Ethanolic extract of nilavembu answers pyrexia

http://www.ncbi.nlm.nih.gov/pubmed/22014740

read the above link.

Varmam protocol

Cervical spondylosis Research Protocol

http://www.webmedcentral.com/article_view/2968



Read the above said link for varmam protocol

safety studies in metallic drugs

Safety Issues in Metallic drugs
Dr. P. Sathiyarajeswaran
Dr. M. Kannan
Dr. K. Gopakumar
Central Council for Research in Siddha
Rationale in using metallic drugs
When
How much
How long
What are all the Do’s and Dont’s
What are all the possible side effects
How to choose a efficacious, non toxic SOP
Which anti dotes are available in literature


Safety studies - Protocol
Pre clinical – Standardization
Animal studies – Efficacy testing
Toxicological profiling
Phase 1 trial
Enzymatic
Sonologic
Tissue Biopsy
Bone teeth
LFT/KFT

Phase 1
Safety
Human healthy volunteers
Males are preferred
Non addictive
Occupationally hazard free subject
CTRI Compulsory
Out come socially Viable
Standardization
Traditional standards- Organoleptic / Five senses
PLIM Standards
Develop our own standards for High order Forms
Nano studies
Chemical Profiling(XRD/SEM/TEM/ICPMS/ICPOES)
Ligands / Chelating testing
Purification understanding
First step in drug manufacturing.
Select the best process.
Is purification alone enough.
Detoxification.
Ore dressing.
Debris removal

Understanding the risk in Phase I
IEC Approval
Pre screening
Consent
Compensation
Out come benefit


Our experience
Linga chenduram
Aya veeram
Aya brinuga raja karpam
Phase II
Minimal sample size
No enzymatic / Sonological Changes
Quality of life Prospectively observed.
To end with
Half baked cakes reach the masses.
Physicians have to think out of the box.
A Metallic study without Clinical trial testing will end up in Bias results.
Use metallic drugs as much you can but Rationally.

Thank you all…

Antibacterial activity of Vallarai Chooranam against Human Pathogens

International Journal of PharmTech Research
CODEN (USA): IJPRIF        ISSN : 0974-4304
                                                                                                              Vol.4, No.1, pp 162-168,          Jan-Mar 2012
Antibacterial activity of Vallarai Chooranam
against Human Pathogens
T.G.Nithya
1
, S.Sivakumar
2
, K.Sangeetha
1
1
Department of Biotechnology, Faculty of Science and Humanities, SRM University,
Kattankulathur,India.
2
Department of Gunapadam , National institute of Siddha, Tambaram Sanitorium,
Chennai,India.
*Corres. Author: nithya_245@yahoo.co.in
Mobile : +919841404193
Abstract:Vallarai chooranam is a polyherb,comprised of 10 different herbs,used for treatment of diabetics,urinary
tract infection,leucorrhea,veneral disesase and also to improve memory power.The present investigation was
carried out to study the unexplored area of the Vallarai  chooranam towards their antibacterial activity against both
Gram positive and Gram negative organisms by Disc diffusion method.Aqueous and solvent extracts of the
chooranam were tested against selected human pathogens viz. S.aureus, P.aeruginosa, B.subtilis ,K.pneumoniae
and E.coli.   B o t h   t h e   e x t r a c t s   w e r e   f o u n d   t o   b e   m o r e   e f f e c t i v e   a g a i n s t   a l l   t h e   t e s t   p a t h o g e n s .B.Subtilis was more
susceptible to the aqueous extracts among the tested organisms.The results of antibacterial activity revealed that
both the extracts exhibited good inhibitory activity against all test pathogens.The presence of phytochemicals in the
formulation were also assayed.The results showed positive response for significant secondary metabolites.
Key words: Polyherbal, antibacterial,Qualitative analysis,Aqueous extract,Solvent extract,Vallarai Choornam.
Introduction:
Herbal medicines derived from plant extracts
are being increasingly utilized to treat a wide variety of
clinical diseases, mainly in developing countries, for
p r i m a r y   h e a l t h   c a r e   b e c a u s e   o f   b e t t e r   c u l t u r a l
acceptability, better compatibility with the human
body and fewer side effects. Nowadays multiple drug
resistance has developed due to the indiscriminate use
of commercial antimicrobial drugs commonly used in
the treatment of infectious disease
1
. In addition to this
problem, antibiotics are sometimes associated with
adverse effects on the host including hypersensitivity,
immune-suppression and allergic reactions
2
.
It has been reported that there has been an
alarming increase in number of diseases and disorders
caused by synthetic drugs prompting a switch over to
traditional herbal medicine
3
.The earliest record of
human civilization and culture of China, Egypt,
Assyria, and Indies valley reveals that the elders and
w i s e   m e n   o f   t h o s e   t i m e s   u s e d   h e r b s   t o   t r e a t   v a r i o u s
diseases. Many of the indigenous medicinal plants are
used as spices for cooking
4
.The ancient use of plants
for healing purposes forms the origin of much of
modern medicine. Many traditional drugs originate
from plant sources: a century ago, most of the effective
drugs were plant based
5
.
It is estimated that there are 250,000 to 500,000
species of higher plants on earth. But relatively smallT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 163
percentage (5-15%) has been systematically
investigated for the presence of bioactive compounds
6
.
Microorganisms are the causative agents of almost all
kinds of acute and chronic diseases.
Plants based antimicrobials have enormous therapeutic
potential. They are effective in the treatment of
infectious diseases while simultaneously mitigating
m a n y   o f   t h e   s i d e   e f f e c t s   t h a t   a r e   o f t e n   a s s o c i a t e d   w i t h
synthetic antimicrobials.The use of plant extracts with
known antimicrobial properties, can be of great
significance in therapeutic treatments.Although
hundreds of plant species have been tested for
antimicrobial properties, the vast majority of them
have not been adequately evaluated
7
.Many efforts have
been made to discover new antimicrobial compounds
from various kinds of sources such as micro-organisms,
animals,and plants.One of such resources is folk
medicines. Systematic screening of them results in the
discovery of novel effective compounds
8
.The
increasing prevalence of multidrug resistant strains of
bacteria and the recent appearance of strains with
reduced susceptibility to antibiotics raises the specter
of untreatable bacterial infections and adds urgency to
the search for new infection-fighting
strategies
9
.Considering the vast potentiality of plants
as sources for antimicrobial drugs with reference to
antibacterial agent, a systematic investigation was
undertaken to screen the polyherbal formulation
Vallarai chooranam for its antibacterial activity
against selected human pathogens.
 This polyherbal formulation is a composition
of 10 different herbs viz.,(In tamil) Vallarai, Jaadhikai,
Jaadhipathiri,Lawangam,Elakkai,Thaaleesapathiri,Ma
asikkai,Kadukkai,Nellikai,Thaantrikkai.(Table 1).The
powder form of this sidhha  Chooranam is used to treat
diabetics,Urinary tract infection,Leucorrhea,Veneral
disesase and also used to improve memory power and
blood purification
10
.The use of alchocol or water as
solvent  is efficient in extracting a wide variety of
active components. Therefore, we analysed the
Vallarai chooranam for the presence of
phytochemicals and evaluated the antimicrobial
activity against selected human pathogens.
Materials and Methods:
Preparation of Formulation:
The ingredients were procured from reputed
commercial Siddha supplier (Dr.Sivakumar and
Dr.Juliet, Selaiyur.Chennai) and authenticated [Table
1]. All the ingredients were shade dried, powdered and
mixed thoroughly in same proportion.The mixture was
further boiled in distilled water at 100ºC for 60
minutes and filtered.The filtrate was evaporated to
dryness, used for subsequent experiments and they are
designated as Chooranam since they are comprised of
multiple herbs.
Solvent extract preparation:
1 0   g r a m s   o f   a i r   d r i e d Vallarai chooranam
powder was extracted with 100ml of Organic solvent
(Ethanol) and kept on rotary shaker at 190-220 rpm for
24 hours.The supernatant was collected and solvent
was evaporated to make the final volume one – fourth
of the original volume and stored at 4
0
C in air tight
bottles
11
.
Aqueous extract preparation:
The aqueous extract is prepared by soaking
100grams of Vallarai chooranam powder in 200 ml of
distilled water for 12hours.The extracts were filtered
using Whatman filter paper (125 mm).
12
Table 1:Polyherbal formulation of Vallarai chooranam
S.No Siddha Name Botanical Name Quantity Family Name
1 Vallarai Centella asiatica 50gms Mackinlayaceae
2 Jaadhikai Myristica fragrans 10gms Myristicaceae
3 Jaadhipathiri Myristica fragrans 10gms Myristicaceae
4 Lawangam Syzgium aromaticum 10gms Myrtaceae
5 Elakkai Eletteria cardamomum 10gms Zingiberaceae
6 Thaaleesa pathiri Taxus beccata 10gms Taxaceae
7 Maasikkai Quercus infectoris 10gms Fagaceae
8 Nellikai Emblica officinalis 10gms Phyllanthaceae
9 Kadukkai Terminalia chebula 10gms Combretaceae
10 Thaantrikkai Terminalia belerica 10gms CombretaceaeT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 164
Phytochemical screening :
The qualitative tests were carried out in both
the extract of Vallarai chooranam using standard
procedures
13-15
.Both the extacts were analysed for the
presence of significant secondary metabolites viz
Alkaloids, Tannins, Flavonoids ,Cardiac glycosides,
Steroids and Saponins.
Growth and Maintenance of Test
Microorganism for Antimicrobial Studies:
Bacterial cultures of Bacillus subtilis (B.
subtilis), Escherichia coli (E. coli), Pseudomonas
aeruginosa (P.aeruginosa),Staphylococcus aureus (S.
aureus) and Klebsiella pneumoniae
(K.pneumoniae)were obtained from  Department of
Microbiology, SRM Medical College, India, and the
studies were performed at Department of
Biotechnology,FSH,SRM University.The bacteria
were maintained on Nutrient Agar(NA) slants at
4°C.For further study, cultures have been grown in
Nutrient Broth (NB) for 24hrs as overnight cultures.
Disc diffusion Method:
 The antibacterial assay of aqueous and
ethanolic extracts was performed by Disc diffusion
method
16
. T h e   N u t r i e n t   a g a r   m e d i a   ( 2 0 m l )   w a s   p o u r e d
into sterilized petri dishes and left to solidify at room
temperature. The overnight bacterial cultures have
b e e n   s p r e a d   p l a t e d   o n   t h e s e   p e t r i d i s h e s   u s i n g   s t e r i l e   L
rod.Taking the crude extract concentration as
100%,different concentration of aqueous and ethanolic
extracts was prepared viz., 20%,40%, 60%, and 80%.
W h a t t m a n ’ s   N o . 1   f i l t e r   p a p e r   d i s c s   ( 3 m m )   w e r e
soaked in 0.1 ml of Ethanol extract of varying
concentrations  from 20% -80%.The similar procedure
was carried out for aqueous extract with varying
concentration from 20% -80% simultaneously.
T h e   f i l t e r   p a p e r   d i s c s   w e r e   p l a c e d
equidistantly on inoculated media and diffusion of
solution was allowed to occur for 30 minutes at room
t e m p e r a t u r e .   P l a t e s   w e r e   i n c u b a t e d   a t   3 7˚C   f o r   2 4
hours. The average zone of inhibition was recorded.
Sterile distilled water and Ethanol were maintained as
control. The diameters of the inhibition zones were
measured in mm.
Table 2 :Antibacterial screening of the extract showing the range of zone of inhibition (mm)
Table 3 :Zone of inhibition of Ethanolic extract (mm)
 Zone of Inhibition (mm) by varying concentration(%)
Test Organism 20% 40% 60% 80%
E.coli 3.12±0.12 6.22±0.15 8.27±0.13 11.22±0.12
S.aureus 6.53±0.11 10.16±0.10 11.13±0.16 12.15±0.19
P.aeruginosa 2.32±0.10 3.85±0.17 5.61±0.13 7.16±0.18
B.subtilis 7.21±0.13 9.45±0.20 11.33±0.19 14.33±0.16
K.pneumoniae 4.33±0.20 6.27±0.14 9.13±0.10 12.33±0.12
Test
Organism
Ethanol
Extract
Aqueous
Extract
E.coli 3 to 11 2 to 11
S.aureus 6 to 12 3 to 10
  P.aeruginosa 2 to 7 2  to  6
B.subtilis 7 to 14 7  to 14
K.pneumoniae 4 to 12 4 to   9T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 165
Ethanolic extract Zone of inhibition
0
5
10
15
20 40 60 80
P e r c e n ta g e   o f   e th a n o l i c   Ex t r a c t
Inhibition (mm)
E . c o l i S . a u r e  u s P . a e  ru  g i  n  o s a
B . s u b t i l i s K . p n e  u m o n i a e
Graph 1:
Table 4 : Zone of inhibition by Aqueous extract (mm)
 Zone of Inhibition (mm) by varying concentration(%)
Test Organism 20% 40% 60% 80%
E.coli 2.75± 0.11 4.51±0.14 6.13±0.13 11.24±0.16
S.aureus 3.87 ± 0.14 5.31±0.15 9.19±0.11 10.16±0.17
P.aeruginosa 2.37 ± 0.20 4.22±0.12 5.31±0.12 6.15±0.18
B.subtilis 7.62±0.15 8.32±0.12 10.14±0.14 14.31±0.20
K.pneumoniae 4.53±0.14 5.12±0.15 7.16±0.13 9.56±0.15
 GRAPH 2:
Aqueous  extrac t  Zone  of  inhibition
0
5
10
15
20 40 60 80
Percentage of Aqueous Extract
Inhibition in mm
E.coli S.aureus P.aeruginosa
B.subtilis K.pneumoniaeT.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 166
GRAPH 3:
Comparision of Antibacterial activity of Ethanolic and
Aqueous extract
0
5
10
15
20E 20A 40E 40A 60E 60A 80E 80A
Percentage of Extracts %
Inhibition(mm)
E.coli S.aureus P.aeruginosa
Table 5: Phytochemical screening results of
Vallarai Chooranam
Compound Result
Alkaloids +
Cardiac glycosides +
Flavonoids +
Steroids +
Saponins +
Tannins +
+  =  Positive
Results :
Antimicrobial activity results obtained in the
present study revealed  that the tested extracts posses
potential antibacterial activity against B. subtilis, S.
aureus,E. coli,K.pneumoniae ,P.auregenosa (Table
2).The diameters of the inhibition zones against all the
tested bacteria were measured in mm. The results
showed that increase in concentration of extract
increased the zone of inhibition .When tested by the
disc diffusion method, the Ethanolic extracts of
Vallarai Chooranam showed significant activity
against S.aureus, K.pneumoniae  around 12mm.The
zone of inhibition for E.coli was observed as 11
mm.The highest antibacterial  activity of 14 mm was
observed in B. subtilis and least activity was  recorded
in P.auergenosa  o f   7 m m.The range of zone of
inhibition by ethanolic extracts against pathogens were
in the following order of higher to
lower,viz.,B.subtilis,S.aureus, E.coli, K.pneumoniae,
P.auregenosa(Table:3&Graph1).
The antimicrobial results for aqueous extract
of Vallarai Chooranam showed maximum activity
against B. subtilis o f     1 4   m m   a n d     l e a s t   a c t i v i t y
observed in P.aueregenosa of 6 mm .Inhibitory
activity against E.coli. S.aureus, K.pneumoniae was
around 9-11mm ( T a b l e   4   &   G r a p h   2 ) . T h e   r e s u l t s
elucidated that the activity was higher against Gram
+ve strains than Gram –ve pathogens.In classifying
the antibacterial activity as Gram-Positive or Gramnegative, it would generally be expected that a much
greater number would be active against Gram-positive
than Gram-negative bacteria
17
, however, in our study
b o t h   G r a m   p o s i t i v e   a n d   G r a m   n e g a t i v e   b a c t e r i a   w e r e
inhibited by the extracts of the Vallarai Chooranam.
On comparison of both the extracts the activity against
pathogens showed similar results with effective and
significant inhibitory action(Graph 3).The aqueous and
s o l v e n t     e x t r a c t s   w e r e   u s e d   t o   i d e n t i f y   t h e   p r e s e n c e   o f
various phytochemicals .Standard test for Flavonoids,
Alkaloids, Saponins, Cardiac glycosides, Tannins and
Steroids showed positive response(Table 5 ).
Discussion:
Ayurveda is a traditional Indian Medicinal
System practiced for thousands of years. Considerable
research on pharmacognosy, chemistry, pharmacology
and clinical therapeutics has been carried out on
ayurvedic medicinal plants. The polyherbal
formulations described in Ayurveda have been the
basis of treatment of various human diseases.
Biological evaluation of herbal formulations based on
their  medicinal uses forms the basis for development
of new drugs from plants
18
. Historically, plants have
provided a source of inspiration for novel drug
compounds,as plant derived medicine have made large
contribution to human health and well-being.
The combination of multiple herbs has
increased the efficacy of the Vallarai Chooranam to a
greater extent, since they possess medicinal values
exerted by the presence of phytochemicals.Flavonoids
a r e   k n o w n   t o   b e   s y n t h e s i z e d   b y   p l a n t s   i n   r e s p o n s e   t o
microbial infection.Hence it should not be surprising
that they have been found to be effective as
antibacterial substances against a wide array of
infectious agents
19
.Tannins (commonly referred to as
tannic acid) are also known as antimicrobial
agents.They are water-soluble polyphenols and
precipitated proteins present in many plant foods.T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 167
Tannins have been reported to prevent the
development of microorganisms by precipitating
microbial protein.The growth of many fungi, yeasts,
bacteria, and viruses were inhibited by this Tannins.
20
According to World Health Report on
infectious diseases 2000, overcoming antibiotic
r e s i s t a n c e   i s   t h e   m a j o r   i s s u e   o f   t h e   W H O   f o r   t h e   n e x t
millennium.Hence,the last decade witnessed an
increase in the investigations on plants as a source of
human disease management
21
and not many reports are
available on the exploitation of plants for
the management of plant diseases
22
.This is mainly due
to lack of information on the screening and evaluation
of diverse plants for their antibacterial
potential.Therefore, in the present investigation of
Vallarai Chooranam,a multiherbal formulation was
evaluated for its antibacterial potential for the first
time against selected human pathogenic bacteria which
are known to cause many infectious diseases.
Conclusion:
In the present investigation Vallarai
Chooranam was evaluated for its antibacterial potential
for the first time against selected human pathogenic
bacteria which are known to cause many infectious
diseases and also the phytoanalysis revealed the
presence of medicinally active constituents. The
antibacterial activity of this polyherbal formulation
would help for development of a new alternative
medicine system which has no side effects. Both the
extracts of this formulation possess a broad spectrum
of activity and open the possibility of finding new
clinically effective antimicrobial compounds.Hence
the study has provided  biochemical basis for
ethanopharmacological claims of the Chooaranam in
treatment and prevention of various diseases and
disorders.
References:
1) Ahmad I, Mehmood Z, Mohammad F.Screening
of some Indian medicinal plants for their
antimicrobial properties. J Ethnopharmacol 62:
183-193, 1998. 3
2) C l a r k   A M   N a t u r a l   p r o d u c t s   a s   r e s o u r c e   f o r   n e w
drugs. Pharm Res 13: 1133-1141
3) Ghule ST, Patil DK. Kisan World. 2001; 28: 33-
34.
4) Okwu DE , Phytochemicals and vitamin content
of indigenous spices of South   Eastern Nigeria.
J. Sustain. Agric. Environ. 6: 30-34.
5) Andrew Vickers, Herbal medicine:  W e s t   J   M e d .
2001 August; 175(2): 125–128.
6) Taylor, L. (2000), Plant Based Drugs and
Medicine, Raintee Nutrition Inc.,pp. 1-5.
7) B a l a n d r i n ,   M . F . ,   J . A .   K l o c k e ,   E . S .   W u r t e l e   a n d
2006. Phytochemical analysis and   antibacterial
W.H. Bollinger, 1985. My. Science., 1: 72-78.
Science, 228: 1154-1160
8) Tomoko.N. Takashi A., Hiromo T., Yuka I.,
(2002): Antibacterial activity of    extracts
preparated from tropical and subtropical plants
on methicillin-resistant Staphylococcus aureus. J.
Health Sci., 48: 273–276
9) Sieradzki K., Roberts R.B.., . (1999):
Thedevelopment of vancomycin resistance  ina
patient with methicillin-resistantStaphylococcus
aureus infection. N. Engl.J. Med., 340: 517–523.
10) Winston, D., Maimes, S., Adaptogens: Herbs
F o r   S t r e n g t h ,   S t a m i n a ,   a n d   S t r e s s   R e l i e f ,   2 0 0 7 ,
pp. 226-7
11) Harborne JB;Phytochemical Methods: A Guide
To Modern Technique of Plant Analysis.3rd
ed .London, England: Chapman and Hall;
1998:114-118.
12) H . O .   E d e o g a ,   D .   E .   O k w u ,     P h y t o c h e m i c a l
constituents of some Nigerian medicinal Plants;
African Journal of Biotechnology Vol. 4 (7), pp.
685-688 July 2005
13) Sofowora AE (1993). Medicinal Plants and
Traditional Medicines inAfrica.. 2nd edition.
Spectrum Books, Ibadan, Nigeria. p. 289.
14) Trease E.G., Evans W.C 1978. Pharmacognosy.
11th Edition, Balliere Tindall, London. 115-222.
15) Harborne JB;Phytochemical Methods: A Guide
To Modern Technique of Plant Analysis.3rd
ed .London, England: Chapman and Hall;
1998:114-118.
16) Bauer, A.W., Kirby, W.M.M., Sherris, J.C.,
Turck, M., 1966. Antibiotic susceptibility testing
by a standardized single disk method. Am. J.
Clin. Pathol. 45: 493-496.
17) McCutcheon AR, Roberts TE, Gibbons E, Ellis
S M ,   B a b i u k   L A ,   H a n c o c k   R E W ,   T o w e r s   G H N
(1995). Antiviral screening of British Columbian
medicinal plants. J. Ethnopharmacol. 49: 101-
110.T.G.Nithya et al /Int.J.PharmTech Res.2012,4(1) 168
18) Krishnan Kannabiran; Antibacterial activity of
saponin isolated from the leaves of  Solanum
trilobatum Linn; Journal of Pharmacy
Research, Vol 2, No 2 (2009)
19) Jasmine. R, Daisy.P and Selvekumar. B.N., 2007.
In vitro Efficacy of Flavonoids from Eugenia
jambolana Seeds Against ESL-Producing
Multidrug-Resistant Enteric Bacteria. Research
Journal of Microbiology. 2 (4):369–374.
20) P r a s a d ,   N .   R . , e t   a l . ,   2 0 0 8 .   P r e l i m i n a r y
phytochemical screening and antimicrobial
activity of Samanea saman . Journal of
Medicinal Plants Research. 2: 268-270.
21) Aiyelaagbe et al. 2000. Fitoterapia. 72: 544–546.
22) Satish S., Raveesha K. A. and Janardhana G. R.
1999. Lett. Applied Microbiol.28: 145–147.
*****

ICDS – Scope of Siddha in Enhancing the Scheme

Traditional medical knowledge  twined with culture and community health   are inherited or spread transplacentally. Usage of certain herbal combinations in safe delivery and compound drugs in lactation enhancement are the scientific Secrets which emerged from Grandmas Pandora box.  Adapting the same in life is the secret behind less statistics of Anemia in Tamilnadu when compared with other states. The knowledge may be transferred and translated by products to a successful completion of ICDS.
                        Immune Enhancement:
Seinei and Uraimathirai should be fed from day 1 up to 1 yr of child growth.
Dosage: 100 mcg of uraimathirai daily/ 0.5 ml ofSeinei.
Vit A Supplementation
This can be achieved through Karisalaikarpam tab and Murungailai chooranam.
 Dosage: 250 mg tab of Karisalaikarpam / 500 mg of Murungai ilai chooranam.
Ante natal care drugs
 Advocacy of Antenatal drugs as mentioned in the text.
 Usage of Pavana panchagulathylam – Internally.
 Application of Ulunthu thylam externally.
               Ayabringa raja karpam / Satavari lehyam – Iron & Folic acid supplementation.    
            For prevention of Vomiting
Mathulai Manappagu   - 10 ml Daily Two times a day
           Post natal care
               Sowbakya chuntee – 10 gms daily from the day 2 of delivery.
              Thaneer vittan kilangu nei /lehyam for promoting lactation.
              Thripala chooranam for douching in excess lochia discharge.
      Anaemia Eradication
              Advocacy of Ayabringarajakarpam - 200 mg –b.i.d for 90 days.
            Advocacy of Anna bedhi Chenduram – 200 mg –Cap for 30 days.
            Advocacy of Muthuchippi parpam -200 mg for calcium supplementation
           Advocacy of  Nellikai lehyam for Vit C – benefit and enhancement of Iron absorption.

Compatible nutrition package
        1.Pancha mutti Kanchi
       2.Pea nut / Ellu mittai
       3.Ulunthu kali / Ulunthu kanchi
       4.Athipalam
       5.Pasumpal .

Saya chooranam -Immuno modulator

Santhammal et al, JAPHR 2011, Vol 1 Issue 3 Research Article
8
Content list available at: www.iaphp.in
Journal of Advances in Pharmacy and Healthcare Research
Journal home page: http://www.japhr.iaphp.in
ISSN 2231-6817
Evaluation of Immunomodulatory Activity of Saya Churnam (A Poly Herbal Formulation) on Albino rats.
Y. Santhammal1, S. Arthika1, N. Sharly Elgal1, R. Ganesan2, P. Satyarajeswaran2, S.N. Gaidhani2, G.Pramod Reddy2* and T. Anadan2
1Department of Bio-technology, Bishop Heber College, Tirucharappalli, T.N. India.
2Department of Pharmacology, Siddha Central Research Institute, Chennai, T.N., India.
Key words:
Saya churnam, Immunomodulation, Antibody titer,
Delayed type hyper sensitivity, Ig E.
Corresponding Author:
Dr. G. Pramod Reddy, Department of Pharmacology, Siddha Central Research Institute, Anna hospital Campus, Arumbakkam, Chennai, T.N., India.
gpramod01@gmail.com
Abstract:
The immunomodulatory activity of Saya churnam (a poly herbal formulation) on Albino rats was evaluated by using Cyclophosphamide as an immunosuppressant. The Poly herbal formulation was administered orally at the dosage levels of 200mg/kg/day and 400mg/kg/day body weight of rat. The assessment of immunomodulatory activity on specific and non- specific immunity were studied by Hemagglutination antibody (HA) titer, delayed type hypersensitivity (DTH), Identification of Ig E, hematological , biochemical analysis. Induction of immune suppression in rats was achieved by using Cyclophosphamide (CP) (100mg/kg/day, p.o). Oral administration of Saya Churnam (SC) showed a significant increase in the production of circulating antibody titer in response to sheep red blood cells (SRBCs). A significant (p< 0.001) increase in the both primary and secondary HA titer was observed when compared to control group, where as in Cyclophosphamide treated group Saya Churnam showed significant (p<0.01) increase in HA titer. Saya churnam showed significant (p<0.01) Delayed type hypersensitivity (DTH) reaction by facilitating the foot pad thickness response to SRBCs in sensitized rats. Also biochemical and hematological analysis showed a significant (p<0.001) increase in Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and WBCs. The study demonstrates that Saya churnam triggers both specific and non-specific responses to a greater extent. The study showed significantly increased results in Heme agglutination (HA) titer, DTH response, Ig E test, biochemical, hematological analysis.
INTRODUCTION
Indian traditional systems of medicines like Siddha and Ayurveda which generally involve herbal formulations have suggested the body’s natural resistance to disease (Sagrawat and Khan, 2007). Recent studies with plants have revealed many compounds with potent antioxidant, antineoplastic, antiulcer, anti-inflammatory and immunostimulating potential (Wanger, 1990). Immune activation is an
effective as well as protective approach against emerging infectious diseases, and this immune system known to be involved in etiology and pathophysiological mechanisms of several diseases. It is believed that the immunomodulatory drugs promote positive health and maintain organic resistance against infections by establishing body’s equilibrium and conditioning the body tissues (Dasgupta et
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al., 1998). The restorative and rejuvenating power of these herbal remedies might be due to their action on the immune system and thereby responsible for the protection of the organism from extraneous substance and maintaining homeostasis.
Plant and plant products are being used as a source of medicine since long. Non – toxic herbal preparations are used to improve the general health by stimulating body’s immunity. In this poly herbal formulation (Terminalia chebula, Piper longum, Piper cubeb, Alpinia galangal, and Myristica officinalis) each herb have a capacity to induce immune response and each herb have anti-inflammatory, anti-ulcer, and other activities.
The Siddha system of medicines not only provides hat alternative, but also scores over the side effects and cost factors of allopathic medicine. Herbal preparation can be more effective and safer (Vinothapooshan et al., 2011) than conventional medicines. Non-toxic herbal drug preparations that are used to improve general health by stimulating body’s immunity (Kumar et al., 1999).
Herbal medicine has become an integral part of standard health care (Agarwal et al., 2010). This formulation of Saya churnam was obtained from the ancient Siddha text (Agasthiar, 1995). In Siddha normally this Saya churnam is prescribed for Tuberculosis, Jaundice, Anemia, Diabetes mellitus, Arthritis. On the basis of literature there are no reports on Saya Churnam for immunomodulatory system.
The present study was undertaken to investigate and to validate the Immunomodulatory activity of Saya churnam.
MATERIALS AND METHODS
Site of the experiment: The experiment was conducted at the Department of Pharmacology, Siddha Central Research Institute, Chennai, in February, 2011.
Plant material: Fruits and rhizome of the plants were procured from local market and it was authentified by Sasikala Ethirajulu, Assistant Director, Department of Pharmacognosy, Siddha Central Research Institute, Chennai.
Preparation of poly herbal formulation: Fruits of Terminalia chebula, Piper longum, Piper cubeb, Myristica officinalis and Rhizome of Alpinia galangal was collected. It was cleaned without any dust particles or other unwanted materials. Each herb is finely powdered and it was mixed in different ratio. The collected fruits and rhizome are fried little and powdered as mentioned in the ancient text. Then the poly herbal powder was used for the further studies. Now this formulation is used for the evaluation study.
Drug and chemicals: All the drugs and chemicals are analytical grade while the other drugs procured were Cyclophosphamide (Biochem pharmaceutical, Mumbai), ELISA commercial kit from Biotran Diagnostics, U.S.A.
Animals: Healthy Wistar albino rats both male and female (180-230 gm) were used for the study. All the animals were housed which were under standard conditions of temperature (25 ± 2? C), 12h light/ dark cycles and fed with standard pellet food and water. The animals were divided into four groups consisting of six animals each. A group of
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six un- treated rats were taken as control (group I). Group II animals received standard drug Cyclophosphamide on day 9th and 16th orally in a dosage range of 100mg/kg/day. The Saya churnam was fed orally for 21 days at a dosage of 200 mg/kg/day (group III), 400) mg/kg/day, (group IV) mg/kg/day for assessment of immunomodulatory activity.
Test compound formulations: The different ratio of all the fruit and rhizome powder was measured and prepared in honey with water in the ratio of 7:3 prior to oral administration of animals. Freshly prepared drug solution was used. The vehicle alone served as control.
EXPERIMENTAL PROCEDURE
Antigenic material: Preparation of sheep RBCs (SRBCs): Sheep blood was collected in sterile Alsever’s solution in 2:1 proportion of Alsever’s solution (Freshly prepared). Blood was kept in the refrigerator and processed, for the preparation of SRBCs batch, by centrifugating at 2000rpm for ten minutes and washing with physiological saline 4-5 times and then suspending into buffered saline for further use (Kirtikar et al., 1961).
Heamagglutinating Antibody (HA) Titer: The rats of all groups are pre-treated with drug for 21 days. And each rat immunized with 0.1X109 SRBC/rat by i.p. route, including control rats. The immunization (Agarwal et al., 1999) was given on day 7th and 14th. The animals were treated with Saya churnam for 21 days. The titer was determined by titrating serum dilutions with SRBCs. The micro titer plates were incubated at room temperature for one hour and examined visually for agglutination has been expressed as HA titer.
Delayed Type Hypersensitivity (DTH) Response: Each group of animals (group I-IV) was immunized with SRBCs on day 7th and 14th. On day 21st Rats are administered 0.1ml of 1% SRBCs in the left hind foot pad by subcutaneous (Fulzele et al., 2003) injection, in the right hind foot pad administered 0.1 ml of 0.9% of normal saline was injected and the increased level of paw volume was measured by plethysmometer (Gayathri et al., 2005) in three different time (0 hour, 1 hour, 3 hour) intervals. The thickness between left and right hind paw volume was measured.
Identification of Ig E: On 21st day serum was obtained from all the group of animals. 25µl of serum was added to all the wells, add 100µl of Ig E biotin reagent then allowed to incubation of an hour. Discard the contents and add 300 µl of wash buffer and discarded that it was carried out four times. Then added 100 µl of Ig E enzyme reagent allowed it for incubation of half an hour. Discard the contents and added 300 µl of wash buffer and discarded it was carried out 4 times after that.1 ml of working substrate solution was added allowed for 15 minutes incubation then added 0.05ml of stop solution read the absorbance at 450 nm.
Liver function and blood parameters test: Activities of Serum Glutamate Oxalate Transaminase (Sharififa et al., 2009) (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and Hematological parameters (RBC, WBC, Hb, PLT) were estimated by using kits (Span Diagnostics, India). For this purpose four groups of animals (one control+three
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Table 1: Effect of Saya Churnam on primary and secondary antibody titer.
Treatment
Primary antibody titer
Secondary antibody titer
Control
6.88±0.83
7.66 ± 0.98
CP 100mg/kg
5.23±0.63**
4.86 ± 1.04**
SCLD 200mg/kg
5.72 ± 1.04***
6.12 ± 0.89***
SCHD 400mg/kg
7.76 ± 1.32***
8.56 ± 0.98***
The values are expressed as (Mean ± S.D), n=6,**p<0.01 and *** p<0.001
Treatments) as described above were used and treated with drug for 21 days.
Statistical analysis: The statistical analysis was performed by using student t Test followed by one way analysis (ANOVA).
RESULTS
Heamagglutination antibody (HA) Titer: Effect of Saya churnam low dose (SCLD) and Saya churnam high dose (SCHD) on primary and secondary antibody response on HA titer is shown in (Table 1). Primary antibody response on day 14th in saya churnam (200mg, 400mg/kg/p.o) treated group with normal immune status showed significant increase (p<0.01) in HA titer when compared with the control group. A significant decrease in the antibody titer was observed in the Cyclophosphamide–treated group when compared with the control group. In immunosuppressed groups, where the immunity was suppressed by administration of Cyclophosphamide on day nine, Saya churnam (400mg/kg/p.o) administration produced a significant (p<0.01) rise in the antibody titer when compared with the Cyclophosphamide – treated group. Secondary antibody titer on twenty first day in Saya churnam both low dose and high dose treated groups with normal immune status group showed a significant rise (p<0.01) in the antibody titer when compared with the control group. In the immunosuppressed groups where the immunity was suppressed by administration of Cyclophosphamide on day sixteenth Saya churnam both low dose and high dose showed a significant rise (p<0.01) in HA titer when compared with the Cyclophosphamide group.
Delayed type hypersensitivity: Effect of Saya churnam on cell mediated immune response by DTH induced foot pad oedema is shown in (Table 2). In the all groups of rats with normal immune status, of Saya churnam low dose (200mg/kg/p.o) and Saya churnam high dose(400mg/kg/p.o) showed significant (**p<0.01,*P<0.05) potentiated DTH response in terms of increase in the mean difference of paw thickness when compared with control group. The drug treated group of rats showed significant (p<0.01) potentiated DTH response in terms of increase in the mean difference of paw thickness. Heightened delayed type hypersensitivity reaction suggests activation of cellular immune system.
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Table 2: Effect of Saya churnam treatment on cell mediated immune response by delayed type hypersensitivity induced footpad oedema.
Treatment
SRBC’s (in left hind paw)
Saline (in right hind paw)
0 hrs
1 hrs
3 hrs
0 hrs
1 hrs
3 hrs
Control
0.81±0.14
1.14±0.32
2.04±0.33
0.81± 0.15
0.91±0.19
0.96±0.36
SCLD200mg/kg
0.78±0.20**
1.30±0.42**
2.35±0.45**
0.88±0.16*
0.87±0.15*
0.86±0.23**
SCHD400mg/kg
0.98±0.12**
1.36±0.32**
3.64±0.36**
1.00±0.21*
0.65±0.15*
1.05±0.40**
The values are expressed as (Mean ± S.D), n=6.*P<0.05 and **p<0.01.
Identification of Ig E: The administration of Saya churnam both low dose and high dose (200mg/kg/day and 400mg/kg/day) showed significantly increased (*p<0.05) levels of immunoglobulin level when compared to control in the animal serum sample
Table 3: Effect of Saya churnam on Ig E
Treatment
Ig E
Control
16±4.5
SCLD 200 mg/kg
13.6±2.8*
SCHD 400mg/kg
14.3±4.9*
The values are expressed as (Mean ± S.D), n=6.*p < 0.05.
Liver function and test: There was a significant elevation in SGOT, SGPT, ALP as a result of treatment with Saya churnam on both low dose and high dose in this study (***p<0.001,*p<0.01) (Table 4). The drug treated group compared with control group which showed significantly increased values.
Hematological parameter test: In hematological analysis observed significant differences in hematological parameters and drug treated groups are compared with control and Cyclophosphamide treated group. Result shows increased level of WBC, RBC, HGB, PLT on low dose and high dose treated group when compared with control group.
DISCUSSION
Immunostimulatory agents of plant and animal origin enhance the (Fulzele et al., 2003) immune responsiveness of an organism against a pathogen by activating the immune system. However these
poly herbal formulations should be subjected to systematic studies to substantiate the therapeutic
claims made with regard to their clinical utility (Joshi et al., 2003). Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions; if it results in an enhancement of immune reaction it is named as immunostimulative drug which primarily implies stimulation of specific and non- specific system. Immunomodulation through natural substances may be considered an alternative for the prevention (Zhang et al., 2011) and cure of disease. Immuno-suppression implies mainly reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factor (Makare et al., 2001). The results obtained in the present study indicate that Saya churnam is a potent immunostimulant, stimulating specific and non-specific immune mechanisms.To evaluate the effect of Saya churnam on humoral response, its influence was tested on sheep erythrocyte specific
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HA titer in rats (Benaceraf et al., 1978). Cyclophosphamide showed significant inhibition in
Table 4: Effect of Saya churnam on liver enzymes.
Treatment
SGOT U/L
SGPT U/L
ALP U/L
Control
56.42 ± 11.9
19.18 ± 3.72
88.8 ± 16.1
CP 100mg/kg
111.3 ± 18.42***
31.58 ± 9.11**
120.02 ±14.1***
SCLD 200mg/kg
59.73 ± 11.12***
36.68 ± 10.7 **
150.55 ± 16.88 ***
SCHD 400mg/kg
62.85 ± 10.98 ***
47.58 ± 7.06**
270.9 ± 73.65 ***
The values are expressed as (Mean ± S.D), n=6.**p<0.01 and ***p<0.001.
antibody titer response, while saya churnam counteract the suppression of both primary and secondary humoral (Kulkarni et al., 2007)
responses induced by Cyclophosphamide. It indicates that the Saya churnam has the significant effect on humoral antibody response. In DTH test, the DTH response, which directly correlates with cell mediated immunity (CMI), was found to be the highest at maximum dose of 400mg/kg/day tested in this study. Increase in the DTH response indicates that Saya churnam has stimulatory effect on lymphocytes and accessory cell types (Mitra et al., 1999) required for the expression of the reaction Cell mediated immunity the mechanism behind this elevated DTH during the CMI responses could be due to sensitized T- lymphocytes. When challenged by antigen they are converted to lymphoblast and secrete variety of molecules including pro-inflammatory lymphokines, attracting more scavenger cells to the site of reaction (Fulzele et al., 2003).
Increase in the DTH response indicates that the drug has a stimulatory effect on lymphocytes and accessory cell types required for the expression of the reaction (Mitra et al., 1999). Foot volume was enhanced after Saya churnam treatment suggesting cell (Sen et al., 1992) mediated immune enhancement. Immunoglobulin level is a direct measure to detect the humoral immunity. Serum immunoglobulin refers to a group of serum molecules produced by B- lymphocytes, they are soluble and secreted from of B-cell receptors and are produced to a maximum level to counter the invasion by an antigen, and hence they are also called as (Ismail and Asad, 2009) antibodies.
Identification of Ig E also showed the presence Ig E in the tested animals. The results shown significantly increased level of immunoglobulin in Saya churnam treated group when compared with control and Cyclophosphamide treated group. Estimation of LFT enzymes did not show any toxicity effect which was concomitant with any
Table 5: Effect of Saya churnam on Hematological parameters
Groups
WBC
RBC
HGB
PLT
Control
6.7± 3.5
5.32±1.11
9.0 1 ±1.93
406.9±108.3
CP 100mg/kg
3.81± 4.01*
6.96±2.81*
12.42±5.4 *
169.5±200.1**
SCLD 200mg/kg
5.52±2.34 ***
6.61±2.52***
11.32±4.46***
267.2±105.4***
SCHD 400mg/kg
7.63±2.07 ***
6.69±2.33***
11.56±4.43***
342.1±155.7***
The values are expressed as (Mean ± S.D),n=6.*P<0.05, **p<0.01and ***p<0.001.
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significant increase in relative weight of liver. High dosage 400mg/kg/day showed increased level of SGPT, SGOT, and ALP. And the results of the presence study revealed that significant difference in the blood parameters. Findings of the present study showed an overall stimulatory effect of Saya churnam on humoral as well as cell-mediated immunity in rats.
CONCLUSION
On the basis of the results obtained in the current study, it can be concluded that the saya churnam has the potential to stimulate cell-mediated and humoral immunity and it may be a potential therapeutic candidate in several immunosuppressed clinical conditions. However, more exhaustive work needs to be performed to substantiate the claim.
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