Evaluation of Antioxidant and Pharmacological Properties of Psychotria nilgiriensis Deb & Gang

Food Sci. Biotechnol. 21(5): 1421-1431 (2012)

DOI 10.1007/s10068-012-0187-x

Evaluation of Antioxidant and Pharmacological Properties of

Psychotria nilgiriensis Deb & Gang

Murugaiyan Iniyavan, Devadoss Sangeetha, Shanmugam Saravanan, and Thangaraj Parimelazhagan

Received: 4 April 2012 / Revised: 8 May 2012 / Accepted: 15 May 2012 / Published Online: 31 October 2012

© KoSFoST and Springer 2012

Abstract The purpose of the present study was to

investigate the antioxidant and pharmacological properties

of fruit, stem, and leaf extract of Psychotria nilgiriensis.

Acetone extract of P. nilgiriensis fruit was found to have

highest total phenolics (505.74 mg GAE/g extract), tannin

(460.78 mg GAE/g extract), and flavonoid (67.78 mg RE/

g extract) content. In vitro antioxidant studies revealed that

the acetone extract of fruit posses significant antioxidant

activity in DPPH radical scavenging, ABTS•+, and ferric

reducing antioxidant power (FRAP) assays. In vivo studies

revealed that P. nilgiriensis fruit (400 mg/kg) showed good

analgesic activity in hot plate method (72%), acetic acid

induced writhing test (53%) and also showed significant in

carrageenan induced inflammation (73.54%). GC/MS

analysis of fruit showed the presence of sesquiterpenes.

The study highlights the significant medicinal value of the

plant.

Keywords: antioxidant, analgesic, anti-inflammatory,

Psychotria nilgiriensis

Introduction

During the last few decades there has been an increasing

interest in the study of medicinal plants and their traditional

use in different parts of the world. The chemical novelty

and diversity associated with medicinal plant products are

higher than that of any other source. Hence, it is accepted

that plants are useful in their crude or advanced form of

drugs. Medicinal plants are known to contain innumerable

biologically active compounds.

A free radical is any atom (e.g., oxygen, nitrogen) with

at least 1 unpaired electron in the outermost shell, and is

capable of independent existence. The interest in reactive

oxygen species (ROS) on recent years in biology and

medicine is evident because of their strong relationship

with many common and life threatening human diseases.

ROS are implicated in the development of many chronic

disorders, such as cancer, diabetes mellitus, atherosclerosis,

nephritis, rheumatism, cardiovascular diseases, gastrointestinal

tract disorders, and inflammatory injury. Antioxidants are

vital substances that play an important role in disease

prevention and protecting the body from damage caused

from free radical-induced oxidative stress owing to their

abilities to remove free radicals. Earlier studies reported

that plants have potent antioxidant and represent an

important source of natural antioxidants. The identification

of new effective antioxidants is a topic of interest; several

plant extracts or secondary metabolites have shown

potential to protect against oxidant-induced damage (1).

Inflammation is the tissue reaction to infection, irritation,

or foreign substance. It is a part of the host defense

mechanisms that are known to be involved in the

inflammatory reactions such as release of histamine,

bradykinin, and prostaglandins. The development of non-

steroids in overcoming human sufferings such as rheumatoid

arthritis has evoked much interest in the extensive search

for new drugs with this property (2). Carrageenan induced

paw edema is widely used for determining the acute phase

of inflammation.

The genus Psychotria of Rubiaceae family contains

medicinally valuable indole alkaloids namely psychotridine

and brachycerine. These alkaloids are widely used to cure

problems in central nerves system of human. Ethnobotanical

and chemotaxonomical studies on species of Psychotria

Murugaiyan Iniyavan, Devadoss Sangeetha, Shanmugam Saravanan,Thangaraj Parimelazhagan (�)Bioprospecting Laboratory, Department of Botany, Bharathiar University,Coimbatore-641 046, Tamil Nadu, IndiaTel: +919750006025; Fax: E-mail: [email protected]

RESEARCH ARTICLE

1422 Iniyavan et al.

resulted in the discovery of a set of novel bioactive

monoterpinoid indole alkaloids (MIAs), some of them with

clear pharmaceutical potential. Few reports have been

published on antioxidant activities of the crude extracts or

compounds isolated from Psychotria. Tender fruit of

Psychotria nilgiriensis (commonly called as odai kaapi

patchilai in Tamil) is consumed along with honey for its

action against rheumatism. It is being used by Kanikkar

tribes of Kalakad, Mundanthurai at Tirunelveli district, and

Irula tribes of Thottabeta at Nilgiris district, Tamilnadu,

India (3). Even though the plant has been reported for the

medicinal property and uses, very limited reports are

available regarding the phytochemical and pharmacological

aspects. Therefore, this study was designed to analyze

radical scavenging activity and pharmacological property

of different solvent extraction from the P. nilgiriensis fruit,

stem, and leaves.

Materials and Method

Collection of plant materials The fresh plant parts of

Psychotria nilgiriensis were collected from Thottabetta,

Nilgiris, Tamil Nadu during the month of February-April,

2011. The collected plant material was identified and its

authenticity was confirmed by comparing the voucher

specimen at the herbarium of Botanical survey of India,

Southern circle Coimbatore, Tamil Nadu. Freshly collected

plant material was cleaned to remove adhering dust and

then dried under shade. Fruit, stem, and leaves of the

samples were separated and dried in shade for 16-25 days.

The dried plant samples were powdered and used for

further studies.

Chemicals DPPH, ABTS, Trolox, linoleic acid, Folin-

Ciocalteu reagent, ammonium thiocyanate, aluminum

chloride, thiobarbituric acid (TBA), TPTZ, polyvinyl

polypyrrolidone (PVPP), ferrous ammonium sulfate, EDTA

disodium salt, metaphosphoric acid, 2,6-dichoroindophenols

(DIP). Chemicals are purchased from Sigma-Aldrich (St.

Louis, MO, USA), Merck and Himedia (Mumbai, India).

All other reagents used were of analytical grade.

Successive solvent extraction The air dried powdered

plant materials of P. nilgiriensis were extracted separately

in Soxhlet extractor successively with petroleum ether,

chloroform, acetone, and methanol. Each time before

extracting with the next solvent, the material was dried in

hot air oven below 40oC. Finally, the material was

macerated using hot water with occasional stirring for 48 h

and the water extract was filtered. The different solvent

extracts were concentrated by rotary vacuum evaporator

(RE300; Yamato, Tokyo, Japan) and then air dried. The

dried extract obtained with each solvent was weighed. The

percentage of yield was expressed in terms of air dried

weight of plant material. The extracts thus obtained were

used directly for the estimation of total phenolics and also

for the assessment of antioxidant potential through various

biochemical assays. The extracts were freeze dried and

stored in desiccators until further analysis.

Determination of total phenolic and tannin contents

The total phenolics of the plant extracts were determined

by Folin-Ciocalteu method. Using the same extract the

tannins were estimated after treatment with PVPP. The

amount of total phenolics and tannins were calculated in

gallic acid equivalents (GAE) as described by Siddhuraju

and Becker (4).

Estimation of flavonoids Flavonoid content of a plant

material was measured by the aluminum chloride colorimetric

assay (5). Samples were analyzed in triplicates and the

amounts of flavonoids were expressed in rutin equivalents

(RE).

Determination of ascorbic acid (vitamin C) The

ascorbic acid was determined using the DIP method with

simple modification (6). The concentrations of the ascorbic

acid in the sample were determined through comparison

with the absorbance of standard ascorbic acid at different

concentration.

In vitro antioxidant assays

DPPH radical scavenging activity: DPPH radical

scavenging activity was assessed according to modified

method of Blios (7) was used to determine the free radical

scavenging activities of plant extract. In this method, a

commercially available and stable free radical DPPH

soluble in methanol was used. Sample extracts at various

concentrations were taken and the volume was adjusted to

100 µL with methanol. Four mL of 0.1 mM freshly prepared

methanolic DPPH solution was added. The reaction mixture

was vortexed well and kept at room temperature for 20 min.

The resulting solutions were read at 517 nm using a UV

spectrophotometer (UV 1800; Shimadzu, Kyoto, Japan).

Methanol was used as the blank. Negative control was

without any inhibitor or extract. Commercial antioxidants

butylhydroxy anisole (BHA), butylhydroxy toluene (BHT),

quercetin, and rutin dissolved in methanol were used as the

positive control under the same assay conditions in all

tests. The free radical scavenging activities of the samples

were expressed as IC50 value (i.e., the concentration of the

sample required to inhibit 50% of DPPH concentration).

ABTS•+ scavenging activity: The total antioxidant activity

of the samples was measured by ABTS radical cation

decolorization assay according to the method of Re et al.

Phytochemical and Pharmacological Properties of P. nilgiriensis 1423

(8). Seven mM ABTS aqueous solution was added with

2.4 mM potassium persulfate and this mixture was

incubated at dark for 12-16 h to produce ABTS•+. Prior to

assay, this solution was diluted in ethanol (about 1:89 v/v)

and equilibrated at 25oC to give an absorbance of 0.700

±0.02 at 734 nm. About 1 mL of diluted ABTS solution

was added to about 30 µL sample solution and 10 µL of

Trolox (final concentration 0-15 µmol) in ethanol. One mL

of diluted ABTS solution was mixed with 30 µL of ethanol

served as the negative control. All the test tubes were

vortexed well and incubated exactly for 30 min at room

temperature. After incubation the absorbance of samples

and standards (BHT and rutin) were measured at 734 nm

against the ethanol blank. The results were expressed as the

concentration of Trolox having equivalent antioxidant

activity expressed as µmol/g sample extracts

Assay of superoxide radical scavenging activity: The

assay was based on the capacity of various extracts to

inhibit formazan formation by scavenging the superoxide

radicals generated in riboflavin-light-NBT (nitroblue

tetrazolium) system (9). Each 3 mL reaction mixture

contained 50 mM sodium phosphate buffer (pH 7.6), 20 µg

riboflavin, 12 mM EDTA, 0.1 mg NBT, and 100 µL

sample solution. Reaction was started by illuminating the

reaction mixture with sample extract for 90 s. Immediately

after illumination the absorbance was measured at 590 nm.

The entire reaction assembly was enclosed in a box lined

with aluminum foil. Identical tubes with reaction mixture

kept in the dark served as blanks. The percentage inhibition

of superoxide anion generation was calculated.

% Inhibition

=[(control OD–sample OD)/control OD]×100

Determination of metal chelating activity: The chelation

of ferrous ions was estimated by method of Dinis et al.

(10). Briefly, 50 µL of 2 mM FeCl2 was added to 1 mL of

different concentrations of the extract (50, 100, 150, 200,

and 250 µg/µL). A 0.2 mL of 5 mM ferrozine was added in

the test tubes to initiate the reaction. The mixture was

vigorously shaken and left to stand at room temperature.

After 10 min, the absorbance was read spectrophoto-

metrically at 562 nm. One mL of deionised water, instead

of sample, was used as a control. All the reagents without

addition of sample extract were used as negative control.

EDTA (20 mg/mL) was used as standard. The results were

expressed as mg EDTA equivalent/g extract.

Ferrous reducing antioxidant power assay (FRAP): The

antioxidant capacities of extracts were estimated according

to the procedure described by Pulido et al. (11). The FRAP

reagent contained 2.5 mL of 20 µmol/L TPTZ solution in

40 µmol/L HCl, 2.5 mL of 20 µmol/L FeCl3·6H2O, and 25

mL of 0.3 mol/L acetate buffer (pH 3.6). Freshly prepared

FRAP reagent was incubated at 37oC. To the 90 µL of

distilled water and 30 µL of test sample or methanol (for

the reagent blank), 900 µL of FRAP reagent was added.

The prepared test samples and reagent blank were again

incubated in water bath at 37oC for 30 min. At the end of

incubation, readings of colored product (ferrous tripyridyl-

triazine complex) were taken immediately at 593 nm.

Lipid peroxidation inhibitory activity: The lipid peroxidation

inhibitory activity of the extracts was determined according

to the method of Duh et al. (12). Briefly, egg lecithin was

homogenized (1%, w/v) in 10 mM phosphate buffer (pH

7.4) and blend well with blender machine to ensure proper

liposome formation. Test samples of different concentrations

(0.1-0.9 mg/mL) were added to liposome mixture (1 mL);

the control was without test sample. Lipid peroxidation

was induced by adding FeCl2 (10 µL, 400 mM) and L-

ascorbic acid (10 µL, 200 mM). After incubation for 1 h at

37oC the reaction was stopped by adding HCl (2 mL, 0.25

N) containing trichloroacetic acid (150 mg/mL) and TBA

(3.75 mg/mL). The reaction mixture was subsequently

boiled for 15 min, cooled, centrifuged at 1,000×g for

15 min and the absorbance of the supernatant was measured

at 532 nm and compared with that of BHA and ascorbic

acid (10 mg/mL). Percentage of radical scavenging activity

was calculated using the following formula:

% Inhibition

=[(Abscontrol–(Abssample–Abssample blank/Abscontrol]×100

In vivo studies

Selection of animal’s: Swiss albino mice weighing 20-30

g and Wistar albino rats of 200-250 g were used for the

pharmacological studies. They were housed in a clean

polypropylene cage and maintained under standard laboratory

conditions (temperature 25±3ºC with dark/light cycle 12/

12 h; 35-60 humidity). They were fed with standard pellet

diet (VRK Nutritional solutions, Sangli, Maharastra) and

water ad libitum. The studies were carried out at Nandha

College of Pharmacy and Research Institute, Perundurai,

Tamil Nadu, India. The experimental protocol was

subjected to the scrutiny of the Institutional Animal Ethics

Committee, and was cleared by same before beginning the

experiment (688/02/C/CPCSEA).

Acute toxicity: Acute oral toxicity study was performed

according to acute toxic class method. Swiss albino mice

(n=6) of either sex selected by random sampling technique

were used for acute toxicity study. The animals were kept

fasting for overnight providing only water, after which

extracts were administered by gavages at different doses

from 50 to 2,000 mg/kg BW, which were increased

progressively so that each dose was 50% higher than the

preceding (13). The treated animals were free to access

water ad libitum. Observations were made at 2, 4, 8, up to

48 h for treatment related behavioral changes like apathy,

reduced locomotor behavior. If mortality was observed in


4 out of 6 animals, then the dose administered was

assigned as toxic dose. If mortality was observed in 2

animals, then the same dose was repeated again to confirm

the toxic dose.

Analgesic activity

Hot plate method: The hotplate method was used to

measure response latencies according to the method described

by Eddy and Leimbach (14) with minor modification.

Animals of either sex were randomly selected and divided

into 6 groups consisting of 5 mice in each group. Each

group received a particular treatment i.e., control (untreated),

standard (diclofenac sodium 10 mg/kg), and the acetone

extract of fruit and stem (200 and 400 mg/kg). The animals

were positioned on Eddy’s hot plate kept at a temperature

of 55±0.5oC. The time taken by the animals to lick the fore

or hind paw or jump out of the place was taken as the

reaction time. The latency was recorded at the time of 0

(just before any treatment) and 30, 60, 120, and 240 min

after oral administration of samples and intraperitoneal

administration of standard. A latency period of 15 s was

observed to avoid damage to the paw. The percentage

thermal pain stimulus relief or protection was determined

by applying the formula:

% Protection against thermal pain stimulus

=[(test mean–control mean)/test mean]×100

Acetic acid-induced writhing in mice: The test was

performed as described by Koster et al. (15). Swiss albino

mice of either sex were divided into 6 groups of 5 mice

each. Group I served as control (acetic acid 1.0 mL/kg i.p.)

while group II (positive control) was administrated with

standard drug- aspirin at a dose of 150 mg/kg. Based on the

acute toxicity studies, the doses for acetone extract of P.

nilgiriensis fruit and stem were decided to be 200 and 400

mg/kg and was given to the Group III, IV, V, and VI

respectively. Thirty min later all the groups were treated

with 0.75% acetic acid at the dose of 0.1 mL/10 g BW.

Mice were placed in individual cages. The numbers of

abdominal contractions were counted 5 min after acetic

acid injection for a period of 10 min. Percentage inhibition

of writhing was obtained using the formula:

Inhibition (%)

= ×100

Anti-inflammatory study

Carrageenan-induced hind paw edema: The anti-

inflammatory property of acetone extract of P. nilgiriensis

fruit and stem were studied against carrageenan-induced

acute paw edema as described by Ghosh (16). The animals

were divided into 6 groups of 6 animals each and were

fasted for a period of 24 h prior to the study. Group 1 as

control; Group 2 received indomethacin 10 mg/kg suspended

in 1% sodium carboxymethyl cellulose (SCC) as standard.

Group 3 and 4 were treated with 200 and 400 mg/kg of

acetone extracts of P. nilgiriensis fruit, Group 5 and 6 were

treated with 200 and 400 mg/kg of acetone extracts of P.

nilgiriensis stem suspended in 1% SCC. Oedema was

induced by injecting 0.1 mL of 1% solution of carrageenan

in saline into the subplantar region of the right hind paw of

the rats. The vehicle, extracts and the standard drugs were

administered 60 min prior to the injection of the phlogestic

agent. The volumes of edema of the injected and the

contralateral paws were measured at 1, 2, 3, 4, and 5 h after

the induction of inflammation. The inflammation was

measured by using an electronic vernier caliper (CD-6

CSX; Digimatic caliper, Mitutoyo, Japan) and calculates

the percentage of paw oedema inhibition.

GC/MS analysis The GC/MS analysis of the P. nilgiriensis

acetone extract was carried out at the South India Textile

Research Association (SITRA), Coimbatore. The GC was

carried out by using Thermo-GC Trace Ultra Version 5.0

equipment with running time of 38.94 min and the MS was

carried out by using Thermo MS-DSQ II equipment (Thermo

Fisher Scientific Inc., Waltham, MA, USA). Auto sampler

and GC interfaced to a MS instrument employing the

following conditions: column TR 5-MS capillary standard

non-polar column (30 M, i.d.: 0.25 mm, FILM: 0.25 µm)

composed of 5% phenyl polysilphenyle-siloxane; helium

(99.999%) was used as carrier gas at a constant flow of

1 mL/min; and an injection volume of 0.5 EI was employed

(split ratio of 10:1) injector temperature 250oC; ion-source

temperature 280oC. The MS of the unknown component

was compared with the spectrum of the known components

stored in the National Institute of Standard Technology

(NIST) library. The name, molecular weight and structure

of the components of the test materials were ascertained.

Statistical analyses All the experiments were done in

triplicates and the results were expressed as mean±standard

deviation (SD). The data were statistically analyzed using

one way analysis of variance (ANOVA) followed by

Duncan’s test for antioxidant studies and by Dunnet’s t-test

for analgesic and anti-inflammatory studies. Mean values

were considered statistically significant when p<0.05,

<0.01, and <0.001

Results and Discussion

Extract yield percentage The acetone extract of fruit

showed higher amount of yield 16.30%. The yield of leaf

mean no. of writhings

control( )⎝ ⎠⎛ ⎞ mean no. of writhings

test( )⎝ ⎠⎛ ⎞–

mean no. of writhings (control)-----------------------------------------------------------------------------------------------------------------------


(13.6%) and stem (10.4%) were high in methanol extract.

On the other hand, petroleum ether extract of stem (1.05%)

showed lower percentage compare to the other solvents.

Total phenolics, tannins, flavonoids, and ascorbic acid

Total phenolics, tannins, flavonoids, and ascorbic acid

content of various solvent extracts of P. nilgiriensis plant

parts were showed in Table 1. The highest levels of total

phenolic (505.74 mg GAE/g extract) and tannin (460.78

mg GAE/g extract) contents were found in acetone extract

of P. nilgiriensis fruit when compared to other extracts. The

petroleum ether extract of fruit showed very poor content

of phenols (25.91 mg GAE/g extract) and tannin (1.54 mg

GAE/g extract). In our study, phenolics and tannin content

of P. nilgiriensis fruit was showed a significant positive

correlation with the antioxidant activity of the plant extracts.

The enrichment of phenolic compounds within plant extracts

is correlated with their enhanced antioxidant activity. A

strong relationship between total phenolic content and

antioxidant activity in fruits, vegetables, and grain products

has been reported by Dorman et al. (17). Recently, it has

been reported that the high molecular weight phenolics

such as tannins have more ability to reduce or scavenge

free radicals. Tannins interfere with iron absorption

through a complex formation with iron when it is in the

gastrointestinal lumen which decreases the bioavailability

of iron. The role of flavonoids as antioxidants has been

well established and there have been numerous reports on

structure-activity relationship in the last decade. The

highest levels of avonoid contents were found in acetone

extract of P. nilgiriensis fruit (67.78±5.03 mg RE/g

extract), while avonoid contents of petroleum ether extract

of fruit were very low (1.52±0.09 mg RE/g extract). Many

avonoid are found to be strong antioxidants capable of

effectively scavenging the ROS because of their phenolic

hydroxyl groups (18). The results revealed that the acetone

extract of fruit contain more avonoid than other parts of the

same plant. It is well known that vitamin C has been

considered by some scientists as a ‘universal panacea’ due

to its broad biochemical and pharmacological functions.

Vitamin C acting as a highly effective antioxidant it can

protect indispensable molecules in the body from damage

by free radicals and ROS. More recently, vitamin C as

potent tyrosinase inhibitor has been studied in detail by

Zeng et al. (19). The results suggested that the acetone

extract of P. nilgiriensis fruit have maximum amount of

vitamin C content (4.15%). Moreover, fruits contain

vitamin C which expresses antioxidant activity.

In vitro antioxidant assays

DPPH assay: In the DPPH assay, the antioxidants were

able to reduce the stable DPPH radical to the yellow

colored diphenyl-picrylhydrazine. The free radical-scavenging

activities of different parts of P. nilgiriensis samples along

with standards such as α-tocopherol, rutin, BHA, and BHT

were determined by the DPPH radical scavenging assay.

The highest free radical scavenging activity was exerted by

acetone extract of fruit (20.0 µg/mL). Chloroform extract

of leaf was found to be least radical scavenging activity

(847.50 µg/mL) (Fig. 1). The relatively stable DPPH

radical has been widely used to examine the ability of

compounds to act as free radical scavengers or hydrogen

donors and thus to evaluate the antioxidant activity.

Acetone extract of the P. nilgiriensis of fruit (20.0 µg/mL),

Table 1. Total phenolics, tannin, flavonoids, and vitamin C content of P. nilgiriensis

Plant sample SolventTotal phenolics

(mg GAE/g extract)Tannins

(mg GAE/g extract)Flavonoids

(mg RE/g extract)Vitamin C(g/100 g)

Fruit

Petroleum ether 1)25.91±1.351) 1.54±0.64 1.52±0.09 1.19±0.06

Chloroform 77.31±5.88 30.81±4.250 2.22±0.38 1.33±0.02

Acetone 0505.74±82.38* 460.78±80.70* 67.78±5.03* 04.15±0.03*

Methanol 0225.91±12.34* 182.63±15.00* 5.33±0.12 1.73±0.04

Hot water 48.04±6.12 5.18±6.73 3.24±0.03 02.64±0.10*

Stem

Petroleum ether 50.70±5.84 15.83±4.210 20.24±1.99* 1.27±0.01

Chloroform 97.62±2.34 21.57±6.790 6.25±0.14 1.32±0.01

Acetone 0216.11±16.57* 150.14±15.86* 21.05±1.96* 1.33±0.01

Methanol 178.71±8.66* 129.27±8.02*0 5.50±0.12 1.81±0.01

Hot water 62.18±4.39 20.31±6.010 3.00±0.17 02.18±0.01*

Leaf

Petroleum ether 57.84±8.92 26.75±7.950 10.50±0.390 1.32±0.01

Chloroform 71.71±3.10 20.17±8.280 10.21±0.220 02.09±0.04*

Acetone 0469.33±50.92* 408.54±51.81* 7.71±0.28 02.89±0.01*

Methanol 66.11±5.13 8.82±5.93 11.09±0.530 02.05±0.00*

Hot water 56.16±2.31 14.99±3.180 2.83±0.410 1.94±0.59

1)Values are mean of triplicate determination (n=3)±SD; Statistically significant at *p<0.05


stem (30.90 µg/mL), and leaf (103.30 µg/mL) showed

spectacular value in DPPH assay than the other solvent

extracts. The acetone extract of plant parts were possibly

contained some substrates, which were electron donors and

could react with free radicals to convert them to more

stable products and terminate the radical chain reaction.

IC50 value of fruit extract (20.0 µg/mL) was unconditionally

significant than the standards α-tocopherol, rutin, and

BHT. Phenolic content profile of this plant may be

endorsed to scavenge the free radicals. The antioxidant

potential of the plant to scavenge free radicals was ensured

in the DPPH assay.

ABTS radical scavenging activity: The Trolox equivalents

antioxidant capacity (TEAC) was measured using the

improved ABTS•+ radical decolorization assay; one of the

most frequently employed methods for antioxidant capacity,

which measures the ability of a compound to scavenge

ABTS•+ radical. Hagerman et al. (20) have reported that

the high molecular weight phenolics (tannins) have more

ability to quench free radicals (ABTS•+) and that effectiveness

depends on the molecular weight, the number of aromatic

rings and nature of hydroxyl groups substitution than the

specific functional groups. ABTS•+ assay is an excellent

tool to determine the antioxidant activity of hydrogen-

donating antioxidants (scavenging aqueous phase radicals)

and of chain breaking antioxidants (scavenging lipid

peroxyl radicals). Acetone extract of P. nilgiriensis fruit

showed significant result (41,343.51 µmol TE/g extract)

against ABTS free radical. Root (35,437.30 µmol TE/g

extract), stem (7,451.96 µmol TE/g extract), and leaf

(7,296.71 µmol TE/g extract) extracts also confirm moderate

result in radical scavenging and ensure their place in free

radical scavenging (Table 2). Petroleum ether extract of

leaves (189.00 µmol TE/g extract) failed to produce

considerable result against ABTS•+. Low polarity of the

solvent may be reason for the poor result in petroleum

either extracts. Higher concentrations of extract were more

effective in quenching free radicals in the system (21).

Earlier reports of Psychotria species showed tremendous

result in ABTS assay the values obtained were meagerly

equivalent to antioxidant standard Trolox.

Superoxide radical scavenging activity: Superoxide anion

plays an important role in formation of ROS. Although

superoxide is a relatively weak oxidant, it decomposes to

form stronger ROS, such as singlet oxygen and hydroxyl

radicals, which initiate peroxidation of lipids. Acetone

extracts of P. nilgiriensis effectively scavenged superoxide

in a concentration-dependent manner. The acetone extract

of leaf (54.90%), fruit (32.97%), and stem (16.91%) has

good superoxide radical scavenging activity (Table 2).

Further, superoxides are also known to indirectly initiate

lipid peroxidation as a result of H2O2 formation, creating

precursors of hydroxyl radicals. These results clearly

suggest that the antioxidant activity of P. nilgiriensis is also

related to its ability to scavenge superoxides.

Metal chelating: Ferrozine can quantitatively form

complexes with Fe2+. In the presence of other chelating

agents, the complex formation is disrupted with the result

that the red color of the complexes decreases. The Fe2+

chelating activity of extracts are shown in Table 2. Ferrozine

can quantitatively form complex with Fe2+. In the presence

of other chelating agents, the complex formation is disrupted

which results in the decreased intensity of the red color of

the complex. Measurement of the rate of color reduction

therefore allows estimation of the chelating activity of the

coexisting chelator. In this assay, both P. nilgiriensis extract

and EDTA interfered with the formation of ferrous and

ferrozine complex suggesting that it has chelating activity

Fig. 1. DPPH radical scavenging activities of P. nilgiriensis. Values are mean of triplicate determination (n=3)±SD; Statisticallysignificant at *p<0.05


and captures ferrous ion before ferrozine. Especially the

acetone extracts of stem (66.67 mg EDTA/g extract), fruit

(65.70 mg EDTA/g extract), and leaf (51.32 mg EDTA/g

extract) showing the higher values of ion chelating capacity.

At the same time petroleum ether extract of fruit was had

very poor metal chelating activity (3.60 mg EDTA/g extract)

when compared to other parts of the plant. While the other

extracts showed the metal chelating activity ranging from

50.46-3.60 mg EDTA/g. Metal chelating capacity was

significant since the extract reduced the concentration of

the catalyzing transition metal in lipid peroxidation. It was

reported that chelating agents, which form δ-bonds with a

metal, are effective as secondary antioxidants because they

reduce the redox potential, thereby stabilizing the oxidized

form of the metal ion (22). Results reveal that P.

nilgiriensis plant extracts has an effective capacity for iron

binding, suggesting that its act as antioxidant.

FRAP assay: The FRAP assay measures the antioxidant

effect of any substance in the reaction medium as reducing

ability. FRAP assay was used by several authors for the

assessment of antioxidant activity of various samples.

Halvorsen et al. (23) suggested most of the secondary

metabolites are redox-active compounds that will be picked

up by the FRAP assay. Antioxidative activity has been

proposed to be related to reducing power. Therefore, the

antioxidant potential of P. nilgiriensis fruit, stem, and

leaves were estimated for their ability to reduce TPTZ-Fe

(III) complex to TPTZ-Fe (II). The ferric reducing ability

of the acetone extracts of P. nilgiriensis revealed excellent

FRAP activity. The acetone extract of fruit resulted greater

value (4,713.33 µmol Fe (II)/mg extract) of ferric reducing

power and the petroleum ether extract of fruit resulted least

value (85.56 µmol Fe (II)/mg extract) (Table 2). Ferric

reducing antioxidant power of the plant showed greater

variability according to the part used and solvent. The

results on reducing powers demonstrate the electron donor

properties of P. nilgiriensis extracts thereby neutralizing

free radicals by forming stable products.

Lipid peroxidation: Lipid peroxidation was an oxidative

deterioration process of polyunsaturated fatty acids which

is induced by radical. One of the degradation products of

lipid peroxidation is malondialdehyde which causes cell

damage can form a pink color chromogen with TBA acid.

The antioxidant compounds present in the extract scavenged

the hydroxyl radicals generated in the Fenton reaction in

the egg yolk. Among different extracts P. nilgiriensis the

acetone extract of leaf (69.66%), fruit (60.67%), and stem

(52.25%) showed maximum activity (Table 2). The acetone

extract of leaf showed significant activity compared to

other extracts and positive control (ascorbic acid and BHA).

High lipid peroxidation inhibitions showed by acetone

extract could be related to the presence of phenolic

compound, which has been shown to be correlated to the

antioxidant activity of natural plant product (24). This

inhibition of lipid peroxidation may either be due to

chelation of Fe ion or by scavenging of the free radicals.

In vivo study

Acute toxicity: The acetone extract of fruit and stem of P.

nilgiriensis was evaluated for its acute toxicity in rats. The

extract did not alter the general behavior and failed to

produce any mortality even at highest dose of 2,000 mg/kg.

Table 2. ABTS, metal chelating, FRAP, superoxide radical scavenging, and lipid peroxidation activities of P. nilgiriensis

Plantsample

Solvent

ABTS(µM Trolox equivalent/g

extract)

Metal chelating activity (mg EDTA

equivalents/g extract)

FRAP(mmol Fe (II)/mg

extract)

Superoxide radical scavenging (%)

activity

Lipid peroxidation (%) activity

Fruit

Petroleum ether 1)173.47±14.221) 3.95±2.45 85.56±1.92 4.52±1.15 7.87±2.97

Chloroform 712.12±26.04 3.60±3.25 129.21±11.76 8.48±0.79 35.39±3.420

Acetone 41,343.51±58.46*0, 65.70±1.698 4,713.33±2.89*0, 36.94±2.47* 060.67±11.24*

Methanol 4,775.60±88.27*, 29.43±1.750 1,411.48±1.28*0, 23.64±2.42* 31.65±4.51*

Hot water 1,427.62±74.92*, 50.46±2.15* 516.35±13.04 17.32±1.530 12.36±2.970

Stem

Petroleum ether 139.05±4.680 4.10±1.75 101.75±2.620 6.27±2.58 21.35±13.98

Chloroform 242.32±9.570 9.98±0.75 161.90±4.760 6.33±1.96 25.28±7.040

Acetone 7,451.96±65.09*, 66.67±0.92* 1,490.56±6.31*0, 16.91±4.40* 052.25±10.30*

Methanol 1,287.56±20.46*, 33.49±2.160 859.26±3.39* 32.97±3.56* 30.34±7.04*

Hot water 413.10±18.22 35.76±1.440 316.98±0.990 14.64±2.830 14.04±4.240

Leaf

Petroleum ether 189.00±15.47 4.76±2.32 111.75±0.270 3.35±1.46 14.79±0.320

Chloroform 255.82±3.090 6.08±1.06 167.14±2.470 8.80±3.03 17.42±3.930

Acetone 7,296.71±23.38*, 51.32±0.09* 1,314.44±0.96*0, 54.90±3.78* 69.66±3.51*

Methanol 1,277.43±29.66*, 30.95±3.200 757.04±5.48* 38.05±3.15* 36.52±5.87*

Hot water 404.32±15.33 45.34±0.84* 270.48±0.480 14.90±2.040 10.30±1.970

1)Values are mean of triplicate determination (n=3)±SD; Statistically significant at *p<0.05


In acute toxicity study, the extract was found to be safe at

2,000 mg/kg even after 48 h, and revealed that this plant

might be well thought-out as a wide harmless one.

Analgesic activity/Hot plate method: The hot plate model

has been found to be suitable for the evaluation of centrally

but not of peripherally acting analgesics. This model

measures a complex response to a non-inflammatory and

acute nociceptive input. In this analgesic testing model,

pentazocine significantly prolonged the reaction time of

animals with relatively extended duration of stimulation,

confirming centrally mediated activity. Acetone extract of

P. nilgiriensis fruit and stem showed significant analgesic

activity at 200 and 400 mg/kg (Table 3). Analgesic activity

of the latter dose was often compared with the standard

drug Pentazocine. At the dose level 400 mg/kg and 240

min reaction time fruit extract showed higher of analgesic

activity 7.25±1.25 (72%), than the stem extract 6.5±0.65

(69%). The present ndings of the hot plate test indicate

significant analgesic effect of acetone extract of P. nilgiriensis

fruit and stem through central mechanism. Several Psychotria

species are used medicinally for pain related purpose.

Amador et al. (25) reported that leaves and flowers of

Psychotria colorata were showed very significant level of

analgesic activity in hot plate method. Many of the earlier

studies on Psychotria species proved their analgesic ability.

Acetic acid-induced writhing test: Acetic acid-induced

writhing responses in rats were used to examine the

analgesic effect. This method is not only simple and reliable

but also affords rapid evaluation of peripheral type of

analgesic action. In this test, the animals react with

characteristic stretching behavior, which is called writhing.

The oral administration of acetone extract of P. nilgiriensis

stem and fruit depicted a dose dependent analgesic activity

and the values±SEM for the extract are shown in Table 4.

Injection of acetic acid into the control mice resulted in

78.0±1.08 writhes. Pretreatment with acetone extract of P.

nilgiriensis fruit and stem at doses of 200 and 400 mg/kg

reduced the number of writhes to 21.00±0.63 (64.20%

inhibition) and 33.75±2.16 (53% inhibition), respectively.

Interestingly, the extract dose of 400 mg/kg registered

higher levels of analgesic activity than the standard drug

aspirin (29.00±1.36 writhes; 50.57% inhibition) at a dose

of 130 mg/kg. It was also observed that the onset of writhing

was delayed and duration of writhing was shortened with

extract pretreatment. It was found that the acetone extracts

of P. nilgiriensis fruit and stem significantly (p<0.05)

inhibited the acetic acid-induced writhing response and

potentiated the analgesic activity of aspirin as well. The

abdominal constriction is related to the sensitization of

nociceptive receptors to prostaglandins. In a previous study

reported that extract of P. colorata have possess high

amount of analgesic activity (26). It is, therefore, possible

that fruit and stem of P. nilgiriensis produced analgesic

effect probably due to the inhibition of synthesis or action

of prostaglandins.

Anti-inflammatory activity

Carrageenan-induced paw oedema in rats: Carrageenan-

induced paw edema as an in vivo model for inflammation

has been frequently used to assess the anti-inflammatory

effect of natural products. Carrageenan-induced paw

edema model is thought to be a biphasic event. In early

phase histamine and serotonin are released, while in the

late phase prostaglandins, proteases and lysozymes are

released (27). Carrageenan-induced paw edema remained

even 4 h after its injection into the sub plantar region of rat

paw. Indomethacin as a reference standard drug inhibited

Table 3. Analgesic activity of P. nilgiriensis fruit and stem using hot plate method

GroupDose

(mg/kg)

Reaction time (s)Inhibition

(%)InitialTime after drug administration (min)

30 60 120 240

Control (water) 1 mL 1)1.25±0.251) 2.3±0.25 2.3±0.25 2.5±0.2900 2.00±0.4100 NA

Pentazocine 10 1.50±0.29 04.0±0.41* 006.3±0.63** 07.3±0.48*** 8.00±0.41*** 75.248

Acetone extract of fruit200 1.25±0.25 3.25±0.25* 05.0±0.71* 6.5±0.96** 6.75±0.25** 70.83

400 01.5±0.29 03.0±0.41* 5.5±0.5* 07.0±0.71*** 7.25±1.25*** 72.02

Acetone extract of stem200 1.75±0.25 3.75±0.25* 5.75±0.48* 5.75±0.25**0 5.5±1.19** 51.79

400 02.0±0.41 03.5±0.29* 04.75±0.25** 6.75±0.25*** 006.5±0.65*** 69.11

1)Values are expressed as mean±SEM (n=6); Significantly different at *p<0.05, **p<0.01, ***p<0.001 when compared to control

Table 4. Analgesic activity of P. nilgiriensis fruit and stemusing acetic acid-induced method

GroupDose

(mg/kg)Number of

writhesInhibition

(%)

Control (water) 1 mL 78.0±1.081) NA

Aspirin 150 36.25±2.06*0 53

Acetone extract of fruit200 45.25±6.5500 35

400 32.25±6.70*0 54

Acetone extract of stem200 0.48±6.650 32

400 033.75±2.16*** 531)Values are expressed as mean±SEM (n=6); Significantly different at*p<0.05, ***p<0.001 when compared to control


the edema formation due to carrageenan to an extent of

(5.28±0.1 mm at 4 h) at the dose of 10 mg/kg. The acetone

extract of P. nilgiriensis fruit and stem significantly

inhibited edema formation in rats (p<0.05) in a dose

dependant manner. The acetone extracts at the dose of

400 mg/kg inhibited edema formation to the extent of

(5.45±0.12 and 5.36±0.18 mm, respectively at 4 h) and the

edema was found to be reduced to 1.51±0.12 mm (Table

5). The results obtained indicated that acetone extracts of

fruit and stem has significantly (p<0.05) inhibited the

formation of rat paw edema, both in early and the late

phases. Researchers demonstrated that inflammatory effect

induced by carrageenan is associated with free radical. The

carrageenan induced inflammatory response has been

linked to neutrophil inltration and the production of

neutrophil-derived free radicals, such as hydrogen

peroxide, superoxide, and hydroxyl radicals, as well as the

release of other neutrophil-derived mediators (28). Oral

pretreatment of animals with acetone extracts of fruit and

stem has resulted in a significant inhibition of carrageenan-

evoked hind paw edema because these extracts have also

showed renounced activity in in vitro antioxidant assays.

GC/MS analysis GC/MS chromatogram of acetone extract

of P. nilgiriensis fruit along with their retention time (RT)

are shown in the Fig. 2. Major phytocomponents present in

the P. nilgiriensis fruit along with molecular formula,

molecular weight, peak area, and structure were presented

in the Table 6. The GC/MS chromatogram of acetone

extract of P. nilgiriensis fruit showed the presence of

several active principle compounds. More than 6 peaks

were identified from acetone extract of fruit at retention

time of 38.94 min. The chromatogram obtained from GC

has shown the phytochemical strength of the P. nilgiriensis

extract. The sesquiterpinoid compounds are more specific

to the Rubiaceae family. In GC/MS analysis of P. nilgiriensis

acetone extract of fruit, a sesquiterpinoids quinines compound

known as Nakijiquinones B was present in this plant

which is commonly known for its fungicidal property.

Nakijiquinones, the only natural product known to

selectively inhibit the Her-2/Neu protooncogene which is

associated with cancer treatment (29). 3, 4-Epoxy-7-octen-

2-one is the compound present in this plant which can act

as antioxidant, hypocholesterolemic and anti-inflammatory

agent. 9-Octadecenoic acid (Z)-(CAS) oleic acid with

Table 5. Anti-inflammatory activity of P. nilgiriensis fruit and stem using carrageenan-induced paw edema in rats

GroupDose

(mg/kg)

Oedema induced by carrageenan (mm)

0 h 1 h 2 h 3 h 4 h

Control (water) 1 mL 1)4.86±0.321) 5.49±0.11 5.92±0.32 6.53±0.18 6.96±0.21

Indomethacin 10 5.08±0.17 5.21±0.15 5.26±0.12 05.44±0.10* 05.28±0.19*

Acetone extract of fruit200 4.98±0.10 5.83±0.16 5.48±0.19 5.62±0.19 6.25±0.11

400 4.96±0.14 5.84±0.20 5.16±0.12 05.17±0.13* 05.36±0.18*

Acetone extract of stem200 5.22±0.06 5.67±0.36 5.12±0.38 05.05±0.15* 5.89±0.16

400 5.10±0.21 5.25±0.20 5.15±0.29 05.17±0.21* 05.45±0.12*

1)Values are expressed as mean±SEM (n=6); Significantly different at *p<0.05, when compared to control

Fig. 2. GC/MS chromatogram of acetone extract of P. nilgiriensis fruit.


antioxidant and anti-inflammatory property. Oleic acid

may hinder the progression of adrenoleukodystrophy

(ALD), a fatal disease that affects the brain and adrenal

glands. Oleic acid may be responsible for the hypotensive

(blood pressure reducing) effects of olive oil (30). Several

other compounds were also depicted through GC/MS

chromatogram. They are also having notable medicinal

property. The GC/MS analysis profile has also confirmed

the anti inflammatory and analgesic properties of P.

nilgiriensis.

According to the data derived from the present study, P.

nilgiriensis extracts was found to be an effective antioxidant

in different in vitro assays when compared to standard

antioxidant compounds. In all the in vitro and in vivo

studies and GC/MS analysis, acetone extract of fruit

showed notable radical scavenging, analgesic and anti

inflammatory properties. Hence further studies should be

considered for isolating bioactive compounds from P.

nilgiriensis which will pave a way in promoting natural

drugs for various health diseases.

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Table 6. GC/MS analytical report for major phytoconstituents in acetone extract of P. nilgiriensis

Name of the compoundRetentiontime (min)

Molecularweight

% ofArea

Structure Uses

2-Hydroxy-2,N-dimethyloctanoic acid amide octanamide, 2-hydroxy-N,2-dimethyl- (CAS)

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C10H21NO2

Pharmaceutical raw materials & food additives

(Z)-2-Methylhex-4-en-3-yl N-phenylcarbamate

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C8H12O2

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C26H28O2Sn

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C26H37NO

Anti-diabetic and anti-inflammatory activity


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Evaluation of Antioxidant and Pharmacological Properties of Psychotria nilgiriensis Deb & Gang

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