40. RPB14150073015..

377 | P a g e International Standard Serial Number (ISSN): 2319-8141
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International Journal of Universal Pharmacy and Bio Sciences 3(2): March-April 2014
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES
IMPACT FACTOR 1.89***
ICV 5.13***
Bio Sciences Research Article ……!!!
MICROMETRIC EVALUATION OF CULTIVATED AND WILD VARIETIES
OF RAPHANUS SATIVUS LINN LEAVES
Pankaj goriya *, Harisha C.R 1
, Shruti ladani 2
, Galib R3
. Prjapati P.K 4
*M.pharm (Ayu) Scholar, 1.Head, Pharmacognosy, 2. M.pharm (Ayu) Scholar, 3. Assistant
Professor, Dept. RS&BK, 4. Professor & Head, Dept. of RS&BK. IPGT & RA, Gujarat Ayurved
University, Jamnagar, Gujarat, India 361 008.
KEYWORDS:
Cruciferaceae, Mulaka,
Pharmacognosy, Raphanus
sativus, Stomata.
For Correspondence:
Pankaj Goriya *
Address: Dept. of RS&BK.
IPGT & RA, Gujarat Ayurved
University, Jamnagar, Gujarat,
India 361 008.
Email:
goriyapankaj222@gmail.com
Mob: 9408134055.
ABSTRACT
Medicinal plants occupied a distinct place in human life right from the
primitive period to until date. Raphanus sativus Linn., commonly known
as Mulaka in Sanskrit, used in Ayurveda therapeutically in the treatment
of fever, laxative, colic pain, Piles, cough, leprosy, urinary stone etc. Till
date no pharmacognostical work has been reported on this drug.
Considering availability of two varieties (cultivated and wild) of Mulaka
in the market, an attempt has been made to develop pharmacognostical
characters of leaf of both cultivated and wild varieties. Materials and
Methods: Macroscopic, microscopic, micrometric characters including
histochemical analysis of leaf were carried-out. Results:
Pharmacognostical evaluation and powder microscopy of both varieties
shows the presence of annular vessels, spiral vessels, fragments of
anomocytic stomata, warty simple trichome, and starch grain etc.
Conclusion: The current work appears to be the first of its kind and can
be considered as reference standard for further studies.

INTRODUCTION:
Pharmacognostic study gives the scientific information regarding the purity and quality of the
plant drugs.1
It is the preliminary step in the standardization of crude drugs.2
Detailed
pharmacognostical evaluation gives valuable information regarding the morphology,
microscopical and physical characteristics of the crude drugs.3
Raphanus sativus Linn is popularly known as Mulaka in Sanskrit and Mulo in Gujarati.4
It
belongs to the family Cruciferaceae. It is cultivated in plains throughout India up to 3000 m in
the Himalayas and other hilly regions.5
There are many traditional medicinal uses for different
parts of R. sativus Linn. The leaves posses therapeutic uses like Sita jvara (fever caused by
cold)6
, Grahi (therapeutic absorptive measure)7
and Swarasa of Mulaka was used in the recipe of
Hapusadya Ghrita in the treatment of Anaha (abdominal distension), Shoola (colic pain),
Yonivyapata (disease of female genital tract), Arsha (piles), Grahani dosa (sprue syndrome),
Duha shvasa (asthma), Svasa-Kasa (cough), Aruchi (anorexia), Jvara (fever),
Bastihyatpasrshvashoola (pain in the region of urinary bladder including kidney) etc.8
Though, this is an important herb; till date no pharmacognostical work is reported on any of the
parts of R. sativus of wild or cultivated varieties. Considering this, an attempt has been made to
evaluate pharmacognostical characters of leaf that include macroscopic, microscopic,
micrometric characters.
Materials and methods:
Collection & Authentification of raw drug:
Raphanus sativus Linn. Leaves of both varieties were collected in Nov, 2013 from Jamnagar,
Gujarat, India. Pharmacognostical authentification was done in pharmacognosy lab, IPGT & RA.
Pharmacognostical analysis:
Macroscopic:
Collected samples were identified and authenticated by studying their characters systematically
as per the methods described in the textbooks of pharmacogosy and other Botanical texts and
floras. The specimen was observed as such with naked eye.9

Organoleptic:
Organoleptic evaluations of both raw samples were done by their characters like, colour, texture,
odour, taste etc.9
Microscopic:
Free hand sections of the leaves observed under the microscope for the presence of primary and
secondary metabolites like starch grains were confirmed by staining with iodine. The sections cleared
with chloral hydrate to observe various ergastic cell contents like, crystals of calcium oxalate, calcium
carbonate, and silica if present any. The nature of these crystals were also confirmed by performing
tests like solubility of them in acids (HCl). The sections then stained with Phloroglucinol and HCl for
detecting lignified elements. Microphotographs were taken under carl-zeiss trinocular microscope
attached with camera.x
Surface study
Both upper and lower surfaces of leaf was scientifically peeled off and cleared with chloral hydrate,
studied under the microscope. Stomata index, length and width etc. were measured.xi
Powder microscopy:
Organoleptic characters:
Powders of the both samples were subjected to the organoleptic characters i.e. Colour, taste and
touch.xii
For powder microscopy, slides were prepared by using water, chloral hydrate as a clearing agent,
stained with phloroglucinol and conc. HCL. Microphotographs were taken under carl-zeiss trinocular
microscope attached with camera.xiii
Quantitative Microscopy
Micrometric evaluation
Quantitative leaf microscopy to determine palisade ratio, stomata index and vein-islet number were
carried out. The leaf epidermal studies were carried out on fresh specimens. Peels were removed
mechanically through epidermal peeling off and stomatal index (SI) was calculated. The vein islet
number and palisade ratio of lamina were determined according to the standard method.xiv
Histochemical evaluation:
Thick sections were treated by various reagents to detect chemicals i.e. tannin, starch, lignin etc. by
Fecl3, Iodine etc.xv

RESULTS AND DISCUSSIONS:
Morphological characteristics of the both leaf
The plant was identified as R. sativus (cultivated or wild variety), based on its morphological [Plate-1,
2 figure1, 13]. [Table 1]
Organoleptic characteristics
Fresh cultivated leaves are smooth to the touch, green in color, bitter in taste, and offensive. Wild
leaves also possess similar qualities with strong bitter taste. [Table 2]
Transverse section of Leaf through mid rib (Cultivated variety)
Diagramatic T.S. of leaf show that dorsiventral epidermis differentiated into upper and lower
epidermal through mid rib shows three distinct vascular bundles.
Detail transverse section showed upper epidermis and lower epidermis, single layer epidermal cells
with thin cuticle. Some of the epidermal cells were interrupted by stomatal openings. Mesophyll made
up of upper 2 to 3 layer compactly arranged palisade parenchyma enriched by chlorophyll pigment.
Some of the palisade parenchyma consist oil globules and prismatic crystals of calcium oxalate. Lower
layer arranged in to 6 to 8 layers of spongy parenchyma between the palisade spongy parenchyma the
vascular strands are passed. Through mid rib shows below the upper epidermis and lower epidermis 1
or 2 layers of collenchymatous layer are present. Ground tissue made up of parenchyma cells rarely
filled with oil globules and crystal, three distinct vascular bundles middle large adjust and small in
enclosed by endodermis. Endodermis single layer with parenchymatous cell 2 to 3 layers of pericyclic
fibre situated of the lower portion of the vascular bundle followed by phloem. Phloem made up of
phloem fibres and some sieve elements. Metaxylem towards lower epidermis, protoxylem towards
upper epidermis made up of xylem fibres and its parenchyma. [Plate 1 figure 2 to 10]
Transverse section of Leaf through mid rib (Wild variety)
Leaf dorsiventral shows epidermis differentiated into upper and lower epidermis through mid rib
shows distinct three vascular bundles.
Detail transverse section shows upper epidermis and lower epidermis, single layered epidermal cells
with thin cuticle. Some of the epidermal cells were interrupted by stomatal openings and epidermal
cells with pink coloured material along with simple trichomes with oil content. Mesophyll made up of
upper 3 to 5 layered compactly arranged palisade parenchyma enriched by chlorophyll pigments. Some
of the palisade parenchyma consist oil globules and prismatic crystals. Lower portion occupied by 5 to
6 layered of spongy parenchyma. Between the palisade and spongy parenchyma the vascular strands
are passed out. Through mid rib shows below the upper epidermis and lower epidermis 3 to 4 layered
of collenchymatous celled layer is present. Ground tissue made up of parenchyma cells rarely filled

with oil globules and crystal, three distinct vascular bundles middle large adjust and small in enclosed
by endodermis. Endodermis single layered with parenchyma cells. 2 to 3 layers of pericyclic fibres
enclosed the vascular bundle. Vascular bundle made up of lower phloem. Phloem made up of phloem
fibres and some sieve elements. Xylem radially arranged metaxylem towards lower epidermis,
protoxylem towards upper epidermis made up of xylem fibres and its parenchyma. [Plate-2 figure 14
to 20]
Surface study & Micrometric evaluation of leaves
Properly peeled of both upper and lower surfaces of leaves are studied under the microscope.
The results were depicted in [table 3] [Plate-1, 2 figure 11, 12 and 21 to 24]
Organoleptic characters of the powder
Organoleptic characters i.e. colour, odour, taste, touch etc. of both leaves were observed by sensory
organs. The results were depicted in [table 4].
Powder microscopy of wild variety
Wild leaves powder showed that annular vessels, spiral vessels, fragments of anomocytic stomata,
warty simple trichome, starch grain with parenchyma, fragments of spongy parenchyma, trichomes
filled with oil, prismatic crystal of calcium oxalate, oil globules, fragments of palisade parenchyma
and blotched trichomes, lignified parenchyma cells. [Plate-4 figure 37 to 47]
Powder microscopy of cultivated variety
Cultivated leaves powder showed that anomocytic stomata, annular vessels, spiral vessel, prismatic
crystal, loosely arranged parenchyma cells, trichome without oil, simple fibbers, fragments of spongy
parenchyma, lignified fibber, oil globule, starch grain and tannin content. [Plate-3 figure 25 to 36]
Histochemical evaluation
Both leaves were subjected to various chemical constituents i.e. lignin, tannin, etc. which were present
in the leaves by using various reagents, results are depleted in table no. 5
Discussion:
To get original quantity of constituents of plant from the mother traits due to eradication to get
quantity wise product changes the morphology, histology, and also the percentage of the end product.
Pharmacognostical study reveals that the morphology of wild plant leaf is 10 – 15 cm long, lythrate, 8
– 9 division lower division having large as compare to cultivated var. midrib color was pinkish brown
and petiole with trichomes. Leaf is margin largely crinate with serrate margin. Whereas morphology of
cultivated plant leaf is 20 – 30cm long, 6 – 8 division base too small upper division very large, midrib
color was whitish green and petiole has no trichomes. Margin is less crinate to serrate margin. Leaf
through midrib showed that cultivated verity of leaf shows that mesophyll made up of upper layer

arranged 3 to 5 layered and wild verity of leaf shows 2 to 3 layered compactly arranged palisade
parenchyma. Lower layer occupied by spongy parenchyma of 5 to 6 layered in cultivated were as 6 to
8 layered in wild verity. Through mid rib below the upper and lower epidermis shows 3 to 4 layered of
collenchymatous celled in cultivated and only 1 to 2 layers are present in wild verity. Endodermis
single layered with parenchyma cells. 2 to 3 layers of pericyclic fibres enclosed the vascular bundle.
Vascular bundle made up of lower phloem. Phloem made up of phloem fibres and some sieve
elements. Xylem radially arranged metaxylem towards lower epidermis, protoxylem towards upper
epidermis made up of xylem fibres and its parenchyma in both varieties. Results showed that the
organoleptic characters like odour, taste, touch are same in all both varieties. But colour of powder is
different. In cultivated colour of leaf powder is green while in wild variety leaf powder is yellowish
green in colour. The powder microscopy rather doesn’t show any much difference but the presence
characters are annular vessels, spiral vessels, fragments of anomocytic stomata, warty simple trichome,
starch grain with parenchyma, fragments of spongy parenchyma, trichomes filled with oil, prismatic
crystal of calcium oxalate, oil globules, and fragments of palisade parenchyma and blotched trichomes,
lignified parenchyma cells. In wild verity as compare to cultivated verity all the significant characters
are Cultivated leaves powder shows anomocytic stomata, annular vessels, spiral vessel, prismatic
crystal, loosely arranged parenchyma cells, trichome without oil, simple fibres, fragments of spongy
parenchyma, lignified fibre, oil globule, starch grain and tannin content. Micrometric evaluation
showed that both epidermis with anomocytic type of stomata with slight variation in the stomatal
length and breadth. Stomatal index was 25 in both epidermises in both types of leaves. Palisade ratio
of cultivated leaf showed five, where as in wild was 4. This result showed that the photosynthetic and
physiological activities more in cultivated as compare to wild variety. The stomatal index number is a
constant for the respective species; hence this can also be a useful for species differentiation. The
Histochemical tests showed that presence of lignified elements, starch grains, tannin contents in both
verities.
Conclusion:
Wild plants which are acclimatize very fast to their environmental condition. Whereas the improved
cultivated tissue culture plants are difficult to acclimatize to the environment.
REFERENCES:
1. Dhanabal SP, Suresh B, Sheeja E and Edwin E. Pharmacognostical studies on Passiflora
quadrangularis. Indian Journal of Natural Product,s (2005) 21(1): 9-11.

2. Sumitra Singh, Vijay Naresh, Surendra Kr Sharma. Pharmacognostic Parameters of Salvadora
Oleoides Decne. Leaves- Asian Journal of Pharmaceutical Research and Development. 2013 1
(3). .
3. Sharma SK (2004). Recent approach to herbal formulation development and standardization;
http//pharmainfo.net.
4. Bapalal G. Vaidya, Nighantu Adarsa, Vol-1, Chaukhambha bharati academy, Varanasi; 2007.
pg-76-79.
5. Anonymous (2002), DATABASE ON MEDITIONAL PLANTS USED IN AYURVEDA,
Publication, & Information Directorate, CCRAS, New Delhi, Vol . IV, Pg. 443.
6. Caraka Samhita of Agnivesa, elaborated by Caraka and Drdhabala, Chikitsasthana 3/267,
Charaka Chandrika Hindi Commentary by Dr. Brahmananda Tripathi, Chaukhamba Surbharati
Prakashana, Varanasi reprint 2006. pg-160-161
7. Caraka Samhita of Agnivesa, elaborated by Caraka and Drdhabala, Chikitsasthana 14/93-96,
text with English translation and critical exposition based on Chakrapani datta’s Ayurveda
Dipika Vol 3 by R.K. Sharma Bhagvan dash Chaukhamba Sanskrita series office,
Varanasi;2004. Pg-602.
8. Caraka Samhita of Agnivesa, elaborated by Caraka and Drdhabala, Chikitsasthana 5/ 71-73,98,
Charaka Chandrika Hindi Commentary by Dr. Brahmananda Tripathi, Chaukhamba Surbharati
Prakashana, Varanasi reprint 2006. Pg- 212, 214
9. Trease and Evans, Pharmacognosy, 15th Ed., W.B. Sunders Company Ltd. 1996 pg- 569,570.
10. Wallis TE, Text book of Pharmacognosy, 5th Ed., New Delhi: CBS Publishers &
Distributors2002, pg- 123- 132, 210-215.
11. Evans WC. Cell differentiation and ergastic cell contents, Techniques in microscopy. In:
Trease and Evans Pharmacognosy. 16th ed. New York: Saunders Elsevier; 2009. pg-551-70.
12. Trease and Evans, Pharmacognosy, 15th Ed., W.B. Sunders Company Ltd. 1996 pg-569,570.
13. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 42nd ed. Nirali Prakashan, Pune, 2008.
Pg- 6.3
14. Krishnamurty KV. Method in the plant histochemistry. Madras: vishwanandan Pvt, limited;
1988, Pg-1-74.
15. Krishnamurty K V. Methods in the plant histochemistry. Madras:VishwanandanPvt.
Limited; 1988. Pg- 1-7.

Table 1: morphological characters of Raphanus sutivus Linn. Leaf
Parameter
Observation
Cultivated Leaf Wild Leaf
Condition Fresh Fresh
Type Simple Simple
Size
Length: 20–30 cm
Width: 14-16 cm
Length: 10–15 cm
Width: 7-9 cm
Shape Spoon shape Spoon shape
Margin Crenate Deeply crenate
Apex Obtuse Obtuse
Base Unequal Unequal
Petiole Greenish Pinkish tinged
Venation Reticulate Reticulate
Phyllotaxy Alternate literate Alternate literate
Surface
Upper surface: Smooth
Lower surface: without trichomes
Upper surface: Slight rough
Lower surface: with trichomes
Extra feature Large leaves Short leaves

Table 2: Organoleptic characteristics of leaves of Raphanus sativus
Linn.
(cultivated and wild variety)
Parameter Cultivated leaves Wild leaves
Texture Smooth Smooth
Color
Greenish
Midrib and veins : greenish
white
Greenish
Midrib and vines : brownish
pinkish
Taste Mucilaginous slightly bitter Mucilaginous strong bitter
Odor Offensive More offensive
Table 3: Surface study & Micrometric evaluation of leaves (Cultivated and wild variety)
Characters
Cultivated variety Wild variety
Lower epidermis Upper epidermis Lower epidermis Upper epidermis
Type of
Stomata
Anomocytic
stomata
Anomocytic
stomata
Anomocytic
stomata
Anomocytic
stomata
Stomata
length
7μm 2μm to 6μm 5μm to 6μm 5μm to 6μm
Stomata
breadth
4.5μm to 5μm 4μm to 5μm 3μm to 5μm 3μm to 4μm
Stomata Index 25 25 25 25
Palisade ratio ---- 5 --- 4

Table 4: Organoleptic characters of powder leaf
Characters
Cultivated
variety
Wild variety
Colour Green yellowish green
Odour
Bitter,
characteristic
Strong bitter,
characteristic
Taste Lavana, Kashaya Lavana, Kashaya
Touch Rough Rough
Table 5: Histochemical tests of leaves of cultivated and wild varieties
Sr.
no
Reagent Observation
Characterist
ics
Cultivated
variety
Wild
variety
1.
Phloroglucino
l + Conc. HCl
Red
Lignified
cells
++ ++
2. Iodine Blue Starch grains ++ ++
3.
Phloroglucino
l + Conc. HCl
Dissolved
Calcium
oxalate
crystals
++ ++
4. Fecl3 solution
Dark blue to
black
Tannin cells ++ ++

Plate No. 1- Cultivated variety
Figure 1. Leaf macroscopic
study
Figure 2. Through mid rib Figure 3. central vascular
bundle
Figure 4. Lower epidermis Figure 5. Upper epidermis Figure 6. Palisade Spongy parenchyma
Figure 7. Strained xylem and phloem Figure 8. Vascular strands passing
thhrough mesophyll
Figure 9. Lower phloem and upper
xylem
Figure 10. Protoxylem and Metaxylem Figure 11. Stomatal (Anomocytic)
Bredth
Figure 12. Stomatal (Anomocytic)
Length

Plate No.2- Wild variety
Figure 13. Leaf macroscopic study Figure 14. Through mid rib Figure 15. Central vascular bundle
Figure 16. Lower epidermis Figure 17. Upper epidermis with
trichome
Figure 18. Starch grain
Figure 19. Pericycle phloem and
upper xylem
Figure 20. Stained Xylem and
phloem
Length
Bredth
Length
Bredth

Plate No. 3- Powder microscopy of cultivated variety
Figure 25. types Stomata Figure 26. Prismatic crystal Figure 27. Fragment of palisade cells
Figure 28. simple trichome Figure 29. Fibre with crystal Figure 30. Fibre
Figure 31. Parenchyma cell Figure 32. Lignified fibre Figure 33. Annular and spiral vessel
Figure 34. Spiral vessel Figure 35. Simple starch grain with
hilum
Figure 36. yellow and brown content

Plate No.4- Powder microscopy of wild variety
Figure 37. Anomocytic stomata Figure 38. Covering trichome Figure 39. Warty trichome
Figure 40. Cork cells with starch
grain
Figure 41. Parenchyma with oil
content
Figure 42. Covering trichome with
oil content
Figure 43. Prismetic crystal Figure 44. Simple and Compound
starch grain
Figure 45. Fragment of Annuler
and spiral vessel.
Figure 46. Fragment of spiral
vessel.
Figure 47. Lignified fibres

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