WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Hassan et al.

World Journal of Pharmacy and Pharmaceutical Sciences

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Volume 5, Issue 1, 129-135

Research Article

ISSN 2278 4357

CHEMICAL COMPOSITION AND IN VIVO ANTI-INFLAMMATORY

ACTIVITY OF THE LIPID EXTRACT FROM PREMNA ODORATA
BLANCO CULTIVATED IN EGYPT
Abeer H. El-Mudomya, Hossam M. Hassana*, Elham Amina,b, Waleed A. Mohamedc and
Mona H. Hettad
a

Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, 62514, Egypt.

b

Department of Pharmacognosy, Faculty of Pharmacy, Al Jouf University, KSA.

Department of Biochemistry, Kasr El-Eini Teaching Hospitals, Cairo University, Egypt.

Department of Pharmacognosy, Faculty of Pharmacy, Fayoum University, 63514, Egypt.

Article Received on
04 Nov 2015,

ABSTRACT

Revised on 27 Nov 2015,

Accepted on 20 Dec 2015

tree native to temperate and tropical Asia including the Philippines.

Premna odorata Blanco (Labiatae) popularly known as alagaw is a

Reviewing the literature; nothing could be traced discussing lipid

content or evaluating the biological activity of this plant. This study
*Correspondence for

was aimed to identify the chemical composition of P. odorata lipoidal

Author
Dr. Hossam M. Hassan

extract using GC/MS analysis. and evaluating its anti-inflammatory

Department of

activity by measuring the pro-inflammatory markers (COX-II and IL-

Pharmacognosy, Faculty

6) in Inflamed liver Wister albino rats. Twenty five compounds were

of Pharmacy, Beni-Suef

identified by GC/MS analysis representing 72.15% of total identified

University, 62514, Egypt.

compounds consisted mainly of fatty acids in free and ester forms

(57.18%). Linolenic acid and its ester form were the main identified compounds representing
18.58% and 19.00% of the total identified compounds, respectively. Biological results
showed that treatment of Inflamed liver of rats using a dose of 500mg/kg lipid extract had
insignificant level change in COX-II and IL-6 when compare to normal control group. It
could be concluded that P. odorata lipid content consisted mainly of fatty acids and interferes
with COX-II and IL-6 roles during the inflammation progress disease.
KEYWORDS: Premna odorata; lipoidal; anti-inflammatory; GC/MS; Linolenic acid.

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INTRODUCTION
Premna odorata Blanco (Labiatae) popularly known as alagaw is a tree native to temperate
and tropical Asia including the Philippines. It has many synonyms as P. curranii H. Lam., P.
oblongata Miq.var. puberula H. Lam., P. serratifolia Blanco and P. vestita Schauer. In the
Philippines, the decoction of the leaves was considered as diuretic, carminative and febrifuge
as well as it is used for vaginal irrigation; coughs; beri-beri; abdominal pains and dysentery.
P. odorata is one of seven plants present in a commercialized Philippine herbal preparation
called Pito-Pito which used in a wide variety of folkloric applications as headaches, fever,
cough, colds, migraine, asthma, abdominal pains and diarrhea (Pinzon et al., 2011).
According to Hsu et al., 2013; polyunsaturated fatty acids (PUFAs) can play a role in
modulating the inflammatory responses by regulating the activities of inflammatory cells
which interfere with the production of cytokines and the various balances within the immune
system. Similarly, study suggested that dietary supplementation of PUFAs have beneficial
impact in suppression of inflammation, especially eicosapentaenoic acid (EPA; 20:5 n-3) and
docosahexaenoic acid (DHA; 22:6 n-3) (Fetterman and Zdanowicz, 2009 and Calder, 2006).
Also; Sterols and triterpenes interfere with the expression of pro-inflammatory cytokines by
blocking NF-B activity (Han and Bakovic, 2015). Reviewing the literature; nothing could be
traced discussing P. odorata lipid content or evaluating its anti-inflammatory activity. These
results encourage to identify of the chemical composition of P. odorata lipoidal extract as
well as the evaluation of its anti-inflammatory activity.
MATERIAL AND METHODS
Plant material and extraction
Premna odorata leaves were collected from The Zoo, Giza, Egypt during the period from
March-May 2013. It was kindly identified by Dr. Abd El-Halim A. Mohammed, Horticultural
Research Institute, Department of Flora and Phytotaxonomy Researches, Dokki, Cairo,
Egypt. A voucher specimen (2013-BuPD 45) was deposited at Department of
Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Egypt. Air dried P. odorata
leaves powder was successively extracted twice by cold maceration using n-hexane organic
solvent.
GC/MS analysis conditions
GC/MS spectrometry used in analyzing n-hexane fraction was carried out in an Agilent 6890
gas chromatograph equipped with an Agilent mass spectrometric detector using a direct

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capillary interface and fused silica capillary column PAS-5 ms (30 m 0.32 mm 0.25 m
film thicknesses). Helium was used as a carrier gas at approximately 1 ml/minute, pulsed
splitless mode. The solvent delay was 3 minutes and the injection size was 1.0l. The mass
spectrometric detector was operated in electron impact ionization mode with ioning energy of
70e.V. scanning from 50 to 500 m/z. The ion source temperature was 230C and the
quadrapole temperature was 150C. The electron multiplier voltage (EM voltage) was
maintained at 1250 V above auto tune. The instrument was manually tuned using
perfluorotributyl amine (PFTBA). The GC temperature program was started at 60C for 3
minutes then elevated to 300C at a rate of 5C/minute and injector temperature was set at
300C. Wiley and Nist 05 mass spectral data base was used for the identification of the
separated peaks.
In vivo anti-inflammatory activity in inflamed liver rats
Wister albino rats (140-200gm) were obtained from animal house of National Research
Centre, Dokki Giza, Egypt. Rats were fed on a standard diet and free access to tap water
(International, 2005). The study was approved by Institutional Animal Ethics Committee of
National Research Center that the animal doesn't suffering at any stage of experiment and
maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The
inflamed liver was induced in rats according to Keegan et al., 1996. The rats were divided
into three groups, each contain five rats, treated with a dose of 500 mg/kg/day; as follows:
group I, Normal control; group II, inflammated liver rats (positive control receiving saline);
groups III, inflammated liver rats were orally treated with n- hexane extract. After 20 days,
rats were sacrificed and blood samples were collected by puncture the sublingual vein in
clean and dry test tube. Allow clotting for 10 minutes before centrifuging at 3000 rpm for
serum separation. The separated serum was stored at -80C for further determinations of the
following tests: COX-II was determined according to colorimetric COX (ovine) inhibitor
screening assay kits, item NO. 760111, Cayman Chemical Company, Ann Arbor, and MI and
IL-6 was determined according to Rat IL-6 ELISA for detection of rat IL-6 in sera, plasma,
body fluids, tissue lysates or cell culture supernates, IBL, America.

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Statistical analysis
The data were expressed as mean SE. (standard error). Readings within a group were
compared using the one-way ANOVA followed by Tukey test. Statistical analysis was
performed using SPSS (Version 14). A level of p < 0.05 was considered to be significant.
RESULTS AND DISCUSSION
GC/MS results of n-hexane extract
Results of GC/MS analysis were presented in Table 1. It showed the presence of 25
compounds represented 72.15% of total identified compounds. It is consisted mainly of
2.66% aliphatic and aromatic hydrocarbons, 57.18% fatty acids in free and ester form and
0.77% sterols and triterpenes.
Linolenic acid and its ester form were the major identified compounds in n-hexane fraction
represented 18.58% and 19.00%, respectively. While palmitic acid and its ester form were
represented 11.57% and 1.41, respectively. Also; stearic acid and its ester form were
presented in concentration 0.11% and 5.03%, respectively. Moreover; eicosanoic acid was
presented in acidic form only (1.18%), while pentadecanoic acid was presented in ethyl ester
form only (0.14%).
Table.1. Results of GC/MS analysis of n-hexane extract of Premna odorata
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

Identified compounds
1-hexadecene
benzene , (1-butyl heptyl)benzene , (1-propyloctyl)benzene , (1-ethylnonyl)benzene , (1-pentylheptyl)benzene , (1- butyloctyl)benzene , (1-propylnonyl)benzene , (1-ethyldecyl)1-octadecene
benzene , (1-pentyloctyl)neophytadiene
2-pentadecanone , 6,10,14-trimethyl
pentadecanoic acid (C15:0), ethyl ester
palmitic acid, methyl ester (C16:0)
9-hexadecenoic acid (C16:1), ethyl ester
homofascaplysin C
stearic acid (C18:0)
palmitic acid (C16:0)
linolenic acid (C18:3), ethyl ester

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Vol 5, Issue 1, 2016.

RRT
0.647
0.676
0.682
0.695
0.734
0.737
0.744
0.757
0.773
0.793
0.800
0.805
0.831
0.852
0.875
0.899
0.907
0.947
1.000

M+
224
232
232
232
246
246
246
246
252
260
278
268
270
270
282
284
284
256
306

% area
0.28
0.06
0.13
0.10
0.12
0.10
0.09
0.11
1.02
0.14
0.51
0.43
0.14
1.41
0.16
11.02
0.11
11.57
19.00*

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stearic acid (C18:0), ethyl ester

1.007
312
5.03
linolenic acid (C18:3)
1.037
278
18.58*
eicosanoic acid (C20:0)
1.107
312
1.18
vitamin e
1.383
430
0.09
-sitosterol
1.448
414
0.56
-amyrin
1.476
426
0.21
% of total identified compounds
72.15%
+
No (number); RRT (relative retention time); M (molecular ion peak); *(major compound)
20
21
22
23
24
25

In vivo anti-inflammatory activity in inflamed liver rats

Table 2 and Figure 1 showed the manipulation of COX-II and IL-6 levels in liver tissue
homogenates of normal control and different treated groups. In response to Inflamed liver
state COX-II and IL-6 levels showed significant increase in their levels as compared to
normal control. Treatment of Inflamed liver rats with n-hexane extracts using a dose of
500mg/kg showed insignificant change in COX-II and IL-6 as compare to normal control
group.
Table.2. In vivo anti-inflammatory activity of n-hexane extract of Premna. odorata
Groups
Group I
Group II
Group III

COX-II
IC50 Mm
1.90.5
3.00.2
1.5*0.1

IL-6
Pg/ml
2.70.3
5.00.1
2.0*0.1

COX-II: cycoloxygenase-II; IL-6: Interleukin-6; *: significant different from positive control,

Group I: negative control group; group II: positive control Inflamed liver group; group III: nhexane extract treatment group.
According to the literature; P. odorata n-hexane extract contain compounds which had antiinflammatory activity. It was reported that, sterols and triterpenes interfere with the
expression of pro-inflammatory cytokines by blocking NF-B activity (Han and Bakovic,
2015). Linolenic acid plays role in the production of both thromboxane A1 (TXA1) and
PGE1which chief effects were anti-inflammatory activity (Kapoor, 2003). Palmitic acid was
significantly inhibiting PLA2 enzyme activity that releases acid which modified inflammatory
mediators prostaglandins and leukotrienes (Aparna et al., 2012).

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Figure.1. Anti-inflammatory activity of Premna odorata n-hexane extract in alcoholic

inflamed liver Wister albino rats, 1: negative control group (I); 2: positive control
inflamed liver group (II); 3: n-hexane extract treatment group (III).
CONCLUSION
It could be concluded that, Lipoidal content of Premna odorata cultivated in Egypt consisted
mainly of long chain fatty acids in free and ester forms (57.18%). Linolenic acid and its ester
form were the major identified compounds in n-hexane fraction represented 18.58% and
19.00%, respectively. The n-hexane extract had anti-inflammatory activity which interferes
with COX-II and IL-6 roles during the inflammation progress disease.
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