botanical and phytochemical comparison of three bergenia species

SRIVASTAVA & RAWAT: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE BERGENIA SPECIES 65Journal of Scientific & Industrial Research

Vol. 67, January 2008, pp.65-72

*Author for correspondence

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Botanical and phytochemical comparison of three Bergenia species

Sharad Srivastava* and Ajay Kumar Singh Rawat

Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow 226 001

Received 20 July 2007; revised 15 October 2007; accepted 18 October 2007

Three species of Bergenia [B. ligulata (Wall) Eng., B. ciliata (Royle) Raizada and B. stracheyi Engl.] were evaluated for

botanical, physicochemical and chemical studies. Botanical study of rhizomes revealed that B. ciliata has large number of starch

grains; B. ligulata has maximum calcium oxalate crystals while B. stracheyi has a lesser amount of starch grains. Physicochemical

studies showed that B. stracheyi had highest percentage of all physicochemical parameters (total ash 15.8, alcohol and water

soluble extractives 13.83 and 16.83, sugar 5.5 and tannins 7.86), except starch and acid insoluble ash, which were highest in B.

ciliata. A comparative HPTLC study was also carried out.

Keywords: Bergenia, Bergenin, HPTLC, Pharmacognosy, Pashanbheda

Introduction

High incidence of urinary calculi is reported from

British Isles, Scandinavian countries, northern Australia,

central Europe, India, Pakistan, and Mediterranean

countries1. Calcium-containing stones are the most

common (75% of all urinary calculi), which may be in

the form of pure calcium oxalate (50%) or calcium

phosphate (5%) and a mixture of both (45%). Different

mineral metabolisms are important in the formation of

urinary stones or calculi2. There are many herbs in the

Indian systems of medicine, which play important role

in inhibition and removal of calcium oxalate crystals3,4.

The effect of aqueous extracts of some common

medicinal plants was also studied on the growth and

inhibition of calcium oxalate monohydrate (COM)

crystals5.

Different Bergenia species (Family: Saxifragaceae),

distributed in South and East Asia and European

countries, are important medicinal plants. In India,

rhizomes have been used for centuries in the Ayurvedic

formulations to dissolve kidney and bladder stones, in

leucorrhea, piles, and pulmonary affections6. Alcohol

extract of plant has exhibited significant analgesic, anti-

inflammatory and diuretic properties7. Anti-bacterial,

anti-inflammatory and anti-tussive activity of

B. ciliata8-10, besides anti-viral activity11,12 and antilithiatic

activity13,14 have also been reported. Inhibition of the

growth of urinary calcium hydrogen phosphate dehydrate

crystals with aqueous extract of Tribulus terrestris

(Zygophyllaceae) and B. ligulata has been reported5.

There are reports on ethnobotanical usage of Bergenia

species as an antilithic, for boils and blisters15, in urinary

diseases, as antidiabetic, hemorrhoids, stomach disorders

and opthalmia16,17. Leaves are used for dissolving kidney

stones18. Rhizome is used as tonic, antipyretic,

antidiarrhoel, in opthalmia, kidney stones, and other

urinary disorders19,20. Plant contains β-sitosterol-D-

glucoside, bergenin21 and afzelechin22. A simultaneous

determination of bergenin and gallic acid by HPTLC is

also reported in B. ligulata23.

This study presents comparative botanical and

phytochemical evaluation of B. ligulata (Wall) Eng., B.

ciliata (Royal) Raizada and B. stracheyi Engl.

Materials and Methods

Plant materials were collected in October 2004 from

Almora (Uttrakhand, India), authenticated by Dr A K S

Rawat, matched with herbarium specimen, and stored

in the Institute’s herbarium with following voucher

specimen numbers: LWG 222437BC; LWG 222438BL;

LWG 222439BS, 2004. Rhizomes preserved in 70%

ethanol for histological study. Microtome sections cut

and stained with safranin and fast green, and

photographed with Nikon F70X camera24.

66 J SCI IND RES VOL 67 JANUARY 2008

Physico-chemical and HPTLC Studies

Physico-chemical (Fig. 1) and phytochemical studies

(Fig. 2) were carried out as per standard procedures25-27

using shade dried powdered (100 mesh) plant material.

A densitometric HPTLC analysis (Fig. 3) was also

performed for the development of characteristic

fingerprint profile, which may be used as markers for

quality evaluation and standardization of the drug.

Extraction of Plant Material

Air-dried (45-55°C) powdered rhizomes of three

Bergenia species (each 1.0 g) were extracted with

es

37.2

8

11

1.2

8.8

33 1

1.8

33

3.3

79

39.1

84

4.9

17

40.5

6

9.1

5

0.2

5

5

8.8

33

2.5

73

28.9

38

3.0

47

39.5

3

15.8

0.7

13.8

33 16.8

33

5.5

01

38.1

61

7.8

62

0

5

10

15

20

25

30

35

40

45

Moisture

content

Total ash Acid-insoluble

ash

Alcohol

soluble

extractive

Water soluble

extractive

Sugar Starch Tannins

Parameters

Perc

en

tag

es

B. ciliata

B. ligulata

B. strachyi

s

1.36

0.66

13.98

11.14

8.217

1.420.74

8.76

9.72

7.94

1.72

1

17.92

14.9

9.46

0

2

4

6

8

10

12

14

16

18

20

Hexane Chloroform Acetone Alcohol Water

Solvents

Perc

en

tag

es

B. ciliata

B. ligulata

B. strachyi

Fig. 1—Comparative physico-chemical studies of different Bergenia species

Fig. 2—Successive extractive values of different Bergenia species

-

-

-

-

-

-

-

-

-

-

SRIVASTAVA & RAWAT: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE BERGENIA SPECIES 67

methanol (10 ml). Extracts were concentrated under

vacuum, redissolved in methanol, filtered and finally made

up to 100 ml with methanol prior to HPTLC analysis.

Chromatographic Conditions

Chromatography was performed on Merk HPTLC

precoated silica gel 60GF254

(20 cm x 20 cm) plates.

Methanolic solutions of samples and standard compound

bergenin of known concentrations were applied to the

layers as 6 mm-wide bands positioned 15 mm from the

bottom and 15 mm from side of the plate, using Camag

Linomat 5 automated TLC applicator with the nitrogen

flow providing a delivery speed of 150 nl/sec from

application syringe. These conditions were kept constant

throughout the analysis of samples.

Detection and Quantification of Bergenin

Following sample application, layers were developed

in a Camag twin through glass chamber that had been

pre-saturated with the mobile phase of ethylacetate:

formaldehyde: acetic acid: water (10:1:1:2) till proper

separation of bands up to 8 cm height. After development,

SF

REF BC BL BS REF

Bergenin

REF

BS

BL

BC

REF

Bergenin

Bergenin

O

O

H CH2OH

H

OH

OH

H

H

OH

HO

CH3O

O

(a)

(b)

Fig. 3— Different Bergenia species: (a) reference sample (Under UV- 254) of bergenin; and (b) HPTLC profile of Bergenia rhizome

[REF, bergenin reference; BC, Bergenia ciliata; BL, Bergenia ligulata; BS, Bergenia stracheyi]


Bergenia ciliata

Dried rhizome

T.S. Rhizome showing Cork

(40X)

T.S. Rhizome showing Cork &

Cortical region (10X)

CK

CO

VB

CR

A portion of cortex showing

Rosette crystal (40X)

CR


Starch grains & Crystals (25X)

CR

ST


Starch grains (40X)

Cortex showing

Starch grains (40X)

ST

T.S. Vascular bundle (40X)

PH

XY

T.L.S. Rhizome (10X)

Vessels showing

Thickenings (40X)

VS

CR

ST

Plate-1 Macro and Microscopic characters of B. ciliata rhizome [ICO, Inner cortex; CK, Cork cells; IVB,

Plate 1—Macro and microscopic characters of B. ciliata rhizome [ICO, inner cortex; CK, cork cells; IVB, inner vascular bundle; OVB,

outer vascular bundle; CO, cortex; FR, fibre; ST, starch; VS, vessels; XY, xylem; ED, endodermis; PR, pericycle]


Plate-2 Macro and Microscopic characters of B. ligulata rhizome

Bergenia ligulata



A portion of cortex showing Vascular

bundles, Starch grains & Crystals (25X)



(40X)


Vascular bundle (25X)

Dried rhizome


Rosette crystal (40X)

CR

CK

CO

VB

PH

CR

XY

TR

VB

CO

VS

ST

TR

CR

T.S. Vascular bundle (40X)

PH

VS

TR


VS

TR

Plate 2—Macro and microscopic characters of B. ligulata rhizome [ICO, inner cortex; CK, cork cells; IVB, inner vascular bundle; OVB,



Bergenia stacheyi

Dried Rhizome

CK

CO

VB

CO

CO

PH

CR

ST

VS

CO

TR

PH

VS



T.S. Rhizome showing cortical region

with Starch grains & Crystals (25X)


Vascular bundles, (25X)

T.S. Vascular

bundle (40X)


& Cortical region (40X)


Rosette crystal & Starch Grains (40X)

CR

ST




VS

TR

VS

ST

TR

VS

ST

CR

Plate 3—Macro and microscopic characters of B. stracheyi rhizome [ICO, inner cortex; CK, cork cells; IVB, inner vascular bundle; OVB,



layer was dried with a dryer and bergenin was quanitifed

using Camag TLC scanner model 3 equipped with Camag

Wincats IV software. Following scan conditions were

applied: silt width, 6 mm x 0.45 mm; wavelength, 260

nm; and absorption-reflection mode. To prepare

calibration curves, stock solutions of bergenin (1 mg/ml)

were prepared and various volumes of solutions were

analyzed through HPTLC as mentioned above;

calibration curves of peak area vs. concentration were

also prepared. Bergenin identified at Rf 0.74 using

regression equation (y= 51.790x+7.075) and r2 at 0.996,

was found to be: B. ciliata, 5.68; B. ligulata, 5.73; and

B. stracheyi, 5.99%.

Results and Discussion

Rhizome can easily be differentiated on the basis of

organoleptic characters; odour and taste of rhizome is

quite characteristic and is aromatic with astringent taste

(Table 1, Plates 1-3). B. ciliata has large number of

starch grains; B. ligulata has maximum calcium oxalate

crystals while B. stracheyi has lesser amount of starch

grains. Physicochemical studies showed that B. stracheyi

has almost maximum percentage of all the

physicochemical parameters (total ash, 15.8; alcohol

soluble extracts, 13.83; water soluble extracts 16.83;

sugar, 5.5; and tannins, 7.86%), except starch and acid

insoluble ash, which were highest in B. ciliata.

Successive Soxhlet extraction from non-polar to polar

solvents (hexane, chloroform, acetone, alcohol, and

water) with powdered sample (5 g) of each species

(100 mesh) at 70°C showed that B. stracheyi was having

almost maximum percentage of all extractives as

compared to B. ciliata and B. ligulata, which have

almost similar percentages of extractives. Quantitative

analysis by HPTLC system showed that bergenin was

maximum in B. stracheyi (5.99%) followed by B. ciliata

(5.73%) and B. ligulata (5.68%). Thus, B. ciliata and

B. stracheyi can be a better source of bergenin and will

elicit the desired biological activity better than B. ligulata.

Characters Bergenia ciliata

Bergenia ligulata

Bergenia stracheyi

Macroscopic

Rhizome pieces solid, dark brown, barrel

shaped, cylindrical with ridges and

furrows having rootlets.

Texture hard.

Odour aromatic and taste astringent.

(Plate 1)

Rhizomes are compact, solid

somewhat cylindrical.

The outer surface is wrinkled,

furrowed, ridged and is covered with

root scars.


(Plate 2)

Rhizomes are compact, solid

somewhat cylindrical, covered with

brown leaf scars.

The outer surface is wrinkled,

furrowed, ridged and is covered with

root scars.


(Plate 3)

Microscopic T.S. rhizome shows cork divided into

two zones, outer zone is made of

compressed cells and inner multilayered

zone composed of thin-walled,

tangentially elongated cells followed by

a single layer of cork cambium and few

layers of tangentially elongated,

rectangular cells with intercellular

spaces. Cortical zone is parenchymatous

having few ever rosette crystals, large

number of starch grains and tanniferous

cells. Endodermis and pericycle is not

visible. Vascular bundles are arranged in

a ring and are conjoint, collateral and

open. Xylem consists of vessel elements,

tracheids, xylem parenchyma and xylem

fibres. (Plate. 1)

T.S. rhizome shows cork divided

into two zones, outer zone is made

of compressed cells and inner

multilayered zone composed of thin-

walled, tangentially elongated cells

followed by a single layer of cork

cambium and few layers of

tangentially elongated, rectangular

cells with intercellular spaces.

Cortical zone is parenchymatous,

full of rosette crystals (calcium

oxalate), less number of starch

grains and tanniferous cells.

Endodermis and pericycle is not

visible. Vascular bundles are

arranged in a ring and are conjoint,

collateral and open. Xylem consists

of vessel elements with simple pits,

tracheids with helical thickening,

xylem parenchyma and xylem

fibres. (Plate. 2)

T.S. rhizome shows cork divided

into two zones, outer zone is made

of compressed cells and inner

multilayered zone composed of thin-

walled, tangentially elongated cells

followed by a single layer of cork

cambium and few layers of

tangentially elongated, rectangular

cells with intercellular spaces.

Cortical zone is parenchymatous,

full of rosette crystals (calcium

oxalate), starch grains and

tanniferous cells. Endodermis and

pericycle is not visible. Vascular

bundles are arranged in a ring and

are conjoint, collateral and open.

Xylem consists of vessel elements

with simple pits, tracheids with

helical thickening, xylem

parenchyma and xylem fibres.

(Plate. 3)

Table 1— Macroscopic and microscopic characters of rhizomes of different Bergenia species


Conclusions

Parameters studied are very useful for the

identification of Bergenia species, which may be useful

to pharmaceutical industries for authentication, quality

control and standardization of these species in order to

identify the correct species used in the formulation.

Acknowledgement

Authors thank Director, NBRI for providing all

facilities to conduct this research work.

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botanical and phytochemical comparison of three bergenia species

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