Chapter 2: Metabolite extraction and fingerprinting of Terminalia arjuna...
Transcript of Chapter 2: Metabolite extraction and fingerprinting of Terminalia arjuna...
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Chapter 2: Metabolite extraction and fingerprinting of Terminalia
arjuna extracts 2.1 Introduction 2.2 Materials and Methods
2.2.1 Plant Material Processing:
2.2.2 Pharmacognosy analysis
I. Thin layer chromatography (TLC)
II. Histological studies
2.2.3 Solvent Based Extraction
I. Direct extraction
II. Successive extraction
2.2.4 Metabolic Fingerprinting of the Terminalia arjuna extracts
2.2.5 Quantitative tests for phyto-chemicals
2.3 Results 2.3.1 Pharmacological validation of Terminalia arjuna bark
2.3.2 Extraction of secondary metabolites from the bark of Terminalia
arjuna
2.3.3 Metabolite profiling of different extracts from the bark of Terminalia
arjuna
2.3.4 Quantitative estimation of Terminalia arjuna bark extracts
2.4 Discussion 2.5 References
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2.1 Introduction In recent years there has been considerable increase in the use of plant based
medicines (Ravishankar, 2007). The renewed interest in the Indian system of
medicine is due to growing recognition of these products as naturally derived,
safe therapeutic interventions with less reported side effects. Infact, some of the
plant based drugs such as Quinine-derived from bark of Cinchona officinalis,
Taxol-dervied from bark of Taxus baccata, Colchicine from Gloriosa superba are
most popular for the treatment of malaria, cancer and gout respectively (Druilhe
et al., 1988; Ravishankar, 2007).
In spite of ancient use of plant parts and its extracts in traditional system of
medicine there have been limiting factors that have prevented the growth of this
segment. Some of the major challenges faced by herbal industries include
availability of large quantities of authentic plant materials, detection of
adulteration with spurious plant material(s), absence of available standards for
quantification of bio-actives, estimation of batch-to-batch variability during
commercial productions.
Composite phyto-extract is characterized by mixture of metabolites. The lack of
extraction and resolution of metabolite diversity by commercially available
standards makes its decoding complex. The metabolite fingerprinting methods
commonly employed in the herbal industries are focused on quantitative
estimation of less than 10 metabolites that occur in significant proportions
(>0.1%). This approach makes it difficult to address the batch to batch variability
observed in bioactivity where the minor constituents (< 0.01 %) might also be
responsible for the observed bioactivity.
The term ‘Pharmacognosy’ is defined as the study of drugs derived from natural
sources. This includes scientific and systematic study such as structural,
physical, chemical and biological characters of crude drugs along with their
method of cultivation, collection and preparation.
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The plant parts from which the herbal drugs are derived are available in crumpled
or broken pieces. It therefore becomes difficult to identify them based on their
morphology. Hence microscopic characteristic of these plant materials aids in
confirming the identity of the plants and thereby authenticity of the plant part in
use. In addition to microscopic characters additional characters such as chemical
evaluation using both qualitative chemical tests, chromatographic separation of
the organic compounds of the extracts are also necessary for identification of the
sample for its purity and consistency.
As discussed in the previous (Chapter 1; section 1.3), Terminalia arjuna as a
ingredient has been used in traditional system as therapeutic component in a
range of formulations. Terminalia arjuna has been reported for mitigation of range
of health related disorders due to the presence of diverse class of secondary
metabolites. In effort to isolate these different classes of metabolites different
extraction conditions and solvent systems have been used, which will be
discussed in length in the following section (section 2.3.2).
Extraction of secondary metabolites is classically performed using two different
methods listed below:
I. Direct extraction
II. Successive extraction
Both extraction procedures can be performed at room temperature under
agitation or static conditions. Direct extraction is performed using single solvent
system. Direct extraction is preferred in situations where complete extraction of
metabolites in a particular solvent is required. Direct extraction at room
temperature is done for temperature sensitive bioactive(s) by immersing the plant
material in the solvent of choice under constant agitation. Once the extraction is
complete, the solvent is passed through filters to remove suspended particles
and then centrifuged to remove any un-dissolved fine material.
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Another most common method of choice for extracting phyto-chemicals using
different temperature and solvent systems is the Soxhlet based extraction
procedure. Soxhlet based extraction can be used for extraction of plant
metabolites using single solvent (Direct extraction) or multiple solvents in series
(successive extraction). Soxhlet works on the boiling temperature or vaporization
temperature of the solvent.
In successive extraction, the same plant material is sequentially extracted with a
series of solvents (from non-polar to polar solvents). This helps in extraction and
isolation of the metabolites based on its class and nature. The extended refluxes
with each of this solvent system ensure extraction of components based on their
polarity components minimizing the chances of carryover in the subsequent
solvent.
Direct/successive extracts of Terminalia arjuna bark were obtained and subjected
to High Performance Liquid Chromatography (HPLC) analysis. HPLC is a
technique wherein small quantity of the sample is injected into a reverse phase
HPLC column (C-18 column; Waters Corporation) under high pressure and the
constituents are allowed to separate based on their interaction with the column
and their retention time within the column. The main purpose of HPLC analysis
was to generate a metabolite fingerprint of the different plant extracts.
This chapter will describe the authentication of plant material by pharmacognosy,
extraction of metabolites using different solvents and extraction conditions as well
as metabolite profiling of the different composite extracts of Terminalia arjuna.
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2.2 Materials and Methods 2.2.1 Plant Material Processing:
Bark of Terminalia arjuna was procured from authentic vendor and from Sandur
forest area which is deposited in Avesthagen Herbarium. The bark was powdered
and taken forward for pharmacognosy authentication.
2.2.2 Pharmacognosy analysis
I. Thin layer chromatography (TLC)
• Bark for Terminalia arjuna (3 grams) was extracted in 50ml methanol for 1-
1.5 hrs.
• The resulting methanol extract was filtered and concentrated to 1/10th of its
original volume by evaporation.
• 3µl of the concentrated methanol extract was spotted on the pre-coated
preparative TLC plate (Silica G F254 of 10 x 10 cm; Merck).
• After air drying the spotted TLC plates were placed vertically in pre-
saturated solvent chamber (Toluene:Ethyl acetate; 8:2) such that the
spotted sample does not submerge in the solvent.
• Due to capillary action the solvent will be absorbed on the TLC plates
resulting in the mobilization and separation of the different components of
the extracts. The solvent based separation is based on the difference in
mobility of the different components present in the extract.
• After the solvent has covered 3/4th of the TLC plate, the solvent phase is
marked and the plates are air dried.
• The metabolite fingerprint of the extract is observed in visible range, long
Ultra violet (U.V.) (366nm), short U.V light (255nm) and using detection
reagent consisting of Anisaldehyde, Vanillin Sulphuric acid (H2SO4 ) and
10% H2SO4. The retention factor (Rf)— [Formula: Rf value= Distance
traveled by solute/ Distance traveled by solvent] was determined for each
band representing the metabolites present in the bark extract of Terminalia
arjuna.
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II. Histological studies
• Transverse section of Terminalia arjuna bark were taken and washed with
water to remove debris.
• Sections were stained with Saffranine for 5-10 minutes and treated with
increasing amount of ethanol to remove the moisture.
• The sections were transferred to clean slide, treated with plain clove oil for 5
minutes. Excess clove oil was drained off using tissue paper.
• The stained bark sections are then mounted on the glass slide using
Canada balsam (Wallis, 1957)
2.2.3 Solvent Based Extraction:
I. Direct Extraction
a. Soxhlet based extraction from bark of Terminalia arjuna
• Powdered bark of Terminalia arjuna (100 grams) was taken into the Soxhlet
extractor. The top was covered with cotton. The level of plant material was
kept one inch below the vapor inlet tube.
• 1000 ml of extraction solvent was taken in to the round-bottomed flask and
was placed on the mantle, temperature was set at 65°C. In order to prevent
bubbling of the solvent few ceramic chips were added in to the flask.
• The extractor containing the bark powder was placed on the solvent
containing round bottom flask. The extractor was then connected with the
condenser and the cold water was circulated continuously in the condenser.
• On heating the vapors of the solvent from the round bottom flask passes
through the inlet into the condenser and the extractor. The vapor gets
condensed in the condenser and the solvent drips on top of the plant
material and gets collected in the extractor (body), in this process
metabolites get extracted from the plant material.
• When the extractor containing the plant material gets completely filled with
solvent, the solvent along with the extracted metabolites gets drained in the
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solvent containing round bottom flask. This completes one cycle of
extraction.
• The extraction was continued for 8 hours, with ~4 cycles per hour.
• Finally the extract collected in the round bottom flask was concentrated by
vacuum lyophilization.
• After complete extraction with the given solvent, the plant material was air
dried and taken up for extraction with the next solvent and the entire
procedure was repeated.
• The percentage yield of the extract was calculated using following formula.
% Yield = weight of lyophilized extract * 100
weight of starting plant material
b. Extraction from bark of Terminalia arjuna at room temperature
• Powered bark of Terminalia arjuna (100 grams) was taken into the conical
flask. To it extraction solvent (500 ml) was added and the mouth was
covered with aluminum foil to avoid solvent evaporation.
• The flask was then placed on an orbital shaker at 210 rpm, room
temperature (25°C) for 4hours.
• The supernatant was centrifugation at 1000 rpm for 10 minutes and was
subsequently subjected to lyophilization.
• The plant material was re-extraction with 500ml extraction solvent for 2
hours.
• Again the supernatant obtained was centrifuged at 1000 rpm for 10 minutes.
• The filtrate was subjected to lyophilization.
• Yield of the extract was calculated using the following formula:
% Yield = weight of lyophilized extract * 100
weight of starting plant material
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II. Successive Extractions:
Successive extraction from bark of Terminalia arjuna was carried out using
soxhlet extractor. The solvents used, were based on their sequential polarity
starting from non-polar to polar, viz; Hexane, chloroform, ethyl acetate, acetone,
ethanol, methanol and water at temperature above the boiling point of the
solvents.
The detailed process of successive extraction is given below:
• Powdered bark of Terminalia arjuna (100 grams) was taken into the Soxhlet
extractor. The top was covered with cotton. The level of plant material was
kept one inch below the vapor inlet tube.
• 1000 ml of solvent (non polar to polar solvent) was taken in to the round-
bottomed flask and was placed on the mantle; temperature was set above
the boiling point of the solvent being used for extraction. In order to prevent
bubbling of the solvent few ceramic chips were added in to the flask.
• The extractor containing the bark powder was placed on the solvent
containing round bottom flask. The extractor was then connected with the
condenser and the cold water was circulated continuously in the condenser.
• On heating the vapors of the solvent from the round bottom flask passes
through the inlet into the condensor and the extractor. The vapor gets
condensed in the condenser and the solvent drips on top of the plant
material and gets collected in the extractor (body), in this process
metabolites get extracted from the plant material.
• When the extractor containing the plant material gets completely filled with
solvent, the solvent along with the extracted metabolites gets drained in the
solvent containing round bottom flask. This completes one cycle of
extraction.
• The extraction was continued for 8 hours, with ~4 cycles per hour.
• Finally the extract collected in the round bottom flask was concentrated by
vacuum lyophilization.
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• After complete extraction with the given solvent, the plant material was air
dried and taken up for extraction with the next solvent and the entire
procedure was repeated.
• The percentage yield of the extract was calculated using following formula.
% Yield = weight of lyophilized extract * 100
weight of starting plant material
2.2.4 Metabolic Fingerprinting of the Terminalia arjuna extracts Metabolic fingerprinting of all the direct and successive extracts from Terminalia
arjuna plant parts is done by HPLC. HPLC Fingerprinting:
The samples for HPLC analysis were prepared by dissolving 10 mg of extract in
1 ml of methanol:water. These samples were filtered, collected in HPLC vials and
10 μl of extract was injected and subjected to separation by Waters 2695 HPLC
instrument. Subsequently the metabolite profiles were analyzed using Waters
Millennium32 (Waters Corporation) and MetagridTM software developed in-house
(Avesthagen).
The HPLC column used for separation was Atlantis dC18, 5μ, 4.6x250mm
(Waters Corporation). The column temperature was maintained at 250 C and the
solvent flow rate was set at 1.0ml per min. HPLC conditions included Gradient
chromatography. Solvents used for metabolite separation were Acetonitrile
(solvent A), Methanol (solvent B), Water (HPLC Grade)+ 0.1%TFA (Solvent C
and D).
Method standardization was done for the maximum separation of the metabolites
of Terminalia arjuna direct and successive extracts. Following are the conditions
that were used for separation and metabolite fingerprinting of respective extracts.
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Sample-AV016BaDi (65) 04(100) Time Program: Time Flow %A %B %C %D Curve
1 0.01 1.00 10.0 0.0 90.0 0.0 6
2 1.00 1.00 10.0 0.0 90.0 0.0 6
3 15.00 1.00 30.0 0.0 70.0 0.0 6
4 30.00 1.00 40.0 0.0 60.0 0.0 6
Sample-AV016BaDi (65) 04(80) Time Program: Time Flow %A %B %C %D Curve
1 0.01 1.00 10.0 0.0 90.0 0.0 6
2 1.00 1.00 10.0 0.0 90.0 0.0 6
3 15.00 1.00 30.0 0.0 70.0 0.0 6
4 30.00 1.00 40.0 0.0 60.0 0.0 6
Sample-AV016BaSu (65) 09(100) Time Program:
Sample-AV016BaSu (65) 01(100) Time Program: Time Flow %A %B %C %D Curve
1 0.01 1.00 0.0 0.0 100.0 0.0 6
2 75.00 1.00 25.0 0.0 75.0 0.0 6
3 90.00 1.00 100.0 0.0 0.0 0.0 1
Sample-AV016BaSu (65) 04(100) Time Program Time Flow %A %B %C %D Curve
1 0.01 1.00 10.0 0.0 90.0 0.0 6
2 1.00 1.00 10.0 0.0 90.0 0.0 6
3 15.00 1.00 30.0 0.0 70.0 0.0 6 4 30.00 1.00 40.0 0.0 60.0 0.0 6
Time Flow %A %B %C %D Curve 1 0.01 1.00 10.0 0.0 90.0 0.0 6 2 65.00 1.00 30.0 0.0 70.0 0.0 6 3 70.00 1.00 100.0 0.0 0.0 0.0 6 4 75.00 1.00 100.0 0.0 0.0 0.0 6 5 75.01 1.00 10.0 0.0 90.0 0.0 1 6 90.00 1.00 10.0 0.0 90.0 0.0 1
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Sample-AV016BaSu (65) 06(100) Time Program Time Flow %A %B %C %D Curve
1 0.01 1.00 10.0 0.0 90.0 0.0 6 2 1.00 1.00 10.0 0.0 90.0 0.0 6
3 15.00 1.00 30.0 0.0 70.0 0.0 6
4 30.00 1.00 40.0 0.0 60.0 0.0 6
Sample-AV016BaSu (105) 08(100) Time Program Time Flow %A %B %C %D Curve
1 0.01 1.00 0.0 0.0 100.0 0.0 6
2 80.00 1.00 25.0 0.0 75.0 0.0 6
3 90.00 1.00 100.0 0.0 0.0 0.0 1
2.2.5 Quantitative tests for phyto-chemicals: Determination of total phenols
Weigh 10 mg of extract to falcon tube and dissolve it in 10 ml of 50% methanol.
Dilute 1:10 of this solution to get 0.1 mg/ml (100 μg/ml).
a. Preparation of standard Gallic acid:
Weigh 10 mg of Standard Gallic acid to falcon tube and dissolve it in 10 ml of
50% methanol. Dilute 1:10 of this solution to get 0.1 mg/ml (100 μg/ml).
b. Preparation of reagents:
• Folin-Ciocalteu (FC) reagent: 5 ml of FC reagent in 95 ml of 50%
methanol
• 7.5% of Sodium carbonate solution: weigh 7.5 gms of sodium carbonate in
100 ml water.
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c. Procedure:
• Pipette out the different concentration of extracts ranging 2 to 20 μg/ml
into microfuge tube.
• Make up the volume to 200 μl with methanol.
• Add 1000 μl of the FC reagent to each microfuge tubes
• Incubate for 5 min at 37°C
• Add 800 μl of the 7.5% Sodium carbonate to all tubes.
• Incubate for 30 min at room temperature
• Read absorbance at 750 nm
d. Calculation:
OD of sample
% Of Total phenol content: -------------------- X 100
OD of standard
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2.3 Results 2.3.1 Pharmacognostic validation of Terminalia arjuna bark Bark of Terminalia arjuna was authenticated by Department of Forestry and
Environmental Sciences, GKVK, Bangalore. Further as an in-house quality
check, Pharmacognostic analysis was carried out as per Ayurvedic
Pharmacopoeia of India (API) (Anonymous, 2001).
As reported earlier (Anonymous, 2001), the bark of Terminalia arjuna was found
to be pinkish in colour, with fibrous texture. It had a bitter and astringent taste.
Further to validate the raw material TLC and histological analysis was performed.
TLC based metabolite fingerprint of the Terminalia arjuna bark methanolic extract
(Chapter 2; section 2.2.2) was performed to assign a reference metabolic
fingerprint. As seen in Figure 2.1, metabolites with the natural pigment failed to
separate using the Toulene:Ethyl acetate (8:2) solvent system (Figure 2.1, lane 1
and 2); using anisaldehyde developing reagent three distinct band at Rf value
0.04, 0.31 and 0.91 were obtained (Figure 2.1, lane 3). Further in the short U.V.
range (254 nm) there were no observed bands (Figure 2.1, lane 4); whereas at
the longer U.V. wavelength (365nm) only one band at Rf value of 0.72 was
obtained (Figure 2.1, lane 5).
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Figure 2.1:
Figure 2.1: TLC based metabolic finger printing of Terminalia arjuna bark. TLC slide representing mobility of methanolic extract of Terminalia arjuna bark powder. Lane 1
[A] represents profile under visible light, lane 2 [B] represents profile after spraying with 10%
Sulphuric acid, lane 3 [C] represents profile after spraying with Anisaldehyde reagent, lane 3 [D]
represents profile under short UV (254 nm) and lane 5 [E] represent profile under long UV (366
nm).
Further as mentioned in API (Anonymous, 2001), histological analysis of
Terminalia arjuna bark was done with saffranine staining (Section 2.2.2).
Transverse section of the bark shows in cortical zone medullary rays, phloem
fibers and calcium oxalate crystals present in parenchyma cells alternating with
fibers. Phloem fibers, which are distributed in rows and present in groups (Figure
2.2).
A B C D E E
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Figure 2.2:
Figure 2.2: Histological analysis of Terminalia arjuna bark. Transverse section of Terminalia arjuna bark stained with Saffranine. Transverse section shows
medullary rays, phloem fibers, calcium oxalate crystals similar to that reported for bark of
Terminalia arjuna in Ayurvedic Pharmacopoeia of India.
2.3.2. Extraction of secondary metabolites from the bark of Terminalia
arjuna Metabolite extraction from bark of Terminalia arjuna was carried out using both
direct extraction and successive extraction method. Direct extraction was
performed at room temperature as well as in soxhlet apparatus. Successive
extraction was performed in soxhlet based extraction using solvent from non-
polar to polar range. The extraction conditions and protocol are mentioned in
section 2.2.3.
Calcium Oxalate crystal Phloem fibers
Medullary rays
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The extracts were coded as per following nomenclature:
Avesthagen AV 016Ba Su/Di (Temp. ext) 01(20)
1. AV- first two letters represents Avesthagen.
2. Plant Name: The Plants used and in use are assigned with unique 3-digit
number, 016 represents Terminalia arjuna.
3. Part of the plant /Tissue: There is a two letter ID for each plant part used.
Here Ba stands for Bark.
4. Solvents: The solvents used for extraction are also assigned with two
digit numbers 01 for Acetone, 02 for Benzene, 03 for Chloroform, 04 for
Ethanol, 05 for Hexane, 06 for Methanol, 08 for water, 09 for ethyl acetate. Percentage of solvents used for extraction is given within
bracket (20) for 20 % of that solvent. For example if 20% of Ethanol was
used for extraction, 04(20).
5. Method of Extraction: Successive extraction is referred to as Su
whereas direct extraction is referred to as Di, temperature for extraction is written in bracket. For example, Su(65) represents
successive extraction at 65 ºC.
The characteristics of extracts using direct extraction with 20% ethanol at room
temperature and soxhlet based extraction using absolute ethanol are
represented in Table 2.1. Similarly, Table 2.2 lists the characteristics of soxhlet
based successive extraction with non-polar to polar solvents.
Plant No &
Solvent No. & % Solvent
Type of extraction: Ext. t
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Table 2.1: Characteristics of direct extracts using 100% and 20% ethanol
Table 2.2: Characteristics of successive extracts using solvents ranging from non-polar to polar solvents
Sl. No Extract ID Solvent
used Extract color
Extract Texture
1 AV016BaSu(65)05(100) Hexane Yellowish Sticky
2 AV016BaSu(65)03(100) Chloroform Greenish Sticky
3 AV016BaSu(65)09(100)
Ethyl
acetate
Orangeish
brown powder
4 AV016BaSu(65)01(100) Acetone
Reddish
brown powder
5 AV016BaSu(65)06(100) Methanol Brownish powder
6 AV016BaSu(65)04(100) Ethanol
Reddish
brown powder
7 AV016BaSu(65)08(100) Water Brownish powder
Sl. No Extract ID Solvent
used Extract color
Extract Texture
1 AV016BaDi(65)04(100) Ethanol
Dark
Brown powder
2 AV016BaDi(25)04(20)
Hydro-
alcohol Brownish powder
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2.3.3 Metabolite profiling of different extracts from the bark of Terminalia
arjuna
Metabolomics is defined as comprehensive metabolite profiling of a given
biological system. Metabolite profiling of Terminalia arjuna was a technically
challenging task due to diversity of the metabolites along with its varying
concentrations. Absence of single analytical method that is capable of extracting
and detecting all metabolites at once makes the task even more challenging.
Currently, the most widely used technique involves reverse phase - HPLC
coupled with photodiode array (PDA), with or without mass spectrometry. Till date
this is the most sensitive method enabling the detection of hundreds of
compounds from a given extract.
To decipher metabolite fingerprints of different Terminalia arjuna bark extracts, I
have used MetaGridTM. MetaGridTM is a reproducible analytical technique for
metabolite fingerprinting, developed at Avesthagen Limited, Bangalore. This
technique consists of reverse phase liquid chromatography coupled with
photodiode array (PDA). The outputs of these results were analyzed using an in-
house developed software; where the results are depicted in the form a
metabolite signature. To obtain the metabolite fingerprint of different Terminalia
arjuna extracts, individual extracts were subjected to reverse phase HPLC based
separation. Each of the extract was standardized with respect to the mobile
solvent system and the run conditions for maximum separation of the metabolites
in composite extracts (section 2.2.4). Three HPLC runs are performed per extract
to generate a metabolite profile. HPLC profiles of different extracts are
represented in form of PDA chromatogram. Here, the absorption maxima of the
different metabolites present in respective composite extracts are recorded from
200 to 700nm at an interval of 10nm. Millenium32 software (Waters Corporation)
is used to generate this PDA chromatogram. PDA chromatogram is a 3-
dimensional chromatogram, where the retention times of the data points are
represented by x-axis, the wave-length (nm) of analysis on z-axis and the
intensity of nm absorption on y-axis.
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Further, the PDA chromatogram is used as an input data for MetaGridTM
software, which in turn represents the data in form of metabolite fingerprint. Here,
the x-axis represents the absorption range from 200-700nm and y-axis
represents the retention time. Additionally, the MetaGridTM software also gives
percentage conservation of metabolites between three individual HPLC runs for
each extract.
Direct and successive extracts from bark of Terminalia arjuna were subjected to
above mentioned HPLC based fingerprinting and MetaGridTM analysis. A
representative PDA chromatogram of each Terminalia arjuna extract at 254nm is
shown in Figure 2.3A-2.9A. Metabolite profile of Terminalia arjuna direct extracts
(Figure 2.3B-2.4B) and successive extracts (Figure 2.5B-2.9B) at 254nm gives a
representative metabolite signature of each extract.
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Figure 2.3: A.
AU
0.00
0.10
0.20
0.30
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
B.
Figure 2.3: HPLC profile and Metabolite finger printing of Direct 100% ethanol extract (AV016BaDi(65)04(100)). (A) Representative HPLC profile of the
extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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Figure 2.4: A.
B.
Figure 2.4: HPLC profile and Metabolite finger printing of Direct 20% ethanol extract (AV016BaDi(65)04(20)). (A) Representative HPLC profile of the extract
at 254 nm. (B) Profile of metabolite fingerprint for extract.
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 min-100
0
100
200
300
400
500
600
700 mAU 254nm4nm (1.00)
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Figure 2.5:
A. Metabolite profile of the extract at 254 nm
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00
B.
Figure 2.5: HPLC profile and metabolite finger printing of Successive 100% ethyl acetate extract (AV016BaSu(65)09(100)). (A) Representative HPLC profile of
the extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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Figure 2.6: A.
AU
0.00
0.20
0.40
0.60
Minutes0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
B.
Figure 2.6: HPLC profile and metabolite finger printing of Successive 100% acetone extract (AV016BaSu(65)01(100)). (A) Representative HPLC profile of the
extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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Figure 2.7: A.
AU
0.00
0.05
0.10
0.15
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
B.
Figure 2.7: HPLC profile and metabolite finger printing of Successive 100% ethanol extract (AV016BaSu(65)04(100)). (A) Representative HPLC profile of the
extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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Figure 2.8: A.
AU
0.00
0.10
0.20
0.30
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
B.
Figure 2.8: HPLC profile and metabolite finger printing of Successive 100% methanol extract (AV016BaSu(65)06(100)). (A) Representative HPLC profile of the
extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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Figure 2.9: A.
AU
0.00
0.10
0.20
Minutes0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00
B.
Figure 2.9: HPLC profile and metabolite finger printing of Successive 100% water extract (AV016BaSu(105)08(100)). (A) Representative HPLC profile of the
extract at 254 nm. (B) Profile of metabolite fingerprint for extract.
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2.3.4 Quantitative estimation of Terminalia arjuna bark extracts In absence of available standards, concentration of the class of metabolites is
used as parameter for representing reproducibility of extracts in commercial
batches. Standardized commercial phyto-extracts from green tea, grape seeds,
and other plant extracts are represented by presence of minimum concentration
of class of metabolites like total phenols. With the subsequent fractionations and
identifications of active molecule(s), using standards, the extracts are then
represented as minimum concentration of bioactive, eg catechins in green tea
extracts (Khokhar and Magnusdottir, 2002). Additionally, there as been positive
relationship between the total phenol content and free radical scavenging
potential. In addition to metabolic fingerprinting, Terminalia arjuna direct and
successive extracts were also estimated for total phenol content to account for
batch to batch variability. Gallic acid was used as standard and total phenol
content of Terminalia arjuna bark successive and direct extracts were
represented as gallic acid equivalent. Standard graph of gallic acid (Figure 2.10)
was made with concentrations ranging from 2 to 20 μg/ml following procedure
mentioned in section 2.2.5.
Figure 2.10:
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14 16 18 20
Concentration (μg/ml)
Abs
orba
nce at 750
nm
Figure 2.10: Standard curve with Gallic acid for determination of total phenol. Graph representing Gallic acid standard curve. X-axis represents concentration of
gallic acid represented in micrograms per milliliter. Y-axis represents absorbance at 750
nanometers.
45
It was found that the different Terminalia arjuna extracts showed total phenol
concentration on the range from 33 to 75% (Table 2.3).
Table 2.3: Total Phenol content of direct and successive extracts from bark of Terminalia arjuna
Sr. No.
Phyto-extract codes Name Of The
Phyto-extract Total Phenol
( % Equivalent To Gallic Acid)
1 AV016BaSu(65)09(100) Successive
Ethyl acetate 32.88 2 AV016BaSu(65)04(100) Ethanol 63.37 3 AV016BaSu(65)07(100) Methanol 55.94 4 AV016BaSu(105)08(100) Water 45.04
5 AV016BaDi(65)04(100) Direct 100%
ethanol 75.17
6 AV016BaDi(65)04(20) Direct 20%
ethanol 66.85
46
2.4 Discussion One of the challenges in herbal industry is to have quality control checks leading
to standardized methods for validation of the raw materials and the metabolites
extracted from plants. In plants without know standards this becomes even more
challenging.
In this study Terminalia arjuna bark under study was analyzed for its authenticity
by morphological, organoleptic, histological and chromatographic studies. Result
obtained reported desired characteristic of Terminalia arjuna bark (section 2.3.1;
Figure 2.1 & Figure 2.2). The bark was then taken for further solvent based
extraction. Direct and Successive extracts of Terminalia arjuna bark were profiled
by HPLC and Metagrid software. The MetagridTM generated metabolite profiles
for all the composite extracts, which became a reference fingerprint for quality
control and for checking variability between samples and extraction procedures.
The extracts under study showed metabolite conservation across HPLC runs
(section 2.3.3; Figure 2.3- 2.9).
The composite successive and direct extracts from bark of Terminalia arjuna
were further taken forward for activity validation using cell free and cell based
assay systems is discussed in Chapter 3
47
2.5 References :
Anonymous (2001). The Ayurvedic Pharmacopoeia of India Vol 1-4 (Delhi: The
Controller of Publications ).
Druilhe, P., Brandicourt, O., Chongsuphajaisiddhi, T., and Berthe, J. (1988).
Activity of a combination of three cinchona bark alkaloids against Plasmodium
falciparum in vitro. Antimicrob Agents Chemother 32, 250-254.
Khokhar, S. and Magnusdottir, S. G. M. (2002). Total Phenol, Catechin, and
Caffeine Contents of Teas Commonly Consumed in the United Kingdom. J.
Agric. Food Chem, 50 (3), 565–570
Ravishankar, B. a. S., V.J. (2007). INDIAN SYSTEMS OF MEDICINE: A BRIEF
PROFILE. African Journal of Traditional, Complimentary and Alternative
Medicines 4, 319-337
Wallis, T. E. (1957). Crystals in the leaf of Lobelia inflata Linn. J Pharm
Pharmacol 9, 663-665.