chapter 5 result and discussion 5 result and discussion...

Chapter 5 RESULT AND DISCUSSION

Bhavik Ph. D Dissertation 123

5 RESULT AND DISCUSSION

5.1 Identification and Authentification of Plants

Bhunimbadi churna consists of ingredients viz., Swertia chirata (whole plant),

Holarrhena antidysenterica (seed), Zingiber officinale (rhizome), Piper nigrum

(fruit), Piper longum (fruit), Cyperus rotundus (rhizome), Picrorrhiza kurroa

(rhizome), Plumbago zeylanica (root), and Holarrhena antidysenterica (stem bark),

These plants were identified by organoleptic and morphologic charcters and were

authenticated by Dr. M. S. Jangid, Professor, Department of Botany, Sir P. T. Science

College, Modasa. Photographes of plants used in churna are given in Fig 5.1and

herbarium sheet of plants are given in Fig 5.2.

1) Chirayata 2) Indraju 3) Sunth

(Swertia chirata) (Holarrhena antidysenterica) (Zingiber officinale)

4) Mari 5) Pippali 6) Nagarmoth

(Piper nigrum) (Piper longum) (Cyperus rotundus)

7) Katuki 8) Chitrak 9) Kada chhal

(Picrorrhiza kurroa) (Plumbago zeylanic) (Holarrhena antidysenterica)

Fig 5.1: Photograph of plants used in Bhunimbadi churna



Fig 5.2(A): Herbarium sheet of Swertia chirata (Whole plant)

Fig 5.2(B): Herbarium sheet of Holarrhena antidysenterica (Seed)



Fig 5.2(C): Herbarium sheet of Zingiber officinale (Rhizome)

Fig 5.2(D): Herbarium sheet of Piper nigrum (Fruit)



Fig 5.2(E): Herbarium sheet of Piper longum (Fruit)

Fig 5.2(F): Herbarium sheet of Cyperus rotundus (Rhizome)



Fig 5.2(G): Herbarium sheet of Picrorrhiza kurroa (Rhizome)

Fig 5.2(H): Herbarium sheet of Plumbago zeylanic (Root)



Fig 5.2(I): Herbarium sheet of Holarrhena antidysenterica (Stem bark)

5.2 Evaluation of Quality Control Parameters for Raw Material

5.2.1 Organoleptic parameters

Organoleptic characters like colour, odour and taste of all ingredients used in powder

form in Bhunimbadi churna were carried out. Organoleptic characters like colour,

odour and taste of all ingredients used in powder form in Bhunimbadi churna is

shown in Table 5.1

Table 5.1: Organoleptic characters of Bhunimbadi churna ingredients

Sr

no.

Name of the

ingredients Part used

Description

Colour Odour Taste

1 Chiryata Whole plant Yellowish green Pleasant Extremely

Bitter

2 Indraju Seed Brownish yellow Characteristic Bitter

3 Sunth Rhizome Yellowish white Aromatic Pungent

4 Mari Fruit Greyish black Aromatic Pungent

5 Pippali Fruit Deep green Aromatic Pungent

6 Nagarmoth Rhizome Brown Pleasant Bitter

7 Katuki Rhizome Dark brown Pleasant Bitter

8 Chitrak Root Greyish yellow Characteristic Bitter

9 Kada chhal Stem Bark Light yellow Characteristic Bitter



Photograph of powders of all ingredients of churna is shown in Fig 5.3.

1) Chirayata 2) Indraju 3) Sunth

(Swertia chirata) (Holarrhena antidysenterica) (Zingiber officinale)

4) Mari 5) Pippali 6) Nagarmoth

(Piper nigrum) (Piper longum ) (Cyperus rotundus)

7) Katuki 8) Chitrak 9) Kada chhal

(Picrorrhiza kurroa) (Plumbago zeylanic) (Holarrhena antidysenterica)

Fig 5.3: Organoleptic characteristics of ingredients used in Bhunimbadi churna

The powder of chiryata was yellowish green, indraju was brownish yellow, sunth was

yellowish white, mari was greyish black, pippali was deep green, nagermoth was

brown, katuki was dark brown, chitrak was grayish yellow and kada chhal was light

yellow. Chiryata, nagarmoth and katuki was pleasant; sunth, mari and pippali was

aromatic and indraju, chitrak and kada chhal was with characteristic odour. Indraju,

nagarmoth, katuki, chitrak and kada chhal was bitter; sunth, mari and pippali powder

was pungent taste and chiryata was extremely bitter.



5.2.2 Powder microscopic characteristics

Ayurvedic pharmacopoeia committee constituted by the department of AYUSH has

introduced certain quality parameters for the plants that are used in Indian system of

medicine. The determination of the correct identity of the genuine source of the plant

material required to be used in various preparations is one of the most important

criteria for the assurance of quality and reliability. Microscopic evaluation of

powdered drugs is the easiest and cheapest method for detecting the correct

authenticity of them. Powder microscopy of all ingredient of Bhunimbadi churna was

carried out.

5.2.2.1 Swertia chirata (whole plant)

Powder microscopy of S. chirata whole plant was carried out and was observed under

normal and polarized light for calcium oxalate crystals and the photograph of the

same given in the fig 5.4

Parenchymataous cells Fibers

Vessels Calcium oxalate crystal

Fig 5.4: Powder characteristics of S. chirata (Whole plant)

Microscopic study of S. chirata whole plant powder showed flattened and tangentially

elongated parenchymatous cells; simple pits fibers; and simple and bordered pitted

vessels. Abundance, minute acicular calcium oxalate crystals were found in secondary

cortex and phloem region.



5.2.2.2 Holarrhena antidysenterica (Seed)

Powder microscopy of H. antidysenterica seed was carried out and observed under

normal and polarized light for calcium oxalate crystals. The photograph of the same

given in the fig 5.5

Endosperm Oil globules

Crystals of calcium oxalate (prismatic & rosette)

Fig 5.5: Powder characteristics of H. antidysenterica (Seed)

Microscopy of H. antidysenterica seed showed light yellowish-brown fragments of

endosperm, oil globules, prismatic and rosette crystals of calcium oxalate.

5.2.2.3 Zingiber officinale (Rhizome)

Microscopy Z. officinale rhizome powder was carried out and was observed under

normal light and the photograph of the same given in the fig 5.6

Cortex cells Vessels

Starch grains Fibres

Fig 5.6: Powder characteristics of Z. officinale (Rhizome)



Microscopic study of rhizome of Z. officinale shown parenchymatous thin-walled

cortex cells, reticulate or spiral vessels, septate fibers and flattened, rectangular and

ovate starch grains.

5.2.2.4 Piper nigrum (Fruit)

Microscopy P. nigrum fruit powder was carried out and was observed under normal

light and the photograph of the same is given in the fig 5.7

Epicarp Stone cells

Perisperum

Fig 5.7: Powder characteristics of P. nigrum (Fruit)

Microscopical study of P. nigrum fruit powder showed, single layered epicarp;

slightly sinuous, tabular cells forming epidermis; endocarp composed of slightly

elongated and beaker-shaped stone cells; perisperm with parenchymatous cells having

a few oil globules and packed, abundant, oval to round, simple and compound starch

grains having few aleurone grains and oil globules.

5.2.2.5 Piper longum (Fruit)

Microscopy P. longum fruit powder was carried out and was observed under normal

light and the photograph of the same is given in the fig 5.8

Microscopic study of P. longum fruit powder showed deep moss green fragments of

parenchyma, oval to elongated stone cells, vessels, oil globules and round to oval,

starch grains.



Fragments of parenchym Stone cells

Vessels Oil globules and starch grains

Fig 5.8: Powder characteristics of P. longum (Fruit)

5.2.2.6 Cyperus rotundus (Rhizome)

Microscopy C. rotundus rhizome powder was carried out and was observed under

normal light and the photograph of the same is given in the fig 5.9

Epidermis cells Vessels

Fibers

Fig 5.9: Powder characteristics of C. rotundus (Rhizome)

Microscopic study of C. rotundus rhizome powder showed epidermis with circular to

oval, thin-walled, parenchymatous cells with small intercellular spaces, narrow,

simple, reticulate and scalariform thickening and oblique pore vessels and fiber like

closely packed sclerified cells.



5.2.2.7 Picrorrhiza kurroa (Rhizome)

Microscopy of P. kurroa powder was carried out and observed under normal light

and the photograph of the same is given in the fig 5.10

Cork cells Parenchyma cells

Vessels Fibres

Fig 5.10: Powder characteristics of P. kurroa (Rhizome)

Microscopic study of rhizome of P. kurroa powder showed dusty grey, groups of

fragments of tangentially elongated suberised cork cells, thick-walled parenchyma,

pitted vessels and aseptate fibers.

5.2.2.8 Plumbago zeylanica (Root)

Microscopy P. zeylanica root powder was carried out and was observed under normal

light and the photograph of the same is given in the figure 5.11.

Cork cells Trachieds Fibers

Starch grains within cells

Fig 5.11: Powder characteristics of P. zeylanica (Root)



Microscopic study of rhizome of P. Zeylanica root powder revealed dark brown,

cubical to rectangular cork cells, pointed ends and narrow lumen trachieds, large

polygonal to tangentially elongated parenchymatous cells varying in size and shape,

containing starch grains and some cells with yellow contents and fibers scattered

singly or in groups.

5.2.2.9 Holarrhena antidysenterica (Stem bark)

Microscopy H. antidysenterica stem bark powder was carried out and was observed

under normal and polarized light for calcium oxalate crystals and the photograph of

the same is given in the fig 5.12.

Cork cells Fiber Calcium oxalate prism

Stone cells

Fig 5.12: Powder characteristics of H. antidysenterica (Stem bark)

Microscopic study of rhizome of H. antidysenterica stem bark powder showed

tangentially elongated cork cells, non-lignified pericyclic fibres, rectangular to oval

stone cells and numerous pits often containing prismatic crystals of calcium oxalate.

5.2.3 Physical parameters of raw material

Physicochemical parameters like moisture content and pH, water and alcohol soluble

extractive and ash value of raw material of all ingredient of Bhunimbadi churna was

carried out. All observation were taken thrice (n=3) and values are given as Mean ±

SEM.

A. Moisture content and pH: Moisture contents and pH of all the ingredients of

churna were carried out and results are given in the Table 5.2.



Table 5.2: Moisture content and pH of churna ingredients

Moisture content was found maximum in Katuki (9.34±0.04%w/w), while minimum

in Chiryata powder (6.11±0.02%w/w). Moisture content of all the ingredients were

less than 10%w/w, which help in free flowing property of churna and prevent

microbial or fungal growth. pH of all ingredients were between 5.82 to 7.30 suggested

palatable formulation. Results indicated that raw materials were reliable.

B. Water and alcohol soluble extractives: Water/alcohol extractive value and

API limits of extractive were given in the Table 5.3.

Table 5.3: Water and alcohol soluble extractives of churna ingredients.

Sr

no.

Ingredients

of churna

Water soluble Alcohol soluble

(%w/w) * API limit (%w/w) * API limit

1 Chiryata 14.58±0.87 > 10 11.03±0.15 > 10

2 Indraju 24.97±0.92 ---- 18.55±0.07 > 12

3 Sunth 15.02±0.84 > 10 3.48±0.19 > 3

4 Mari 9.76±0.97 > 6 7.88±0.08 > 6

5 Pippali 8.43±0.75 > 7 7.67±0.12 > 5

6 Nagarmoth 13.66±0.85 > 11 5.67±0.12 > 5

7 Katuki 22.71±0.94 > 20 16.19±0.14 > 10

8 Chitrak 15.45±0.98 > 12 12.84±0.05 > 12

9 Kada chhal 22.78±0.30 > 10 19.32±0.10 > 18

Water soluble extractive value was found maximum in indraju powder

(24.97±0.92%w/w), while minimum in pippali powder (8.43±0.75%w/w). Alcohol

Sr

no.

Ingredients

of churna

Moisture content

(%w/w)

PH

1 Chiryata 6.11±0.02 5.90±0.02

2 Indraju 9.10±0.01 6.35±0.01

3 Sunth 8.25±0.04 6.22±0.05

4 Mari 7.67±0.05 7.30±0.03

5 Pippali 6.56±0.02 6.40±0.02

6 Nagarmoth 8.97±0.01 5.88±0.01

7 Katuki 9.34±0.04 6.33±0.02

8 Chitrak 7.45±0.03 6.41±0.04

9 Kada chhal 6.97±0.04 5.82±0.02



soluble extractive were maximum in kada chhal powder (19.32±0.10%), while

minimum in sunth powder (3.48±0.19%). Water soluble extractives were higher than

alcohol soluble extractive in all ingredients of churna. Results were complies with

The Ayurvedic Pharmacopoeia of India, New Delhi.

C. Ash value

Total ash, acid insoluble ash and water soluble ash of all ingredients of churna were

carried out and results are express in Table 5.4.

Table 5.4: Different ash value of ingredients present in Bhunimbadi churna.

Sr

no.

Ingredients

of churna

Total ash Acid insoluble ash Water soluble

ash %w/w %w/w API Limit %w/w API Limit

1 Chiryata 2.75±0.02 < 6 0.74±0.03 < 1 1.20±0.01

2 Indraju 4.89±0.04 < 8 1.99±0.03 < 3 2.40±0.07

3 Sunth 3.97±0.01 < 6 1.02±0.04 < 1.5 1.40±0.02

4 Mari 4.18±0.02 < 5 0.22±0.07 < 0.5 0.52±0.03

5 Pippali 5.88±0.09 < 7 0.39±0.03 < 0.5 0.57±0.03

6 Nagarmoth 6.81±0.08 < 8 2.36±0.06 < 4 3.08±0.08

7 Katuki 4.51±0.02 < 7 0.35±0.05 < 1 0.49±0.09

8 Chitrak 2.43±0.04 < 3 0.48±0.02 < 1 0.67±0.02

9 Kada chhal 5.17±0.05 < 7 0.69±0.06 < 1 0.97±0.04

Total ash value, acid insoluble ash and water soluble ash were found maximum in

nagarmoth (6.81±0.08%w/w), (2.36±0.06%) and (3.08±0.08%) respectively. Total

ash value was found minimum in chitrak powder (2.43±0.04%w/w). Acid insoluble

ash and water soluble ash were minimum in mari powder (0.22±0.07%) and

(0.52±0.03%) respectively. Amount of inorganic material present in the plant is

represented as total ash, salt of sodium, potassium are soluble in water is represented

as water soluble ash, while silica like material remain insoluble in acid is represented

as acid insoluble ash. They are constant in plant and may help to diagnose the quality

of plant material. Results for all ingredients complies the limits of The Ayurvedic

pharmacopoeia of India indicating the material were of reliable quality.

5.3 Physical Parameters for Bhunimbadi churna.

Physical parameters like description, bulk density, tapped density, Hausner ratio,

Carr's index, angle of repose, pH, moisture content, water soluble, alcohol soluble,



total ash, water soluble ash, acid soluble ash of churna were carried out and results

are given in Table 5.5. All observation were taken thrice (n=3) and values are given as

Mean ± SEM.

Table 5.5: Physical parameters for Bhunimbadi churna.

Parameters Observations

Colors Grayish yellow

Odour Characteristic

Taste Bitter

Bulk density 0.3692g/cm3

Tapped density 0.5641g/cm3

Hausner ratio 1.528

Carr's index 34.55

Angle of repose 46.17º

pH 5.35±0.11

Moisture content 8.11±0.13%w/w

Extractive value

Water soluble

Alcohol soluble

Methanol soluble

Chloroform soluble

21.62±0.18%w/w

12.42±0.13%w/w

19.38±0.15%w/w

3.45±0.24%w/w

Ash value

Total ash 5.26±0.02%w/w

Water soluble ash 1.68±0.06%w/w

Acid insoluble ash 0.49±0.01%w/w

Results indicated that churna was greyish yellow, with characteristic odour and bitter

taste. Bulk density, tapped density, Hausner ratio and Carr's index indicated very poor

flow ability. Angle of repose indicated poor-must agitate, vibrate flow property. pH of

Churna were 5.35±0.11 and Moisture content of churna were 8.11±0.13%w/w. Water

soluble extractive 21.62±0.18%w/w was higher than others. Methanol soluble

extractive 19.38±0.15%w/w values was more than alcohol soluble extractive

12.42±0.13%w/w. Chloroform extractive 3.45±0.24%w/w values was the lowest than

others in Bhunimbadi churna. Total ash value was 5.26±0.02%w/w, water soluble ash

was 1.68±0.06%w/w and acid soluble ash was 0.49±0.01%w/w.



5.4 Qualitative Phytochemical Evaluation of Plant Material and Bhunimbadi

churna

All the ingredients were subjected for the various phytochemical tests carried out.

Results are given in Table 5.6

Table 5.6: Phytochemical tests for Bhunimbadi churna and ingredients

Sr

No.

Test

Ch

iryata

Ind

raju

Su

nth

Mari

Pip

ali

Narm

oth

Katu

ki

Ch

itra

k

Kad

a

chh

al

Bh

un

imba

di

chu

rna

1 Alkaloids

Dragandorff’s + + + + + + - - + +

Mayer’s - + + + + + - - + +

Wagner’s + + + + + + - - + +

2 Glycoside

General test + + + + + + + + + +

Foam test + + + - - + + + + +

3 Flavanoid

Shinoda test + + + - - + + + + +

Lead acetate test + + + + + - + + + +

4 Tannins

FeCl3 test + + + + + + + + + +

Gelatin test + + + - - - + + + +

5 Steroids and

Triterpenoids

Liberman burchard + + + + + + + + + +

Salkowaski’s + + + - - + + + + +

6 Phenolic

FeCl3 test + + + + + + + + + +

Acetic acid test + + + + + - + + + +

7 Carbohydrates

Molish test + + + + + + + + + +

Fehling’s test + + + + + + + + + +

8 Proteins and

Amino acids

Millon’s Test - - - - - - - - - -

Ninhydrin Test - - - - - - - - - -

Except katuki and chitrak all ingradients of churna had shown the presence of

alkaloid. All powders had shown the presence of glycoside, flavanoid, steroids &

triterpenoids, tannins and carbohydrate, while absence of protein and amino acid.

Bhunimbadi churna had shown the presence alkaloid, glycoside, flavanoid, steroids &

triterpenoids, tannins, carbohydrate while protein and amino acid were absent

indicating the presence of all ingredients in churna.



5.5 TLC of Raw Material and Churna with Laboratory Standard.

TLC provides a chromatographic finger print of drug. It is, therefore, suitable for

monitoring the identity and purity of drugs and for detecting adulteration and

substitution. TLC of all ingredient of churna and churna were carried out using silica

Gel 60F254 plate; samples in respective solvent, (alcohol extract of S. chirata , Z.

officinale and P. longum; methanol extract of P. nigrum, C. rotundus and P. kurroa;

chloroform extract of P. zeylanica and H. antidysenterica seeds and stem barks

methanol extract treated with 2ml 30% ammonium solution; respective solvent

system; derivatization by spraying reagents and heating the plate) for about 10min at

105°C. Results indicated all major spots corresponding to raw material as well as

churna are given in Figure 5.13 to 5.21.

Sample: Alcohol extract

Mobile phase: Toluene: Ethyl acetate:

Formic acid (5:4:1)

Spraying reagent: Anisaldehyde-

Sulphuric acid

Major spots at Rf: 0.25 (light violet),

0.40 (orange), 0.48 (violet), 0.68 (blue).

Fig 5.13: TLC of Swertia chirata



Sample: Methanol extract treated with

2ml 30% ammonium solution


Diethyl ether (70: 20: 10)

Spraying reagent: Dragendorff’s reagent

Major spots at Rf: 0.30, 0.45, 0.50 and

0.62 (all orange).

Fig 5.14: TLC of Holarrhena antidysentrica


Mobile phase: Toluene: Ethyl acetate

(93:7)

Spraying agent: Vanillin sulphuric acid

reagent

Major spots appear at Rf: 0.16, 0.22,

0.28(all violet) 0.50(red), 0.54(brown),

0.65, 0.68 and 0.78(light violet).

Fig 5.15: TLC of Zingiber officinale



Sample: Methanol extract


(7:3)

Spraying reagent: Vanillin sulphuric

acid

Major spots at Rf: 0.20(light blue),

0.48(green) 0.50, 0.52 and 0.68(light

blue)

Fig 5.16: TLC of piper nigrum



(90:10)

Spraying reagent: Vanillin Sulphuric

acid

Major spots at Rf: 0.28(green), 0.30,

0.42, 0.52 and 0.80(light brown).

Fig 5.17: TLC of piper longum





(9:1)

Spraying reagent: Vanillin sulphuric

acid

Major spots at Rf: 0.34(Brown),

0.74(light brown).

Fig 5.18: TLC of Cyperus rotundus


Mobile phase: Chloroform: Methanol

(95:5)

Spraying reagent: Anisaldehyde

sulphuric acid


0.48(blue) and 0.70(violet)

Fig 5.19: TLC of Picrorrhiza kurroa



Sample: Chloroform extract


(3:1)

Spraying reagent: Anisaldehyde

sulphuric acid


0.35(light green), 0.45(violet) and

0.84(yellow).

Fig 5.20: TLC of plumbago zeylanica

Sample: Methanol extract treated with

2ml 30% ammonium solution


Diethyl ether (70: 20: 10)

Spraying reagent: Dragendorff reagent

Major spots at Rf: 0.32, 0.45, 0.52 and

0.62 (all orange).

Fig 5.21: TLC of Holarrhena antidysenterica

The spots present in individual plant ingredient of churna were also present in TLC of

churna at same Rf and with identical colour, indicating incorporation of authentic

plant ingredient in churna.



5.6 HPTLC of Raw Material and Churna with Marker Compound.

CAMAG TLC scanner 3 and CAMAG Automatic TLC Sampler-4 spotting device

densitometry evaluation system with winCATS software was used for scanning of

thin layer chromatogram objects in reflectance or transmission mode by absorbance or

by fluorescence at 254 or 366nm or after derivatization, respectively. Rf value of

sample was evaluated using following formula

𝐑𝐟 =𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐚𝐦𝐩𝐥𝐞 𝐟𝐫𝐨𝐦 𝐛𝐚𝐬𝐞𝐥𝐢𝐧𝐞

𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐯𝐞𝐧𝐭 𝐟𝐫𝐨𝐦 𝐛𝐚𝐬𝐞𝐥𝐢𝐧𝐞

5.6.1 Estimation of mangiferin from Swertia chirata and churna by HPTLC

The standard solution of mangiferin 1mg/ml was prepared. Graded concentrations of

0.016, 0.027, 0.054, 0.080, and 0.107 µl volume of mangiferin were applied on a pre-

coated TLC silica gel 60 F254 plate (20cmx10cm). The concentration of the

mangiferin was 16, 27, 54, 80 and 100 ng/spot respectively. The mobile phase used

was ethyl acetate: methanol: water: formic acid (10:1:1:0.5). The photographs of

HPTLC plate of mangiferin, raw material of S. chirata and churna at 254nm and

366nm were shown in figure 5.22 and 5.23.

Track-1: 0.016 µg/µl of std mangiferin. Track-2: 0.027 µg/µl of std mangiferin.

Track-3: 0.054 µg/µl of std mangiferin. Track-4: 0.080 µg/µl of std mangiferin.

Track-5: 0.107 µg/µl of std mangiferin. Track-6: 5 µg/µl of swertia chirata

Track-7: 10 µg/µl of swertia chirata Track-8: 5 µg/µl of churna.

Track-9: 10 µg/µl of churna

Fig 5.22: Photograph of HPTLC plate mangiferin, S. chirata and churna at 254 nm



Fig 5.23: Photograph of HPTLC plate mangiferin, S. chirata and churna at 366 nm

HPTLC densitometric chromatogram of standard mangiferin, swertia chirata and

churna were shown in figure 5.24.

Fig 5.24: HPTLC densitogram mangiferin, S. chirata and churna



The peak areas of mangiferin for each concentration were recorded. Calibration curve

of mangiferin was prepared by plotting peak area against applied concentration of

mangiferin and shown in figure 5.25.

Fig 5.25: Calibration curve of mangiferin

A spot at Rf 0.39 was observed in the HPTLC chromatogram of the standard

mangiferin and raw material of S. chirata and churna. There was no interference with

other components present in the raw material and churna because they appear at

significantly different Rf values. The calibration curve of mangiferin was found to be

linear dependent on the concentration against peak area. The results of linearity

equation (y=10248x+1009) and correlation coefficient (R2=0.992) showed that within

the concentration range indicated, there was good correlation between peak area and

the corresponding concentration of mangiferin. The total concentration of mangiferin

in the raw material of S. chirata and churna were determined using calibration curve

of mangiferin. Raw material of S. chirata and churna were found to contain

mangiferin 0.21%w/w and 0.076%w/w respectively.

5.6.2 Estimation of 6-gingerol from Zingiber officinale and churna by HPTLC

The standard solution 1mg/ml 6-gingerol was prepared. Graded concentrations of

0.077, 0.128, 0.256, 0.384, and 0.512µl volume representing 77, 128, 256, 384 and

512ng/spot of 6-gingerol respectively were applied on a pre-coated TLC silica gel 60

F254 plate (20cmx10cm). The mobile phase was used n-hexane: ethyl acetate (50:50).

The photographs of HPTLC plate of 6-gingerol, raw material of Z. officinale and

churna at 254nm, 366nm and after derivatization were shown in figure 5.26.



At 254nm

At 366nm

After derivatization

Track-1: 0.077 µg/µl 6-gingerol. Track-2: 0.128 µg/µl 6-gingerol.

Track-3: 0.256 µg/µl 6-gingerol. Track-4: 0.384 µg/µl 6-gingerol.

Track-5: 0.512 µg/µl 6-gingerol. Track-6: 5 µg/µl Z.officinale

Track-7: 10 µg/µl of Z. officinale Track-8: 5 µg/µl churna.

Track-9: 10 µg/µl of Churna

Fig 5.26: HPTLC Photograph plate of 6-gingerol, Z. officinale and churna



The separated bands on the HPTLC plates were scanned over the wavelength of 400–

800 nm. The source of radiation utilized was tungsten lamp. The maximum

absorbance was found to be at 533 nm. HPTLC densitometric chromatogram of

standard 6-gingerol, zingiber officinale and churna are shown in figure 5.27.

Fig 5.27: HPTLC densitogram of 6-gingerol, Z. officinale and churna.

A spot of 6-gingerol was observed at Rf 0.60 in the HPTLC chromatogram of the

standard, raw material of Z. officinale and churna. There was no interference with

other components present in the raw material and churna because they appear at

significantly different Rf values. The peak areas of 6-gingerol for each concentration



were recorded. Calibration curve of 6-gingerol was prepared by plotting peak area

against applied concentration of 6-gingerol and is shown in figure 5.28.

Fig 5.28: Calibration curve of 6-gingerol

The calibration curve of 6-gingerol had given linearity equation (y=6191x+239) and

correlation coefficient (R2=0.993) indicating calibration curve was found to be linear

dependent on the concentration against peak area. The results of showed that within

the concentration range indicated, there was good correlation between peak area and

the corresponding concentration of 6-gingerol. The total concentration of 6-gingerol

in the raw material of Z. officinale and Bhunimbadi churna was determined using

calibration curve of 6-gingerol and were found to be 0.54%w/w and 0.06%w/w.

5.6.3 Estimation of piperin from Piper nigrum, Piper longum and churna by

HPTLC

The standard solution 1mg/ml piperin was prepared. Graded concentrations of 0.4,

0.8, 1.2, 1.6, 2.0 µl volume of Piperin indicating 400, 800, 1200, 1600 and 2000

ng/spot Piperin respectively were applied on a pre-coated TLC silica gel 60 F254

plate (20cm x 10cm). The mobile phase was used Toluene: Ethyl acetate: Formic acid

(3:1:0.1). The photographs of HPTLC plate showing Piperin in varying concentrations

and in raw material of p. nigrum, p. longum and churna at 254nm and 366nm are

shown in figure 5.29 and 5.30.



Fig 5.29: Photograph of HPTLC plate of piperin, P. nigrum, P. longum and

churna at 254 nm.

Track-1: 0.04 µg/µl Piperin. Track-2: 0.06 µg/µl Piperin.

Track-3: 0.08 µg/µl Piperin. Track-4: 0.10 µg/µl Piperin.

Track-5: 0.12 µg/µl Piperin. Track-6: 2 µg/µl of P. nigrum.

Track-7: 4 µg/µl P. nigrum. Track-8: 2 µg/µl P. longum.

Track-9: 4 µg/µl P. longum. Track-10: 2 µg/µl churna.

Track-11: 4 µg/µl churna.

Fig 5.30: Photograph of HPTLC plate of piperin, P. nigrum, P. longum and

churna at 366nm.



The separated bands on the HPTLC plates were scanned over the wavelength of 400–

600 nm. The source of radiation utilized was D2 lamp. The maximum absorbance was

found to be at 334 nm. HPTLC densitometric chromatogram of standard piperine, P.

nigrum, P. longum and churna were carried out and shown in figure 5.31.

Fig 5.31: HPTLC densitogram of piperin, P. nigrum, P. longum and churna.



A spot of Piperine was observed at Rf 0.28 in the HPTLC chromatogram of the

standard piperin and raw material of p. nigrum, p. longum and churna. There was no

interference with other components present in the raw material and churna because

they were separted at significantly different Rf values. The peak areas of Piperin for

each concentration were recorded. Calibration curve of piperin was prepared by

plotting peak area against applied concentration of piperin and shown in figure 5.32.

Fig 5.32: Calibration curve of piperin

The calibration curve of piperin was with linearity equation (y=11539x+136.4) and

correlation coefficient (R2=0.986) indicated the calibration curve of piperin was

linear and dependent on the concentration against peak area within the concentration

range indicated. There was good correlation between peak area and the corresponding

concentration of piperin. The total concentration of piperin in the raw material of P.

nigrum, P. longum and churna was determined using calibration curve of piperin and

were found to be 2.87%w/w,1.53%w/w and 0.24%w/w respectively.

5.6.4 Estimation of conessine in H. antidysenterica seed & stem bark and

churna by HPTLC

The standard solution 1mg/ml conessine was prepared. Graded concentrations of 0.2,

0.4, 0.6, 0.8, 1.0µl volume of conessine repesenting 200, 400, 600, 800 and 1000

ng/spot conessine respectively were applied on a pre-coated TLC silica gel 60 F254

plate (20cmx10cm). The mobile phase toluene: ethyl acetate: diethyl amine

(7.0:2.0:1) was used.



The photographs of HPTLC plate showing conessine in varying concentation and in

raw material of H. antidysenterica seed & stem bark and churna at 254nm, 366nm

and after derivatization are shown in figure 5.33.

At 254.

At 366.

After derivatization.

Track-1: 0.04 µg/µl conessine. Track-2: 0.06 µg/µl conessine Track-3: 0.08 µg/µl

conessine. Track-4: 0.10 µg/µl conessine. Track-5: 0.12 µg/µl conessine. Track-6: 2

µg/µl H.seed. Track-7: 4 µg/µl H. seed. Track-8: 2 µg/µl H. stem bark. Track-9: 4

µg/µl of H. stem bark. Track-10: 2 µg/µl churna.Track-11: 4 µg/µl churna.

Fig 5.33: HPTLC plate of conessine, H. antidysenterica seed & stem bark and churna



The separated bands showing conessine on the HPTLC plates were scanned over the

wavelength of 400–800 nm. Tungsten lamp was utilized as a source of radiation. The

maximum absorbance was found to be at 520nm. HPTLC densitogram of conessine in

varying amount and in raw material of H. antidysenterica seed & stem bark and

churna were carried out and is shown in figure 5.34.

Fig 5.34: HPTLC densitogram conessine, raw material of H. antidysenterica seed,

stem bark and churna

A spot of conessine was observed at Rf 0.86 in HPTLC chromatogram of the standard

conessine, raw material of H. antidysenterica seed, stem bark and churna. There was

no interference with other components present in the raw material and churna because



they appear at significantly different Rf values. The peak areas of conessine for each

concentration were recorded. Calibration curve of conessine was prepared by plotting

peak area against applied concentration of conessine and shown in figure 5.35.

Fig 5.35: Calibration curve of conessine

The calibration curve of conessine was found with linearity equation (y=280x+1653)

and correlation coefficient (R2=0.995) indicated calibration curve of conessine was

linear and dependent on the concentration against peak area. There was good

correlation between peak area and the corresponding concentration of conessine. The

total concentration of conessine in the raw material of H. antidysenterica seed, stem

bark and churna was determined using calibration curve of conessine and were found

to be 0.19%w/w,0.90%w/w and 0.083%w/w.

HPTLC methods developed for estimation mangiferine, 6-gingerol, piperin and

conessine in individual plant and combination as churna was accurate, linear, rugged,

simple and rapid. Total concentration of mangiferine, 6-gingerol, piperin and

conessine estimated was well complies with the reference standard. This method can

be utilize the estimation of plant containing mangiferine, 6-gingerol, piperin and

conessine individual and in combination. Result showed that HPTLC technique can

be successfully used for estimation of mangiferine, 6-gingerol, piperin and conessine

in Bhunimbadi churna.



5.6.5 HPTLC finger printing of Bhunimadi churna

HPTLC finger printing of Bhunimadi churna was carried out using silica Gel 60F254

plate; churna in methanol extract using toluene: ethyl acetate: formic acid (5: 1.5: 0.5

v/v) solvent system. Plate was observed under UV-light at 254nm and 366nm.

Derivatization by anisaldehyde sulphuric acid spraying reagents and heating the plate

for about 10min at 105°C. Solvent system was modified by trial and error to give

separation of maximum compounds. Photographs of plate and chromatogram of

churna is given in Figure 5.36.

Fig 5.36: HPTLC fingerprinting of Bhunimbadi churna



Compounds separated in HPTLC fingerprinting were observed under UV light at

254nm and 366nm and after derivatization are tabulated with their Rf value in Table

5.7.

Table 5.7: Peaks of HPTLC of Bhunimbadi churna

Peak At 254 At 366 After Derivatization

Max Rf Max Rf Max Rf

1 0.12 0.25 0.05

2 0.25 0.37 0.09

3 0.33 0.44 0.19

4 0.45 0.56 0.25

5 0.56 0.62 0.45

6 0.59 0.73 0.56

7 0.62 0.83 0.62

8 0.73 0.88 0.68

9 0.75 0.73

10 0.82 0.82

11 0.85 0.95

12 0.87

Result indicated that HPTLC finger printing of Bhunimadi churna under UV light at

254 nm, had showed 13 blue colour spots at Rf 0.25, 0.33, 0.45, 0.56, 0.59, 0.62, 0.73,

0.75, 0.82, 0.85, 0.87, 0.95. UV light at 366nm had showed 8 spots at Rf 0.25, 0.37,

0.56, 0.62 were purple; 0.44, 0.56 were dark green and 0.73, 0.83, 0.88 were black.

After derivatization with the anisaldehyde-sulphuric acid reagent had showed 11 spots

at Rf 0.05, 0.68 were dark purple; 0.09 was yellow; 0.19 was light purple; 0.25 was

purple; 0.45 was green; 0.56 was dark green; 0.62 was purple; 0.73, 0.95 were black

and 0.82 was light pink.

Human fingerprint is unique and will not match with others. Similarly HPTLC

fingerprinting of plant and their formulation are also unique and not match with other

plants and other formulations. Hence HPTLC fingerprinting helps in identification,

establishing uniformity, detection of adulteration, addition and omission of ingredient

of formulation and determination of quality of raw material used in the formulation.

5.7 Heavy Metals Analysis:

Analysis of heavy metal in Bhunimbadi churna like Lead (Pb), Cadmium (Cd),

Arsenic (As), Mercury (Hg), were carried out using Labindia Inductively coupled

plasma-optical emission spectroscopy (ICP-OES). Results are shown in Table 5.8



Table 5.8: Heavy metal analysis

Sr No. Heavy metal Bhunimbadi churna (ppm) API limit (ppm)

1 Lead (Pb) 9.40 10

2 Cadmium (Cd) 0.3 0.3

3 Arsenic (As) 2.15 10

4 Mercury (Hg) Absent 1

Heavy metals after entering in the human body can’t be removed easily. On long

usage they accumulated in the body ultimately produce toxicity by hindering the

normal physiological activity. Hence WHO and various pharmacopoeias have

prescribed the limit for heavy metals. Results showed that concentration of lead and

cadmium in Bhunimbadi churna were 9.40ppm and 0.3ppm respectively and

concentration of arsenic was 2.15ppm and mercury was absent in Bhunimbadi churna.

The presence of heavy metal in Bhunimbadi churna were less than the limit

prescribed in The Ayurvedic Pharmacopoeia of India.

5.8 Microbial Analysis

Microbial analysis of Bhunimbadi churna included total viable aerobic count, total

yeast and mould, E. coli, S. Typhi count. The results are given in Table 5.9

Table 5.9: Microbial analysis

Sr No. Microbial analysis Bhunimbadi churna API Limit

1 Total aerobic viable count 410 cfu/gm 105 /gm

2 Total yeast and mould Absent 103 /gm

3 E. coli Absent Absent

4 Salmonella Sp Absent Absent

Pathogenic bacteria in vegetative and non-vegetative forms are harmful to human

body because they produce diseases. Hence WHO and pharmacopoeia of the advance

countries have prescribed the limit for total aerobic viable count, total yeast and

mould, E. coli and Salmonella species. Results indicated that Total aerobic viable

count was 410 CFU/gm and Total yeast and mould, E. coli and Salmonella species

were absent in Bhunimbadi churna. This indicated that total aerobic viable count, total

yeast and mould, E. coli and Salmonella Species in Bhunimbadi churna were lower

than the limit prescribed in The Ayurvedic Pharmacopoeia of India.



5.9 In-vitro Antioxidant Activity of Bhunimbadi churna.

In-Vitro the antioxidant activity of the methanol extract of the churna was carried out

by DPPH-Free radical scavenging activity, H2O2 scavenging activity and Ferric

reducing antioxidant power antioxidant models, using ascorbic acid as reference

standard.

5.9.1 DPPH-Free radical scavenging activity

The absorbance maximum of a stable DPPH radical in methanol was measured at

517nm. The decrease in absorbance of DPPH caused by antioxidants (ascorbic acid,

methanol extract of churna) was determined at 517nm. The reaction results in the

scavenging of free radical by hydrogen donation change the color from purple to

yellow. Results are illustrated in Figure 5.37. Values are express as means± SD of

triplicate analyses and Significance P<0.05 compared to control

Fig 5.37: DPPH radicals scavenging activity of methanol extract of churna

The results indicated that methanol extracts of churna and ascorbic acid had showed

significant dose dependents reduction in free radical to the corresponding hydrazine in

the antioxidant principles of DPPH. The odd electron of DPPH radical becomes

paired with hydrogen from a free radical scavenging antioxidant to form the reduced

DPPH-H. The resulting decolorization is stoichiometric with respect to number of

electrons captured. This indicated antioxidant activity of Bhunimbadi churna.



5.9.2 Hydrogen peroxide scavenging activity

Absorbance of H2O2 at 230nm was determined with different concentrations of

methanol extract of churna and standard ascorbic acid and also after 10min against a

blank solution containing phosphate buffer without H2O2. The percentage inhibition

activity was calculated. H2O2 scavenging activity was graphical presented by plotting

graph using concentration vs. absorbance and shown in Figure 5.38. All the tests were

performed in triplicate and the graph was plotted with the average of three

observations.

Fig 5.38: H2O2 scavenging activity of methanol extract of churna

Result demonstrated that methanol extract of churna and ascorbic acid had shown

significant concentration dependant scavenging of H2O2. H2O2 is an intermediate

during endogenous oxidative metabolism and mediates radical oxygen formation such

as OH, which may be used to predict the scavenging capability of antioxidants in

biological systems. H2O2 has only a weak activity to initiate lipid peroxidation, but its

activity as an active oxygen species comes from its potential to produce the highly

reactive hydroxyl radical through the Fenton reaction. Thus, removal of H2O2 is very

important for protection of food systems.

The antioxidant activity of churna was expressed as IC50 which is defined as the

concentration (μg/ml) of methanol extract of churna inhibits the formation of DPPH

radicals by 50%. IC50 in DPPH and H2O2 models was calculated by linear regression

and results are given in table 5.10.



Table 5.10: IC50 of methanol extract of churna in DPPH and H2O2 models.

Sr No. Antioxidant model IC50 (µg/ml)

Ascorbic acid Methanol extract of

Bhunimbadi churna

1 DPPH model 14.26 62.80

2 H2O2 model 35.64 75.45

The IC50 values in DPPH model were found to be 62.80 and 14.26 µg/ml for

methanol extracts of churna and ascorbic acid respectively. IC50 values in Hydrogen

Peroxide scavenging model were found to be 35.64 and 75.45 μg/ml for Ascorbic acid

and Methanol extract of churna respectivily. Methanol extract of churna had exibited

higher antioxidant activity in DPPH and H2O2 models.

5.9.3 Ferric reducing antioxidant power activity

Methanol extracts of churna was subjected to Ferric reducing power activity.

Reducing power acitivity was determind by increase in absorbances. Concentration

vs. absorbance of methanol extracts of churna and ascorbic acid were graphically

plotted and given in Fig 5.39. Values are means± SD of triplicate analyses.

Fig 5.39: Ferric reducing antioxidant activity of methanol churna

The results showed that methanol extract of churna and ascorbic acid had

demonstrated dose dependent antioxidant activity in ferric reducing power model.

Antioxidant activity of methanol extract of Bhunimbadi churna in DPPH, H2O2 and

ferric reducing antioxidant model may attributed due to presence of phytoconstituents

like; alkaloid, glycoside, flavanoid, phenolic compounds and tannins.



5.10 Antidiabetic Activity of Churna in Alloxan Induced Diabetic Rat Model.

Bhunimbadi churna, prepared as per ‘Bruhad Nighantu Ratnakar’ and was subjected

to assess its antidiabetic potential. Alloxan, when it is administered parenterally,

intravenously, intraperitoneally or subcutaneously, causes a massive reduction in

insulin release by the destruction of β-cells of the islets of Langerhans and induces

hyperglycaemia, hence Alloxan induced diabetics in rat model was selected to

establish the efficacy of churna.

5.10.1 Acute toxicity: Acute toxicity of Bhunimbadi churna was carried out in rat.

Four hrs. fasted animals were orally administered 100-2000mg/kg body weight

Bhunimbadi churna in 5% v/v Tween 80 solution in five animal with different dose.

Animals were observed individually during the first 30minute after dosing, every 4hr

during the first 12hr, and thereafter for 3 days for behaviour change. Neurological

change and autonomic change. Results are given in table 5.11.

Table 5.11: Acute toxicity of Bhunimbadi churna

Profile 30min 4hrs 8hrs 12hrs 3days

Behavioral Alertness NC NC NC NC NC

Restlessness NC NC NC NC NC

Irritability NC NC NC NC NC

Fearfulness NC NC NC NC NC

Neurological Spontaneous activity NC NC NC NC NC

Reactivity NC NC NC NC NC

Touches response NC NC NC NC NC

Pain response NC NC NC NC NC

Gait NC NC NC NC NC

Autonomic Defecation NC NC NC NC NC

Urination NC NC NC NC NC

Mortality Death NC NC NC NC NC

NC= No change

Acute toxicity study revealed the non-toxic nature of churna up to 2000mg/kg. All the

animals were alive, healthy and active during the observation period. No lethality or

single toxic reactions were found at any of the doses selected until the end of the



study period. Considering this, 200mg/kg and 400mg/kg doses were selected for

further study.

For antidiabetic study animals were divided into five groups. Normal control group

(Group I) received laboratory diet and water ad libitum. Rests of the rats were fasted

18hrs and were intraperitonialy given a single dose 100mg/kg, body weight Alloxan

monohydrate. Animals were maintained for two days in diabetic condition for well

establishment of diabetes. Diabetic control group (Group II) was fed with Standard

Laboratory Diet and water ad libitum. Reference standard group (Group III) was daily

treated orally with 5mg/kg body weight Glibenclamide in water for 14 days. Group IV

and Group V were daily treated with dose of 200 and 400mg/kg b.w Bhunimbadi

churna respectively for 14 days. Blood samples were collected from the retro-orbital

plexus of 8hr fasted and anesthetized animals by slight exposure to ether on first, 7th

and 14th

days and biochemical parameters like; body weight, blood glucose, serum

total cholesterol, serum triglycerides, serum HDL, serum VLDL, serum LDL, serum

total protein and albumin proteins, serum creatinine and urea were determined.

Results were statistically analysed using one-way analysis of variance (ANOVA)

followed by Dunnett’s test using GraphPad Instat3 software and expressed as Mean ±

SEM (n=6), Group II was compared with Group I, Group-III, IV and groupV.

**P<0.01, *P<0.05.

5.10.2 Body Weight: Body weight of animal in all the groups were checked at the

end of 1st, 7

th and 14

th days and results are given in Figure 5.40 and 5.41.

Fig 5.40: Antidiabetic activity of Bhunimbadi churna by rat body weight



Fig 5.41: Antidiabetic activity of Bhunimbadi churna by rat body weight

Result showed that after first, 7th

and 14th

days, body weight in normal control group,

reference standard group, group-IV and V had significantly increased (p<0.05). While

diabetic control group showed significantly (p<0.05) decreases in body weight. It was

concluded that induction of diabetes with Alloxan is associated with a characteristic

loss of body weight, which is due to increased muscle wasting and loss of tissue

proteins. Diabetic rats treated with the Bhunimbadi churna showed significant dose

dependent increase in body weight, which may be due to its effect in controlling

muscle wasting; by reversal or antagonizing. This was supported by the result in

reference standard group receiving Glibenclamide which significantly prevent the loss

of body weight in experimental diabetic rats.

5.10.3 Blood glucose level: Blood glucose level of animal in all the groups were

corded on first, 7th

and 14th

days and results are graphically represented, day wise in

Figure 5.42 and group wise in Figure 5.43.

Alloxan used as an agent for the development of experimental diabetes to induce

selective dysfunctioning of pancreatic β-cells. In-vitro studies have shown that

Alloxan is selectively toxic to pancreatic β -cells, leading to the induction of cell

necrosis. The cytotoxic action of alloxan is mediated by reactive oxygen species, with

a simultaneous massive increase in cytosolic calcium concentration, leading to a rapid

destruction of β-cells characterized by reduced circulating concentration of insulin;

poor insulin sensitivity or insulin resistance and poor glucose tolerance resulting in

high blood glucose level.

*

**** **

**

***

**

***

130

140

150

160

170

180

190

200

Group I Group II Group III Group IV Group v

gm

Treatment in Days

Body weight (gm)



Fig 5.42: Antidiabetic activity of Bhunimbadi churna by rat blood glucose level

Fig 5.43: Antidiabetic activity of Bhunimbadi churna by rat blood glucose level

Result showed that after first, 7th

and 14th

days, blood glucose level in diabetic control

group animals was significantly increased. While diabetic animals receiving

Bhunimbadi churna 200 and 400mg/kg of body weight and Gibenclamide

significantly decreased blood glucose level (p<0.05). Bhunimbadi churna in Alloxan

induced rats produced significant dose dependent decreased in blood glucose level

(p<0.05) indicating antidiabetic activity.

******

**

**

**

**

**

**

**

50

100

150

200

250

300

350

400

Group I Group II Group III Group IV Group v

mg/

dl

Treatment in Days

Blood glucose Level (mg/dl)



5.10.4 Lipid profile: Lipid profile of serum cholesterol level, serum triglyceride,

serum HDL, VLDL and LDL animal in all the groups was checked at the end 14th

days and results are graphically presented in Figure 5.44.

Fig 5.44: Antidiabetic activity of Bhunimbadi churna by rat lipid profile

Results indicated that after 14th

days serum cholesterol level significantly (p<0.05)

dose dependently decreased in diabetic animal treated with Bhunimbadi churna.

There was also significant (p<0.05), and dose dependent decreased in the serum

triglyceride level in diabetic animal receiving Bhunimbadi churna. There was

significant (p<0.05), dose dependent increased in the serum HDL level in diabetic

animal treated with Bhunimbadi churna. There was significant (p<0.05), dose

dependent decreased in the serum VLDL level and serum LDL level in diabetic

animal treated with Bhunimbadi churna. Gibenclamide had also significantly

normalized Alloxan induced diabetic animal by decreasing serum cholesterol, serum

triglyceride, serum VLDL and serum LDL and increasing serum HDL (p<0.05).

Results indicated the Bhunimbadi churna had shown antidiabetic activity by

significant (p<0.05) and dose dependent decreasesing the serum cholesterol, serum

triglyceride, serum VLDL, serum LDL and increasesing the serum HDL level in

alloxan induced diabetic rats.

The levels of serum lipids are usually elevated in diabetes mellitus and such an

elevation represents a risk factor for coronary heart disease. This abnormal high level

of serum lipids is mainly due to the uninhibited actions of lipolytic hormones on the



fat depots mainly due to the action of insulin. Under normal circumstances, insulin

activates the enzyme lipoprotein lipase, which hydrolyses triglycerides. However, in

diabetic state lipoprotein lipase is not activated due to insulin deficiency resulting in

hyper-triglyceridemia and insulin deficiency is also associated with hyper-

cholesterolemia due to metabolic abnormalities. Hence, the diabetic rats showed

hyper-cholesterolemia and hyper-triglyceridemia and the treatment with churna

significantly (p<0.05) and dose dependently lowered cholesterol and triglyceride

levels.

5.10.5 Serum urea: Serum urea level is one of the parameter of renal profile. Serum

urea level was checked in the animals of all the groups on 14th

days and results are

graphically presented in Figure 5.45.

Fig 5.45: Antidiabetic activity of Bhunimbadi churna by rat serum urea

Results indicated that there was a significant rise in serum urea level (p<0.05) in

Diabetic control rats. Bhunimbadi churna (200 and 400 mg/kg) and Glibenclamide

significantly reduced the elevated serum urea level (p<0.05).

5.10.6 Serum creatinine: Serum creatinine level is also one of the parameter for

renal profile. Serum creatinine level was measured in the animals of all the groups on

14th

days and results are graphically presented in Figure 5.46.



Fig 5.46: Antidiabetic activity of Bhunimbadi churna by rat serum creatinine

There was also significant rise in serum creatinine level in alloxan induced rats

(p<0.05). Bhunimbadi churna (200 and 400 mg/Kg) and Glibenclamide significantly

reduced the elevated serum creatinine level (p<0.05).

Serum urea and creatinine is metabolic end product of amino acid and protein, in the

blood that would be normally excreted in the urine by kidneys’ nephrons vessels.

When blood glucose levels are consistently too high affects the kidneys ability to

filter and filtration rate in glomeruls, cause End Stage Renal Disease (ESRD). Urea

formation is influenced by a number of factors such as liver function, protein intake

and rate of protein catabolism. Elevation of serum urea and creatinine level in alloxan

induced diabetic rat and significantly normalize by application of glibenclamide and

Bhunimbadi churna in Alloxan induced diabetic rats.

5.10.7 Serum albumin and total protein: Similar to serum urea and serum

creatinine, serum albumin and total protein are also the parameters for the renal

profile. Serum albumin and total protein level were measured in the animals of normal

control group, diabetic control group, standard reference group and Bhunimbadi

churna 200 and 400 mg/kg body weight treated group on 14th

days and results are

graphically presented in Figure 5.47.



Fig 5.47: Antidiabetic activity of Bhunimbadi churna by rat albumin and total

protein.

Results indicate that there was a significant decreased in serum albumin and total

protein level (p<0.05) in Diabetic control rats. Bhunimbadi churna (200 and 400

mg/kg) and Glibenclamide significantly elevated serum albumin and total protein

level (p<0.05).

Low levels of albumin (microalbuminuria) in the urine cause incipient nephropathy.

Microalbuminuria is a sensitive and early marker of diabetic nephropathy, other forms

of renal dysfunction, and endothelial permeability throughout the vascular tree. It

predicts progression of kidney disease leading to End Stage Renal Disease,

development of cardiovascular disease, retinopathy, peripheral vascular disease and

total mortality. Decreased in serum albumin and total protein level in Alloxan induced

diabetic rat and significantly normalize by administration of Glibenclamide and

Bhunimbadi churna in Alloxan induced diabetic rats.

Bhunimbadi churna improved renal profile in Alloxan induced diabetic rats by

decreasing serum urea and creatinine level and increasing serum albumin and total

protein indicating renal protecting activity of Bhunimbadi churna.

**

**

***

**

**

****

0

1

2

3

4

5

6

7

8

g/

dl

Serum albumine Serum Total Protein

Renal profile (gm/dl)



Antidiabetic activity of Bhunimbadi churna was concluded by exhibiting regaining

body weight, lowering blood glucose level, improving lipid profile by decreasing

serum cholesterol, serum triglyceride, serum VLDL & serum LDL level and

increasing serum HDL level and improving renal profile by decreasing serum urea

and creatinine level and increasing serum albumin and total protein.

Bhunimbadi churna was shown the presence alkaloid, glycoside, flavanoid, steroids

and triterpenoids, tannins and carbohydrate which conformed by phytochemical

analysis. Antioxidant activity, antidiabetic activity, improvement in lipid profile and

renal profile exhibited by Bhunimbadi churna may be due to phytoconstituents

present like; alkaloid, glycoside, flavanoid, steroids and triterpenoids, tannins and

carbohydrate. Flavonoids, steroids/ triterpenoids, alkaloids and phenolics are known

to be bioactive antidiabetic principles. Flavonoids are known to regenerate the

damaged beta cells in Alloxan induced diabetic rats.

Present investigation justifies the traditional therapeutic claim as antidiabetic activity

of Bhunimbadi churna. However further phytochemical investigation may provide

isolation and identification of specific compound/s from Bhunimbadi churna which

may be responsible for antioxidant activity, antidiabetic activity and improving lipid

and renal profile.

chapter 5 result and discussion 5 result and discussion...

Documents