Pharmacognostical studies on Oroxylum indicum (Linn.)Vent

Indian Journal of Natural Products and Resources

Vol. 2(4), Dec. 2011, pp. 472-478

Pharmacognostical studies on Oroxylum indicum (Linn.)Vent. stem bark

Anupam Bisht*1

, K. Zaman2, Mamta Singh

1, Richa Gupta

1 and Vinod Singh

1

1Department of Pharmaceutical Sciences, Sardar Bhagwan Singh P.G. Institute of Biomedical Sciences & Research, Balawala,

Dehra Dun-248 161, Uttarakhand, India 2Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh-786004, Assam, India

Received 12 January 2011; Accepted 12 April 2011

Oroxylum indicum (Linn.)Vent. is one such plant which is extensively used in the Indian systems of medicine as an

important ingredient of “Dashmula” and in the treatment of many diseases. Proper identification of the drug is desired for

obtaining its complete therapeutic effects. It is with this aspect in view the present study dealing with pharmacognostical

study and some other related studies of the stem bark of the species O. indicum (Linn.)Vent. were done. The stem bark is

characterized by the well developed cork region; phelloderm region consisting number of stone cells and fibres,

ceratenchyma is also present in inner phloem region, medullary rays are heterogenous and multiseriate, minute starch grains

up to 5 µm in diam. are present in secondary phloem region. Powder of stem bark shown fragments of cork cells, stone cells

and sclereids, fragments of fibres, parenchymatous cells, filled with black brown content, acicular crystals and starch grains

were also found present. TLC of various extracts showed spots in different solvent system and phytochemically the plant

was found to contain flavonoids, saponin, phenolic and tannins.

Keywords: Ceratenchyma, Flavonoids, Oroxylum indicum, Bignoniaceae, Dashmula.

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

Introduction The Bignoniaceae is a family of about 100 genera

and 800 species, having mainly climbing plants.

Oroxylum Vent. is a genus of trees found in

Indo–Malaysian region and China. One species

Oroxylum indicum (Linn.)Vent. (Shyonaka) is found

in India1. It is a small to medium sized deciduous

tree, up to 12 m in height, branched at top, bark light

brown, soft and often with numerous corky lenticles.

Leaves large, up to 1.5 m long, pinnate and bipinnate.

Flowers numerous in large erect racemes, fleshy, dark

lurid reddish purple outside, dull or pale pinkish

yellow within. Fruit is large, flat sword shaped, 1 m

long, seeds many, flat with broad silvery wings2,3

.

It grows in the foothills of tropical India. Most parts

of this tree are used as medicine. The Ayurvedic drug

O. indicum or Shyonaka is mentioned in various

classical texts of Ayurvedic System of Medicine, viz.

Charak, Sushrut and other treaties like Bhava Prakash.

The root, bark, stem and leaf are prescribed for

snake-bite. The stem and wood for scorpion-sting

(Sushruta)1. O. indicum is extensively used in Indian

System of Medicine as an important ingredient of

Dashmula which is a compound decoction of 10 roots.

It is a medicine of repute in the treatment of remittent

fever, otorrhoea, bronchitis, leucoderma, diarrhoea,

inflammation and in acute rheumatism. Powdered

stem bark or its infusion is diaphoretic4.

Phytochemical studies on leaves shown presence

of baicalein-6-glucuronide, baicalein-7-glucuronide,

scutellarein, scutellarein-7-glucuronide5, alo-emodin

6

and stem bark contains oroxylin-A, baicalein,

scutellarein, ρ-coumaric acid7. Root bark contains

oroxylin-A and ellagic acid8; heart wood contains

prunetin and ß-sitosterol9, Seeds contain oroxindin,

baicalein-6-glucoside, tetuin, a glucoside and

fixed oils10-12

. Studies have shown that the oral

administration of concentrated aqueous extract

of the root bark provided symptomatic relief as

well as absence of Entamoeba histolytica cysts

in stool of patients13

. The aqueous extract of the

stem bark possesses anti-inflammatory activity14

and dichloromethane extract exhibited antifungal

activity against dermatophytes and wood rot15

.

Literature survey revealed that the pharmacognostic

details on root bark has been already worked out16,17

——————

*Correspondent author:

E-mail: [email protected];

Phone: 09411726538 ( Mob.)

BISHT et al.: PHARMACOGNOSTICAL STUDIES ON OROXYLUM INDICUM STEM BARK

473

but the information on the stem bark is minimal.

Therefore, the present study is focused on the

structural features of stem bark of O. indicum

and preliminary phytochemical screening,

fluorescence analysis and TLC fingerprinting, etc.

of the stem bark were done for the purpose

of standardization of the crude drug.

Materials and Methods

Plant material

The stem bark was collected from Dibrugarh

University campus, Assam and was identified by

Dr. Vivek Kumar, Taxonomist, Pharmacognosy &

Ethnopharmacology Division, National Botanical

Research Institute, Lucknow. A herbarium

No.DU/PSc/HRB-03/2005 of this plant was

deposited in the Department of Pharmaceutical

Sciences, Dibrugarh University, Assam. The bark

material was dried in shade, powdered and stored

in air-tight container for further study. Macroscopy

Macroscopy i.e. evaluation of the drug for the

confirmation of its identity, determination of quality

and purity and detection of adulteration was done by

visual appearance by the naked eyes. Other sensory

characteristics like odour, taste, and feel of the drug

to the touch were also observed. Microscopic features

For microscopic studies the plant material was

fixed in the solution of formaldehyde, glacial acetic

acid and ethanol (90 ml of 70% ethanol + 5 ml

of glacial acetic acid + 5 ml of formaldehyde).

The section was cut transversely and stained

with safranin and mounted following the usual

plants microtechniques18

. Photomicrographs were

taken with LeicaUSA (Leica2000ATCmodel) Digi 3

Photomicrographic unit at 10 × 10 × magnification.

For powder drug study, the dried stem bark

was grinded in pestle–mortar then passed through

60 mesh sieve. The sieved powder was mounted

in chloral hydrate and Glycerin solution (1:1) and

seen under microscope at 15 × 10 × magnification.

Majority of the cell structures were studied and

drawn directly from the microscope by the help

of Camera lucida. Physicochemical study

Physico-chemical constants like ash values,

extractive values, crude fibre content and moisture

content were determined as per methods given in

IP 199619

.

Fluorescence analysis

For fluorescence analysis of the powder sample

it was treated with different chemical reagents to

observe various colour reactions which may help to

ascertain the purity of the drugs20

.

Preliminary phytochemical screening

Dried, coarsely powdered stem bark was

extracted successively with petroleum ether

(60-80°C), chloroform, acetone, methanol and

water using Soxhlet apparatus. All the four

extracts were screened for the presence of

phytoconstituents21

.

TLC finger print profile

For TLC fingerprinting, methanolic extract

was prepared by extracting 2g. of the powdered

stem bark with methanol (3 × 10 ml) at room

temperature, the extracts were combined, filtered

and evaporated on water bath. 10 mg of this dried

extract was re-dissolved in 1 ml of methanol and

15µl of it was implicated with Camag Linomet

IV applicator on a precoated silica gel G 60 F254

TLC plate (Merck) of uniform thickness of 0.2 mm.

The plate was developed in solvent system

Chloroform: Methanol: Formic acid (8.8:0.5:0.2)

and visualized under UV 254 nm, UV 366 nm

and under visible light after spray with Vanillin-

sulphuric acid reagent followed by heating at

110°C for 5-10 minutes in order to develop the

chromatogram. The finger print profiles were

documented with the help of CAMAG Switzerland

photo documentation unit Reprostar 322

.

Results

Macroscopic features

Stem bark is 0.3-1 cm thick, curved, outer surface,

rough and warty due to the presence of lenticels,

buff or whitish black in colour, algae filaments are

attached on the outer surface which appears green

or black in colour, inner surface light yellow, finally

longitudinally striated, odourless, slightly sweetish

and astringent in taste. Fracture is medium and coarse

(Plates 1, 2). Microscopic features

The T.S. of the stem bark shows outer most 10-15

layered cork cells. The cork is 171-190 µm thick

INDIAN J NAT PROD RESOUR, DECEMBER 2011

474

and are arranged in radial rows, suberized, some

cells become lignified and appear as stone cells.

Cork followed by 2-4 layered cork cambium which is

19.0-28.5 µm thick. Phelloderm region vary narrow,

15-25 cells broad. The phelloderm is 285 µm-380 µm

thick. Number of stone cells and fibres were

embedded in this region. Stone cells are much smaller

in size as compared to phloem region. Rest of the

section is made of secondary phloem. The phloem is

divided into two parts, outer and inner region. In outer

phloem plenty of stone cells are present in large

groups and some fibres. While in inner phloem

the condition is vice versa that is more fibre groups

are presents and stones cells are very less in number.

Ceratenchyma is also observed in inner phloem

region. Most of the parenchymatous cells are filled

with brownish-black content. Medullary rays are

multiseriate and heterogenous and the cells of

medullary rays are much smaller compared to

the other phloem parenchymatous cells. Acicular

crystals are embedded in medullary rays cells

and parenchymatous cells of phloem. Starch grains,

very minutes up to 5 µm in diam. are present in

the secondary phloem region (Plates 3, 3a-f).

Diagnostic character of powder

The powder was yellowish-green in colour,

without any characteristic odour, astringent in

taste. Under microscopic observation the powder

shows fragments of cork cells in surface and

tangential view. In surface view the cells are

hexagonal and polygonal in shape. Stone cells

and abundant sclereids, isolated or fairly in large

groups, thick walled, pitted showing a considerable

variation in size and shape. Few are elongated

(fibre sclereids). Paranchymatous cells filled with

black–brown content, patches of parenchymatous

cells. Fragments of fibres in groups, isolated bast

fibre, thick walled with uneven lumen with

tapering ends, septate and non-septate fibre

with varying shapes and sizes. The abundant

acicular crystals were found scattered as such

and embedded in medullary ray cells. Very minute

starch grains were scattered into the powder.

Parenchymatous cells were attached to the medullary

rays (Plate 4).

Physicochemical properties

Various physico-chemical constants like loss

on drying, total ash, acid insoluble ash, water soluble

ash, sulphated ash, water soluble extractive and

alcohol soluble extractive were determined and are

given in Table 1.

Fluorescence analysis Powdered drug was treated with different reagents

and was examined under UV light (254 & 366 nm)

emitted various colour radiations which help

in identifying the drug in powder form are given

in Table 2.

Preliminary phytochemical studies

Phytochemical test shows the presence of

carbohydrates in alcohol and water extracts. Phenolic compounds and tannins in acetone, alcohol and water extracts. Flavonoids in chloroform, acetone, alcohol and water extracts whereas saponins were observed in water extracts and sterols in petroleum ether extracts only.

TLC finger print profile

Thin layer chromatography of the methanol extract

was done using Chloroform: Methanol: Formic acid (8.8:0.5:0.2) as mobile phase and the Rf value were recorded (Plate 5 a-c and Table 3).

Plate 1—Fresh stem bark of Oroxylum indicum

Plate 2—Dried stem bark of Oroxylum indicum


475

Plate 3—Microscopic features of stem bark of Oroxylum indicum (at10×10× magnification,); Plate 3a—T.S. of stem bark showing cork,

cork cambium, phelloderm, stone cells, phloem, medullary rays; Plate 3b—7:T.S. of stem bark showing outer phloem region;

Plate 3c—T.S. of stem bark showing fibers and phloem; Plate 3d—T.S. of stem bark showing ceratenchyma, fibres and medullary rays;

Plate 3e—T.S. of stem bark showing ceratenchyma, fibres, stone cells and medullary rays


476

Plate 4—Microscopic examination of powdered stem bark of Oroxylum indicum

Table 1—Quantitative standards for the stem bark of

Oroxylum indicum

Serial No. Parameter Value* (%)

1 Ash Values

a) Total ash 17.38

b) Acid–insoluble ash 2.34

c) Water–soluble ash 4.21

d) Sulphated ash 18.01

2 Extractive Values

a) Alcohol soluble 26.09

b) Water soluble 28.38

3 Loss on drying 18.91

4 Crude fibre content 25.5

*Average of three readings

Table 2—Fluorescence analysis of the powdered stem bark of

Oroxylum indicum

Serial

No. Treatment

Day

Light

Short UV

Light

Long UV

Light

254 nm 365 nm

1 Powder as such Yellow Dark

brown Yellow

2 Powder + 1N NaOH (aqueous)

Yellowish–green

Dark brown

Fluorescent green

3 Powder+1N NaOH (alcoholic)

Straw colour

Dark brown

Greenish–yellow

4 Powder + 1N Hcl Straw colour

Chocolate brown

Green

5 Powder+50% H2SO4 Yellow Dark

brown Yellowish–

green

6 Powder+Conc.HNO3 Light Orange Green Green


477

Plate 5—TLC finger print of methanolic extract of the stem bark

of Oroxylum indicum

Table 3—TLC fingerprints of methanolic extract of stem bark of

Oroxylum indicum

Detection Rf Colour

0.04 Dark Brown

0.21 Brown

0.46 Dark Purple

0.53 Faint Blue

0.70 Dark Brown

0.83 Blue

0.90 Fluorescent Blue

Under UV 366

0.98 Pink

0.03 Yellow

0.09 Marone

0.25 Marone

0.30 Mustard

0.33 Blue

0.38 Blue

0.65 Purple

0.72 Purple

Under Visible

Light after Spray

with Vanillin–

sulphuric acid

0.98 Pink

Discussion Previous work done on this bark reported that the

stem bark is rough, greyish yellow in outer surface

and smooth golden yellow in inner surface with short

and fibrous fracture, mucilaginous and sweetish taste.

T.S. of stem bark showed cork is 12-20 layered,

arranged in tangentially elongated rectangular cells

with wavy walls. Phelloderm consists of parenchyma

and large number of groups of stone cells. In phloem

sieve plates are prominent, showing callus deposition.

In inner phloem region medullary rays are bi to

tetraseriate while in outer phloem region rays are

multiseriate24

. In our studies stem bark is curved,

warty, buff or whitish black in colour, fracture

medium and coarse, odourless, astringent in taste.

The T.S of stem bark showed that the cork is 10-15

layered, arranged in radial rows, suberized. Cork is

followed by 2-4 layered cork cambium. 15-25 cells

broad phelloderm region consisting number of stone

cells and fibre. The stone cells are much smaller

in size as compared to phloem region; secondary

phloem consist number of stone cells and

fibres; presence of ceratenchyma in inner phloem

region; heterogenous and multiseriate medullary rays,

acicular crystals embedded in medullary rays cells

and parenchymatous cells of phloem and minute

starch grains up to 5 µm in diam. are present

in secondary phloem region.

In our finding ceratenchyma was observed but

no callus deposition was found in sieve plates, cells of

multiserriate medullary rays consist starch grain and

acicular calcium oxalate crystals. Parenchymatous

cells were filled with black brown content. Under

microscopic observation powder shows fragments of

cork cells, stone cells and sclereids, fragments of

fibres in groups, parenchymatous cells filled with

black brown content, acicular crystals, medullary

rays attached with parenchymal cells and starch

grains. Medullary rays attached with parenchymal

cells and starch grains. Various physicochemical

parameters, viz. loss on drying, ash values,

extractive values, TLC of different extracts,

fluorescence analysis and phytochemical screening

of various extracts were performed to substantiate

standardization data on O. indicum.

Conclusion

The present study concludes that the complete

pharmacognostic parameters of the stem bark of

O. indicum would be useful in identifying the stem

bark for safe applications in drug manufacturing.

Acknowledgements Authors duly acknowledge Dr. AKS Rawat (Head)

and Dr. Sayyada Khatoon, Scientist, Pharmacognosy

and Ethnopharmacology Division, NBRI, Lucknow

for their kind help and thanks are due to the

management SBSPGI for the support.

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Pharmacognostical studies on Oroxylum indicum (Linn.)Vent