Indian Journal of Natural Products and Resources

Vol. 1(1), March 2010, pp. 22-28

Pharmacognostical studies on Flickingeria nodosa (Dalz.) Seidenf. stem

and pseudobulbs – A botanical source of the Ayurvedic drug Jivanti

V Madhavan1, Gajendra Singh Tomar

1, S N Yoganarasimhan

1* and M R Gurudeva

2

1Department of Pharmacognosy, M S Ramaiah College of Pharmacy, MSRIT Post, Bangalore-560 054, Karnataka, India 2Department of Botany, V V Pura College of Science, Bangalore-560 004

Received 9 September 2008; Accepted 6 May 2009

The pharmacognostical evaluation of Flickingeria nodosa (Dalz.) Seidenf. stem and pseudobulb which are considered as

one of the botanical sources of the important Ayurvedic drug Jivanti is presented. The study comprises taxonomic details,

macro- and microscopical characters of parts used, physico-chemical details, HPTLC profile of aqueous and alcohol

extracts. This study will help in laying down pharmacopoeial standards for F. nodosa.

Keywords: Flickingeria nodosa, Pharmacognosy, HPTLC, Jivanti, Ayurvedic drug, Stem, Pseudobulb.

IPC code; Int. cl.8—A61K 36/898, A61K 125/00

Introduction

Flickingeria nodosa (Dalz.) Seidenf. (Orchidaceae) is

one of the botanical sources of the Ayurvedic drug

Jivanti1. Some species, viz. Leptadenia reticulata

(Retz.) Wt. & Arn., Leptadenia pyrotechnica

(Forssk.) Decne., Holostemma ada-kodien Schultes

(all of Asclepiadaceae) and Trema orientalis Blume

(Ulmaceae) are also used as Jivanti2,3

. However,

Leptadenia reticulata Wt. & Arn. is considered as the

accepted botanical source of Jivanti4. Jivanti is one of

the important rasayana drugs in Ayurveda and is used

as an ingredient in formulations like Jivantadya

ghrita, Jivantayadi rasa, Jivantadyadi taila,

Ashwagandhadi ghritha, Anuthaila, Chandanadi

thaila which are used in treatment of tuberculosis,

emaciation, fever, haemorrhage and cardiac ailments

to mention a few5,6

. The main phytoconstituents of F.

nodosa are alkaloid Jibentine and α and β-Jivantic

acids3. Important therapeutic properties of Jivanti are:

alterative, aphrodisiac, astringent, cooling, diuretic

and used as a tonic in debility due to seminal

discharges, also useful in asthma, bronchitis and

throat trouble3. Such pharmacognostical investigation

with macerate and powder study details on stem and

pseudobulb of F. nodosa along with HPTLC studies

which help in identification of crude drug besides in

establishing the biomarker/bioactive compound is not

available and hence the present study was undertaken.

Materials and Methods

Plant material investigated in the present study was

collected from a coffee estate, near Mercara, Kodagu

district, Karnataka, on 13th March, 2006. Voucher

herbarium specimen (Gajendra Singh Tomar 010),

was prepared and preserved along with sample of

crude drug in the herbarium and museum of MSRCP,

Bangalore7. The plant material was identified and

authenticated at the herbarium of the Regional

Research Institute (Ayurveda) (RRCBI), Bangalore 8,9

. For pharmacognostical studies, a small quantity of

stem and pseudobulb in fresh condition were

preserved in 70% alcohol. Pharmacognostical

evaluation including histochemical, macerate and

powder studies were carried out by taking free hand

sections following Johansen10

, Wallis11

and Trease

and Evans12

. Safranin (4%) was used to stain

transverse sections. Reagents like potassium iodide,

ferric chloride, Sudan III, ruthenium red, concentrated

HCl and phloroglucinol with dilute HCl were used for

histochemical tests. Concentrated nitric acid (50%)

with pinch of potassium chlorate crystals was used as

the macerating fluid. Photomicrographs were obtained

by observing free hand sections of drug under

compound binocular microscope (Olympus-CH20i

model) with built in analogue camera (CMOF, 1.4

mega pixel). Computer images were captured using

AV-Digitaliser having Grand VCD 2000-Capture

Guard. Measurements of cells and tissues were

carried out using Micro Image Lite Analysis Software

——————

*Correspondent author:

E-mail: dr_yogan@yahoo.co.in

MADHAVAN et al : PHARMACOGNOSTICAL STUDIES ON FLICKINGERIA NODOSA

23

(Cybernetics, Maryland, USA). Physicochemical

constants, fluorescence studies, organic analysis and

thin layer chromatography were carried out from

shade-dried powder following prescribed

methods13-16

. HPTLC studies were carried out using

Camag HPTLC system equipped with Linomat V

sample applicator, Camag TLC scanner3 and CATS 4

software for interpretation of data. An aluminium

plate (5×10 cm) precoated with silica gel 60F254

(E Merck) was used as adsorbent. The plates were

developed using n-butanol:glacial acetic acid:water

(5:1:2) and tolulene:ethyl acetate (4:1) for aqueous

and alcohol extracts, respectively in a Camag twin

through chamber to a distance of 8cm each.

Results

Botanical description

Flickingeria nodosa (Dalz.) Seidenfaden in Dansk

Bot Ark, 34, 41 1980; F. macraei (Lindl.) Bennet,

J Econ Taxon Bot, 5, 452, 1984; Dendrobium

nodosum Dalz., Hooker`s J Bot, Kew Gard Misc, 4,

292, 1852; Dendrobium macraei Lindl., Gen Sp Orch,

75, 1830; Desmotrichum fimbriatum auct. non Blume,

1925; Fischer in Gamble, Fl Pres Madras, 3, 987,

1957 (repr. ed.); Ephimeranthae macraei (Lindl.)

Hunt. & Summerh, Taxon, 10, 105, 1961.

Epiphytes with creeping, annulate rhizome,

branches ending in pseudobulbs. Pseudobulbs 3.5×

1.5 cm, oblong or fusiform, compressed, furrowed,

yellow. Leaves 10-12×1-2 cm, solitary and sessile on

pseudobulbs, coriaceous, oblong-elliptic. Flowers ca

1 cm long, white, 1 to 3 originate from leaf base.

Sepals 3; lateral sepals adnate to column-foot forming

a mentum. Petals equal to or slightly shorter and

narrower than sepals. Lip 3-lobed; midlobe elongated

and expanded above lateral lobes into 2-winged erect

structures. Column oblong. Anther 1-2-celled; pollinia

4, in 2 pairs; caudicle absent; stigmatic surface broad

(Figs. 1-2). Distributed in South-East Asia, occurring

in India in Meghalaya, Sikkim Himalayas and the

western Ghats, up to an altitude of 2100 m.

Parts used: Drug consists of stem and pseudobulbs. Macro- and microscopical characters of stem and pseudobulb

Stem slender, greenish, turning yellow, smooth,

annulate, profusely branched; branches polygonal,

smooth, shining, yellowish, ending in pseudobulbs;

internodes to 6 cm apart; last internode terminating

into a pseudobulb. It has no characteristic taste and

odour (Fig. 3).

Transverse section is oval in outline. Epidermis

covered by a distinct shining yellow cuticle, cells

thick walled, rectangular to barrel shaped, 5-9-16µ,

contain cell contents. Next to epidermis is 3 to 5

layers of sclerenchyma, cells 8-11-13µ, followed by a

broad parenchymatous ground tissue. Ground tissue

cells 24-42-50µ, isodiametric, with small intercellular

spaces, some pitted, while others contain

mycorrhizae. Vascular bundles scattered in ground

tissue, closed, collateral, 46-94-166µ, enclosed by

sclerenchymatous sheath. Phloem consists of

companion cells and sieve tubes, 12-24-26µ while

xylem consists of vessels, tracheids, fibres and xylem

parenchyma (Figs. 4-8). Macerate exhibits fragments

of thick cuticle with impressions of epidermal cells,

thick walled pitted epidermal cells and with cell

contents, parenchyma cells of various size and

shape,120-135-150µ; fibres with broad or narrow

lumen and pointed ends, sometimes forked with one

short arm and one long arm, 159-24-443µ; xylem

fibres with wavy margin and with shining crystals

found along depressions; tracheids with scalariform

thickenings; vessels narrow with spiral thickenings,

123-143-155µ (Figs. 9 to 24). Pseudobulb 7-8×2-3cm,

consists of swollen and fleshy internode, smooth,

shining, yellow, slightly compressed, narrowly

fusiform, 5 to 8 in each aerial stem, without

characteristic odour or taste (Fig. 3). Transverse

section oval in outline, covered by thick yellow

cuticle. Epidermis single layered, thick walled, cells

rectangular with cell contents, 6-9-11µ. Outer 3 to 4

layers of ground tissue made up of thickened cells

appearing as sclerenchymatous, 14-17-19µ; remaining

layers parenchymatous, sometimes with intercellular

spaces, raphide bundles found in some cells while

simple starch grains 3-7-13µ (diameter) are present in

other cells. Vascular bundles distributed throughout

ground tissue, collateral, closed, 19-25-37µ, capped

with sclerenchymatous bundlesheath on both sides.

Phloem found externally and xylem internally

(Figs. 25 to 31).

Macerate exhibits fragments of cuticle with

impressions of epidermal cells, epidermal cells of

different size and shape, thick walled, pitted and with

cell contents, parenchyma cells of different size and

shape, some pitted, 290-320-370µ, bundles of acicular

raphides, fibers long, with wavy margin, lumen narrow

and with tapering ends, 138-165-206µ, tracheids long,

narrow, with pitted thickenings, 160-340-530µ, vessels

with spiral thickenings (Figs. 32 to 56).

INDIAN J NAT PROD RESOUR, MARCH 2010

24

Figures 1 - 24— Pharmacognostical studies on the Ayurvedic drug Jivanti – Flickingeria nodosa.

1. Habit (×0.06); 2. Clump of plant in situ (×0.1); 3. Crude drug (×0.2); 4. T.S. of stem (× 285); 5, 6. Portion of ground tissue enlarged

(×430, ×1200); 7, 8. Portion of ground tissue with vascular bundle (both ×1200); 9. Cuticle with impression of epidermal cells (×1300);

10, 11. Epidermal cells (both ×1300); 12. Sclerenchyma cell (×1300); 13. Xylem parenchyma (×1300); 14, 15, 16. Parenchyma cells

(×1590, ×1680, ×1600); 17, 18, 19, 20, 21. Fibres (×380, ×1730, ×1750, ×1650, ×1600); 22. Vessel (×1760); 23. Vessel with spiral

thickenings (×1700); 24. Vessel (×1785)

MADHAVAN et al : PHARMACOGNOSTICAL STUDIES ON FLICKINGERIA NODOSA

25

Figures 25 - 56 — Pharmacognostical studies on the Ayurvedic drug Jivanti – Flickingeria nodosa

25. T.S. of pseudobulb (×85); 26, 27. Portion of ground tissue enlarged (×760, ×260); 28. Ground tissue showing intercellular space

(×850); 29. Vascular bundle enlarged (×1900); 30. Ground tissue showing raphide bundle (×950); 31. Ground tissue showing starch

grains (treated with iodine) (×900); 32, 33, 34. Cuticle with epidermal cells and their impressions (×1430, 1420, ×1430); 35, 36.

Epidermal cells (both ×2140); 37, 38, 39, 40, 41, 42. Sclerenchyma cells (×1605, ×1660, ×1590, ×1666, ×1650, ×1620); 43, 44.

Parenchyma cells (×600, ×525); 45, 46. Xylem parenchyma (×1032, ×1000); 47, 48. Raphides (×1850, ×1300); 49, 50, 51, 52. Fibres

(×700, ×760, ×9090, ×700); 53, 54, 55. Tracheids (×178×1430, ×1400); 56. Vessel (×1785);

INDIAN J NAT PROD RESOUR, MARCH 2010

26

Histochemical tests

Sections of drug when treated with different

reagents gave different colour reactions (Table 1)

indicating presence or absence of respective cell

contents.

Powder study

Powder yellowish brown, with no characteristic

odour or taste. When powder was treated with

chloralhydrate, phloroglucinol and HCl and stained

with safranin for 5 to 10 minutes, lignified fibres,

parenchyma cells, vessels, tracheids, groups of

epidermal cells were observed; with iodine solution,

turned blue indicating presence of starch.

Physico-chemical studies

Physico-chemical constants of aerial stem and

pseudobulb were found to be: moisture content 7.90,

total ash 2.54 and acid insoluble ash 0.62%. The % of

alcohol extractive value was 19.32 and that of water

extractive 11.64.

Preliminary organic analysis

Successive Soxhlet extractive values, colour and

consistency of extracts of aerial stem with pseudobulb

were found to be: pet. ether (4.16, black, sticky mass);

benzene (2.32, dark green, sticky mass); chloroform

(3.45, yellowish-brown, sticky mass) and water (2.37,

light brown, semi solid), respectively. Preliminary

phytochemical analysis revealed presence of

alkaloids, carbohydrates, glycosides, fixed oils, fats,

phenolic compounds, tannins, saponins, flavonoids,

gums and mucilage in alcohol extract while in

aqueous extract similar constituents except fixed oils

and fats were present whereas in acetone extract

carbohydrates, glycosides, phenolic compounds,

tannins and flavonoids were detected.

Chromatographic studies

HPTLC studies of aqueous extract revealed ten

phytoconstituents having Rf values 0.01, 0.15, 0.27,

0.31, 0.50, 0.58, 0.64, 0.80, 0.91, 0.97 with a most

pronounced spot of maximum area at Rf 0.58 (Fig. 57)

while alcohol extract gave 12 phytoconstituents

having Rf 0.02, 0.04, 0.12, 0.16, 0.19, 0.26, 0.37, 0.44,

0.59, 0.70, 0.76, 0.97 with a most pronounced spot of

maximum area at Rf 0.59 (Fig. 58).

Ultra-violet analysis

Powdered drug of stem and pseudobulb under UV

and ordinary light when treated with different

reagents emitted various colour radiations (Table 2)

which help in distinguishing drug in powder form.

Table 1— Histochemical tests of stem and pseudobulb of F. nodosa

Material Reagent Test for Colour change Result

Section Iodine Starch Blue Present

Section Ferric chloride solution (10%) Tannin Black Present

Section Con. H2SO4 Cellulose Green Present

Section Dilute HCl + Pinch of phloroglucinol Lignin Magenta colour Present

Section Concentrated HCl Calcium oxalate crystals Effervescence Present

Fig. 57— HPTLC profile of aqueous extract

Fig. 58 — HPTLC profile of alcohol extract

MADHAVAN et al : PHARMACOGNOSTICAL STUDIES ON FLICKINGERIA NODOSA

27

Discussion

F. nodosa plant and drug consisting of

stem and pseudobulb, is identified by the

diagnostic characters.

The drug Jivanti is considered in Ayurveda as a

controversial drug since different botanical sources

are used by the physicians20

. The pharmacognosy of

L. reticuata5,21

, H. ada-kodien

22 and F. nodosa

3 (also

present work) are investigated while no

pharmacognostical work is available on L.

pyrotechnica and T. orientalis17-20

. A comparative

study of the important pharmacognostical characters

and phytochemical constituents of the investigated

species is provided in Table 3 which helps to

differentiate these species used as Jivanti.

Table 2— Ultra-violet powder analysis of stem and pseudobulb of F. nodosa

Treatment Visible light UV light

short wave (254 nm) long wave (365 nm)

Powder as such Cascade green Water green No fluorescence (fl)

Powder + 50% H2SO4 Pale cream Cascade green No fl

Powder + 50% HNO3 Mid buff Pale cream No fl

Powder + 5% KOH Pale cream Mint green No fl

Powder + Methanol Royal crown Water green No fl

Powder + 1N HCl Royal crown Water green No fl.

Powder + 1 N methanolic NaOH Cascade green Mint green No fl

Powder + Ethanol Pale cream Cascade green No fl

Powder + 1N ethanolic NaOH Pale cream Cascade green No fl

Table 3— Comparative distinguishing features of F. nodosa, L. reticulata and H. ada-kodien

Botanical source*

Characters F. nodosa L. reticulata21 H. ada-kodien22,23

Family Orchidaceae Asclepiadaceae Asclepiadaceae

Habit Epiphyte Straggling shrub Twining herb

Part used Pseudobulb and stem Root Root

Latex Absent Present in stem Present in stem

External surface Pseudobulb smooth, stem striated, yellow Root rough with longitudinal

ridges and furrows, white or

buff coloured

Root smooth, yellowish-

brown.

Cuticle Present, thick, glistening yellow Absent Absent

Secondary growth Absent Present Present

Epidermal cells Present with cell content Absent; cork present Absent; cork present

Sclerenchyma/

stone cells

Present in stem and pseudobulb as 3 to 5 layers below

the epidermis

Stone cells in layers in

secondary cortex and

secondary phloem

Stone cells in groups

present in secondary

cortex.

Mycorrhiza Present in ground tissue Absent Absent

Calcium oxalate

crystals/raphides

Shining yellow crystals present along margins of

xylem fibres of stem. Raphide bundles are present

in the ground tissue of pseudobulb

Rhomboidal crystals present

in secondary cortex and

secondary phloem

Druses type of crystals

present in secondary

cortex

Starch grains Present in ground tissue Present in medullary rays Present in secondary

cortex and secondary

phloem

Vessels Spiral thickenings present Pitted thickenings present Pitted thickenings present

Chemical

constituents

Alkaloid Jibantine, α and β-Jivantic acid present

in stem and root3

Sterols in stem and root;

fructosan of inulin type in root

present24

α-Amyrin, lupeol, β-

sitosterol and amino acids

present24

*Note: The other two taxa, viz. Trema orientalis belonging to Ulmaceae is a tree species and contains octacosanoic acid and

1-octacosanyl acetate, simiarenone, simiarenol, episimiarenol and triterpene alcohol trematol in stem bark while Leptadenia pyrotechnica

belonging to Asclepiadaceae, is a straggling shrub and aerial parts contain taraxerol, farenol, β-sitosterol22 and pregnane glycosides22,25,26.

INDIAN J NAT PROD RESOUR, MARCH 2010

28

Conclusion The drug Jivanti has been identified by the above

mentioned diagnostic characters. HPTLC studies of

aqueous and alcohol extracts of stem and pseudobulb

revealed 10 and 12 spots which not only help in

determining the marker compound, but also to

identify it in powder form besides to take up further

isolation and identification of the bioactive principle.

It is interesting to note here that the part used as

Jivanti in Ayurveda is the root while stem and

pseudobulbs are also used by drug manufacturers.

Acknowledgement

The authors are thankful to the authorities of

Gokula Educational Foundation and Principal, V V

Pura College of Science for evincing interest in this

work.

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