In Vitro and Vivo study in Herbal Drug Research

Motivation for Life Academy by Dr Khalid B.M 1
In Vitro And Vivo
Evaluation Techniques Pertaining To
Ayurvedic Research
13-07-2023

2000 BC – Here, eat this root
1000 AD – That root is heathen, say this prayer
1850 AD – That prayer is superstition, here drink this potion
1940 AD – That potion is snake oil, here, swallow this pill
1985 AD – That pill is ineffective, here, take this antibiotics
2000 AD – That antibiotics is dangerous, here, eat this root
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PHYTOCHEMISTRY – AN OVERVIEW
Biological activity
1. Selection of promising plant material Folk medicine
2. Proper collection of selected plants.
3. Authentication of plant
4. Drying of plant
5. Grinding of dried plants
6. Packing, storage & preservation
7. Extraction
8. Purification
9. Identification of bioactive compounds.
Wild plant
Cultivated
Establishing identity by
taxonomy experts
Field botanist
Maceration
Percolation
Digestion
Hot continuous extraction
Super critical fluid
Extraction
Microwave
Solid phase extraction
Chromatography
Simple qualitative analysis
Advanced qualitative /
quantitative analysis
Spectroscopy UV-VIS,
NMR, MS
Chromatography
X ray crystallography Motivation for Life Academy by Dr Khalid B.M 3
13-07-2023

CHEMICAL CONSTITUENTS
ORGANIC INORGANIC
Carbohydrates, Proteins, Amino Acids, Fats Calcium, Magnesium, Sodium, Potassium,
Oils,, Volatile Oil, Glycosides, Steroids Iron, Sulphate, Phosphate, Chloride Etc.
Alkaloids, Phenols Compounds, Flavonoids ,
Tannins, Oxygenic Acids, Enzymes
• Phenols = Amalaki, Fresh grapes, Fruits.
Flavonoids = Quercetin, Rutin.
Alkaloids= Chitraka, Berbarine, Piparine, Atropine.
Steriods = Ashwagandha.
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Plant Extraction
Organic Solvents Water
Percolation Maceration Soxhlet Extraction Infusion Decoction Distillation
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Extraction
1. Percolation
2. Cold maceration: Wt 4gm of coarsely powdered drug in weighing bottle and transfer it to dry 250 ml of
conical flask keep it for 24 hours shaking frequently
3. Hot extraction : Attach reflex condenser to the flask and boil gently for one hour , cool and weigh
4. Soxhlet extraction: Is only required where desired compound has limited solubility in a solvent
5. Maceration: Coarsely powder plant drug is kept in contact with the solvent in a container for defined
period
6. Decoction: This method is used for the extraction of the water soluble and heat stable constituents from
crude drug by boiling it in water for 15 minutes, cooling, straining and passing sufficient cold water through
the drug to produce the required volume
7. Infusion: It is a dilute solution of the readily soluble components of the crude drugs. Fresh infusions are
prepared by macerating the solids for a short period of time with either cold or boiling water.
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Challenges
Selection of
solvents
SL.No Solvent Polarity
index
Nature of
polarity
Secondary metabolite can be extracted
1 Hexane 0.1 Low
polarity
Waxes, fats
2 Cyclohexane 0.2 Low
polarity
Waxes, fats
3 Diethylether 2.8 Low
polarity
Aglycones, alkaloids
4 Dichloromet
hane
3.1 Low
polarity
Terpenoids, flavonoids
5 Chloroform 4.1 Low
polarity
Terpenoids, flavonoids, alkaloids, Aglycones,
6 Ethylacetate 4.4 Low
polarity
Aglycones, alkaloids, glycosides
7 Acetone 5.1 Medium
polarity
flavonoids, alkaloids, Aglycones,
8 Ethanol 5.2 Medium
polarity
Tannins, polyphenols, flavanols, terpenoids,
anthocyanins
9 Methanol 6.6 Medium
polarity
Saponin, tannin, sugars terpenoids, starch,
polyphenols.
10 Water 9.0 High
polarity
Sugars, amino acids, saponins, tanins, terpenoids,
anthocyanins, starch, polypeptides.
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Test Test Applied/Reagent used Observation
Carbohydrates Molisch Test Violet ring formation
Saponin Foam test Foam formation
Phenols Ferric chloride test Bluish black colour
Flavonoids Conc.H2SO4 Test Orange Colour
Steroids Salkowakis test Chloroform layer appear red, acid layer
shows greenish yellow fluorescence.
Alkaloids Dragendroff”s test
Mayer's test
Hager's test
Wagner's test
Red or Orange Brown colour
Anthraquinone Ammonia Test Rose Red colour in aqueous layer
Proteins & Amino Acids Xanthoproteic Test Yellow colour formation
Anthocyanine 2N HCl Test Pink reddish colour formation
Quinone Conc. HClTest Green Colour
Glycoside Alkaline Reagent Test Yellow colour
PHYTOCHEMISTRY
13-07-2023

13-07-2023 Motivation for Life Academy by Dr Khalid B.M 9
Avishkara Central Research Laboratory SJGAMC&H Koppal Phytochemical Screening
Sl.No
Sample Carbohydrate
s
Saponin Tannin Flavonoids Steriods Terpenoids Alkaloids
Proteins &
Amino Acids
Quinone
Anthrquainin
e
Anthocyanine Phenols Glycosides
AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE AE EE
1 Shunti + + + + + - + - + + - - - - + + - - - - + + - - + +
2 Pippali + + + + - + + + + - - - + + + - - - - - - - + - + -
3 Amalaki + + + + + - + - + - + - + + + - - - - - - - + + - -
4 Maricha + + - - - - + + + - - - + + + + - - - - - - - + -
5 Shatavari + - + + + + + - + - + - + + + - - - - - - - - - + -
6 SG Leaf + + + + + + + - + - - - + + - - - + - - - - + + + +
7
Lavanga
Stem&root + - + - - - + - - + - + + - - + - + - - - - - + +
8 Laxman Phal - - + + - - + + - + + - + + - - - - + + - - + + - -
9 Mahagony Seed + - + - - + + + - - - + + + - - - - - - - - - - - -
10 Mahagony Pulp + + + - - - + - + + + - + + + - - - - - - - - - - -
11 Neem - - + + + + + + + + + - + + + - - - - - - - + + + +
12 Guduchi + - + + + + + + + + + + + + - + - - - - - - + + + +
13 Kalamegha + + + + + + + + + - + + + + + - + - - - - + + + -
14 Niligiri Leaves + - + + + + + + + - + + + + - - + + + + - - + + + -
15 Nelanalli + - + + + + + + + - + + + + + + - - - - - - + + +
16 Ramaphal - + + + + + + + + + - + + + + + - - - - - - + + + +
17 Guduchi Root + - + + + + - - + + + + + + + + - - - - - - - + -
18 SG Fruit + - + + + + + + + + + + + + + + - - + + + + + +
19 Kamakasturi + - + + + + + + + - + - + + + - - + - - - - + + + -
20 Madanaphala + - + + + - + + + + - + + + - - - - - - - - + + + +

TESTS FOR VITAMINS
VITAMIN A 1 ml of chloroform and add 5 ml of antimony trichloride
solution, transparent blue colour is produced
VITAMIN C DCPIP Solution, solution will become discoloured
VITAMIN D chloroform and add 10 ml of antimony trichloride
solution
Pinkish red colour appears
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Inorganic analysis
Test Reagent Result
Calcium Ammonium carbonate solution White ppt
Magnesium Ammonium carbonate solution White ppt with ammonium
carbonate sol no ppt with
ammonium carbonate sol
Sodium 2ml potassium pyroanthallolate White ppt
Potassium Few drops of sodium cobalt
nitrate
Yellow ppt
Iron Few drops of potassium
ferrocyanide
Dark blue colour
Sulphate Few drops of 5% Bacl2 sol White crystalline BaSO4 ppt
appears, insoluble in HCL
Phosphate Few drops ammonium
molybdate sol
Yellow crystalline ppt
Chloride 3-5 ml lead acetate sol White ppt soluble in hot water
Carbonate Magnesium sulphate White ppt
Nitrate Sulphuric acid and copper Liberates red fume
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CHROMATOGRAPHY
CHROMATOGRAPHY : Chromo – colour
Graphy – measure or to write.
“Chromatography is an analytical technique used for separation of coloured substance from the
plants is now the most extensive technique of separation of coloured colourless organic
compounds”.
Types
1. Thin layer chromatography
2. HPTLC
3. HPLC
4. Gas Chromatography
5. Column Chromatography
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THIN LAYER CHROMATOGRAPHY
Thin layer chromatography is a basic, versatile and effective chromatographic technique. It is
used to separate mainly nonvolatile compounds from the plant materials
TLC is a solid- liquid from of chromatography where the stationary phase is normally a polar
absorbent & the mobile phase is single solvent or combination of solvents
Rf = Distance travelled by solute / Distance travelled by solvent.
13-07-2023

If starting a new unknown separation
start with
50% Ethyl-acetate/Hexane
if it’s not working try Methanol / Dichloromethane (DCM
last try toluene with acetone, Ethyl-acetate, or
DCM.
Feel free to change the ratio Two solvents from low polarity
(n-hexane: chloroform)
Two medium polarity
(dichloromethane: n-butanol)
One highest polarity
(water)
SELECTION OF SOLVENT FOR TLC
13-07-2023

Analytes Stationary phase Mobile phase
Steroids Silica gel G Hexane: ethylactetae (1:1)
Terpenoides Silica gel G Hexane: acetic acid (9:1)
Flavonoids Silica gel G Toluene: acetic acid(9:2)
Alkaloids Silica gel G Ethylactetate: methanol: water
(2.4:7.2:0.3)
Phenols Silica gel G Toluene: acetone: formic acid(4.5:4.5:1)
Tannins Silica gel G Toluene: ethylactete: formic acid
(3.3:0.8:0.2)
Quinones Silica gel G dichloromethane-n-hexane, 8:2
Saponin Silica gel G Chloroform: glacial acetic acid:
methanol: water (6:2:1:1)
EXAMPLES OF ANALYTES EVALUATE BY TLC
13-07-2023

Aragwadha - Petroleum ether: ethylactetae:
formic acid(15:2.5:0.2)
Bibitaki - Chloroform:ethylactetae: formic
acid(5:4:1)
Rakta
chandana
- Toluene: ethyalactetae (9:1)
Sarpagandha Reserpine Methanolic extract, Chloroform:
methanol (97:3, v/v
Kutaja Conessine Methanolic extract, Toluene: ethyl
acetate: diethyl amine (6.5:2.5:1,
v/v/v)
Amalaki Gallic acid Methanolic extract,
toluene: ethyl acetate: formic acid:
methanol (3:3:0.8:0.2, v/v/v
Shatavari roots Alkaloids, Flavonoids,
Tannins, phenolic
chloroform: methanol (3:7, v/v)
Yastimadhu Glycyrrhizin Methanol: water (7: 3, v/v)
Arjuna bark Gallic acid toluene: acetic acid: ethyl acetate
(1:0.1:1, v/v/v)
13-07-2023

Avishkara Central Research Laboraotry SJGAMC&H Koppal - Thin Layer Chromatography
Sl.No Sample TLC Steriods Terpenoids Flavonoids Alkaloids
1Pippali Solvent front 5.5 5.8 5.9 5.8
Sample 3.7,4.8 1.5 4.2 5.2
Rf 0.67, 0.87 0.25 0.71 0.87
2Shunti Solvent front 5.5 5.6 6.2 5.7
Sample 4.9 3 4 5.2
Rf 0.89 0.53 0.64 0.91
3Maricha Solvent front 5.5 5.9 6 5.5
Sample 3.9 1.2 4.2 4.8
Rf 0.701 0.203 0.7 0.872
4Trikatu Solvent front 5.5 5.9 6 5.5
Sample 3.9 1.2 4.2 4.8
Rf 0.701 0.203 0.7 0.872
5Chritaka Solvent front 5.5 5.5 5.6 5.4
Sample 4 2.8 3.2,3.7 4.5
Rf 0.727 0.509 0.531,0.660 0.833
6Amalaki Solvent front 5.4 Absent 5.5 5.5
Sample 2.4,3.4 1.4 2.2,3.8
Rf 0.44,0.629 0.181, 0.727 0.690,0.4

7Haritaki Solvent front 5.5 5.7 5.8 5.5
Sample 0.6 0.8 1.5,1.2 3.4,0.5
Rf 0.109 0.14 0.258,0.206 0.618,0.09
8Bibitaki Solvent front absent 5.7 5.5 5.5
Sample 0.6 1.6 5.4
Rf 0.105 0.29 0.981
9Pippalimula Solvent front 5.6 5.7 5.4 5.5
Sample 3.2 1.2 4.1 1.6,4.7
Rf 0.571 0.21 0.759 0.290,0.854
10Chavya Solvent front 5.4 5.5 5.4 5.2
Sample 4.1 2.3 3.5,3.7 1.2,3.8
Rf 0.759 0.418 0.648,0.685 0.230,0.730

11Triphala Solvent front 5.6 5.7 5.7 5.6
Sample 1.2,2.5,4.9 2.5 4.5,1.1 3.6,1
Rf
0.214,0.446,0.
875 0.438 0.789,0.192 0.642,0.178
12Shatavari Solvent front 7.3 6.8 7.5, 7.5
Sample 6.8,2.5 6.1,3.7 6.2,5,2 6.4,3.6,
Rf 0.931,0.342 0.897,0.544
0.826,0.666,0.26
6 0.853,0.48
13Simaruba glauca Solvent front absent 5.7 7.6 7.6
Sample 1.5 7.2 6.4,7.3
Rf 0.277 0.947 0.842,0.960
14Argawadha Solvent front 7.1 6.9 6.6 6.7
Sample
4.1,5.6,5.9,6.3,
6.7 1.4,1.8 3.8,4 4.3,4.6,4.9,5.5
Rf
0.57,0.78,0.83,
0.8,0.94 0.20,0.26 0.57,0.60
0.64,0.68,0.73,0.8
2
15Raktachandana Solvent front 6.8 6.6 6.8 6.2
Sample 5,5.9 0.4,0.5 3.2,3.5 5.8
Rf 0.73,0.86 0.06,0.75 0.47,0.51 0.93

IN VITRO PROPAGATION OF TINASPORA CORDIFOLIAAND ESTIMATION OF BERBERINE
CONTENT BY CHROMATOGRAPHIC ANALYSIS
Berberine identification
TLC: solvent system toluene: acetic acid: water ( 5:15:1) was used for separation of berberine
Berberiene was found to be 0.553
* Hardirakanda Curcumin
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13-07-2023

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
HPTLC is a from of TLC that provides superior separation power using
optimized coating material, novel procedures for mobile phase feeding, layer
conditioning, & improved sample application
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13-07-2023

EXAMPLE OF MOBILE PHASE USED IN HPTLC FOR HERBAL COMPOUNDS
Chemical compounds (Herbal) Mobile phase
Polar Compounds Anthraglycosides, Arbutin,
Alkaloids, Cardiac Glycosides, Bitter Principles,
Flavonoids, Saponin
Ethyl Acetate: Methanol: Water [100:13.5:10]
Lipophilic Compounds Essential oils, Terpenes,
Coumarin, Napthoquinons, Velpotriat
Toluene: Ethyl Acetate [93:7]
Alkaloids Toluene: Ethyl Acetate: Diethyl Amine [70:20:10]
Flavonoids Ethyl Acetate: Formic Acid: Glacial Acetic Acid:
Water [100:11:11:26]
Saponin Chloroform: Glacial Acetic Acid: Methanol: Water
[64:32:12:8]
Cardiac Glycosides Ethyl Acetate: Methanol: Water [100:13.5:10] OR
[81:11:8] Terpenes Chloroform: Methanol: Water
[65:25:4]
Triterpenes Toluene: Chloroform: Ethanol [40:40:10]
Essential Oil Toluene: Ethyl Acetate [93:7]
Lignans Chloroform: Methanol: Water [70:30:4],
Chloroform: Methanol [90:10]
Toluene: Ethyl Acetate [70:30]
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LIST OF COMMON DERIVATIZATION AGENTS USED IN HPTLC
Chemical compounds
(Herbal)
Colour reagents Colour
Alkaloids Dragendroff Reagent It forms
complex reaction with some
nitrogen containing compounds
Red - brown Zone (vis)
Flavonoids Natural products Polyethylene
Glycol reagent i.e.
Diphenylboric acid -2-
aminoethyl ester forms
complexes with 3-
hydroxyflavones via
condensation reaction
Intense yellow, Orange and
Green Fluorescent zones in
UV 366 nm
Amino acids, peptides,
amines and amino-
sugars
Ninhydrin Reagent Yellow, brown to pink and
violet
Phenols Sodium carbonate solution, FC
Reagent
Tannin Ferric chloride reagent
13-07-2023

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC was developed in early 1970’s & originally referred as HPLC because, high
pressure was used to
increase the efficiency of separation.
PRINCIPLE:
HPLC is a solid liquid form of chromatography & the separation principle employed
is adsorption
13-07-2023

GAS CHROMATOGRAPHY
Gas chromatography is an analytical technique used for separation of thermally
stable & volatile substances. In GC, the mobile phase used is gas & the stationary
phase is either liquid or solid.
GC is a well-established analytical technique commonly used for the
characterization, quantization and identification of volatile compounds. It can be
used in many different fields such as pharmaceuticals, cosmetics and even
environmental toxins. Since the samples have to be volatile, human breath, blood,
saliva and other secretion containing large amounts of organic volatiles can be
easily analyzed using GC
13-07-2023

GAS CHROMATOGRAPHY – MASS SPECTROSCOPY
GC-MS is an advanced hyphenated analytical instrumental technique that
combines the separation capabilities of gas chromatography with the
identification of compounds by mass-determining capabilities of mass
spectrometry.
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13-07-2023

Asparagus racemosus
Asparagus racemosus belonging to the family Liliaceae also known as Shatavari is considered in Ayurvedha as "Queen of
herbs". The phytochemical screening of Asparagus racemosuswas performed by GC-MS analysis using GC Clarus 500
Perkin Elmer system which has an AOC-20i autosampler and GC interfaced to a mass spectrophotometer instrument. Using
this technique presence of phytochemical constituents like steroids, tannins, phenols, carbohydrates cardiac glycosides,
saponins, and flavonoids was identified. Specifically, 2-furan carboxaldehyde, tetradecyl acid, n-hexadecanoic acid,
oleic acid, and 12-octadecanoic acid had been identified.
Glycyrrhiza glabra
Glycyrrhiza glabra, also known as licorice, is a perennial herb belonging to the Fabaceae family. Glycyrrhiza glabra
consists of major constituents namely, glycyrrhizin, glycyrrhizic acid, isoliquiritin, isoflavones, etc., and their derivatives. A
GC-MS SHIMADZU QP2010 system (GC interfaced to MS) was employed for the quantitative analysis. As a result, the
major bioactive compounds Hymecromone, 7-acetoxy 4-methyl coumarin, Glabridine, 7-hydroxy-8-(-- `dimethyl
allyl) flavanone, Liquiritigenin, Licochalcone A, Licoisoflavone B, 5,7,8, trimethyl di-hydro coumarin were identified
in the plant root extract.
Application of GC-MS in Phytochemical Screening of Traditional Medicinal Plants - A Review:
www.ijppr.humanjournals.com
13-07-2023

Terminalia chebula
Terminalia chebula is called the "Kings of medicine" in Tibet and is always listed first in the Ayurvedic Materia medica since
it has extreme power of healing. In this study, the GC-MS instrument was used to screen the photo components of the
Terminalia chebulais Shimadzu QP2010 PLUS system. Sixty-four constituents were identified in Terminalia
chebulausing GC-MS out of whichaepferol-3-rutinoside which is a flavonoid and vitamin E was detected in the fruits
of this plant. The major component of the extract found is pyrogallol 46.26% using Gc-MS analysis. Other
components like phenol (2.73%), octadecene (3.68%), gallic acid, Chebulic acid, Chebulanin, ellagic acid, etc., were
identified
Tinospora cardifolia
Tinospora cardifoliais also called Giloe, belongs to the family Menispermaceae. In the Ayurveda system of medicine, it is
known as “Guduchi”, “Amrutha” in Sanskrit, and “Gurkha” in Hindi which poses adaptogenic and immune-modulatory
activity. The GC-MS analysis revealed the presence of various components like Isopinocarveol α-ylangene, 1H-3a,7-
Methanoazulene, octa hydro-tetramethyl Caryophyllene, trans-Z-α-Bisabolene epoxide, Benzene, 1-(1,5-dimethyl-4-
hexenyl)-4-methyl- trans-α-Bergamotene, β-Bisabolene,β-Cubebenecubedol Sativen Methyl hexadecatetraenoate,
Alloaromadendrene oxide-(1) α-acorenol, epi-cis sesquisabinene hydrate Octadecadienoic acid, methyl ester, Phenol,
2-methyl trimethylcyclopentyl)-, (S)-Isopropyl-2,8-dimethyl-9-Oxatricyclo decan-7-one, Hexadecanoic acid, ethyl
ester, Octadecenoic acid Nonadecatriene n-Propyl cinnamate, Dasycarpidan-1-methanol, acetate (ester).
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Withania somnifera
Withania somnifera commonly known as Ashwagandha, Indian ginseng, etc., belonging to the family Solanaceae is an
important plant of the Ayurvedic system having a wide range of pharmacological activities like anti-erotogenic, anti-arthritic
and hepatoprotective activity. GC-MS analysis was performed by Agilent 7890 instrument, detector used was Flame
Ionization Detector (FID), and Joel Accu Time of Flight Analyzer (TOF) GCV instrument for MS was used. GC-MS
analysis presence of 12 different bioactive compounds namely, Azetidin-2-one3,3-dimethyl-4-(1-aminoethyl), O-
Bromo atropine, 2-Methox y-4-vinyl phenol, Sucrose, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Hexadecenoic acid,
methyl ester, 17-Octadecynoic Acid, n-Hexadecanoic acid, 9-Octadecenoic acid (Z)-, methyl ester, Phytol, 9-Methyl-Z-
10-tetradecane-1-ol acetate and Oleic Acid.
Zingiber officinale
Also called Zingiber roscoe is a perennial aromatic plant belonging to the family Zingiberaceae. It is widely used in
Ayurvedic medicine as an antiemetic, anti-inflammatory, antipyretic, analgesic, and antiseptic, to treat GI disorders
(Ravindran PN; 1994), etc., GC-MS instrument used was Thermo GC-Trace Ultra VER: 5.0, Thermo MS DSQ II. t. The
spectra of the GC-MS revealed the presence of Furan-3-carboxaldehyde, Benzene-1-(1,5-dimethyl-4-4hexenyl)-4-
methyl, 1-,3-cyclohexadiene-5- (,5 diethyl-4-hexenyl -2-methyl (zingiberene), Alpha farnesene, butylated
hydroxytoluene, C15H24, cyclohexene-3- (1,5-dimethyl-4-hexenyl-6- methylene C15H24, 2-butanone-4-(4-hydroxy-3-
methoxyphenyl C11H14O3, methyl tetra decanoate C15H32O2, n-hexadecanoic acid C16H32O2, 9-octadecenoic acid
methyl ester C9H26O2, Octadec-9-enoic acid, C18H34O2, Gingerol C17H28O4 and Ricinoeic acid C18H24O2
respectively.
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Sl.No MEDICINAL PLANT CONSTITUENTS NO. OF. BIOMOLECULES
IDENTIFIED
1 Asparagus racemosus Alkaloids, Glycosides, Steroids 11
2 Nardostachysjatamansi Terpenoids And Steroids 61
3 Haritaki Flavonoid, Vitamin E, Pyrogallol 64
4 Guduchi Terpenoids, Alkaloids, Lignans 36
5 Shigru Alkaloids, Phenols. 31
6 Nerium oleander Glycosides, Phytosterols 16
7 Vrikshamla (Garcinia
cambogia )
Zanthones, Terpenoids, Anthraquinone 13
8 Bitter gourd
(Momordica charantia)
Vitamin E, Phytosterol, Cardiac
Glycosides
13
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COLUMN CHROMATOGRAPHY
Column chromatography is a separation technique in which a column of
stationary phase is used. The stationary phase used in column
chromatography may be solid or liquid.
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Quantification Of Phenols
Determination Of Total Phenolic Content
Gallic acid
in
microliter
Methanol
in
microliter
Concent
ration in
FC in
ml
7.5%
Sodium
carbona
te in ml
Absorba
nce at
760 nm
100 900 100 2.5 2 1.3326
200 800 200 2.5 2 1.6523
300 700 300 2.5 2 1.7523
400 600 40 2.5 2 2.0021
500 500 500 2.5 2 2.2092
600 400 600 2.5 2 2.2654
y = 0.0019x + 1.2103
R² = 0.9694
0
0.5
1
1.5
2
2.5
0 200 400 600 800
Absorbance
Concentration
Calibration Curve of Gallic acid
Series1
Linear (Series1)
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Sample Total Phenols
Date palm 25.6mg/g Gallic acid equivalent
Simaruba Glauca (Leaf) 5.20 mg/g Gallic acid equivalent
Simaruba glauca (fruit) 408.3 mg/g Gallic acid equivalent
Ashwagandha 210.89 mg/g Gallic acid equivalent
Lakshmana phala 73.05 mg/g Gallic acid equivalent
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Determination of Total Flavonoids
Rutin
in ml
Distill
ed
water
in ml
Conce
ntratio
n in
mg/ml
5%
NaN
O2
in
ml
Stand
the
saolut
ion
for 5
min
10
%
Al
Cl3
in
ml
Mix
thourou
ghly
and
allow to
stand
for 1
min
1M
NaO
H in
ml
Absorban
ce at 510
nm
0.4 2 40 0.3 0.3 1 2.1952
0.6 2 60 0.3 0.3 1 2.2951
0.8 2 80 0.3 0.3 1 2.4211
1.0 2 100 0.3 0.3 1 2.5502
1.2 2 120 0.3 0.3 1 2.6101
Samp
le
2 0.3 0.3 1 1.997
y = 0.0054x + 1.9804
R² = 0.9885
2.15
2.2
2.25
2.3
2.35
2.4
2.45
2.5
2.55
2.6
2.65
2.7
0 50 100 150
Absorbance
Concentration
Rutin
Series1
Linear (Series1)
13-07-2023

Sample Total Flavonoids
Date palm 3.0740 mg/g Rutin
Equivalent
Kalamegha 50.16mg/g Rutin
Equivalent
Neem 184.68 mg/g Rutin
Equivalent
13-07-2023

SPECTROPHOTOMETRIC ESTIMATION OF TOTALALKALOIDS USING
BROMOCRESOL GREEN
Atropine in
ml
pH4.7
Phosphate
buffer in ml
BCG
Solution in
ml Taken in
separating
funnel &
mixture
was shaken
with
extract
1,2,3,4ml of
chloroform
Concentrati
on in ml
Absorbance
at 470nm
0.4 5 5 40 0.0927
0.6 5 5 60 0.114
0.8 5 5 80 0.1533
1 5 5 100 0.1678
1.2 5 5 120 0.199
Sample 5 5 0.1472

y = 0.0013x + 0.0388
R² = 0.9856
0
0.05
0.1
0.15
0.2
0.25
0 50 100 150
Absorbance
Concentration
Atropine
abs
Linear (abs)
Extract
Chitraka
mg of Atropine
Equivalent of extract
Ethanol
83.42

Estimation Of Protein By Lowry’s Method
Volume
of
standar
d
BSA(ml
)
Volum
e of
distille
d
water
(ml)
Concentrati
on of
protein (µg)
Volum
e of
reagen
t
C(ml)
Incuba
te at
room
temp
for 10
min
Volum
e of
Reage
nt
D(ml)
Incuba
te at
room
temp
for 30
min
Absorban
ce (660)
0.0 1.0 0.0 5 ml 0.5 0.0856
0.2 0.8 40 5 ml 0.5 0.1689
0.4 0.6 80 5 ml 0.5 0.2284
0.6 0.4 120 5 ml 0.5 0.2974
0.8 0.2 160 5 ml 0.5 0.3566
1.0 0.0 200 5 ml 0.5 0.4680
y = 0.3639x + 0.0856
R² = 0.9901
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.2 0.4 0.6 0.8 1 1.2
Absorbance
Concentration
BSA of Protein
Series1
Linear (Series1)
Sample Total protein
Shigru 11.97
Shobhanjana Yusha 3.45
13-07-2023

Estimation of Total Carbohydrate
Glucose
solution in
ml
Distilled
water in
ml
5% Phenol
solution in
ml
96%
Sulphuric
acid in ml
Concentrati
on mg/ml
Absorbanc
e
0 1 1 5 0 0.1347
0.2 0.8 1 5 0.020 0.2466
0.4 0.6 1 5 0.040 0.2789
0.6 0.4 1 5 0.060 0.3578
0.8 0.2 1 5 0.080 0.3854
1 0 1 5 0.10 0.4654
Sample-0.2 0.8 1 5 0.3730
y = 2.6895x + 0.1848
R² = 0.9775
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 0.02 0.04 0.06 0.08 0.1 0.12
Absorbance
Concnentration
Calibration curve of Glucose
Series1
Linear (Series1)
Sample Total carbohydrate
Atibala 34.95
13-07-2023

Maltose
solution in
ml
Distilled
water in
ml
DNS in
ml Heat in
boiling
water
bath for
5 min
Distille
d water
in ml
Concentrati
on mg/ml
Absorbanc
e
0 1 1 8 0 0.0711
0.2 0.8 1 8 0.020 0.2794
0.4 0.6 1 8 0.040 0.3908
0.6 0.4 1 8 0.060 0.5029
0.8 0.2 1 8 0.080 0.5721
1 0 1 8 0.10 0.6961
Estimation of Total Maltose
y = 5.0735x + 0.1839
R² = 0.9942
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.02 0.04 0.06 0.08 0.1 0.12
Absorbance
Concentration
Maltose Curve
Series1
Linear (Series1)
Sample Total Maltose
Vidarikanda 26.45%
13-07-2023

ANTIOXIDANT

Concentrat
ion in
µg/ml
Methano
l in µl
DPPH in
ml
Control
absorban
ce
Sample %RSA IC50
20 2980 1 0.3034 0.2454 19.11
77.52
40 2960 1 0.3034 0.2056 32.23
60 2940 1 0.3034 0.1754 42.18
80 2920 1 0.3034 0.1456 52.01
100 2900 1 0.3034 0.11214 59.98
concentration
(μg)
Amalaki Ascorbic
acid
20 19.11 20
40 32.2 36
60 42.18 50
80 52.01 60
100 59.98 70
In vitro Antioxidant study
DPPH Assay
13-07-2023

13-07-2023

IN VITRO ANTIDIABETIC STUDY
1. Alpha Amylase Enzymes Assay
2. Alpha Glucosidase Assay
13-07-2023

13-07-2023

SAMPLE IC 50
Avartaki 37.28
Arjuna 48.75
Chitraka 68.66
Mustaka 89.54
Vacha 133.6
Nagakeshara 146.8
Darvi 177.9
Ativisha 323.1
ALPHAAMPYASE INHIBITION OF PLANT EXTRACTS
SCREENING OF NINE HERBAL PLANTS FOR IN VITRO a-AMYLASE INHIBITION Asian J Pharm Clin Res, Vol 7,
Issue 4, 2014
13-07-2023

IN VITRO ANTIMICROBIAL STUDY
ANTIMICROBIAL STUDY
Diffusion Dilution Diffusion and dilution
Agar Disk Diffusion Method Agar Dilution Method E test
Agar Well Diffusion Method (liquid – should not be turbid)
Agar Plug Diffusion
Cross Streak Method
Agar Diffusion Method
(should not be oily)
13-07-2023

ANTI MICROBIALASSAY
Anti – Against to microorganism
Used in research purpose to check Minimum Inhibition Concentration of an Antimicrobial agent
Plants – Produce metabolites & this effect over microorganism. The root, leaf, fruit prevents the
growth of wide range of gram positive and gram negative bacteria, Metabolites come out not allow to
grow microorganism has inhibitory effect called clearing zone minimum inhibition concentration
Types of extract using
Aqueous extract
Methanol extract
Chloroform extract
Hexane
In different extract we will check antimicrobial activity
The area up to which will the antibiotic shows its effect, is referred as zone of inhibition or clearing
zone or which concentration is having highest antimicrobial activity

AGAR WELL DIFFUSION METHOD
Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants
or microbial extract. Similarly to the procedure used in disk-diffusion method, the agar
plate surface is inoculated by spreading a volume of the microbial inoculum over the
entire agar surface. Then, a hole with a diameter of 6 to 8 mm is punched aseptically
with a sterile cork borer or a tip, and a volume (20–100 mL) of the antimicrobial agent
or extract solution at desired concentration is introduced into the well. Then, agar plates
are incubated under suitable conditions depending upon the test microorganism. The
antimicrobial agent diffuses in the agar medium and inhibits the growth of the microbial
strain tested
13-07-2023

AGAR DISK-DIFFUSION METHOD
In this well-known procedure, agar plates are inoculated with a standardized inoculum of the test
microorganism. Then, filter paper discs (about 6 mm in diameter), containing the test compound at a
desired concentration, are placed on the agar surface.
The Petri dishes are incubated under suitable conditions. Generally, antimicrobial agent diffuses into the
agar and inhibits germination and growth of the test microorganism and then the diameters of inhibition
growth zones are measured.
13-07-2023

AGAR PLUG DIFFUSION METHOD
Agar plug diffusion method is often used to highlight the antagonism between
microorganisms and the procedure is similar to that used in the disk-diffusion
method.
It involves making an agar culture of the strain of interest on its appropriate culture
medium by tight streaks on the plate surface. During their growth, microbial cells
secrete molecules which diffuse in the agar medium. After incubation, an agar-plot or
cylinder is cut aseptically with a sterile cork borer and deposited on the agar surface
of another plate previously inoculated by the test microorganism. The substances
diffuse from the plug to the agar medium. Then, the antimicrobial activity of the
microbial secreted molecules is detected by the appearance of the inhibition zone
around the agar plug
13-07-2023

13-07-2023

ANTIMICROBIAL GRADIENT METHOD (ETEST)
In the procedure, a strip impregnated with an increasing
concentration gradient of the antimicrobial agent from one end to the
other is deposited on the agar surface, previously inoculated with the
microorganism tested
13-07-2023

CROSS STREAK METHOD
Cross streak method is used to rapidly screen microorganisms for antagonism. The
microbial strain of interest is seeded by a single streak in the center of the agar plate.
After an incubation period depending upon the microbial strain, the plate is seeded
with the microorganisms tested by single streak perpendicular to the central streak.
After further incubation, the antimicrobial interactions are analyzed by observing
the inhibition zone size.
13-07-2023

POISONED FOOD METHOD
Poisoned food method is mostly used to evaluate the antifungal effect against molds.
The antifungal agent or the extract is incorporated into the molten agar at a desired
final concentration and mixed well. Then, the medium is poured into Petri dishes. After
overnight pre-incubation, the inoculation can be done by a mycelia disc ranging from 2
to 5 mm, which is deposited in the center of the plate. After further incubation under
suitable conditions for the fungal strain tested, the diameters of fungal growth in
control and sample plates are measured, and the antifungal effect is estimated by the
following formula: Antifungal activity % Dc Ds /Dc 100
13-07-2023

THIN-LAYER CHROMATOGRAPHY (TLC)–BIOAUTOGRAPHY
In 1946, Goodall and Levi combined paper chromatography method (PC) with
contact bioautography to detect different penicillins for their determination.
Thereafter, Fischer and Lautner introduced TLC in the same field. This technique
combines TLC with both biological and chemical detection methods. Several works
have been done on the screening of organic extracts, mainly plant extracts, for
antibacterial and antifungal activity by TLC– bioautography. As shown below, three
bioautographic techniques, i.e., agar diffusion, direct bioautography and agaroverlay
assay, have been described for the investigation of antimicrobial compounds by this
approach.
13-07-2023

AGAR DIFFUSION
Also known as agar contact method, it is the least-employed one of the
techniques. It involves the transfer by diffusion of the antimicrobial agent
from the chromatogram (PC or TLC) to an agar plate previously
inoculated with the microorganism tested. After some minutes or hours to
allow diffusion, the chromatogram is removed and the agar plate is
incubated. The growth inhibition zones appear in the places, where the
antimicrobial compounds contact with the agar layer
13-07-2023

DIRECT BIOAUTOGRAPHY
Direct bioautography is the most applied method among these three methods. The
developed TLC plate is dipped into or sprayed with a microbial suspension. Then,
bioautogram is incubated at 25 °C for 48 h under humid condition [45]. For
visualization of the microbial growth, tetrazolium salts are frequently used. These salts
undergo a conversion to corresponding intensely colored formazan by the
dehydrogenases of living cells [46,47]. p-Iodonitrotetrazolium violet is the most suitable
detection reagent [44,48]. These salts are sprayed onto the bioautogram, which is
reincubated at 25 °C for 24 h [49] or at 37 °C for 3–4 h [5]. The Mueller Hinton Broth
supplemented with agar has been recommended to give a medium sufficient fluid to
allow a best adherence to the TLC plate and maintain appropriate humidity for
bacterial growth [50].
13-07-2023

AGAR OVERLAY BIOASSAY
Also known as immersion bioautography, it is a hybrid of the both previous methods.
TLC plate is covered with a molten seeded agar medium. In order to allow a good
diffusion of the tested compounds into the agar medium, the plates can be placed at low
temperature for few hours before incubation
13-07-2023

Broth Dilution Method
13-07-2023
Liquid media
Optical density (Absorbance) of turbidity
*extract should be clear

MINERALS AND METALS
1. Petrological Microscope
2. Physicochemical analysis
3. Inorganic analysis
4. Flame Photometer : Sodium, Potassium, Calcium
5. Atomic absorption spectrophotometer: Identification of heavy metals
13-07-2023

IN VIVO STUDIES
13-07-2023

EXPERIMENTAL ANIMALS
Rodents: Mouse, Rat, Guinea Pigs, Gerbil, Hamster Etc.
Non Rodents: Rabbit, Monkey, Dog, Cat Pig Etc.
Miscellaneous: Frog, Pigeon, Zebrafish, Chicken Etc.

13-07-2023

CENTRAL NERVOUS SYSTEM
Aim: TO DEMONSTRATE MUSCLE RELAXANT PROPERTY OF
DIAZEPAM IN MOUSE USING ROTA ROD APPARATUS
Rotarod test is used to evaluate fore and hind limb
Motor coordination of rodents
13-07-2023

Aim: TO DEMONSTRATE MUSCLE RELAXANT PROPERTY
OF DIAZEPAM IN MOUSE USING CHIMNEY TEST.
13-07-2023

Aim: TO DEMONSTRATE ANTI ANXIETY EFFECT OF DIAZAPUM IN RAT
USING ELEVATED PLUS MAZE APPARATUS .
This instrument is made for a novel test for the selective identification of
anxiogenic and anxiolytic drug effects in rodents. It also used to evaluate the
learning and memory in rodents in presence or absence of a drug.
The plus maze apparatus consists of two open(16*5cm for mice and 50*10cm for
the rats) and two closed arms(16*5*12cm for mice and 50*10*40cm for rats) and
an open roof with the entire maze elevated(25cm for mice and 50cm for rats) from
the floor. The animals are placed individually at the centre of the elevated plus
maze with their head facing the open arm. During the 5 minutes test the preference
of the animal for the first entry, the numbers of entries into the open and closed
arms entries reflect the relative safety of closed arms compared with the relative
fearfulness of open arms. Anxiolytics would be expected to increase the frequency
of entries and time spent into open arms. It is also good screening method for
learning and memory in rodents.
13-07-2023

Aim: TO DEMONSTRATE AMNESIC EFFECT OF DIAZEPAM IN
RAT USING MORRIS WATER MAZE APPARATUS .
This instrument is designed to evaluate learning and memory in
rodents and to evaluate effect of drugs. The test apparatus
consists of a circular fiberglass tank (130cm in diameter,50cm in
depth, dimension for rat). The pool is filled to a height of 30cm
with water at room temperature, 21-22.c. the pool is divided into
four vertical quadrants (Q1,Q2,Q3 and Q4) of equal surface area.
A transparent escape platform (10cm in diameter,29cm in height)
is placed in a fixed location in the tank in the centre of one of a
quadrant, 1cm below the water surface. The platform is not visible
from just above water level. Many extra maze cues surrounding
the maze are available for the rats to use in locating the escape
platform. The minimum cut off time for learning and memory is
300s.
13-07-2023

Aim: TO DEMONSTRATE THE ANTI CONVULSION ACTIVITY OF
PHENYTOIN AGAINST MAXIMUM ELECTROSHOCK (MES)
INDUCED CONVULSION IN RATS
This instrument is used to induce convulsions experimentally
which are hypothesized to originate from the forebrain or
brainstream.
Instrument and its parts:Electroshock is applied through a pair of
ear clip electrodes or corneal electrode, using a sinusoidal maximal
current of 150mA, 50Hz for 0.2sec for rat and 12mA, 50Hz for
0.2sec for mice. Animals is housed in acrylic cage, 1min before
current application, and observed for an additional 2 min period.
Recording may be videotaped and watched for all stages of
seizure. It only application is the screening, if the antiepileptic
drug action on tonic-clonic convulsion and animal should be
screened one week prior to the experimental day.
13-07-2023

This instrument used for screening of inflammation or edema in
mouse or rat. Or to demonstrate the anti-inflammatory property of
indomethacin against carrageenan induced paw edema.
The principle of screening of anti-inflammatory agents is based on the
reduction of edema caused by any irritant or phlogistic agent( agent
that may induce inflammation or fever). The edema is measured by an
instrument known as the plethysmograph which is used to measure the
change in volume. In the experiment it measures the volume of the rat
paw in the presence and absence of irritant and after the treatment of
anti-inflammatory drug.
Precautions before experimentation
Animals should be marked properly, to avoid mixing in two group
Clean the paw with wet cotton
Mark the paw( insertion part must be the same every time)
ANTI INFLAMMATORY STUDY
Aim: TO DEMONSTRATE ANTI INFLAMMATORY PROPERTY BY
PAW EDEMA
13-07-2023

Aim: TO DEMONSTRATE THE EFFECT OF ANY
GIVEN LOCALANESTHETIC USING GUINEA PIG.
13-07-2023

Punch line
RETURN TO NATURE
13-07-2023

TAKE HOME MESSAGE
1. YUGAANUSARA RUPA SANDARBHAM
2. ERA OF EVIDENCE BASE MEDICINE
3. INTEGRATIVE APPROACH WITH ALLIED SCIENCE IS NEED OF
THE HOUR
4. क्वचित् धर्म क्वचित् र्ैत्री क्वचित् अर्म क्वचित् यश!
कर्ममभ्यमसं क्वचितश्चेचत चिचकत्सम नमस्ती चनष्फलम!!
13-07-2023

SEASON MAY COME AND SEASON MAY GO
EVERY THING IN DO THE DEW
BUT STILL I HAVE LOVE AND AFFECTION FOR
YOU
THANK YOU

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