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Jour nal of Pharmacy Research Vol.9 I ssue 4.Apr il 2015

Y.C. Tri pathi et al. / Jour nal of Pharmacy Research 2015,9(4),271-277

271-27

Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

*Corresponding author.Y.C. Tr ipathi

Chemistry D ivision

Forest Research I nstitu te,

P.O. New For est

Dehradun – 2486008 (I ndia)

Evaluation of Total Polyphenol and Antioxidant Activity of

Leaves of Bambusa nutans and Bambusa vulgari s

Y.C. Tripathi*, Zayd Jhumka and Nishat Anjum

Chemistry Division, Forest Research Institute, PO New Forest, Dehradun-248006 (India)

Received on:26-02-2015; Revised on: 05-05-2015 ; Accepted on:11-05-2015

ABSTRACT

Background: Leaves of several bamboo species have been used to treat a variety of diseases for thousands of years. The medicinal effec

of bamboo leaves are mostly attributed to their bioactive polyphenol constituents. However, different classes of polyphenol have differe

degree of solubility in various solvents, a single solvent will not fully extract all phenolic compounds, thus may exhibit varying antioxida

potency. Methods: The present study was designed to examine extractive values of leaves of Bambusa nutans and Bambusa vulgaris usin

chloroform, acetone and methanol successively and to evaluate the total polyphenols contents and free radical scavenging capabilities

each extract. The total phenolic content of the various extracts was determined spectrophotometrically using a modified Folin-Ciocalte

method and antioxidant efficacy by following DPPH radical scavenging protocol. The extracts were also subjected to preliminary screenin

for presence or absences of various phytochemical constituents. Results: The results showed that B. nutans produced better yield o

polyphenol [methanol (15.3542 ± 0.1576 mg/ml GAE/100mg extract) > acetone (11.7992 ± 0.5502 mg/ml GAE/100mg extract) > chlorofor

(10.1618 ± 0.3284 mg/ml GAE/100mg extract)] than corresponding solvent extracts of B. vulgaris [methanol (12.7976 ± 0.4878 mg/ml GAE

100mg extract) > acetone (10.1328 ± 0.2135 mg/ml GAE/100mg extract) > chloroform (8.8549 ± 0.1747 mg/ml GAE/100mg extract)]. The DPP

radical scavenging assay showed that methanol extracts leaves of both the Bamboo species exhibited highest TPC and radical scavengin

activity whereas leaves of B. nutans found to be superior to B. vulgaris in term of TPC and radical scavenging potency. Preliminar

phytochemical screening of leaf extracts of the two bamboo species indicated the presence of major classes of phytochemicals. Conclusion

The study evidently showed that leaves of B. nutans and B. vulgaris are rich sources of phenolic compounds and natural antixodants an

they could be used as natural antioxidant. The study further indicated methanol as the appropriate extractant for better yield of polypheno

and leaf extract of Bambusa nutans is superior to that of B. vulgaris in term of TPC and antioxidant efficacy.

KEYWORDS: Bambusa nutans, B. vulgaris, Leaf extracts, Polyphenol, Antioxidant activity.

INTRODUCTION

Bamboos are members of the sub-family Bambusoideae within the

grass family Poaceae. More than 1400 species of bamboos are distrib-

uted all over the world, but the major species diversity occurs in

Southeast Asia1-3. Bamboos are among the fastest growing species

on Earth. They have played a significant role in human society since

time immemorial because of their multipurpose use and today con-

tribute to the subsistence needs of over a billion people worldwide4.

Bamboos have over 1500 documented uses5 ranging from being purely

decorative to being, raw materials for construction, wood substitute,and paper and pulp for industry6. They are also known to be a highly

nutritive food source7-9 and their medicinal properties have been ex-

tensively documented. Bamboos have been used in traditional Chi-

nese medicine for over 1000 of years10.

All parts of the bamboo plant such as rhizomes, culms and bark shav

ings, resin, shoots, leaves, and seeds have clinical applications11-

In particular, the leaves are used for treatment of fever, hypertensio

arteriosclerosis, detoxification, stomach heat, respiratory disease

chest inflammation, oedema, restlessness and diarrhoea, vomiting an

excessive thirst10,11,18-21. In addition, several in vitro and in vivo e

periments on animals have confirmed important biological and m

dicinal properties of bamboo leaves extracts such as: antioxidan

antimicrobial, haemolytic activities, anti-inflammatory, anti-ulceantihelminthic, anti-diabetic16,19,20,22-28. The medicinal effects of bam

boo leaves are mostly attributed to their antioxidant capacity29.

Antioxidants protect cell from damage caused by free radicals. Exce

sive free radicals and resulting oxidative stress in the body has bee

implicated in the onset and progression of several diseases such a

aging, atherosclerosis, cancer, inflammation, etc30-34. Polyphenols a

known for their exceptionally high antioxidative capabilities. So fa

only a limited number of bamboo species have been investigated f





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polyphenols and antioxidant capabilities. It has also been reported

that two main groups of polyphenols are responsible for most of the

biological activities of bamboo leaves. These are C-glycoside fla-

vonoids, represented by orientin, homoorientin, isoorientin, vitexin,

homovitexin and tricin and phenolic acids, such as p-coumaric acid,

chlorogenic acid, caffeic acid and ferulic acid 35-39.

Studies on polyphenol content vis-à-vis antioxidant activity of leaves

of some bamboo species have so far been conducted with extracts

derived using only one or two solvents. However, as a matter of fact,

different classes of polyphenols have different degree of solubility in

different solvents. For instance, more polar phenolic compounds and

glycosides are readily soluble in water, ethanol and methanol but less

polar aglycones such as isoflavones, flavonones and methoxylated

flavones tend to be more soluble in non aqueous solvents. As such,

the successive use of different solvents with increasing order of

polarity will extract the maximum amount of polyphenols. In view of

the foregoing reports and facts, the present study was designed to

examine extractive values of leaves of Bambusa nutans Wall. ex Munro

and Bambusa vulgaris Schrad. ex J.C. Wendl.

using solvents of

varying polarity (chloroform, acetone and methanol) successively

and to evaluate the total polyphenols contents of each extract and

their DPPH radical scavenging capabilities.

MATERIALS AND METHODS:

Collection of plant materials

Fresh healthy bamboo leaves of Bambusa nutans and Bambusa

vulgaris were collected from the Bambuseteum of the Forest

Research Institute (FRI), Dehradun, India during the second week of

September 2013 and authenticated by Systemic Botany Section of

Botany Division, FRI. Voucher specimen of the collected plant mate-

rial has been preserved in the Chemistry Division, FRI, Dehradun.

The fresh leaves sample of each species were thoroughly cleaned

and cut into small pieces ranging in size from 0.5 to 1.0 cm and dried

under shade at room temperature (25°C) for 10 days and then pow-

dered to a coarse consistency in a grinder mill. The powder was

passed through 50# mesh particle size and stored in an air tight con-

tainer at room temperature.

Preparation of extracts

The dried and powdered leaves of Bambusa nutans and B. vulgaris

were successively extracted with chloroform, acetone and methanol

in the order of increasing polarity. 200 g of dried leaves of each spe-

cies was separately soaked in 2000 ml 100% chloroform for 48 hours.

The mixture was mechanically stirred for 6 hours daily and left to

macerate the rest of the time during the 48 hours period. The mixtur

was then filtered with Whatmann filter paper (No.1) at room temper

ture and the residue was left to macerate for an additional 24 hours

chloroform and filtered. This procedure was repeated twice and aft

which all the chloroform extracts were pooled together and evap

rated under pressure at 50oC to a constant weight. 100% acetone an

100% methanol were then used successively to obtained aceton

extract and methanol extract from the residue following the same pr

cedure. All the extracts were stored at 4oC until required for the phy

tochemical screening analysis and for evaluation of the total polyph

nols and DPPH radical scavenging capabilities.

Determination of leave extractive value

The percentage yield of all solvent extracts of the two bamboo leav

on dry weight basis was calculated as follows:

Yield (%) = [ EW / DW ] ×100

Where EW is the weight of the extract after solvent evaporation an

DW is the dry weight of the plant material used for extraction.

Preliminary Phytochemical Screening:

The presence or absence of the phytochemical constituents of th

various evaporated leave extracts of Bambusa nutans and Bambus

vulgaris was analyzed using the following methodologies wa

adapted Harborne and Parekh and Chanda40-41.

Carbohydrates: Molish’s Test: The extract (100mg) was dissolved

5 ml of water and filtered. To 2 ml of filtrate, two drops of alcohol

solution of α-naphtol was added, the mixture was shaken well an

1 ml of concentrated sulphuric acid was added slowly along the sid

of the test tube and allowed to stand. A violet ring indicated th

presence of carbohydrates.

Reducing sugars: Fehling’s Test:The extract (100mg) was dissolve

in 5 ml of water and filtered. Then 1 ml of filtrate was boiled on wat

bath and treated with Fehling solutions. A red precipitate indicate

the presence of sugar.

Proteins: Biuret Test: The extract (100 mg) was dissolved in 10 ml

distilled water and filtered. An aliquot of 2 ml of filtrate was treate

with one drop of 2 % copper sulphate solution. To this, 1 ml of eth

nol (95%) was added, followed by excess of potassium hydroxiod

pellets. Pink colour in the ethanolic layer indicated the presence

proteins.

Free amino acids: The aqueous extract is taken in a test tube and





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few drops of phenolphthalein were added to it. Then very little dilu-

tion of sodium hydroxide is added drop by drop until the solution

turns pink. Formation of pink coloration indicated the presence of

free amino acids.

Alkaloids: Wagner’s test: 50mg of solvent free extract is stirred with

a few ml of dilute HCL acid and filtered. To a few ml of filtrate, a few

drops of Wagner’s reagent were added by the side of the tube. A

reddish-brown precipitate confirmed the test as positive.

Terpenoids: Salkowski test. 5ml of aqueous extract was mixed in 2ml

of chloroform and 3ml Conc. Sulphuric acid was carefully added to

form a layer. A reddish brown colouration at the interface was formed

show positive result for the presence of terpenoid.

Phenolics: Lead Acetate Test: A small portion of the extract was

dissolved in a few ml of distilled water and 3 ml of 10% lead acetate

solution was added. A bulky white precipitate indicated the presence

of phenolic compounds.

Tannins: A small quantity of extracts was mixed with water; heated,

filtered and ferric chloride was added. Presence of tannin is indicated

by appearance of blue or greenish black or dark red colour with po-

tassium ferrocyanide and ammonia.

Flavonoids: 5ml of dilute ammonia solution was added to a portion of

extract followed by addition of conc. sulphuric acid. A yellow

colouration observed in the extract indicated the presence of fla-

vonoid. The yellow colour disappeared on standing.

Flavonol Glycosides: Zn/HCl or Mg/HCl reduction: A small portion

of extract was dissolved in alcohol and a few fragments of magnesium

ribbon or zinc powder and concentrated HCl acid was added (drop

wise). The presence of flavonol glycosides was inferred by the

development of pink to crimson colour.

Determination of total polyphenol content

Total polyphenolic content (TPC) of the chloroform, acetone and

methanol extracts of Bambusa nutans and Bambusa vulgaris were

determined using a slightly modified Folin-Ciocalteu colorimetric

method. Stock solutions for each extract were prepared by dissolving

0.0913g of extract into 3 ml 100% methanol. For increase accuracy and

reliability in total polyphenol estimation, four different dilutions (20,

40, 60 and 80 times) of each stock solution were prepared. One ml of

for 5 minutes. A gallic acid standard curve was made using gallic ac

concentration (5 - 180 ppm) with Folin-Ciocalteu reagent and 75%

sodium carbonate. After incubation the tubes were allowed to cool

darkness and absorbance was read at 740 nm. Calculation of tot

polyphenol in each extract was made with respect to the equation

the standard curve of Gallic acid. The total phenolic content wa

expressed as as mg/ml of gallic acid equivalents (GAE) per 100 mg o

leaves extract.

Determination of DPPH radical scavenging activity

To determine the radical scavenging capacity of the different extrac

of Bambusa nutans and Bambusa vulgaris, a modified 1,1-Dipheny

2-picrylhydrazyl (DPPH) radical scavenging methodology adapte

from42. A stock solution of 1 mg/ml solution of each extract in meth

nol was prepared. Sixteen serial dilutions of the extract stock solutio

and one blank sample (control) was made in methanol. 2 ml of fresh

prepared 1 mg/ml stock solution of DPPH was added to each te

tube. The sample was thoroughly mixed and incubated in the dark

28-30 oC. The absorbance (A) was read at 517 nm after 25 minute

The percentage DPPH radical scavenging was determined by th

formula:-

Inhibition (%) = [(AControl

- ASample

) / (AControl

)] x 100.

The results of each extract was expressed IC50

(50% inhibiting con

centration value), that is concentration of the extract that causes 5

% loss of the DPPH activity (scavenge the initial DPPH concentrtion by 50%). IC

50 value was expressed in µg/ml. Lower IC

50 indica

better DPPH radical scavenging ability. Linear regression analys

were carried out in SPSS and the IC50

values were estimated fro

linear regression equations.

RESULTS AND DISCUSSION

Extractive value

The yield of evaporated solvent extracts of leaves Bambusa vulgar

and Bambusa nutans is shown in Table 1. The yield of extract wa

higher in Bambusa vulgaris. In addition, it was observed that th

yield of extract increases in each successive solvents i.e. chloroform

acetone and methanol.. The polyphenols with attached sugars, suc

as glycosides are generally more soluble in polar solvents and le

polar aglycones such as isoflavones, flavonones and highl

methoxylated flavones and flavonols tend to be more soluble in le

polar solvents. Since higher weight of extracts was obtained with th

more polar solvents it can be inferred that the leaves samples con

tained more polyphenol glycosides than the less polar aglycones.

each diluted stock solution, 0.5 ml Folin-Ciocalteu reagent, 4 ml 75%

sodium carbonate was added consecutively in each test tube. The

tubes were then capped, mixed thoroughly and incubated at 50-55oC





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Table 1. Extractive valuesB. vulgari s andB. nu tans leaves in differ-

ent solvents

Species Extractive Values (%)

Chloroform Acetone Methano l Total Extract

Bambusa nutans 2.63 3.15 4.57 10.34

Bambusa vulgari s 3.06 3.84 6.04 12.94

Preliminary phytochemical screening:

The result of the preliminary phytochemical test for the presence of

carbohydrates, proteins, free amino acids, reducing sugars, alkaloids,

terpenoids, phenolics, tannins, flavonoids, flavonol glycosides in

the various extracts of Bambusa nutans and Bambusa vulgaris is

summarized in Table 2. Among the important bioactive polyphenolics

components, phenolics, flavonoids, flavonol glycosides and tannins

were detected in all the extracts.

Table 2. Results of preliminary phytochemical screening

Phytochemicals Bambusa nutans Bambusa vulgar is

Chloroform Acetone Methanol Chloroform Acetone Methanol

Carbohydrates - + + - + +

Proteins - + + - + +

Free amino acids + + + - + +

Reducing sugars + + + + + +

Alkaloids - + + + - +

Terpenoids + + + + + +

Phenolics + + + + + +

Tannins + + + + + +

Flavonoids + + + + + +

Flavonol

Glycosides + + + + + +

(+) Present (-) Absent

Determination of Total phenolic content

The total polyphenol of Bambusa vulgaris and Bambusa nutans for

the different solvent extracts are summarized in table 3. The total

phenolic content was expressed as mg/ml of gallic acid equivalents

(GAE) per 100 mg of evaporated leaves extract. The results also are

graphically illustrated in Figure (1) along with their relationship with

corresponding DDPH IC50

values. The sum of total polyphenol (TP)

obtained from Bambusa nutans and Bambusa vulgaris was 37.3151

mg/ml and 31.7853 mg/ml of GAE per 100 mg of evaporated leaves

extracts respectively.Table 3. Total polyphenolics of leaves extracts of B. nu tans and

B. vulgari s

Extract Total Polyphenol (mg /ml GAE per 100 mg extract)

Bambusa nutans Bambusa vu lgar is

Chloroform Extract 10.1618 ± 0.3284 8.8549 ± 0.1747

Acetone Ex tr ac t 11.7992 ± 0.5502 10 .1328 ± 0.2135

Methanol Extract 15.3542 ± 0 .1576 12.7976 ± 0 .4878

*Each value is a mean of quadrupli cate determinati ons ± standard

deviation

The results showed that each successive solvent extract produce

better yield of polyphenol. In addition, B. nutans produced bett

yield of polyphenol [methanol (15.3542 ± 0.1576 mg/ml of GAE p

100 mg of extract) > acetone (11.7992 ± 0.5502 mg/ml of GAE per 10

mg of extract) > chloroform (10.1618 ± 0.3284 mg/ml of GAE per 100 m

of extract)] than corresponding solvent extracts of B. vulgaris [meth

nol (12.7976 ± 0.4878 mg/ml of GAE per 100 mg of extract) > aceton

(10.1328 ± 0.2135 mg/ml of GAE per 100 mg of extract) > chlorofor

(8.8549 ± 0.1747 mg/ml of GAE per 100 mg of extract)]. The percentag

difference in total polyphenolic yield between Bambusa nutans tha

Bambusa vulgaris were: 6.87 %, 7.60 %, 9.8 % for acetone, chlorofor

and methanol.

To investigate whether the there were any significant different diffe

ences in the mean total phenolics and whether there was any intera

tions between species and solvent type on total poplyphenol a two

way ANOVA was applied (statistical analysis was carried out in SPS

ver. 17.0).

Tests of Between-Subjects Effects

Dependent Variable:Total Polyphenol

Source Type III Sum df Mean F Sig.

of Squares Square

Species 20.386 1 20.386 163.193 .000

Extracts 87.089 2 43.545 348.582 .000

Species * Extracts 1.656 2 .828 6.626 .007

The results showed that species ( B.vulgaris and B.nutans) and sovent type (chloroform, acetone and methanol) as well as their intera

tion significantly (p < .0005) affect the total polyphenol yield for bo

independent variables. The differences in the total phenolic for th

two species and extract types imply that different or different propo

tions of the various classes of polyphenols are present in B. nutan

and B. vulgaris. A number of studies of bamboo have yielded info

mation about the phenolics constituents of bamboo leaves, but ver

few systematic evaluations has been carried out. All the major phe

nolic groups (including phenolic acids, flavonoids, flavonol

flavonones etc.) have been reported from bamboo leaves extract

However, the main bioactive phenolics in bamboo leaves are flvonoids, phenolic acids and coumaric lactones and include the com

pounds orientin, homoorientin, vitexin, isovitexin, naringin-7

rhamnoglucoside, quercetin, luteolin, rutin, tricin, caffeic acid, chl

rogenic acid and phydroxy coumaric acid16,35,43,21. Phenolic compound

are also known to contribute directly to antioxidative action.

DPPH radical scavenging assay

The scavenging ability of 1,1-diphenylpicrylhydrazyl (DPPH) radic

by the different bamboo leaf extracts of B. nutans and B. vulgar

Table 4: Table summarizing the results of the two-way ANOVA





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were evaluated. The assays were carried out in methanol and the

results expressed as IC50

(50% inhibiting concentration). Linear re-

gression analyses were carried out in SPSS and the IC50

values were

estimated from linear regression equations (Table 5). Lower IC50

indi-

cated better DPPH radical scavenging ability. The results showed

that extracts of B. nutans were better radical scavengers [chloroform(320.24 µg/ml), acetone (205.94 µg/ml), methanol (123.24 µg/ml)] than

corresponding extract of B. vulgaris [chloroform (389.23 µg/ml), ac-

etone (300.55 µg/ml), methanol (262.90 µg/ml)].

Table 5. Regression equations and IC50

values of different extract

of Bambusa nutans andB. vulgari s

Species Extract R Square Regression Equation IC50

(µg/ml)

B. nutans Chloroform 0.943 Y = 1.003 + 0.153 (X) 320.24

Acetone 0.811 Y = -1.278 + 0.249 (X) 205.94

Methanol 0.718 Y = 29.754 + 0.164 (X) 123.45

B. vulgari s Chloroform 0.767 Y = 3.293 + 0.120 (X) 389.23

Acetone 0.842 Y = 3.715 + 0.154 (X) 300.55

Methanol 0.806 Y = 14.772 + 0.134 (X) 262.90

Figure 1 illustrates the relationship between DPPH IC50

concentra-

tions in relation to total polyphenol. It can be seen that for both B.

nutans and B. vulgaris, the IC50

values decreases with increasing

total polyphenol levels. Since lower IC50

values indicates better radi-

cal scavenging capabilities, this implies that the radical scavenging

activity (hence antioxidant activity) increases with increasing total

polyphenol level. Also, it can be observed B.nutans had an overall

better radical scavenging capability than B. vulgaris. This is prob-

The relationship between the total polyphenol contentand radic

scavenging activity was confirmed by a correlation analysis (r

-0.936, p < 0.005). The strong inverse relationship between the twvariable indicated that radical scavenging activity (hence antioxida

activity) increases with total polyphenol.

Previous studies have shown that bamboo leaves extracts has th

ability of either inhibiting free radical formation or itself be a fre

radical scavenger 23,44. The above results highlight the ability of di

ferent bamboo leaves extract to scavenge DPPH free radicals by the

proton-donating ability. Consistent with previous research, the r

sult obtained here, indicates that anti-oxidant activity of the variou

extract of B. vulgaris and B. nutans increases with total polyphenol

content.

In bamboo leaves two main groups of polyphenols have bee

reported to be responsible for most of the biological activities o

bamboos leaves. These are C-glycoside flavonoids, represented b

orientin, homoorientin, isoorientin, vitexin, homovitexin and tricin an

phenolic acids, such as p-coumaric acid, chlorogenic acid, caffe

acid and ferulic acid16,35-39. Since, the C-glycoside flavonoids are mo

soluble in more polar solvent, this provides some explanation for th

superior total polyphenolic and anti-oxidant activity of methanol an

acetone extracts.

ably because for all the different extract B. nutans produces bett

yield of total polyphenol than B. vulgaris.

Figure 1. Relationship between Total Polyphenol Content and DPPH IC50

values





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CONCLUSION

Excessive free radicals production generally leads to oxidative stress

which is responsible for the onset and progression of several dis-

eases. Natural anti-oxidants have the ability to protect organisms

from damage caused by free radical-induced oxidative stress and

therefore can be used to fight a variety of ailments and diseases.

Bambusa vulgaris has been traditionally used in folkloric medicine

and most of the medicinal properties have been attributed to its anti-

oxidant activities. Since, this study revealed that Bambusa nutans

has higher polyphenolic content and antioxidant activity than

Bambusa vulgaris. Therefore, it can be speculated that Bambusa

nutans can also be potentially useful as a natural source of antioxi-

dant or in medicine.

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evaluation of total polyphenol and antioxidant activity of leaves of bambusa nutans and bambusa...

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