Chapter 7: Phytochemical examination of Phellinus rimosus

TABLE OF CONTENTS

7.1. INTRODUCTION

7.2. MATERIALS AND METHODS

7.2.1. Preparation of the extract

7.2.2. Phytochemical evaluation of the aqueous ethanol extract

7.2.2.1. Preliminary phytochemical screening

7.2.2.1.1. Test for carbohydrates/ Glycosides: (Molisch’s test)

7.2.2.1.2. Test for Saponins

7.2.2.1.3. Test for Alkaloids

7.2.2.1.3.1. Dragendroff 's test

7.2.2.1.3.2. Wagners' test

7.2.2.1.3.3. Mayer's test

7.2.2.1.4. Test for Steroids/Terpenoids (Liberman- Burchard test)

7.2.2.1.5. Test for Flavnoids (Shinoda’s test)

7.2.2.1.6. Test for Phenolics

7.2.2.1.7. Test for Coumarins

7.2.2.1.8. Test for tannins

7.2.3. Estimation of total carbohydrate

7.2.3.1. Estimation of total carbohydrate in the extract by Anthrone method

7.2.3.2. Estimation of total carbohydrate in the extract by phenol-sulphuric acid method

7.2.4. Estimation of protein in the extract by Lowry’s Method

7.2.5. Thin layer Chromatography (TLC) analysis of the extract

7.2.5.1. Preperation of spray reagents

7.2.5.1.1. 10% Ethanolic KOH

7.2.5.1.2. 10 % Methanolic ferric chloride

7.2.5.1.3. Anisaldehyde-Sulphuric acid reagent

7.2.6. HPTLC Analysis

7.3. RESULTS

7.4. DISCUSSION

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7.1. INTRODUCTION

Phytochemistry is the branch of chemistry concerned with the enormous variety of

complex chemical compounds found in plant kingdom and deals with the biosynthesis,

metabolism, natural distribution, isolation, structure elucidation and biological functions of

these substances. It is an interdisciplinary subject related to chemistry, botany and

pharmacology. Chemical characteristics of mushroom have been noted and received

considerable attention in recent years. The development of newer and faster screening

techniques based on chromatography has led to the rapid identification of large number of

compounds which in turn and has led to natural products receiving more attention from

chemists and pharmacologists (Ohsaki et al., 1999).

Mushrooms have long been valued as delicious and nutritional foods in many

countries. Mushrooms are appreciated, not only for texture and flavour but also for their

chemical and nutritional characteristics (Manzi et al., 1999). A large number of secondary

metabolites from mushroom such as lectins, alkaloids, terpenes, and antibiotics have an

established history of application in medicine (Wasser, 2010). On a dry weight basis, they

are considered to be good sources of digestible proteins (10–40%), carbohydrates (3–21%)

and dietary fibre (3–35%). Mushrooms contain all the essential amino acids and are

limiting in the sulfurcontaining amino acids, cysteine and methionine (Breene, 1990;

Chang, 1991). Although mushrooms contain all the main classes of lipids, including free

fatty acids, mono-, di- and tri-glycerides, sterol esters and phospholipids, their levels are

low at approximately 2–8% (on dry weight basis) and the calorific value of most

mushrooms is also low. Mushrooms are excellent sources of thiamine (vitamin B1),

riboflavin (vitamin B2), nicotinic acid (vitamin B3), biotin and ascorbic acid (vitamin C).

Edible mushrooms in cooked or other processed forms are nutritionally sound and good

dietary component for vegetarians (Breene, 1990) and are suitable for diabetic and heart

patients. Mushrooms are not only sources of nutrients but have also been reported as

therapeutic foods, useful in preventing diseases such as hypertension,

hypercholesterolemia, cancer (Bobek et al., 1995; Bobek and Galbavy, 1999). Some

recently isolated and identified compounds, originating from mushrooms, show other quite

significant medical properties, such as immunomodulatory, cardiovascular, liver

protective, anti-fibrotic, anti-inflammatory, anti-diabetic, anti-viral and antimicrobial

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activities (Gunde-Cimerman, 1999; Ooi and Liu, 1999; Wasser and Weis, 1999a, 1999b;

Ooi, 2000).

The investigations carried out in our research lab showed that the aqueous-ethanol

extract of P. rimosus possessed significant antioxidant, antitumor, hepatoprotective,

nephroprotectiv, radio protective and antidiabetic activity. Since the biological activities

were directly related to the bioactive constituents, investigations were carried out to

examine the major constituents of aqueous-ethanol extract of Phellinus rimosus and the

findings are presented in this chapter.

7.2. MATERIALS AND METHODS

7.2.1. Preparation of the extract

Aqueous-ethanol (70%) extract of P. rimosus was prepared as described in section

2.2.1.

7.2.2. Phytochemical evaluation of the aqueous ethanol extract

7.2.2.1. Preliminary phytochemical screening

The aqueous ethanol extract of P. rimosus was analysed for the presence of

secondary metabolites by standard methods (Harborne, 1973; Wagner et al., 1984; Daniel,

1991) as follows,

7.2.2.1.1. Test for carbohydrates/ Glycosides: (Molisch’s test)

5 mg of extract was mixed with 0.5 ml of distilled water and add 2 drops of 10 %

α-naphthol in ethanol. To this mixture 1 ml of concentrated H2SO4 was added slowly

through the sides of the test tube so that it forms a ring at the middle of the two solutions.

The formation of a reddish violet ring indicates the presence of carbohydrate/ glycosides.

7.2.2.1.2. Test for Saponins

A little of the extract was shaken thoroughly with distilled water and the formation

of froth in the test tube, which persists for a few min, shows the presence of saponins.

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7.2.2.1.3. Test for Alkaloids

7.2.2.1.3.1. Dragendroff 's test

The Dragendroff 's reagent was prepared by mixing solution A (containing 0.6 g of

bismuth subnitrate in 2 ml of concentrated HCl and 10 ml of distilled water), solution B

(containing 6 g of KI in 10 ml of water) together with 7 ml of concentrated HCl and 15 ml

distilled water and the whole solution was diluted with distilled water to 100 ml to form

Dragendroff 's reagent. Many alkaloids give a brown precipitate with Dragendroff 's

reagent.

7.2.2.1.3.2. Wagners' test

1.72 g of Iodine and 2 g of KI were dissolved in 5 ml of distilled water and the

solution was made up to 100 ml with distilled water to form Wagner's reagent. Many

alkaloids give a brown flocculent precipitate with Wagner's reagent.

7.2.2.1.3.3. Mayer's test

The Mayer's reagent was prepared by mixing solution A (1.36 g of HgCl2 in 60 ml of

distilled water) and solution B (5 g of KI in 10 ml of distilled water) and then the mixture

was made up to 100 ml with distilled water. Since this reagent reacts only with salts of

alkaloids, the alkaloidal solution should be made distinctly acidic with HCl or H2SO4

before adding the reagent. The reagent was added drop wise. A white precipitate indicates

the presence of alkaloids.

7.2.2.1.4. Test for Steroids/Terpenoids (Liberman- Burchard test)

A little of the extract was mixed with CHCl3, to this adds 1 ml of acetic anhydride

and mixed well. 1 ml of concentrated H2SO4 was added through the sides of the tube. A

green colour indicates the presence of steroids and pink colour indicates the presence of

terpenoids.

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7.2.2.1.5. Test for Flavnoids (Shinoda’s test)

Extract was dissolved in methanol and magnesium turnings or ribbons were added

followed by concentrated HCl drop by drop. Pink colour indicates the presence of

Flavonoids.

7.2.2.1.6. Test for Phenolics

10% alcoholic solution of ferric chloride was prepared. The extract was mixed with

this solution and the formation of a green colour with alcoholic ferric chloride indicates the

presence of phenols.

7.2.2.1.7. Test for Coumarins

Extract was dissolved in methanol and alcoholic NaOH is added. A yellow colour

appears which later disappears on adding drops of concentrated HCl indicate the presence

of coumarins.

7.2.2.1.8. Test for tannins

To 2ml of the extract, a few drops of the lead acetate solution were added. The

formation of white precipitate indicates the presence of tannins in the extract.

7.2.3. Estimation of total cabohydrate

7.2.3.1. Estimation of total carbohydrate in the extract by Anthrone method

Principle:

Carbohydrates are first hydrolysed into simple sugars using dilute hydrochloric

acid. In hot acidic medium, glucose is dehydrated to hydroxymethyl furfural. This

compound forms a green coloured product with anthrone (absorption maximum at 630

nm).

Procedure:

100 mg of the sample is hydrolysed by 5 ml of 2.5 N HCl in a boiling water bath

for 3 hrs. It was neutralized with solid sodium carbonate until the effervescence ceases,

made up to 100 ml and centrifuged. Collected 0.5 and 1 ml of the supernatant and was

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used for the analysis. The standards are prepared by selecting 0.2, 0.4, 0.6, 0.8 and 1 ml of

working glucose standard. Volume in each tube (including the sample tube) is made up to

1 ml with distilled water. Four ml of anthrone reagent (Anthrone reagent: Dissolve 200 mg

anthrone in 100 mL of ice-cold 95% H2SO4. Prepare fresh before use) added to all the

tubes and heated for eight min in boiling water bath. Read the green to dark colour at 630

nm. The amount of carbohydrate in the sample was calculated from the calibration graph.

7.2.3.2. Estimation of total carbohydrate in the extract by phenol-sulphuric acid

method

Quantitative examination of carbohydrate was carried out according to Dubois et al.

(1956) method using glucose as standard

Principle:

In hot acidic medium glucose is dehydrated to hydroxymethyl furfural. This forms

a coloured complex with phenol and has absorption maxium at 490 nm.

Procedure:

0.1 and 0.2 ml of the test solution having unknown concentration was made up to 1

ml with distilled water and added 1 ml of 5% phenolic solution. Then 5 ml of 97% H2SO4

was added through the side of the tubes without disturbing the tubes and mixed well. After

10 min, the contents in the test tubes were kept in boiling water bath for 10 min and the

O.D was noted at 490 nm. The concentration of carbohydrate in the test solution was

calculated from the standard graph.

7.2.4. Estimation of protein in the extract by Lowry’s Method

The amount of protein present in the extract was determined by the method of

Lowry et al. (1951) using bovine serum albumin as the standard as described in the section

2.2.3.

7.2.5. Thin layer Chromatography (TLC) analysis of the extract

Principle:

Partition chromatography is the distribution of the solute between two liquid

phases, which is based primarily on the solubility differences. The position of each

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component of a mixture is determined by calculating the distance travelled by the

component relative to the distance travelled by the solvent, relative mobility (Rf value).

The Rf value for a substance is constant for a certain set of experimental conditions.

Procedure:

TLC anlysis of the extract was performed on a pre-coated silica gel plate purchased

from Merk India Ltd. Pre-coated silica gel plate was kept in hot air oven for 1 hr at 1100 C

for activation. The sample (70 % aqueous - ethanol extract) was dissolved in small amount

of methanol and spotted on the TLC plates 2cm above the base of the plates. Solvent

systems (Toluene: Ethyl acetate: Formic acid 5:5:0.3 for the spray reagent 10 % ethanolic

KOH and methanolic ferric chloride and Chloroform:Methanol 8:2 for spray reagents

anisaldehyde sulphuric acid and Vanillin sulphuric acid ) were poured in to a TLC jar and

left undisturbed for half an hour for saturation with vapours. The plate was then placed in

the solvent at 450 angles and allowed the solvent to run. After complete solvent run the

plate was removed from the jar, the solvent front was marked and allowed to dry at room

temperature. The plates were examined under UV for detecting fluorescent spots. The

plates were then sprayed with specific spray reagents to detect different constituents.

7.2.5.1. Preperation of spray reagents

7.2.5.1.1. 10% Ethanolic KOH

10g of KOH was dissolved in 100 ml of ethanol. Formation of red colour after

spraying with this reagent indicates the presence of quinones

7.2.5.1.2. 10 % Methanolic ferric chloride

10g of FeCl3 was dissolved in 100 ml of methanol. Formation of green colour after

spraying with this reagent indicates the presence of Phenolic compounds.

7.2.5.1.3. Anisaldehyde-Sulphuric acid reagent

0.5 ml anisaldehyde is mixed with 10 ml glacial acetic acid, followed by 85ml

ethanol and 5ml concentrated sulphuric acid in that order (The reagent has only limited

stability, and is no longer usable when the colour has turned to red violet). The plate is

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sprayed with anisaldehyde-Sulphuric acid reagent, heated at 1000C for 5-10 minutes, then

observed in visible or in UV 365 for the detection of the terpenoids (Wagner et al, 1984).

7.2.6. HPTLC Analysis

Ten mg of the extract was dissolved in 10 ml methanol and used for analysis. The

samples (5 μl) were applied as bands using microsyringe on precoated silica gel 60 F254

plates (E. Merck). The plates after sample application were developed in twin trough

chambers. Toluene : ethylacetate : methanol : formicacid (50: 50 : 5 : 5) was used as the

solvent system. The plates were air dried after development and scanned under UV (254

nm) or sprayed with ethanolic KOH reagent and then scanned. Camag TLC scanner 3 was

used for scanning. HPTLC profile was obtained with Desaga Video Documentation Unit

III.

7.3. RESULTS

Phytochemical screening of the aqueous ethanol extract of P. rimosus showed the

presence of polysaccharides, proteins, terpenoids, saponins, phenols, flavonoids and

quinones in the extract (Table 7.1). Steroids, alkaloids, coumarins and tannins are found to

be absent in the extract.

TLC is one of the simplest method for detection of metabolite constituents

(Hostettmann and Wolfender, 1997). The detection on TLC plates depends on the

compound and based on the natural colour, reaction with spray reagents, fluorescence at

UV-365 and 254 nm. TLC analysis of the aqueous-ethanol extract of P. rimosus showed

many spots under UV. The use of selective or universal chemical spray reagents to

determine the classes of compounds has also widely reported (Wagner et al., 1984). TLC

plates showed two red coloured spots with ethanolic KOH spray reagent and this indicated

the probable presence of quinone in the extract. However the extract when sprayed with

methanolic ferric chloride showed the presence of one green coloured spot indicates the

presence of phenolic compounds.

Table 7.1: Phytochemical screening of aqueous-ethanol extract of P. rimosus

Class of compound

P. rimosus

Polysaccharide

+

Proteins

+

Terpenoids

+

Steroids

-

Alkaloids

-

Saponins

+

Flavanoids

+

Coumarins

-

Phenolics

+

Quinone

+

Tannins

-

+ indicates presence of compound - indicates absence of compound

Table 7.2: HPTLC analysis of the P. rimosus extract using Toluene : ethylacetate :

methanol : formicacid (50: 50 : 5 : 5) as the solvent system.

Extract

Rf values

Aqueous ethanol extract

0.07, 0.20, 0.27, 0.32, 0.42, 0.46, 0.53, 0.74, 0.76.

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HPTLC analysis showed blue and yellow coloured bands under UV. However the

HPTLC analysis revealed that the extract contains a large number of minor components

(Fig 7.1.A, Fig 7.1.B and Fig 7.1.C). Nine spots with different Rf values as described in

Table 7.2 were detected by HPTLC analysis. Fig 7.2 shows the HPTLC profile of the

extract with solvent system Toluene: ethylacetate: methanol: formic acid (50: 50: 5: 5).

The extract on reaction with anthrone reagent formed a green coloured mixture

which indicated the presence of protein bound polysaccharides. Further, the quantification

using anthrone reagent showed the extract contains 15% total carbohydrate content. The

phenol-sulphuric acid method also formed a green coloured reaction mixture which

confirmed the presence of polysaccharide and the amount of carbohydrate in the extract by

the phenol-sulphuric acid method was 13%. The amount of protein present in the extract

was estimated to 35% by the Lowry’s method.

7.4. DISCUSSION

Natural products have been used by man since antiquity for a number of

applications, such as drugs, pigments and flavours. The development of newer and faster

screening techniques based on chromatography led to the rapid identification of large

number of compounds which in turn and has led to natural products receiving more

attention from chemists and pharmacologists (Ohsaki et al., 1999). This has led to more

advanced phytochemical investigations of their biological properties which are an

important prerequisite in the screening of plants for new drugs, cosmetics and

nutraceuticals (Kubitzki, 1984).

The phytochemical analysis of P. rimosus extract revealed the presence of

flavonoids, quinones, terpenes, phenolics, saponins, carbohydrate and proteins. Flavonoids

and tannins, a heterogeneous group of ubiquitous plant polyphenols, exhibit different

pharmacological activities, including hypolipidemic and anti-atherogenic effects (Del Bas

et al., 2005). Numerous preclinical and some clinical studies suggest that flavonoids have

potential for the prevention and treatment of several diseases. Flavonoids include free

radical scavenging, strong antioxidant activities in preventing the oxidation of low-density

lipoproteins (LDL), inhibition of hydrolytic and oxidative enzymes (phospholipase A2,

cyclooxygenase, lipoxygenase). Flavanoids are known to have anti-inflammatory,

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anticarcinogenic (Caltagirone et al., 2000), Antiproliferative effects (Kandaswami et al.,

1991), Inhibition of cell cycle progression and other cancer pathways.

Plant saponins are widely distributed amongst plants and have a wide range of

pharmacological properties (Estrada et al., 2000), such as anti-inflammatory (Sirtori,

2001), Antifungal (Sindambiwe et al., 1998), Antibacterial (Killeen et al., 1998),

Antiparasitic (Traore et al., 2000), anti tumour (Kuroda et al., 2001) and antioxidant

(Huong et al., 1998) activities. Several studies also show that saponins possess

hypolipidemic activity and this has been reported to increase the lipoprotein lipase activity,

which helps to remove free fatty acids from circulation, causing decrease in cholesterol

level (Oakenful and Sidhu, 1990).

More than 30,000 terpenoids have been isolated from plants, microorganisms and

animals. It was well documented that triterpenes are one of the major constituents isolated

from Ganoderma. Terpenes possess promising biological activities including,

antimicrobial activity (Dorman and Deans, 2000), antiviral activity (Chiang et al., 2005),

antioxidant activity (Burits and Bucar, 2000), analgesic activity (Barocelli et al., 2004),

anti-tinflammatory (Amabeoku et al., 2001), digestive activity (Meister et al., 1999; Shen

et al., 2005;), and anticarcinogenic activities (Greenwald, 2001).

Phenolics are suggested to be the major bioactive compounds for health benefits.

Phenolics are one of the groups of nonessential dietary components that have been

associated with the inhibition of atherosclerosis and cancer. The bioactivity of phenolics

may be related to their ability to chelate metals, inhibit lipoxygenase, and scavenge free

radicals (Mallavadhani et al., 2006). Hispolon, a polyphenolic compound was first found in

Inonotus hispidus in 1996 (Ali et al., 1996b). Hispolon and hispolon derivatives were also

isolated from the fungus Phellinus igniarius (Mo et al., 2004). Hispolon has been reported

to induce apoptosis in human epidermoid KB cells (Chen et al., 2006b) and suppress

mitogen-induced proliferation of spleen lymphocytes in mice (Ali et al., 1996).

The biological activity of the mushrooms can be directly co-related with the

chemical constituents present in them. An excellent example is Ganoderma lucidum, the

sporocarp of which has been reported to contain nearly 400 chemical constituents (Wasser,

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2005). Several mushrooms belonging to the genera Inonotus and Phellinus, such as

Inonotus obliquua, Phellinus linteus, Phellinus ribis and Phellinus igniarius have been

used as traditional medicines for the treatment of gastrointestinal cancer, cardiovascular

disease, tuberculosis, liver or heart diseases, fester, bellyache, bloody gonorrhea, stomach

ailments, and diabetes (Nakamura et al., 2004). Polysaccharides, especially β-glucan, are

considered to be responsible for their biological activity.

Earlier investigations showed that ethyl acetate and methanol extracts of P. rimosus

possessed antioxidant, antitumor, and hepatoprotective activities (Ajith and Janardhanan

2001, 2002 and 2003). Recent investigations have also demonstrated the profound

antioxidant, anti-inflammatory, and antiarthritic activities of polysaccharide protein

complex (PPC-Pr) isolated from the aqueous extract of P. rimosus (Meera et al., 2009a;

2009b). Different biological properties observed in the current studies assumed to be due

to the large number of bioactive chemical components present in it. The HPTLC analysis

supports this conclusion.