pharmacognostic studies on the flower of mesua ferrea l

Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 211-226. C) Printed in India.

Pharmacognostic studies on the flower of Mesua ferrea L.

U S H A SHOME, S H A N T A M E H R O T R A and H P S H A R M A Pharmacognosy Laboratory, National Botanical Research lnstitute, Lucknow 226 001, India

MS received 15 June 1981

Abstract. Stamens of Mesua ferrea L. constitute the genuine 'Nagkeshara' of Ayurveda, a drug considercd to be astringent, stomachic and expectorant. The present communication deals with detailed pharmacognosy of the drug and includes morpho- logical, anatomical as well as certain phytochemical characters of the floral parts of Mesuaferrea. Some of the distinguishing characters are : cortical fibres, numerous resin canals and calcium oxalate crystals in the cortex and pith of the pedicel; anamocytic, anisocytic of paracytic stomata on sepals and petals ; and 3-(4-) zonocolporate pollen grains with reticulate exine surface. Fluorescence analysis, behaviour.of the drug with certain chemical reagcnts and thin layer chromatography were also carried out.

Keywords. Mesuaferrea ; flower ; pharmacognosy ; cluseaceae.

1. Introduction

Mesua ferrea Linn. belonging to family Cluseaceae is considered the genuine "Nagkeshara ' (Chunekar 1960, Dyrnock et al 1885 ; Mudal iar 1957 ; Vaidya 1971; Waring 1868). It is known as Nagpushpa champeya in Sanskr i t ; Peela nagkeshara in Hindi ; Nageshwara in Bengali ; Nagchampa in Madhya Pradesh ; Pilu-nagkeshara in Gu j r a t i , Viluha champkan in T a m i l ; I ron wood tree and Cobras Saffron in English.

Stamens of M. ferrea constitute the actual drug (Sharma 1978). I t finds mention in all the important ancient Ayurvedic literature (Bhavmishra 1949 ; Charaka 1949; Sushruta 1952) to be used as an astringent stomachic and expectorant. Flowers are given in bleeding piles, in the form of a paste made with butter and sugar. These are also applied for relief in burning sensation in the feet (with old a n d a hundred times washed 'Ghee ' ) (Chakradat ta , See Nadkarni 1937; Kir t ikar and Basu 1933). A syrup o f the flower buds (1 in 10) is said to cure dysentery (Nadkarni 1937). The drug was also adopted by Arab and Unani physicians into their Materia Medica (Anonymous 1970) according to which it has a depressent action on uterine muscles, is digestive, diaphoretic, antidysenteric and an emmenagogue. It is used a s a constituent of about 11 compound Unani formulations (Anonymous 1970).

N.B.R.I. Research Publication No. 139 (New Series)

211

212 Usha Shome, Shanta Mehrotra and H P Sharma

Mesua ferrea L. is a large, evergreen tree with a short trunk, often buttressed at the base. It is found in the Himalayas from Nepal eastwards ascending to an altitude of 1500 m. I t i s also cultivated in the gardens for its beautiful flowers and attractive foliage (Anonymous 1962). Detailed pharmacognostic work on the flower of Mesua has not been carried out so far and hence the present study.

1.1. Previous Work

Several workers have carried out market surveys of Nagkeshara. Satkopan and Thomas (1967) surveyed Gujrat markets and found only one sample from Surat as genuine 'Nagkeshara'. These authors have also tried to identify other samples being sold as Nagkeshara (Satkopan and Thomas 1967a,b; 1968). Ali (1967) surveyed the South Indian Markets. However, he found none of the samples as Mesuaferrea L.

The presente of an essential oil and two bitter substances has been reported in the flowers by Boorsma in 1904 (see Chakraborty et al 1959). Subramanyam eta l (1975) have isolated mesuanic acid, a new carboxylic acid, from the acetone extract of Mesuaferrea stamens. Subramanyam and Subba Rao (1969) had earlier isolated mammeisin from the seeds of" Mesua ferrea L. Bala and Seshadri (1971) isolated mammeigin and mesuol as the main phenolie components from two different samples of seed oil. Dutt et al (1940), Chakraborty aud Bose (1960) and Chakraborty and Das (1966)isolated two crystalline antibiotie prineiples mesuol and mesuone from the seed kernel oil. Chakraborty et al (1959) investigated anti-bacterial activity of mesuol and mesuone. None of the eonstituents, however, was found to be antifungal. Bhattacharya et al (1979) have synthesized an antibiotic mesuagin from Mesua ferrea.

2. Material and methods

Fresh material was collected from the Forest Research Institute, Dehra Dun in the month of May and preserved in form acetic-alcohol. Hand sections stained with safranin were used for the present study. Phloroglucinol, iodine and terric chloride were used to test lignin, starch and tannin respectively. For whole mounts petals were treated with a dilute solution of nitric acid followed by clearing in chloral hydrate. Physicochemical studies were performed with the powdered, shade dried material.

3. External characters of the flower

The flowers are fragrant ; creara coloured ; ebracteate ; pedicellate ; pedicel short, axillary or terminal ; solitary or in pairs and 2.5-7.5 cms in diameter (figure 1). The buds are subglobose.

Sepals 4, large persistent, boat shaped, 2 outer slightly shorter than the inner ones and depressed at the base, Petals 4, large, cream coloured, spreading, euneate, margins undulating, cadueous ; stamens indefinite, golden-yellow, united

Pharmacognostic studies o f Mesua ferrea L. 213

Figure 1. Mesua ferrea LŸ : Two twigs with flowers • 0.425.


Figures 2-12. 2, 3. T. S. pedicet (Diagrammatic) 4. A portion of transverse section through the pedicel region showing details. 5. Upper epidermis of a sepal showing stomata and striations. 6. Upl:er epidermis o f a sepal showing elongated narrow cells over the vein. 7. Lower epidermal cells of the sepal showing trichome bases and stomata. 8. Lov, er epiderrnal cells showing trichomes only. 9 and 10. uni and multicellular uniseriate trichomes. 11. T. S. sepal (Diagrammatic). 12. A portion of the same showing detaiis. Abbreviations : ASP, airspace ; BS, bundle sheath; COL, collenchyma ; COR,

cortex ; CR, crystal ; CU, cuticle ; EP, epidermis ; GT, ground tissue ; HYP, hypodermis ; LEP, lower epidermis ; MXY, metaxylem ; PH, phloem ; P1, pith ; PXY, protoxylem ; RC, resin canal ; S, starch grains ; SCL, sclerenchyma, ST, stomata ; STC, stone cells ; TR, trichome ; TRB, trichome base ; UEP, upper epidermis ; VB, Vascular bundle ; XY,Xylem.

Pharmacognostic studies of Mesua ferrea L. 215

at the base and forming a fleshy basal sheath, tilaments small, anthers oblong ; ovary superior, bicarpellary, syncarpous, style twice as long as stamens, stigma eapitate, style and stigma persistent in young fruit but are shed away later on.

Fruit conical when young, ovoid to almost round with a prominent beak when mature, pericarp hard, warty, 2-valved after dehiscence. Seeds 1-4, angular, smooth and ehestnut brown.

4. Histology

4.1 Pedicel

A transverse section of the pedicel is almost circular in outline. The epidermis is highly cuticularised and is formed by narrow eiongated cells bearing one- to five- celled, uniseriate hairs. The cortex is 19 or 20 layered and is composed of rounded, collenchymatous cells. Cells of the outer cortical layers have characteristic semi-lunar, lignified thickenings on their walls. The inner cortical cells have fairly large starch grains as eragastic inclusions. As the pedicel matures sclerencbymatous fibres develop in the form of radially elongated patches outside the phloem. The phloem is well developed and forros a cortcentric cylinder. It cotxsists of sieve tubes and phloem parenchyma. The xylem is endarch and comprises of vessels, fibres and xylem parenchyma.

The pith is weU developed and collenchymatous like the cortex. Resin canals (which ate lineal by numerous elongated secretory cells) and Ca-oxalate crystals of rosette and prismatic types are abundant in both cortex and pith (figures 2-4).

4.2 Sepals

In surface view cells of the upper epidermis ate larger as compared to those of the lower one (figures 5-8). Stomata ate anisocytic or paracytic (figures 5-7) and those on the upper surface ate sparse and larger in size. The guard cellshave parallel striations radiating out from the stomata (figures 5 and 6).

Cells over the veins are narrow and elongated (figure 6). Uni- to multiceUular, uniseriate trichomes (figures 9 and 10) are present on both the surfaces but are more abundant on the lower one.

A transverse seetion of the sepal is semi-lunar in outline. Cells of the upper epidermishave a thick cuticle on the outside ; are larger, and broader than long and have lignified walls and sunken stomata (figures 11 and 12). The epidermis is followed by a well demarcated hypodermis, again with lignified large rounded cells. In contrast the lower epidermis comprises of small, narrow, vertically elongated and unlignified cells.

The ground tissue is many layered and has rounded collenchymatous cells. Up to four layers of these have greatly thickened cells similar to those in the pedicel cortex. The vascular bundles ate scattered in the middle of the ground tissue. These ate usuaUy amphicribral and are surrounded by a bundle sheath formed of two to three layered sclerenchymatous fibres. Resin canals, lined by a layer of narrow epithelial cells ate present throughout the ground tissue. The


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17 14 21 Figures 13-21. 13. Upper epidermal cells of a petal. 14. A port[on from figure ]3 enlarged to show the details. 15-17. Cells of the lower epidermis of a petal from apex, middle and base respectively. 18. Epidermal cells of a petal showing stomata arranged at right angle to the cells. 19 and 20. T. S. of petal from lower and middle portions. (Dia- grammatic). 21. A portion from figure 19 magnified to show the details. Abbreviations : CC, cell contents ; CU, cuticle ; GT, ground-tissue ; LEP, lower

epidermis ; PH, phloem ; RC, resin canal ; ST, stomata ; UEP, upper epidermis ; XY, xylem.

d iameter o f the canals is var iable , be ing sometimes even larger than the vascular bundles . Dise shaped Ca-oxa la te crystals ate also present in abu•dance.


4.3 Petals

The cells of the upper epidermis are uniform, laterally elongated, have anisocytic or paracytic stomata and are larger than those of the lower side (figures 13 and 14). The lower epidermal cells, on the other hand, vary considerably in shape and size from base to apex (figures 15,18). Epidermal cells in the apical region are squarish with wavy anticlinal waUs (figure 15). In the lower region, however, these are narrow elongated and thick walled with comparatively smaller stomata (figures 16 and 17). The stomata are either oriented in the same plane of may be arranged at right angles to the epidermal cells (figures 17 and 18).

The transverse section of a petal near the basal region shows a thick central portion and narrow curved margins (figure 19). The upper epidermis is undulating and has a thick cuticle on the outside. Sometimes even the anticlinal inner walls may also be thickened. A little below the epidermis one or two layers of cells show conspicously brown tannin containing cells. This layer is absent at higher levels (figure 20). The lower epidermis is smooth but cuticularised and is formed of comparatively smaller cells. The ground tissue is multilayered and is formed of parenchymatous cells with irregular outlines (figure 21). Resin canals are very few at lower levels but at higher levels these are quite abundant and ate larger than the vascular bundles (figure 20). The vascular bundles are arranged almost in a row, some large and some small. They are collateral and formed of a few xylem and phloem elements.

4.4 Androecium

The stamens are numerous, have short filaments and somewhat thick, elongated anther lobes. These are tetra-sporangiate (figures 22 and 23). Some fused stamens (figures 24 and 25), alongwith their respective vascular supplies, have also beeu observed. Occasionally the fusion may involve the anther lobes as well. In such eases, however, fusion of the anther lobes is not complete (figure 24). Auother stamen in which only connectives are fused in the middle can be seen in a transverse section (figure 25).

A semi diagrammatic representation of a transverse section of dehisced anther lobe can be seen in figure 26 and details of the vascular bundle in figure 27. The cells of the connective are elongated and papillate (figure 28). They are lignified and are positive to p h l o r o g l u c i n o l - cono. hydrochloric acid stain~ The epidermis of the auther lobe is formed of large colourless highly cuticularised cells followed b y a fibrous endothecium (figures 26, 29).

4.5 Pollen grains

Pollen grains are 3-(4-) zono-colporate with average size 35.89 • 48.71~m (range 30.77 - - 48.71 X 35.89 - - 58.97/~m) ; shape oblate to suboblate ; exine surface reticulate ; thiekness 1--2/~m, muriduplibaculate, colpus measures 13 • 33~m (figures 30-32).


Table 1. Mesuaferrea Linn. : Behaviour ofpowder with different chemical reagents.

$1. No. Treatment Observations

Flower Stamen

1. Powder

2. Powder

+ IN NaOH in methanol

+ Pieric aeid (saturated)

3. Powder + Acetic acid 4. Powder +Conc. HCI 5. Powder + Cono. HNOs 6. Powder + Iodine (5%) 7. Powder + Selq rcagent 8. Powder + Ferrie chloride (5%)

9. powder + 40% NaOH + a few drops of 10% lead acetate

10. Powder + Sudan III (alcoholic)

11. Powder +Conc. HNOa + ammonia.

Blackish brown Brown with yellow tinge

Yellowish Yellowish orange orangr Brown Orange yellow Brown Brown Reddish orange Reddish orange Blackish brown Blackish brown Yellowish brown Yellowish brown Dark blackish- Dark brown brown with with green green tinge tinge Blackish- Light brown brown with with yellowish yellowish white ppt. whlte ppt. Dark reddi.~h Reddisb orange orange Orange with Orange with yellow ppt. whitish

yellow ppt

4.6 Gynaecium

The ovary is superior, bicarpellary and syncarpous (figures 33 and 34). Epidermal cells of the ovary are small, hexagonal in surface view and have s tomata measuring 21.09 X 11.59 (range 19.00 - - 26.60 • 9.50 - - 14.25~tm (figure 35).

In transection the ovary is circular in outline (figures 36-38). The placentae are swollen and united at the base (figure 36). But at higher levels they separate (figures 36-39). The ovules arise one f rom each margin in each of the locules (figure 36)~ The placentation is thus axile tending to become parietal at higher levels. A transverse section passing through the style shows a stylar canal in the centre and three bundles, a dorsal flanked by two laterals on either side (figure 40). Resin canals a.re abundant even at this level.

5. Powder study

The powder of the whole flower is greyish brown in colour, pleasant smelling and slightly astringent in taste. The powder of stamens is golden brown in colour pleasant smelling and astringent to taste. The powder of whole ¡ was sieved through No. 40 mesh, cleared in chloral hydrate and mounted in glycerine. A microscopic examinat ion revealed the following elements. (i) Pieces of t r ichomes (figures 41 A-F). (ii) Pieces of tissues showing s tomata (figure 41 G), simple pits (figure 41 H-I) . (iii) abundant pollen grains in different planes (figures 41 J and K).

The behaviour of the powdered drug with different chemical reagents was also studied and is presented in table 1.


Figures 22-41. 22. A stamen showing one antherlobe. 23. T. S. of the antherlobe showing tetra sporangiate condition (Diagrammatic). 24. A stamen showing two anther lobes partially fused. 25. T. S. of the fused antherlebe. (Diagrammatic). 26. T. S. of dehisced antherlobe-(Semi-dia~,rammatic). 27. Vascular bundle from figure 26 magnified to show the details. 28. Cells from the connective (surface view). 29. A port ion of antherlobe wall magnified showing cutieularised epidermis and endothecium. 30. A pollen grain showing wall stratification and ornamentat ion. 31. A palynogram of pollen. 32. L O pattern of pollen ornamentat ion. 33 and 34. Diagrammatic representat ion of very young and slightly mature ovaries. 35. Epidermal cells from ovary showing stomata in surface view. 36-38. Serial T. S. of ovary from base to apex (Diagrammat ic ) 39. L. S. of ovary (Diagrammatic) . 40. T. S. of style (Diagrammatie) . 41. A-K. Different tissues from the drug powder. For details refer to the text. Abbreviations : AL, antherlobe ; C, connective ; CO, Colpus ; CU, cuticle ; EP.

epidermis ; EX, exine ; END, endothecium, FI, filament ; O, ovary ; OV, ovu le ; OW, ovary wal l ; PG, pollen grain, PH, phloem ; PL, placentum ; RC, resin canal ; SC, stylar cavity ; ST, stomata ; STI, stigma ; VB, vascular bundle ; VS, Vascular supply ; XY, Xylem.

Pharmacognostic studies of Mesua ferrea L.

Table 2. Mesua ferrea L. : Fluorescente analysis

221

SI. No. Treatment Fluorescence under UV light

Stamen Flower

1. Drug as such 2. Powder + Nitro-cellulose in amylacetate 3. Powder + IN HCI 4. Powder + 1N HCI + Nitro-cellulose

in amylacetate 5. Powder + aq. IN NaOH 6. Powder + aq. IN NaOH + Nitro-

cellulose in amylacetate 7. Powder 4- 1N NaOH in Methanol 8. Powder + IN NaOH in Methanol +

Nitrocellulose in amylacetate 9. Powder + 50% Nitric acid

10. Powder + 50% Sulphuric acid

Purplish brown Blackish brown Olive green P/~rplish brown Reddish brown Blackish brown Dirty green Dark brown

with purple tinge Dark brown Dark brown Bleckish brown Blackish brown

Blackish brown Greenish brown

Blackish brown Greenish brown

Reddish brown Dark brown Greenish brown Reddish brown with red tinge

The fluorescence analysis was carried out according to the method described by Chase and Prat t (1949) and Kokoski et al(1958). The observations ate recorded in table 2.

6. Phytoehemieal studies

Phytochemical characters of the drug were studied separately for the whole flower and stamens respectively. The determination of ash values, sugar, tannins, alcohol and water soluble extractives were made from air dried material . The procedures recommended by Anonymous (1966)were followed for calculating total ash, acid insoluble ash, alcohol and water soluble extractive percentages, whereas for calculating taunins, total and reducing sugars Fohlin-Denis reagent and Shaffer's Somogyi micromethods prescribed by AOAC (Anonymous 1965) were followed. (1) Ash Flowers Stamens

(a) Total ash 9.297% 2.370% (b) Acid insoluble ash 3.730% 0.230%

(2) Tannins 4.4% 3.76%

(3) Sugars (a) Total sugars 1.244% 1.852% (b) Reducing sugars 0.960% 1.160% (c) Non-reducing sugars 0.350% 0.692%

(4) Volatile oil 0 .25% 1.75%

(5) Alcohol soluble extractive 21.57% 25.31% (I.P. Method)

(6) Water soluble extractive 14.86% 15.40% (I.P. Method)

N N N

Tab

le3.

Mes

uafe

rrea

L.P

reli

min

ary

phyt

oche

mic

alte

sts.

s:H

exan

eex

trac

tive

Ben

zene

extr

acti

veC

hlor

ofor

mex

trac

tive

Eth

anol

extr

acti

veA

queo

usex

trac

tive

;:;..

Cl

Par

amet

ers

~Fl

ower

Stam

enF

low

erSt

amen

Flow

erS

tam

enFl

ower

Sta

men

Flo

wer

Stam

en

~T

otal

perc

enta

ge30

.92%

13.5

2%2.

197%

1.5%

0.46

9%0.

784%

0.79

3%1.

4%9.

958%

1.53

%,!

\

byw

eigh

t~

Phys

ical

appe

ar-

Bro

wni

shB

righ

tB

row

n,Y

ello

wis

hY

ello

wis

hY

ello

w.

Bro

wn,

Bro

wn,

Bro

wn,

Bro

wn

:¥

ance

and

cons

is-

yello

w,

yello

w,

stic

kybr

own,

brow

n,st

icky

stic

kyst

icky

pow

dery

pow

dery

is

tenc

yoi

lyoi

lyst

icky

ticky

iA

lkal

oids

--

--

2s+

++

--

-F

lavo

noid

s-

--

--

--

--

-:::

Red

ucin

gsu

gars

-2

++

3+

j+

Cl

--

--

-s

Res

ins

4+

4+

3+

2+

2+

2+

2+

2+

--

I:l...

Sap

onin

s-

--

--

--

-3

++

::t:St

eroi

ds-

--

--

--

--

-~

Tri

terp

enoi

ds4

+4

+4

+4

+-

--

--

-~

Tan

nins

--

--

--

2+

2+

3+

3+

Cl ~ Cl

Pharmacognostic studies 0l e Mesua f errea L. 223

. . . . . . . . 6 . . . . . .

| o 0

Q

Q O

O

4 2

+

i

+

- ~ ~ - - - ' 1 P o 0 ~

e

e e t e !

t e

I

h i " (~'Y

HE,XAHE E~T 4 ~

p~

w

, . J

| �9

i ~ t :

�9 ~ ~

B~IZENE EXT

!

q ~

2

, e

!

! !

- ~ , ' -9 -"~-~ ,~ a

,o

Q

Y t ; o+, �9 0 ~~

A � 9 1

4 5

F i g u r e s 4 2 - 4 7 .

,...+' o ~ ' s '++, y O +

0 ++Q, +

~+4m+ | ',+~ , - ,0 | :,."

e +~ /', , �9 (~ +

CN LOROFOP~ EXT

A - - Stamen extractive B - - Flower extractive

'i a

0 | 0 8 e S

S | I I | } | O

~ 0 S

bV

�91 |

~ B

EXT. 4 7

) ~

~ 5

o

BR, brown ; BY, bright yellow ; D.BR, dark brown ; GY, greenish yellow ; LBR, light bro,~n ; LP, ltght purplr ; LRY, light reddish yellow ; LY, light yellow ; P, purple ; PY, purplish yellow; RO, reddish orange ; Y, yellow ; YB,Yellowish brown.

Beside the above 15 gres of air dried powdered flowers and stamens were extracted separately in a Soxhlet apparatus with hexane, benzene, chloroform, alcohol and water successively and percentagr of each extractive calculated after evaporation of respective solvents. These were further screened for steroids, and triterpenoids (L.B. test - Peach and Tracy 1955) ; flavonoids (Shinoda's tes t - Loc.Cit.) ; alkaloids (Mayer's reagont- Loc.Cit.) ; and tannins (ferric chloride test-Loc.Cit.). The results obtained are presented in table 3.


The hexane and benzene extractives of both the parts show presence o f tri terpenoids and resins. Percentage of reducing sugars in aleoholic extractivr in the case of flower, was found to be quite high.

Water soluble portion of both the parts show presente of tannins and saponins. Concentra t ion of saponins, however, was quite high in the case of flower.

Thin layer chromatography of the above extractives was also carried out (figures 42-47). The hexane soluble extractive of stamens shows a larger number o f discrete spots as compared to that o f the flower (figure 42). With hexane-ethyl acetate (72 : 29) as solvent system the hexane extractive separates into spots with shades of yellow and brown in the case of flower (figure 43b). Those of the stamens are, however, purple (figure 43a).

The benzene extraetives (figure 44) are quite comparable. Again, chloroform extractive resolved into 4 spots each with toluene-acetone (35 �9 15) as the solvent system, Of these the two upper most and the lowermost on Rf0 .96 , 0.82 and 0.35 are comparable . However, ah oval yellow pa tch at R f 0.43 a n d a brown spot at R f 0.65 in stamen and flower extracts respectively ate different. The same extractive resolved into 5 spots in flower and 6 spots in stamens with solvent system ch loroform : acetone : methanol , 32.5 : 15 : : 2.5 (figure46). It is interesting to note that the alcoholic portion of the stamens gave 8 spots while the flower port ion gave only 2 spots (figure 47) (Solvent system : formic acid-ethyl formate- toluene (1 ". 4 : 5). The R f values o f different extractives have been recorded in a tabular form (table 4).

Table 4. Mesuaferrea Linn : TLC Rf. Values

Extractive Solvent system Rf Values

Flower Stamen

Hexane Hexane : Benzene 0.067,0.12,0.19,0.82, 0.04,0.10,0.17,0.25, soluble (30 : 70) 0.93 0.64,0.89,0.95 extractive

Hexane Hexane : Ethyl- 0.76,0.83,0.91 0.79,0.83,0.91,0.99 soluble acetate (72 : 29) extractive

Benzene Benzene : Chloroform : 0.46,0.63.0.80,0.95 0.50,0.63,0.79,0.99 soluble Acetonr extractive (3 : 1 : 0.05 cc.)

Chloroform Toluene : Acetone 0.35,0.65,0.83,0.96 0.35,0.43,0.81,0.97 soluble (35 : 15) extractive

Chloroform Chloroform : Acetoue : 0 .28 ,0 .43 ,0 .59 , 0.28,0.41,0.68,0.76, soluble Methanol 0.78,0.90 0.00,0.93 extractive (32.5 : 15 : 2.5)

Alcohol Formlc acid : 0.19,0.36,1.0 0.03.0.06.0.08, soluble Ethyl formate : 0.23,0.29,0.35 extractive Toluene 0.4,0.48,0.99

( 1 : 4 : 5 ) $praying reagent 2% H2SO4 Underlined Rfs denote spots which are distinctive.


Acknowledgements

The authors ate extremely grateful to Dr T N Khoshoo , Direc tor , N a t i o n a l Botanical Research lns t i tu te , for his con t inued and keen interest in the progress o f this work. The late Mr K M Vaid was great ly helpful in p rocur ing p lan t mater ia l for us for which we ate sincerely t h a n k f u l to himo We are also t hank fu l to Dr Mithi lesh Chaturvedi for her helpful suggestions regarding the pol len study. Our thanks are also extended to Messrs A Jha, R S Ojha and M K T a n d o n for technical assistance.

References

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