Phytochemical and
Pharmacological Evaluation of Martynia annua for Immunomodulatory
Potential
Vivekanand Katare, Chandra
Kishore Tyagi
Sri Satya Sai University of
Technology and Medical Sciences, Sehore-466001, Madhya Pradesh, India.
*Corresponding Author E-mail:
kishore198012@gmail.com
ABSTRACT:
Ethnopharmacological
relevance: Martynia annua L (Martyniaceae) is a well-known aquatic plant
which has been used for the treatment of several disorders including skin
disease, cough, inflammation, fever and many other disorders. Aim of the
study: To explore the immunomodulatory activity of extract of (MEMA) and
(MEAC) of the plant. Materials and methods: The immunomodulatory
activity of MEMA and MEAC was evaluated using various in vivo models
including the total and differential leukocyte count (TLC and DLC),
nitroblue-tetrazolium reduction (NBT) test, neutrophil adhesion test,
phagocytic response and delayed type hypersensitivity (DTH) reaction. Sheep red
blood cells (SRBC, 5 × 109 cells/ml) were used to immunize the animals. NNRE
and NNSE at the doses of 100 and 300 mg/kg were administrated. Result: The
TLC and lymphocyte count increased significantly but the neutrophil count was
decreased for MEMA and MEAC treated groups compared to the control. A
dose-dependent potentiation of DTH reaction induced by SRBC was observed from
the extracts. The percentage of neutrophil adhesion to the nylon fiber was
increased in MEMA treated groups (63.22 and 62.91%) compared to the MEAC
treated group (54.86 and 54.23%). A potential phagocytic response was seen on
treatment of the extracts, and significant changes were observed in the
formation of formazone crystals. Conclusion: This finding suggests that
the extract of rhizome and seed Martynia annua stimulate defense
system by modulating several immunological parameters.
KEYWORDS: Phyochemical,
Pharmacological, Evaluation, Immunomodulatory Potential.
INTRODUCTION:
Natural products and folklore
medicines are the main contributors of the leads in the design and development
of therapeutic agents. Several plant derived compounds have been identified
over the years for their immunomodulatory characteristics(1).
Numerous illnesses can be
alternatively treated by immunomodulation using medicinal plants, instead of
chemotherapy. The discovery and isolation of more specific immunomodulatory
agents from plant origin possesses potential to counteract the side effects and
high cost of synthetic compounds. This highlights the significance of medicinal
plants as producers of immunomodulatory molecules of very varied chemistries
with possible uses in animal and human health(2). The challenges
encountered by the application of plant derived immunomodulators need to be
addressed. Though, the path from traditional medicines to western
pharmaceutical practices is not always easy. The inconsistency of responses of
phytomedical practices can be clarified in terms of the typically strong reliance
of plant secondary metabolite profiles on environmental signals that can
disturb reproducibility of results with extracts. This can be decreased if the
principles of standardization of extracts and enriched fractions are thoroughly
applied(3).
MATERIALS AND
METHODS:
Plant material
Collection and authentication:
The fresh bark of Martynia
annua were collected from the field area of Bhopal district M.P. India. For
identification and taxonomic authentication, plant material was submitted in
Department of Botany, Saifia College, Bhopal, India. Its authenticity was
confirmed and authenticated by Dr. Zia-Ul-Hasan. Collected plant materials were
shade-dried and coarsely powdered.
Preparation of
extract:
Shade-dried and
coarsely powdered 100gm powder from bark of Martynia annua were soaked
in 500ml of methanol [methanol/drug mass ratio 5:1] separately. It was kept at
room temperature for 48 hours with intermittent mixing. Methanol extract of
plants (MEMA) obtained after 48 hours of soaking was filtered using Whatman
paper. The extracts, which was thus obtained, was evaporated to make it into
the powder form to re-dissolve in methanol.
Drugs:
Cyclophosphamide
(Endoxan from Cadila Healthcare Limited) 50mg/kg.b.w was used intraperitoneally(5).
Methanol extract of Martynia annua (MEAC), was used at a dose of 150 and
300mg/kg p.o.(6) was used at a dose of 800 and 1200mg/kg, p.o.(7).
Cyclophosphamide and plant extracts doses and dosing schedules were based on
published report.
Test animals:
Healthy mice
(25-30g) of either sex were selected for the study. They were kept in the
Animal House of Faculty of Pharmacy, College of Pharmacy, SSSUTMS, Sehore in
colony cages at an ambient temperature of 25 ± 2°C and relative humidity 45–55%
with 12 h light/dark cycles after initial acclimatization for about 1 week.
They had free access to standard rodent pellet diet and water ad libitum. The
experimental protocol and animal house has been approved by the institutional
ethical committee with approval no. COP/Pharm/Ph.D./CPCSEA/12/06.
Antigenic
materials:
For the present
study, the antigenic material used was sheep RBCs (SRBC). Fresh blood was
collected from sheep sacrificed in the local slaughter house. It was mixed with
Alsever’s solution in 1:1 proportion and was stored at 4ºC in the refrigerator.
Table 1. Composition
of Alsever’s Solution
|
Contents |
% W/ V |
|
Glucose |
2.05 |
|
Sodium Chloride |
0.42 |
|
Sodium Citrate |
0.80 |
|
Citric Acid |
0.55 |
During the
experimentation, from the above stock solution (i.e. SRBCs, stored in Alsever’s
solution), an enough quantity of blood was taken and was allowed to stand at
room temperature. It was washed three times with pyrogen free normal saline
(0.9% w/v NaCl). Using Neubauer's chamber, the RBC count of this suspension was
determined by hemocytometer. The known amount of RBCs (0.5x109 cells/ml/100g)
was injected intraperitoneally to the mice as an antigenic challenge.
Immunosuppressant:
In the present
study cyclophosphamide (CP) was used as immunosuppressing agent(8).
Dosing schedule:
Animals were
divided into six groups (I-VI). Each group comprised of a minimum of six
animals. Group I (control) animals received normal saline for 7th
consecutive days; group II (CP) animals were injected with a single dose of CP
on 6th day of initiation of experiment. Group III (MEMA 1) animals in MEBC
treatment and group III, IV (MEAC 1 and MEAC 2) animals in MEAC treatment,
received plant extract treatment for 7th consecutive days. Group V, VI animals
(MEBC 1+ CP and MEBC 2 + CP), in MEBC treatment and V, VI animals in MEAC
treatment (MEAC 1+ CP, and MEAC 2 + CP), were given plant extract treatment for
7th days along single injection of CP on 6th day of initiation of
experiment. For humoral response animals of all groups were challenged with
0.2ml of 10% SRBC i. p. on the 5th day. It was perform using the
procedure of Bin-Hafeez et al. (2001) with some modifications. Cell Mediated
Immunity was assayed by footpad reaction method. On the 7th day,
SRBC was injected in right hind paw of animals of all groups. While 0.9% saline
was injected into the left hind paw of the mice of all groups. Blood parameter
was assessed in the blood, withdrawal from tail veins. The mice were
decapitated under ether anesthesia 24 hr. after the last dose, for body weight
determination.
Table 2. Dosing
Schedule of MEBC Treatment
|
GROUP |
TREATMENT |
TREATMENT SCHEDULE |
|
I |
Control |
1 to 7th day Normal saline |
|
II |
Cyclophasphamide (CP) |
1 to 7th day saline, SRBC on 5th day, CP on 6th day |
|
III |
MEBC1 |
1 to 7th day MEBC1 |
|
IV |
MEBC2 |
1 to 7th day MEBC2 |
|
V |
MEBC1+CP |
1 to 7th day MEBC1, SRBC on 5th day, CP on 6th day |
|
VI |
MEBC2+CP |
1 to 7th day MEBC2, SRBC on 5th day, CP on 6th day |
Table 3. Dosing
Schedule of MEAC Treatment
|
GROUP |
TREATMENT |
TREATMENT SCHEDULE |
|
I |
Control |
1 to 7th day Normal saline |
|
II |
Cyclophasphamide (CP) |
1 to 7th day saline, SRBC on 5th day, CP on 6th day |
|
III |
MEAC1 |
1 to 7th day MEAC1 |
|
IV |
MEAC2 |
1 to 7th day MEAC2 |
|
V |
MEAC1+CP |
1 to 7th day MEAC1, SRBC on 5th day, CP on 6th day |
|
VI |
MEAC2+CP |
1 to 7th day MEAC2, SRBC on 5th day, CP on 6th day |
Immunization
schedule:
All the above
groups’ mice were antigenically challenged with SRBC (0.5x109 cells/ml/100 g)
on the 5th day intraperitoneally (9).
Humoral immune
response model:
By using the method
of Bin-Hafeez et al. (2001) with some modification, measurement of antibody
titer by hemagglutination reaction was performed. Anesthetic ether was used to
anaesthetize mice. With the help of a fine capillary gently inserted into the
lower angle of the eye at 45o, the blood was obtained from
retro-orbital plexus. The blood was collected into the vial and centrifuged for
separating serum. The mice serum was used for analysis of hemagglutination
titer. Microtitration plate having 96 cups was used for carrying out titration.
Each cup was filled with 25±l μl of normal saline. 25±l μl of serum
obtained from mice blood was added to 1stcup and was mixed with 25±l μl of
normal saline present in microtitration plate. By this method, two-fold serial
dilutions of serum were prepared. To each cup 25±l μl of 1% v/v SRBC was
added. The plate was incubated at 37oC for one hr. and then was observed for
agglutination. The antibody titer was expressed in terms of maximum dilution,
which gave positive hemagglutination reaction (10, 11).
Cellular immune
response model:
Footpad reaction
test was done for cell-mediated immune response. On 7th day, after
measuring the volume of footpad of both legs, SRBC (0.025x109 cells) was
injected in right paw and 0.025 ml of saline was injected into the left paw of
animals of all groups. On 8thday after 24 hours, the paw volume was measured
again to check the increase or decrease in volume. The increase in paw volume
was considered as an index of cell-mediated immunity (delayed type
hypersensitivity) (12,13).
Blood Parameters:
For the detection
of blood parameters, blood withdrawn from the above antigenically challenged
mice were used to check hematological parameters (hemoglobin, RBCs, and WBCs)(14).
Relative organ
weight Determination:
For relative organ
weight determination, animals of all groups were sacrificed 24 hr after the
last dose. Relative organ weight (organ weight/100g of body weight) of the
liver, kidney, and spleen were determined for each animal(15).
Assessment of
antioxidant parameters:
In all group
animals, Spleen was collected after the scarification and washed immediately
with cold saline to remove blood. Spleen tissues of mice were homogenized (10%)
in phosphate buffer (pH 7.4). The homogenate was centrifuged at 12000g for 20
min at 4°C to obtain the supernatant, and it was used for the estimation of
LPO, reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD).
Assay of TBARS:
Lipid peroxidation
is a free radical settled event. The primary products of such damage are a complex
mixture of peroxides that then break down to produce carbonyl compounds. The
MDA (malondialdehyde) is one such carbonyl compound, which forms a
characteristic chromogenic adduct with two molecules of TBA. The colorimetric
reaction of TBA with MDA, a secondary product of lipid peroxidation, has been
widely accepted for measuring lipid peroxidation. The total protein that was
present in the homogenate was estimated by following the method that was
described by Lowry et al. (16) The TBARS assay was performed
according to earlier reported method(17). 1 ml of homogenate was
combined with 2ml of TCA-TBA, HCl reagent and mixed thoroughly the solution was
then heated on a boiling water bath for 15 min. then the mixture was cooled and
centrifuged for 15 min. The supernatant absorbance was read at 535nm against a
blank solution. TBARS activity was determined using a molar extinction
coefficient of 1.56×105 M−1 cm−1. The units
of TBARS activity expressed in terms of nmoles MDA/mg protein.
Assay of
Glutathione:
This
spectrophotometric procedure was based on the method of Ellman. DTNB [5,
5'-dithiobis-(2-nitrobenzoic acid)] is reduced by –SH groups to form one mole
of 2-nitro-5- mercaptobenzoic acid per mole of –SH. The GSH activity unit was
expressed in terms of μg/mg protein (18).
Assay of SOD:
The assay of SOD
was carried out, based on the ability of the enzyme to inhibit the
auto-oxidation of pyrogallol as described by McCord with some modification. The
total protein that was present in the homogenate was estimated by the method
that was described by Lowry et al. (19). The units of the SOD
activity which were determined were expressed in terms of Units /mg protein (20).
Assay of Catalase:
Catalase activity
was determined using Aebi’s method with some modifications. In the UV range, H2O2
shows a continuous increase in the absorption with decreasing wavelength. The
decomposition of H2O2 can be followed directly by the decrease
in the absorbance at 240 nm. The difference in absorbance (ΔA) per unit
time is a measure of the catalase activity. The molar extinction coefficient of
H2O2, 43.6 M−1 cm−1 was
used to determine the catalase activity. The units of the CAT activity which
were determined were expressed in terms of nmol H2O2/mg
protein(21).
Determination of
TNF-α, and IL-6 level:
The concentrations
of TNF- α and IL-6 in the mice serum were determined using specific
quantitative sandwich ELISA kits according to the instruction of the
manufacturer purchased from Pierce Biotechnology, Rockford, IL, USA (22).
STATISTICAL
ANALYSIS:
All the results
were expressed as means ±SEM. Data was analyzed using one-way Analysis of
Variance (ANOVA) followed by Tukey-Karmer multiple comparison tests to
determine significant differences in data of various groups. P values less than
0.05 were considered statistically significant.
RESULTS AND
DISCUSSION:
Haemagglutinating
antibody (HA) titer:
The HA titer was
used to assess humoral immune response. The effect of Martynia annua extract
on humoral immune response showed that the administration of both the plant
different doses in group 3rd and 4th group’s animals
produced a significant dose-related increased in H.A titer value when compared
to control group animals (Table 2). While in negative control group animals,
cyclophosphamide treatment produced a significant (P<0.001) decreased in
titer value when compared to control group animals. When cyclophosphamide
treatment was given along with different dose of both plants in group 5th
and 6th groups animals, significant (P<0.001) recovery of
immunosuppressive effect of cyclophosphamide (CP) was observed by increasing
the titer value as compared to CP treated group. Hence, the plant extract
showed the protective effect over humoral immunity.
Delayed type
hypersensitivity (DTH) reactions:
The effect of test
extract on DTH response showed that the test extract in both plant different
doses (group 3rd and 4th animals) produced a significant
(P<0.001) dose-related increased in DTH reactivity in mice when compared to
control animals (Table-3). It showed the stimulatory effect of test extracts on
T-cells. Potentiation of DTH response was also observed in cyclophosphamide-treated
animals because it has damaged short lived suppressor T-cells in the immune
system. When comparing MEBC (150mg/kg) treatment along with CP (group 5th)
animals, to cyclophosphamide treated group, elevation in DTH reactivity was
found but it was not statistically significant, while in group 6th
when plant extract treatment at higher dose (MEBC 300 mg/kg) was given with CP
a significant (P<0.001) elevation in DTH response was found as compared to
CP alone treated group. On the other side when treatment of MEAC different
doses along with CP was given in group 5th and 6th group
animals no significant elevation in DTH reactivity was observed as compared to
cyclophosphamide alone treated group. Thus, alteration of DTH reactivity in
mice in response to T-cell dependent antigen (SRBCs) revealed the stimulatory
effect of MEBC and MEAC extracts on T cells.
Effect on Relative
organ weight:
The effect of MEBC
and MEAC different doses (group 3rd and 4th group animal)
on relative organ weight showed no significant relative weight difference on
liver, kidney and spleen of the animals of different group when compared to
control group (Table-4). But CP injection in group 2nd animals
caused a significant reduction in relative organ weight of spleen as compared
to control group animals. No significant recovery of spleen weight was observed
in plant extract and CP treated animals (group 5th and 6th
group) in both plants.
Effect on
Hematological parameters:
Effect of plant
extract on hematological parameters showed that MEBC significantly (p<0.05)
increased the white blood cell count at the dose level 150mg/kg and 300mg/kg as
compared to control group animals. While MEAC treatment also showed
significantly increased in white blood cell count at the dose level 800mg/kg
(p<0.05) and 1200mg/kg (p<0.01) as compared to control group. But CP
injection caused a significant (p<0.001) reduction in white blood cell count
as compared to normal control group animals (Table-5). Combined treatment of CP
and MEBC (150mg/kg) showed significant (p<0.05), and MEBC (300mg/kg) showed
significant (p<0.001) restoration of bone marrow activity as compared with
cyclophosphamide alone treated mice. While combined treatment of CP and MEAC
(800mg/kg) showed significant (p<0.01) and MEAC (1200mg/kg) showed
significant (p<0.0001) restoration of bone marrow activity as compared to
cyclophosphamide treated group. But no significant effect was observed in RBC
and Hb count in various groups of animals in plant extract treatment
(both plants) when compared to normal control group animals, and no significant
protective effect was observed in RBC and Hb count in plant extract
and CP treated groups in both plants when compared to CP alone treated animals.
Effect of MEBC and
MEAC on antioxidant enzymes:
The oxidative
stress marker study revealed (Table- 6) that the administration of CP
significantly increased (p<0.001) the level of LPO, decreased the activity
of SOD (p<0.001), CAT (p<0.001) and reduced the content of GSH
(p<0.001) as compared to control group animals. While CP treatment along
with different doses of extract significantly decreased the LPO (p<0.001)
level as compared to CP exposed group, and a significant elevation in CAT
(p<0.001), GSH and SOD were observed in comparison to the CP-treated group
in both plants. When compared to MEBC and MEAC extract effects, on each enzyme,
the overall result justified that the MEBC was more effective antioxidant than
the MEAC.
Effect of test
drugs on pro-inflammatory cytokine:
Plant extract effects
on pro-inflammatory cytokines level showed that the secretion of TNF-α and
IL-6 significantly decreased (P<0.001) in the negative control group when
compared to normal control groups animals. While TNF-α and IL-6 level were
up-regulated significantly in MEBC different dose treatment, MEBC (150mg/kg)
showed significant (p<0.001) and MEBC (300mg/kg) showed significant
(p<0.001) up-regulation as compared to normal control animals.
Co-administration of CP and MEBC different dose showed significant (P<0.001)
increased in cytokines level as compared to CP alone treated group. While in
case of MEAC different dose treatment increased was found in TNF-α and
IL-6 level but it was not statistically significant when compared to control
groups animals and upon co-administration of CP and MEAC different dose,
increased was found in TNF-α and IL-6 cytokines level but also it was not
statistically significant when compared CP exposed group.
Table 4: Effect of
Methanol Extract of Martynia annua (MEMA) on Humoral Immune Response.
|
Group |
Treatment
|
Mean antibody titer a (in terms of rank of cups of titer plate) ± S.E.M. |
|
Group I |
Control (Normal saline) |
9.25 ± 0.30 |
|
Group II |
Normal saline + CP |
3.17b ± 0.16 |
|
Group III |
MEBC 150 mg/kg |
11.2b ± 0.30 |
|
Group IV |
MEBC 300 mg/kg |
13.1b± 0.20 |
|
Group V |
MEBC 150 mg/kg + CP |
6.83c ± 0.30 |
|
Group VI |
MEBC 300 mg/kg + CP |
8.5c ±0.22 |
aValues are expressed
as mean ± S.E.M. of 6 mice, bP<0.001 Statistical
significance versus Group I, cP<0.001 Statistical significance
versus Group II.
Table 5: Effect of
Methanol Extract of Martynia annua (MA) Extract on Humoral Immune
Response.
|
Group |
Treatment
|
Mean antibody titer a (in terms of rank of cups of titer plate) ± S.E.M. |
|
Group I |
Control (Normal saline) |
9.25 ± 0.30 |
|
Group II |
Normal saline + CP |
3.17b ± 0.16 |
|
Group III |
MEAC 800 mg/kg |
10.5c ± 0.22 |
|
Group IV |
MEAC 1200 mg/kg |
11.25b ± 0.25 |
|
Group V |
MEAC 800 mg/kg + CP |
5.3d ± 0.21 |
|
Group VI |
MEAC 1200 mg/kg + CP |
7.6d ± 0.20 |
aValues are expressed
as mean ± S.E.M. of 6 mice, bP<0.001 Statistical
significance versus Group I, cP<0.01 Statistical
significance versus Group I, dP<0.001 Statistical significance versus
Group II.
Table 6: Effect of
Methanol Extract of Martynia annua on Delayed Type Hypersensitivity
Response.
|
Group |
Treatment |
Mean of right food pad thickness a (mm) ± S.E.M |
|
Group I |
Control-Normal saline |
0.80 ± 0.05 |
|
Group II |
Normal saline + CP |
1.078b ± 0.03 |
|
Group III |
MEBC 150 mg/kg |
1.16b ± 0.02 |
|
Group IV |
MEBC 300 mg/kg |
1.195b ± 0.03 |
|
Group V |
MEBC 150 mg/kg + CP |
1.097 ns± 0.02 |
|
Group VI |
MEBC 300 mg/kg + CP |
1.37 c ± 0.01 |
aValues are expressed
as mean ± S.E.M of 6 mice, bP<0.001 Statistical
significance versus Group I, cP<0.001 Statistical
significance versus Group II.
Table 7. Effect of
Methanol Extract of Martynia annua Extract on Delayed Type
Hypersensitivity Response.
|
Group |
Treatment |
Mean of right food pad thickness a (mm) ± S.E.M. |
|
Group I |
Control-Normal saline |
0.80 ± 0.05 |
|
Group II |
Normal saline + CP |
1.078b ± 0.03 |
|
Group III |
MEAC 800 mg/kg |
1.15c ± 0.01 |
|
Group IV |
MEAC 1200 mg/kg |
1.147c ±0.08 |
|
Group V |
MEAC 800 mg/kg + CP |
1.09 ns ± 0.03 |
|
Group VI |
MEAC 1200 mg/kg + CP |
1.11ns ± 0.04 |
aValues are expressed
as mean ± S.E.M. of 6 mice, bP<0.01 Statistical significance
versus Group I, cP<0.001 Statistical significance versus
Group I.
Table 8: Effect of Martynia
annua Extract on Relative Organ Weight.
|
Group |
Treatment |
Relative organ weighta (g) ± S.E.M. |
||
|
Liver |
Kidney |
Spleen |
||
|
Group I |
Control (Normal saline) |
5.14± 0.01 |
1.367± 0.45 |
0.622±0.05 |
|
Group II |
Normal saline + CP |
4.82ns ±0.17 |
1.115ns ±0.26 |
0.334b ±0.06 |
|
Group III |
MEBC 150 mg/kg |
5.21ns ±0.05 |
1.225ns ±0.27 |
0.511ns ±0.01 |
|
Group IV |
MEBC 300 mg/kg |
5.25ns±0.06 |
1.391ns± 0.46 |
0.620ns ±0.08 |
|
Group V |
MEBC 150 mg/kg + CP |
4.85ns±0.1 |
1.31ns ±0.35 |
0.452ns ±0.02 |
|
Group VI |
MEBC 300 mg/kg + CP |
4.92ns ±0.12 |
1.30ns ±0.02 |
0.552ns ±0.05 |
aValues
are expressed as mean ± S.E.M. of 6 mice, bP<0.01
Statistical significance versus Group I.
Table 9: Effect of Martynia
annua Extract on Relative Organ Weight.
|
Group |
Treatment |
Relative organ weighta (g) ± S.E.M. |
||
|
Liver |
Kidney |
Spleen |
||
|
Group I |
Control (Normal saline) |
5.14± 0.01 |
1.367± 0.45 |
0.622± 0.05 |
|
Group II |
Normal saline + CP |
4.82ns ±0.17 |
1.115 ns ±0.26 |
0.334b ±0.06 |
|
Group III |
MEAC 800 mg/kg |
5.04ns ±0.74 |
1.325ns ±0.02 |
0.500ns ±0.01 |
|
Group IV |
MEAC 1200 mg/kg |
5.12ns ±0.06 |
1.330ns ±0.26 |
0.611 ns ±0.02 |
|
Group V |
MEAC 800 mg/kg + CP |
4.89ns ±0.11 |
1.352ns ±0.25 |
0.451 ns ±0.07 |
|
Group VI |
MEAC 1200 mg/kg + CP |
4.90ns±0.07 |
1.34ns ±0.53 |
0.511ns ±0.10 |
aValues are expressed
as mean ± S.E.M of 6 mice, bP<0.05 Statistical
significance versus Group I.
Table 10: Effect of Martynia
annua Extract on Hematological Parametersa
|
Group
|
Treatment
|
RBC (× 106/mm3) |
WBC (× 103/mm3) |
Hb (g/dl) |
|
I |
Control-Normal saline |
9.26±0.60 |
6.26±0.40 |
13.76±0.48 |
|
II |
Normal saline + CP |
9.22 ±0.28ns |
1.52±0.29b |
12.52 ±0.78ns |
|
III |
MEBC 150 mg/kg |
9.64±0.34ns |
7.98±0.46c |
13.71 ± 0.92ns |
|
IV |
MEBC 300 mg/kg |
9.59±0.47ns |
8.21±0.38c |
14.01 ± 0.51ns |
|
V |
MEBC 150 mg/kg + CP |
9.35±0.44ns |
3.52±0.43d |
12.67 ± 0.28ns |
|
VI |
MEBC 300 mg/kg + CP |
9.40±0.31ns |
4.31±0.29e |
12.73 ± 0.31ns |
aValues are expressed
as mean ± S.EM. for 6 mice, bP<0.001 Statistical
significance versus Group I, cP<0.05 Statistical
significance versus Group I, dP<0.05 Statistical
significance versus Group II, eP<0.001 Statistical
significance versus Group II RBC (million/mm3), WBC (thousand/mm3),
hemoglobin(g/dl).
Table 11: Effect of
Methanol Extract of Martynia annua on Hematological Parameters a
|
Group
|
Treatment
|
RBC (× 106/mm3) |
WBC (× 103/mm3) |
Hb (g/dl) |
|
I |
Control-Normal saline |
9.26±0.60 |
6.26±0.40 |
13.76±0.48 |
|
II |
Normal saline + CP |
9.22±0.28ns |
1.52±0.29b |
12.52±0.78 ns |
|
III |
MEAC 800 mg/kg |
9.53±0.14 ns |
7.65±0.24c |
13.36±0.92 ns |
|
IV |
MEAC 1200 mg/kg |
9.47±0.38 ns |
7.93±0.40d |
13.65±0.51 ns |
|
V |
MEAC 800 mg/kg + CP |
9.24±0.21 ns |
3.21±0.15 e |
12.33±0.28 ns |
|
VI |
MEAC 1200 mg/kg + CP |
9.29±0.57 ns |
3.99±0.30f |
12.39±0.31 ns |
aValues are expressed
as mean ± S.E.M. for 6 mice, bP<0.001 Statistical
significance versus Group I, cP<0.05 Statistical
significance versus Group I, dP<0.01 Statistical
significance versus Group I, eP<0.01 Statistical
significance versus Group II, fP<0.001 Statistical
significance versus Group II, RBC (million/mm3), WBC (thousand/mm3),
Hemoglobin (g/dl).
Table 12. Effect of Martynia
annua Extract on Oxidative Stress Parameters a
|
Group |
Treatment |
LPO (nmol MDA/mg protein) |
GSH (μg/mg protein) |
SOD (units/mg protein) |
CAT (nmol H2O2 /mg protein) |
|
I |
Control (Normal saline) |
3.79±0.04 |
3.26±0.02 |
3.34±0.02 |
28.21±0.09 |
|
II |
Normal saline + CP |
7.38 ±0.01b |
1.87 ±0.05b |
2.70 ±0.03b |
19.83 ±0.03b |
|
III |
MEBC 150 mg/kg |
4.13 ±0.08b |
2.72 ±0.09b |
2.93 ±0.01b |
26.82 ±0.02b |
|
IV |
MEBC 300 mg/kg |
4.30 ±0.01b |
2.95 ±0.04d |
3.11 ±0.0f |
27.78 ±0.05b |
|
V |
MEBC 150 mg/kg + CP |
5.95 ±0.06c |
2.19 ±0.03e |
3.17 ±0.05c |
23.84 ±0.05c |
|
VI |
MEBC 300 mg/kg + CP |
6.31 ±0.06c |
2.52 ±0.03c |
3.22 ±0.08c |
24.14 ±0.03c |
MEBC= Methanol
extract of Martynia annua bark, CP=cyclophosphamide, LPO=lipid
peroxidation, SOD=superoxide dismutase, GSH=reduced glutathione, CAT=catalase.
aValues are mean ±
S.E.M. of 6 mice, bP<0.001 Statistical significance versus Group
I, cP<0.001 Statistical significance versus Group II, dP<0.01
Statistical significance versus Group I, eP<0.01
Statistical significance versus Group II, fP<0.05
Statistical significance versus Group I.
Table 13. Effect of Martynia
annua Extract on Oxidative Stress Parameters a
|
Group
|
Treatment |
LPO (nmol MDA/mg protein) |
GSH (μg/mg protein) |
SOD (units/mg protein) |
CAT (nmol H2O2 /mg protein) |
|
I |
Control (Normal saline) |
3.79±0.04 |
3.26±0.02 |
3.34±0.02 |
28.21± 0.09 |
|
II |
Normal saline + CP |
7.38±0.01b |
1.87±0.05b |
2.70±0.03b |
19.83±0.03b |
|
III |
MEAC 150 mg/kg |
3.24±0.04b |
2.31±0.03b |
2.85±0.06b |
26.23±0.09b |
|
IV |
MEAC 300 mg/kg |
3.97±0.03c |
2.49±0.04b |
3.01±0.06b |
27.12±0.04b |
|
V |
MEAC 150 mg/kg + CP |
4.10±0.01d |
2.11±0.05d |
3.07±0.03d |
23.21±0.23d |
|
VI |
MEAC 300 mg/kg + CP |
4.33±0.06d |
2.27±0.02d |
3.10±0.04d |
23.94±0.05d |
MEAC= methanol
extract of Martynia annua leaves, CP=cyclophosphamide, LPO=lipid
peroxidation, SOD=superoxide dismutase, GSH=reduced glutathione, CAT=catalase, aValues
are mean ± S.E.M. of 6 mice, bP<0.001 Statistical significance
versus Group I, cP<0.05 Statistical significance versus
Group I, dP<0.001 Statistical significance versus Group
II.
Table 14. Effect of Martynia
annua Extract on Proinflammatory Cytokines (pg/ml)
|
Group |
Treatment |
TNF-α |
IL-6 |
|
Group I |
Control (Normal saline) |
23.4±0.13 |
34.5±0.1 |
|
Group II |
Normal saline + CP |
9±0.21b |
12±0.35b |
|
Group III |
MEBC 150 mg/kg |
26.98±0.45b |
35.87±0.05b |
|
Group IV |
MEBC 300 mg/kg |
28.0±0.82b |
36.04±0.07b |
|
Group V |
MEBC 150 mg/kg + CP |
14.43±0.55c |
15.73±.0.21c |
|
Group VI |
MEBC 300 mg/kg + CP |
17.97±0.71c |
17.09±0.07c |
Data were expressed
as the mean ± S.E.M., n = 6 animals per group. Concentration was expressed in
pg/ml. bP<0.001 Statistical significance versus Group I, cP<0.001
Statistical significance versus Group II.
DISCUSSION:
The shade-dried
roots of M. annua (0.75Kg) were extracted with petroleum ether (40-60°C)
followed by chloroform, acetone and ethyl alcohol. The extracts were
concentrated in vacuum to remove the solvent. The concentrated acetone extract
was fractionated into hexane and dichloromethane soluble fractions. The hexane
extract did not yield any compound. The dichloromethane extract was concentrated
under vacuum to give a semi-solid (1.5g) which was made into slurry with silica
gel (4g). The extract was chromatographed over a column of silica gel (100g)
packed in hexane. The column was eluted with a) hexane, b) hexane:
dichloromethane mixtures with increasing amounts of dichloromethane, c)
dichloromethane. Fractions of 100 mL were collected each time, distilled off
the solvent and the resulting residues were examined on TLC by using different
solvent systems and similar fractions were mixed together. The identification
of the compound was done by spectroscopic techniques like UV-Visible, IR
spectroscopy, NMR spectroscopy, HPLC and GC-MS. To predict the antiviral
property of the isolated compound was docked against HIV-1 protease. In
addition to anti-viral properties.
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Received on 11.04.2020
Modified on 26.04.2020
Accepted on 11.05.2020 ©AandV
Publications All right reserved
Res. J. Pharmacognosy and
Phytochem. 2020; 12(2): 94-100.
DOI: 10.5958/0975-4385.2020.00021.7