isolation and characterization of antidiabetic...

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Chapter 6: Isolation and Characterization of Antidiabetic Compound

Ph.D. Thesis 2013 University of Delhi 115

CHAPTER 6

ISOLATION AND CHARACTERIZATION OF ANTIDIABETIC COMPOUND

6.1 INTRODUCTION Review of literature afforded that some fractions/ compounds isolated from different parts of the banana plants have antidiabetic action.(Ross, 2001). Unsaponifiable fraction of flower of Musa sapientum when applied to a column of alumina gave five crystalline fractions. The third fraction with m.p. 129-31, existing in form of shining white needles, exhibited maximum degree of hypoglycaemia (Jain SR,1968). Pectin isolated from inflorescence stalk of Musa sapientum, had hypoglycemic as well as hypolipidemic effect (Gomathy et al, 1990). Active proteinous compounds isolated from the fruit which have approx 12566 Dalton of molecular weight showed the hypoglycemic activity.(Ahmed YT, et al, 2008). Some bioflavonoids compounds isolated from the flower of Musa sapientum,were effective in treating diabetes. Mechanism of action appeared to be by increasing insulin receptor tyrosine kinase activity. (Ganugapati et al, 2012).

It is evident from preliminary studies described in chapter 4- that aqueous extract of Musa sapientum has significant antidiabetic effect. There is no report on isolation and characterization of antidiabetic compounds from central stem of Musa sapientum. This chapter describes procedure for isolation of antidiabetic compound from central stem of Musa sapientum, it’s structure determination and results on it’s activity. 6.2. RESULTS 6.2.1. Phytochemical screening of Aqueous Extract Central Stem of Musa

Sapientum The preliminary phytochemical analysis of aqueous extract of stem revealed the presence of phenols, saponins, pectins terpenes and steroids, as shown in Table 6.1.



Table 6.1: Phytochemical screening of Aqueous extract of stem Musa sapientum Phytochemicals AqMS

Phenols +

Saponins +

Pectins +

Terpenes +

Steroids +

6.2.2. Total Phenolic Content The total phenolic content were determined by Folin-ciocalteau quantitative analysis of AqMS showed phenolic ccontent to 50 mg /100 gm extract, which shows it has high phenolic content. 6.2.3. Purification Aqueous extract was prepared from stem of Musa sapientum and lyophilized as described in chapter 3. Lyophilized extract was insoluble in ethanol but dissolved in methanol. 100g of lyophilized extract was extracted in methanol using soxhelet apparatus at 12 hrs for 40 0C, then filtered through whatman paper no. 1. Filterate was vapourized under reduced pressure using rotary evaporator. This resulted in formation of a brown residue, which is referred to as methanolic extract. (yield 0.11%).

Treatment regime-To assess the hypoglycemic activity of different doses of Methanolic fractions after 15 days treatment, rats were divided into 5 groups of six rats each as follows: Group I- Healthy control rats; Group II- Diabetic control; Group III- Diabetic rats treated with Methanolic fraction (10mg/kg/day); Group IV- Diabetic rats treated with Methanolic fraction (20mg/kg/day); Group V- Diabetic rats treated with Methanolic fraction (40mg/kg/day).

The diabetic control rats had a significantly higher FBG and PPG levels compared to healthy controls. The methanolic fraction produced dose dependent fall in FBG and PPG. Fall in FBG increased from 16 to 23% when the dose of drug was increased from 10 mg/kg to 20 mg/kg and fall in PPG increased from 24% to 32% , but

Figure 6.1: Peak fragmentation and Gas chromatogram of methanolic fraction from stem of Musa sapientum



increasing the dose to 40 mg/kg had no additional benefit. This shows maximum effective dose of methanolic fraction is 20 mg/kg. (Table 6.2).

Table 6.2: Effect of 15 days treatment with Methanolic fraction obtained from aqueous extract of central stem of Musa sapientum in diabetic rats

Groups Blood glucose values

0th day FBG

15th day FBG

% fall

FBG 0th day PPG

15th day PPG

% fall PPG

Group I (Healthy control) 78±2.2 82± 2.1 - 134±2.3 135±2.6 - Group II (Diabetic control) 162±2.8 217±2.5 - 298±3.3 345±3.2 -

Group III (Diabetic +Methanolic fraction

10mg/kg) 169±2.1 142±2.7 a 16 306±3.4 234±2.6a 24

Group IV (Diabetic + Methanolic fraction 20mg/kg) 171±2.2 130±1.8a 23 304±2.5 206±2.1a 32

Group V (Diabetic + Methanolic fraction 40mg/kg) 177±2.4 134±1.8 a 24 298±2.3 204± 3.1

a 32

Values are mean ± S.E.M of six rats in group; a p<0.01 0 day blood glucose value compared to 15th day

6.2.4 Gas Chromatography and Mass Spectrometry (GC-MS) Analyses of Methanolic Fraction of AqMS

Methanolic fraction then applied on Gas chromatography and Mass spectrometry. GC-MS analysis obtained on a Shimadzu GC-MS-QO-2010 system. Methanolic fraction was dissolved in methanol and applied on Rtx-5 column (60 m X 0.25 mm i.d., film thickness 0.25 µm) to get the chromatogram. Helium as carrier Injector, Ion source temperature and inter phase temperatures were varied from 250 to 2800C, respectively. Column temperature programmed from 100 to 2500C at 100C/min. with hold time of 5 min and from 250 to 3000C at 150C/min. with hold time 20 min. respectively. The flow rate of carrier gas was 1.2 ml/min and split ratio was 10:1. EI source at 70 eV and mass range was 40-750 amu. Compounds were identified using Willey and NIST libraries.



Gas Chromatography of Methanolic fraction from stem of Musa sapientum The Gas chromatography and mass spectroscopic studies suggested that the presence of sterols is high in methanolic extract. GC-MS analysis of extract showed that extract was mixture consisting of a series of sterols and % area was very high (89%) with sterol rich compounds at retention time of 54.04 with molecular weight of ranging from 140 Da to 430Da according to Willey and Nist library.(Figure 6.1) 6.2.5. Column Chromatography of Methanolic Fraction

Methanolic fraction was partitioned between Petroleum ether and Chloroform. Chloroform formed upper layer and lower layer was of petroleum ether. Maximum amount of extract came in petroleum ether fraction. Both fractions were evaporated to dryness, suspended in water and hypoglycemic activity was assessed in diabetic rats. Petroleum ether extract showed hypoglycemic activity and was further applied on different columns. We have used many type of columns (LH-20 and silica A-60) for column chromatography. LH-20 column gave better seperation. 6.2.5.1. Preparation of LH-20 Column

25 g of LH-20 soaked in 100 ml of methanol and swallon gel was packed in a glass column up to 40 cm. The elution solvent was petroleum ether with flow rate of 6 to7 drops/min. The eluent of 5 ml were collected, eluent were pooled on the basis of colour. This afforded us three fractions. These fractions (Fraction1 (light yellow), Fraction 2 (Dark yellow) & Fraction 3 (white froth). Fractions were evaporated to dryness. Dried fraction suspended in water were fed to diabetic rats for one week and blood glucose levels estimated. 6.2.6 Assessment of Hypoglycemic Activity of Different Fractions Treatment regime-To assess the hypoglycemic activity of different chromatographic fractions (eluted from gel chromatography). Rats were divided into 5 groups of 6 rats each and treated with fractions for seven day as follows: Group I- Healthy control rats; Group II- Diabetic control; Group III- Diabetic rats treated with Fraction I (light



yellow); Group IV- Diabetic rats treated with Fraction II (Dark yellow); Group V- Diabetic rats treated with Fraction III (White Dense froth).

There was 17 % and 19.4 % fall in FBG and PPG respectively after 7 days treatment with fraction 3. Whereas fraction 1 and 2 showed no significant hypoglycemic activity. So fraction 3 was further purified.(Table 6.3)

Table 6.3: Hypoglycemic activity of different chromatographic fractions obtained from methanolic fractions of Musa sapientum after 7 days treatment

Groups Fasting Blood Glucose (mg/dl) Postprandial glucose (mg/dl) 0th day 7th day % fall 0th day 7th day % fall

Group I (Healthy control) 81±1.8 83±2.3 134±2.3 132±2.6

Group II (Diabetic control) 159±2.4 204±3 272±2.8 342±2.5

Group III (Diabetic + Light yellow fraction)

150± 3.2 201±2.7 - 255±2.9 335± 2.7 -

Group IV (Diabetic+ Dark yellow fraction)

155±3.1 200±2.3 - 251±3.5 325±2.4 -

Group V (Diabetic+ White

dense froth fraction) 153±2.3 128± 2.1a 17 266±2.6 167± 2.1a 19.4

Values are mean ± S.E.M of six rats in group; ap<0.001 Diabetic treated rats compared to diabetic control.

6.2.7. Thin Layer Chromatography of Active Fraction 3 Fraction 3 obtained from column chromatography was subjected to TLC. We have taken different solvent systems like (Chloroform: Methanol/ Methanol: Pethroleum ether/ Hexane: choloroform / Petroleum ether: ethyl acetate) with different ratio. Petroleum ether: ethyl acetate (1:9) gave good separation. Glass plates (20 x 20 cm) coated with silica gel G (mesh 60-120; 40g in 80 ml of distilled water) at a thickness of 1mm were activated for 1 hr at 110 0C.The active fraction was applied on to the silica gel plate in the form of band at 1.5 cm above the edge of the plate. The separation was carried out in Shandon TLC tank (10 x 24 x24 cm) using Petroleum ether: ethyl acetate (1:9 v/v).after completion of the run, the plates were air-dried and developed by keeping them in iodine saturated chamber for few minute. Fraction 3



gave three bands at different Rf values. The bands seen (FIII a (0.65), FIIIb (0.42) & FIIIc (0.30) were scraped from TLC plate. Material in bands extracted in water and given to the diabetic rats for assessment of hypoglycemic activity. These result are shown in table 6.6. 6.2.8. Assessment of hypoglycemic activity of different bands separated after TLC Treatment regime-To assess the hypoglycemic activity of different bands obtained from Thin layer chromatography (TLC), rats were divided into 5 groups of six rats each and treated for seven days as follows: Group I- Healthy control rats; Group II- Diabetic control; Group III- Diabetic rats treated with F3a; Group IV- Diabetic rats treated with F3b; Group V- Diabetic rats treated with F3c.

Band F3c (Rf-0.30) showed hypoglycemic activity. It decreased fasting blood glucose from 159±3.2 to 128±1.7 mg/dl (19 %) and post prandial blood glucose values 201±2.5 to 156±2.2 (22%).other two bands FIIIa and FIIIb did not produce appreciable fall in FBG and PPG. The active hypoglycemic component obtained from TLC was named MSH-3. MSH-3 so obtained was subjected to TLC again to get a compound with maximum purity and then subjected to gas chromatography and mass spectroscopy.(Table-6.4)

Table 6.4 Hypoglycemic activity of different TLC bands of active hypoglycemic fraction 3 (purified from gel chromatography) after 7 days Treatment

Values are mean ± S.E.M of six rats in group; ap<0.001 diabetic control rats compared to diabetic treated rats.

Groups

Fasting Blood Glucose (mg/dl) Postprandial Glucose (mg/dl) 0th day 7th day % fall 0th day 7th day % fall

Group I (Healthy control)

82±2.1 85±2.6 128±2.2 131±2.4

Group II (Diabetic control)

156 ±2.1 208±2.7 198±2.4 268± 2.8

Group III (Diabetic + FIIIa)

158±2.7 204±2.3 - 201±2.4 260±2.7 -

Group IV (Diabetic + FIIIb)

152±2.7 200±2.1 - 205±3.1 254±2.6 -

Group V (Diabetic + FIIIc)

159±3 128±1.7ab 19 201±2.5 156±2.2ab 22

0.65

0.42

0.30

Solvent front

Figure 6.2: Thin layer chromatogram of active hypoglycemic fraction 3 (purified from gel chromatography)

Figure 6.3: Flow chart diagram of purification processs of central stem of Musa sapientum



6.2.9. Gas chromatography and Mass Spectrometry of TLC Purified Fraction

The active TLC band (3) was applied for 2 to 3 times on TLC (ReTLC) with same solvent system and get the single band at Rf (0.30). This ReTLC purified fraction was subjected to GC-MS (above mentioned system) it gave single peak in Gas chromatography and its % area and mass distribution was found in mass spectrometry. This active purified fraction which was obtain by ReTLC and had single peak this peak was eluted out for structural analysis and to confirm activity in diabetic rats.

Gas Chromatogram analysis of MSH-3 showed a single peak and % area was very high (99.57%) with highest purity at retention time of 27.06 with molecular weight of approximately 414 Da according to Willey and Nist library. 6.2.10. Structural Analysis of Purified Compound MSH-3 by Nuclear Magnetic

Resonance Technique (NMR) To elucidate structure of MSH-3, NMR experiments like proton (1H), carbon (13C), dept 135 NMR were performed using a Bruker Avance 400 MHz FT- NMR spectrometer.All the NMR spectra were recorded by dissolving the active compound, MSH-3, in NMR solvent with a total volume of 0.5 ml in 5mm NMR tube.

Figure 6.4a shows the 1H NMR spectrum. It was acquired with the following experimental parameters: 32768 data points, spectral width of 10.3 kHz with a relaxation delay of 2S of total run time 15 min.

1H NMR (500MHZ, CDCl3) ∂ 5.38 (d, 1H, H11), ∂ 5.16 (1H, H24, dd, J= 8.5 Hz), ∂ 5.17 (1H, H23,dd, J= 8.5 Hz), ∂ 3.55 (1H, {br}, H22), ∂ 2.30-2.31(1H, {M},H25), ∂ 2.04-2.19 (3H,{br}, H21), ∂ 2.23-2.33 (2H, {M},H28), ∂ 1.86-1.88 (2H,{M},H10), ∂ 1.69-1.74 (1H,{M},H19), ∂ 1.42-1.55 (11H,{M},H2+5+6+7+8+9+16), ∂ 1.12-1.18(3H,{br}, H20), ∂ 1.03-1.07 (8H,{M}, H3+4+14+15+17), ∂ 0.814-0.876 (9H, {M},H27+29+30), 0.72 (3H,{br},H18), ∂ 0.97-0.99 (1H, {M},H26).



Figure 6.5 demonstrated the 13C NMR spectrum. It was acquired with the following experimental parameters: spectral width of 29.76 kHz, 765432 data points, pulse width 8.75, relaxation delay 2s, 1024 free- incubation decays were accumulated to obtain a reasonably good signal-to-noise ratio, line broadening of 1.0 Hz was used before Fourier transformation.

13C NMR (500MHZ, CDCl3) ∂ 42.22 (C1), ∂ 58.67(C 2+7), ∂ 31.66 (C3), ∂ 51.25 (C4), ∂ 21.23(C5), ∂ 40.52 (C6), ∂28.94 (C8), ∂ 25.43 (C9), ∂ 37.26 (C10), ∂ 121.73 (C11), ∂ 140.76 (C12), ∂ 36.52 (C13), ∂ 39.68 (C14), ∂ 31.90 (C15), ∂ 71.80 (C16), ∂ 50.15 (C17), ∂ 12.06 (C18), ∂ 42.31 (C19), ∂ 18.99 (C20), ∂ 19.41 (C21), ∂ 138.84 (C23), ∂ 129.24 (C24), ∂ 55.95 (C25), ∂ 31.92 (C26), ∂ 21.08 (C27+30), ∂ 24.38 (C28), ∂ 12.27 (C29) DEPT NMR spectrum of MSH-3 also shown in figure 6.6. 6.2.11. Properties of Purified Active Compound, MSH-3 � Appeareance:- It is white in colour. � Molecular weight:- its molecular weight is approximately 414. � Nature of compound: Sterol derivative � Molecular formula-C29H50O � Postulated structure of MSH-3:-

Figure 6.7

6.3 DISCUSSION Plants have various phytochemicals like sterols, glycosides, phenols, terpenes, saponins etc. which are known to have antidiabetic effect. (Tiwari and Rao, 2002) Aqueous extract of stem of Musa sapientum showed the presence of terpenes, sterols, pectin and phenols. Total phenolic content of stem was 50 mg of gallic acid

Retention time- 27.06 Concentration of compound -99.76% Line#.2 R.Time 27.06 (Scan#:3013) Raw Mode: Averaged 27.00-27.012 (3012-3014) Basal Peak:43.05 BG Mode: Calc. from Peak Group 1-Event 1 Hit#.2 Entry: 145790 Library : NIST05.L1B SI:74 Formula: C29H50O CAS:0-00-0 Mol Weight:414 Ret Index:2703

Figure 6.4: Gas chromatogram and mass spectrometry of TLC purified fraction (MSH-3)

Figure 6.4- 1H NMR spectrum of MSH-3

Figure 6.4b-13C NMR spectrum of MSH-3

Figure 6.4C-Dept -135 NMR of MSH-3



equivalent /100 gm of stem. Lyophilized aqueous extract was further extracted in methanol. The GC-MS studies found that the presence of plant sterols is high in methanolic fraction of central stem of Musa sapientum. Most effective dose of methanolic fraction of central stem of Musa sapientum was found to be 20 mg /kg. It produced significant fall in fasting as well as postprandial blood glucose level. (p<0.05) Methanolic fraction was further purified by column chromatography and gave three fractions out of which one fraction (fraction III) showed hypoglycemic effect. Further purification of fraction 3 was carried out by thin layer chromatography. Thin layer chromatography of this fraction resulted in separation of 3 bands (FIIIa, FIIIb and FIIIc). Band FIIIc with Rf value 0.30 was found active. GC-MS studies revealed a single peak of hypoglycemic compound at retention time 27.06 possess 99.76% purity of the compound and structural analysis of this compound by nuclear magnetic resonance (NMR) revealed that it is a sterol derivative with molecular weight of 414 approximately. This compound is amphipathic in nature. It is a white powder. It was active at a very small dose of 2 mg/kg. Sterols are found in plant families like Brassica, Musaceae, Solanaceae, Liliaceae and Scrophylariaceae (Gracia et al, 2011). Musa sapientum var. paradisiaca used in this study is belonging to family Musaceae. The compound MSH-3 isolated from central stem of Musa sapientum, MSH-3 is a sterol derivative as revealed by its structure elucidated by NMR spectral analysis. A survey of literature revealed that sterol or their derivative have been isolated from various plants as well as from fruit of Musa sapientum (Akihisa et al, 1986). Sterol class of compounds are known for their antihyperglycemic potential (panda et al, 2009). Sterols also exhibit Hypolipidemic activity. (Ahirwar et al, 2003).

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