Постинг
29.12.2016 21:44 -
Най-доброто средство за унищожаване на грипа
Автор: apollon
Категория: Изкуство
Прочетен: 5059 Коментари: 4 Гласове:
Последна промяна: 01.05.2017 12:09
Прочетен: 5059 Коментари: 4 Гласове:
10
Последна промяна: 01.05.2017 12:09
Всички в края на този декември са болни. Това се случи през 2016 лето Господне. Само аз и неколцина останаха здрави по една истинска случайност. Приемах препарата неокс, на който му минаваше срока и който съдъжаше ситно смлени частици от семената на сминдух. Латинското име на растението е Trigonella foenum-graecum L. Семената от бобовете на тази тревиста билка се оказва, че действат силно токсично на цял ред насекоми, за които те биха били достъпна храна и следователно никой бръмбар на земята, нито неговите ларви не могат да се развият или хранят от тях. В това се крие биологическия смисъл на растението. Освен това растението намалява кръвната захар, действа апетитовъзбуждащо,, разхлабващо и увеличава мъжката потентност. При жените увеличава лактацията. Намалява холестерола и липидите. Действа противовъзпалително на стомаха и червата.
Други два люти метода за предпазване от грип са употребата на чай от джинджифил или резени за смучене, както и резени от скилидка чесън, защото действат вирусоцидно в устната кухина, откъдето заразата навлиза в организма най-вече по въздушно-капков път.
На български билката се казва още гръцко сено.
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Статия за активиране на имунната система при мишки от водни екстракти на гръцко сено:
Immunomodulatory effects of fenugreek
(Trigonella foenum graecum L.) extract in mice
Bilal Bin-Hafeez, Rizwanul Haque, Suhel Parvez, Suwarna Pandey,
Iqbal Sayeed 1, S. Raisuddin*
Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard University, New Delhi 110 062, India
Received 19 April 2002; received in revised form 20 July 2002; accepted 13 December 2002
Abstract
Immunomodulatory activity of aqueous extract of Trigonella foenum graecum L., a widely used medicinal and dietary
herb, was evaluated in male Swiss albino mice. Mice were treated with three doses of extract (50, 100 and 250 mg/kg body
weight per os) for 10 days. Body weight, relative organ weight, cellularity of lymphoid organs, delayed type of
hypersensitivity (DTH) response, plaque-forming cell (PFC) assay, haemagglutination titre (HT), quantitative haemolysis of
SRBC (QHS) assay, phagocytosis, and lymphoproliferation were studied in various groups of animals. At doses of 50 and
100 mg/kg, a significant increase ( p < 0.05) in relative organ weight of thymus was observed but there was no effect on
kidney and spleen weights. Liver weight also increased significantly at doses of 100 and 250 mg/kg. However, no elevation
in the levels of liver function test (LFT) enzymes was observed. As regards lymphoid organ cellularity, spleen recorded no
significant increase at any dose, whereas cellularities of thymus and bone marrow were significantly increased. T. foenum
graecum extract elicited a significant ( p < 0.001) increase in the DTH response at doses of 50 and 100 mg/kg, but the change
at higher dose of 250 mg/kg was not statistically significant. Humoral immunity as measured by PFC showed an elevated
response at a dose of 100 mg/kg, but at 50 and 250 mg/kg, no significant effect was observed. In the HT test, plant extract
also showed modulatory effect at all the doses. Plant extract elicited a significant increase in phagocytic index and
phagocytic capacity of macrophages. Stimulatory response of plant extract was also observed in lymphoproliferation assay
but the response was weak. Overall, T. foenum graecum showed a stimulatory effect on immune functions in mice. As it is
used for a variety of medicinal purposes, its immunostimulatory effect, as reported in this study, strengthens the rationale of
its use in several Ayurvedic and Unani drugs.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Trigonella foenum graecum; Immunomodulation; Traditional medicine systems; Immunosuppression
1. Introduction
A large number of plants and their isolated
constituents have been shown to potentiate immunity
[1,2]. Some medicinal plants have been shown
to exert anti-inflammatory, anti-stress and anti-can-
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1567-5769(02)00292-8
* Corresponding author. Tel.: +91-11-26059688x5568;
fax: +91-11-26059663.
E-mail address: sraisuddin@hotmail.com (S. Raisuddin).
1 Present address: Department of Neurology, University of
Magdeburg, Leipziger Str., 44d-39120, Magdeburg, Germany.
www.elsevier.com/locate/intimp
International Immunopharmacology 3 (2003) 257–265
cer effects by modulating the immune functions
[3,4]. We have reported protective effect of Cassia
occidentalis and Emblica officinalis Gaertn against
cyclophosphamide (CP)-induced suppression of
humoral immunity in mice [5,6]. It was also found
that C. occidentalis, E. Officinalis and Cassia
cinnamon have antimutagenic effects against benzo[
a]pyrene and CP-induced mutagenicity [7–10].
Besides source of medicine, these plants are used as
dietary constituents in India and other Afro-Asian
countries.
Fenugreek (Trigonella foenum graecum L.; family
Leguminoceae) is one such plant whose seeds
and leaves are used not only as food but also as an
ingredient in traditional medicine. Seeds of fenugreek
are used as a condiment and as a supplement
to wheat and maize flour for bread making and as a
constituent of the daily diet of general population in
Indian subcontinent. Its leaves are consumed widely
in India as a green leafy vegetable and are a rich
source of calcium, iron, h-carotene and other vitamins
[11]. Seeds of T. foenum graecum contain
tannic acid, fixed and volatile oils and a bitter
extractive, diosgenin, alkaloids trigonelline, trigocoumarin,
trigomethyl coumarin, and steroidal saponin
such as gitogenin and traces of trigogenin and
vitamin A [12,13].
Some of the therapeutic uses of T. foenum
graecum include its use as hypoglycemic, antiulcerogenic,
hypocholesterolemic and antihypertensive
agent [11,14,15]. In Ayurvedic and Unani
systems of medicine, fenugreek is used for the
treatment of epilepsy, paralysis, gout, dropsy,
chronic cough and piles [16]. In the present communication,
we report its immunomodulatory effects
in mice.
2. Materials and methods
2.1. Plant extract
Total aqueous extract of T. foenum graecum in
semisolid form was procured from the Plant Extract
Division of Saiba Industries, Mumbai. A certificate of
authenticity was provided along with the extract. The
chemical analysis report provided by the manufacturers
showed no presence of endotoxin.
2.2. Chemicals
Antibiotic antimycotic solution (100), bovine
serum albumin (BSA), fetal bovine serum (FBS),
Hank’s balanced salt solution (HBSS), Histopaque-
1077, phosphate-buffered saline (PBS) and RPMI-
1640 medium were purchased from Sigma, St. Louis,
MO. Petroleum jelly was obtained from Hi-Media
Labs, Mumbai, conconvalin A (Con A) from Banglore
Genei (India) and solvents from BDH, Mumbai;
tritiated [3H]-thymidine was purchased from Amersham
Pharmacia Biotech UK, Bukinghamshire.
2.3. Animals
Study was conducted in Swiss albino male mice
(30–35 g). Female guinea pigs (250 g) were used for
the preparation of complement for plaque-forming cell
(PFC) assay. The Central Animal House Facility of the
University provided the animals. The animals were
bred and maintained under standard laboratory conditions
(temperature 25F2 jC; photoperiod of 12 h).
Commercial pellet diet and water were given ad
libitum.
2.4. Dosage
The plant extract was suspended in normal saline
and was administered orally for 10 days at doses of 50,
100 and 250 mg/kg body weight. The dose volume
was 0.2 ml. Control animals received the same volume
of normal saline.
2.5. Body weight, lymphoid organ weight and
cellularity
Animals were divided into four groups (I–IV).
Each group comprised of a minimum of five animals.
Group I (control) received normal saline;
group II, plant extract 50 mg/kg body weight; group
III, plant extract 100 mg/kg; and group IV, plant
extract 250 mg/kg. The animals were humanized 24
h after the last dose. Body weight gain (percentage)
and relative organ weight (organ weight/100 g of
body weight) of kidney, liver, spleen and thymus
were determined for each animal. Single cell suspensions
were prepared in RPMI-1640 medium from
bone marrow (from femur), spleen and thymus for
258 B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265
cell count. Cell counting was done by using the
Neubauer chamber [17].
2.6. Assessment of humoral immune functions
Animals of all the groups treated in the abovedescribed
manner were challenged with 0.2 ml of 10%
sheep red blood cells (SRBC), i.p. on the 10th day of
initiation of experiment. The following parameters of
humoral immunity were studied in those animals.
2.6.1. Haemagglutinin titre (HT) assay
HT assay was performed using the procedure of
Bin-Hafeez et al. [5]. On the fifth day after immunization,
blood was collected from orbital plexus of
each mouse for serum preparation. Serial twofold
dilution of serum was made in 50 Al of PBS (pH 7.2)
in 96-well microtitre plates (Tarsons Products, Calcutta)
and mixed with 50 Al of 1% SRBC suspension in
PBS. After mixing, plates were kept at room temperature
for 2 h. The value of antibody titre was assigned to
the highest serum dilution showing visible haemagglutination.
2.6.2. Plaque-forming cell assay
The PFC assay was performed using the method of
Raisuddin et al. [18]. The animals were humanized on
the fifth day of immunization with SRBC. The spleen
was removed, cleaned free of extraneous tissues, and a
single cell suspension of 106 cells/ml was prepared
from it in RPMI-1640 medium. For PFC assay, the
SRBC were prepared at a cell density of 5108 cells/
ml in PBS. One milliliter of SRBC in medium along
with 0.5 ml of diluted guinea pig complement (1:10
diluted with normal saline) was added to 1 ml of
spleen cell suspension. Cuningham chambers were
prepared using glass slide, coverslips and doublesided
tape (Scotch Brand, St. Paul, MN). The chambers
were loaded with a known volume of assay
mixture, sealed with petroleum jelly and incubated
at 37 jC for 1 h. The plaques were counted under a
light microscope (Olympus BX50) and expressed as
PFC per 106 spleen cells.
2.6.3. Quantitative haemolysis of SRBC (QHS) assay
QHS assay was performed using the methods of
Simpson and Gozo [19] with some modification [5].
Spleens were removed and a cell suspension of
1106/ml was prepared in PBS. One milliliter of
0.2% SRBC and 1 ml of 10% guinea pig serum were
mixed with cell suspension and incubated for 1 h at
37 jC. After centrifugation at 3000 rpm for 3 min,
optical density of the supernatant was measured at
413 nm using a spectrophotometer (Shimadzu UV-
1201).
2.7. Assessment of cellular immune function
For assessment of cellular immune function, delayed
type of hypersensitivity (DTH) response was
evaluated in the extract-treated mice. Four groups of
animals were treated as described above.
2.7.1. Delayed type of hypersensitivity response
The DTH response was determined using the
method of Raisuddin et al. [18]. On the day of
termination of the treatment, animals were immunized
with 1109 SRBC, subcutaneously. On the fifth day
of immunization, all the animals were again challenged
with 1108 cells in the left hind footpad. The
right footpad injected with the same volume of normal
saline served as trauma control for nonspecific swelling.
Increase in footpad thickness was measured 24 h
after the challenge using dial caliper (Guanglu,
China).
2.8. Assessment of nonspecific immune functions
For assessment of nonspecific immune functions,
phagocytic activity of macrophages was studied. Four
groups of animals were treated as described above.
2.8.1. Phagocytic activity of macrophages
The phagocytic activity of peritoneal macrophages
was evaluated using the suspension assay as described
by Fujiki and Yano [20] with some modification.
Briefly, 0.1 ml of aliquot of cells having density of
10106 cells/ml was mixed with 0.1 ml RPMI-1640
medium containing 20% FCS and 100106 cells/ml
heat-treated yeast (Saccharomyces cereviceae) cells.
The mixture was incubated at 37 jC for 1 h with
occasional shaking. After incubation, 50 Al of this
mixture was smeared on the glass slide, air dried and
stained with Wright–Giemsa stain. The slides were
observed under light microscope using oil immersion.
At least 500 cells were counted. The phagocytic activ-
B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265 259
ity was expressed as phagocytic index (PI) and phagocytic
capacity (PC) using the following formulae.
PI ј AB
where A is the percentage of yeast-ingesting phagocytes
and B is the number of yeast-ingested per phagocytes;
PC is the mean percentage of cells that
engulfed z4 yeast cells.
2.8.2. Lymphocyte proliferation assay
For assessment of lymphocyte proliferation assay,
four groups of mice were treated as described above.
On completion of treatment blood was withdrawn
from retro-orbital plexus of mice. Lymphocytes were
isolated from the blood by density gradient centrifugation
on Histopaque-1077. The mononuclear cells
from the buffy coat were carefully pipetted out with a
sterilized Pasteur pipette and pelleted at 400g for
10 min. The cell pellet was suspended in 1 ml of
growth medium (RPMI-1640) containing 10% FBS
and recommended concentration of antibiotic and
antimycotic solution. The cell viability was assessed
by the trypan blue exclusion method and the cells
were plated in 96-well culture plates (Tarsons) at a
cell density of 1106 cells/well. ConA was used as a
mitogenic agent. The culture plate was incubated at
37 jC in CO2 (5%) incubator (Forma Scientific,
Ohio), with water-saturated air for 6 days. After 6
days the cells were pulsed with 1 ACi of [3H]-methylthymidine.
Cells were harvested after 18 h of radioactive
pulse and the amount of [3H]-thymidine incorporated
into DNA was determined by liquid
scintillation spectroscopy [21].
2.8.3. Liver function tests
Activities of serum glutamate oxalate transaminase
(SGOT) and serum glutamate pyruvate transaminase
(SGPT) were estimated by using the kits (Span
Diagnostics, Surat, India). For this purpose, four
groups of animals (one control + three treatments) as
described above were used and treated for 10 days
with respective doses of plant extract.
2.9. Statistical analysis
Data were statistically analyzed using Student’s ttest
to determine significant differences in the data of
various groups. P values less than 0.5 were considered
significant. The values are expressed as means
FS.E.
3. Results
3.1. Effect of plant extract on body weight, lymphoid
organ weight and cellularity
None of the doses of Trigonella extract showed
toxicity or mortality in the extract-treated animals.
No significant body weight gain differences were
recorded in various groups of animals (Table 1). No
effect was observed in the spleen weight at any
dose when compared with control (normal salinetreated)
animals (group I). At doses of 50 and 100
mg/kg, a significant increase ( p < 0.05) in relative
organ weight of thymus was observed but there was
no effect at a dose of 250 mg/kg in its weight. As
regard the relative organ weight of liver, a significant
( p < 0.001) increase was observed at doses of
Table 1
Effect of T. foenum graccum on the relative organ weights of mice
Group Relative organ weight (meansFS.E.) in grams
Spleen Thymus Liver Kidney
(I) Control 0.64F0.01 0.25F0.01 4.88F0.23 1.39F0.08
(II) Plant extract (50 mg/kg) 0.67F0.02 0.27F0.01a 5.29F0.14 1.31F0.04
(III) Plant extract (100 mg/kg) 0.72F0.08 0.28F0.01a 6.20F0.22b 1.36F0.03
(IV) Plant extract (250 mg/kg) 0.62F0.03 0.24F0.01 6.14F0.07b 1.36F0.05
Values are meansFS.E. of five mice.
a p < 0.05 when compared with group I (significantly different).
b p < 0.001 when compared with group I (significantly different).
260 B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265
100 and 250 mg/kg. This increase in liver weight
did not reflect any abnormality as assessed by LFT.
There was no significant elevation in the levels of
SGOT and SGPT as a result of treatment with T.
foenum graecum at any of the doses used in this
study.
Lymphoid organ cellularity (Table 2) data indicate
no significant increase in the spleen cellularity at any of
the doses. In the case of bone marrow, doses of 50 and
100 mg/kg showed a significant ( p < 0.01 and p < 0.05)
increase in cellularity. A significant (p
Други два люти метода за предпазване от грип са употребата на чай от джинджифил или резени за смучене, както и резени от скилидка чесън, защото действат вирусоцидно в устната кухина, откъдето заразата навлиза в организма най-вече по въздушно-капков път.
На български билката се казва още гръцко сено.
.jpg)
.jpg)
.jpg)
.jpg)
Статия за активиране на имунната система при мишки от водни екстракти на гръцко сено:
Immunomodulatory effects of fenugreek
(Trigonella foenum graecum L.) extract in mice
Bilal Bin-Hafeez, Rizwanul Haque, Suhel Parvez, Suwarna Pandey,
Iqbal Sayeed 1, S. Raisuddin*
Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard University, New Delhi 110 062, India
Received 19 April 2002; received in revised form 20 July 2002; accepted 13 December 2002
Abstract
Immunomodulatory activity of aqueous extract of Trigonella foenum graecum L., a widely used medicinal and dietary
herb, was evaluated in male Swiss albino mice. Mice were treated with three doses of extract (50, 100 and 250 mg/kg body
weight per os) for 10 days. Body weight, relative organ weight, cellularity of lymphoid organs, delayed type of
hypersensitivity (DTH) response, plaque-forming cell (PFC) assay, haemagglutination titre (HT), quantitative haemolysis of
SRBC (QHS) assay, phagocytosis, and lymphoproliferation were studied in various groups of animals. At doses of 50 and
100 mg/kg, a significant increase ( p < 0.05) in relative organ weight of thymus was observed but there was no effect on
kidney and spleen weights. Liver weight also increased significantly at doses of 100 and 250 mg/kg. However, no elevation
in the levels of liver function test (LFT) enzymes was observed. As regards lymphoid organ cellularity, spleen recorded no
significant increase at any dose, whereas cellularities of thymus and bone marrow were significantly increased. T. foenum
graecum extract elicited a significant ( p < 0.001) increase in the DTH response at doses of 50 and 100 mg/kg, but the change
at higher dose of 250 mg/kg was not statistically significant. Humoral immunity as measured by PFC showed an elevated
response at a dose of 100 mg/kg, but at 50 and 250 mg/kg, no significant effect was observed. In the HT test, plant extract
also showed modulatory effect at all the doses. Plant extract elicited a significant increase in phagocytic index and
phagocytic capacity of macrophages. Stimulatory response of plant extract was also observed in lymphoproliferation assay
but the response was weak. Overall, T. foenum graecum showed a stimulatory effect on immune functions in mice. As it is
used for a variety of medicinal purposes, its immunostimulatory effect, as reported in this study, strengthens the rationale of
its use in several Ayurvedic and Unani drugs.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Trigonella foenum graecum; Immunomodulation; Traditional medicine systems; Immunosuppression
1. Introduction
A large number of plants and their isolated
constituents have been shown to potentiate immunity
[1,2]. Some medicinal plants have been shown
to exert anti-inflammatory, anti-stress and anti-can-
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1567-5769(02)00292-8
* Corresponding author. Tel.: +91-11-26059688x5568;
fax: +91-11-26059663.
E-mail address: sraisuddin@hotmail.com (S. Raisuddin).
1 Present address: Department of Neurology, University of
Magdeburg, Leipziger Str., 44d-39120, Magdeburg, Germany.
www.elsevier.com/locate/intimp
International Immunopharmacology 3 (2003) 257–265
cer effects by modulating the immune functions
[3,4]. We have reported protective effect of Cassia
occidentalis and Emblica officinalis Gaertn against
cyclophosphamide (CP)-induced suppression of
humoral immunity in mice [5,6]. It was also found
that C. occidentalis, E. Officinalis and Cassia
cinnamon have antimutagenic effects against benzo[
a]pyrene and CP-induced mutagenicity [7–10].
Besides source of medicine, these plants are used as
dietary constituents in India and other Afro-Asian
countries.
Fenugreek (Trigonella foenum graecum L.; family
Leguminoceae) is one such plant whose seeds
and leaves are used not only as food but also as an
ingredient in traditional medicine. Seeds of fenugreek
are used as a condiment and as a supplement
to wheat and maize flour for bread making and as a
constituent of the daily diet of general population in
Indian subcontinent. Its leaves are consumed widely
in India as a green leafy vegetable and are a rich
source of calcium, iron, h-carotene and other vitamins
[11]. Seeds of T. foenum graecum contain
tannic acid, fixed and volatile oils and a bitter
extractive, diosgenin, alkaloids trigonelline, trigocoumarin,
trigomethyl coumarin, and steroidal saponin
such as gitogenin and traces of trigogenin and
vitamin A [12,13].
Some of the therapeutic uses of T. foenum
graecum include its use as hypoglycemic, antiulcerogenic,
hypocholesterolemic and antihypertensive
agent [11,14,15]. In Ayurvedic and Unani
systems of medicine, fenugreek is used for the
treatment of epilepsy, paralysis, gout, dropsy,
chronic cough and piles [16]. In the present communication,
we report its immunomodulatory effects
in mice.
2. Materials and methods
2.1. Plant extract
Total aqueous extract of T. foenum graecum in
semisolid form was procured from the Plant Extract
Division of Saiba Industries, Mumbai. A certificate of
authenticity was provided along with the extract. The
chemical analysis report provided by the manufacturers
showed no presence of endotoxin.
2.2. Chemicals
Antibiotic antimycotic solution (100), bovine
serum albumin (BSA), fetal bovine serum (FBS),
Hank’s balanced salt solution (HBSS), Histopaque-
1077, phosphate-buffered saline (PBS) and RPMI-
1640 medium were purchased from Sigma, St. Louis,
MO. Petroleum jelly was obtained from Hi-Media
Labs, Mumbai, conconvalin A (Con A) from Banglore
Genei (India) and solvents from BDH, Mumbai;
tritiated [3H]-thymidine was purchased from Amersham
Pharmacia Biotech UK, Bukinghamshire.
2.3. Animals
Study was conducted in Swiss albino male mice
(30–35 g). Female guinea pigs (250 g) were used for
the preparation of complement for plaque-forming cell
(PFC) assay. The Central Animal House Facility of the
University provided the animals. The animals were
bred and maintained under standard laboratory conditions
(temperature 25F2 jC; photoperiod of 12 h).
Commercial pellet diet and water were given ad
libitum.
2.4. Dosage
The plant extract was suspended in normal saline
and was administered orally for 10 days at doses of 50,
100 and 250 mg/kg body weight. The dose volume
was 0.2 ml. Control animals received the same volume
of normal saline.
2.5. Body weight, lymphoid organ weight and
cellularity
Animals were divided into four groups (I–IV).
Each group comprised of a minimum of five animals.
Group I (control) received normal saline;
group II, plant extract 50 mg/kg body weight; group
III, plant extract 100 mg/kg; and group IV, plant
extract 250 mg/kg. The animals were humanized 24
h after the last dose. Body weight gain (percentage)
and relative organ weight (organ weight/100 g of
body weight) of kidney, liver, spleen and thymus
were determined for each animal. Single cell suspensions
were prepared in RPMI-1640 medium from
bone marrow (from femur), spleen and thymus for
258 B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265
cell count. Cell counting was done by using the
Neubauer chamber [17].
2.6. Assessment of humoral immune functions
Animals of all the groups treated in the abovedescribed
manner were challenged with 0.2 ml of 10%
sheep red blood cells (SRBC), i.p. on the 10th day of
initiation of experiment. The following parameters of
humoral immunity were studied in those animals.
2.6.1. Haemagglutinin titre (HT) assay
HT assay was performed using the procedure of
Bin-Hafeez et al. [5]. On the fifth day after immunization,
blood was collected from orbital plexus of
each mouse for serum preparation. Serial twofold
dilution of serum was made in 50 Al of PBS (pH 7.2)
in 96-well microtitre plates (Tarsons Products, Calcutta)
and mixed with 50 Al of 1% SRBC suspension in
PBS. After mixing, plates were kept at room temperature
for 2 h. The value of antibody titre was assigned to
the highest serum dilution showing visible haemagglutination.
2.6.2. Plaque-forming cell assay
The PFC assay was performed using the method of
Raisuddin et al. [18]. The animals were humanized on
the fifth day of immunization with SRBC. The spleen
was removed, cleaned free of extraneous tissues, and a
single cell suspension of 106 cells/ml was prepared
from it in RPMI-1640 medium. For PFC assay, the
SRBC were prepared at a cell density of 5108 cells/
ml in PBS. One milliliter of SRBC in medium along
with 0.5 ml of diluted guinea pig complement (1:10
diluted with normal saline) was added to 1 ml of
spleen cell suspension. Cuningham chambers were
prepared using glass slide, coverslips and doublesided
tape (Scotch Brand, St. Paul, MN). The chambers
were loaded with a known volume of assay
mixture, sealed with petroleum jelly and incubated
at 37 jC for 1 h. The plaques were counted under a
light microscope (Olympus BX50) and expressed as
PFC per 106 spleen cells.
2.6.3. Quantitative haemolysis of SRBC (QHS) assay
QHS assay was performed using the methods of
Simpson and Gozo [19] with some modification [5].
Spleens were removed and a cell suspension of
1106/ml was prepared in PBS. One milliliter of
0.2% SRBC and 1 ml of 10% guinea pig serum were
mixed with cell suspension and incubated for 1 h at
37 jC. After centrifugation at 3000 rpm for 3 min,
optical density of the supernatant was measured at
413 nm using a spectrophotometer (Shimadzu UV-
1201).
2.7. Assessment of cellular immune function
For assessment of cellular immune function, delayed
type of hypersensitivity (DTH) response was
evaluated in the extract-treated mice. Four groups of
animals were treated as described above.
2.7.1. Delayed type of hypersensitivity response
The DTH response was determined using the
method of Raisuddin et al. [18]. On the day of
termination of the treatment, animals were immunized
with 1109 SRBC, subcutaneously. On the fifth day
of immunization, all the animals were again challenged
with 1108 cells in the left hind footpad. The
right footpad injected with the same volume of normal
saline served as trauma control for nonspecific swelling.
Increase in footpad thickness was measured 24 h
after the challenge using dial caliper (Guanglu,
China).
2.8. Assessment of nonspecific immune functions
For assessment of nonspecific immune functions,
phagocytic activity of macrophages was studied. Four
groups of animals were treated as described above.
2.8.1. Phagocytic activity of macrophages
The phagocytic activity of peritoneal macrophages
was evaluated using the suspension assay as described
by Fujiki and Yano [20] with some modification.
Briefly, 0.1 ml of aliquot of cells having density of
10106 cells/ml was mixed with 0.1 ml RPMI-1640
medium containing 20% FCS and 100106 cells/ml
heat-treated yeast (Saccharomyces cereviceae) cells.
The mixture was incubated at 37 jC for 1 h with
occasional shaking. After incubation, 50 Al of this
mixture was smeared on the glass slide, air dried and
stained with Wright–Giemsa stain. The slides were
observed under light microscope using oil immersion.
At least 500 cells were counted. The phagocytic activ-
B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265 259
ity was expressed as phagocytic index (PI) and phagocytic
capacity (PC) using the following formulae.
PI ј AB
where A is the percentage of yeast-ingesting phagocytes
and B is the number of yeast-ingested per phagocytes;
PC is the mean percentage of cells that
engulfed z4 yeast cells.
2.8.2. Lymphocyte proliferation assay
For assessment of lymphocyte proliferation assay,
four groups of mice were treated as described above.
On completion of treatment blood was withdrawn
from retro-orbital plexus of mice. Lymphocytes were
isolated from the blood by density gradient centrifugation
on Histopaque-1077. The mononuclear cells
from the buffy coat were carefully pipetted out with a
sterilized Pasteur pipette and pelleted at 400g for
10 min. The cell pellet was suspended in 1 ml of
growth medium (RPMI-1640) containing 10% FBS
and recommended concentration of antibiotic and
antimycotic solution. The cell viability was assessed
by the trypan blue exclusion method and the cells
were plated in 96-well culture plates (Tarsons) at a
cell density of 1106 cells/well. ConA was used as a
mitogenic agent. The culture plate was incubated at
37 jC in CO2 (5%) incubator (Forma Scientific,
Ohio), with water-saturated air for 6 days. After 6
days the cells were pulsed with 1 ACi of [3H]-methylthymidine.
Cells were harvested after 18 h of radioactive
pulse and the amount of [3H]-thymidine incorporated
into DNA was determined by liquid
scintillation spectroscopy [21].
2.8.3. Liver function tests
Activities of serum glutamate oxalate transaminase
(SGOT) and serum glutamate pyruvate transaminase
(SGPT) were estimated by using the kits (Span
Diagnostics, Surat, India). For this purpose, four
groups of animals (one control + three treatments) as
described above were used and treated for 10 days
with respective doses of plant extract.
2.9. Statistical analysis
Data were statistically analyzed using Student’s ttest
to determine significant differences in the data of
various groups. P values less than 0.5 were considered
significant. The values are expressed as means
FS.E.
3. Results
3.1. Effect of plant extract on body weight, lymphoid
organ weight and cellularity
None of the doses of Trigonella extract showed
toxicity or mortality in the extract-treated animals.
No significant body weight gain differences were
recorded in various groups of animals (Table 1). No
effect was observed in the spleen weight at any
dose when compared with control (normal salinetreated)
animals (group I). At doses of 50 and 100
mg/kg, a significant increase ( p < 0.05) in relative
organ weight of thymus was observed but there was
no effect at a dose of 250 mg/kg in its weight. As
regard the relative organ weight of liver, a significant
( p < 0.001) increase was observed at doses of
Table 1
Effect of T. foenum graccum on the relative organ weights of mice
Group Relative organ weight (meansFS.E.) in grams
Spleen Thymus Liver Kidney
(I) Control 0.64F0.01 0.25F0.01 4.88F0.23 1.39F0.08
(II) Plant extract (50 mg/kg) 0.67F0.02 0.27F0.01a 5.29F0.14 1.31F0.04
(III) Plant extract (100 mg/kg) 0.72F0.08 0.28F0.01a 6.20F0.22b 1.36F0.03
(IV) Plant extract (250 mg/kg) 0.62F0.03 0.24F0.01 6.14F0.07b 1.36F0.05
Values are meansFS.E. of five mice.
a p < 0.05 when compared with group I (significantly different).
b p < 0.001 when compared with group I (significantly different).
260 B. Bin-Hafeez et al. / International Immunopharmacology 3 (2003) 257–265
100 and 250 mg/kg. This increase in liver weight
did not reflect any abnormality as assessed by LFT.
There was no significant elevation in the levels of
SGOT and SGPT as a result of treatment with T.
foenum graecum at any of the doses used in this
study.
Lymphoid organ cellularity (Table 2) data indicate
no significant increase in the spleen cellularity at any of
the doses. In the case of bone marrow, doses of 50 and
100 mg/kg showed a significant ( p < 0.01 and p < 0.05)
increase in cellularity. A significant (p
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Предишен постинг
Чесън е лекарството против грип!
Поздрави!
цитирайПоздрави!
Това е неоспорима пасторална класика - аз се чудя защо високопланинските кози не боледуват от грип, а те хрупали планински чесън
цитирайаз ти разправям, че шарената сол те спасява от грип, ти пак си знаеш за чесъна. Поздрави Индиррамм.
цитирайhttp://www.ijabbr/article_7851_bbd8fa7701b237d7746306a9df24e736.pdf
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