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Structure elucidation of Rauwolfia Serpentina Benth. Water soluble seed polysaccharide by periodate oxidation.

Introduction

Rauwolfia serpentina Benth. plant belongs to family Apocynaceae and called as Sarpagandha. It is an evergreen plant, perennial, glabrous and errect under shrub grows to the height of 15-45 cm. It is widely distributed in sub-Himalayan tract from Punjab eastwards to Nepal, Sikkim, Bhutan, Assam, in the lower hills of the Gangetic plains, Eastern and Western ghats, in central India and in Andamans. Beyond India, the plants distributed in East Pakistan, Srilanka, Myanmar, Malaysia, Thialand and Java.

Roots are tuberous with pale brown cork. Leaves are in whorls of three, elliptic lanceolate or obovate, bright green above, pale green below, tip acute or acuminate, base slender and petioles long. Flowers are in many flowered irregular corymbose cymes, white or violet coloured tinge.

Rauwolfia serpentina Benth. is a medicinal plant and is gifted with unique alkaloids that have remarkable medicinal properties. Reserpine alkaloids are present in root, stem, leaves and seeds of plant and percentage of alkaloids depends on the geographical place from where the plant is collected. The Rauwolfia alkaloids extracted from the roots, leaves and seeds, medically it is important therapeutic agent both as antihypertensives and as sedatives. It is also used for relief of various nervous system disorders both psychic and molar including anxiety status, excitement, maniacal behaviors associated psychosis intensity and epilepsy. Roots extract are employed for the treatment of intestinal disorders, diarrhea, dysentry and anthelmentic. It is believed to stimulate uterine contraction and recommended for the uses in child birth. Juice of leaves has been used as remedy for corneal opacity.

Rauwolfia alkaloids, pharmacologically can be divided into reserpine and ajmaline groups, while reserpine is used for sedative action in mild anxiety states chronic psychoses and in treatment of chronic schizophernic patients. Seeds also contains alkaloids, fresh seeds are heavier than water, contain 0.2-0.3% alkaloids. Hindus used this plant for centuries as febrifuge and as an antidote to the bite of poisonous snakes. It is used to treat dysentery and other painful intestinal infections. In ayurvedic system of medicine product ranges as confide (Speman Forte), Lukol, Serpina etc.

Material & Methods

Isolation of polysaccharide

Seeds (250gm) of Rauwolfia serpentina Benth. were collected from F.R.I. Dehradun, then washed the seeds with water dried and crushed to a greyish powder. Powdered seeds (100gm) were dissolved in distilled water (800ml) for 24 hrs. The contents were stirred thorough with the help of mechanical stirrer then the viscous solution was filtered through muslin cloth then it was again filtered by sharpel's super centrifuge to remove all finely suspended particles. Filtrate was precipitated with ethanol to precipitate out all polysaccharide in light brown form. At the time of precipitation of polysaccharide was well stirred with the help of mechanical stirrer. The precipitate of polysaccharide was filtered through sintered funnel G-3 under suction and dried in vaccum at 60[degrees]c after washing with acetone and petroleum ether. It was obtained as a greyish powder (8.67 gm) had sulphated ash 1.84% and optical[{[alpha]}.sup.25.sub.D] + 31.2[degrees]c [H.sub.2]O.

Periodate oxidation of the polysaccharide

In periodate oxidation studies of the purified polysaccharide (450gm) from Rauwolfia Serpentina Benth. seeds (Abdel & Smith, 1951) was dissolved in 100 ml water and sodium metaperiodate solution (0.025M, 100ml) and make up the volume to 250ml with distilled water. The reaction mixture was kept in dark at 4-8[degrees]c in refrigerator for 60hrs. A blank was also set up in the similar way. Aliquotes (5ml) was pippeted out from the reaction flask at different time intervals for the determination of amount of periodate consumption and formic acid liberation.

The periodate oxidation reaction was first discovered by Malaprade (1928), Fluery and Lange (1933), have given a better method for the more extensive use of periodic acid glycol. Perlin (1954) has given the two important reagents as periodic acid and lead tetra acetate. Kumar (1976), Chatterjee (1976) and Sarkar (1976) have used periodate oxidation to determine the seeds polysaccharide structure. The central atom of the oxidation reagent must be able to co-ordinate at least two hydroxyl groups and glycol groups undergo cyclic ester formation with oxidant. The periodate oxidation reaction is to be a dialdehyde type of oxidation.

Determination of Periodate Consumption

The periodate oxidation method proposed by Fluery & Lange, (1933) was used for the periodate oxidation of seeds polysaccharide (0.190 gm) and suspended in 50ml distilled water and 250 ml sodium metaperiodate solution (0.25M) then the volume of reaction flask were made upto 250ml with distilled water. The measuring flask was shaken to form a colloidal solution and kept in dark in refrigerator to maintain low temperature range of 4-8 [degrees]c. The reaction mixture (5ml) was pipetted out at different time intervals and excess periodate was estimated by arsenic method (Perlin, 1959) with sodium arsenide solution (0.01N, 25ml). To each aliquot, 2ml standard solution of sodium bicarbonate and 2 ml of 20% potassium iodide solution were added. The reaction mixture was shaken for 1 hr and kept in dark for 15 min and then iodine solution (0.01N, 5ml) was added to it. The excess of iodine was titrated against sodium thiosulphate (hypo) solution (0.1N), using starch as an indicator near the end point. A blank titration was also carried out in a similar way. The difference between blank and experiment gives the values of periodate consumption of 2.72 moles for 60hrs and results are given in table- 1 and plotted curves are shown in Fig. 1.

Determination of Formic Acid Liberation

During the periodate oxidation, the released formic acid was determined by the procedure proposed by Halsall et. al.,1947, & Blecher, 1957 and Brown, 1948. The aliquots 5ml were taken out from each flask at different time intervals. To each aliquot about 10ml of freshly distilled ethylene glycol was added to destroy the excess of periodate present in the reaction mixture for 30 minutes. The formic acid liberation was estimated by titration against sodium hydroxide solution (0.12 N) using methyl red dye as an indicator. A blank was also carried out in the similar way for the estimation of formic acid. The titer values became constant after 60hrs which corressponds to 0.108 moles of formic acid per mole of anhydrohexos sugar as shown in fig. 2 and results are given in table 2.

Reaction of periodate oxidation of polysaccharide

The study showed the consumption of 2.72 moles of periodate ions per anhydrohexose sugar unit as determined by volumetrically. The probable reaction by which the periodate consumption of polysaccharide (glucopyranose) are occurs as follows:

This reaction showed that the 90% of D-glucopyranose units were containing adjacent free hydroxyl groups which resulting in the consumption range of periodate ions during periodate oxidation reaction. It is conducted from the above mentioned facts that the probably one branching point occurs per repeating units of the glucomannan, which constituting the non-ionic polysaccharide of Rauwolfia Serpentina Benth. Graph between time of oxidation and moles of periodate (oxidant) consumed indicated that on increasing the time of oxidation from 10 hrs to 50 hrs, the consumption of moles of periodate increases from 1.14 to 2.72, but from 50 hrs to 60 hrs, the consumption of moles of periodate becomes constant (2.72 moles).

The formic acid appears is to be originating from the reducing as well as nonreducing terminal units of the D-glucopyranose as shown in the following.

From the above reaction it may be concluded that the terminal D-glucopyranose units of the seeds polysaccharide are not substituted. The graphs between time of oxidation and amount of formic acid released during periodate oxidation reaction indicates that the on increasing the time of oxidationfrom 10 to 50 hrs, the amount of released formic acid increases from 0.46 to 1.08, but from 50 to 60hrs the amount of release formic acid becomes constant (1.08 moles).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Result & Discussion

The periodate oxidation study showed that the consumption of 2.72 moles of periodate ion per anhydrohexose sugar units are determined volumetrically. The probable reaction of periodate consumption of Rauwolfia serpentina Benth. seed polysaccharide (glucopyranose) are occures as follows:

This reaction showed that the 90% of D-glucopyranose units were containing adjecent free hydroxyl groups. Resulting in the consumption range of periodate ion during periodate oxidation reaction. It is concluded above mentioned facts that probably one branching point occures per repeating units of the glucomannan, The formic acid appears to be origenating for reducing as well as non-reducing terminal units of D-glucopyranose units.

It may be concluded that the terminal D-glucopyranose units of the seeds polysaccharide from the above reaction liberate about 1.08 moles of formic acid per anhydrohexose sugar units after 60 hrs.

Rauwolfia serpentina Benth. seed polysaccharide was oxidised with sodium meta periodate solution (0.025M) at 4-8[degrees]c in refrigerator. The periodate consumption and formic acid liberation were estimated at different time intervals and found to be 2,72 moles of periodate consumption with simultaneous liberation of 1.08 moles of formic acid per mole of anhydrohexose unit after 60 hrs.

References

[1] Abdel, A.M. and Smith, F., (1951) J.Amer. Chem. Soc., 73:994.

[2] Baker, S.A., Bourne, B.J. and Whiffen, O.H.(1950) Method in Biochemical Analysis,3: 213-216.

[3] Brown. F., (1948) J. Chem. Soc, 27.

[4] Chadha, Y.R., (1976). The Wealth of India, Raw materials, Publication and Information, CSIR, New Delhi, India, 10 (SP-W): 62

[5] Chatterjee, B.P., (1976) Indian J Chem., 14B: 914.

[6] Fluery, P. and Lange, J.; (1933) J.Pharm. Chem., 17: 107.

[7] Halsal, T.G.; Hirst, E.L. and Jones, J.K.N., (1947) J. Chem, Soc, 1427.

[8] Krishnamurthi, A., (1969) The Wealth of India, Raw materials, Publication and information directorate, CSIR, New Delhi, India, 8 (Ph-Re) 376-390.

[9] Kolthoff, J.M. and Blecher, R.; (1957) Volumetric Analysis, Intersciences Pub. Inc, New York, 3: 276

[10] Kumar, P., (1976) J.Int Chem. (India), 48: 192..

[11] Malaparde, L., (1928) Bull. Soc. Chem., 43: 683.

[12] Pathak, D.K., (2009). Chemical structure determination of "Rauwolfia Serpentina Benth." Seeds Polysaccharide., Ph.D thesis. Dept. of Chemistry, Agra University, India.

[13] Pathak, D.K, Yadav, R. and Pandita.P., (2009) Chintan, Vol.2(1): 59

[14] Perlin., A.S., (1957) J. Amer. Chem. Soc. 76: 4101.

[15] Perlin., A.S., (1959) Adv. Carbohydr. Chem. 14-B.

[16] Perlin, A.S., (1976) Int. JChemtracks, 9:14b.

[17] Sarkar, M., (1976) Indian J. Chem., 14-B: 919.

[18] Singh, R.B., Singh, S.P. and Jindal, V.K., (1992) Acta Ciencia Indica, Chemistry. 18-C(4):307.

[19] Singh, R.B., (2002) Oriental J.Chem., 18(3): 509-512.

[20] Whistler, R.I., (1965) Methods of /Carbohydrate Chemistry, Academic Press, London. 5: 296-299.

(1) Dhiresh Kumar Pathak, (2) H.S. Chaudhary, (3) Reena Yadav and (1) Prarina Pandita

(1) COP, IILMAHL, Knowledge Park II, 17&18, Greater Noida, 201306, India -Dept. of Chemistry, Amardeep Degree College, Firozabad, 283203, India Corresponding Author E-mail: dhiresh.pathak@gmail.com
Table 1: Periodate oxidation of Rauwolfia Serpentina
Benth. Seeds Polysacchride.

S.No. Sugar Present

1 Periodate consumption of
 anhydrohexose sugar units (moles/mole)

S.No. Time (hours)

1 10 20 30 40 50 55 60
 1.14 1.52 1.96 2.34 2.64 2.72 2.72

Table 2: Formic Acid Liberation of Rauwolfia Serpentina Benth. Seeds
Polysacchride.

S.No. Sugar Present

1 Formic acid liberation of anhydrohexose
 sugar units (moles/mole)

S.No. Time (hours)

1 10 20 30 40 50 55 60
 0.46 0.87 0.94 0.98 1.02 1.08 1.08
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Author:Pathak, Dhiresh Kumar; Chaudhary, H.S.; Yadav, Reena; Pandita, Prarina
Publication:International Journal of Applied Chemistry
Date:Sep 1, 2011
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