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:



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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.



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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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


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

Documentation

Text Box

34

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.

36

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.

37

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.

38

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)

39

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.

40

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


41

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


42

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


43

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


44

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.

chapter 2: metabolite extraction and fingerprinting of terminalia arjuna...

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