,2,4- and 1,3-linked galactose residues. Arabinose residues are terminal, 1,5-, 1,3,5-linked. These polymers contain acetyl groups and give viscous solution in water.

Naringi crenuiata (Roxb.) Nicolson (Fam. Rutaceae), a tree with green fol iage, grows abundantly in different parts of Indi a. The therapeutic effect of the fruits, leaves and roots of this plant in the treatment of different tropical diseases is well documented in folk medicine lo3 . Different parts of this plant has been investigated for chemical constituents to some extent, and found to contain various types of chemical compounds. It has been known that root-bark of this plant contains coumarins4

.5 and anti-fungal compounds6, whereas its stem bark contains alkaloids7.8 and has insecticidal propert/ . The leaves of this plant afforded coumarins, triterpenoids and steroids 10. A new tyramine derivative was obtained from the fruits of Naringi creflulata ll

• However, the fruits of Naringi crenulata have been less investigated for the polysaccharide contents before l2 . Many polysaccharides from natural origin have been found to have biological activit/ 301 6. Since structure and functions are intimately related knowledge of the structure of isolated compound is a prerequisite for better understanding and therefore modification, if

any, of the activity. The aim of the present research was to isolate the different polysaccharides present in this fruit, to fractionate them and to analyze their structures.

Results and Discussion Pectic polysaccharides were isolated from the

pericarp of Naringi crenulata fruit by extraction with water (PH 5.5) at 35°C. The water extract, which contains 12.5% protein, was subjected to graded precipitation with ethanol. The less soluble fraction (LSF) contained 32.5% neutral sugar and 38.2% uronic acid. The other i.e., the more soluble fraction (MSF) was enriched in neutral sugar (61.4%) and contained lesser amount of uronic acid (17.3%) compared to LSF. Sugar analysis revealed that arabinose (31.8 mol %), galactose (38.5 mol %), and galacturonic acid (19.4 mol %) were the main sugars found in MSF fractions together with smaller quantities of xylose (4.5 mol %), mannose (2.5 mol %), glucose (2.1 mol%), rhamnose (1.1 mol %), and traces of fucose. The presence of high amount of galacturonic acid (confirmed by TLC analysis) in both the fractions suggests the presence of pectic polysaccharides. Neither glucuronic nor any uronic acids other than galacturonic ac id were detected by this method. Arabinose was found at a higher concentration than galactose in the LSF fraction , where it probably originated from arabinose rich side chainl7. GLC analysis and colorimetric estimations also showed that the ratio of acidic to neutral sugars in LSF was around 50 galacturonic acid molecules for 50 neutral sugar residues (two rhamnose inside the chain and 48 arabinose, galactose and other sugars in lateral branches). But MSF fraction contains 19 galacturonic acid molecules for 81 neutral sugar residues (one rhamnose inside the chain and 80 arabinose, galaclose and other sugars in lateral branches). These results revealed that the water-soluble material comprises population of polysaccharides differing in neutral sugars (NS)/uronic acid (UA) ratio. MSF fraction was hydrolyzed and the liberated acetic acid and methanol were measured by HPLC. The degrees of acetylation of MSF and LSF were 6.7% and 3.4% respectively. The actual positions of the acetyl groups are not

438 INDIAN J. CHEM., SEC B, FEBRUARY 2003

known. The acety l groups are usually ass igned to the galacturonic ac id res idues, though other sites of attachment are known to ex ist. These data show that less branched pectic materi als with low degree of acety lat ion were precipitated at lower ethanol concentration while the more highly branched pectic polymers had hi gher acetylation degrees and precipitated at higher ethanol concentration . The degree of methylation (OM ) of the LSF fraction was 11.1 %. No phenolic acids were detected. Monosaccharide residues in the furanose form are hydro lyzed under mild conditions compared to residues in the pyranose form. Appearance of arab inose as the only detectable sugar in weak ac id hydrolysate indicates the presence of Arar res idues. If, however, the hydro lys is is continued upto 12 hr in the same condition the hydro lysate contains arabinose as the major sugar along with minor quantities of galac tose, xylose, and glucose residues. The water un­extractab le residue (PWUR) contained a significant amount of pectic polysaccharides. Glucose is the most importan t sugar in thi s fraction and most of the glucose present here are of cellulosic origin.

DEAE-cellulose chromatography of LSF fraction resolved it into five (PF I, PF2, PF3, PF4 and PFn) ac idic fractions. Based on the mass of the composite sugar residues, 96% of the neutral and 91 % of the uronic acid injected onto the column was eluted with various inorganic eluants. The yield and sugar compositions of the fractions obtained are shown in Figure 1 and Table J, respectivel y. The sugar composition of the pectic fractions (PF2, PF3 and PF4) is qualitatively similar, but quantitatively there is a difference. With regard to the neutral sugar composition, arabinose and galactose dominated in vary ing ratios. Further neutral sugars found were xylose (1.9 - 24.7 mol%), g lucose (trace - 1.1 mol %), man nose (trace - 3.2 mol%), rhamnose (1.4 - 2.4 mol %) and fucose (0.3 mol %). Uronic acid is the major sugar found in all fractions except PF J. The sugar composition of various pectic fraction s indicates the probable presence of rhamnogalaturonan. The varying ratio of uronic acid to rhamnose found in different fractions indicates the presence of heterogeneity in pectic polymers isolated (assuming no degradation occurred during extraction and fractionation). Lateral chains of glycans consisting mainly of arabinose and galactose residues play a major role in fractionation; the number of rhamnose residue intercalated in the galacturonan chains decreasing slightly from fraction PF2 to

90

80

70

60

~ 0 50 '0 'iii 40 >=

30

20

10

0

Fractions

~ Yield (%) UA

Yield (%) NS

PFn

Figure 1- Yield (%) of neutral sugar and uronic ac id of fractions obtai ned from an ion exchange chromatography of LSF fraction [see resu lts and discussion for the identifi cat ion of fractions].

Table I - Yie lds and sugar composition of fracti ons recovered from DEAE-ccllulose chromatography of

the LS F fraction (see experimen tal fo r identifica tion of fractions).

PFI PF2 PF3 PF4

Yield NS' 24.8 56.7 12.8 4.0

Yield UAh 4.6 83.4 5.8 3 .2

NSc 17.7 22.6 28.9 7. 1

UAc 0 .6 30.4 22. 1 nd

Rhad 2 .3 2.4 1.4

Fucd 0.9 0.3 0.3

Arad 20.7 20.6 24.5 37.2

Xyld 5.9 1.9 7.7 24.7

Mand 12.3 3.2 tr Ir

Gald 32.6 12.8 12.3 38. 1

Glcd 18.2 l.l 0.6 tr

UAd 6.4 57.5 53. 1 nd

Ara/Gal 0.63 1.6 1.99 0 .98

UA/Rha 2.8 24 37.9 nd

• Percentage weight of total neutral sugar recovered, b Percentage weight o f total uronic acid reco vered,

PFn

1.7

2.9

12.8

11.2

17.4

6.9

tr

14.2

18.4

43.1

1.22

Nd

C Percentage weights of fraction dry weight , d Percentage mol , nd : not determined, tr: trace, -: not detected

fraction PF4 . PF2, the major fraction, accounted for 56.7% and 83.4% of the neutral sugars and uronic acid recovered from the anion exchanger, respectively . The fraction obtained with 400 mM-

NOTES 439

buffer concentrations had an aralgal ratio higher than all other fractions .

The major fraction (PF2) was further characterized by its molecular weight distribution as determined by gel permeation chromatography. Figure 2 shows the chromatogram for this fraction obtained using Sephadex G-200 column. As indicated by the Kav . values at the front and tail ends of the chromatograms (Kav . 0 to 0.7), the PF2 fraction elutes well within the fractionation range of the column. Gel permeation chromatography of PF2 yielded two subfract ions: PF2A (fraction no 5 to 9) and PF2B (fraction no 16 to 21). Both the subfractions had practically identical monosaccharide compositions (similar to that of PF2). The on ly difference between these two subfractions, as judged by gel permeation chromatography, seems to be the molecular weight. The fractionation range of thi s column was 1,000 - 200,000 for dextrans. Subfraction PF2A was elu ted almost completely at the excl usion limit whereas the polymers in subfraction PF2B had molecular weights lower than the exclusion limit of the column (i.e. in the range of 40,000 -70,000 Oa based on calibration with standard dextrans).

The FT-IR spectra of various fract ions are given in Figure 3. The broad band between 3600 and 3000 cm" , corresponding to vibrations of the hydroxylic groups appeared to be si milar in all the spectra. Methyl and methylene group vibrations appeared around 2927 cm-' and were present in the spectra of all fractions. The pectic polymers present in the water extracted material (Figure 3a) and the pericarp itself (Figure 3b) show a band in the region 1739 cm" related to the >C = 0 stretching of the ester group' 8. After de-esterification a reduction of the absorbance band at 1739 cm" was observed (data not shown). Structural features arising from particular conformations around the glycosidic bond of the pectin are observable in the 850- 1200 cm" region ' 9.2'.

The bands at 1014 to 1101 cm" are characteristic of the galacturonic ac id res idues of the pectic polysaccharides (Figures 3a and 3c).

In order to have an idea about the mode of linkages of the polysaccharide present in the LSF, the native polymer (LSF) and its carboxyl-reduced derivative (LSF-R) were subjected to methylation analysis. The results of methylation analysis (Table II) indicated that rhamnose is 1,2-linked with branching via C-4. Reduction of the galacturonic ac id res idues resulted in increased values for 1,4-linked Galp residues owing to the reduction of galacturonic ac id .. The slight

o t5 ~

400 --------

300

----, 1.2

3l 200

0.8 UA Rec ..... 0.6 NS

o Rec

c o a. (J) Q)

a:

100

Kav

0.4 .....

0.2

o l....iESL-____ jhTTT'/'1'T1'1~ 0

5 9 13 17 21 25 29

Elution volume

Figure 2 - Gel permeation chromatography of PF2 fraction. isolated from the peri carp of Naril1gi crelluiara fruit , on Sephadcx G-200 colum n.

55 .-------------~=--------------------,

/\,

if' .. ' . fv\ j '\'/i Y' "", , ~ .\ f \!~'. ~

%T

;" ,', ),;, \: .. . ; " . : . ,

. .

30 (e ) . .. .... :

20

: '.' :

. """, I 11=-__ ~ __ ~~--~--~~--~--~~--~~ 4000 3000 2000 1000 200

WAVE NUMBER (cm-1)

Figure 3 - FJ'-JR spectra of (a) the pericarp of Naril1gi crenuiala fruit (P). (b) the water extract (LSF), and (c) the Pr2 fraction [see experimental fo r the identification of fractions).

increase in the 1,2- and 1,2,4-linked Rhap residues was probably due to better de-polymerization of reduced than that of the nati ve polymer. Indeed, the aldobiouronic acid, ~-O-GaIA-(l ,4)-L-Rha, in pectin is resistant to acid hydrolysis but after reduction of the polymer, the reduced galacturonic ac id and rhamnose

440 INDIAN J. CHEM ., SEC B, FEBRUARY 2003

Table II - The linkage (mol%) of LSF fraction determined by reduction, methylation and GC/MS. LSF-R is the

carboxy l reduced derivative of LSF fraction.

Parti ally methy lated LSF LSF-R Linkage alditol acetate'

2,3-Mc2-Xy l 6.7 4. 1 1,4

2,3,5-Mer Ara 13.2 5.4 Terminal

2,3-Mer Ara 15.1 9 .3 1,5

2-Me-Ara 7.2 4.9 1,3,5

3,4-MeT Rha 1.2 1,2

3-Me-Rha 1.5 1.5 1,2,4

2,3,6-Glc 1.2 1.8 1,4

2,3,4,6-Me4-Gal 7.8 4.2 Terminal

2,4,6- MeJ-Gal 2 2 1,3

2,3,6-Mer Gal 13.8 5 1.1 1,4

2.3 ,4-Mer Gal 7.1 7.5 1,6

3,6-MerGal 3.2 1.5 1,2,4

2,4-Me2-Gal 10 .5 2 .3 1,3,6

2.3,6- Mer Man 4.7 3.2 1,4

' 2,3,5 -Mer Ara = 2,3,5-tri-O-methyl-1 ,4-d i-O-acetyl arabinitol etc.

_h = not detccted.

linkage is hydro lyzed more eas ily and thus a better recovery of rhamnose is obtained. Arabinoses are in furanose form (Ara!> and are 1,5-, 1,3,5- and terminally linked. About 16.9% of the galactose res idues occupy a terminal position, and 29.7% is branched. The presence of 1,6- and 1,3,6-linked galactosy l, and 1,3- and terminal arabi nosyl residues suggests the presence of type II arabinogalactan. Besides, nearly 33% of the galactose residues is 1,4-linked, and a small portion (6.4%) is 1,2,4- linked. Galactopyranosyl (Galp) or Araf groups terminated most of the branched res idues.

To verify the results of methylation analysis, the polysaccharide (PF2) was subjected to periodate oxidation. Alditol acetates deri ved from the periodate oxidi zed sample included arabin itol , galactitol and a tetritol. The occurrence of tetritol indicated the presence of 1,4-linked pyranosy l and/or 1,5-linked furanosyl units without substituents at positions 0-2 and 0-3. Survival of some arabinose residues established the presence of 1,3,5-linked arabi no­furanosyl units The presence of galactitol confirmed the results of methylation analysis that some of the galactopyranose residues are 1,3- and 1,2,4-linked and therefore resistant to periodic acid oxidation. Although the galacturonic acid residues are l,4-linked

but a significant amount of this residue survived periodate oxidation.

Conclusion Pectic polysaccharides isolated from the pericarp of

Naringi crenulata fruit yield fractions consisting of population of polysaccharides differing in neutral sugars/uronic acid ratio. These pectic substances consisted of a range of structurally related polymers that differed widely in their behavior towards anion exchanger. These polymers gave viscous solution, were lightly esterified, and contained acetyl groups.

Experimental Section Plant material and polysaccharides. Pericarps of

the fruits from Naringi crenulata were the same as that used previousl/ 2

• Water-soluble polysaccharides were obtained from the dried powdered pericarp (P, 1 g) by extraction with water pH -5 .5 as follows . The fruit powder (1 g) was di spersed in 150 mL of deioni zed water and stirred continuously using a mechanical stirrer over 4 hr period. The supernatant was removed by centrifugation (15 min at 16,000 g) and the pellet re-extracted with ISO mL water. The combined supernatant dialyzed exhaustively agai nst water and then concentrated to give a VISCOUS

solution . Graded precipitation with ethanol. The con­

centrated dialysate (-200 mL) was then submitted to graded precipitation with dehydrated ethanol. The concentration of alcohol was increased in steps of 20%. Each mixture was placed in the refrigerator for 3 hr. The precipitate obtai ned at 60% ethanol concentration was collected by centri fugation, washed thoroughly with 80% ethanol, 95% ethanol, acetone and diethyl ether, and finally dried in vacuum over P20 S (LSF; yield 21 mg). The second precipitate obtained at 80% ethanol concentration was isolated therefrom by centrifugation WllS d ried by solvent exchange as described above (MSF; yie ld 52 mg). The water un-extracted residue was also dried by solvent exchange as described (PWUR ; yield 605 mg).

Anion exchange chromatography. Approxi­mately 340 mg of LSF was fractionated on a column (2.2x51 cm) of DEAE-Sepharose CL-6B that was previously equi librated with 10 mM NaOAc buffer pH 6.5 . Elution was carried out successively with 10, 100, 300, 400 and 1,000 mM NaOAc buffer pH 6.5 (350 ml each) in a stepwise manner and finally with 200 mM NaOH (300 mL). The collected fractions

NOTES 441

were analyzed for neutral sugar and uronic acid contents and were concentrated, dialyzed, and the concentrated retentate was diluted with 3 vols. of absolute ethanol at room temperature. The precipitate was recovered by centrifugation (20 min at 16,000 g); the pellet was dehydrated by solvent exchange as described above and finally dried, under vacuum, over P20 5.

Gel permeation chromatography. The poly­saccharide (PF2, 3 mg) was dissolved in 400 mM sodium acetate buffer pH 4.0 and passed through a Sephadex G-200 column (1.3 x 30 cm) equilibrated with the same buffer at 15 mlfh. Fractions (1 mL) were collected and assayed for the neutral sugar and uronic acid contents. Elution of polysaccharide from

GPC is expressed as Kav [Kav = (Vc-Vo)/(V,-Vo) with V, and Vo being the total and void volume of the column (determined as the elution volume of glucose and amylopectin respectively) and Ve is the elution

volume of the samples]. The column was calibrated with standard dextrans wi th a molecular-weight range of 10,000 to 500, 000. Fractions 5 to 9 and 16 to 21 (Figure 2) were pooled separately. Each of them was dialyzed, concentrated and lyophi li zed to yield PF2A and PF2B subfractions. Their sugar compositions were determined.

De-esterification. The pH of the polysaccharide solution (2 mg/mL) was increased to 13 using 0.2 M NaOH. After Ihr at 35°C the pH of the solution was lowered to 5 by dropwise addition of glacial acetic acid. Neutralization was carried out over an ice bath and the solution stirred continuously using a magnetic stirrer bar. The solution dialyzed, concentrated and then diluted with 4 vols of ethanol. The de-esterified material was recovered from the retentate by centrifugation and dried after solvent exchange.

Carboxyl reduction of the LSF fraction. This was carried out by the methods of Taylor and Conrad22 as described in refs .23.24 to yield the carboxyl reduced derivative LSF-R.

Methylation analysis. Methylation by the methods of Blakeney et al.25

, conversion to partially methylated alditol acetates (PMAA) and GLC and or GLCIMS on columns of SGE BP I column (25 m x 0.22 mm, 0.25 11m) and OB-225 OW) column (30 m x 0.32 mm, 0.25 11m) as described' 2.' 3.24.

Periodate oxidation. LSF (500 mg) in 0.25 M formic acid, pH adj usted to 3.7 with 1 M NaOH was treated with of Nal04 (2.675 g) as described in ref. 24. The product was isolated after desalting on a

Sephadex G-I0 column (2.0 cm x 2S cm). The recovered material was hydrolyzed and after usual treatments was analyzed by TLC and GLe.

Analytical methods. Chemicals used were analytical grade or best available. All determinations were done at least in duplicate. Protein was estimated by the methods of Lowry et al?6 using bovine serum albumin as standard. Acetic acid and methanol contents were estimated by HPLC according to Voragen et al. 27

• The degree of acety lation (OA) and degree of methylation (OM) were calculated as the molar ratios of acetic acid and methanol to uronic acid. The uronic acid content was determined by the m-hydroxydiphenyl assay as described by Ahmed and Labavitch28 using galacturonic acid as standard. Correction was made for the interference of neutral sugars in the determination of uronic acid. Neutral sugars were estimated by the phenol-sulfuric acid assa/9 using galactose as standard. In this method corrections were made for interference from anhydro­galacturonic acid. For the determination of monosaccharide composition, the polysaccharides in the samples were hydrolyzed using sulfuric acid (2 M, 3 hr at 100°C), preceded by a I hr prehydrolysis at 30°C with aqueous 72% H2S04 for insoluble residues. Neutral sugar compositIon of the hydrolysates was determined by GLC using a SGE BP 225 column (25 m x 0.32 mm, 0.25 11m) operating isothermally at 195°C with H2 as carrier gas, after conversion of the monosaccharides to

alditol acetates as described by Blakeney et al. 30 .

myo-Inositol was used as internal standard, and hydrolytic losses were accounted for by using an external standard31 . Alternatively, the alditol acetates were separated on a OB-225 OW) column (30 m x 0.32 mm, 0.25 11m). The temperature program was ISO°C for 7 min and then IS0o-250°C at SoC Imi n. Phenolic acids were determined by HP'LC after saponification and extraction as described32 .

Identification of uronic acid and neutral sugars by thin layer chromatography

The sugars in the acid hydrolysates were analyzed by TLC on Silica Gel G plate impregnated with 0.5 M NaH2P04. The plate was e luted using ethanol­phenol-pyridine- 0.1 M phosphoric acid (l0:2:2:4) and/or 2-propanol - methanol - water (16: I :3) as developing agent. Sugars were detected by spraying with saturated aniline phthalate and drying at lOO°e.

442 INDIAN 1. CHEM .• SEC B. FEBRUARY 2003

IR spectroscopy. Samples were dried at 35 0- 44°C

in vacuum over phosphorus pentoxide for 72 hr prior to making pellet with KBr. Infrared spectra were recorded on a JASCO FTIR 420 spectrophotometer.

Partial acid hydrolysis. Polysaccharide (LSF) sample was hydrolyzed with 20-mM oxalic acid at 100°e. The hydrolysate removed from the reaction vial at 3 hr and 12 hr, neutrali zed with CaCOJ , de­cationised with Amberlite IR-1 20 (H+), filtered and then concentrated. The sugars present in the hydrolysates were analyzed either by TLC as described above or by GLC of the derived alditol acetates.

Acknowledgement This work was funded in part by the University

Grants Commission, India under DSA project in Chemistry at Burdwan University. The authors are indebted to Dr J -F Thibault, Director, INRA, Nantes, France for instrumental facilities and Mrs M J C and Ms J Vigouroux for DE, OM and phenolic acid estimations and for recording GLe. Thanks are due to RSIC, Lucknow for providing GC-MS analysis.

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