Abstract - Online International Interdisciplinary Research Journal

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Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN 2249-9598, Vol-IV, Jan 2014 Special Issue
Chromatographic Finger Print Analysis, Phyto-Chemical and
Microbicidal Screening, of an Important Medicinal Plant of Vindhya
Region – Santalum Album Linn
Mishra Deepaka, Mishra Pratimab, Awasthi Arpitac
a
Department of Biotechnology, A.K.S. University Satna (M.P.), India
b
Department of Biotechnology, Pentium Point College Rewa (M.P.), India
c
Department of Botany and Microbiology, T.R.S. College, Rewa (M.P.), India
Corresponding author:
Deepak Mishra
Department of Biotechnology, A.K.S. University Satna (M.P.), India
Abstract
In present study Ayurvedic formulated drug Santalum album Linn. was
subjected to phyto-chemical analysis, microbial analysis and chemo profiling to fix the
quality standards of this drug. It is one of the important herbal plant mentioned in
Ayurveda. It is cultivated for its aromatic wood and oil. It is widely distributed in the
Indo-Malesian region and Peninsular India. Sandalwood is used for the treatment of acute
dermatitis, bronchitis, cystitis, eye diseases, gonorrhea, herpes, zoster, infection,
palpitations, sunstroke, urethritis, vaginitis, psychopathic, Skin disorders, Heart ailments,
Anti-pyretic, General weakness, Urinary tract infection and many more. During phytochemical analysis, microbial limit test and chemo profiling churna was prepared as
siddhyog sangrah and the preparation are standardized according to guidelines of
Ayurvedic Pharmacopoiea, Various chromatographic techniques have been carried out
for chemo profiling of drug. These studies on Santalum album (wood) were
reproducible, precise and may be considered as a method for its quality control.
KEYWORDS: standardization, Ayurvedic formulation.
Introduction:
Herbal medicines are basically used as a part of ayurvedic system of medicines.
These are traditional remedies based either on whole plant or plant extracts. In the last
few decades there has been an exponential growth in the field of herbal medicine. It is
getting popularized in developing and developed countries owing to its natural origin and
lesser side effects2. In olden times, vaidyas used to treat patients on individual basis, and
prepare drug according to the requirement of the patient. But the scene has changed now;
herbal medicines are being manufactured on a large scale in mechanical units, where
manufacturers own across many problems such as availability of good quality raw
material, authentication of raw material, availability of standards, proper standardization
methodology of single drugs and formulations, quality control parameters, etc3.
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Page 237
Santalum album Linn. is one of the important herbal plant used in ayurveda for
the treatment of various diseases. It is member of family Santalaceae and commonly
known as sweta chandan. It is widely distributed in throughout the India especially in
Indo-Malesian region and in the dry regions of peninsular India3. Though it is naturalized
in many parts of India i.e. in Vindhya Mountains southwards, also in Karnataka, Andhra
Pradesh and Tamilnadu; it is cultivated for its aromatic wood and oil4. Sweta chandan is a
small to medium sized, evergreen semi-parasitic tree, with slender branches, sometimes
reaching up to 18 m in height and 2.4 m in girth. Barth reddish or dark grey or nearly
black, rough with deep cracks on old trees; leaves glabrous, thin, elliptic-ovate or ovatelanceolate, 1.5-8 cm * 1.6-3.2 cm, sometimes larger; flowers straw-coloured, brownish
purple, reddish purpleor violet5. Sandal is capable of growing in different kinds of soil
like sand, clay, laterite, loam, black-cotton etc6, 7. It is capable of regenerating profusely
in the absence of fire and grazing8. Sandalwood is used for acute dermatitis, bronchitis,
cystitis, eye diseases, gonorrhea, herpes, zoster, infection, palpitations, sunstroke,
urethritis, vaginitis, psychopathic, Skin disorders, Heart ailments, Anti-pyretic, General
weakness, Urinary tract infection and many more9, 10.
Material & Methodology:
Selection of plant material for study:
In present work Santalum album Linn. (Swetachandan) have been selected for the
study. It has been collected from Chitahara forest (Manjhagawa) of Satna district. The
plant has identified on the basis of different pharmacopeial and botanical standard.
Mostly extract of wood has been used in the study11.
Phyto-chemical Profiling:
Tests for Alkaloid:
1ml of alcoholic extract of the drugs with 1.5% w/v HCL few drops add a few drops
of wagner reagent. A yellow or brown ppt is not formed.
Test for carbohydrate:
In a test tube containing 2ml of aqueous extract of the drug add 2drop of freshly
prepared 20% alcoholic solution of α-napthol the mix pour 2ml cons. Sulphuric acid
so as to form a layer blew the mixture, carbohydrate, it present, produce a red-violet,
which disappears on the addition of an excess of alkali solution.
Tests for Flavonoids:
In a test tube containing 0.5 ml of alcoholic extract of the drug. 5-10 drop of dil.HCL
was added followed by smell of flavonoids a pink, raddish brown or brown colour is
produced.
Tests for Protein:
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To 1ml of hot aqueous extract of the drug add 5-8 drops of 10% w/v sodium
hydroxide solution followed by 1 to 2 drops of 3% w/v copper sulphate -A red or
violet colour is obtained.
Tests for Resins:
Dissolve the aqueous /alcoholic extract in acetone and pour the solution into distilled
water turbidity indicates the presence of resins.
Tests for Saponins:
In a test tube containing about 5ml of an aqueous extract of the drug add a drop of
solution bicarbonate solution. Shake the mixture vigorously and leave for 3minutes.
Formation of honey comb like froth indicates the presence of saponins.
Tests for Tannins:
To 1-2 ml extract of the drug add few drops of 5% neutral FeCl3 solution A green
colour.
Microbial Limit Test of Drug:
The following tests were carried out to determine the microbial load in drug
powder of Pharmaceutical substances.
Total Aerobic Microbial Count:
1ml of the pretreated preparation (inoculums) was taken and about 15ml of
liquefied, lukewarm casein soyabean digest agar was poured in petridish in triplicate and
incubated at 35+_2˚c for 48 hours, obtained in a shorter time. The number of colonies
observed was calculated with the help of digital colony counter.
Tests for Yeast &Mould:
1ml of the pretreated preparation (inoculums) was taken and about 15ml of liquefied,
lukewarm potato dextrose agar was poured in petridish in triplicate. After gentle rotation
and solidification of agar the plates were incubated at 22˚c to 25˚c for 5days, unless a
more reliable count was obtained in shorter times. Then results using plates with not
more than 100 colonies were calculated.
Tests for Specified Microorganism:
Test for E. coli:
1gm of sample taken in 100 ml of sterilized nutrient broth in a sterile screwcapped container, shaken & allowed to stand for 1hour and shaken again. Loosen the cap
& incubated at 37˚ For 18-24hrs.
Primary Test:
1ml enriched culture was inoculated into tubes containing 5ml of macconkey
broth, incubated in a water bath at 37˚c for 48 hrs. Observations were noted.
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Secondary Test:
By means of a inoculating loop, a small portion from the enrichment culture was
streaked on the surface of macconkey medium. Covered & inverted the plates and
incubated at 35+_2.for 24hrs. upon examination, if none of the colonies are brick-red in
colour and have a surrounding zone of precipitated bile the sample meets the
requirements of the test for the absence of the E.coli .
Test for Salmonella:
1g of sample taken 100ml of sterilized nutrient broth in a sterile screw-capped
container, shaken & allowed to stand for 1hour and shaken again, loosen the cap &
incubated at 37˚c for 24hrs.
Primary Test:
1ml enriched culture was inoculated to each of the two tubes containing (a) 10ml of
selenite f broth abd (b) tetrathionate-bile-brillient green broth and incubated at 37˚c for
48hrs. from each of these two cultures a small portion of inoculums was subcultured on
brilliant green agar and xylose lysine-deoxycholate agar. Covered and inverted the plates
and incubated at 37˚For 18-24 hrs. upon examination, if none of the colonies showed
characteristic growth the sample meet the requirements of the test for the absence of the
genus Salmonella.
Colony Characteristic of Salmonella:
Brilliant green agar
Xylose-lysine-deoxycholate agar
Colourless & opaque pinkish/white surrounded
By a pink or red zone.
Red with or without black Centers
Test for Pseudomonas aeruginosa:
1g of sample taken in 100 ml of sterilized soyabean-casein digest broth in a sterile
screw-capped container, mixed & incubated at 37˚For 24-48hrs . a small portion of
culture was streak on the surface of cetramide agar medium each plated on petridish.
Covered and inverted the plates and incubated at 37˚ For 18-24 hours. Upon examination,
if none of the plate contains colonies having greenish fluorescence growth in uv light, the
sample meets the requirements for the freedom from Pseudomonas aeruginosa.
Test for Staphylococcus aureus:
1gm sample taken in 100ml of sterilized soyabean-casein digest broth in a sterile
screw-capped container, mixed & incubated at 37˚ for 24-48 hours. A small portion of
culture was streak on the surface of Vogal-johnson agar each plated on petridishes in
triplicate covered and inverted the plates and incubated at 37˚ for 18-24 hours. Upon
examination, if none of the plate contain no growth having black colonies surrounded by
yellow zones, the sample meets the requirements for the absence of Staphylococcus
aureus.
Chemo profiling of Drug:
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HPTLC-extract the crude samples in alcohol (150 ml×5), concentrate to 5-10 ml
on water bath and carry out High performance thin layer chromatography by applying 4µl
of the sample on pre-treated TLC silica gel plate 60 F254 (from Merck India Ltd,
Germany) and develop the plate to a distance of 10 cm using Toluene: Ethyl acetate:
Formic acid (7:2.5:0.5) as mobile phase. After derivatization allow the plates to dry in
room temperature & examine under ultra violet light at 254 nm; under366 nm; after
derivatization with methanol-sulphuric acid reagent. Measure and record the distance of
each spot from the point of its application and calculate the fro value by dividing the
distance traveled by the spot by the distance traveled by the front of the mobile phase.
Results & Discussion:
In Ayurvedic system of medicine standardization of drugs and formulation is
very important task for assuring uniform quality and optimum therapeutic efficiency13.
Standardization of Santalum album Linn. becomes very helpful in establishing standards
for quality control and drug development. The phyto-chemical parameters identified
represent the specific diagnostic characters of drug. This study facilitates in identifying
the adulterant or substitutes of the ingredients used in the formulation. Chemo profiling
by HPTLC has helped in developing identification tests and finger printing of
constituents of the ingredients used in the formulations.
The study revealed differences in the quality of market products of all the these
formulation. The reason for this could be assigned to variation in the active constituents
in the plant parts used, difference in the method of manufacture adopted and use of
substitute and or adulterants
Microbiological quality assessment is an important parameter that should be
routinely monitored, as gross contamination could jeoparadize the health rather than
curing the condition for which it is used. In case of Santalum album Linn. although the
standard batch compiled with the prescribed standards to respect of microbial and fungal
counts. It is advisable to adopted aseptic technique to prepare this formulation to ensure
total freedom from microbial contamination.
Biological activity studies of formulations reconfirmed the findings in the
Ayurvedic literature about their medical application in ailments for which they are
conventionally recommended.
The acceptability and utility of herbal formulations is many a time eclipsed for
lack of acceptable norms of quality control parameters. These quality parameters /
analytical values developed in the present study can be utilized by in-house quality
control laboratory of an Ayurvedic pharmaceutical unit to evaluate the genuineness and
authenticity of plant material available under the name of Santalum album Linn.
In association of these studies also chemo profiling studies has been conducted. In
which we study about comparative profile of wild drug (A) in respect to market sample
(B):
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A
B
A
A
B
B
Fig. 01:- Chromatographic Figure Print of Sweta Chandana
Table 1: Rf value in Test Solution of Santalum album Linn.
Rf - value
Before derivatization
Rf at 254 nm
Sample A
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Sample B
Rf at 366 nm
Sample A
Sample B
Rf -1
0.43(Black) 0.43(Black)
0.05(blue)
0.05(blue)
Rf -2
0.11(Green)
0.11(Green)
Rf -3
0.15(Green)
0.15(Green)
Rf -4
0.56(Gray)
0.56(Gray)
0.20(Green
0.20(Green
Rf -5
0.63(Gray)
0.63(Gray)
0.24(blue)
0.24(blue)
Rf -6
-
-
0.30(sky
blue)
0.30(sky
blue)
Rf -7
-
-
0.44(blue)
0.44(blue)
Rf -8
-
-
0.48(blue)
0.48(blue)
Rf -9
-
-
0.52(green)
0.52(green)
Rf -10
-
-
0.60(Green)
0.60(Green)
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Rf - value
After derivatization
Rf at Visible light
Sample A
Sample B
Rf -1
0.43(Black)
0.43(Black)
Rf -2
0.48(Black)
0.48(Black)
Rf -3
0.52(Black)
0.52(Black)
Rf -4
0.56(Grey)
0.56(Grey)
Rf -5
0.63(Grey)
0.63(Gray)
Table 2: Photo-chemical Contents (Sweta Chandana)
S.No.
Constituents
Observation
Result
Absent.
Present.
6.1
Alkaloid
6.2
Flavanoid.
6.3
Resin.
A yellow brown ppt is not
formed.
Pink, raddish pink or brown color
is produced
Turbidity present.
6.4
Saponin.
Honey comb like froth is formed.
Present.
6,5
Tannin.
Brown color is obtained.
Present.
6.6
Carbohydrate.
Absent.
6.7
Protein.
Red or brick red ppt is not
formed.
Red or violet color is ontained.
Present.
Present.
Table3: Results of Microbial Limit Test (Sweta Chandana)
S.No.
7.1
7.2
7.3.1
7.3.2
7.3.3
7.3.4
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Microbial test
Total Bacterial Count (TPC)
Yeast & Mould.
E.Coli.
Salmonella sp.
Staphylococcus aureus.
Pseudomonas aeruginosa.
ISSN 2249-9598
Appearance
18 cfu/gm
28 cfu/gm
Absent
Absent
Absent
Absent
API Limits
103 cfu/gm
105 cfu/gm
Absent
Absent
Absent
Absent
Page 243
Plate 1: Plate showing negative result for
Staphyllococcus aureus.
Plate 3:- Plate showing negative results
for Salmonella
Plate 5:- Agar plate showing Yeast
& Mould Counts
Plate 2: Plate showing negative results for
Pseudomonas aeruginosa.
Plate 4: Plate showing negative results
for E. coli.
Plate 6:- Agar plate showing
Aerobic Microbial Counts
Fig.:- Photographs of Microbiological Limit Test of Sweta Chanadana
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Conclusion:
there are not any type of evidence observed in ancient literature of Indian
medicine regarding quality standard of drug however the acceptability and utility of drug
is basically depends upon its quality control parameters14. In the above investigation we
have trying to develop and standardize these parameters and this study can be utilized by
in-house quality control laboratory of an Ayurvedic pharmaceutical unit to evaluate the
genuineness and authenticity of plant material available.
Finally it has been concluded that Herbal drug standardization is an important task
and massively wide15. Quality control of herbal medicines has not only to establish
reasonable analytical methods for analyzing the active constituents in herbal medicines,
but many other factors should be concerned, such as pesticides residue16, aflatoxine
content17, the heavy metals contamination, good agricultural practice (GAP), good
manufacturing practice (GMP), etc18. There is so much to know and so much seemingly
contradictory theories on the subject of herbal medicines among the existing analytical
methods, chromatographic methods are still the mainstream, due to their integrative
evaluation characteristics19.
Bibliography:
1. Caius J F., 1990 The Medicinal and Poisonous Plants of India Indian Medicinal
Plants;2:489.
2. Devi M Vimala, 2000 Quality Control And Assurance Of Indian Medicines
Health Administrator Vol : XX Number 1&2 : 21-25 Pg.
3. Baldovini N., Delasalle C., and Joulain D., 2011 Phytochemistry of the
heartwood from fragrant Santalum species: a review Flavour and Fragrance
Journal; 26:7-26.
4. Samantray Sanghamitra, Upadhyaya Chandni, 2010 Methodological studies
and research on micropropagation of Chandan (Santalum album L.): An
endangered plant International Journal on Science and Technology (IJSAT)
Volume 1, Issue I, 010-018
5. Banerjee S., Ecavade A., and Rao A.R., 1993 Modulatory influence of
sandalwood oil on mouse hepatic glutathione S-transferase activity and acid
soluble sulfhydryl level Cancer Letters; 68: 105-109.
6. Jain S.H., Angadi V.G. and Shankaranarayana K.H., 2003 Edaphic,
Environmental and Genetic Factors associated with Growth and adaptability of
Sandal (Santalum album L) In provenances. Sandalwood Research Newsletter
:17, 6-7
7. Anjum Perveen And Mohammad Qaiser, 2005 Pollen Flora of Pakistan–XLIII
Lythraceae Pak. J. Bot., 37(1): 1-6
8. Fox, J. E., 2000 Sandalwood: the royal tree Biologist (London); 47:31-34.
9. Balachandran P, Govindrajan R., 2005 Cancer- An ayurvedic perspective
Pharmacol Res; 51:19-30.
10. Srinivasan, V. V., Sivaramakrishnan, V. R., Rangaswamy, C. R., Anantha
padmanabha, H. S. and Shankaranarayana, K. H., 1992 Sandal (Santalum
album L.), ICFRE, Dehra Dun,
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11. M. Rajani and Niranjan S. Kanaki, 2008 Phytochemical Standardization of
Herbal Drugs and Polyherbal Formulation Bioactive Molecules and Medicinal
Plants, 10., 349-369
12. Blois M S., 1958 Antioxidant determinations by the use of a stable free radical
Nature; 181:1199-1200.
13. Brahmachari U. N., 2001 The role of science in the recent progress of medicine
Current Science, VOL. 81, NO. 1,
14. Jigna Parekh, Sumitra V. Chanda, 2008 Antibacterial Activity of Aqueous and
Alcoholic Extracts of 34 Indian Medicinal Plants against Some Staphylococcus
Species Turk J Biol 32 63-71
15. Singh Himmat, Mishra Swatantra Kumar and Pande Milind, 2010
Standardization Of Arjunarishta Formulation By TLC Method International
Journal of Pharmaceutical Sciences Review and Research; 2,(1), 25-28
16. Battaglia S., 2003 The Complete Guide to Aromatherapy The International
Centre of Holistic Aromatherapy, Brisbane, Australia.
17. Tambekar D. H. and Dahikar S. B., 2010 Exploring antibacterial potential of
some ayurvedic preparations to control bacterial enteric infections Journal of
Chemical and Pharmaceutical Researc ; 2(5): 494-501
18. Singh Rana Pratap and Singh D.A., 2011 Antibacterial activity of Alcoholic
and Aqueous extracts of some Medicinal Plants International Journal of
PharmTech Research; 3(2), 1103-1106
19. Patra Kartik Chandra, Pareta Surendra K., Harwansh Ranjit K., Kumar K.
Jayaram, 2010 Traditional Approaches towards Standardization of Herbal
Medicines-A Review Journal of Pharmaceutical Science and Technology Vol. 2
(11), 372-379
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Abstract - Online International Interdisciplinary Research Journal

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