Isolation and Characterization of Borassus flabellifer Mucilage

 

Ravi Kumar1*, Rajarajeshwari N.2

1Research Scholar, Shri Jagdish Prasad Jhabarmal Tibrewala University, Rajasthan

2Visveswarapura Institute of Pharmaceutical Sciences, Bangalore.

*Corresponding Author E-mail: ravikumar300@gmail.com

 

ABSTRACT:

The prospects of natural polymers are brighter as synthetic polymers have certain disadvantages such as high cost, toxicity, environmental pollution during synthesis, non renewable sources, side effects and less patient compliance. The endosperm of Borassus flabellifer contains a high proportion of mucilage and it also being used for different therapeutic purposes. However there are no reports on isolation and characterization of mucilage of Borassus flabellifer fruit. Hence, the present study was planned to isolate, purify and to characterize Borassus flabellifer mucilage. This study elucidated the physical, thermal, sorption and functional properties Borassus flabellifer mucilage were characterized viz: elemental analysis, Fourier transmittance infra red analysis, particle size analysis, thermo gravimetric analysis, differential scanning calorimetry, scanning electron microscopy and X-ray powder diffraction. Borassus flabellifer mucilage had a glass transition temperature (Tg) and melting peak of 96.06°C and 292.63°C, respectively. This material showed a 7.1% % loss in weight at 200oC. The sample had peaks at approximately 20o, 19o, and 18o 2θ degrees of 2-theta (θ) in the X-ray powder diffraction pattern. Elemental analysis showed that Borassus flabellifer mucilage contains 40.02, 6.289, and 1.22% of carbon, hydrogen and nitrogen, respectively. The results of isolated mucilage from Borassus flabellifer fruit were very promising, cheap and effective natural excipient that can be used as an effective alternative for the formulation of pharmaceutical formulations.

 

KEY WORDS: Borassus flabellifer, mucilage, natural excipient, swelling index, moisture content.

 


INTRODUCTION:

Plant products serve as an alternative to synthetic products because of local accessibility, eco-friendly nature and lower prices as compared to imported synthetic products. Natural gums and mucilage have been widely explored as pharmaceutical excipients1-4 and have been known since ancient times for their medical uses. In India, natural gums and mucilage are well known for their medicinal uses. They are widely used in the pharmaceutical industry as thickeners, water-retention agents, emulsion stabilizers, gelling agents, suspending agents, binders, film formers, and sustained-release agents5-6. They also are used in the manufacturing of cosmetics, textiles, paints, and paper7. As their demand continues to increase, new sources are constantly being explored. However, large quantities are still imported from Europe to meet the increasing demand8.

Mucilages are polysaccharide complexes formed from sugar and uronic acid units. Mucilages form slimy masses in water, are typically heterogeneous in composition.

 

Upon hydrolysis, arabinose, galactose, glucose, mannose, xylose and various uronic acids are the most frequently observed components. Mucilages are obtained mainly from seeds or other plant parts. Some are obtained from marine algae, and from selected microorganisms9.

 

The Borassus flabellifer is a tall and erect palm, with large, fan-shaped leaves which are quite unlike the pinnate leaves of other palms. Borassus is from a Greek word describing the leathery covering of the fruit and flabellifer means “fan-bearer”. Synonyms of the plant include jaggery palm, Palmyra palm, toddy palm, wine palm. This species is globally distributed from Africa to Australia. Within India, it is found throughout tropical regions, especially along the peninsular coast and in West Bengal and Bihar. It is often cultivated. The Palmyra palm has long been one of the most important trees of Cambodia and India. The different parts of the plant is used for the various ailments like secondary syphilis, antiperiodic, heart burns, liver and spleen enlargement etc. Other than these pharmacological uses the juice of the plant is used in preparation of health drinks, jellies etc. The leaves are use to make baskets, hats and many other useful items. Borassus flabellifer contains albuminoids, fats and the fresh pulp is reportedly rich in vitamins A and C. The fresh sap is reportedly a good source of vitamin B-complex. Male inflorescence constitutes spirostane-type steroid saponins like borassosides and dioscin. It also contains 20 known steroidal glycosides and carbohydrates like sucrose. It also contains bitter compound called flabelliferrins, these are steroidal saponins10-14. The fruits are tender and the seeds contain a soft, sweet, jelly-like endosperm with sap. The endosperm contains a high proportion of mucilage. The two major polysaccharides present in this endosperm are galactomannan and mannan.

The prospects of natural polymers are brighter but even here extensive testing will be required. In present study the fruits of Borassus flabellifer were selected for the isolation and purification of mucilage. However there are no reports on isolation, purification and characterization of Borassus flabellifer fruits mucilage. Hence, the present study was planned to isolate and characterize mucilage of Borassus flabellifer fruits. The data so obtained will be a standardizing parameter for future research work.

 

MATERIALS AND METHODS:

Collection and Authentication of plant material

Borassus flabellifer endosperm was purchased from local market of Udupi District (figure 1), Karnataka, India. It was authenticated by Botanist, Science centre, Pilikula Nisargadhama, Vamanjoor, Mangalore; a specimen sample is kept in the laboratory for future use.

 

Figure 1: Crude Borassus flabellifer fruit endosperm containing mucilage

 

Materials

Acetone, ethanol, methanol, hydrochloric acid, sodium hydroxide and diethyl ether were procured from SD Fine chemicals (Mumbai, India). All other chemicals, reagents and solvents used were of AR grade and double distilled water was used throughout the experiments.

 

Isolation and purification of mucilage from Borassus flabellifer endosperm15

The endosperm of Borassus flabellifer fruit contains mucilage. To increase the yield of the mucilage the endosperm of Borassus flabellifer fruit were extracted by different solvents. The endosperm of Borassus flabellifer were collected, cut into small pieces and dried using tray dryer at 37°C for 24 h at room temperature, made fine powder by crushing in a mixer. The fine powder was soaked in different solvents such as water, hot-water, phosphate buffer solution (PBS) of pH 4.0, 6.8, 9.2, separately for 2-3h and heated up to 80-90°C for 30-45 min for complete release of the water soluble mucilage into the solvents. The mucilage was then extracted by using a multi layer muslin/cheese cloth bag to remove the marc and concentrated viscous solution under reduced pressure at 60-70°C. Acidified ethanol (5% HCl in 75% ethanol) was added to the concentrated viscous solution with constant stirring. The gel like precipitate was formed and separated by filtration. The precipitate was washed 2-3 times with 75% and 95% ethanol. After complete washing of the precipitate with ethanol 95%, brownish white powder was obtained. The powder was dried in an oven at 37°C, collected, grounded, passed through a # 80 sieve and stored in a desiccator till use. The brownish white powder was considered as mucilage for pharmaceutical use (figure 2).

 

Figure 2: Purified Borassus flabellifer mucilage

 

Characterization of Borassus flabellifer mucilage (BFM)

Preliminary phytochemical screening of isolated BFM16

The extracted mucilage was tested for chemical characteristics for identification, test for carbohydrate, test for tannins, test for chloride, test for sulphate, test for uronic acid, test for flavanoids, test for steroids, test for saponins, test for tannins, test for phenols and test for alkaloids. The mucilage was also tested for unwanted chemicals viz., foreign matter, heavy metal and arsenic.

 

Physicochemical characterization of BFM17

The isolated mucilage was evaluated for physical characteristics viz., appearance, odour, solubility, percentage yield, average particle size, melting point, swelling ratio, viscosity, microbial load, surface tension, weight loss on drying, pH, density, total ash, acid insoluble ash, water soluble ash, thermal stability, moisture content, and bio burden. All these values were tested in triplicate.

 

Micromeritic properties of BFM18

The BFM was tested for the flow properties viz., angle of repose, bulk densities, compressibility index and Hausner’s ratio. All these evaluations were carried out as per procedures described in official books. All these experiments were conducted for five times.

Evaluation of Toxicity

Toxicity studies were carried out according to the method of Knudsen and Curtis19 and OECD guidelines no.423. The animals used in the toxicity studies were sanctioned by the Institute Animal Ethical Committee (Approval No:KCP/IAEC/Ph.Ceutics/05/2011-2012). The male albino rats of Wistar strain weighing 160-200g were divided into different groups comprising of six animals each. The control group received normal 0.5%CMC solution (20ml/kg i.p). The other groups received 500, 1000, 2000, 3000, 4000 and 5000 mg/kg of mucilage suspension in normal saline orally. The animals were observed continuously for the behavioral changes for the first 4 hours and then observed for mortality if any for 72 hours. Since no mortality, no toxic manifestations were observed and behavioural pattern was unaffected. In chronic toxicity studies, 22 animals were used, divided in to two groups, 6 as control and 16 as test animals. In the test group a dose of 500 mg/kg was administered daily for a period of 30 d. body weights were recorded for both the groups at an interval of 10d. And at the end of 30 days, hematological and biochemical parameters were studied in both the groups and after 30 days of chronic toxicity study the animals were scarified and subjected to histopathological studies.

 

Thermogravimetric Analysis

Thermo gravimetric analysis (TGA) was performed in a Shimadzu TGA apparatus Japan. Sample (1.78 mg) was heated at a rate of 10°C/min from ambient temperature to 200°C. Nitrogen was used as the purge gas at a flow rate of 20 mL/min.

 

Differential scanning calorimetry study

Thermal properties of BFM were characterized using a differential scanning calorimetry (DSC- 60, Shimadzu limited, Japan). Nitrogen, at the rate of 20 mL/min, was used as purge gas; 2.7 mg of powdered material were sealed in aluminium pan and heated from 30°C up to 500°C at the rate of 10°C/min, followed by a cooling cycle back to 30°C at the same rate.

 

Fourier transform-infra red spectral study

The Fourier transform-infra red (FT-IR) spectrum of the sample was recorded in an IR spectrometer (FTIR 8300, Shimadzu, Japan), using potassium bromide (KBr) discs prepared from powdered samples mixed with dry KBr in the ratio 1:200. Triplicate measurements were made, and the spectrum with the clearest identifiable peaks was chosen.

 

X-ray powder diffraction study

X-ray diffraction (XRPD) patterns of the BFM were analyzed using a Siemens D5000 Xray diffractometer (Siemens, Munich, Germany). Powder sample, packed in rectangular aluminium cells, illuminated using CuKα radiation (λ = 1.54056 Å) at 45 kV and 40 mA. Samples were scanned between diffraction angles of 5° to 45°C 2θ. Scan steps of 0.1 were used and the dwell time was 15.0 sec. A nickel filter was used to reduce the Kβ contribution to the X-ray signal. The ‘d’ spacing was computed according to Bragg’s law of diffraction. Triplicate measurements were made at ambient temperature.

 

Microstructure studies

The morphological features of the BFM were studied with a JSM-5600 LV scanning electron microscope (SEM, JEOL, Tokyo, Japan). The dried sample was mounted on a metal stub and sputtered with gold in order to make the sample conductive, and the images were taken at an accelerating voltage of 20kV with magnifications of ×170, ×200 and ×1000. The samples providing most meaningful information for purposes of our analysis were obtained at × 170 magnifications.

 

Elemental analysis

Elemental analysis of carbon, hydrogen and nitrogen was carried using a Thermo Finnigan FLASH EA 1112 CHNS-2000 analyser.

 

RESULT AND DISCUSSION:

Plant products serve as an alternative to synthetic products because of local accessibility, environment friendly nature and lower prices compared to imported synthetic products. Herbs are non-polluting renewable resources for sustainable supplies of cheaper pharmaceutical products. Today, we have a number of plant-based pharmaceutical excipients. A number of researchers have explored the utility of plant-based materials as pharmaceutical excipients.

 

The mucilage was extracted using solvents such as   distilled/demineralised water, hot water, PBS pH 4.0, pH 6.8 and pH 9.2 and the yield of the dry water soluble mucilage was varied depends upon the solvents used. Percent yield of the dry water soluble mucilage was 45%, 60%, 22%, 30% and 35% in distilled/demineralised water, hot water, PBS pH 4.0, PBS pH 6.8, and PBS pH 9.2 respectively. The solvents like distilled/demineralised water, hot water and phosphate buffer pH 9.2 could be used for extraction for better yield.

 

Physiochemical characterization of BFM

The extracted mucilage was characterized by various organoleptic, morphological and physical evaluatory studies such as colour, odour, taste, shape, nature, touch and texture. The results of organoleptic characteristics of the mucilage indicated that isolated mucilage was slight brownish white color, acceptable and characteristic odour, mucilaginous taste and amorphous in nature.

 

The solubility behaviour of the mucilage indicated that it is quickly soluble and forms neutral, viscous colloidal solution in warm water, sparingly soluble in cold water, whereas insoluble in ethanol, methanol, acetone, chloroform and ether.

 

The moisture content of BFM was found to be 2.5% was found to be within official limit suggesting its suitability in formulations containing moisture sensitive drugs.

 


 

Figure 1:  Thermogravimetric curve of BFM

 

Figure 2: Differential scanning calorimetry curve of BFM

 


Under given suitable temperature moisture will lead to the activation of enzymes and the proliferation of micro organisms, thereby affecting the shelf life of most routine formulations. It is important to investigate the moisture content of a material because the economic importance of an excipient for industrial application lies not only on the cheap and ready availability of the biomaterial but the optimization of production processes such as drying, packaging and storage. The ash values such as total ash, acid insoluble ash and water–soluble ash of BFM was found to be 2.75± 0.20, 0.2± 0.01 and 1.21±0.02 respectively. Ash values reflect the level of adulteration or handling of the drug. Adulteration by sand or earth is immediately detected as the total ash is normally composed of inorganic mixtures of carbonates, phosphates, silicates and silica. Therefore, the low values of total ash and acid insoluble ash obtained in this study indicate low levels of negligible amount of sandy material, contamination, during gathering, processing and handling of crude mucilage. The swelling characteristic of BFM was studied in different media; 0.1N hydrochloric acid, phosphate buffer (pH 7.4) and water. The swelling was highest in water followed by phosphate buffer and least in 0.1N HCl. Generally, the results show that BFM has high swelling index suggesting that the mucilage may perform well as binder/disintegrant/ matrixing agent. The mucilage is a pH responsive polymer, it is therefore a “smart polymer,” and may find application in controlled release dosage formulations. The relatively higher swelling index obtained for BFM at pH 7.4 implies that unlike tragacanth, the mucilage may be useful as a matrix former in controlled drug release. Swelling is a primary mechanism in diffusion controlled release dosage   form. The swelling ability of any mucilage depends upon its water retention capacity or water absorption capacity. The water absorption capacity of BFM was found to be 13 ml. A 1% w/v suspension of BFM in water gave a pH of 6.5. The near neutral pH of BFM implies that when used in uncoated tablets, it may be less irritating to the gastrointestinal tract. It may also find useful application in formulation of acidic, basic and neutral drugs.

 

Table 1: Results of Physicochemical characterization of BFM

Parameters

Result

pH (1%w/v)

6.5

Moisture content (%)

2.5

Ash value (%)

2.75± 0.20

Water-soluble ash (%)

1.21± 0.02

Acid insoluble ash (%)

0.2± 0.01

Swelling index

 

In distil water

In 0.1 N HCl

In Phosphate Buffer pH 7.4

-

 

25

07

18

Total bacterial count :Microbial Load

Bacteria:(CFU/g)

Fungi: (CFU/g)

 

10

01

E.coli

Absent

Salmonella typhi

Absent

S.aureus

Absent

Pseudomonas  aeruginosa

Absent

Yield (% w/w)

60

Water absorption capacity

13 ml

Surface tension (0.1%w/v)

85.52 ± 0.21

Average particle size (μm)

165.18±8.54

Test for foreign matter (%)

NMT 0.1

Test for Arsenic

<1 ppm

Test for heavy metal (lead)

15 ppm

Melting point (°C)

290±6.851

Viscosity (0.5 % dispersion of BFM)

325 cps

 

Knowledge of the pH of an excipient is an important parameter in determining its suitability in formulations since the stability and physiological activity of most preparations depends on pH. The surface tension of BFM was found to be 85.52 dynes/cm. Melting point of mucilage was found to be 290±6.851oC, which is a characteristic of most of the mucilage. The mucilage has negligible bio burden. The average particle size of dried mucilage was 165.18±8.54μm (assessed by microscopy). The average particle size of dried mucilage was found to be uniform. The extracted and purified BFM were evaluated for microbial load, BFM showed 10 CFU per gram of BFM for bacteria and 1CFU per gram of BFM for fungi which shows mucilage were under microbial limit, it also proved that BFM does not support microbial growth and is free from all the pathogen organisms. The foreign matter in this mucilage was found to be not more than 0.1 and the heavy metal as lead were found to be 15 ppm Arsenic was found to be Less than 1 ppm all the values were found to be within the limits. The viscosity of the extracted dried mucilage was 325 cps for 0.5 % solution. It can be concluded that mucilage has a viscosity of such type that is suitable for fast dissolving drug delivery. The results of physicochemical characterisation of BFM are summarized in table 1.

 

Thermo gravimetric analysis

The thermogravimetric analysis (TGA) was used to determine the weight loss of the material on heating. Transitions involving mass changes are detected by TGA as a function of temperature and time. The TG curve for the BFM showed a one stage weight loss corresponding to loss of water around 50-400°C.The curve shows that the BFM did not decompose before 400°C. It has been reported that water is formed by intra and intermolecular condensation of polymeric hydroxyl groups which are the main product of decomposition at temperatures below 300°C. The BFM underwent 7.1% weight loss at 200°C. This is low compared to weight loss reported for other polymers such as starch. It implies that BFM has excellent thermal stability. The TG curve for BFM is shown in figure 1.

 

Differential scanning calorimetry

Differential scanning calorimetry (DSC) was used to measure the occurrence of exothermal or endothermal changes with increase in temperature. DSC, because of its sensitivity and accuracy, has been extensively used to study the phase transitions of polymers. The thermogram for BFM showed that the mucilage has both amorphous and crystalline portions. Glass transition (Tg) temperature occurred at 96.06°C while a melting peak was observed at about 292.63°C.

 

Table 02: X-ray powder diffraction data for 3 strongest peaks of BFM

Peak No

Angle 2- ()

d value (Ǻ)

Intensity (%)

12

20.2480

4.38220

100

11

19.5000

4.54858

88

10

18.8540

4.70295

79

 

Table 03: Element composition data for BFM

Element

Composition (%)

Carbon

40.02

Hydrogen

6.289

Nitrogen

1.22

 


 

Figure 3: FTIR spectrum of BFM

 

Figure 4: XRD pattern of BFM

 


Table 04: Results of phytochemical screening of BFM

 

Tests

Observation

1.  

Test for Carbohydrates( Molisch’s test)

+

2.  

Test for Tannins and phenolic compounds (Ferric chloride test)

-

3.  

Test for proteins ( Ninhydrin test)

-

4.  

Test for alkaloids (Mayer’s test)

-

5.  

Test for glycosides(Keller– Killaini test)

-

6.  

Test for mucilage ( Ruthenium red test)

+

7.  

Test for flavonoids (Shinoda test)

-

8.  

Test for reducing sugar (Felhing’s test)

+

9.  

Mounted in 95% alcohol

Transparent angular masses under microscope

10.

Mounting in the iodine

Particles not stained blue

11.

Test for chlorides( silver nitrate test)

-

12.

Test for sulphates (barium chloride test)

-

13.

Test for Uronic acid

+

14.

Tests for saponins  (Foam test)

-

15.

Tests for steroids  (Salkowski test)

-

16.

Tests for triterpenoids (Salkowski test)

-

     *+ Present; - Absent

Two exothermic peaks and one endothermic peak are exhibited by the sample corresponding to its glass transition, recrystallisation and melting respectively. The onset, peak and conclusion temperatures of phase transition were observed to be high. The continuous (broad) endothermic transition that followed the glass transition is indicative of crystallite melting occurring over the glass transition range. The glass transition temperature (Tg) was also observed to be high, indicating a high degree of crystallinity of the mucilage.

 

This has been shown to provide structural stability and made granules more resistant to heat. It has also been reported that materials of low Tg have low crystallinity. The knowledge of Tg is essential in production processes and storage as Tg is affected by moisture and other additives, facilitating conversion to the rubbery state and hence facilitating crystallization through molecular rearrangement. The mucilage was also observed to have low enthalpy; this is attributed to the presence of regular small and oval granules. The DSC thermogram for BFM is shown in figure 2.

 

 

Fourier transform-infra red spectral analysis

The IR spectrum of BFM is shown in Figure 3. The finger print region of the spectrum consists of two characteristic peaks between 871 and 1406 per cm, attributed to the C-O bond stretching. The band at 1652 per cm was assigned to the O-H bending of water. There are absorptions (weak) in the 1730 per cm area that indicate carbonyls. The absence of significant aromatic stretches in the 1660-1690 per cm region and the weakness of the stretches, imply that there is a modest amount of peptidic cross linking by amide bond formation.  The sharp band at 2931 per cm is characteristic of methyl C-H stretching associated with aromatic rings. The broad band at 3396 per cm is due to the hydrogen-bonding that contributes to the complex vibrational stretches associated with free inter and intra-molecular bound hydroxyl groups which make up the gross structure of carbohydrates. This is all consistent with a polysaccharide structure that is neither a starch nor cellulose, but does have some peptide cross links and some amino-sugars. The essentially neutral pH of this material leads us to conclude that there can be very few free carboxyl groups to contribute to hydrogen bonding.

 

X-ray powder diffraction analysis

The X-ray diffractogram of BFM is shown in figure 04. The Bragg reflection angle, 2θ, along with the interplanar spacing, d, and the relative intensity of the peaks were calculated and results are tabulated in table 2. The interplanar spacing has been calculated using Bragg’s equation given as; nλ = 2d sinθ, where θ is one half the angle read from the diffractogram. The sample shows peaks at approximately 20°, 19°, and 18°, 2θ. However, other peaks are very weak and unresolved. The result of XPRD corroborates that of the DSC which shows that, BFM is of low moisture and exhibit both amorphous and crystalline portions.

 

Surface Characteristics of BFM by Scanning Electron Microscopy (SEM)

The biological and botanical source of a pharmaceutical material serves as a determining factor in the granule shape, size and morphology. As a result, these characteristics not only help to differentiate between various materials but also give an indication of the processing parameters. The SEM of BFM is shown in figure 5. It exhibits fairly irregular, fragmented tiny granules and slightly elongated with rugged appearance.

 

 

Figure 5: SEM Photograph of BFM

 

Elemental analysis

The quantitative elemental analysis of BFM is shown in table 03. The results showed that BFM contains 40.02, 6.28 and 1.22 % of carbon, hydrogen and nitrogen respectively. The low level of nitrogen is suggestive of amino acid (peptide) crosslink in the sample. The ratio of carbon to hydrogen is just over 7:1 indicating, along with the ratio of carbon to oxygen, a good number of unsaturation due to aromatic rings and/or polysaccharide composition.

 

 

Phytochemical screening of BFM

The purity of mucilage was determined by prescribed phytochemical tests, which indicated the absence of alkaloids, steroids, flavonoids, saponins, glycosides, tannins, chloride, sulphate and phenols. On treatment of mucilage with ruthenium red, it showed red colour confirming the obtained product was mucilage. A violet ring was formed at the junction of two liquids on reaction with Molisch’s reagent indicating the presence of carbohydrates. The mucilage gave positive test for uronic acid which is general constituent of mucilage. Mucilage could reduce Fehling’s solution, so the sugars present were reducing sugars. Only carbohydrates were found to be present, which confirms the purity of mucilage. The results of phytochemical screening of mucilage are summarized in table 4

 

 

 

Table 05: Micromeritic properties of BFM

Parameters

Result

Angle of repose(0)

25.8±1.240

Loose Bulk density (g/cm3)

0.53±0.05

Tapped bulk density(g/cm3)

0.67±0.05

Carr’s Index (%)

20.9±0.04

Hausner’s ratio

1.27±0.03

True density (g/cc)

1.058±0.018

 

 

 

 

Table 6: Hematological values of male rats receiving BFM for 3 months

SL. No.

Parameters

Control*

Test**

1.      

Hematocrit (%)

45.21±1.34

49.21±3.41

2.      

RBC (×106 cells/mm3)

4.86±0.21

4.92±0.37

3.      

Hemoglobin (g/dl)

15.87±0.26

16.26±0.98

4.      

MCV (μm3/red cell)

55.25±1.21

56.54±1.21

5.      

MCH (pg/red cell)

18.21±0.21

18.21±0.48

6.      

MCHC (g/dl RBC)

31.47±0.27

31.67±0.24

7.      

WBC (×103 cells/mm3)

2.18±0.26

2.17±0.25

8.      

Platelet (×103 cells/mm3)

942±36

959±29

9.      

Neutrophil

15.0±3.81

17.0±4.58

10.   

Eosinophil (%)

2.0±0.46

1.0 ±0.56

11.   

Lymphocyte (%)

67.90±6.21

69.0 ±4.54

12.   

Monocyte (%)

9.0±5.35

11.0±3.72

13.   

Basophil (%)

3.0±1.27

4.00±1.53

*Data represents as the mean ±SD of 6 animals; **Data represents as the mean ±SD of 16 animals

 

 

 

 


Table 7: Biochemical values of male rats receiving BFM   for 3 months.

SL.No.

Parameters

Control*

Test**

1.      

SGOT (IU/ml)

55.6 ± 1.0

53.1 ± 2.1

2.      

SGPT(IU/ml)

12.4 ± 0.7

11.0 ± 0.2

3.      

LGOT(IU/100 mg of liv. tissue)

108.12 ± 4.21

104.24 ± 5.30

4.      

LGPT(IU/100 mg of liv. tissue)

129.24 ± 0.91

126.28 ± 3.1

5.      

GSH(μ mole/g wet tissue)

8.97 ± 0.28

8.79 ± 0.21

6.      

Mean Body Weight(mg)

29.5 ± 1.02

28.9 ± 1.27

*Data represents as the mean ±SD of 6 animals; **Data represents as the mean ±SD of 16    animals

 


Micromeritic properties of BFM

The angle of repose of the dried BFM powder was 25.8±1.240° indicates an excellent flow property (25-30°). The bulk and tapped densities give an insight on the packing arrangement of the particles and the compaction profile of a material. The loose bulk density and tapped bulk density values were 0.53±0.05 and 0.67±0.05 g/cm3 respectively. These Bulk density values were considered for calculating compressibility index and Hausner's ratio. The compressibility index of the dried mucilage was 20.9±0.04% indicates fair flow properties (18-21%) and Hausner's ratio was found to be 1.27±0.03. The true density of mucilage was found to be 1.058±0.018. This is important in scale up processes involving this material as an excipient in a pharmaceutical formulation. Modification of formulations containing this mucilage for the improvement of flow properties during process development will therefore be minimal (e.g., inclusion of glidants or agents to aid in feeding). The results of Flow properties of BFM are summarized in Table 05.

 

Toxicity Study

To determine the safety level of the extracted mucilage, acute and chronic toxicity studies were conducted according to OECD guidelines no.423. In both toxicity studies the extracted mucilage revealed no behavioral changes, no changes in body weight for first four hours and no mortality; no toxic syndromes were observed even at the dose level 5g/kg body weight after 24 hours, indicating the safety of the mucilage. To assess the suitability of mucilage for the oral delivery we have recorded the body weight profile for the animals during the chronic toxicities at regular intervals of 10 d. it was found that the body weight of both test and control and rate of increase were also comparable. Hence it is concluded that chronic administration of the mucilage might not influence either the food intake or growth. Hematological and biochemical parameters that were determined at the end of 30 d of continuous administration were also found to be comparable to that of control rat. The effect of mucilage on hematological and biochemical parameters is summarized in table 6 and 7 respectively. Histological examination of the main organs like liver, kidney, heart and brain were carried out at the end of 30days of chronic toxicity study. From this study it was revealed that there was no sign of pathological changes in both control and in treatment group. The results are shown in figure 6.

 

 


 

Control                                                                                                           Test

 

Brain                                                                                                     Brain

  

Heart                                                                                                              Heart

   

Kidney                                                                                                             Kidney

       

Liver                                                                                                                   Liver

Figure 6: Histological sections of vital organs after treatment of BFM for 30 days

 

 

 


CONCLUSION:

From the present data, it can be concluded that the mucilage isolated from Borassus flabellifer has good physicochemical characteristics with good flow properties. The work can be further extended for evaluation of its suitability as a suspending agent, gelling agent, binding agent, disintegrating property, emulsifying agent and other similar pharmaceutical applications considering the easy and ample availability of the plant. In conclusion, BFM opens a new avenue for future research as functional polysaccharide for pharmaceutical applications. As natural substances are cheap, biocompatible, biodegradable and easy to manufacture, they can be used as pharmaceutical excipient in place of currently marketed synthetic pharmaceutical excipients.

 

ACKNOWLEDGEMENTS:

The authors  are  thankful  to  ICMR  for  the  financial  support  for  this  research  project  (21/12/17/09/HSR, dated: 24/06/2010). The authors are also thankful to Mart Laboratory Pvt. Ltd. Hyderabad, India for carrying out CHNS, DSC, TGA, IR, SEM & XRPD analysis of the sample.

 

REFERENCE:

1.       Monif T, Malhotra AK, Kapoor VP. Cassia fistula seed galactomannan: potential binding agent for pharmaceutical formulation. Indian Journal of Pharmaceutical Sciences. 54(6); 1992: 234-40.

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Received on 13.07.2012       Modified on 23.07.2012

Accepted on 08.08.2012      © RJPT All right reserved

Research J. Pharm. and Tech. 5(8): August 2012; Page 1093-1101