Download 14. Narayanan S. et al. Antioxidant activities of Seabuckthorn

“Anticancer effect of methanol extract of Hippophae rhamnoides
Linn. seed alone and in combination with Cyclophosphamide”
M. Pharm Dissertation Protocol Submitted to
Rajiv Gandhi University of Health Sciences, Karnataka
Bangalore– 560 041
By
Mr. Chirag A. Patel B.Pharm
Under the Guidance of
Dr. Divakar Goli M.Pharm. Ph.D
Professor
2008-09
Department of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road, Bangalore – 560 090
1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the candidate &
Address.
Mr. Chirag A. Patel
At & Post-Aritha,
Ta-Sami, Dist-Patan, Patelvas,
Gujarat-384240
2.
Name of the Institution.
3.
Course of the study
& subject
M.Pharm (Pharmacology)
4.
Date of admission.
05/06/2008
5.
Acharya & B.M. Reddy College Of Pharmacy
Soldevanahalli, Hesaraghatta Road,
Chikkabanavara Post,
Bangalore-560090.
Phone No: 080 65650815
Fax No: 080 28393541
“Anticancer effect of methanol extract of
Title of the Topic
Hippophae rhamnoides Linn. seed alone and
in combination with Cyclophosphamide”
6.
7.
Brief resume of intended work
6.1 Introduction and need of the work
Enclosure I
6.2 Review of Literature
Enclosure II
6.3 Aim and Objective of the study
Enclosure III
Materials & Methods
7.1 Source of data
Enclosure IV
2
7.2 Methods of collection of data
Enclosure V
7.3 Does the study require investigation
on animals?
a. If yes give details
Enclosure VI
7.4 Has ethical clearance been obtained
from your institution in case of 7.3
Yes (Copy Enclosed)
8.
List of references ( About 1 – 6)
9.
Signature of the candidate
Enclosure VII
Remarks & Signature of the guide
10.
11.
12.
Name & Designation of Guide
Head of the Department
Signature of HOD
13.
Dr. Divakar Goli M.Pharm. Ph.D
Professor
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road Bangalore – 560 090
Dr. Kalyani Divakar M.Pharm. Ph.D
Professor & Head Dept of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road Bangalore– 560 090
Remarks of the Principal
14.
Signature of Principal
Dr. Divakar Goli M.Pharm. Ph.D
Principal
Department of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli, Chikkabanavara (Post)
Hesaraghatta Main Road Bangalore – 560 090
3
ENCLOSURE-I
6. BRIEF RESUME OF INTENDED WORK
6.1 Introduction and need of work:
Neoplasia literally means “new growth”. A neoplasm as defined by Willis is “an
abnormal mass of tissues, the growth of which as same of the normal tissue and persist in the
same excessive manner after the cessation of the stimuli which evoked the change”.1
In common medical usage, neoplasm is often referred to as tumor, and the study of
tumors is called oncology. Characteristic of benign & malignant neoplasm include 1)
Differentiation & Anaplasia 2) Rate of growth 3) Local invasion and 4) Metastasis.
Malignant tumors are collectively referred to as cancer. Cancer is not one disease but many
disorders that share profound growth deregulation, some cancers such as Hodgkin’s
lymphoma is curable whereas cancer of pancreas has high mortality.1
Cyclophosphamide is an alkylating chemotherapeutic agent with immunosuppressive
activities. The alkylating metabolite can bind to a variety of molecules including amino acids,
proteins, and peptides, but the most important binding site is DNA where cross-linking
2
occurs. It is effective in the treatment of cancer (lymphoma, acute and chronic leukemia’s,
multiple myeloma) and non-malignant disease states such as rheumatoid arthritis and
vasculities. It is inactive until metabolized in the liver by p450 mixed function oxidase. The
important side effects are nausea, vomiting and bone marrow depression.4
In fact, one of the most striking medical practices of the 21st century is the
chemoprevention of cancer.5 Many plant species are known to elicit antimutagenesis and thus
have a full range of prospective application in human health. Even for populations which use
herbs traditionally, encouraging the use of species with chemopreventive actions could be
helpful as part of life expectancy improvement strategies: costs are significantly low, herbs
4
have usually little or no toxicity during long-term oral administration and are relatively
available at large scale.6
Seabuckthorn (Hippophae rhamnoides L. Elaegnaceae) is thorny nitrogen fixing
deciduous shrub, native to Europe and Asia. All parts of the plant are considered to be a good
source of a large number of bioactive substances. The ripe fruit has been reported to be a rich
source of Vitamins A, C, E, and K, carotenoids, and organic acids. Many medicinal effects of
Seabuckthorn against antihypertensive, inflammatory disease, cardiovascular diseases,
mucosal injuries, and skin disorders have been suggested to be due to the high contents of
antioxidant substances present in this plant.7
The present study is planned to evaluate anticancer activity of methanol extract of
Seabuckthorn seeds alone and in combination with Cyclophosphamide.
5
ENCLOSURE-II
6.2 Review of Literature:
Plant:
Name of the plant selected for the present study is “Hippophae rhamnoides L”,
belonging to family Elaeagnaceae.
Description of plant:
Seabuckthorn is a dioecious multi-branched, thorny shrub, reaching 2 to 4 m in height
with stout branches forming a round often symmetrical head. It has brown or black rough
bark and a thick grayish-green crown. Staminate and pollinate flowers are inconspicuous
appearing before the leaves. Leaves are alternate, narrow 4 to 6 cm long, and lanceolate with
a silver-grey color on the upper side. Flower buds are formed mostly on 2-year-old wood,
differentiated during the previous growing season. Fruit is subglobose, 6 to 10 mm long and 4
to 6 mm in diameter, turning yellow to orange when mature in mid September. The root
system is characterized by nitrogen fixing nodules. The male inflorescence consists of four to
six apetalous flowers. The female inflorescence usually consists of one single apetalous
flower with one ovary and one ovule.8-9
Distribution of plant:
Seabuckthorn (Hippophae rhamnoides) is one of the important natural resources of
the mountainous regions of China and Russia. The plant grows naturally in sandy soil at an
altitude of 1,200-4,500 meters (4,000-14,000 feet) in cold climates, though it can be
cultivated at lower altitudes and into temperate zones. Seabuckthorn is a widely distributed
throughout the temperate zone of Asia and Europe. The distribution ranges from Himalayan
regions including India, Nepal, Bhutan, Pakistan
and Afghanistan to China, Mongolia,
Russia, Kazakhstan, Hungary, Romania, Switzerland, Germany, France and Britain. The
wide distribution of Seabuckthorn is reflected in its habit-related variation not only in
6
morphology, yield, growth rhythms and cold hardiness, but also in berry related characters
such as fresh weight, chemical and sensory attributes.10
Chemical Constituents:
Seabuckthorn berries are among the most nutritious and vitamin-rich fruits found in
the plant kingdom. The vitamin C concentration in berries varies from 360 mg/100g of
berries and Vitamin E concentration can be up to 160 mg/100g of berries. Seabuckthorn is
also rich in flavonoid and contains appreciable amounts of water soluble and fat soluble
vitamins. Seabuckthorn berries contain up to 13% soluble sugars, mainly glucose, fructose,
xylose and 3.9% organic acids, mainly malic and succinic acid. A total of 18 amino acids
have been found in Seabuckthorn fruit. There are at least 24 chemical elements present in
Seabuckthorn juice; e.g. Nitrogen, Phosphorous, Iron, Manganese, Boron, Calcium,
Aluminum, Silicon and others. Oil from the juice and pulp is rich in palmitic and palmitoleic
acids (C16:0 and C16:1), while the oil from the seed contains unsaturated fatty acids of C18
type oils (linoleic and linolenic acid). Oils from the seed and juice also contain Vitamin E and
carotenes. In addition, sea-buckthorn berries, leaves and bark contain sitosterol, tocopherol
and many other bioactive compounds.10
Traditional Uses:
Different parts of H. rhamnoides L. have been used for the treatment of diseases in
traditional medicine in various countries of the world. It has long been used for relieving
cough, aiding digestion, invigorating blood circulation and alleviating pain since ancient
time. The extracts of H. rhamnoides L. branches and leaves are used to treat colitis in humans
and animals. Branches and leaves are also used in the treatment of diarrhea. Leaves are used
in gastrointestinal and dermatologic disorders and have been applied as compress form in
rheumatoid arthritis in the middle Asia flowers of H. rhamnoides L. are used as skin softener
7
in Tajikistan. For its haemostatic and anti-inflammatory effects, fruits of the plant are added
to prescriptions in pulmonary, gastrointestinal, cardiac, blood and metabolic disorders in
Indian and Tibetian medicine. After reviewing the ancient literature Li and Guo point out that
sea buckthorn is a mild drug with the characteristic effects such as lowering fever,
diminishing inflammation, counteracting toxicity and abscesses, treating cough and colds,
keeping warm, easing respiration, clearing sputum, having mildly laxative effect, treating
tumors, especially of the stomach and the esophagus, and treating different kinds of
gynecological diseases in Tibetian medicine. Oil extracts obtained from fruits are used in
liver diseases, inflammatory processes, absorption disorders in the gastrointestinal system,
and are applied externally in hemorrhage. Juice, syrup, and oil of the fruits have been used as
pain killer, to heal wounds, in ulcer and other diseases of the stomach, cancer, and as a
metabolism regulator in traditional medicine. The freshly pressed juice is used in the
treatment of colds, febrile conditions, and exhaustion. Oil from fruits and seeds is used in the
treatment of eczema, lupus erythematosus, chronic wounds that are difficult to heal,
inflammatory diseases, erosion of the cervix uteri, in the treatment of burns and frozen parts
of the body. Also, its berry oil is reported to treat skin disease and thrombosis. Oil extracts
are used externally in dermatologic diseases such as eczema, psoriasis, lupus erythematosus,
and chronic dermatoses. In ophthalmology, they are used in the treatment of keratitis,
trachoma, injuries or burns of eye lid, conjunctivitis.11
8
Therapeutic uses with scientific support:

Antioxidant activity was performed using alcoholic extracts of leaves, fruits and
berries of Seabuckthorn (SBT) against chromium (VI), sodium nitroprusside, and
hypoxia induced oxidative stress in albino rats. The results showed that the leaves,
fruits and berries of Seabuckthorn (SBT) have marked anti-oxidant activity.12-14

Antioxidant and antibacterial activities were conducted using methanol extracts of
various Seabuckthorn (H. rhamnoides L.) seeds. The results indicated that antioxidant
and antimicrobial effects of the extract implicate its potential for natural
preservation.15-16

Anti-platelet aggregation activity was conducted using supercritical CO2 extracted
Seabuckthorn berry oil (SBO) and Total flavones of Hippophae Rhamnoides L (TFH).
The study reveled that the beneficial effects of SBO on blood Clotting.17-18

Anti-mutagenic activity was conducted using methanol extract of Seabuckthorn (H.
rhamnoides Linn) berries with Ames assay. The results reveled that Seabuckthorn
berries has Anti-mutagenic activity.19

In vitro anti-tumor activity conducted using isorhamnetin flavonol aglycone isolated
from Hippophae rhamnoides L. against BEL-7402 cells. The treatment results
indicated that in the appearance of a hypodiploid peak (sub-G0/G1 peak), probably
due to the presence of cells in apoptosis and apoptotic bodies with DNA content less
than 2n.20

Anti-inflammatory activity was conducted using leaves and berries extract of
Seabuckthorn (SBT). The observations suggest that the SBT leaves extract has a
significant anti-inflammatory activity and had the potential for the inflammatory
diseases.21-23
9

Anti ulcer activity conducted using CO2-extracted seed and pulp oils of Sea buckthorn
(Hippophae rhamnoides L.) on experimental models of rats. The results suggested
that Seabuckthorn seed and pulp oils have both preventive and curative effects against
experimental gastric ulcers in rats.24

Seabuckthorn extract appraised the effect on liver fibrosis by significantly decreasing
the serum levels of LN (Laminin), HA (Hyaluronic acid), collagen types III and IV,
total bile acid (TBA). So Seabuckthorn may be a hopeful drug for prevention and
treatment of liver fibrosis.25

Antihypertensive effect was conducted using total flavones extracted from seed
residues of Hippophae rhamnoides L. (TFH-SR) and its underlying mechanism in
chronic sucrose-fed rats by evaluating its ability to regulate insulin and angiotensin-II
levels.26

Cardiac function and hemodynamic effects of total flavones of Hippophae rhamnoids
L (TFH) performed on healthy human subjects. The results suggested that TFH may
not only improve myocardial contractility and strengthen cardiac pump function, but
also may decrease peripheral vessels resistance and increase vessels elasticity.27

Healing activity was performed using seed oil, total flavones, procyanidins and leaf
extract of Seabuckthorn (Hippophae rhamnoides) against hypoxia induced cerebral
vascular injury, stress of patellar tendon, acetic acid-induced lesions in the rat
stomach and cutaneous excision-punch wound model respectively. The results suggest
that Seabuckthorn promotes wound healing which may be due to increased
antioxidant levels in the granulation tissue.28-31

Glycometabolism activity performed using flavonoid from the seed residue and fruit
residue of Hippophae rhamnoides L. (FSH and FFH) in mice. The results indicated
10
that FSH and FFH can decrease the levels of blood glucose and lipid in normal mice,
and also control of glyconeogenesis.32

Radioprotection activity conducted using berries and herbal preparation of Hippophae
rhamnoides against whole body lethal Irradiation in mice. The results indicated that
the H. rhamnoides protects mitochondrial and genomic DNA from radiation-induced
damage.33-35

Effect of dietary supplementation with Seabuckthorn (Hippophae rhamnoides) seeds
and pulp oils on fatty acid composition of skin glycerol phospholipids of patients with
atopic dermatitis. The results show that the fatty acid composition of skin glycerol
phospholipids is well buffered against short-term dietary modification.36

Protective effects of seed oil and fruit extracts of Seabuckthorn (Hippophae
rhamnoides L.) against injury induced by sulfur dioxide inhalation and the toxic
effects of arsenic in mice respectively.37-38
11
ENCLOSURE-III
6.3 Aim and Objectives of the study
Aim:
The present study is intended to investigate the anticancer effect of methanol extract
of Hippophae rhamnoides Linn. seeds alone and in combination with Cyclophosphamide.
Objectives:
1. To collect and prepare the methanol extract of seeds of Hippophae rhamnoides L.
2. To perform preliminary phytochemical screening of methanol extracts of seeds
Hippophae rhamnoides L.
3. To study the acute toxicity study of methanol extract of seeds of Hippophae
rhamnoides L.
4. To study the anticancer effect of methanol extract of seed Hippophae rhamnoides L.
alone and in combination with Cyclophosphamide through:
a. MTT assay
b. Clonogenic Assay
c. Cell viability test
d. DNA fragmentation test
e. Glutathione estimation
f. Ames Assay
12
ENCLOSURE-IV
7. MATERIALS AND METHODS
7.1 Source of Data:
Data will be obtained from laboratory based studies by finding outA) Phytochemical analyses of methanol extract of seed Hippophae rhamnoides Linn.
B) Evaluations of the anticancer effect of methanol extract of seed Hippophae
rhamnoides Linn. alone and in combination with Cyclophosphamide by conducting:
a. Glutathione estimation (in Swiss albino mice).
b. MTT assay, Clonogenic assay and Viability test on HL-60 Cell lines
c. DNA fragmentation test on HL-60 Cell lines
d. Ames assay.
C) National & International Journal
D) Text books.
E) Internet.
13
ENCLOSURE-V
7.2 Method of Collection of Data
Drug: Cyclophosphamide will be purchase from pharmacy store.
Plant: The seed of Hippophae rhamnoides L. will be collected and dried. They will be then
coarsely powdered & stored in an air tight container.
Animals: Swiss albino mice weighing 22-25 g will be selected for glutathione estimation.
The animals will be housed in standard environmental condition and provided with food and
water at ad libitum.
Solvent: Methanol is selected for extraction of the seed powder.
Methodology:
Acute toxicity study of methanol extract of seeds of Hippophae rhamnoides L: - Acute
toxicity study will be conduct according to OECD guideline 425.
Preparation of Extraction: - Powdered seed of Hippophae rhamnoides L will be extracted by
using methanol for 18 h with Soxhlet extractor. The dried extract will be used for
determination of the anticancer activity.
Pharmacological studies: - Evaluations of the anticancer effect of methanol extract of seeds
of Hippophae rhamnoides L. alone and in combination with Cyclophosphamide by
conducting following methods:
a. MTT assay
b. Clonogenic Assay
c. Cell viability test
d. DNA fragmentation test
e. Glutathione estimation
f. Ames Assay
14
MTT Assay:-39
The 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a
common method used to assess cell proliferation and cytotoxicity. Briefly, 1x104
exponentially growing cells will be exposed to various concentrations of drug and incubated
and MTT will be added and incubated at 37oC for 4 h. The precipitated Formosan salt will be
dissolved in DMSO and the samples will be read at 570 nm. The 50% inhibitory
concentration (IC50) of drug will be calculated.
DNA fragmentation analysis:- 39
HL-60 cells will be incubated with 0, 5, 10, 15, 20 and 25 μg/ml of drug for 24 h at 37oC. The
DNA will be isolated and precipitated with ethanol, air-dried and dissolved in TE buffer (5
mM Tris-HCl (pH 8.0) and 20mM eidetic acid. DNA fragmentation will be analyzed by
agarose gel electrophoresis.
Cell viability:-39
The effect of drug on the HL-60 cancer Cell will be determined by means of tryphan blue
exclusion technique. Briefly, 3x104 cells will be seeded per well in a 24 well and then treated
with various concentrations of drug. The wells will be incubated at 37oC and the number of
cultured cells in the different wells will be counted using a hemocytometer after staining with
0.4% tryphan blue every 24 h to calculate the doubling time.
Clonogenic Assay:- 40
Tumor colony forming units will be cultured in Dulbecco's modified Eagle's medium (Gibco)
supplemented with 0.3% agar and 20% fetal calf serum. The cultures will be incubated at
37°C in a fully humidified atmosphere containing 10% CO2 in air. Colonies (greater than 40
cells) will be scored after 10-20 days using a dissecting microscope at ×32. A linear relation
of the number of cells plated and colonies could be established.
15
Ames assay:- 19
The salmonella point mutation assay proposed by Maron and Ames was followed with little
amendments as suggested by Bala and Grover to verify the inhibitory activity of the extract.
Constant concentration of two direct acting mutagens, NPD (20 µg/0.1 ml/plate), sodium
azide (2.5 g/0.1 ml/plate) and S9 dependent mutagen, 2AF (20 µg /0.1 ml/plate) will be used
to positive control. 0.1 ml each of bacterial culture (about 1-2 X108 cells/ml), mutagen and
extract will be added to 2 ml of top agar. Equal quantity of the mutagen and the extract will
be blended and allowed to stand for 30 min at 37˚C under incessant shaking and 0.2 ml of
this will be added to 2 ml of soft agar with 0.1 ml of fresh bacterial culture. Soft agar will be
pour on minimal glucose agar plate at 37˚C for 48 h. concurrently, a positive control and a
negative control will also be set. All the test sample and mutagen, i.e. NPD and 2AF, will be
dissolved in dimethylsulfoxide and sodium azide in distilled water. The activity of each
extract will be expressed as percentage decrease of reverse mutations
Glutathione estimation:- 2
7
The tumor will be maintained in vivo by serial intraperitoneal (i.p.) transplantation of 1 × 10
tumor cells (0.25 ml in phosphate-buffered saline, pH 7.4) per mice.
Drugs treatment schedule and antitumor activity:-
Tumor-transplanted mice will be randomly divided into four groups, (I-V) of 20 mice each.
Group-I mice will be serve as control and received normal saline. Group-II and Group-III
mice will be given 1/3 of LD50 and 1/7 of LD50 of methanolic extract of Seabuckthorn from
th
th
5 day to the 10 day of tumor transplantation respectively. Group-IV mice will be given a
th
single dose of CP, i.p., on the 10 day post-tumor transplantation. Group-V mice will be
th
given methanolic extract of Seabuckthorn and, then, administered with CP on the 10 day
post-tumor transplantation. The survival patterns of the hosts will be determined and deaths,
if any, in different groups, will be recorded daily. After 1, 3, 6 and 12 days of treatment, liver,
16
kidneys, spleen, and tumor tissue will be dissect out for biochemical investigations. The
antitumor efficacy of different treatments is reported as percent increase in life span (ILS)
and calculated as per the following formula:
(T/ C x 100) – 100, where T and C are the mean survival days of treated and control
mice, respectively.
Glutathione estimation:Total GSH (TGSH) and non-protein thiol (NPSH) content will be determined using the
method of Sedlak and Lindsay. Briefly, 5% homogenates of tissues will be prepared in 0.02
mol/ L EDTA, pH 4.7 in a motor-driven Teflon–pestle homogenizer. TGSH will be
determined by adding 100 ml of the homogenate to 1.0 ml of 0.2 mol/L Tris-EDTA buffer,
pH 8.2 and 0.9 ml of 0.02 mol/L EDTA, pH 4.7 followed by 20 μl of Elliman’s reagent (10
mmol/L DTNB in methanol). For the determination of NPSH, 500 μl of the homogenate will
be precipitate with 500 μl of 10% trichloroacetic acid and centrifuged at 3000 × g for 15 min.
To 800 μl of the supernatant, 1.6 ml of 0.4 mol/L, Tris-EDTA buffer, pH 8.9, will be add
before the addition of 25 μl of Elliman’s reagent. After 30 min of incubation at room
temperature, the reaction mixture will be centrifuge and the absorbency of supernatants will
be read against a reagent blank at 412 nm using a Spectrophotometer.
Statistical analysis
The significance of difference between groups will be test using one-way ANOVA followed
by post-hoc Fisher’s LSD test. P<0.05 was considered significant.
Animals Required:
12 Swiss albino mice will be used for acute toxicity studies.
100 Swiss albino mice will be used for Glutathione estimation test.
17
Group
I
After treatment
No. of animal
1st day
5
3rd day
5
6th day
5
12th day
5
1st day
5
1/3 of LD50 of methanolic extract of
3rd day
5
Seabuckthorn
6th day
5
12th day
5
1st day
5
1/7 of LD50 of methanolic extract of
3nd day
5
Seabuckthorn
6th day
5
12th day
5
1st day
5
3rd day
5
6th day
5
12th day
5
1st day
5
Methanolic extract of Seabuckthorn +
3rd day
5
Cyclophosphamide
6th day
5
12th day
5
Treatment
Normal saline.
II
III
IV
Cyclophosphamide
V
18
ENCLOSURE-VI
7.3 Does the study require any investigation or intervention to be conducted on patients
or other humans or animals? If so, please describe briefly.
The above study requires investigation on Swiss albino mice (112 no.) for acute
toxicity study and anticancer activity.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The study is cleared from Ethical Committee of the institution. (Certificate enclosed).
19
ENCLOSURE-VII
8. Reference:
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with ascorbic acid against murine ascites Dalton’s lymphoma. Ind J Pharmacol 2006
Aug;38(4):260-5.
3. Abraham P, Sugumar E. Increased glutathione levels and activity of PON1 (phenyl
acetate esterase) in the liver of rats after a single dose of Cyclophosphamide: A
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21
18. Johansson AK, Korte H, Yang B, Stanley JC, Kallio HP. Sea buckthorn berry oil
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J of Chinese Clinical Med 2007 Aug;2(8):
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isorhamnetin isolated from Hippophae rhamnoides L. against BEL-7402 cells.
Pharmacol Res 2006;54:186–194
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leaves. Inter Immunopharmacol 2005; 5:1675–84.
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inflammatory response in murine macrophages. Inter Immunopharmacol 2006;6:
646– 52.
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