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INDEX – GJRMI - Volume 3, Issue 3, March 2014
MEDICINAL PLANTS RESEARCH
Bio-technology & Botany ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID ETHYL ESTER FROM
THE METHANOLIC EXTRACT OF THE AERIAL PARTS OF ANISOMELES MALABARICA (L).
R.BR.
Ismail Shareef M, Leelavathi S, Gopinath S M 67–74
Bio-technology ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL PLANTS
Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal, Sreekanth B,
Purushotham K M
75–79
Review Article GENE SILENCING AND ITS APPLICATIONS IN CROP IMPROVEMENT & FUNCTIONAL
GENOMICS
Jemal Ali, Pagadala Vijaya Kumari 80–90
Review Article CALLICARPA MACROPHYLLA: A REVIEW OF ITS PHYTO-CHEMISTRY, PHARMACOLOGY,
FOLKLORE CLAIMS AND AYURVEDIC STUDIES
Pandey Ajay Shankar, Srivastava Bhavana, Wanjari Manish M, Pandey Narendra Kumar, Jadhav Ankush D
91–100
Short communication PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND HEALING PLANTS
Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal, Purushotham K M
101–104
INDIGENOUS MEDICINE
Ayurveda – Rasa Shastra A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS STANDARDIZATION
Wavare Ramesh, Yadav Reena, Sheth Suchita, Sawant Ranjeet 105–111
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER OF CEIBA PENTANDRA (L.) GAERTN., OF THE
FAMILY MALVACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT,
KARNATAKA, INDIA
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID
ETHYL ESTER FROM THE METHANOLIC EXTRACT OF THE AERIAL
PARTS OF ANISOMELES MALABARICA (L). R.BR.
Ismail Shareef M
1*, Leelavathi S
2, Gopinath S M
3
1Assistant Professor, Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli,
Acharya Post (O), Bangalore-560 107, Karnataka, India 2Professor, DOS in Botany, Manasagangotri, University of Mysore, Mysore-570 006, Karnataka, India
3Professor and Head, Department of Biotechnology, Acharya Institute of Technology, Soladevanahalli,
Acharya Post (O), Bangalore-560 107, Karnataka, India
*Corresponding Author: E-mail:[email protected]; Mobile: +91-9538833786, Fax No.:080-
23700242
Received: 09/02/2014; Revised: 22/02/2014; Accepted: 28/02/2014
ABSTRACT
Anisomeles malabarica (L). R.Br., (Lamiaceae) is distributed in major parts of India, especially
in South India it is known as a traditional medicinal plant reported to possess anti-spasmodic, anti-
inflammatory properties and is used in Rheumatoid arthritis. The preliminary phytochemical
investigation on the methanolic extract of the aerial parts of the plant revealed the presence of
carbohydrates, phytosterols and triterpenoids. The aim of the current study was to isolate and
characterize the bioactive compounds from the aerial parts of Anisomeles malabarica as the plant is
reported to possess potent anti-inflammatory properties and is also used in the treatment of
Rheumatism. For isolation purpose, the dried powder of was extracted with methanol using Soxhlet
apparatus continuously for 16 hours. The extract was dried under reduced pressure to evaporate the
solvent and the dried mass was taken for the isolation work. Pentadecanoic acid ethyl ester was
isolated by column chromatography from the methanolic extract of aerial parts of Anisomeles
malabarica. The structural elucidation of the isolated compound was on the basis of spectroscopic
analysis.
KEYWORDS: Anisomeles malabarica; pentadecanoic acid ethyl ester; rheumatoid arthritis;1H-
NMR; 13
C-NMR; LC-ESI-MS
Research Article
Cite this article:
Ismail Shareef. M, Leelavathi. S, Gopinath. S. M (2014),
ISOLATION AND CHARACTERIZATION OF PENTADECANOIC ACID ETHYL ESTER
FROM THE METHANOLIC EXTRACT OF THE AERIAL PARTS OF ANISOMELES
MALABARICA (L). R.BR., Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 67–74
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Today, Ayurvedic, Homeo and Unani
Physicians utilize numerous species of
medicinal plants (Mujumdar AM et al., 2000).
Many compounds used in today's medicine
have a complex structure and synthesizing
these bioactive compounds chemically at a low
price is not easy (Madhava C, 1998). The
increasing awareness about side effects of
drugs had made the western pharmaceutical
industries to turn towards the plant based
Indian and Chinese medicine (Balandrin MJ &
Klocke JA 1988). Anisolmeles malabarica (L).
R.Br., (Lamiaceae) is distributed in major parts
of India and especially in South India as a
traditional medicinal plant commonly known as
Peymarutti (Tamil), Gouzaban (Hindi),
Chodhara (Marathi), Karithumbi (Kannada)
and Malabar catmint (English)(Kritikar KR &
Basu BD, 1935). The herb is reported to
possess anti-spasmodic, anti-periodic properties
and used in Rheumatoid arthritis (Nadkarni
KM, 2006). It is used for the traditional
treatment of snakebite as antidote and plant
leaves are used as carminative, astringent,
stomachic, rheumatism and diaphoretic in
Coimbatore district and also used as dentifrice
to cure various problems (Kalyani K et al.,
1989). The methanolic extract of aerial parts of
Anisomeles malabarica (L). R.Br., (AmA)
produced significant anti-rheumatic activity in
a dose-dependent manner (200 mg/Kg and 400
mg/Kg body weight) to that of standard drug
indomethacin (10 mg/Kg). The extract
exhibited inhibitory effect in carrageenan
induced hind paw oedema in rats with all the
doses used when compared to the control
group. The data obtained indicate that the crude
extracts of the aerial parts of the plant AmA
possess potential anti-rheumatic activity by
supporting the folkloric usage of the plant to
treat various inflammatory conditions (Setty
AR, 2005).
AmA extract was tested for cytotoxicity in
RAW and L-929 cell lines and was found to be
non-toxic. Based on the results, non-toxic doses
of extracts were tested for their inhibitory
activity against LPS induced TNF-α
production. AmA showed better activity by
reducing the LPS induced TNF-α production by
38.75 % (Ismail SM et al., 2012). So based on
the various in-vivo and in-vitro studies
conducted, it can be concluded that the plant
AmA possesses potent immuno-modulatory
and anti-rheumatic properties. With the above
findings, the present work was carried to isolate
and characterize the bio-active phyto-
constituents present in AmA.
MATERIALS AND METHODS
Collection of plant material
Fresh leaves of Anisomeles malabarica free
from disease was collected from different
regions in & around Bangalore and were
authenticated by taxonomists & the Voucher
specimen was deposited in the department for
future reference.
a) Chemicals
Hexane, ethyl acetate, chloroform,
methanol and silica gel of mesh size 60–120
and 200–400 was purchased from Sd fine
chemicals, Mumbai, India. Column length was
100 cm and column diameter was 3 cm.
b) Extraction procedure
Dried powder of AmA was extracted with
methanol using Soxhlet extraction unit for 18
hours as per standard procedure (Mukherjee
PK, 2010) the extract was dried under reduced
pressure to evaporate the solvent and dried
mass (20 gm) was taken further for isolation
work.
Isolation of phytochemicals
I. Column Chromatography Purification Of
Methanolic Extract
a) Adsorption of sample on silica gel
The methanolic extract of AmA (dried mass ;15
gms) was adsorbed on dry silica and the
adsorbed sample was kept for complete drying
and later used for coloumn elution.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
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b) Loading of column (wet packing)
Column was packed with silica gel slurry of
mesh size 60–120 with hexane. Column length
was 100 cm and diameter was 3 cm. On top of
silica bed activated sample was loaded and
cotton was placed on top of it to avoid any
disturbance to the sample bed.
c) Elution of the column
Initially Hexane solvent was eluted in small
quantity for correct distribution of activated
sample in the column and later eluted with two
solvent combinations with increasing order of
polarity.
Based on preliminary TLC observations,
column elution was started with hexane, ethyl
acetate and methanol combinations. Fractions
were collected in 50 ml portions.
Pattern of column elution:
1. Hexane
2. Hexane: Ethyl acetate: 7:3
3. Ethyl acetate
4. Ethyl acetate : Methanol : : 5 : 5
5. Methanol
From the above elution process, the
fractions were pooled as per their mobile phase.
From the above fractions, Ethyl acetate:
Methanol: 5:5 was further processed for
purification through column chromatography.
The other fractions were not used due to their
low yield.
II. Column Chromatography Purification Of
Ethyl Acetate: Methanol: : 5 : 5
a) Adsorption of sample on silica gel
The ethyl acetate : methanol : : 5: 5 fraction
(dried mass; 5–8 gms, brown colour powder)
was adsorbed on dry silica and the adsorbed
sample was kept for complete drying and later
used for column elution.
b) Loading of column (wet packing)
Column was packed with silica gel slurry of
mesh size 200–400 with chloroform. On top of
silica bed, activated sample was loaded and
cotton was placed on top of it to avoid any
disturbance to the sample bed.
c) Elution of the column
Initially chloroform was eluted in small
quantity for correct distribution of activated
sample in the column and later with two
solvent combinations with increasing order of
polarity.
Column elution was started with
chloroform and methanol combinations.
Fractions were collected in 15 ml portions.
d) Evaporation of fractions
Based on TLC profiles of the eluted
fractions, they were pooled and evaporated.
Chloroform: methanol : : 7 : 3 fractions were
dried under reduced pressure and then
subjected for preparative TLC for purification.
e) Preparative TLC of chloroform :
methanol : : 7 : 3 fractions
The dried fraction was purified by
preparing the TLC plate with silica gel G, a
mobile phase of Chloroform : methanol : : 7.5 :
3 was used for TLC. After TLC separation, the
plate was air dried and observed under UV
light. A yellow fluorescent band was scrapped
off and the band was eluted by mixing with
methanol and later centrifuged. The solvent
was collected, dried and later checked for
purity by TLC and the compound (under
investigation) was sent for spectral analysis i.e.,
IR, MASS, C13
NMR &1H NMR for structural
elucidation.
RESULTS
Spectral studies
The Compound in its ESI-MS (positive
mode) spectrum exhibits a peak at m/z 279 for
an ion [M+Na] +
suggesting a molecular weight
of 256.
In its 1H-NMR spectrum (Figure 1–4) it
showed peaks at δ 0.80 showing the presence
of methyl groups in the compound. The large
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
singlet at δ 1.15 and the signals at δ 1.80 were
due to the long chain methylene groups. The
signal at δ 1.90 is due to a methylene adjacent
to a carbonyl group. The signal at δ 3.40 may
be due to the protons attached to oxygen
function.
In the 13
C-NMR (Figure 5) the signals at δ
20.00 are due to methyl group, at δ 25, 28.00 to
31.00 are due to the methylene carbons. The
signal at δ 70.00 is due to the carbon attached
to the oxygen function. The signal at δ 172.00
confirms the presence of a carbonyl group. The
results of LC-ESI-MS is depicted in Figure 6.
Based on the above data the structure of the
compound is Pentadecanoic Acid Ethyl Ester
(Figure 7).
Figure 1.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br. at 279 MHz
Figure 2.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Figure 3: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br.
Figure 4.: 1H-NMR of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br.
Figure 5.: 13
C-NMR of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Figure 6.: LC-ESI-MS of bio-active compound from the aerial parts of Anisomeles malabarica
R.Br.
Fig.7. Pentadecanoic Acid Ethyl Ester
CH3- (CH2)13- COO-CH2- CH3
Molecular formula: C17H34O2
Synonym: Ethyl N-Pentadecanoate
DISCUSSION
Anisomeles indica L., and Anisomeles
malabarica R. Br. Ex Sims, is found growing
wild in India. The chemical composition and
antibacterial activity of the essential oils from
Anisomeles indica L and A. malabarica were
investigated together. The aerial parts (Stem,
leaves, flowers and fruit) of hydrodistilled
essential oils were analyzed by gas
chromatography-mass spectrometry (GC-MS),
and antibacterial activity was individually
evaluated against Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and
Bacillus pumilus using a paper disc diffusion
method. Collectively more than forty
compounds were identified in A. indica and A.
malabarica, representing 98.29–97.88% of the
total essential oil, respectively. The major
constituents of essential oils obtained from the
A. indica, were linalyl acetate (15.3%), and α-
thujone (11.9%). The most abundant
compounds in essential oils of A. malabarica,
were - α-thujone (17.6%), terpenyl acetate
(16.45%) and, δ-cadinene (11.5%). All tested
G+ ve& G-ve were inhibited by essential oil
samples. The GC-MS results of both plants
indicated the essential oil is rich in
monoterpenes and terpenoids, which have been
implicated antibacterial activity, comparable to
gentamycin, it was used as a positive probe.
The current findings also help to differentiate
the valuable Anisomeles species for phyto-
pharmaceuticals (Ushir Y & Patel K, 2011).
Seven fatty acids were identified from the
methanolic extract of Anisomeles indica L., and
Anisomeles malabarica L. R. Br. Ex Sims
aerial parts. The extracted fatty acids were
methyl-esterified and then analyzed by GC-
MS. The relative contents of the fatty acids
were calculated with Area normalization.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Seven fatty acids amounting to 77.778% in A.
indica and 68.027% in A. malabarica of the
total contents were detected. The major fatty
acids found in A. indica were palmitic acid
(23.334%), stearic acid (22.749%), lignoceric
acid (21.54%) and, in A. malabarica, palmitic
acid (35.252%), stearic acid (21.43%). The
results the content of fatty acids was abundant
in Anisomeles species, and it had a great range
of potential utilities and a prospect of
development in foods, medical and health care
(Ushir Y et al., 2011).
Based on the above findings by (Ushir Y et
al., 2011) it can be concluded that Anisomeles
malabarica R.Br. possesses potent bio-active
compounds, both reported in literature and yet
to be reported. So it was investigated to
evaluate the in vivo and in vitro activity of the
isolated compound namely, Pentadecanoic
Acid Ethyl Ester from the aerial parts of AmA.
CONCLUSION
The current investigation from the
methanolic extract of aerial parts of the plant
Anisomelos malabarica has revealed the
presence of Pentadecanoic Acid Ethyl Ester.
Also the plant is reported to posses’ inhibition
of invitro TNF-α production and possesses anti-
rheumatic and immuno-modulatory properties.
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aromatic and industrial materials from
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Inhibition on in vitro TNF-α production
by Anisomeles malabarica R.Br.
reinforces its anti-rheumatic and
immunomodulatory properties. Proc.
Natl. Acad. Sci., India, Sect. B Biol..83,
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Ismail SM, Leelavathi S, Thara SKJ, Sampath
KKK (2012): Evaluation of in-vivo anti-
rheumatic activity of Anisomeles
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(1989): Medico-Botanical Survey of
plants in Marudhamalai Hills of
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Medicinal plants. 2nd Edition,
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Dehradun, India.,2011–2012.
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Edition, Popular Prakashan Pvt., Ltd.,
Mumbai, India.,114–115.
Narayana R, Thammanna K. (1987): Medicinal
plants of Tirumala hills, department of
garden, tirumalatirupatidevasthanams,
Tirupati, India.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 67–74
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Perumalswamy R, Maung TM,
Gopalakrishnakone P, Ignacimuthu S
(2008): Ethno Botanical survey of folk
plantsfor the treatment of Snakebites
inSouthern part of Tamil Nadu, India.J
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Ushir Y, Patel K, Sheth N (2011): Analysis of
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and antibacterial activity of essential oil
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Source of Support: NIL Conflict of Interest: None Declared
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL
PLANTS
Jagan Mohan Reddy P1, Ismail Shareef M
2*, Gopinath S M
3, Dayananda K S
4,
Ajay Mandal5, Sreekanth B
6, Purushotham K M
7
1,2,3,4
Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107,
Karnataka, India. 5Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore-
560 107, Karnataka, India. 6Department of Chemical Engineering, SDMCET, Dharward-580 002, Karnataka, India.
7 Institute of Animal health & Veterinary Biological, Hebbal, Bangalore, Karnataka 560024, India
*Corresponding author: [email protected]; Mobile: +91 9916836390
Received: 03/02/2014; Revised: 25/02/2014; Accepted: 05/03/2014
ABSTRACT
The evolution and spread of antibiotic resistance, as well as the evolution of new strains of
disease causing agents, is of great concern to the global health community. Our ability to effectively
treat disease is dependent on the development of new pharmaceuticals, and one potential source of
novel drugs is traditional medicine. The present study explores the antibacterial properties of plants
used in traditional medicine. We tested the hypothesis of 95% ethanol and showed the inhibitory
effect against gram-positive and gram-negative bacteria. The extracted medicinal plants used to treat
symptoms often caused by bacterial infection would show antibacterial properties in laboratory
assays, and that these extracts would be more effective against moderately virulent bacteria than less
virulent bacteria. The striking feature in most of the aromatic plants enlisted in the indigenous system
of medicine is attributed to their essential oil contents in them which exert their marked therapeutic
potency. The large volume of work accumulated so far, obviously justifies the importance of
medicinal activity of the aromatic plants; the antimicrobial activity being credited to their essential
oil fraction only.
KEYWORDS: Medicinal plants, antibacterial activity, crude ethanolic extract, Blumea lacera
Research Article
Cite this article:
Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M,
Dayananda K S, Ajay Mandal, Sreekanth B, Purushotham K M (2014),
ANTIBACTERIAL ACTIVITY OF SOME INDIGENOUS MEDICINAL PLANTS,
Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 75–79
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Indigenous plants, widely used for folk
medicinal purposes, are numerous and diverse.
In India, about 500 plant species have been
identified as medicinal plants because of their
therapeutic properties In the meantime, a large
number of industries (400 herbal factories)
have been established in this country for
producing Ayurvedic and Unani medicines.
Medicinal use of plants is the oldest form of
healthcare known to mankind. It has been
estimated that India has a market of about
rupees 100 crore worth herbal products
annually (Tyler V, 1994). The total size of the
medicinal plant market at wholesale prices was
estimated at some US$ 14 million per annum,
which corresponds to 17000 tonnes of products.
Local supply accounts for about 70 % by
volume and 40 % as value. It has been
estimated that 12,500 tonnes of dried medicinal
plant produce is sold in India. These products
are approximately worth Rs 255 per million to
rural economy. At the factory level, 5000
tonnes of imported medicinal plants cost
around 480 million rupees (Gopinath.S.M et
al., 2012). Although modern medicinal science
has developed to a greater extent, many rural
people of India still depend on plant products
and herbal remedies for treating their ailments.
Being naturally gifted by a suitable tropical
climate and fertile soil, India possesses a rich
flora of tropical plants. About 5000 species of
Phanerogams and Pteridophytes grow in its
forests, jungles, wastelands and roadsides as
indigenous, naturalized and cultivated plants.
Out of them more than a thousand have been
claimed to possess medicinal and/ or poisonous
properties, of which 546 have recently been
enumerated with their medicinal properties and
therapeutic uses (Annapoorani Chockalingam
et al., 2007). In addition of possessing various
other medicinal properties, 257 of these
medicinal plants have been identified as
efficacious remedies for diarrhoeal disease and
47 for diabetes. A large number of plants in
different locations around the world have been
extracted and semi-purified to investigate
individually their antimicrobial activity
(Dranghon, 2004). The aim of work was to
collect these indigenous plants to investigate
antibacterial activity of the leaves and
identification of particular bioactive compound
as potential drug for the medicinal applications.
MATERIAL AND METHODS
Plant materials
The Plants Callicarapa arborea,
Lanneacorom andelica, Ficus racemosa,
Streblus asper, Lawsonia inermis, Holarrhena
antidysenterica, Mentha arvensis, Enhydra
fluctuans, Blumea lacera, Glinus oppositifolius,
Chenopodium album, Hemidesmus indicus,
Coccinea cordifolia, Cuscuta reflexa, Capparis
zeylanica and Kalanchoe pinnata were
collected from in and around Bangalore district,
Karnataka, India which were used for the
treatment of various infectious diseases by
people. The plants were authenticated by
taxonomists & voucher specimen was stored in
the department for future reference. The plant
materials were oven-dried at 40ºC and then
ground into coarse powder.
Extraction
20 g of coarse powder of all plant
materials Callicarapa arborea, Lannea
coromandelica, Ficus recemosa, Streblus
asper, Lawsonia inermis, Holarrhena
antidysenterica, Mentha arvensis, Enhydra
fluctuans, Blumea lacera, Glinus oppositifolius,
Chenopodium album, Hemidesmus indicus,
Coccinea cordifolia, Cuscuta reflexa, Capparis
zeylanica and Kalanchoe pinnata were
extracted with ethanol for a week at room
temperature. The extracts were then filtered off
through Whatman filter paper number-1 and
the solvent was removed under vacuum at 30ºC
until dry mass were obtained by Buchirota
vapour.
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Table 1. Plants details collected with the name of locality
Serial
No.
Name of plant Plant parts Family locality
1 Callicarapa arborea Leaf Verbenaceae Kolar, Karnataka, India.
2 Lannea coromandelica Leaf Anacardiaceae Kolar, Karnataka, India.
3 Ficus racemosa Leaf Moraceae Kolar, Karnataka, India.
4 Streblus asper Leaf Moraceae Kolar, Karnataka, India.
5 Lawsonia inermis Leaf Lythraceae Kolar, Karnataka, India.
6 Holarrhena
antidysenterica
Leaf Apocynaceae Kolar, Karnataka, India.
7 Mentha arvensis Aerial parts with
flowers
Lamiaceae Mallur, Karnataka, India.
8 Enhydra fluctuans Leaf and root Asteraceae Mallur, Karnataka, India.
9 Blumea lacera Aerial parts Asteraceae Mallur, Karnataka, India.
10 Glinus oppositifolius Leaf Molluginaceae Mallur, Karnataka, India.
11 Chenopodium album Leaf Chenopodiaceae Mallur, Karnataka, India.
12 Hemidesmus indicus Root Periplocaceae Mallur, Karnataka, India.
13 Coccinea cordifolia Leaf and rhizome Cucurbitaceae Mallur, Karnataka, India.
14 Cuscuta reflexa Leaf Convolvulaceae Kolar, Karnataka, India.
15 Capparis zeylanica Leaf Capparidaceae Mallur, Karnataka, India.
16 Kalanchoe pinnata Leaf Crasselaceae Mallur, Karnataka, India.
Antibacterial Activity Test
Microorganisms
The bacteria used included: Shigella
dysenteriae, Salmonella typhi, Salmonella
paratyphi, Bacillus cerus, Bacillus subtilis,
Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Vibrio cholera and
Bacillus megaterium. Bacterial cultures were
maintained on Nutrient agar media. All cultures
were sub cultured monthly and subsequently
stored at 4°C (Gopinath. S. M., 2011).
Screening for Antimicrobial Activities
Disc diffusion method (Gopinath. S. M.,
2011) was used to test the antimicrobial activity
of the extractives against ten bacteria. Dried
and sterilized filter paper discs (6 mm
diameter) were then impregnated with known
amount of the test substances dissolved in
ethanol (40 μg/ml) using micropipette and the
residual solvents were completely evaporated.
Discs containing the test material (20μg/disc)
were placed on nutrient agar medium uniformly
seeded with the test microorganisms. Standard
disc of kanamycin (30μg/disc) and blank discs
(impregnated with solvents followed by
evaporation) were used as positive and negative
control, respectively. These plates were then
kept at low temperature (4°C) for 24 hours to
allow maximum diffusion of test samples. The
plates were then incubated at37°C for 24 hours
to allow maximum growth of the organisms.
The test materials having antimicrobial activity
inhibited the growth of the microorganisms and
a clear, distinct zone of inhibition was
visualized surrounding the disc. The
antimicrobial activity of the test agents was
determined by measuring the diameter of zone
of inhibition in millimetre. The experiment was
carried out in triplicate and the average zone of
inhibition was calculated (Ahmed, A.M.A.,
Rahman, M.S., and Anwar, M.N. 1999)
RESULT AND DISCUSSION
During this study, 16 plants were selected
which were used for the treatment of infectious
diseases by peoples. The aforesaid are
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summarized in Table1. From primary
Screening, it was found that only 5 plants
exhibited antibacterial activity against more
than 6 test organisms (Table 2). Blumea lacera
showed moderate to good (11–23 mm in
diameter zone of inhibition) antibacterial
activity against all organisms except
Pseudomonas aureuginosa. Enhydrus fluctuans
and Mentha arvensis showed moderate activity
(7–10 mm in diameter) against all test
organisms except Bacillus cereus in case of
Mentha arvensis. Salmonella paratyphi,
Bacillus megaterium, chenopodium album and
Glinus oppositifolius showed comparatively
better activity (9–13mm in diameter). The
largest zone of inhibition (23 mm in diameter)
was recorded against Bacillus cereus with the
leaf of Blumea lacera.
Table 2. Antibacterial activity of alcoholic extracts of five plants
Average zone of inhibition in diameter (mm)
Bacterial test organisms
Nam
e of
pla
nts
Part
s of
pla
nt
use
d
Sh
. dys
ente
riae
Salm
on
ella
typ
hi
Pse
udom
on
as
sp
Baci
llu
s ce
reu
s
Salm
on
ella
para
typh
i
Vib
rio c
hole
rae
Baci
llu
s m
egate
riu
m
E.c
oli
Baci
llu
s su
bti
lis
Sta
ph
yloco
ccu
s au
reu
s
Blumea lacera Aerial parts 11 11 _ 23 11 9 19 13 21 15
Chenopodium album Leaf 13 _ 9 10 _ 11 9 13 9 11
Enhydra fluctuans Leaf and rhizome 9 7 10 11 10 10 9 8
Mentha arvensis Aerial parts with flowers 7 9 10 _ 7 10 9 7 10 8
Glinus oppositifolius Leaf 13 9 9 10 11 11 11 9 _ 13
Similar antibacterial activity of other plant
extracts has been reported previously
(Harborne JB., 1973; Jamine.R.Daisy, 2007;
Gopinath S M. et al., 2012). The present
investigation ensures that crude extracts of 5
plants contain antibacterial properties, which
are used by local people. During the study it
was observed that gram-positive bacteria are
more sensitive than gram negative bacteria.
From our results, it appeared that the crude
extracts of some traditional medicinal plants
has good inhibitory effect against selected
bacterial strains. Among the medicinal plants
tested herein, Blumea lacera showed most
promising antibacterial properties indicating
the potential for discovery of antibacterial
principles
CONCLUSION
It was found that out of 12 different plant
materials only 5 plants exhibited antibacterial
activity against more than 6 test organisms
have potential application as therapeutic agents
and bioactive compounds. Plant extracts that
exhibits the exploitation of the pharmacological
properties involves further investigation of the
active ingredients of an implementation
technique of extraction, purification,
separation, crystallization and identification
and can further use as potential drug.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 75–79
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REFERENCES
Ahmed, A.M.A., Rahman, M.S., and Anwar,
M.N. (1999). Antimicrobial activity of
extracts and crude alkaloids
ofPolyalthia longi folia (Sonn.) Thw.
Stem bark. The Chittagong University
Journal of Science, 23(10): 53–56
Alam, M.K., Chowdhury, J.U. and Hasan,
M.A. (1996). Some folk formularies
from Bangladesh. Bangladesh Journal
of Life Science, 8(1): 49–63
Annapoorani Chockalingam, Dante S.
Zarlenga, Douglas and D. Bannerman.
(2007). Antimicrobial activity of bovine
bactericidal permeability-increasing
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negative bacteria isolated from the milk
of cows with clinical mastitis. American
Journal of Veterinary Research. 68 (11):
pp: 1151 –59
Bauer, A.W., Kirby, M.M., Sherris, J.C. and
Turck, M. (1966). Antibiotic
susceptibility testing by a standardized
single disc method. American Journal
of Clinical Pathology, 45: 493–496
Bartner, A. and Grein, E.(1994). Antibacterial
activity of plant extracts used externally
in traditional medicine. Journal of
Ethnopharmacology, 44:35–40
Dranghon, F.A. (2004). Use of botanicals as
biopreservatives in foods. Food
Technol.58:20–28.
Ghani, A. (2000).Vheshaja Oshudh (Herbal
Medicine), Bangla Academy Dhaka,
Bangladesh.
Gopinath, S.M., Suneetha, T. B., Singh, Sumer
(2012). Evaluation of effect of
methanolic and aqueous extracts of
Punica granatum against bacterial
pathogens causing bovine mastitis,
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Gopinath, S.M., Suneetha, T. B., Mruganka,
V.D. (2011), chemical prophylaxis and
antibacterial activity of Methanolic and
aqueous extracts of some medicinal
plants against bovine mastitis,
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Methods. London: Chapman and Hall;.
Methods of Plant Analysis; p. 132.
Jasmine, R and P.Daisy, (2007). Effect of crude
extract and fractions from Elephantopus
scaber on hyperglycemia in streptozotocin-
diabetc rats.Int.J.Biol.Chem., 1:111–116.
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and Anwar, M.N.(1998). Antimicrobial
activity of Holarrhena antidysenterica
against Salmonella typhi. The
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Rojas, A., Hernandez, L., Pereda-Miranda, R.
and Mata, R.(1992).Screening for
antimicrobial activity of crude drug
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Mexican medicinal plants.Journal of
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Source of Support: NIL Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
GENE SILENCING AND ITS APPLICATIONS IN CROP IMPROVEMENT &
FUNCTIONAL GENOMICS
Jemal Ali 1, Pagadala Vijaya Kumari
2*
1Department of Biotechnology, Gondar University, GONDAR, Post Box 196 – Ethiopia.
2Department of Biology, AMBO University, AMBO , Post Box 19 – Ethiopia.
*Corresponding Author: E-mail: [email protected];
Received: 11/02/2014; Revised: 27/02/2014; Accepted: 01/03/2014
ABSTRACT
Gene silencing is a technique used to turn down or switch off the activity of a gene. The
mechanism of switching off a gene could be used for different purposes. Therefore the present work
was initiated with the objective to introduce some gene silencing mechanisms occurring in plants and
to introduce the applications of gene silencing in crop improvement and functional genomics. In this
review work three different articles were reviewed; In the first article: Maize dwarf mosaic virus
(MDMV), a widespread pathogenic virus of maize is targeted to be suppressed by the RNAi using a
hpRNA expression vector containing a sense arm and an antisense arm of 150 bp sequence and the
second deals with suppression of α-zein protein subfamilies of either the 19- or 22-kD using RNAi
technology to obtain moderately increased total lysine content and the third deals with inactivation of
the 587bp sized Bcp1 gene of Arabidopsis thaliana which is responsible for fertile pollen
development. The results of suppression of MDMV showed that the disease index of the transgenic
plant line h2 had no significant difference from the highly resistant control line H9-21. The
suppression of α-zein protein subfamilies displayed a reduced accumulation of both the 19- and 22-
kD α -zeins from the 26 of the 29 events for those transformed with pMON73567 construct and total
amino acid analysis showed that there is an increase in the lysine content. Finally, for the last article,
49 out of 58 Arabidopsis lines transformed with RNAi construct containing Bcp1 sequences were
male sterile. In conclusion, gene silencing is a promising science for identification of unknown genes
and for the treatment of different diseases.
KEY WORDS: RNA interference, Maize dwarf mosaic virus, Male sterility, α -zeins
Review Article
Cite this article:
Jemal Ali, Pagadala Vijaya Kumari., (2014), GENE SILENCING AND ITS
APPLICATIONS IN CROP IMPROVEMENT & FUNCTIONAL GENOMICS,
Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 80–90
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 80–90
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Most plants and animal cells have unusual
forms of RNA that can naturally inactivate
gene expression. Understanding how these
RNA molecules function and how to
manipulate these novel RNA molecules is a
challenge but very essential for new discoveries
(Scanlon, 2004). Gene silencing is a technique
used to turn down or switch off the activity of
genes. It is a powerful technology for gene
discovery and determining gene function in
humans, animals, and plants. In plants RNA
mediated gene silencing, especially the
homology dependant gene silencing (HDGS) is
used to develop new crop varieties and holds
tremendous promise as a therapeutic agent to
control different diseases (Charagonda, 2008).
Now a day, the exploitation of genomic
sequences of animals, plants, fungi and
microorganisms had given us many
opportunities in predicting the genes contained
in an organism. However, this wealth of genetic
information opens new challenges in
deciphering the complete list of protein-
encoding genes (Scanlon, 2004). This is due to
a transcriptional event such as RNA splicing
and post-translational modifications that make
it difficult to predict the exact number of genes
or proteins. With this degree of complexity,
monitoring the entire proteome expression
levels as a means of elucidating the functions
of genes and proteins became significant
challenges for the biotech industry (Myers and
Ferrell, 2005).
Prior to the discovery of RNA mediated
gene silencing, scientists applied various
methods such as insertion of T-DNA elements,
and transposons, treatment with mutagens and
irradiation to generate loss-of-function
mutations in studying the functions of a gene or
gene family of interest in an organism. Apart
from being time-consuming, the above methods
did not always work satisfactorily. For
instance, transposons and T-DNA elements
were found to occasionally insert randomly in
the genome resulting in highly variable gene
expression. Furthermore, in many instances the
particular phenotype or a trait could not be
correlated with the function of a gene of
interest (Williams et al., 2004). As a result of
this the discovery of gene silencing is of
paramount importance for various areas of
genetic studies such as functional genomics,
drug discovery, for the development of plants
with virus resistance and high nutritional value.
Therefore, the present work was initiated with
the objective to introduce some gene silencing
mechanisms occurring in plants and to
introduce the applications of gene silencing in
crop improvement and functional genomics.
RNA Interference-Based Transgenic Maize
Resistant to Maize Dwarf Mosaic Virus by
Zhang et al, 2010
Maize dwarf mosaic virus (MDMV) is a
widespread, worldwide pathogenic virus that
causes chlorosis, stunting, and serious loss of
yield in maize (Zea mays). Strategies for the
management of viral diseases normally include
control of the vector population using
insecticides, adjusting seedtime, and the use of
virus-free propagating material and appropriate
cultural practices. However, these methods are
not effective because of the non-persistent
model of virus transmission by aphids. The use
of resistant germplasm is an environmentally
sustainable and effective way for controlling
viral diseases of maize but the conventional
breeding method is time consuming because
identification and development of resistant
inbred lines or hybrids needs lots of time to
respond, furthermore there will be a year-to-
year inconsistency of viral disease pressure.
Therefore RNAi triggered by hairpin RNA
(hpRNA) transcribed from the transgenic
inverted-repeat sequence provides a straight-
forward natural defense mechanism against
invasive viruses and has been proved to be
more efficient. This study was done with the
objective of: suppressing the P1 protein
(protease) of maize dwarf mosaic virus
(MDMV) using the inverted repeat of sense and
antisense arms of p1 protein.
1. MATERIALS AND METHODS
Target selection and the gene construct: A
150-bp specific fragment of the P1 protein
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(protease) gene was selected from the genomic
sequence of MDMV and amplified by PCR.
The amplified fragments were then inserted
into the pSK vector in sense and antisense
orientations, separated by introns of the maize
actin gene, to construct an hpRNA expression
vector. Then the hpRNA expression construct
was cloned into the plant expression vector
pCAMBIA1300 under the control of the
ubiquitin promoter and nos terminator,
generating the hpRNA expression vector
pASP150 (fig. 1). For selection, the
hygromycinphosphotransferase gene conferring
hygromycin B resistance was used under the
control of the cauliflower mosaic virus 35S
promoter (P-35S) and 35S terminator (T-35S).
Finally the hpRNA expression vector,
pASP150 was introduced by electroporation
into the disarmed Agrobacterium tumefaciens
strain EHA105.
LB left border, RB right border,
Hpthygromycinphosphotransferase gene, P-35S
cauliflower mosaic virus 35S promoter, T-35S
cauliflower mosaic virus 35S terminator, P-Ubi
ubiquitin promoter, T-nos terminator of
nopaline synthase, intron of maize actin gene,
P150 150-bp fragment of MDMV P1 protein
(protease) gene.
Figure 1: The T-DNA regions of hpRNA expression vector pASP50.
Transformation and Selection: Once the
Agrobacterium is ready, it was cultured for
some time, Maize immature embryos cultured
in darkness for callus isolation. Embryonic calli
were screened, sub cultured, and transformed
by co-cultivation with the transformed
Agrobacterium. After cultivation for 7 days, the
calli were transferred to selection medium
containing hygromycin B and cultured for 20
days. Then, the screened resistant calli were
transferred to regeneration medium. Plantlets
with fully grown shoots and roots were
transplanted to greenhouse and allowed to
acclimatize for 2–3 weeks in greenhouse, and
then transplanted into the field for self
pollination to produce T0 seeds.
After planting in the field, a leaf blade was
collected from each regenerated plant and used
for DNA extraction. And PCR was made to
amplify 150-bp fragment of the P1 gene for
screening the putative transgenic plants.
Southern blotting was also made with the
genomic DNA extracted from the leaf samples
of 13 T1 lines derived from the fertile T0 plants
positive in PCR to identify the stable
integration of the transgene into the maize
genome and to evaluate the transgene copy
number.
The transgenic T2 plant lines derived from
the T1 lines positive in Southern blotting,
together with non-transformed controls of a
highly resistant line, a highly susceptible line ,
and the non transformed control line 18-599
were grown in the field and mechanical
inoculation was done twice within 1 week at
the three- to four-leaf stage, using inoculums
prepared from the leaf sap of maize plants
systemically infected with MDMV. The disease
incidence and symptom scale were investigated
at the adult stage according to the standard
proposed by Lin(1989). The disease index was
calculated as:
The virus titer of the transformed lines was
quantified by DAS-ELISA using an MDMV
DAS ELISA kit (AC Diagnostics Inc., USA)
In most cereal grains, including maize,
lysine is usually the most limiting essential
amino acid for animal nutrition and lysine
supplementation is required when corn is used
as a major component of feed. Therefore, the
need for development of high lysine corn lines
is clear. During the 1960s and the 1970s, many
naturally occurring high lysine maize mutants
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were identified. Among them, opaque-2 (o2)
and floury-2 (fl2) are the two best-
characterized mutants. It is believed that the
higher lysine content in these two mutants is
caused by a general decrease in zein synthesis
and an increase in accumulation of non-zein
proteins. Zeins, which make up about 60% of
total maize seed proteins, are rich in glutamine,
proline, alanine, and leucine, but are almost
completely devoid of lysine and tryptophan.
Recently through recombinant DNA
technology, transgenic maize plants were
produced with reduced α-zein protein
subfamilies of either the 19- or 22-kD but with
moderately increased total lysine content.
These two subfamilies of α -zeins, which
comprise 40% and 20% of the total zein
fraction respectively, are almost entirely devoid
of lysine and tryptophan residues. It is possible
that by reducing both subfamilies of α-zeins,
the lysine content could be further enhanced.
This approach relies on the efficient and
concurrent suppression of multiple genes that
encode α-zeins by RNAi technology.
2. MATERIALS AND METHODS –
High lysine and high tryptophan transgenic
maize resulting from the reduction of both
19- and 22-kD α-zeins, by Huang et al., 2006
In most cereal grains, including maize,
lysine is usually the most limiting essential
amino acid for animal nutrition; Opaque-2 (o2)
and floury-2 (fl2) are the two best-characterized
mutants due in zein synthesis and an increase in
accumulation of non-zein proteins. By
Recombinant DNA technology, transgenic
maize plants were produced with reduced α-
zein protein with moderate increase of Lysine
content. Thus this approach relies on the
efficient and concurrent suppression of multiple
genes that encode α-zein by RNAi technology
(Mertz et al., 1964).
Target selection and the gene construct: Two
different constructs (pMON73567 and
pMON73566) were used in this research and
ligated on Agrobacterium based vector (fig. 2)
for making the pMON73567 construct, four
segments of 19-, 22-kD α-zeins were ligated.
The first two segments were partial cDNA
sequences of 19- & 22-kD α-zeins in antisense
orientation, which paired with the later two
segments in sense orientation driven by the
maize gamma zein endosperm specific
promoter, Z27. The 3’ untranslated region of
pea RbcS2 gene with a 643-bp fragment is used
as a terminator (E9) terminator. The selectable
marker, epsps-cp4, for glyphosate resistance is
ligated next to the right border on the
Agrobacterium based vector.
Figure 2: The T-DNA regions of the plasmids used in the transformation maize.
The plasmids contain two different designs
of α-zein reduction cassettes next to the
selectable marker, epsps-cp4, which confers the
glyphosate resistance. The designations of the
genetic elements are as follows: RB, right
border; LB, left border; pZ27, the promoter of
27-kD γ-zein gene; 19AS, a partial 19-kD α-
zein gene in antisense orientation; 22AS, a
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partial 22-kD α-zein gene in antisense
orientation; 22S, a partial 22-kD α-zein gene in
sense orientation; 19S, a partial 19-kD α-zein
gene in sense orientation; tE9, the 3¢ region of
pea RbcS2 gene.
In pMON73566, the third segment
corresponding to the sense region of 22-kD α-
zein gene was removed, leaving only the 19-kD
α-zein sequence to form dsRNA and the 22-kD
α-zein sequence as the unpaired loop region.
The selectable marker gene (epsps-cp4) and the
rest of the genetic elements in the binary vector
were similar to those previously described.
Once the constructs were made, the vectors
with the construct were electroporated into
Agrobacterium tumefaciens ABI strain and
introduced into maize embryos by
Agrobacterium mediated transformation.
To determine the transgene copy number of
resulting R0 plants, genomic DNA was
extracted from leaves, copy number determined
and single copy R0 transgenic plants were self-
pollinated to produce R1 seeds. Total RNA was
isolated from developing kernels (25 days after
pollination) and northern hybridization was
done with PCR labeled DNA fragments
corresponding to the coding sequences of 19-,
22-kD α-zeins and 27-kD γ as the probes.
Special kind of mass spectrometry called
Matrix-assisted laser desorption ionization
time-of- flight mass spectrometry (MALDI-
TOF MS) method was used for analyzing zein
proteins. To obtain free amino acid
accumulation data, extracts of ground meal
samples were filtered, diluted and analyzed by
o- phthaldialdehyde (OPA) derivatization. OPA
derivatized samples were injected onto a C18,
reverse phase HPLC column, detection with an
Agilent HPLC (model 1100) equipped with a
fluorescence detector was used. For total
tryptophan analysis, base hydrolysis was
performed prior to OPA derivatization (Huang
et al., 2006).
Male sterile mutants are of agricultural
importance for the production of hybrids and to
prevent the spread of foreign gene products via
pollen in transgenics. The emasculation of
anthers is very labor intensive, and makes it
very difficult to manage on a large scale. There
are also some natural mechanisms that breeders
can use to develop male sterile plants, (e.g.,
cytoplasmic nuclear (genetic) and genetic-
cytoplasmic) but they are not available for
many crops. Therefore engineered male
sterility is an alternate method in cases where
natural male sterility is not available. RNAi
targeted to some genes in pollen development
could be used to produce male sterile plants.
Bcp1 gene is a 587bp sized gene of
Arabidopsis thaliana responsible for fertile
pollen development and active in both diploid
tapetum and haploid microspores. Perturbation
of this gene in either tapetum or microspores
prevents production of fertile pollen. Thus,
mature anthers contain dead shriveled pollens.
Therefore the current study is initiated with the
objective of producing male-sterile lines by
specific down-regulation of the anther-specific
gene Bcp1 of Arabidopsis by RNAi.
3. MATERIALS AND METHODS –
Development of male sterility by silencing
Bcp1 gene of Arabidopsis through RNA
interference by Tehseen et al, 2010
Male sterile mutants are of agricultural
importance for the production of hybrids. It is
the alternate method to engineer these male
sterile lines. RNAi targeting is one of the
methods used; Bcp 1 gene is a 587bp
in Arabidopsis thaliana responsible for pollen
fertility. Therefore the study initiates with the
objective of producing male-sterile lines by
specific down-regulation of anther specific Bcp
1 (Tehseen et al, 2010).
Target selection and the gene construct: Total DNA was isolated by the CTAB method and both the sense and antisense copy of the Bcp1gene was amplified by PCR using primers designed to have restriction endonuclease recognition sequences for easier cloning of the amplified fragments into appropriately digested vector (Fig 3). Then BCP1 RNAi cassette was made by ligating the sense (BCP1s) and antisense (BCP1as) DNA copies of 163 bp
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 80–90
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region of Bcp1 gene in a reverse order on an agrobacterium based vector pFGC5941 with the help of restriction sites present in the amplified PCR products. The inverted sense
and antisense constructs were separated by a spacer region or introns of petunia Chalcone synthase gene A to increase the efficacy of PTGS.
Figure 3: Production of the Bcp1 RNAi cassette.
Two pairs of primers were used to PCR-amplify a portion of the 163 nucleotide of Bcp1 gene, yielding fragments with BamHI-XbaI and NcoI-XhoI ends. These fragments were directionally cloned into the RNAi vector pFGC5941, yielding the final pTA29RNAi cassette. Shown are the Bcp1 sense (Bcp1s) and antisense (Bcp1as) amplification fragments, the Agrobacterium tumefaciens promoter (PMAS) and polyadenylation signal (PA, the Caulifower mosaic virus 35S promoter (P35S), the petunia chalcone synthase A gene intron (C intron), the Agrobacterium tumefaciens polyadenylation signal, gene encoding BASTA (BAR), T-DNA left border (LB) and T-DNA right border (RB).
The Agrobacterium based vector, pFGC5941 has two multiple cloning sites, bar gene for resistance to the herbicide Glufosinate Ammonium which will be regulated by the Agrobacterium tumefaciens promoter (PMAS) and 35S constitutive promoter (P35s) within left and right borders of the T-DNA region. Then the gene construct Bcp1/pFGC was transformed in Agrobacterium tumefaciens strain LBA4404 by electroporation and it was cultured in LB supplemented with 50 µg/ml acetosyringon, 50 µg/ml kanamycin and 50 µg/ml rifampicin. The transformants were confirmed through PCR using forward and reverse primers.
The next step was Agrobacterium mediated transformation of the construct in to Arabidopsis thaliana, For these leaves were taken from a three weeks seedlings of Arabidopsis, cut in to discs (leaf explants)
finally immersed in to the 50 ml of diluted bacterial suspension and incubated for 5–10 min with gentle shaking, 2 days of dark incubation, washed with cefotaxime and transferred to culture jars supplemented with growth regulators and incubated at 22°C under white light conditions with a 3 weeks of subculturing interval till the shoots reach enough lengh. They were then finally transferred to rooting, acclimatized in the greenhouse. The cefotaxime kills the Agrobacterium and glufosinate ammonium (5µg/ml) that reduced glutamine and increased ammonia levels in the plant tissues and thus only leaf discs having transgene were survived.
Polymerase chain reaction (PCR) and Southern hybridization were employed for the confirmation of the putative transgenic plants through bar gene specific primers and probes and their fertility was recorded (Tehseen et al., 2010).
1. RESULTS AND DISCUSSION
From the 800 pieces of embryonic calli transformed by co-cultivation, 98 (12.3%) pieces of resistant embryonic calli were obtained after hygromicin B selection. A total of 46 (46.9%) plantlets were regenerated after the recovering subculture and the multiplication. Of the 46 regenerated plants, 18 (39.1%) were detected as positive by specific PCR amplification and certified as putative transgenic plants (Fig. 4). Out of these 18 plants, 13 (72.2%) grew to reproduce seeds of the T1 generation. Out of the 13 T1 lines, nine (69.2%) were shown by Southern blotting to have stable transgene integration.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 80–90
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Figure 4: PCR detection of the regenerated plants.
Lane M is 50-bp DNA ladder marker, lane 1 is non-transformed control 18-599, lane 2 positive control of expression
vector pASP150, lanes 3–20 transformed plants 3, 5, 9, 10h, 10L, h1, h2, 12, 13, 14, m-1, 15, 16, 18, 20, 21, 24, and 25,
respectively
Virus Resistance of T2 Plant Lines: After the
pollination stage, systemic infection of MDMV
was observed in the non-transformed control
(18–599), the susceptible control (Mo17), and
in some T2 plant lines. The disease indexes of
the different T2 plant lines and the controls
matched each other in the two environments
(Table1). The non-transformed control (18–
599) was evaluated as susceptible (S) to
MDMV with a disease index between 40.1%
and 60.0%, the susceptible control (Mo17) was
evaluated as highly susceptible (HS) with a
disease index >60.1%, while the resistant
control (H9-21) was evaluated as resistant with
a disease index between 10.1% and 25.0%.
Of the nine transgenic T2 plant lines derived
from the T1 lines positive in Southern blotting,
lines h2, 13, and h1 were judged to have
intermediate resistance to MDMV with a
disease index between 25.1% and 40.0%,
showing no systemic infection. This resistance
is increased significantly when compared with
the non-transformed control line 18–599, but
was not significantly different from the highly
resistant control line H9-21.
The DAS ELISA result also showed an
increment in the viral protein as we go from a
resistant control to the highly susceptible
(Table 2).
Table 1. MDMV resistance of T2 transformed plant lines
T2 plant line and
Control
Disease incidence Disease index (%) Resistance
grade Xinzhou Ya’an Average Xinzhou Ya’an Average
H 9-12 (resistant
control)
56.2 48.1 52.2 22.9 17.9 20.4a R
h2 26.0 29.5 27.8 25.4 27.5 26.5a I
13 45.3 38.2 41.8 33.3 27.1 30.2ab I
h1 52.2 38.6 45.4 34.1 28.8 31.5ab I
9 6.5 65.2 63.4 42.7 46.0 44.4bc S
5 72.0 65.6 68.8 47.8 49.3 48.6c S
3 82.9 78.4 80.7 55.1 46.7 50.9c S
18-599 (non-
transformed control)
93.7 100.0 96.9 48.6 55.8 52.2c S
21 88.7 83.5 86.1 60.4 57.4 58.9c S
10L 100.0 95.7 97.9 85.4 72.7 79.1d HS
10h 100.0 100.0 100.0 86.8 81.2 84.0d HS
MoI7 (Susceptible
control)
100.0 100.0 100.0 85.5 83.5 84.5d HS
In the column of average disease index, the same lowercase letters indicate non-significance, and the different lowercase
letters indicate significance at possibility level of 0.05 R resistance, I intermediate, S susceptible, HS highly susceptible
to MDMV
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Table 2: Absorbance value at 405 nm in DAS-ELISA of the T2 plant lines and controls
T2 plant line and control Absorbance at 405nm
H 9-21 (resistant control) 0.201
h2 0.207
I3 0.285
H1 0.311
9 0.503
5 0.692
3 0.778
21 0.896
18-599 (non transformed control) 0.904
10L 1.002
10h 1.323
Mo17 (susceptible control) 1.580
In summary, the disease index of the
transgenic plant line h2 had no significant
difference from the highly resistant control line
H9-21. The underlying reason for the lack of
resistance in some of the transgenic lines
remains to be clarified. But the effective length
of the expressed hpRNA constructs to trigger
RNAi in transgenic plants is 300–800 bp, and
the short limit is ≈98 bp. The 150-bp hpRNA
expression construct introduced into the maize
genome might be a little too short to trigger
efficient RNAi.
2. RESULTS AND DISCUSSION
It was found that in 26 of the total 29 events
produced from pMON73567, the R1 seeds
showed reduced zein content. All 26
pMON73567 events displayed a reduced
accumulation of both 19- and 22-kD α-zeins.
Of the 14 events produced from pMON73566,
2 of the 14 had a reduction in both 19- and 22-
kD α-zeins. The majority of these pMON73566
events, 10 of the 14, showed reduction in 19-
kD α-zein accumulation.
For Conformation of zein gene suppression
Northern blot analysis was done where two
pMON73567 events, M80442 and M82186,
along with two pMON73566 events, M80780
and M80791, were chosen to advance to
subsequent generations for further analyses.
Six R2 kernels per event were analyzed and
because of the dominant nature of the transgene
these kernels should segregate 3:1 of transgenic
vs. wild-type. It was observed that apparent
reduction of both 19- and 22-kD α-zein
transcripts in M80442 kernels and the
reductions of 19-KD α-zein transcripts in
M80780 kernels (Fig 5). In M80791 kernels,
the reduction of 19-kD α-zein transcripts was
clearly observed along with some reduction in
22-kD α-zein transcripts (Fig 5).
Figure 5. Northern blot analysis of developing transgenic kernels.
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Equal amounts of RNA isolated from
individual kernels were separated through
electrophoresis on three separate agarose gels.
After blotting, each was probed with one of
three probes corresponding to the coding
sequences of 19-, 22-kD α-zeins or 27-kD γ.
Six segregating R2 kernels were used per
event. M80442 and M80791 events show
reduced accumulation of 19- and 22-kD α-zein
transcripts and M80780 event shows reduced
accumulation of 19-kD α-zein transcripts.
The result of total amino acid analysis
showed that there is an increase in the lysine
content from 2438 ppm in wild-type kernels to
4035, 5003, 4533 and 4800 ppm in kernels
from M80780, M80791, M80442 and M82186
events, respectively. Similarly, the tryptophan
content increased from 598 ppm in wild-type to
877, 1087, 940 and 1040 ppm in M80780,
M80791, M80442 and M82186,
correspondingly. Generally in this study, Up to
5.62% of lysine and 1.22% of tryptophan were
achieved in transgenic lines compared with
2.83% of lysine and 0.69% of tryptophan in
wild-type.
An important factor underlying the
significant increases in lysine and tryptophan
levels in zein reduction kernels was the
synthesis of non-zein proteins. The observation
that the average protein content across the
transgenic zein reduction lines is unchanged
relatively to wild-type suggests that the zein
proteins are being replaced with non-zein
proteins.
Figure 6. Correlations between the protein content and the total lysine or tryptophan contents
in zein reduction ears.
Linear correlations between the lysine (A) or tryptophan (B) content and the protein content among pooled kernels
of zein reduction ears were observed from the transgenic lines. The graphs indicate an incremental increase of 723-ppm
of lysine and 157-ppm of tryptophan for every percentage of protein accumulation in these ears.
Figure 7. Correlations between the protein content and the free amino acid levels in zein
reduction ears.
Linear correlations were observed between the total free amino acid (A) or asparagine (B) levels and the protein
content among pooled kernels of zein reduction ears from transgenic lines.
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The results on the above graphs confirm
that the increases in lysine and tryptophan in
the zein reduced kernels are the result of the
replacement of lysine-poor zeins with more
amino acid-balanced, lysine-containing non-
zein proteins.
The simplest explanation of the substantial
upsurge in the free amino acid level in zein
reduction kernels is that during kernel
development excess amino acids, unable to be
incorporated into zeins were either diverted to
other seed proteins or remained as free amino
acids at maturity. In such a case, one would
expect an inverse correlation between the free
amino acid level and the overall protein content
in zein reduced kernels.
3. RESULTS AND DISCUSSION
They found that the dsRNA interfere the
Bcp1 gene function in the transgenic
Arabidopsis plants and consequently male
sterile plants were obtained. About 49 out of 58
Arabidopsis lines transformed with RNAi
construct containing Bcp1 sequences were male
sterile (Table 3).
Table 3. Transformation of Arabidopsis thaliana with Agrobacterium based plasid and
evaluation of regenerated plants. Agrobacterium tumefaciens (LBA4404) bearing plasmid
containing 163 bp region of male fertility (Bcp1) gene under 35S promoter.
Batch
no.
Leaf discs
treated
(no.)
Explants
regenerated on
selected media*
(no.)
Plants which
developed
roots (no.)
Positive
plants for
bar
genes**
Number
of
sterile
plants
Seed
production up
on cross
pollination
1 71 49 28 20 17/20 Yes
2 84 51 22 17 12/17 Yes
3 60 43 27 21 19/21 Yes *Containing 10mg/l BASTA ®
** Analysed by PCR using bar gene specific pair of primers
Transgenic plants were phenotypically
indistinguishable from non-transgenic plants
except for aborted or malfunctioning pollen
grains. These transgenic plants were used as
female plants for crossing with wild type non-
transgenic plants to produce hybrid seeds. In
these plants there were non-viable pollens to
fertilize their own female partners on self-
pollination. By using the same strategy, it will
be easy to produce hybrid seeds on large scale,
for higher yield and quality of the crop. From
this study, it is concluded that silencing of
Bcp1 through RNAi is responsible for male
sterility in Arabidopsis. A sequence homology
in other crop species like Brassica (Bhalla,
P.L and Singh, 1999) of this gene also showed
that this method can be employed to obtain
hybrid seeds of commercially high valued
crops.
CONCLUSION
From the research results above, it could be
seen that the discovery of gene silencing has
great impact for various areas of genetic studies
such as functional genomics, for the
development of plants with virus resistance and
high nutritional value. Thus gene silencing is a
promising and emerging branch of Plant
Biotechnology that can be used as a tool for
identification of unknown genes and for the
diagnosing of different kinds of plant diseases.
ACKNOWLEDGEMENTS
We greatly acknowledge Department of
Biology – AMBO and Department of
Biotechnology – GONDAR ETHIOPIA, for
there constant encouragement.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 80–90
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REFERENCES
Appasani, K. (2005). RNA Interference
Technology.From basic science to drug
development. Cambridge University
Press, United States of America, New
York.
Bhalla, P.L and Singh, M.B. (1999) Molecular
control of male fertility
in Brassica.Plant Cell, Tissue and
Organ Culture. 56: 89–95.
Charagonda, R. (2008). Antisense Suppression
of σ-Cadinene Synthase Gene in
Cotton.Unpublished Master’s Thesis,
University of Agricultural Sciences,
Dharwad.
Huang, S; Frizzi, A; Florida, C. A.; Kruger, D.
E. and Luethy, M. H. (2006).High
lysine and high tryptophan transgenic
maize resulting from the reduction of
both 19- and 22-kD α-zeins. Plant
Molecular Biology. 61:525–535.
Lin, K (1989) Studies on the resistance of corn
inbred lines and hybrids to maize dwarf
mosaic virus strain B. Sci Agric Sin
22:57–61
Mertz, E.T., Bates, L.S. and Nelson, O.E.
(1964).Mutant gene that changes
protein composition and increases
lysine content of maize endosperm.
Science 145: 279–280.
Myers, J. W and Ferrell, J.E.(2005). Dicer in
RNAi: Its roles in vivo and utility in
vitro. In: Appasani, K.RNA Interference
Technology: From Basic Science to
Drug Development. Cambridge
University Press.New York, U.S.A.
pp.20–54.
Scanlon, K. (2004). Anti-Genes: siRNA,
Ribozymes and Antisense. Current
Pharmaceutical Biotechnology. 5: 1–6
Tehseen, M; Imran, M; Hussain, M; Irum, S;
Ali, S; Mansoor, S and Zafar, Y.
(2010). Development of male sterility
by silencing Bcp1 gene of Arabidopsis
through RNA interference.African
Journal of Biotechnology. 19: 2736–
2741.
Source of Support: NIL Conflict of Interest: None Declared
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Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
CALLICARPA MACROPHYLLA: A REVIEW OF ITS PHYTO-CHEMISTRY,
PHARMACOLOGY, FOLKLORE CLAIMS AND AYURVEDIC STUDIES
Pandey Ajay Shankar1, Srivastava Bhavana
2*, Wanjari Manish M
3,
Pandey Narendra Kumar4, Jadhav Ankush D
5
1Senior Research Fellow (Pharmacognosy), National Research Institute for Ayurveda-Siddha, Human
Resource Development, Gwalior-474009 MP, India. 2Research Officer, Dept. of Chemistry, National Research Institute for Ayurveda-Siddha, Human Resource
Development, Amkho, Gwalior-474009, MP, India. 3Research Officer, Dept. of Pharmacology, National Research Institute for Ayurveda-Siddha, Human
Resource Development, Amkho, Gwalior-474009, MP, India. 4Research Officer, Dept. of Botany, National Research Institute for Ayurveda-Siddha, Human Resource
Development, Amkho, Gwalior-474009, MP, India. 5Research Officer incharge S-4 (Ayu), National Research Institute for Ayurveda-Siddha, Human Resource
Development, Amkho, Gwalior-474009, MP, India.
*Corresponding author: E-mail: [email protected]; [email protected]; Phone: (+91)7489814440
Received: 29/01/2014; Revised: 20/02/2014; Accepted: 05/03/2014
ABSTRACT
Callicarpa macrophylla, commonly known as Priyangu is a useful medicinal plant for the
treatment of various disorders like tumour, polydipsia, diarrhoea, dysentery, diabetes, fever, etc. In
Ayurvedic system of medicine, the plant is also known as Phalawati and used for obstetric
conditions. As the plant is very important because of its therapeutic potential, research on its phyto-
chemistry, pharmacology, folklore claims and Ayurvedic studies are reviewed in this article to
present comprehensive information on this plant, which might be helpful for scientists and
researchers to focus on the priority area of research that are yet to be discovered and to find out new
chemical entities responsible for its claimed traditional uses.
KEYWORDS:
Callicarpa macrophylla, Priyangu, Phyto-chemistry, Pharmacology, Folklore claims, Ayurvedic
studies.
Review Article
Cite this article:
Pandey Ajay S., Srivastava Bhavana, Wanjari Manish M,
Pandey Narendra K., Jadhav Ankush D (2014), CALLICARPA MACROPHYLLA: A REVIEW
OF ITS PHYTO-CHEMISTRY, PHARMACOLOGY, FOLKLORE CLAIMS, AND
AYURVEDIC STUDIES, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 91–100
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Callicarpa macrophylla Vahl. (syn.
Callicarpa incana Roxb.) belonging to family
Verbenaceae, commonly known as Priyangu in
Sanskrit & Hindi is an important Ayurvedic
drug used in treatment of various ailments. In
Ayurvedic system of medicine, the plant is also
known as Phalawati and used for obstetric
conditions. It also forms one of the ingredients
of a compound drug - Lodhrasa; used for
gyanocological and skin diseases. There are
two varieties of the plant, described in
Samhitas, as Priyangu and Gandha Priyangu.
The second variety is a fragrant one. As a folk
medicine the plant is useful in the treatment of
various disorders viz. tumour, polydipsia,
diarrhoea, dysentery, diabetes, fever, etc
(Anonymous, 1992). An extensive review was
carried out to explore the hidden potentials and
to enumerate the benefits of the Priyangu.
Parts used
Root, Bark, Leaves, Flowers, Fruits.
Vernacular / Tribal / Common names
The plant Callicarpa macrophylla is known
as Perfumed cherry and Beauty berry in
English; Priyangu, Daya, Dhaiya and
Fulprayangi in Hindi; Priyangu, Priyanguka,
Priyaka, Gandhaphali, Gandhipriyangu,
Phalini, Vanita, Kaantaa, Shyaamaa and
Anganapriya in Sanskrit and in Ayurvedic
literature; Habb-ul-mihlb in Unani texts;
Baramala, Mathara and Mattranja in Bengali;
San-natadagidda, Kadu-edi, Priyangu and
Navane in Kannada; Gyazhalpoo and Nalal in
Tamil; Prenkhanamu in Telugu; Bonmala and
Tong-lotti in Assamese; Sumali in Punjabi;
Ghaunla and Priyango in Gujrati; Nazhal,
Kadurohini, Njazhal, Jnazhal and Chimpompil
in Malyalam; Gauhala, Gahula and Priyangu
in Marathi and Priyangu in Oriya language
(Khare, 2007; Anonymous, 2008).
GEOGRAPHICAL DISTRIBUTION
The plant is found in open and secondary
forests in upper Gangetic plains, West Bengal
Plains, Eastern and Western Himalaya region
(Gupta et al., 2008), Kashmir to Assam,
Arunachal Pradesh and northern Andhra
Pradesh, up to an altitude of 1800 m
(Anonymous, 1992). In other parts of world it
is distributed across Nepal, Bhutan, Myanmar,
South East Asia and china (Billore et al., 2005).
BOTANICAL DESCRIPTION
The plant is an erect undershrub, 1.5–2.5 m
tall. Leaves are elliptic-oblong to lanceolate or
ovate to ovate–lanceolate, 12–25 × 5–11 cm,
acute or acuminate at apex, acute or cuneate at
base, glabrescent, crenate-dentate; densely
stellate–tomentose beneath; petiole 4–12 mm
long; Inflorescence axillary, solitary or often
corymbose–cyme; Flowers purplish; Calyx 5,
4-toothed, bell-shaped, persistent
gamosepalous, covering almost half of the fruit
sometimes attached; corolla lilac or purple,
about 3 cm long; lobes 4, ovate, subacute.
Fruits globose, drupes or berries, white to
yellowish-brown with or without fruit stalk,
fresh being succulent, 1–3 mm in dia. Intact
fruits are smooth and brownish in colour and
exhibit centrally located bilocular carpel and 4
nutlets each embedded with a yellowish white
seed, in a transversely cut surface of a fruit;
Flowering & fruiting: June–Dec. Fruits taste at
first somewhat sweet, later bitterish; odour
fragrant specially after slight bruising the fruit
(Anonymous, 2008; Gupta et al., 2008; Mudgal
et al., 1997). The fruits of Callicarpa
macrophylla are edible (Dangol, 2008; Mehta
et al., 2010).
MICROSCOPY
The stomata found on the leaves of C.
macrophylla are anomocytic (Mathew & Shah,
1981). The transverse section of the dried fruits
irregularly circular in outline with undulated
margin showing a layer of epidermis, wide
parenchymatous mesocarp traversed with
vascular strands, stony endocarp and centrally
located exalbuminous seeds with
sclerenchymatous coat and oil celled layer.
Detailed transverse section of the fruit shows
thin epicarp, forms skin of fruit consisting of
outer epidermal cells covered with thin cuticle,
a few epidermal cells elongate to form short
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stalked, disc-shaped, 2–4 celled glandular hairs;
some other epidermal cells form stellate hairs;
mesocarp is parenchymatous composed of 5–8
layered, radially elongated irregular shaped
large sized, thin walled cells, traversed with
vertically and tangentially running obliquely
cut vascular strands and microcluster crystals
of calcium oxalate, the innermost few layers of
mesocarp are embedded with volatile oil
globules. Endocarp hard and stony, lignified
and shows compactly arranged 4–5 rows of
spherical small sized discontinuous thick and
thin walled stone cells, peripherally studded
with small prismatic crystals of calcium
oxalate, encircling the centrally located seed,
seeds four in each fruit; seed exhibits a thin
layer of seed coat and radially elongated thick
walled palisade like sclerenchymatous band
lying underneath it; followed by layer of oil
cells embedded with volatile oil. Endosperm 2–
6 layered consisting of isodiametric cells;
cotyledons 2, consisting of isodiametric cells
filled with fixed oil and aleurone grains
(Anonymous, 2008; Gupta et al., 2008).
Powder microscopy shows plenty of
lignified stellate and branched trichomes, their
broken fragments, sessile glandular trichomes
with one to many celled head from the pedicel
and sepals of the fruit; epidermal cells with
anomocytic stomata of sepal; fragments of
straight walled, lignified cells of seed coat; oval
to elongated, elliptical endocarp cells in surface
view; single and isolated groups of elongated,
thick and thin walled, oval to rectangular,
lignified pitted stone cells having concentric
striations, radial canal, with narrow lumen;
microcluster crystals of calcium oxalate
scattered as such or embedded in the cells of
cotyledons and mesocarp; fragments of
cotyledons embedded with oil globules and
aleurongrains (Anonymous, 2008;
Gupta et al.,
2008).
PHYTOCHEMISTRY
Qualitative/quantitative analysis
Alcoholic extract of stem showed the
presence of glycosides, flavonoid, tannins,
carbohydrates, steroids and absence of
alkaloids, saponins, proteins, and amino acids
while aqueous extract of the stem showed the
presence of glycosides, flavonoids, saponins,
carbohydrates and tannins and absence of
alkaloids, steroids, proteins, and amino acids
(Yadav et al., 2012a). In another study
ethanolic extract of plant (excluding roots)
showed the presence of tannins (Atal et al.,
1978).
The leaf and fruit oils found rich in selinene
derivatives. The fruit oil is comprised of 41.6%
beta-selinene and 6% alpha-selinene.
Dendrolasin, a potential perfumery natural
furanoidsesquiterpenoid is reported in leaf and
fruit essential oils (Singh et al., 2010). The
content of luteolin increased gradually with the
growth of plants and reached the peak at the
end of growth period (Zhou et al., 2011). Total
flavonoid accumulation of plant changed along
with the growth of the plants, i.e. the contents
increased gradually in the trunk and root,
decreased in leaves (Liu et al., 2010).
High performance thin layer
chromatography (HPTLC) and Reverse phase-
High performance liquid chromatography (RP-
HPLC) with UV detection can be used for
quantitative determination of calliterpenone
and calliterpenone monoacetate, the two major
plant growth promoters in Callicarpa
macrophylla (Verma et al., 2009). RP-HPLC
method is also suitable for the quality control
of C. macrophylla because betulinic acid can
be well separated from other compounds in the
plant by RP-HPLC method (Pan et al., 2008).
Thin layer chromatography
Thin layer Chromatography (TLC) of the
alcoholic extract of fruit on Silica gel 'G' plate
using n-butanol : acetic acid: water (4:1:5)
shows under ultra violet (UV) light (366 nm)
one conspicuous fluorescent spot at Rf 0.82
(sky blue). On exposure to iodine vapours two
spots appear at Rf 0.82 and 0.92 (both
yellowish brown). On spraying with ferric
chloride (10% aqueous solution) two spots
appear at Rf 0.82 and 0.92 (both greyish brown)
(Anonymous, 2008). TLC of methanol extract
of fruit on silica gel 60F254 plates using toluene:
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ethyl acetate (70:30) using oleanolic acid as
standard; after spraying vanillin sulphuric acid
as detecting reagent showed under visible light
five spots at Rf 0.11and 0.35 (Blue), 0.52
(Purplish grey), 0.61 (Brown) and 0.69
(Blackish blue) (Gupta et al., 2008). T.L.C. of
the alcoholic extract of the stems collected
from Tarikhet, Uttrakhand using mobile phase
hexane: chloroform: ethyl acetate (2:1:1)
showed two spots with Rf value 0.84 and 0.91
(Yadav et al., 2012a).
Phyto-chemicals
Two tetracyclic diterpenes, calliterpenone
and calliterpenone monoacetate have been
isolated from the petrol extract of the aerial
parts (Chatterjee et al., 1972). Ursolic acid, β-
sitosterol and 5,4′-dihydroxy 3'-7-3′-trimethoxy
flavone have been isolated from the petroleum
ether extract of leaves (Chaudhary et al., 1978).
Methanol extract of the deposit of the water
extract obtained after distillation of the
essential oil of the leaves yielded 16α,17-
isopropylideno-3-oxo-phyllocladane
(isopropylidenocalliterpenone) along with
calliterpenone and its monoacetate (Singh &
Agrawal, 1994). The structure and absolute
configuration of calliterpenone has been
established as 3-oxo-13β-kaurane-16α,17-diol
(Fujita et al., 1975). 16,17-dihydroxy-3-
oxophylloladane,16-hydroxy-17-acetoxy-3-
oxophyllocladane, β-amyrin and β -sitosterol-3-
O-β-D-glucoside have been isolated from
fruits.
α-Amyrin, ursolic acid, 2α,3α,19α-
trihydroxyurs-12-en-28-oic acid, betulinic acid,
β-sitosterol, daucosterol have been isolated
from plant by column chromatography on silica
gel, Sephadex LH-20
(Pan & Sun, 2006).
Chung et al., (2005) have isolated acyclic
triterpene callicarpenol from plant (Chung et
al., 2005). Phyllocladane diterpenoids
calliterpenone and calliterpenone monoacetate
have been isolated from Callicarpa
macrophylla Vahl. in shoot cultures of
Rauwolfia serpentina (Goel et al., 2007).
Identity, Purity and Strength
For Callicarpa macrophylla fruit, foreign
matter should not be more than 2 % w/w, total
ash, acid-insoluble ash should not exceed 6.5 %
w/w, 1 % w/w respectively, alcohol-soluble
extractive value should not be less than 3 %
w/w and water-soluble extractive value should
not be less than 10 % w/w (Anonymous, 2008).
For inflorescence of this plant foreign matter
should not be more than 2 % w/w, total ash,
acid-insoluble ash should not exceed 8 % w/w,
2 % w/w respectively, alcohol-soluble
extractive value should not be less than 10 %
w/w and water-soluble extractive value should
not be less than 14 % w/w (Anonymous, 1999).
Stems collected from Tarikhet, Uttrakhand
showed 3.5% total ash, 1% acid insoluble ash,
0.3% water soluble ash, 14.0% alcohol soluble
extractive, 9.8% water soluble extractive and
8.75% moisture. Inorganic elements like
potassium, phosphates, iron and sulphates are
also found in these stems (Yadav et al., 2012a).
PHARMACOLOGICAL PROPERTIES
Analgesic, digestive, diuretic (Chunekar &
Pandey, 1999), antipyretic, antiemetic,
antipoisoning, blood purifier and anti burning
(Zarkhande & Mishra, 2004).
Leaves
The ethanolic and aqueous extracts of
leaves at the dose of 200 mg/kg and 400 mg/kg
showed dose dependent anti-inflammatory
action when evaluated by carrageenan induced
rat paw edema method using diclofenac sodium
as standard drug (Yadav et al., 2011). Ethanolic
extracts of leaves at the concentration of 200
and 400 μg/disc showed significant anti-fungal
activity against Gibberella fujikoroi,
Cryptococcus neoformans, Candida albicans,
Myrothecium verrucaria, Aspergillus niger,
Neurospora crassa and Rhizopus oligosporus
fungal strains when evaluated using disk
diffusion method using fluconazole as standard
drug while aqueous extract showed no
antifungal activity
(Yadav et al., 2012d).
Aqueous extract of leaves (200 and 400 µg/ml)
showed significant analgesic activity compared
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
to ethanolic extract (200 and 400 µg/ml) when
evaluated by tail immersion method in rats
using pentazocine as standard and also
exhibited better anti-pyretic potential at same
dosage than ethanolic extract when evaluated
by Brewer’s yeast induced pyrexia model using
paracetamol as standard (Yadav et al., 2012b).
Stem
Ethanolic extract of stem at concentration
of 200 µl/disc and 400 µl/disc exhibited in-
vitro antibacterial activity against various gram
positive bacterial strains like Streptococcus
pyogens, Bacillus cereus, Micrococcus luteus,
Staphylococcus epidermidis, Clostridium
sporogens, Streptococcus faecalis,
Staphylococcus aureus and Bacillus subtilis
and gram negative bacterial strains like
Agrobacterium tumifaciens, Klebsiella
pneumonia, Salmonella typhimurium,
Pseudomonas aeruginosa, Serratia marcesens,
Enterobacteria aerogens, Proteus vulgaris and
Escherichia coli when compared with the
standard drug ciprofloxacin. Aqueous extract
was found inactive against all the bacterial
strains (Yadav et al., 2012c).
Bark
Extract of bark was shown to inhibit lipid
peroxidation in biological membranes (Kumar
& Muller, 1999).
Flower
The alcohol extract of flowers, at the dose
of 100 and 200 mg/kg, was found to exhibit
significant dose dependent antidiabetic activity
along with reduction in hyperlipidemia in
dexmethasone induced insulin resistance and
streptozotocin induced diabetes in rats (Patel,
2011).
Whole plant
Ethanolic extracts of the plant was found to
lack cytotoxic activity against KB cells (a
subline of the ubiquitous KERATIN-forming
tumor cell line HeLa) (Bhakuni et al., 1971;
Dhar et al., 1973; Suffness et al., 1988).
Folklore claims
The plant is reported to be useful to stop
internal and external bleeding and to treat burns
(Bensky et al., 1986). According to the tribal
people of Sikkim, India, the plant is bitter in
taste and useful in blood dysentery, sweating,
burning sensation and fever due to its cold
potency. This is the best medicine for bleeding
disorders and it reduces the bad smell from
body (Panda, 2007). In Bangladesh, Tripura
tribes use this plant as a tonic, as antidote to
poison, in the treatment of dermatitis and
cancer
(Rahmatullah et al., 2011). In a
preparation the plant is used in combination
with other herbs to treat diarrhea, dysentery,
intestinal worms, and skin disorders and to
purify the blood and eliminate toxins (Khare,
2004).
Roots are useful in the treatment of
pneumonia (Gautam, 2013), stomach disorders
and rheumatic pain (Rai, 2003). Tripura tribe of
Bangladesh use the decoction of roots for the
cure of frequent diarrhea, heart palpitation and
in frequent defecation
(Rahmatullah et al.,
2011; Hossan et al., 2009). About 10 ml
decoction is drunk twice a day for fifteen days
to cure bronchitis
(Rai, 2004). In Dibru-
Saikhowa biosphere reserve of northeast India
the root powder is used for the cure of
hydrophobia (Purkayastha et al., 2005). The
people of Chamoli district of Uttarakhand India
use root powder in the treatment of urinary
complaints and for regularizing menstruation
(Dangwal et al., 2011).
The Bark is used in the treatment of
rheumatism and gonorrhoea (Das et al., 2012).
Bark extract is used orally to treat fever by
Chakma tribe of Bangladesh (Rahman et al.,
2007). Aromatic oil from the roots is reported
to be useful to treat disordered stomach
(Talapatra et al., 1994).
Leaves are used for the treatment of
stomach disorders (Rai, 2003). In the Apatani
of Ziro valley in Arunachal Pradesh, leaves are
used for treatment of headache (Kala, 2005)
and tribal people of Jaunsar area of Garhwal
region in Himalaya use leaves for the treatment
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
of rheumatic pain (Bhatt & Negi, 2006; Uniyal
& Shiva, 2005). Tribal people of Mizoram
apply paste of leaves in bone fracture (Rai &
Lalramnghinglova, 2010). Decoction of fresh
leaves is useful as a regular mouthwash for
recovery from sores and gingivitis (Arya &
Agarwal, 2008). People of Chamoli district of
Uttarakhand India use warmed leaf infusion for
the treatment of pain in arthritis (Dangwal et
al., 2011). Leaf extract is reported to be useful
for the treatment of rheumatism (Talapatra et
al., 1994). Juice of tender leaf buds mixed with
Drumaria diandra BL., Oxalis corniculata L.
and Cheilanthus albomarginata C.B. Cl. is
reported to be given in case of acidity and
gastric troubles (Manandhar, 1993).
In Tamil Nadu, the flowers and fruits are
used to treat diabetes (Jeyachandran & Mahesh
2007). Tribal people of Jaunsar area of
Garhwal region in Himalaya use fruits for the
treatment of rheumatic pain (Bhatt & Negi,
2006). Local vaidyas in Ukhimath block,
Uttarakhand use fruit juice to treat fever
(Manandhar, 1995), while the fruit extract to
treat rheumatic pain and mouth ulcers (Semwal
et al., 2010; Samal et al., 2004). The seeds are
reported to be useful for the treatment of oral
infections and intestinal complaints (Ahmad et
al., 1976).
Medicinal properties of the plant in
Ayurveda
For fruits the taste is sweet, bitter &
astringent; physical properties are cold, heavy
and dry, potency is cold, taste of fruit after
digestion is pungent. Actions of fruit include
alleviation of vital pitta & vata and as blood
purifier (Anonymous, 2008). For inflorescence
taste is bitter and astringent, physical property
is dry, cold in potency, taste after digestion is
pungent. Actions of inflorescence include
alleviation of vital pitta & vata, refrigerant, anti
diarrheal, diuretic, jointer, wound healer and as
blood purifier (Anonymous, 1999).
Recommended dose of fruit is 1–2 g
(Anonymous, 2004) and inflorescence is 1–3 g
in powder form (Anonymous, 1999).
Therapeutic uses
Fruits are used in the therapeutics of
burning sensation in the body, fever, vomiting,
blood disorders, vertigo, nervous system and
rheumatic diseases (Anonymous, 2008) while
inflorescence are used in the therapeutics of
burning sensation in the body, fever, blood pitta
diseases, amoebic dysentery and hyperhydrosis
(Anonymous, 1999).
Safety Aspects
The drug used traditionally in prescribed
doses may be considered safe (Gupta et al.,
2008).
Important Ayurvedic formulations
Important Ayurvedic formulations of fruits
include Jirakadi Modaka, Brhatphala Ghrta,
Brhatcchagaladya Ghrta, Vyaghri Taila
(Anonymous, 2008) while Ayurvedic
formulations of inflorescence include
Khadiradi Gutika, Eladi curna, Kanaka Taila,
Kunkumadi Taila and Nilikadya Taila
(Anonymous, 1999).
CONCLUSION
The above discussion clearly indicates that
Callicarpa macrophylla is an important
medicinal plant with diverse pharmacological
spectrum. The plant shows the presence of
many chemical constituents which are
responsible for varied pharmacological and
medicinal property. The literature claims that
there is vast potential in this plant in view of
therapeutics. Chemists and pharmacologists
must explore this plant for the potent phyto-
constituents and their pharmacological
properties by which new chemical entities can
be established to meet the challenges of
pharmaceutical profession to fight the
frightening diseases of the day and future.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 91–100
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Source of Support: NIL Conflict of Interest: None Declared
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND
HEALING PLANTS
Jagan Mohan Reddy P1, Ismail Shareef M
2*, Gopinath S M
3, Dayananda K S
4,
Ajay Mandal5, Purushotham K M
6
1,2,3,4Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore- 560 107,
Karnataka, India. 5Research Scholar, Department of Biotechnology Engineering, Acharya Institute of Technology, Bangalore-
560 107, Karnataka, India. 6Institute of Animal health & Veterinary Biological, Hebbal, Bangalore, Karnataka 560024, India
*Corresponding author: [email protected]; Mobile: +91 9916836390
Received: 06/02/2014; Revised: 27/02/2014; Accepted: 03/03/2014
ABSTRACT
Present investigation has been evaluated to find out active constituents of some indigenous plants
such as Calotropis procera, Ricinus communis and Mentha piperita potent against ectoparasite. The
results revealed that the % yield of Calotropis procera, Ricinus communis and Mentha piperita was
10.23, 22.79, 14.46 respectively and physical characteristic was sticky solid, dark greenish, agreeable
in Calotropis procera, semi-solid, dark greenish, agreeable in Ricinus communis however, sticky
semi solid, dark greenish, characteristic organic in Mentha piperita. The plants having alkaloids,
saponins, flavonoids, glycosides, carbohydrates, fixed oils and fats, tannins and phenolic compounds
as main active constituents in all the three plants which is very useful for preparing drug
development against wound healing.
KEY WORDS: alkaloids, glycosides, % yields, saponins, flavonoids, Calotropis procera, Ricinus
communis, Mentha piperita
Short communication
Cite this article:
Jagan Mohan Reddy P, Ismail Shareef M, Gopinath S M, Dayananda K S, Ajay Mandal,
Purushotham K M (2014), PHYTOCHEMICAL ANALYSIS OF SOME INDIGENOUS WOUND
HEALING PLANTS, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 101–104
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Since time immemorial plants have been
used for the treatment of various ailments.
Even today several important drug used in the
modern system of medicine are obtained from
plants. The use of medicinal plants has figured
in several ancient manuscripts like the Rigveda,
the Bible, the Iliad, the Odyssey and the history
of Herodotus. As far back as 4000 B.C. the
ancient Chinese were using drug plants. The
earliest reference to the use of medicinal plants
as a cure for disease is found in the manuscript
of Eber Papyrus written in 1600 B.C. (Abu
Hanifah Y., 1990) with the advancement of our
knowledge; such superstitions were gradually
lost. In India, earliest reference to medicinal
plants appears in the Rigveda, written between
3500 and 1600 B.C. in Artharvanaveda too,
detailed descriptions of several medicinal
plants were given. Most of the drug plants are
wild and only a few of them have been
cultivated (Aldridge K. E., 1994). Studies of
medicinal plants based on ancient literature and
its investigation in modern light is under
process. The medicinal importance of a plant is
due to the presence of some substances like
alkaloids, glycosides, resins, volatile oils,
gums, tannins etc. these active principles
usually remain concentrated in the storage
organs of the plants, viz., roots, seeds, bark,
leaves etc. Considering all these facts, the
present investigation is designed to find out
phytochemical analysis of some indigenous
plants which are potent against wound healing.
MATERIALS AND METHODS
Selection of plants
Three local plants Ricinus communis,
Calotropis procera and Mentha piperita were
selected on the basis of their medicinal
properties against ticks and lice as reported in
various literatures. These plants were identified
and verified with taxonomical studies as
reported by (Aldridge K. E., 1994).
Collection of plant material
The plants were collected from different
regions of Bangalore, Karnataka, India.
Preparation of plant extract
Plant material was kept for drying for about
2 weeks, away from direct Sun light below 45°
C (shade dried). The dried material was
crushed in an electric grinder to coarse powder
consistency. About 500 gm of the powder
material was uniformly packed into a thimble
of a soxhlet extractor. It was exhaustively
extracted with methanol for a period of about
48 hrs or 22 cycles or till the solvent in the
siphon tube of an extractor becomes colourless.
The completion of the extraction was
confirmed by taking the solvent from the
thimble and evaporated to check the absence of
residue. The extract was taken out, filtered and
distilled to concentrate to get the syrupy
consistency in rotary evaporator. The residue
was dried over anhydrous sodium sulphate to
remove traces of alcohol. The extracts were
preserved in airtight container to avoid loss of
volatile principles (Annoni, F et al., 1989).
Physical characteristic of plant
The physical characters of the extract were
noted and the percentage yield was calculated
on such basis. The extracts were preserved in
an airtight container to avoid loss of volatile
principles for further studies.
Solubility of plant extract
All the extracts were dissolved in different
solvents for checking the solubility of extracts.
Phyto-chemical analysis of plant extract
The extracts were tested for the presence of
some active chemical compounds such as
alkaloids, flavonoids, glycosides, fixed oils &
fats, proteins, tannins and phenolic compounds,
carbohydrate, saponins. The analysis was
conducted as per universal methods.
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
RESULTS
The physical characteristics observed in the
crude extracts of all the 3 plants are depicted in
Table 1.
The percentage yield of various extracts of
Ricinus communis, Calotropis procera and
Mentha piperita was calculated as 10.23%,
22.79% and 14.46% respectively (Table 2).
The phyto-chemical analysis of methanolic
crude extract of Ricinus communis, Calotropis
procera and Mentha piperita was found
positive for saponins, flavonoids, fixed oils and
fats in common. None of the 3 plants reported
the presence of Protein & amino acids.
Carbohydrates was absent in Calotropis
procera & Glycosides were absent in Mentha
piperita whereas Alkaloids, Carbohydrates,
tannins & phenolic compound were absent in
Ricinis communis (Table 3).
Table: 1. Physical characteristics of different crude extract of plants
Plant extracts Consistency Colour Colour
Calotropis procera (Leaves) Sticky semi solid Dark white Agreeable
Ricinus communis (Leaves) Semi solid Dark greenish Agreeable
Mentha piperita (Leaves) Sticky semi solid Dark greenish Characteristics organic
Table: 2. Percentage yield of different plants extract
Plants Weight of dry
powder (gm)
Weight of dry
extract (gm)
Yield
(%)
Calotropis procera (Leaves) 242.20 24.80 10.23
Ricinus communis (Leaves) 217.60 49.60 22.79
Mentha piperita (Leaves) 300.00 43.40 14.46
Table: 3. Qualitative determinations of active ingredients in crude extract of different Plants
Phyto-chemicals Calotropis procera
(Leaves)
Ricinus communis
(Leaves)
Mentha piperita
(Leaves)
Alkaloids + − +
Saponins + + +
Flavonoids + + +
Glycosides + + −
Carbohydrate − − +
Fixed oils and fats + + +
Tannins & phenolic
Compound
+ − +
Proteins & amino Acids − − −
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 101–104
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
DISCUSSION
The search for “natural remedies” for a
common disorder such as wounds has drawn
attention to herbals From ancient times, herbals
have been routinely used to treat wounds and
in many cultures and their use in traditional
medicine. Plants are more potent healers
because they promote the repair mechanism in
the natural way. This study exposed that
traditional medicines are still used by tribal
peoples & it is established the value of a great
number of plants used in tribal medicine
especially for wound healing. Seemingly much
still unknown information about plants to treat
various disease including wounds. So far, very
few studies have been carried out on medicinal
plants which present the wound healing
activity. The aim of the review was to list out
the medicinal plants which is reported already.
However, there is a need for scientific
validation, standardization and safety
evaluation of plants of the traditional medicine
before these could be recommended for healing
of the wounds.
CONCLUSION
The study demonstrates that the crude
methanolic extracts of Ricinus communis,
Calotropis procera and Mentha piperita exhibit
phyto-chemical principles of therapeutic value
and this has provided scientific basis for its
folkloric use in the treatment of various
infectious conditions and wound healing. The
wound healing potential which was confirmed
by the in vivo experiments further supports the
ethno-medicinal uses of the plants.
REFERENCES
Abu Hanifah, Y. (1990). Post-operative
surgical wound infection. Med.
J.Malays. 45:293–297.
Aldridge, K. E. (1994). Anaerobes in
polymicrobial surgical infections:
incidence, pathogenicity and
antimicrobial resistance. Eur. J. Surg.
Suppl. 573:31–37.
American Diabetes Association. (1999).
Consensus Development Conference on
Diabetic Foot Wound Care. Diabetes
Care 22:1354.
Annoni, F., M. Rosina, D. Chiurazzi, and M.
Ceva. (1989). The effects of a
hydrocolloid dressing on bacterial
growth and the healing process of
legulcers. Int. Angiol. 8:224–228.
Armstrong, D. G., and K. A. Athanasiou.
(1998). The edge effect: how and why
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Clin.Podiatr. Med. Surg. 15:105–108.
Armstrong, D. G., and L. A. Lavery. (1998).
Evidence-based options for off-loading
diabetic wounds. Clin.Podiatr. Med.
Surg. 15:95–104.
Armstrong, D. G., L. A. Lavery, and T. R.
Bushman. (1998). Peak foot pressures
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foot ulcers treated with total contact
casts. J. Rehabil. Res. Dev. 35:1–5.
Source of Support: NIL Conflict of Interest: None Declared
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal
A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS
STANDARDIZATION
Wavare Ramesh1, Yadav Reena
2*, Sheth Suchita
3, Sawant Ranjeet
4
1Associate Professor & Head, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu Ayurved
Mahavidyalaya, Charni road, Mumbai. 2P. G Scholar, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved
Mahavidyalaya, Charni road, Mumbai. 3Assistant Professor, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu, Ayurved
Mahavidyalaya, Charni road, Mumbai. 4Assisitant Professor, Department of Rasashastra, Kamaladevi Gauridutta Mittal Punarvasu Ayurved
Mahavidyalaya, Charni road, Mumbai.
*Corresponding Author: Email: [email protected]; Phone:+91 9922900872, +918108269466.
Received: 03/02/2014; Revised: 25/02/2014; Accepted: 05/03/2014
ABSTRACT
Throughout most of recorded history Manikya (ruby) has been the world’s most valued
gemstone, which consists of Aluminium oxide, chromium, titanium. Ruby, the rarest of gemstones is
grouped under ratna varga in Rasashastra texts. As per Ayurveda, Manikya has the properties like
such as appetizer, aphrodisiac, bhutaghna, papaghna. Also it balances vitiated vata, pitta and kapha
hence its use leads to generate mental and spiritual powers. Manikya Pishti is a unique preparation
mentioned in Rasashastra texts of Ayurveda. The objective of the study was to prepare Manikya
Pishti (dosage form) and standardize it with different physical, chemical and instrumental analysis.
The prepared Pishti was subjected to ancient as well as modern analytical tests mainly X Ray
diffraction analysis of raw ruby showed Aluminum oxide as the principal component. Mean particle
size of raw ruby was 107.58 nm and that of pishti was 52 nm. The immense decrease in particle size
of ruby in pishti form concludes it will be useful as a nanomedicine, hence it can be said that it will
have a better dissolution rate.
KEYWORDS: Manikya Pishti, bhavana, trituration, particle size, XRD.
Research Article
Cite this article:
Wavare Ramesh, Yadav Reena, Sheth Suchita, Sawant Ranjeet (2014),
A PHARMACEUTICAL APPROACH ON MANIKYA PISHTI TOWARDS
STANDARDIZATION, Global J Res. Med. Plants & Indigen. Med., Volume 3(3): 105–111
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
INTRODUCTION
Ayurveda is not merely the science of
disease and drugs whereas it has every aspect
of life in its sphere. The main aim of Ayurveda
is to maintain good health as well as to promote
a healthy life span. Hence Rasashastra
(Iatrochemistry) had used almost all the gems
for the purpose of inducing longevity of life in
human body (Vaidyopadhyaya, 1983). Also
internal use of gems can cure several diseases
(Vaidyopadhyaya, 1983). According to
Ayurveda, Manikya has been grouped under
ratna varga. Manikya Pishti preparation from
precious gem Manikya is a famous Ayurvedic
preparation. It is a versatile drug having
properties like memory enhancing, aphrodisiac
and specially recommended in erectile
dysfunction, general debility, and it has best
rasayana (antioxidant) as well as aphrodisiac
property. Though Ruby is known for a range of
therapeutic effects, it has been observed that
practitioners donot prevalently use it due to a
non-standardized pharmaceutical process
involved in processing of Ruby. So an attempt
was made to prepare and standardize Manikya
Pishti to facilitate its use in Ayurvedic
therapeutics.
MATERIALS AND METHODS
Materials
Raw ruby (Voucher No. -
SAS/10/7188/2013) was authenticated through
microscopic examination at gems testing lab,
Mumbai. An herbal drug that is rose water was
authenticated at pharmacognosy dept. of
Nicholas Piramal Lab, ltd Mumbai.
Methods
Manikya processing was performed using
standard procedures and includes steps namely
Manikya Shodhana (purification) (Sharma,
1979; Gopalbhatt, 2006 and Kaleda, 177) and
Manikya Pishti (trituration) (Kaleda, 177).
Preparation of Manikya Pishti:
1. Purification of Manikya
a. by swedana (specialized process of
steaming) in lime juice (Sharma, 1979)
b. by swedana in Kulattha Kashaya
(Decoction of Dolichos biforus Linn.)
(Gopalbhatt, 2006)
c. by quenching of ruby in rose water
(Kaleda, 177).
2. Preparation of Manikya pishti (Kaleda, 177)
Purification of Ruby
Materials - Crude Ruby, lime juice, Kulattha
Kashaya
Method - The sample was wrapped in a cloth
and tied to an iron rod in a suspended manner,
which was kept horizontally over a steel vessel.
Lime juice was taken in the steel vessel and
Ruby was immersed in the lime juice in such a
way that, it neither touches the sides nor the
bottom of the container. The vessel was heated
for 3 hrs under medium heat. Similarly
procedure was repeated with the decoction of
Dolichos biflorus instead of lime juice.
Nirvapana (process of reducing red hot
elements in liquid media) of Ruby in Rose
water was carried out for 101 times to make it
more brittle and to convert it into a fine
powder.
Manikya Pishti preparation [MP]
Materials - Manikya, Rose water
Method - Shodhit Manikya was made into fine
powder and triturated with rose water for 15
days [3hrs/day].
Method of Analysis of Manikya & Manikya
pishti using parameters described in
Ayurveda texts
Raw Ruby - Raw Ruby was subjected to
Grahyalakshana (selection criteria mentioned
in Ayurvedic text) of Manikya mentioned in
Rasashastra text (Khare, 1992).
Prepared Pishti
Prepared Pishti was subjected to bhasma
pariksha mentioned in Rasashastra text.
(Khare, 1992)
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Methods for analysis of Ruby & Manikya
Pishti on Modern parameters
Dynamic light scattering (DLS) Instrument - Malvern Mastersizer, Ver. 2000 5.31 Place: IIT, Pawai, Mumbai. Method: A rolling table with a partly adjustable inclined piece was constructed from hardboard and mounted on a steel frame. It was varnished with ordinary wood varnish to present a smooth surface along which movement could take place. Glass jars of various sizes were filled with different masses of the various materials and rolled from a fixed position with inclined part down the rolling table. Still on the flat part of the table readings were recorded and noted. Each measurement was performed 3 times.
Nanoparticle tracking analysis (NTA) Instrument - Nanoparticle tracking analyzer. Place - Institute of Science, Churchgate, Mumbai Method: The powders were placed in a container and dispersed in liquid media. The light scattered by the particles is captured using a CCD or EMCCD camera over multiple frames. Computer software is then used to track the motion of each particle from frame to frame. The rate of particle movement is related to a sphere equivalent hydrodynamic radius as calculated through the Stokes–Einstein equation.
X-ray diffraction (XRD) Instrument: Philips Holland XRD system Place: IIT Institute, Powai, Mumbai Method: The powdered sample was spread on to a double side tape with spatula, which was then placed on an aluminum sample holder. All
the peaks were recorded on the chart, and the corresponding 2 theta values were calculated. The strongest peak identified in the sample was corundum
Elemental Analysis by (ICP-AES) Method: Inductively coupled plasma (ICP) can be generated by directing the energy of a radio frequency generator into ICP argon gas. Coupling is achieved by generating a magnetic field by passing a high frequency electric current through a cooled induction coil. This inductor generates a rapidly oscillating magnetic field oriented in the vertical plane of the coil. Ionization of the flowing argon is initiated by a spark from a Tesla coil. The resulting ions and their associated electrons from the Tesla coil then interact with the fluctuating magnetic field. This generates enough energy to ionize more argon atoms by collision excitation. The electrons generated in the magnetic field are accelerated perpendicularly to the torch. At high speeds, cations and electrons, known as eddy current, will collide with argon atoms to produce further ionization which causes a significant temperature raise. Within 2 ms, a steady state is created with a high electron density. Plasma is created in the top of the torch. A long, well-defined tail emerges from the top of the high temperature plasma on the top of the torch. This torch is the spectroscopic source. It contains all the analyte atoms and ions that have been excited by the heat of the plasma.
RESULTS
Analysis of Manikya using modern
parameters Raw Ruby sample was tested in gem lab for
its gentility showing certified natural ruby qualities [Table 1].
Table 1: Showing Certified natural ruby qualities
Shape Rough
Cut Uncut
Isotropic/Anisotropic Anisotropic
Weight 4.78cts
Dimension 5.00 length approx.
Mounted / Unmounted Unmounted
Colour Milky pinkish brownish
Fluorescence Inert
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Sample of Manikya passed all the criteria of
genuine Manikya (Ruby). Raw Ruby and MP
was also analysed using the technique like
Dynamic light scattering (DLS), Nanoparticle
tracking analysis (NTA), X Ray Diffraction
(XRD).
Prepared Pishti
The prepared pishti showed following
characters
1) Rekhapurnatva: A pinch of pishti was
taken in between the thumb and index
finger and rubbed. It was observed that the
pishti entered into the lines of the finger
and was not easily washed out from the
cleavage of the lines.
2) Nishandratva: The pishti was taken in a
petri dish and observed for any lustre in
daylight through magnifying glass. No
lustre was observed in the pishti.
3) Varitaratva: A small amount of the
prepared pishti was sprinkled over the still
water in a beaker. It was found that the
pishti particles floated over the surface of
water.
Dynamic light scattering (DLS)
DLS analysis of raw sample of Ruby for
particle size estimation showed following
results [Table 2] & [Figure 1] The average
particle size of Raw Ruby was 1071.58 nm.
Nanoparticle tracking analysis NTA:
Manikya pishti when subjected to NTA
yielded following results [Table 3] & [Figure
2].
X-ray diffraction (XRD)
XRD analysis of raw Manikya as well as its
pishti showed following results [Table 4 & 5]
[Figure 3 & 4].
Elemental Analysis by (ICP-AES)
ICP-AES analysis of raw ruby and Manikya
pishti shown following results [Table 6]
Table 2: Particle size of Raw Ruby by DLS
Sample Test below Observation
Raw Sample of
Ruby
10% particles 134.717
50% particles 706.819
90% particles 1445.421
100% particles 1999.400
Figure 1: Particle size of Raw Ruby by DLS
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Table 3: Particle Size of Manikya Pishti BY NTA
Sample Particle size
MP 52
Figure 2: Particle Size of Manikya Pishti [MP] BY NTA
Particle size of MP (Manikya Pishti) was in nano scale. Hence it can be researched for its efficacy in Nano-medicine.
Table 4: X-ray diffraction of raw ruby
2-Theta value d-spacing intensity
25.580 3.479 100
26.621 3.346 10.7
27.480 3.243 6.1
35.140 2.552 84.3
37.780 2.379 35.5
43.360 2.085 97.5
52.580 1.739 64.2
Table 5: X-ray diffraction of Manikya Pishti
MP
2-Theta value d-spacing Intensity
25.620 3.4741 513
25.800 3.4503 3971
28.00 3.1840 600
35.380 2.5349 1504
37.940 2.3969 1088
38.060 2.3624 600
43.500 2.087 1904
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
Figure 3: X-ray diffraction of raw Ruby
Figure 4: X-ray diffraction of Manikya Pishti
Table 6: Showing results of elemental analysis of Raw ruby, Manikya Pishti [MP]
Elements Unit Raw Ruby MP
Al (%) 51.2 46.9
Cu mg/Kg (ppm) 21.6 74.0
Fe mg/Kg (ppm) 1600 25776
Cr mg/Kg (ppm) 2884 3928
K mg/Kg (ppm) 274 951
Na mg/Kg (ppm) 52 3372
Ca mg/Kg (ppm) 174.4 12092
Mn mg/Kg (ppm) ND 186
Mg mg/Kg (ppm) 1004 5752
As mg/Kg (ppm) ND ND
S mg/Kg (ppm) ND ND
Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 3 | March 2014 | 105–111
Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||
DISCUSSION
The process of trituration involves
breakdown of the material by rubbing action
between two surfaces, i.e. surface phenomena,
it is also called as attrition. When stress in the
form of attrition is applied, the particle surfaces
chip and produce small particles. Bhavana
(levigation) is given by grinding with some
liquid media, so it may be considered as wet
grinding and it is observed interestingly that
finer size of particles can be achieved by wet
grinding than dry grinding. For Pishti
preparation trituration is done with rose water,
which serves as liquid media for wet grinding
of material and facilitates easy and smooth
grinding and even eliminates hazards of dust.
It is also found interestingly in practice that
the finer particle size can be achieved by wet
grinding than by dry grinding. All elements
are found in human body in trace levels, but not
to be absorbed in to body in their elemental
form, plants are having capacity to transform
them in to readily absorbable form.
Trituration may help in reduction of particle
size of Manikya as it has hardness 9 it needs
more grinding effect. Mardana (trituration
process) also helps in loosening the
molecular cohesiveness and helps the drug to
break into fine particles during the subsequent
processing (Suneeta, 2011) (Mehta, 2010).
CONCLUSION
In the present study Manikya Pishti was
successfully prepared according to the methods
mentioned in Rasashastra texts and
standardized with help of Ayurvedic as well as
modern parameters. It will be helpful for
further researchers and manufacturers to
prepare the pishti according to mentioned
standard process & parameters which will also
facilitate its usage in Ayurvedic therapeutics.
REFERENCES
Chintamani Khare, (1992).
Rasaratnasamucchaya, Sanskrit
Commentary by, 4th
ed. Chapter 4,
Verse 4, Anadashram Publications,
Pune, India. p. 67
Gopalbhatt (2006) Rasendra Sara Sangrah with
‘Rasavidyotini’ Hindi Commentary by
Indradev Tripathi, 4th
ed. Chapter 1,
Verse 378-379, Chaukhamba Orientalia
publications, Varanasi, India,. p. 94
Krishna Gopalaji kaleda (1991) Rasatantra Sara
va siddaprayoga sangraha, Vol.1 13th
ed, Krishna gopala Ayurveda bhavana.
Gujarat, India. p. 177
Mehta R. M. (2010), Pharmaceutics part 2, ed.
3rd
, Chapter 4, p.77
Ramprasad Vaidyopadhyaya (1983) Rasendra
Puran, Hindi Commentary,1st ed.
chapter 28, Verse 7, Laxmi
Venkateshwar Press, Mumbai, India, p.
391-392
Sharma S. (1979) Rasa Tarangini. Hindi &
Sanskrit Commentary by Kashinath,
11th
ed. Chapter 23,Verse 46, Motilal
Banarasidas Publication, Delhi, India, p.
609
Suneeta M. (2011) Pharmaceutico Analytical
Study of Manikya Bhasma.
Source of Support: NIL Conflict of Interest: None Declared
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