Pharmacognostical studies on the root and rhizome of...

Indian Journal of Natural Products and Resources

Vol. 3(3), September 2012, pp. 371-385

Pharmacognostical studies on the root and rhizome of Nymphoides hydrophylla

(Linn.) O. Kuntze –An alternate source for Tagara drug

V Madhavan1, M Jayashree

1, S N Yoganarasimhan

1*, M Gurudeva

2, R Deveswaran

3 and R Mythreyi

1

1Department of Pharmacognosy, MS Ramaiah College of Pharmacy, Bangalore-560 054, India 2Department of Botany, VV Pura College of Science, Bangalore

3Department of Pharmaceutics, MS Ramaiah College of Pharmacy, Bangalore

Received 13 October 2011; Accepted 19 January 2012

Tagara is an important drug used in Ayurvedic medicine for the treatment of several diseases. The accepted botanical

source of Tagara is Valeriana jatamasni Jones, although different species of Nymphoides Hill are used by the physicians.

The pharmacognostical evaluation of the root and rhizome of Nymphoides hydrophylla, a potential alternative source for

Tagara is presented in this paper. Important details like morphology of the plant, macro-, microscopical characters,

macerate, histochemical tests, UV studies of the root and rhizome along with physico-chemical constants, phytochemical

analysis and HPTLC finger print profile are presented, all of which will be useful in the standardization of this drug.

Isolation of β-sitosterol, betulinic, salicylic and tannic acids are reported for the first time from N. hydrophylla. The

pharmacognostical and phytochemical studies help in the identification of N. hydrophylla from other species used as Tagara.

Keywords: Nymphoides hydrophylla, Pharmacognosy, Root, Rhizome, HPTLC, Tagara.

IPC code; Int. cl. (2011.01)—A61K 36/00

Introduction Tagara is an important drug used in Ayurvedic

medicine for the treatment of diseases like anaemia

(pandu), epilepsy (apasmara), fever (jwara), jaundice

(kamala), mental disorders (unmada), tuberculosis

(yakshma) and also as a general and brain tonic1.

Valeriana jatamansi Jones (Valerianaceae) is the

accepted botanical source of Tagara which constitutes

as one of the ingredients in ayurvedic formulations like

Bala taila, Bilvadi gutika, Dasanga lepa, Jatiphaladya

curna, Karpuradyarka, Maha kalyanaka ghrita, Maha

narayana taila, Puga khanda, Sahacaradi taila,

Srikhandasava, to name a few2. In South India, a drug

under the name Granthika tagara (Kannada),

botanically identified as Nymphoides macrospermum

Vasudevan (Menyanthaceae) is used in place of

Tagara (Valeriana jatamansi) for the same ayurvedic

preparations, under the same formulations supporting

the fact that Granthika tagara (N. macrospermum) may

have similar therapeutic properties to that of Tagara

(V. jatamansi); furthermore, it is often found that

Granthika tagara is an admixture of different species

of Nymphoides Hill found in S. India, viz.

N. aurantiacum (Dalz.) O. Kuntze, N. hydrophylla

(Lour.) O. Kuntze, N. indica (Linn.) O. Kuntze and

N. macrosperumum Vasudevan3. N. indica is used as

an antiscorbutic and febrifuge, and as a substitute for

chiretta [Swertia chirayita (Roxb. ex Flem.) Kars.] to

treat fever and jaundice4. Literature review revealed

that while pharmacognostical studies have been carried

out on the roots and rhizome of N. macrospermum5 and

N. indica6, no work is available on N. hydrophylla

7-9.

Hence, the present investigation on the root and

rhizome of N. hydrophylla was undertaken.

Materials and Methods

Plant material was collected from a water tank in the

vicinity of Nagercoil, Kanyakumari district, Tamil

Nadu, during November 2007, processed into

herbarium specimen10

; voucher herbarium specimen

(Jayashree 025) and a sample of the crude drug are

preserved at the herbarium and crude drug museum of

the Department of Pharmacognosy, M S Ramaiah

College of Pharmacy, Bangalore (MSRCP). The plant

material was identified following local floras11-13

and

authenticated by S N Yoganarasimhan, Plant

Taxonomist. For pharmcognostical studies, a small

quantity of rhizome and root were preserved in 70%

alcohol; free hand sections were taken for macro- and

microscopical observations14,15

. Photomicrographs

were obtained by observing the free hand sections

under a compound binocular microscope (Olympus-

___________

*Correspondent author:

E-mail: [email protected]; [email protected]

Phone: +91-80-23608942

INDIAN J NAT PROD RESOUR, SEPTEMBER 2012

372

CH20i model) with a built-in analogue camera

(CMOF, 1.4 mega pixel). Computer Images were

captured using AV Digitaliser fitted with a Grand VCD

2000–Capture Guard. Measurements of cells and

tissues (in µm) of the transverse section and macerate

were carried out using Micro Image Lite Image

Analysis Software (Cybernetics, Maryland, USA) and

are provided in parenthesis. Physicochemical studies,

ultra-violet and chromatographic studies were carried

out following standard methods16-20

. Vernacular names

are provided following Gurudeva21

.

Isolation of

β-sitosterol was carried out as per Gerhardt et al22

. The

total phenolic compounds were determined following

Folin-Ciocalteace method23

and HPTLC was carried

out using a Camag HPTLC system with Linomat V

sample applicator, a Camag 3 TLC Scanner and

WINCATS 4 software for interpretation of data. An

aluminum plate (20 × 10 cm) pre-coated with silica gel

60F254 (E Merck) was used as adsorbent. The plates

were developed using toluene:chloroform:ethanol

(28.5:57:14.5) for alcohol and aqueous extracts in a

Camag twin trough chamber and scanned under 254,

366 and 425 nm. The developed plate was derivatised

by dipping in Libermann Burchard’s reagent and

drying at 110°C in a hot air oven for 10 min, scanned

under all 3 wavelengths and photo documented both

before and after derivatisation using a Camag 3

Reprostar. For the identification of phenolic

compounds, 10 µl of tannic acid solution and 5 µl of

salicylic acid solution were applied and developed with

ethyl acetate: benzene: formic acid (9:11:0.5) and

scanned under 254, 366 and 425 nm. The developed

plate was photo documented at 254 nm. For the

identification of betulinic acid, both extracts were

developed with n-hexane: ethyl acetate: glacial acetic

acid (7:3:0.03), derivatised with Anisaldehyde

H2SO4(Ref.24)

, scanned at 425 nm and photodocumented.

For the identification of β-sitosterol, toluene:diethyl

ether (40:40) was used, derivatised with Vanillin-

H2SO4, dried at 105°C(Ref. 25)

, scanned at 425 nm and

photo documented. The Rf values and peak area of the

extracts and the isolated sample were interpreted by

using a WINCATS 4 software. The developed plates

were photo documented under 425 nm using a Camag

3 Reprostar. All the solvents used were of HPLC grade.

Results Plant morphology

Nymphoides hydrophylla (Linn.) O. Kuntze, Rev.

Gen. Pl. 429. 1891. Menyanthes cristata Roxb. Pl. Cor.

2: 3. t. 105, 1798; Limnanthemum cristatum Griseb.

Gen. and Spec. Gent. 342. 1839; Clarke in Hook. f., Fl.

Brit. India, 4: 131. 1883; Gamble, Fl. Pres. Madras, 2:

883, 2005 (reprint); Menyanthes hydrophylla Lour., Fl.

Cochinch. 1:129, 1790.

Aquatic herbs with long floating stem (stolon).

Rhizome short, erect, with petiole-like branches

reaching the surface of water and producing a node

from which arise a tuft of adventitious roots, a cluster

of flowers, a single floating leaf and a single branch,

which again proceeds similarly. Leaves orbicular,

deeply cordate at base, often purplish, to 10 cm in

diameter, lobes rounded, faintly crenate, pale green

above, glandless and with prominent nerves beneath;

petioles to 3.5 cm long. Flowers white, with yellow

centre, delicate, with a ring of white hairs round the

throat, numerous, in dense clusters; pedicels to 6 cm

long, unequal. Calyx divided almost to the base,

oblong-lanceolate, obtuse. Corolla white, when

expanded; lobes obovate, rounded at apex, glabrous,

with a broad longitudinal crest down the middle of

each lobe, margins not ciliate. Capsule broadly ovoid

or subglobose. Seeds numerous with prominent, small,

slightly glochidiate tubercles, scabrous, pale yellowish-

brown (Plates 1, 2).

Nymphoides hydrophylla usually flowers and fruits in

the winter season. In this species polystely occurs in the

cortical region of the stolon and in the petiole of the

leaves. It is very common throughout India and is

gregarious in habit, generally growing near the margins

of tanks and lakes. Also found in Sri Lanka, China and

Malaysia26

.

Vernacular names

Tamil: Akacavalli; Sanskrit: Kumudvathi, Seethali,

Kalanu saariva, Tagara; Kannada: Anthara thaavare;

Marathi: Kolare chikal; Hindi: Tagarmul, Cumuda,

Ghainchu, Kumudini, Lodh, Baank; Telugu: Anthara

thaarama, Chiri alli, Pitta kaluva; Bengali: Panchuli,

Chandmalla; Malayalam: Tsjeroea–citamber;

Manipuri: Tharo–macha.

Macro- and microscopical characters of the root and rhizome

Macroscopical characters of the root

Roots arise in tufts all around the rhizome, running

vertically downwards, very slender, spongy, brownish,

measuring to 32 cm long when fresh, and has no

characteristic odour or taste. Young fresh roots are

minutely hairy and white to brownish while dry roots

are slender, brownish (Plates 3, 4).

Macroscopical characters of the rhizome

The rhizomes are slender, running horizontally,

cone-like with many leaf bases, rough, brittle, brownish

MADHAVAN et al: PHARMACOGNOSY OF NYMPHOIDES HYDROPHYLLA

373

without, white within, with several fragments of roots

arising from different points, measuring to 9 cm long;

it possesses no characteristic odour or taste; dry

rhizomes are blackish with a few slender roots and

covered by several leaf bases (Plates 5, 6); when

transversely cut, it shows a broad whitish cortex and a

narrow creamy-white central stele (Plate 7).

Microscopical characters of the root

A transverse section of the root is wheel-like and

exhibits an epidermis, an outer, middle and inner

cortex, a stele region with 7 vascular strands (Plate 8).

Epidermis is single layered, made up of compactly

arranged rectangular cells, measuring 14-18-21 × 16-

21-30 µm; a few root hairs arise from the epidermal

Plates 1-14Macro and microscopical characters of Nymphoides hydrophylla root and rhizomes —1. Habit , 2. Flower, 3. Vertical roots,

4. Young roots , 5. Dried roots, 6. Dried rhizome, 7. Rhizome cut open, 8. T.S. showing different regions, 9. Portion showing epidermis and

outer cortex, 10. Cortex with intercellular spaces, 11. Cortex showing plug-like structures, 12. Portion showing middle cortex and

aerenchyma, 13. Portion showing inner cortex and stele, 14. Stele enlarged showing pith. AC-air chamber; AER-aerenchyma; CAM-cambium;

COR-cortex; CT-conjunctive tissue; END-endodermis; EP-epidermis; HY-hypodermis; IC-inner cortex; ICS-intercellular space; MC-middle

cortex; MXY-metaxylem; OC-outer cortex; PC-pericycle; PH-phloem; PI-pith; PLS-plug-like structure; PXY-protoxylem; RH-root hair; SCD-

sclereid; SG-starch grain; ST-stele; VB-vascular bundle; VT-vascular trace; XY-xylem


374

cells. Next to the epidermis is the outer cortex which

is 5 to 6 layered, consisting of compactly arranged

parenchymatous cells with intercellular spaces,

measuring 26-49-65 × 23-36-48 µm (Plates 9, 10),

followed by a broad zone of middle cortex comprising

of vertically arranged layers of parenchymatous cells,

traversed by large air cavities comprising the

aerenchyma, measuring 20-32-51 × 16-32-46 µm

(Plate 11); the aerenchyma cells in young roots are

interconnected by plug-like structures (Plate 12). Next

to the aerenchyma lies 3-4-layered inner cortex

consisting of compactly arranged oval shaped

parenchymatous cells measuring 27-45-66 × 17-34-33

µm (Plate 13). Endodermis cells measure 16-24-33 ×

16-15-25 µm and pericycle cells measure 5-7-8 × 9-

11-14 µm, single layered which is followed by the

stele. Vascular bundles are radially arranged; xylem is

exarch, cells measure 5-10-15 × 10-14-18 µm, with a

central parenchymatous pith, cells measuring 6-8-11 ×

7-11-16 µm (Plate 14). Starch is absent in pith cells.

Macerate of the root exhibited: 1. Parenchyma cells

which are oval and compactly arranged, measuring

40-63-80 × 301-38-50 µm (Plates 15, 16). 2. Vessels

with reticulate and spiral thickenings, measuring

84-105-122 × 5-9-12 µm) (Plates 17, 18).

Microscopical characters of rhizome

Transverse section of rhizome exhibits epidermis

which is one layered, made up of narrow, tangentially

elongated parenchymatous cells, measuring 15-20-25

× 21-28-40 µm. It is followed by 4-5-layered

hypodermis, made up of compactly arranged

parenchymatous cells, measuring 20-29-38 × 29-35-

42 µm. Next to the hypodermis lies a broad

aerenchymatous cortex, cells measuring 14-21-33 ×

10-15-23 µm and large air cavities, measuring 13-20-

31×18-24-29 µm (Plates 19-21). Sclereids are present

in the cortex, protruding into the air cavities (Plate

22). The innermost few layers of the cortex consists

of compactly arranged cells, without air cavities; a

few root traces are found in the cortex (Plate 23)

giving the appearance of individual stele which

represent a pseudo polystele condition. Stele consists

of concentrically arranged vascular bundles, with

conjunctive tissue found in between. Vascular bundles

are conjoint, collateral, open, endarch; metaxylem

cells measure 166-242-323 × 68-98-124 µm;

cambium is multilayered, cells measuring 5-10-20 ×

3-8-15 µm (Plates 24-26). Pith is large,

parenchymatous, cells measuring 20-27-41 × 10-15-

18 µm (Plate 27). Starch grains are found in the

cortex and pith; they are simple, solitary or

aggregated, oval or rounded (Plates 28, 29).

Macerate of the rhizome exhibited: i. Epidermal

cells which are compactly arranged, measuring 13-21-

28 × 10-15-28 µm (Plate 30); ii. Parenchyma cells

which are thin walled, measuring 23-33-51 × 16-22-

24 µm (Plate 31); iii. Fibers which are long and with

pointed ends, having a broad lumen, measuring 331 ×

9 µm (Plate 32); iv. Sclereids of different sizes and

shapes- U-, Y-, Z-, and H-shaped, some are with thick

walled arms, some with fragmented arms, measure

55-106-121 × 9.5-12.5-16.6 µm (Plates 33 to 39); v.

Tracheids which are long, narrow and with pitted

thickenings, measure 505 × 20 µm (Plate 40); vi.

Vessels of different size and shape, linear or drum

shaped, with reticulate and spiral thickenings,

measure 30-72-133 × 13-16-24 µm (Plates 41 to 43). Histochemical tests

The sections of root and rhizome when treated with

phloroglucinol and HCl turned pink indicating the

presence of lignin; with ferric chloride turned black,

indicating presence of tannin; with iodine solution,

rhizome sections turned blue indicating presence of

starch whereas root sections did not turn blue,

indicating absence of starch.

Powder studies

The powder is greenish-brown, possesses no

characteristic odour and taste. When treated with

phloroglucinol and HCl, stained with safranin,

following elements were observed as: i. Fragments of

epidermal peel showing compactly arranged cells

Plates 15-18Macerate of root— 15, 16. Parenchyma cells, 17, 18. Vessels with reticulate and spiral thickenings.


375

(Plate 44); ii. Aerenchyma cells with intercellular spaces

(Plate 45); iii. Sclereids of different size and shape, some

with thick walled arms, some with fragmented arms

(Plates 46 to 49); iv. Fragments of vessels with reticulate

and spiral thickenings (Plate 50, 51).

Ultra-violet studies

The powder exhibited the following fluorescence

under 425 nm, 254 nm, and 365 nm, respectively when

treated with different reagents. Powder as such

emitted royal crown, dark green, nickel grey; with dil.

Plates 19 to 29Microscopical characters of rhizome —19. Portion showing epidermis, hypodermis and cortex, 20. Cortex showing

aerenchyma and air cavity, 21. Cortex showing intercellular spaces, 22. Portion showing sclereid in cortical cells, 23. Portion showing

vascular trace and inner cortex, 24, 25. Portion showing stele, 26. Portion showing phloem and vascular cambium, 27. Pith region, 28.

Starch grains in cortical cells, 29. Starch grains in pith cells. AC-air chamber; AER-aerenchyma; CAM-cambium; COR-cortex; CT-

conjunctive tissue; END-endodermis; EP-epidermis; HY-hypodermis; IC-inner cortex; ICS-intercellular space; MC-middle cortex; MXY-

metaxylem; OC-outer cortex; PC-pericycle; PH-phloem; PI-pith; PLS-plug-like structure; PXY-protoxylem; RH-root hair; SCD-sclereid;

SG-starch grain; ST-stele; VB-vascular bundle; VT-vascular trace; XY-xylem


376

Ammoniacal solution gave teak brown, dark green,

aquamarine; with ethanol (70%) gave brown, french

blue, satin blue; with 1N methanolic NaOH gave teak

brown, dark iced tea, light iced tea; with 1N ethanolic

NaOH gave teak brown, wild purple, wild lilac; with

50% H2SO4 royal crown, walnut brown, bottle green;

with 50% HNO3 gave mid buff, walnut brown, satin

brown; with 5% KOH gave teak brown, walnut

brown, satin brown; with 1N HCl gave timber brown,

satin grey, smoke grey; with acetone gave teak brown,

Plates 30 to 43Macerate of rhizome—30. Epidermal cells, 31. Parenchyma cells, 32. Fibre, 33 to 39. Sclereids, 40. Tracheid, 41 to 43. Vessels.

Plates 44 to 51Powder studies— 44. Epidermal peel, 45. Aerenchyma cells, 46 to 49. Sclereids, 50, 51. Vessels with reticulate and spiral

thickenings.


377

royal blue, grey (the colours are based on the Asian

paints limited, Mumbai).

Physico-chemical studies

The moisture content percent of the drug was found

to be 10.35, total ash 8.5, acid insoluble ash 7.6, water

soluble ash 0.3, sulphated ash 10.0, alcohol soluble

extractive 12.0 and water soluble extractive 22.0.

Successive Soxhlet % extractive values, and the

colour and consistency were found to be: petroleum

ether (60-80ºC) (olive-green, solid, 2.2), chloroform

(walnut brown, solid, 1.48), acetone (brown, sticky

mass, 4.24), ethanol (brown, solid, 3.7) and aqueous

extract (walnut brown, solid, 6.66).

Phytochemical analysis

The petroleum ether (60-80ºC), benzene,

chloroform, acetone, ethanol (95%) and chloroform-

water (2%) (aqueous) extracts were subjected to

preliminary phytochemical analysis. It was found that

carbohydrates, glycosides, phenolic compounds,

tannins and saponins were all present in ethanol and

aqueous extracts, coumarins in ethanol extract, fixed

oils and fats in pet. ether and benzene extracts, phenols,

tannin and flavonoids in acetone, ethanol and water

extracts, saponins, phytosterols in pet ether, benzene,

acetone and ethanol extracts whereas alkaloids,

flavonoids, gums and mucilage, proteins and amino

acids were absent in all extracts; besides traces of

volatile oil was found to be present on hyrodistillation.

Determination of total phenolic compounds

The total polyphenolic compounds found in the

aqueous extract was 1.11 (±0.318) (mg/g) and 1.80

(± 0.35) (mg/g) (mean ± SEM) in alcohol extract

(Figure 1).

Chromatographic studies

HPTLC profile of aqueous and alcohol extract

Before derivatisation, the chromatogram was

developed in toluene:chloroform:ethanol

(28.5:57:14.5). The aqueous extract revealed 10

phytoconstituents having Rf 0.13, 0.14, 0.20, 0.22,

0.28, 0.31, 0.40, 0.47, 0.58, 0.86 (Figure 2a) and the

alcohol extract gave 10 phytoconstituents having Rf

0.13, 0.16, 0.25, 0.28, 0.31, 0.35, 0.40, 0.47, 0.76,

0.87 under 254 nm (Figure 2b). At 366 nm, aqueous

extract revealed 1 phytoconstituent having Rf 0.39

(Figure 2c) and alcohol extract revealed 3

phytoconstituents having Rf 0.37, 0.53, 0.65

(Figure 2d). At 425 nm, aqueous extract revealed 2

phytoconstituents having Rf 0.31, 0.42 (Figure 2e) and

alcohol extract revealed 7 phytoconstituents having

Rf 0.12, 0.15, 0.28, 0.37, 0.53, 0.68 and 0.75 (Figure

2f). The chromatograms of both aqueous and alcohol

extracts were observed under 254 nm (Figure 3a, 3b),

366 nm (Figure 3c, 3d) and 425 nm (Figure 3e, 3f)

and Rf values of prominent spots are represented in

respective figures.

The plate was derivatised with Libermann-

Buchardt reagent and scanned under all the three

wavelengths. At 254 nm, aqueous extract revealed 17

constituents having Rf 0.11, 0.15, 0.16, 0.20, 0.24,

0.28, 0.34, 0.41, 0.46, 0.48, 0.51, 0.52, 0.61, 0.65,

0.68, 0.82, 0.86 (Figure 4a) and alcohol extract

revealed 13 phytoconstituents having Rf 0.14, 0.19,

0.23, 0.27, 0.30, 0.36, 0.38, 0.45, 0.50, 0.73, 0.81,

0.87, 0.99 (Figure 4b). At 366 nm, aqueous extract

revealed 5 phytoconstituents having Rf 0.32, 0.33,

0.40, 0.63, 0.86 (Figure 4c) and 11 phytoconstituents

in alcohol extract having Rf 0.10, 0.15, 0.23, 0.27,

0.32, 0.38, 0.45, 0.50, 0.59, 0.81, 0.87 (Figure 4d). At

425 nm, aqueous extract revealed 8 phytoconstituents

having Rf 0.11, 0.17, 0.21, 0.26, 0.32, 0.33, 0.42, 0.50

(Figure 4e) and the alcohol extract revealed 11

phytoconstituents having Rf 0.14, 0.23, 0.28, 0.30,

0.36, 0.38, 0.45, 0.50, 0.62, 0.81, 0.87 (Figure 4f).

The chromatograms of both aqueous and alcohol

extract were observed under 254 nm (Figures 5a, 5b),

366 nm (Figures 5c, 5d) and 425 nm (Figures 5e, 5f)

and Rf values of prominent spots are represented in

respective figures.

Identification of phenolic compounds

To identify the presence of phenolic compounds in

aqueous and alcohol extracts, phenolic compounds

such as tannic acid and salicylic acid were used as

standards. Standard tannic acid was identified having

Rf 0.42 (Figure 6a) and standard salicylic acid was

detected at Rf 0.57 (Figure 6b) at 254 nm. Both

Figure 1Calibration curve for total phenolic contents.


378

aqueous and alcoholic extracts at 254 nm resolved into

15 and 14 phytoconstituents having Rf 0.42, 0.45, 0.48,

0.50, 0.53, 0.55, 0.57, 0.59, 0.62, 0.64, 0.67, 0.70, 0.71,

0.73, 0.75 (Figure 7a) and having Rf 0.42, 0.44, 0.48,

0.54, 0.55, 0.57, 0.61, 0.62, 0.65, 0.67, 0.69, 0.70, 0.72,

0.73 (Figure 7b), respectively in ethyl acetate: benzene:

formic acid (9:11:0.5) solvent system. The peaks and

spots having Rf 0.42 and 0.57 corresponded to that of

tannic acid and salicylic acid, respectively in both the

extracts which was further confirmed by overlay

spectrum of standard phenolic compounds (Figures 8-10). Identification of betulinic acid

The Rf of standard betulinic acid24

is 0.59 which was

detected in both the aqueous and alcohol extracts under

425 nm (Figures 11a & b, 12).

Figure 3a-fChromatograms before derivatisation—a &

b-aqueous and alcohol extract at 254 nm; c & d- aqueous and

alcohol extract at 366 nm; e & f- aqueous and alcohol extract

at 425 nm.

Figure 2a-fHPTLC profile before derivatisation—a & b- Aqueous and alcohol extract at 254 nm; c & d- Aqueous and alcohol extract at

366 nm; e & f- Aqueous and alcohol extract at 425 nm.


379

Isolation and identification of β-sitosterol

The % yield of the isolated compound was found to

be 24% and the melting point 136-137°C. It was

subjected to IR analysis and the peaks obtained were

recorded. The peaks of the isolated compound was

observed to be as follows: Peak 3200-3550 and 1330-

1420/cm was due to O-H stretching , peak 2937 and

675-900/cm-was due to aromatic C-H bending, while

peak 1608-1456/cm was due to aromatic C-C

stretching (Figure 13). HPTLC profile of the aqueous

extract gave a peak having Rf 0.56 (Figure 14a),

alcohol extract (Rf 0.55) (Figure 14b), chloroform

extract (Rf 0.56) (Figure 14 c) and isolated β-sitosterol

(Rf 0.56) (Figure 14d) at 425 nm. Rf of standard

sitosterol was found to be 0.55(Ref. 25)

. Rf 0.54 was

detected in both aqueous and chloroform extracts and

Rf 0.53 in alcohol extract which were identified as

β-sitosterol (Figure 15) and confirmed by IR

spectra (Figure 13).

Diagnostic characters

N. hydrophylla plant and the drug consisting of

roots and rhizome are identified by the following

characters:

Figure 4a – fHPTLC profile after derivatisation — a & b- Aqueous and alcohol extract at 254 nm; c & d- Aqueous and alcohol extract

at 366 nm; e & f- Aqueous and alcohol extract at 425 nm.

Figure 5a-fChromatograms after derivatisation— a & b- aqueous

and alcohol extract at 254 nm; c & d- aqueous and alcohol extract at

366 nm; e & f- aqueous and alcohol extract at 425 nm.


380

Figure 6a-bHPTLC of standard Tannic acid at 254 nm (Rf 0.42) and standard Salicylic acid at 254 nm (Rf 0.57).

Figure 7a-b—HPTLC of aqueous extract at 254 nm (Rf 0.42-tannic acid) and alcohol extract at 254 nm (Rf 0.42-tannic acid and 0.57-

salicylic acid).

Figure 8—Overlay spectrum of tannic acid, aqueous and alcohol extracts.


381

1. Aquatic free-floating habit; 2. Presence of corolla

lobes which are entire, white and seeds with

glochidiate tubercles; 3. Presence of large

aerenchyma; 4. Presence of different types of

sclereids in the rhizome; 5. Vessels of spiral and

reticulate thickenings in both the root and rhizome.

Discussion

The stagnant and depleting ayurvedic materia

medica is replenished by bringing into light new plant

sources for some useful and established ayurvedic

drugs like Tagara, whose accepted botanical sources

are either becoming endangered or are not available

in adequate quantities for medicinal preparations26

.

Nymphoides Hill is a genus of aquatic flowering

plants belonging to the family Menyanthaceae. It

consists of 20 species, out of which N. hydrophylla

(Lour.) O. Kuntze, N. macrospermum Vasudevan, N.

aurantiacum (Dalz.) O. Kuntze, N. indica (Lour.) O.

Kuntze, N. parvifolium (Griseb.) O. Kuntze, N.

peltata (S.G. Gmel.) O. Kuntze, N. sivarajanii Joseph

and N. krishnakesara Joseph and Sivar. are found in

India27,28,29

. The accepted botanical source of Tagara

is Valeriana jatamansi Jones. In South India,

Granthika Tagara (Kannada), botanically identified

as Nymphoides macrospermum, is used as Tagara in

several therapeutic preparations. Granthika Tagara is

often found as an admixture of above mentioned

species of Nymphoides occurring in South India. Also,

species like V. hardwickii Wall. var. arnottiana (Wt.)

C.B. Clarke, V. beddomei C.B. Clarke, V. hookeriana

Wt., V. leschenaultiana DC. and Cryptocoryne

spiralis (Retz.) Fisch. ex Wydl., are also treated as

Figure 10—Chromatogram of phenolic compounds (alcohol and

aqueous extracts, standard and salicylic acid at 254 nm).

Figure 9—Overlay spectrum of salicylic acid, aqueous and alcohol extracts at 254 m.


382

potential candidates of Tagara30,31

.

The different

species of Nymphoides, viz.. N. macrospermum, N.

hydrophylla and N. indica does not exhibit much

differences exomorphologically. The corolla is pure

white in N. macrospermum and N. hydrophylla

whereas it is yellow in N. indica. The seeds are larger

in size in N. macrospermum while they are very small

in N. hydrophylla and N. indica. The roots are of

spongy and stout type in N. macrospermum while it is

of only spongy type in N. hydrophylla and N. indica.

The rhizome and root of all the three species show

similar characters microscopically5,6,29

. Ultra-Violet

analysis of the powder gave characteristic

fluorescence which helps to distinguish the drug from

other species considered as Tagara. Preliminary

phytochemical analysis of N. hydrophylla revealed the

presence of carbohydrates and glycosides, coumarins,

phytosterols, saponins, phenolic compounds and

tannins and traces of volatile oil (present work)

whereas steroids, phenols, tannins, saponins, sugars,

iridoid or monoterpenic derivative, known as

valepotriates, an iridoid ester glycoside and essential

oils are some of the phytoconstituents present in V.

jatamansi32

which is the accepted source of Tagara.

In the alternate sources of Tagara, viz. N.

macrospermum, steroids, phenols, tannins, saponins,

sugars and in N. indica, glycosides, phytosterols,

saponins, phenolic compounds, gums and mucilage

and traces of volatile oil have been reported33

.

N. macrospermum gave 3 phytoconstituents in

petroleum ether extract, while chloroform extract

gave 4 phytoconstituents; the alcoholic extract of N.

indica gave 7 phytoconstituents, while the aqueous

extract gave 9 phytoconstituents5, 6

; both aqueous and

alcoholic extracts of N. hydrophylla gave 10

phytoconstituents before derivatisation (present

work). A triterpenoid compound, β-sitosterol reported

in the alcohol extract of roots and rhizomes of

N. macrospermum was also found in N. hydrophylla.

Betulinic acid from the aqueous and alcohol extracts

and β-sitosterol from aqueous and chloroform extracts

were also detected in N. hydrophylla using HPTLC.

The sedative and anticonvulsant property in

N. macrospermum34

and the anticonvulvasant

property in N. indica35

are reported which are also

assigned to Tagara in Ayurveda1.

Conclusion

The taxonomical characters dealing with the

exomorphology of N. hydrophylla, a potential source

of the drug Tagara in Ayurveda, is described to helps

in the identification of the plant in the field. Other

studies, viz. macro- and microscopical studies of the

drug (root and rhizome), chromatographic studies,

Figure 11—HPTLC of aqueous extract of Betulinic acid at 425 nm, alcohol extract of Betulinic acid at 425 nm.

Figure 12—Chromatogram of Betulinic acid in alcohol and

aqueous extracts at 425 nm.


383

phytochemical analysis, etc carried out during this

study will help in evolving diagnostic characters, in

determining pharmacopoeial parameters,

standardisation of therapeutic formulations, besides in

the isolation and identification of bioactive

principles/biomarkers. HPTLC fingerprinting of

tannic acid and salicylic acid was carried out in

alcohol and aqueous extracts and betulinic acid from

aqueous and alcohol extracts and β-sitosterol from

aqueous and chloroform extracts were detected for the

first time in this species. N. hydrophylla which is an

allied species of N. macrospermum and N. indica may

Figure 13—IR spectra of β-sitosterol.

Figure 14(a-d) — HPTLC of β-sitosterol in aqueous extract at 425 nm, in alcohol extract at 425 nm, in chloroform extract at 425 nm, isolated

β-sitosterol in alcohol extract at 425 nm


384

serve as a substitute source of Tagara as it may have

similar therapeutic properties.

Acknowledgements

The authors are thankful to the authorities of the

Gokula Education Foundation and V V Pura College of

Science, Bangalore for providing necessary facilities

and encouragement. They are also thankful to

Mr. V Chelladurai of Tirunelveli for help in providing

plant material.

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