Pharmacognostic Studies on the Leaves of Anisomeles indica Linn.

Abstract

Pharmacognostical parameters for the leaves of Anisomeles indica Linn. (Labiatae) were studied with the aim of drawing the Pharmacognostical standards for this species. Macroscopical and microscopical characters, physio-chemical constants, quantitative
microscopy parameters, extractive values with different solvents, fluorescence analysis of extracts, its reaction after treatment with chemical reagents under visible light and UV light at 254 nm and 366 nm. Preliminary phytochemical screening on the leaves
Anisomeles indica was also studied. Caryophyllaceous stomata (in the upper and lower epidermis), glandular, non-glandular trichomes and starch grains were identified. The determination of these characters will help future researchers in their Phytochemical
as well as Pharmacological analysis of this species.

Full text

Phcog J. [A Supplement to Phcog Mag.]
Vol, 1, Issue 1, 44-48, 20 09
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Open Access
PHCOG J: Research Article
Pharmacognostic Studies on the Leaves of Anisomeles indica Linn.
(Labiatae)
Y. V. Ushir*1 , V. R. Chidrawar 1, K. N. Patel 2, A. U.Tatiy a3, and S. J. Surana3
1Departme nt of Pharmacog nosy and Phytochemistry, Shree H. N. Sh ukla Institute of Pharmac e uti cal Education and Research, Rajkot-
360001 (GS), India
2 Department of Pharmacognos y and P hytoc hemistry, Arihant Sc h ool of Pharmacy and Bio-Rese ar ch Institute, Gandhinagar (GS),
India
3 Department of Pharmacognos y and P hytoc hemistry, R.C. P atel College of Pharmacy,
Karw and Naka, Shirp ur-425405, (M S), India
* Corr espondence: ushir29 @gmail.com
ABST RAC T
Pharmacog nostical parameters for the leave s of Anisomeles indica Li nn. (Labiatae) were studied wi th th e aim of drawing the
Pharmacog nostical stand ards for this species. Macroscopical and microscopical characters, physi o-che mical constants, quantitative
micr oscop y par ameters, e x tr ac tive value s w ith d ifferent solv ents, fl uores cence analysis of extr acts, its r eaction af ter treatment with
ch emical reagents under vi sible light and UV light at 254 nm and 366 nm. Preliminary phytochemical screening on th e leaves
Anisome les indi ca was also stud ied. Caryophyllaceous stomata (in the upper and lower epidermis), glandular, non-glandular
trich omes and star ch grains were identified. The determination of these characters will h elp future researchers in their P hy tochemical
as w ell as Phar macological analysis of this specie s .
KEY WORDS
Anisome les indica Linn., Chemo-microscopy , Evaluation, M acrosc opy, Micr ometry, Pharmacognosy
INTRODU CT ION
Anisome les indi ca Linn. (Family: Labiatae) is one of the
important medicinal plant found in sa tpuda vally,
speci fi cally in T oranmal (altitude 1800m), Maharashtr a
(India ). It’s an erect, camphor -scented, varia bl e herb or
woody under shr ub, 1-2 m in height; stems acutely
qua d rangula r , softly pubescent (1).
Anis omeles i ndica are used in folk medicine all over the
world. It is popul arly known as ‘Jirnya’ in nor theastern
part of India, where it receives wi d espread us ed as folk
medicine, predominantly in the tr eatment of intestinal
disorders and intermi ttent fever. Ani s omeles indica have
anti-micr obial, astringent, carminative etha nolic extract
(50%) of the herb show ed hypother mic a ctivity and w hen
burnt acts as a mosquito repel lant. The essentia l oil
present in the herb is useful i n uterine affections (2, 3).
This study is intended to establis h, ma croscopical,
microscopical, chemo-microscopical, qua ntitative-leaf
microscopical characters as well as quantitative evaluation
of the pow dered and fresh leaves of the plant to be us ed as
diagnostic featur es in the identification, eva luation a nd
monograph preparation of the plant (4, 5).
MATERIALS AND METHO DS
Plant collection and identification
Whole plant of Anisome les indica was col lected from
Toranmal forest (Satpuda Vall ey), M aha rashtra , Ind ia in
the month of Aug-Sep 2006. Plant sample w as identified
and authenticated by Dr. D.A.Pa til taxonomist, SS V P’ S
Science College, Department of Bota ny, Dhule (M S), India.
Voucher speci men of the pl ant material has been
deposited at Institute level (RCPCOP/AI-0 6).
Chemicals and instr uments
Compound micros cope, simple microscope, glass slides,
cover slips, w atch gl a ss and other common glassware
were the basic appara tus and instruments used for the
study. Photomicroscope pr ovided with MOTIC IM AGES
PLUS 2.0 software. Some crystals, starch grains and
lignified cell slides w ere taken und er projection
microscope. Solvents viz. petr oleum ether, chloroform,
methanol, ethanol a nd rea gents viz . phloroglucinol ,
glycerin, Hcl , Iod ine and potassium hydroxide w er e
procured from Loba Chemicals, M umbai, India.
Macr oscopical examinations
For morphological observations, fresh young l eaves
(approx. 3-7 cm i n length) were used. The macro-
morphological features of the pla nt parts (leaves) were
observed und er magnifying lens a nd simple microscope
(6).
Micr oscopical examinations
Fresh leaves of the pla nt w ere stud ied transversely and
longi tudinal ly, using surfa ce preparations a nd sections.
The differ ent parts of leaf like la mina and midrib w ere
studied according to the methods of Brain and T urner (7 ).
For the micr oscopica l stud ies, cros s sections w ere
prepared and s tained as per the procedure of K. R.
Khandelwal (8). Qua ntitative evaluations and
qua nti tative-leaf microscopy w ere also carried out as
outl ined by W allis T. E. (9, 10).
Chemo-mi croscopical examinations were also carried out,
following thorough clearing of the powd ered lea ves with
potassium hydroxide soluti on and a subsequent mounti ng
with dilute glycerol on a microscope slide, a nd tested w ith
vari ous detecting rea gents. Various ergastic substances/
chemical constituents were identified in accordance with
(11).
Preliminary phytochemicals investig ation
The i nvestiga tion wa s carri ed out by using stand ard
procedures (12 ).
Quantit at ive evaluat ions of t he crude drug - Moisture
content of the pow dered leaves determi ned based on the

© Phcog.Net 2009 | w ww .phcog.net 45
loss of d rying method (1 3). The ash values (Total ash, acid-
insoluble ash a nd water-soluble ash) wer e d etermined , to
find out about the physiological state and level of
extra neous ma tter . Total a sh of the drug was subjected for
testing d ifferent inorga nic constituents (1 4, 15). E xtractive
values (ether , methanol, chloroform , alcohol a nd w a ter)
were d etermined according to the official methods
prescribed in Ayurved ic pharma copoeia (16 ). Fluorescence
analysis of powdered leaf was done by s tandard method
of Chase and Pratt (17).
RESU LTS
Macr oscopic ex amination (Figure 1)
The ma cr o-morphological characteristic of the leaves of
Anisome les indica identified w ere acute apex, crenate
margin, asymmetric base, reticulate venation a nd hairy to
softl y pubescent shap e. Lea ves surface thick, with
dimension 3.8-10 x 5.5-6 cm. Color is gr een to yellow ish
green; taste is slightly a stringent with characteristic od or.
The plant shows covering trichomes more on the lower
surface of the leaves.
Micr oscopic examination
Transverse section of leaf (Figure 2)
It is a dorsi ventral leaf. Follow ing tissues a re present in
la mina a nd midrib:
Lamina
The Upper epidermal cell s are compactly arranged with
no inter cellular spaces except stomata. E pidermis is made
up of single layer of spherical to polygona l cells with
straight to sli ghtly bead ed anti clinal walls wavy in shape
(Figure 2a). M ucilage is detected in epid ermi s. M esophyll
is differentiated i nto palisade and spongy parenchyma.
Palisade formed from compactly arranged elo ngated ,
narr ow columnar cells w ith beaded anti clinal walls.
Triseriate layer has been continuous over the mid rib
region (Figure 2b). Palisade cells were filled with
chlorophyll. Spongy parenchym a made up of
parenchymatous cells w ith varying size a nd shape. Lo wer
epidermis is made up of si ngle la yer of polygonal cells
with straight to slightly beaded a nticlinal walls.
Chlorophyll is present in epiderma l cells, numerous
caryophyllaceous or diacytic stomata (Figure 2 c), and
multicellular clothing trichomes as well a s gl andula r
trichomes were observed on the both epidermi s.
Midrib (Figure 2d )
Midrib present both surface with different degree of
concavity. Dorsal surface i s more convex than ventral
surface. Low er palisade is replaced by patch of
collenchymatous cells, which is about 3 7.5 µm in diameter.
Colla tera l vascular bundle is promi nent, occupying the
centr a l portion of the midrib. Xyl em vessels ar e cover ed by
xylem fibers. Xylem pa renchym a is mad e up from s emi-
rectangular, lignified cells. The phloem is non-lignified
and collenchyma tous in nature. The micr ometric analysis
is ta bulated in Ta ble 1.
Powder characteristic
The leaf powder is gr eenish black in color with astringent
taste. No greasy stain was observed when powd er press
betw een Whatmann’s filter paper No. 40 indicates absence
of fixed oi l. But the strong a r omatic odor of the filter paper
is a mark of presence of essential oil. The behavior of
powder with different chem ical reagent is shown in Table
2. On microscopically examination, the powd er showed
Trichomes - T wo types of trichomes; one is non-glandular
multicellular trichomes (Fi gure 3a ) and another wa s
glandular unicellular stalk with multicellular head type
(Figure 3b). After addition of small qua ntity of water in
powder it shows the follow ing elements.
Xylem fibe rs (Figure 3 c) - Scaliform with ladder like
thickening.
Mesophylls – Fragments of lea f showing spongy
parenchyma cells.
Starch grains are simple granules, spherical or irr egularly
ovoid w ith centric helium and less prominent striations.
(Figure 3d ). L arge lignified cell entire or scattered for ms
(Figure 3e).
Quantit at ive Micr oscopical parameters of leaves
Pertaining to the stomata l i ndex, stoma tal number, vein-
islet number and V ein-termination number data given in
Table 3.
Chemo-microscopical examination
The counter id ea about pr esence of phytoconstituents is
obtai ned through this study like phenol ic compound in
palisade as indicated by brownish black stain on ferric
chloride solution treatment (Ta ble 4).
Preliminary phytochemicals investig ation
Revealed the presence o f prima ry and s econdary
metaboli tes as carbohydrates, mucilage, Tannins,
Terpenoid, Gl ycosides, Alkaloids and Phytosteroi ds (Ta bl e
5)
Table 1: Micr ometr y of cellular elements
Table 2 : Behavior of powdered leaves
Reagent
Color /
ppt.
Constituent
Pow der + Sulphuri c
Acid
Reddish
Steroi ds +ve
Pow der + Aqs. Ferric
Bla ckish
Tannins
*
+ve
Cellular elements / cell content
Measurement (µ)
Upper epidermal cell
Length: 3 2.1
–
47.0
–
60.5 ; Wi d th: 20 .5
–
29.0
–
38.4
Lower epidermal cell
Length: 1 3.9
–
23.4
–
29.6 ; Width: 6 .60
–
1
4.9
–
21.7
Palisade cell
Length: 92.5
–
118
–
140 ; Width: 39.7
–
48.4
–
63.0
Upper collenchymatous cell
20.6
–
28.5
–
42.4
Lower collenchymatous cell
24.8
–
37.5
–
50.7
Parechymatous cell
55.1
–
79.3
–
98.6
Phloem cell
8.10
–
15.5
–
28.6
Xylem vesse
ls
14.1
–
16.9
–
19.8

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chloride sol.
Pow der + Iod ine sol.
Blue
Star ch +ve
Pow der + Picr ic
Aci d
sol.
Yellow
color
Alkaloid
*
+ve
Pow der + Aqs. Silver
Nitra te sol.
No
change
Protein +ve
Pow der + Aqs.
Pota ssium hyd roxide
sol.
No color
change
Antraquinone
Glycoside -ve
*may be present in powd er
drug; ‘+’ p resence; ‘
’ abse nce
*may be
present in
powder dr ug; ‘ +’
presence; ‘
Table 3: Quant it ative parameter s of leaves
Leaf Constants
Size (
µ
)
D
iameter of stomata
U
pper Surface: 3 5.0
-
37.0
-
40.0 ;
Lower Surface: 34.0 - 36.0 - 38 .0
S
tomatal number
U
pper Surface: 4 8.0
–
55.0
–
62.0
; Lower Surface: 24.6 - 25.5 - 26.5
S
tomatal i ndex
U
pper Surface: 5 6.0
–
62.0
–
68.0
; Lower Surface: 21.9 - 22.9 - 24.1
Vein
-
islet number
11.5 sq.mm
Vein
-
termi nation
number
21.9 sq.mm
Table 4 : Chemo-microchemical tests of Aniso meles indica leaves
Rea
gent
Col or
Test for
His tological zone
Phloroglucinol + Hcl
Pink
Lignin
Vascular bundle
Wea k iodine solution
Blue
Starch
Mesophyll region
Ferric chloride solution
Brownish bla ck
Phenolics
In palisad e cell region
Libermann
-
Burchardlt r ea gent
Greenish
St
eroids
Mesophyll region
Dragend orff’s reagent
Orange
Alkaloids
Lamina
Million’s rea gent
Blue
Proteins
Vascular bundle
Table 5: Preliminar y phytochemicals investigation
Phytoconstituent
Pet. ether
Chloroform
Methanol
Ethanol
W ater
Steroids
+
+
+
+
-
C
arbohydrates
-
-
-
-
+
Alkaloids
-
+
-
-
-
Glycosides
-
-
+
+
+
Reducing suga r
-
-
-
-
+
Phenolics
-
-
+
+
+
Tannins
-
-
+
-
-
Proteins
-
-
-
-
-
Amino aci d
-
-
-
-
-
Mucilage
-
-
-
+
+
‘+’ presence; ‘-’ absence
Table 6: Quantitative evaluations
Parameter
Value s % (w/w)
*
± SD
Total ash
11.87 ± 0.32
Acid insoluble as h
2.70 ± 0 .10
Water soluble ash
6.17 ± 0 .15
Sulphated ash
8.31 ± 0 .10
Moisture content
5.37 ± 0 .06
Extra ctive s:
Petroleum ether soluble
3.48 ± 0 .24
Chloroform soluble
9.23
± 0.15
Methanol soluble
13.63 ± 0.15
Ethanol soluble
11.20 ± 0.26
Water soluble
19.18 ± 1.01
* values expressed as mean of three r eadi ngs; SD-
Standard Deviation
Table 7: Qualitative analysis of elements present in
total ash
Element
Inf erence
Alu
minum
--
Calcium
++
Pota ssium
++
Magnesium
++
Sodi um
++
Iron
--
Zinc
--
Antimony
--
Chlorides
--
Sulphate
--
Phosphates
++
Carbonates
--
‘+ +’ Presence of elements, ‘--’ absence of elem ents
Table 8: Fluorescence analysis of extr act s
Extra ct Consistency Color in Day light Color in UV light
254nm
366nm
Petroleum ether
Sticky
Yellowish
Greenish yellow
Dark Brow
Chloroform
Sticky
Brownish
Dark Green
Dark Brown
Methanol
Sticky
Reddish
Green
Brownish

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Ethanol
Solid
Bla ckish
Dark Green
B
rownish
Aqueous
Solid
Bla ckish
Greenish Bla ck
Brownish
Figur e 1: Anisomeles indica Linn.
Figure 2: T. S. of midrib enlarged
[Xyl- Xylem; Phl- Phloem; Par- Parenchyma; L col- Low er
col lenchyma’s; L epi- Low er epid ermis; tri- Trichomes]
Fig ure 2
a: Upper epidermis
[
U epi
-
Upper epid ermis]
Figure 2b
: Palisade cells
[
Pal
-
Pal isade
Figure 2c: Diacytic St omata
[S- Stoma; G 1,2- Guard cells; SC- Subsidiary Cell; EC-
Epithelial Cel l]

© Phcog.Net 2009 | w ww .phcog.net 48
Figure 2d: Vascular Bundles
[Xyl- Xylem; Phl- Phloem]
Quantit at ive evaluations of the crude drug
The moisture content (NM T 5.37 ± 0.06 % w/w) seems to
be low er than necessary to support the gr owth of microbes
to br ing any change in the composition of the dr ugs.
Physical constant as ash value of the drug gives an idea of
the ea rthy matter or the inorganic composition a nd other
impurities present along w ith the drug. E xtractive va lues
are useful for the determination of exhausted or
adulterated drugs. The results of the physical constants of
the drug powder are given in Ta ble 6. The qualitative
analysis of ash ind icated presence of calcium, Potassi um,
magnesium, sodium a nd phosphates (Table 7). The results
of fl uorescence ana lysis of the extracts presented in T a bl e
8.
CONCLU SION
The exhausted l iterature survey conclude before the
present work start there is no pha rmacognostic stud y on
record of this much valued traditional drug. Therefore
present work was taken up with a vi ew to lay down
standards which could be useful to find the authenticity of
this tra ditional medicinal plant. In other words, the
pharmacognostic features examined in the present study
may serve as tool for identi fica tion of the plant for
validatio n of the r aw mater ial and for standardization of
its formulations at Herbal industrial level in the coming
days.
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