Chapter 7 Phytochemical analysis of S. colebrookiana and S. violacea

Contents 7.1. Introduction

7.2. Materials and methods

7.2.1 Phytochemical screening

7.2.1.1. Determination of total phenolics

7.2.1.2. Estimation of total flavonoids

7.2.2. Spectrophotometric analysis

7.2.3. HPLC analysis

7.2.4. HPTLC analysis

7.2.5. Fourier transforms infrared spectrophotometer (FT-IR)

7.3. Results

7.3.1. Qualitative and quantitative analysis of phytochemicals

7.3.2. UV-visible spectra and HPLC analysis

7.3.3. HPTLC fingerprinting profile

7.3.4. FTIR analysis

7.4. Discussion

List of tables

Table: 7.1. Phytochemical screening of chloroform extract of S. colebrookiana (SC) and S. violacea (SV)

Table: 7.2. Quantitative analysis for total phenol and flavonoids present in chloroform extract of S. colebrookiana and S. violacea

Table: 7.3. Chromatographic profile of chloroform extract of S. violacea

Table: 7.4. Chromatographic profile of chloroform extract of S. colebrookiana

Table: 7.5. FTIR spectral peak values and functional groups obtained for chloroform extract of S. colebrookiana

Table: 7.6. FTIR spectral peak values and functional groups obtained for chloroform extract of S. violacea

List of figures

Fig: 7. 1. Calibration curve for the determination of total flavonoids (A) and total

phenolics (B)

Fig: 7. 2. UV-Visible spectrum of chloroform extracts of Scutellaria and standard

baicalein

Fig: 7. 3. HPLC chromatogram of A) S. colebrookiana B) S. violacea and C)

standard baicalein

Fig: 7. 4. HPLC calibration curve of standard flavonoid, baicalein

Fig: 7. 5. HPTLC finger printing of chloroform extract of S. colebrookiana, S.

violacea and baicalein at 366 nm

Fig: 7. 6. HPTLC finger printing of chloroform extract of S. colebrookiana, S.

violacea and baicalein at 254 nm

Fig: 7.7. HPTLC chromatogram of chloroform extract of Scutellaria and baicalein

Fig: 7. 8. FTIR analysis of chloroform extract of S. colebrookiana

Fig: 7.9. FTIR analysis of chloroform extract of S. violacea

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7.1. Introduction

Medicinal plants are abundantly available all over the world and are more focused

than ever because they have the ability to produce many benefits to human society,

especially for the treatment of various types of human ailments. They serve as

therapeutic agents as well as important raw materials for the manufacture of

traditional and modern medicine (Ghani, 2003). They are the richest bio-resource

of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food

supplements, folk medicines, pharmaceutical intermediates and chemical entities

for synthetic drugs (Ncube et al., 2008). In most of the traditional systems of

treatment, the use of medicinal plant include the fresh or dried parts, whole,

chopped, powdered or an advanced form of the plant usually made through

extraction with different solvents play a major role and constitute the backbone of

the traditional medicine (Mukherjee, 1986).

The therapeutic properties of medicinal plants are due to some chemical

compounds they synthesize. These are regarded as secondary metabolites because

the plants that synthesise them may have little need for them (Duraipandiyan et al.,

2006). They are synthesized in all parts of the plant body; bark, leaves, stem, root,

flower, fruits, and seeds (Tiwari et al., 2011). Plant produces these chemicals to

protect itself from herbivores but recent research demonstrates that many

phytochemicals can protect humans against diseases. Different phytocontituents

present in medicinal plants are flavonoids, carotenoids, alkaloids, anthocyanidins,

phenolics and tannins, carboxylic acids, terpenes, amino acids, and inorganic acids

etc (Argal et al., 2006).

Flavonoids have been demonstrated to have anti-inflammatory, anti-allergenic,

anti-viral, anti-aging, and anti-carcinogenic activity (Kuhnau et al., 1976; Havsteen

et al., 1983; Middleton et al., 1984; Cody et al., 1986). Pro-anthocyanidins are also

associated with a number of biological activities, such as anti-inflammatory

(Torras et al., 2005), anti-asthmatic (Lau et al., 2004), anti-cancer (Ray et al,

2005), anti-microbial, anti-allergy, anti-hypertensive and cardioprotective

(Kohama et al., 2004) properties. Beta carotene and other carotenoids are also

believed to provide anti-oxidant protection to lipid-rich tissues (Jacob, 1995; Sies

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et al, 1995). Phenolic compounds are reported to possess anti-oxidant (Lekse et al.,

2001; Braca et al., 2002) and anti-cancer properties (Hirvonen et al., 2001).

Alkaloids have diverse structures and many show a range of pharmacological

activities including antimicrobial activity (Ahmed et al., 1986).

Presence of these phytoconstituents gives specific distinctiveness and properties to

plants (Parekh et al., 2007). Therefore, the analysis of these constituents would

help in determining various biological activities of plants. Knowledge of the

chemical constituents of plant secondary metabolite is also desirable because such

information will be helpful for the synthesis of complex chemical substances. A

variety of techniques can be used to determine the presence of such

phytocontituents in medicinal plants. However, simple, cost-effective and rapid

tests for detecting phytocomponents are necessary. Spectroscopic (UV-Vis, FTIR)

methods together or separate can be used in this sense as well as conventional

methods (Ibrahim et al., 2008). Ultraviolet/visible spectrophotometry (UV/Vis)

related to the spectroscopy of photons in the UV-visible region. UV-visible

spectroscopy uses light in the visible ranges or its adjacent ranges. The colour of

the chemicals involved is directly affects the absorption in the visible ranges.

Molecules undergo electronic transitions in these ranges of the electromagnetic

spectrum (Gunasekaran, 2003).

At present, particularly in phytochemistry, FTIR has been exercised to identify the

concrete structure of certain plant secondary metabolites (Yang and Yen, 2002).

Fourier Transform Infrared Spectrometry (FTIR) spectroscopy is an established,

time‐saving method to identify the structure of unknown composition or its

chemical group, and the intensity of the absorption spectra associated with

molecular composition or content of the chemical group (Surewicz, 1993). FTIR is

a physico-chemical analytical technique that does not resolve the concentrations of

individual metabolites but provides a snapshot of the metabolic composition of a

tissue at a given time (Griffiths and Haseth, 1986). The FT-IR method measures

the vibrations of bonds within chemical functional groups and generates a

spectrum that can be regarded as a biochemical or metabolic “fingerprint” of the

sample. By attaining IR spectra from plant samples, it might possible to detect the

minor changes of primary and secondary metabolites (Surewicz et al., 1993).

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The optimized chromatographic finger print is not only an alternative analytical

tool for authentication, but also an approach to express the various patterns of

chemical ingredients distributed in the herbal drugs. Chromatographic

fingerprinting is a rational option to meet the need for more effective and powerful

quality assessment to ITM (IndianTraditional Medicine) and TCHM (Traditional

Chinese Herbal Medicine). High-performance liquid chromatography (HPLC) is

proved to be a rapid and sensitive method to analyze the majority of the

constituents in herbal medicines particularly for the detection of those present in

minor or trace quantity. Methods established using HPLC technique facilitates the

convenient and rapid quality control of traditional medicines and their

pharmaceutical preparations (Thenmozhi et al., 2011). Currently, HPTLC is often

used as an alternative to HPLC for the quantification of plant products because of

its simplicity, reliability, accuracy, cost-effectiveness and rapidity (Wasim et al.,

2008). HPTLC fingerprint has better resolution and reasonable accuracy in

estimating active in a shorter time (Pawar et al., 2011). It can serve as a tool for

identification, authentication and quality control of herbal drug (Ram et al., 2011).

Experimental studies have demonstrated the anti-oxidant, anti-inflammatory, anti-

tumour, anti-mutagenic, anti-carcinogenic and anti-cancer properties of chloroform

extract of S. colebrookiana and S. violacea. Hence, the present study has been

made to investigate the phytochemical screening of chloroform extract of S.

colebrookiana and S. violacea using UV-VIS spectrophotometry, HPLC, HPTLC

and FTIR.

7.2. Materials and methods

7.2.1. Phytochemical screening

Phytochemical examinations were carried out to find out the major class of

phytochemicals present in Scutellaria chloroform extracts. Detailed procedure is

given in chapter 2, section 2.2.5.

7.2.1.1. Determination of total phenolics

The amount of phenolics in chloroform extract of S. colebrookiana and S. violacea

was determined with Folin-Ciocalteu reagent. Added 2.5 ml of 10% Folin-

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Ciocalteu reagent and 2 ml of Na2CO3 (2% w/v) to 0.5 ml of each sample (3

replicates) of plant extract solution (1 mg/ml). The resulting mixture was incubated

at45°C with shaking for 15 min. The absorbance of the samples was measured at

765 nm using UV/visible light. The content of total phenol was calculated on the

basis of the calibration curve of gallic acid and the results were expressed as mg of

gallic acid equivalents (GAEs) per gram of extract.

7.2.1.2. Estimation of total flavonoids

Aluminum chloride colorimetric method was used for flavonoid determination in

chloroform extract of S. colebrookiana and S. violacea. Extract (1 mg/ml) was

mixed with 3 ml of methanol, 0.2 ml of10% aluminum chloride, 0.2 ml of 1 M

potassium acetate and 5.6 ml of distilled water and remains at room temperature

for 30 min. The absorbance of the reaction mixture was measured at 420 nm with

UV visible spectrophotometer. The flavonoid content was calculated on the basis

of the calibration curve of quercetin and the results were expressed as mg of

quercetin equivalents per gram of extract.

7.2.2. Spectrophotometric analysis

The extracts were examined under visible and UV light for proximate analysis.

Standard baicalein (1 mg/20 mL), S. colebrookiana and S. violacea extracts (5

mg/25 mL) were dissolved in methanol and centrifuged. The supernatant was

collected, scanned the absorbance from 200 to 900 nm using a UV-visible

spectrophotometer and the characteristic peaks were analyzed using UV win5

software (P.G. Instruments +80, UK).

7.2.3. HPLC analysis

Chloroform extract of S. colebrookiana and S. violaceaand standard baicaleinwere

dissolved in acetonitrile and centrifuged for 5 minutes at 10,000 rpm. The

supernatant was collected, filtered through 0.45 µm nylon filterand injected 20 µl

sample to end capped, purospher Star column rp-18 (5 μm, 250 x 4.60 mm size)

(Merck, Germany). The column was eluted by acetonitrile with flow rate 1 ml/min

using Shimadzu SPD-10AVP HPLC system equipped with UV-VIS detector. The

column temperature was kept at 25°C during running and the absorbance of eluted

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sample was measured at 274 nm. The standard stock solution of baicalein was

prepared in methanol at different concentrations such as 12.5, 25, 50, 100 and 200

µg/ml to plot the calibration curve. The same calibration curve was subsequently

used for quantification of baicalein in S. colebrookiana and S. violacea.

7.2.4. HPTLC analysis

Chloroform extracts of S. colebrookiana and S. violacea along with standard

baicalein were tested to obtain characteristic finger printing profile using HPTLC

(CAMAG Switzerland Instrument). HPTLC was performed on 20 × 10 cm

aluminium backed plates coated with silica gel 60F254 (Merck, Mumbai, India).

Standard solution of baicalein and sample solution were applied to the plates as

bands 8.0 mm wide, 30.0 mm apart, and 10.0 mm from the bottom of the plate and

15 mm from the sides edge of the same chromatographic plate by use of a Camag

(Muttenz, Switzerland) Linomat V sample applicator equipped with a 100-μL

Hamilton (USA) syringe. Ascending development to a distance of 80 mm was

performed at room temperature (28 ± 2°C), with toluene: acetone: formic acid,

4.5:4.5:1 (v/v/v), as mobile phase, in a Camag glass twin-trough chamber

previously saturated with mobile phase vapour for 20 min. After development, the

plates were dried with a hair dryer and then scanned at 254 and 366 nm with a

Camag TLC Scanner with WINCAT software, using the deuterium lamp.

Photographs were recorded by CAMAG TLC visualizer.

7.2.5. Fourier Transforms Infrared Spectrophotometer (FT-IR)

FT-IR is perhaps the most powerful tool for identifying types of chemical bonds

(functional groups). The wavelength of light absorbed is characteristic of the

chemical bond as can be seen in this annotated spectrum. By interpreting the

infrared absorption spectrum, the chemical bonds in a molecule can be determined.

Chloroform extract of S. colebrookiana and S. violacea were used for FTIR

analysis. The powdered extracts were treated for FTIR spectroscopy (Shimadzu, IR

Affinity 1, Japan) with scan range: from 500 to 3500 cm−1 with a resolution of 4

cm−1. The spectra obtained for the extracts was analyzed and interpreted with a

chart for characteristics infrared absorption frequencies of organic functional

groups and carbonyl containing functional groups.

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7.3. Results

7.3.1. Qualitative and quantitative analysis of phytochemicals

Preliminary phytochemical screening of chloroform extract of S. colebrookiana

and S. violacea showed the presence of phytoconstituents like alkaloids,

flavonoids, carbohydrates, phenols, proteins, phytosterols and diterpenes (Table

7.1). Tannins and saponins were totally absent in both of the extracts. The amount

of total phenolic content in the tested extracts was determined by Folin-Ciocalteu

method and expressed as gallic acid equivalents (GAE) from the calibration curve

(R2=0.997) while total flavonoid contents was calculated as quercetin quivalents

(mg QE/g) from the calibration curve (R2= 0.979) (Figure 7.1A and B). Chloroform

extract of S. colebrookiana and S. violacea had total phenolic content of 380 ± 0.23

and 203.7 ± 1.4 mg GAE/g extract, respectively. Total flavonoid content of S.

colebrookiana was 111 ± 0.56 and that of S. violacea was 84.5 ± 0.18 mg quercetin

equivalents (QE)/g (Table 7.2).

7.3.2. UV-visible spectra and HPLC analysis

The spectrophotometric analysis of chloroform extract of S. colebrookiana and S.

violacea extracts scanned within wavelength range 200-900 nm showed three

peaks (210, 276, and 360 λ) which are similar to the standard baicalein (Figure

7.2). The HPLC analysis of S. colebrookiana and S. violacea was conducted along

with baicalein. HPLC chromatogram obtained for chloroform extract of both

Scutellaria species showed similar profiling with a major peak with retention time

of 3.4 min. The identity of the peak of baicalein in the extract chromatograms was

confirmed by spiking with standard baicalein (Figure 7. 3). The retention time of

baicalein (3.4 min) was found to be same to that of peak present in the

chromatogram of extract (3.4 min). HPLC method also enables quantitative

analysis of baicalein in S. colebrookiana and S. violacea. The calibration curve for

baicalein revealed significant linearity between concentration and area (y = 0.174x

–0.182, R2– 0.998). Moreover, the concentration of baicalein in chloroform extract

of S. colebrookiana and S. violacea was determined from the regression equation

as 4.6 and 3.5 % respectively (Figure 7.4).

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7.3.3. HPTLC fingerprinting profile

The chloroform extract of S. colebrookiana and S. violacea were run along with

the standard baicalein for HPTLC analysis. Figure 7.5 and 7.6 showed the

chromatographic bands obtained at 366 and 254 nm for S. colebrookiana and S.

violacea respectively. The identity of baicalein peak in chloroform extract of S.

colebrookiana and S. violacea was confirmed by comparing to that obtained for

standard baicalein. The peak corresponding to baicalein from the extracts had same

retention factor as that for the baicalein standard (Figure 7.7, Table 7.3 and 7.4).

7.3.4. FTIR analysis

The FTIR spectra analysis was utilized to identify the functional group of the

active ingredients on the basis of peak value in the vicinity of infrared radiation.

Results of FTIR peak values and functional groups of S. colebrookiana and S.

violacea chloroform extract are represented in figure 7.8 and 7.9. Chloroform

extract of S. colebrookiana showed characteristic absorption bands at 3460 cm-

1(for a hydroxyl group), 2962 cm-1 (for C-H stretching), 1728 cm-1 (for a carbonyl

group (C=O), 1668 and 1490 cm-1 (C=C ring stretch) and at 1261 cm-1 (C-O

stretching vibration), The characteristic absorption band were exhibited at 3446

cm-1(for a hydroxyl group), 1726 cm-1 (for a carbonyl group, C=O), 1261 cm-1 (C-

O stretching vibration), 1606 cm-1,1508 cm-1,1450 cm-1 and 1415 cm-1 (C=C

stretching) by S. violacea extract (Table 7.3 and 7.4).

7.4. Discussion

In recent times, many medicinal plants have been used as alternative medicine for

treatments or preventions of a number of diseases, including diabetes,

hyperlipidemia, cancer and Alzheimer’s diseases. Medicinal plants have become

very popular because they have very few side effects as compared to synthetic

drugs (Shruthi et al., 2012). The curative properties of medicinal plants are

conceivably due to the presence of a variety of secondary metabolites such as

alkaloids, flavonoids, glycosides, saponins, tannins, phenolic compounds and

steroids etc. They are not indispensable for the plant that contains them; their

production is secondary to plant hence the name secondary metabolites.

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Accordingly, the World Health Organization (WHO) consultative group on

medicinal plants has formulated a definition of medicinal plants in the following

way: “A medicinal plant is any plant, in which one or more of its organ, contains

substance that can be used for therapeutic purpose orwhich is a precursor for

synthesis of useful drugs” (Goldstein, 1974).

The preliminary phytochemical screening of a plant or plant parts might be helpful

in the nature of active principles and sometimes may lead to the discovery and

development of new compounds. The phytochemical analysis conducted on S.

colebrookiana and S. violacea revealed the presence of flavonoids, alkaloids,

polyphenolics, phytosterols and proteins. Quantitative phytochemical analysis

indicated that the chloroform extract of S. colebrookiana and S. violacea contain

significant amounts of phenolics and flavonoids. Phenolics and flavonoids are

ubiquitously seen in most of the plant species and reported to possess a broad

spectrum of biological properties (Nijveldt et al., 2001).

Scutellaria baicalensis is one of the most famous herb comes under Scutellaria

genus and widely used for treating a number of ailments in Traditional Chinese

Medicine (TCM). The active ingredients of S. baicalensis are reported as

flavonoids such as baicalein, baicalin, wogonin and wogonoside. Among these

flavonoids, baicalein is the most active one and reported to have various beneficial

effects such as anti-oxidant, anti-inflammatory, anti-cancer, hepatoprotective, anti-

mutagenic etc. Being members of Scutellaria genus, S. colebrookiana and S.

violacea might contain the above flavonoids. To detect the presence of most active

flavonoid baicalein, run spectrophotometric and HPLC analysis of chloroform

extract of S. colebrookiana and S. violacea along with baicalein.

Interestingly, spectrophotometric analysis between 200 and 900 nm range showed

compound with absorption maxima at 210, 276, and 360 λ which was closely

similar to standard baicalein. No other visible peaks were observed in the spectra

which indicate that a simple soxhlet extraction itself yields baicalein with fewer

impurities. HPLC method facilitates qualitative and quantitative analysis of

baicalein in S. colebrookiana and S. violacea. Peaks obtained for extracts showed

identical retention time to that of baicalein and confirmed its presence in S.

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colebrookiana and S. violacea. To estimate quantity of baicalein, prepared

calibration curve of baicalein and from that curve, quantity of baicalein present in

root extract of both plants could detect.

HPTLC finger printing profile is valuable as a phytochemical marker and also a

good estimation of genetic variability in plant populations. Also it is an economical

method for separation, qualitative identification, or semi-quantitative analysis of

samples and can be used to solve many qualitative and quantitative analytical

problems in a wide range of fields, including medicine, pharmaceuticals,

chemistry, biochemistry, food analysis, toxicology and environmental analysis

(Jain et al., 2009). Here, HPTLC analysis of chloroform extract of S.

colebrookiana and S. violaceawas conducted to figure out active ingredients

present in extracts. From HPTLC studies, it has been found that chloroform extract

of S. colebrookiana and S. violacea contain not a single compound but a mixture of

compounds and so it is established that the pharmacological activity shown by

them are due to the cumulative effect of all the compounds in composite. HPTLC

analysis further confirms the presence of baicalein in chloroform extract of these

two plants. Previous studies detected the pharmacological properties of S.

colebrookiana and S. violacea including anti-oxidant, anti-inflammatory, anti-

mutagenic, anti-tumour etc. Presence of baicalein in S. colebrookiana and S.

violacea might partly responsible for the observed pharmacological activities of

these plants.

FTIR analysis of chloroform extract of S. colebrookiana and S. violacea showed

presence of hydroxyl group which is common in all phenolic compounds.

Scutellaria extracts absorption bands were attributed to (OH) stretching vibrations

from phenols (chemical) containing hydroxyl functional groups (-OH) attached to

an aromatic hydrocarbon. This strongly supports the presence of phenolic

compounds (flavonoids) in chloroform extract of S. colebrookiana and S. violacea

as evidenced by phytochemical analysis.

In conclusion, phytochemicals present in S. colebrookiana and S. violacea

indicates their potential as a source of principles that may supply novel medicines.

UV-Visible spectroscopy, HPLC and HPTLC analysis of extracts confirmed the

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presence of flavonoid baicalein. Further investigation will be desirable to find out

the structural analysis of flavonoid compounds by use of different analytical

methods such as NMR and mass spectrophotometer. So the plant could act as a

potent source of this multiple target flavonoid which can be used for impending

drug development.

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Table: 7.1. Phytochemical screening of chloroform extracts of S. colebrookiana (SC) and S. violacea (SV)

Plant AL CH SA PS PH TA FL PT DT SC + + _ + + _ + + +

SV + + _ + + _ + + +

AL-alkaloids PH-phenols PT-proteins

CH-carbohydrates TA-tannins DT-diterpenes

SA-saponins FL-flavonoids PS-phytosterols

Table: 7. 2. Quantitative analysis for total phenol and flavonoids present in chloroform extract of S. colebrookiana and S. violacea

The values are expressed as mean ± standard deviation.

Chemical constituents Plants S. colebrookiana

S. violacea

Total phenol (mg GAE/gm) 380.5 ± 0.23 203.7 ± 1.4

Flavonoids (mg QE/gm) 111 ± 0.56 84.5 ± 0.18

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Table: 7.3. HPTLC chromatographic profile of chloroform extract of S. violacea

Crude extract Solvent system Detection Rf values

Chloroform

Toluene: acetone: formic acid (4.5:4.5:1)

At 254 and 366 nm in UV light

0.46 0.60 0.67 0.76 0.86

Standard Baicalein Toluene: acetone:

formic acid (4.5:4.5:1)

At 254 and 366 nm in UV light

0.60

Table: 7.4. HPTLC chromatographic profile of chloroform extract of S. colebrookiana

Crude extract Solvent system Detection Rf values

Chloroform

Toluene: acetone: formic acid (4.5:4.5:1)

At 254 and 366 nm in UV light

0.45 0.60 0.67 0.74 0.88

Standard Baicalein Toluene: acetone:

formic acid (4.5:4.5:1)

At 254 and 366 nm in UV light

0.60

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Table: 7.5. FTIR spectral peak values and functional groups obtained for chloroform extract of S. colebrookiana

Table: 7.6. FTIR spectral peak values and functional groups obtained for chloroform extract of S. violacea

Peak values Functional groups

3460 cm-1 OH stretching

2962 cm-1 Aromatic CH stretching

1728 cm-1 Presence of C=O

1668 cm-1

1490 cm-1

C=C ring stretch

C=C ring stretch

1357 cm-1 C-O-H bending

1261 cm-1 C-O stretching vibration

1164 cm-1 Presence of C-CO-C

1095 cm-1 C-O-C Stretching

802 cm-1 Aromatic CH bending

Peak values Functional groups

3446 cm-1 OH stretching

1726 cm-1 presence of C=O

1508 cm-1

1450 cm-1

1415 cm-1

C=C stretching

C=C stretching

C=C stretching

1261 cm-1 C-O stretching vibration

Summary

and

Conclusion