Journal ofSRIV ScientificASTAV &A Industrial& RAWAT Research: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE SPECIES 65 Vol. 67, January 2008, pp.65-72

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 activity13,14 have also been reported. Inhibition of the High incidence of urinary calculi is reported from growth of urinary calcium hydrogen phosphate dehydrate British Isles, Scandinavian countries, northern Australia, crystals with aqueous extract of Tribulus terrestris 5 central Europe, , Pakistan, and Mediterranean (Zygophyllaceae) and B. ligulata has been reported . countries1. Calcium-containing stones are the most There are reports on ethnobotanical usage of Bergenia common (75% of all urinary calculi), which may be in species as an antilithic, for boils and blisters15, in urinary the form of pure calcium oxalate (50%) or calcium diseases, as antidiabetic, hemorrhoids, stomach disorders phosphate (5%) and a mixture of both (45%). Different and opthalmia16,17. Leaves are used for dissolving kidney mineral metabolisms are important in the formation of stones18. Rhizome is used as tonic, antipyretic, urinary stones or calculi2. There are many herbs in the antidiarrhoel, in opthalmia, kidney stones, and other Indian systems of medicine, which play important role urinary disorders19,20. contains β-sitosterol-D- in inhibition and removal of calcium oxalate crystals3,4. glucoside, bergenin21 and afzelechin22. A simultaneous The effect of aqueous extracts of some common determination of bergenin and gallic acid by HPTLC is medicinal was also studied on the growth and also reported in B. ligulata23. inhibition of calcium oxalate monohydrate (COM) This study presents comparative botanical and 5 crystals . phytochemical evaluation of B. ligulata (Wall) Eng., B. Different Bergenia species (Family: ), ciliata (Royal) Raizada and B. stracheyi Engl. distributed in South and East Asia and European countries, are important medicinal plants. In India, Materials and Methods rhizomes have been used for centuries in the Ayurvedic Plant materials were collected in October 2004 from formulations to dissolve kidney and bladder stones, in Almora (Uttrakhand, India), authenticated by Dr A K S leucorrhea, piles, and pulmonary affections6. Alcohol Rawat, matched with herbarium specimen, and stored extract of plant has exhibited significant analgesic, anti- in the Institute’s herbarium with following voucher inflammatory and diuretic properties7. Anti-bacterial, specimen numbers: LWG 222437BC; LWG 222438BL; anti-inflammatory and anti-tussive activity of LWG 222439BS, 2004. Rhizomes preserved in 70% B. ciliata8-10, besides anti-viral activity11,12 and antilithiatic ethanol for histological study. Microtome sections cut *Author for correspondence and stained with safranin and fast green, and Tel: +91 9415082210; Fax: +91 522 2207219 photographed with Nikon F70X camera24. E-mail: [email protected] 66 J SCI IND RES VOL 67 JANUARY 2008

45 - 56 . 53 . 184 . 40 161 . 39 39 40 28 - . 38 37

35 - 938 B. ciliata . 30 - 28 B. ligulata B. strachyi

es 25 g - a t n ce r

e 20 833

- . P 8 . 16 15 833 . 15 13 833

- . 11 11 15 833 833 . . . 9

10 862 8 8 - . 7 501 . 917 . 5 5 4 379 047 5 . . 573 3 .

- 3 2 2 . 7 . 25 1 . 0 0 0 - Moisture Total ash Acid-insoluble Alcohol Water soluble Sugar Starch Tannins content ash soluble extractive extractive Parameters Fig. 1—Comparative physico-chemical studies of different Bergenia specieses

20

17.92 18

16 14.9 13.98 14

12 11.14 s e g a t B. ciliata 9.72 n 10 9.46 e B. ligulata c

r 8.76 e B. strachyi 8.217 P 7.94 8

6

4

1.72 2 1.36 1.42 1 0.66 0.74

0 Hexane Chloroform Acetone Alcohol Water Solvents Fig. 2—Successive extractive values of different Bergenia speciess

Physico-chemical and HPTLC Studies fingerprint profile, which may be used as markers for Physico-chemical (Fig. 1) and phytochemical studies quality evaluation and standardization of the drug. (Fig. 2) were carried out as per standard procedures25-27 using shade dried powdered (100 mesh) plant material. Extraction of Plant Material A densitometric HPTLC analysis (Fig. 3) was also Air-dried (45-55°C) powdered rhizomes of three performed for the development of characteristic Bergenia species (each 1.0 g) were extracted with SRIVASTAVA & RAWAT: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE BERGENIA SPECIES 67 (a)

SF

Bergenin H CH2OH H OH O OH

CH3O OH H H O HO

O

Bergenin

REF BC BL BS REF

Bergenin

(b)

REF

BS

BL

BC

REF

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] methanol (10 ml). Extracts were concentrated under Linomat 5 automated TLC applicator with the nitrogen vacuum, redissolved in methanol, filtered and finally made flow providing a delivery speed of 150 nl/sec from up to 100 ml with methanol prior to HPTLC analysis. application syringe. These conditions were kept constant throughout the analysis of samples. Chromatographic Conditions Chromatography was performed on Merk HPTLC Detection and Quantification of Bergenin Following sample application, layers were developed precoated silica gel 60GF254 (20 cm x 20 cm) plates. Methanolic solutions of samples and standard compound in a Camag twin through glass chamber that had been bergenin of known concentrations were applied to the pre-saturated with the mobile phase of ethylacetate: layers as 6 mm-wide bands positioned 15 mm from the formaldehyde: acetic acid: water (10:1:1:2) till proper bottom and 15 mm from side of the plate, using Camag separation of bands up to 8 cm height. After development, 68 J SCI IND RES VOL 67 JANUARY 2008

CR

Dried rhizome

Bergenia ciliata

A portion of cortex showing

Rosette crystal (40X) CK

ST

CO

VB T.S. Rhizome showing Cork (40X) Cortex showing CR Starch grains (40X)

T.S. Rhizome showing Cork & PH Cortical region (10X)

CR XY

ST

A portion of cortex showing T.S. Vascular bundle (40X) A portion of cortex showing Starch grains (40X) Starch grains & Crystals (25X) VS

CR

ST

T.L.S. Rhizome (10X) Vessels showing Thickenings (40X) 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] SRIVASTAVA & RAWAT: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE BERGENIA SPECIES 69

Dried rhizome CR

Bergenia ligulata A portion of cortex showing Rosette crystal (40X)

CK

PH CO

VS T.S. Rhizome showing Cork (40X) VB

PH TR

CR

XY T.S. Vascular bundle (40X) T.S. Rhizome showing Cork & Cortical region (25X)

TR VS VB

A portion of cortex showing

Vascular bundle (25X) TR

CO VS

ST TR

CR T.L.S. Rhizome (40X)

T.L.S. Rhizome (25X) A portion of cortex showing Vascular bundles, Starch grains & Crystals (25X)

Plate-2 Macro and Microscopic characters of B. ligulata rhizome Plate 2—Macro and microscopic characters of B. ligulata 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] 70 J SCI IND RES VOL 67 JANUARY 2008

Dried Rhizome

Bergenia stacheyi

CK VS

CO TR

VB T.L.S. Rhizome (40X)

CO T.S. Rhizome showing Cork & Cortical region (10X) T.S. Rhizome showing Cork VS & Cortical region (40X) ST CO PH T.L.S. Rhizome (25X) CR TR ST VS VS T.S. Rhizome showing cortical region ST with Starch grains & Crystals (25X) CR CO T.L.S. Rhizome (10X) TR

T.S. Vascular CR PH bundle (40X) ST

VS A portion of cortex showing Rosette crystal & Starch Grains (40X) A portion of cortex showing Vascular bundles, (25X)

Plate 3—Macro and microscopic characters of B. stracheyi 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] SRIVASTAVA & RAWAT: BOTANICAL AND PHYTOCHEMICAL COMPARISON OF THREE BERGENIA SPECIES 71 layer was dried with a dryer and bergenin was quanitifed crystals while B. stracheyi has lesser amount of starch using Camag TLC scanner model 3 equipped with Camag grains. Physicochemical studies showed that B. stracheyi Wincats IV software. Following scan conditions were has almost maximum percentage of all the applied: silt width, 6 mm x 0.45 mm; wavelength, 260 physicochemical parameters (total ash, 15.8; alcohol nm; and absorption-reflection mode. To prepare soluble extracts, 13.83; water soluble extracts 16.83; calibration curves, stock solutions of bergenin (1 mg/ml) sugar, 5.5; and tannins, 7.86%), except starch and acid were prepared and various volumes of solutions were insoluble ash, which were highest in B. ciliata. analyzed through HPTLC as mentioned above; calibration curves of peak area vs. concentration were Successive Soxhlet extraction from non-polar to polar solvents (hexane, chloroform, acetone, alcohol, and also prepared. Bergenin identified at Rf 0.74 using regression equation (y= 51.790x+7.075) and r2 at 0.996, water) with powdered sample (5 g) of each species was found to be: B. ciliata, 5.68; B. ligulata, 5.73; and (100 mesh) at 70°C showed that B. stracheyi was having B. stracheyi, 5.99%. almost maximum percentage of all extractives as compared to B. ciliata and B. ligulata, which have Results and Discussion almost similar percentages of extractives. Quantitative Rhizome can easily be differentiated on the basis of analysis by HPTLC system showed that bergenin was organoleptic characters; odour and taste of rhizome is maximum in B. stracheyi (5.99%) followed by B. ciliata quite characteristic and is aromatic with astringent taste (5.73%) and B. ligulata (5.68%). Thus, B. ciliata and (Table 1, Plates 1-3). B. ciliata has large number of B. stracheyi can be a better source of bergenin and will starch grains; B. ligulata has maximum calcium oxalate elicit the desired biological activity better than B. ligulata.

Table 1— Macroscopic and microscopic characters of rhizomes of different Bergenia species Characters Bergenia ciliata Bergenia ligulata Bergenia stracheyi

Macroscopic Rhizome pieces solid, dark brown, barrel Rhizomes are compact, solid Rhizomes are compact, solid shaped, cylindrical with ridges and somewhat cylindrical. somewhat cylindrical, covered with furrows having rootlets. The outer surface is wrinkled, brown leaf scars. Texture hard. furrowed, ridged and is covered with The outer surface is wrinkled, Odour aromatic and taste astringent. root scars. furrowed, ridged and is covered with (Plate 1) Odour aromatic and taste astringent. root scars. (Plate 2) Odour aromatic and taste astringent. (Plate 3) Microscopic T.S. rhizome shows cork divided into T.S. rhizome shows cork divided T.S. rhizome shows cork divided two zones, outer zone is made of into two zones, outer zone is made into two zones, outer zone is made compressed cells and inner multilayered of compressed cells and inner of compressed cells and inner zone composed of thin-walled, multilayered zone composed of thin- multilayered zone composed of thin- tangentially elongated cells followed by walled, tangentially elongated cells walled, tangentially elongated cells a single layer of cork cambium and few followed by a single layer of cork followed by a single layer of cork layers of tangentially elongated, cambium and few layers of cambium and few layers of rectangular cells with intercellular tangentially elongated, rectangular tangentially elongated, rectangular spaces. Cortical zone is parenchymatous cells with intercellular spaces. cells with intercellular spaces. having few ever rosette crystals, large Cortical zone is parenchymatous, Cortical zone is parenchymatous, number of starch grains and tanniferous full of rosette crystals (calcium full of rosette crystals (calcium cells. Endodermis and pericycle is not oxalate), less number of starch oxalate), starch grains and visible. Vascular bundles are arranged in grains and tanniferous cells. tanniferous cells. Endodermis and a ring and are conjoint, collateral and Endodermis and pericycle is not pericycle is not visible. Vascular open. Xylem consists of vessel elements, visible. Vascular bundles are bundles are arranged in a ring and tracheids, xylem parenchyma and xylem arranged in a ring and are conjoint, are conjoint, collateral and open. fibres. (Plate. 1) collateral and open. Xylem consists Xylem consists of vessel elements of vessel elements with simple pits, with simple pits, tracheids with tracheids with helical thickening, helical thickening, xylem xylem parenchyma and xylem parenchyma and xylem fibres. fibres. (Plate. 2) (Plate. 3)

72 J SCI IND RES VOL 67 JANUARY 2008

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