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Vol. 7(44), pp.

3242-3246, 25 November, 2013


DOI: 10.5897/JMPR09.030
ISSN 1996-0875 2013 Academic Journals
http://www.academicjournals.org/JMPR

Journal of Medicinal Plants Research

Full Length Research Paper

Immunostimulant activity of certain plants used in


Indian traditional medicine
Anju Puri1*, Anuj Srivastava 1, Anubhav Bhardwaj1, Jata Shanker Tandon2 and
Krishan Chand Saxena1
1

Division of Biochemistry, Central Drug Research Institute, Lucknow-226001, India.


Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow-226001, India.

Accepted 20 May, 2010

Certain plants products widely used in the traditional medicine system of India were studied for
immunostimulant activity. In the present study, strong stimulation of antigen specific and non specific
immunity was evidenced by increase in haemagglutinating antibody (HA) titre, plaque forming cells
(PFC) and in macrophage migration index (MMI), in mice fed with 50% ethanolic extract of these plants.
The observation provides scientific basis for wide spread use of these plant products which are likely
to play a role in Indian traditional medicine as tonic for rejuvenation therapy and chronic ailments.
Key words: Macrophage migration index, haemagglutinating antibody, immunostimulant.

INTRODUCTION
Immunostimulant activity has been demonstrated in
several plants used in the traditional medicine of India,
China and European countries for rejuvenation therapy
and treatment of chronic ailments (Atal et al., 1986;
Wagner, 1983, 1984a, b). Strong immunostimulant
activity has been reported from this laboratory in Panax
pseudoginseng (Araliaceae) (Dua et al., 1989), Picrorhiza
kurroa (Scrophulariacae) (Puri et al., 1992), Andrographis
paniculata (Acanthaceae) (Puri et al., 1993), Nyctanthes
arbortristis L. (Nyctanthaceae) (Puri et al., 1994),
Curculigo orchioides (Hypoxidaceae) (Lakshmi et al.,
2004), and certain dry fruits and plant material, namely:
Prunus
amygdalus
(Rosaceae),
Euryale
ferox
(Nymphaceae), Buchanania lanzan (Anacardiaceae),
Phoenix dactylifera (Arecaceae) and Zingiber officinale
(Zingiberaceae) given to mothers after child birth and to
invalids in both of whom the immune status is low (Puri et
al., 2000).
In addition to the immunostimulant activity, P. kurroa

was also shown to provide non specific protection against


Leishmania donovani infection in hamsters (Puri et al.,
1992). A. paniculata and N. arbortristis have also shown
antileishmanial (Tandon et al., 1991) and antimalarial
(Misra et al., 1992), respectively. Pipalli Rasayana an
Aurvedic preparation consisting of Piper longum
(Piperaceae) and Butea monosperma (Fabaceae) was
shown to be effective in the management of giardiasis in
patients (Agarwal et al., 1997). In experimental giardiasis,
a curative effect of the rasayana was accompanied by
stimulation of specific and nonspecific immune responses
(Agarawal et al., 1994). P. Longum, the main constitutent
of Pipalli rasayana was later shown to be effective
against experimental giardiasis accompanied by
stimulation of specific and nonspecific responses in the
animals (Tripathi et al., 1999).
The present communication reports on the
immunostimulant activity of certain other plants used in
the Indian traditional medicine as tonics for invigorating

*Corresponding author. E-mail: anjup4@rediffmail.com. Tel: 91-522-2612411-18. Fax: 91-522-2623938.

Puri et al.

health and for a variety of ailments.


MATERIALS AND METHODS
Collection and extraction of plant material
The seeds, leaves, flowers or stem of various plants were collected
from different localities and identified by the Botany Division of this
Institute. The voucher specimens have been deposited in the
herbarium of the Institute. The materials were shade dried and
extracted separately with 95% ethanol. The ethanol extracts were
concentrated under reduced pressure below 50C in a rotatory
evaporator to yield viscous mass which were dried separately under
high vacuum to remove last traces of the solvent.

Experimental animals and treatment


BALB/c albino mice of either sex, weighing 20 to 25 g were used in
the present study. The animals were kept on a standard pellet diet
(Lipton, Mumbai, India) and water ad libietum. The plant extracts
were fed orally at a dose of 25 mg/kg/day for 7 consecutive days.

3243

for 2 h and then examined for agglutination. The reciprocal of the


highest dilution of the test serum giving 50% agglutination has been
expressed as the HA titre.
Plaque forming cell (PFC) count: The assay was carried out after
the technique of Jerne and Nordin (1963). Spleen cells were
separated from the extract treated and control animals 4 days after
the immunization with SRBC, in RPMI-1640 medium, washed twice
with the medium and suspended in complete RPMI-1640 medium
containing fetal calf serum. Glass petri dishes (25 mm) were
layered with 1.2% agarose in 1.5 M NaCl to form the bottom layer.
A mixture of 2 ml 0.6% agarose in RPMI-1640 medium at 42C, 0.1
ml suspension of 20% SRBC and 1 105 spleen cells in 0.1 ml
were layered over the bottom layer and the petri dishes incubated
at 37C for 45 min. Two milliliters of 1:10 diluted fresh guinea pig
serum was then added and the incubation continued for 45 min,
washed twice with the medium and resuspended in complete RPMI1640 medium containing Fetal calf serum. Glass petri dishes (25
mm) were layered with 1.2% agarose in 1.5 M NaCl to form the
bottom layer. In a mixture of 2 ml 0.6% agarose in RPMI-1640
medium at 42C, the plaques were counted immediately and the
value has been expressed as counts per 105 spleen cells.

RESULTS AND DISCUSSION


Immunostimutant activity
The immunostimulant activity was determined using: (1) macrophage migration index (MMI) which is a correlate of macrophage
activation and cellular immunity, (2) haemagglutination antibody
(HA) titre and (3) plaque forming cell (PFC) count, both of which are
parameter of antigen specific humoral immunity.

Macrophage migration index (MMI)


The test was performed according to the method of Saxena et al.
(1991). Briefly peritoneal exudate cells (PEC) collected from the
extract treated and control mice, packed in a haematocrit capillary
tubes of 7.5 mm length and uniform bore (1.0 to 1.2 mm) were
allowed to migrate in migration chamber filled with complete
Roswell Park Memorial Institute (RPMI-1640). The area of PEC
migration was marked on Whatmann filter paper using Camera
Lucida and weighed separately. The ratio of the migration area of
PEC from the extract treated group (A1) to that from the untreated
group (A2) has been expressed as macrophage migration index.
MMI = A1 / A2

Humoral immune response


Groups of the extract treated and untreated control mice were
injected each with a suspension of sheep red blood cells (SRBC)
using 1 108 cells per mouse intraperitoneally: (a) Haemagglutinating antibody (HA) titre and (b) plaque forming cell (PFC) counts
were determined 4 days after the immunization with SRBC.
Haemagglutination antibody (HA) titre: Two fold dilutions of sera
from the immunized mice were prepared in 0.15 M phosphate
buffer saline (PBS) pH 7.0 and 50 l of each dilution was aliquotted
into 96 well microtitre plates. Twenty-five microlitre of fresh 10%
SRBC suspension in PBS was then dispensed into each wells,
mixed thoroughly and the titre plates were incubated at 25 to 30C

The data on immunostimulant activity of 18 plants studied


have been presented in Table 1 in which the plants have
been arranged in order of their macrophage activating
cellular immune response measured in terms of
macrophage migration index (MMI). As evident an form,
the data in which the animals were treated with the
extracts of the first six plants showed appreciably high
MMI values in the range of 2.3 to 3.2 times higher than
the control untreated animals. The maximum value of 3.2
was recorded for Tinospora cordifolia (Meninspermaceae), Achyranthes asper (Amaranthaceae) Ocimum
sanctum (Lamiaceae), Tephrosia purpuria (Fabaceae),
Eclipta alba (Asteraceae) and Luffa echinata
(Cucurbitaceae) also showed quite high values between
2.8 to 3.0. Cinnamon tamala (Lauraceae), Cichorium
intybus (Asteraceae) and Zizyphus sativa (Rhamnaceae)
showed MMI values between 1.5 and 2.0. The rest of the
plants showed only marginal or no activation of
macrophages.
The humoral immune response in terms of
haemagglutinating antibody (HA) titre was highest for
Cassia occidentalis (Caesalpiniaceae) with mean HA titre
of 1638 followed by O. sanctum, E. alba,, Evolvulus
alisinoides (Convolvulaceae), Zingiber officinale and
Pureria tuberosa, (Fabaceae) which gave mean HA titre
of 1443 each. T. purpurea , L. echinata, Foeniculum
vulgare
(Umbelliferae)
and
Memordica
diocea
(Cucurbitaceae) also showed a high HA titre with mean
value 1228. The titre for T. cordifolia and A. asper
(Compositae) which elicited highest macrophage activity
response gave rather low antibody response in terms of
HA titre and C. tamala and C. intybus (Compositae)
which showed significant macrophage activation failed to

3244

J. Med. Plants Res.

Table 1. Cell mediated and humoral immune response in certain plants used in Indian system of medicine.

S/No.

Plants

Cell (MMI)
mediated immune
response

Humoral immune response


PFC
meanSD
12612.94a

Medicinal uses of plants

Antisheumatic, cough & chest disorders, febrifuge

Tinospora cordifolia

3.2

HA titre
meanSD
614.4228.97

Achyranthus asper

3.0

716.8280.4

12614.47a

Tephrosia purpurea

2.9

1228.8457.94b

9017.67

Ocimum- sanctum

2.9

1433.6560.8a

13023.71a

Stimulant, bronchitis, anti asthmatic fever

1433.6560.8a

13021.5a
9619.18a

Tonic, fever, hepatitis, spleen, enlargement, tetanus, prevents ageing and premature graying of hears.
Antitubercular, jaundice, antiseptic bronchitis

Bitter tonic, general debility, febrifuge anti-rheumatic antigonorrhoeic, jaundice, antileprotic, skin disorder.
Tonic, anti asthmatic, anti rheumatics, bronchitis, diseases of lung and chest, spleen enlargement, liver diseases,
antigonorrhoeic, anti syphilitic eczema

5
6

Eclipta alba
Luffa echinata

2.9
2.8

1228.8457.94b

Evolvulus alisinoides

2.3

1433.6560.8a

12012

8
9

Amoora rohituka
Cinnamon tamala

2.3
2.0

614.4228.97
307.2114.48

8016.55
6510.09

Tumors, spleen and liver disorder anti rheumatic


Stimulant, anti rheumatic, cold, anti gonorrheic

10

Cichorium intybus

2.0

358.4140.21

8017.13

Tonic in fever, spleen and liver disorders, anti rheumatics, gout.

11
12

Zizyphus sativa
Zingiber officinale

1.5
1.3

460.8114.48
1443.6560.8a

9018.54
16048.08a

Tonic, chronic bronchitis, soar throat, rheumatism.


Stimulant, anti rheumatic, affections of threat head and chest.

13

Foeniculum vulgare

1.2

1228.8457.94b

10513.17b

Anti gonorrhoeic, stimulant, cordiotonic cormivative, expectorant.

14

Cassia occidentalis

1.0

1638.4560.86a

19022.06a

Tonic, febrifuge, spleen enlargement jaundice, leprosy.

15
16

Pureria tuberosa
Memordica diocea

1.0
1.0

1443.6560.86a
1228.8457.94b

15035.06a
12514.88a

Tonic, febrifuge, anti rheumatic.


Gout -rheumatism, spleen and liver disorders, febrifuge, jaundice.

17

Solanum nigrum

0.9

716.8280.4

12012.74a

Febrifuge, bronchitis, liver disorders, anti asthmatic

18
19

Hedychium spicatum
Control

0.8
1.0

912.6228.97
307.2114.48

9619.17
6515.47

Febrifuge, antiasthamatic, bronchitis

Stimulant, liver disorders


-

p< 0.01; p < 0.05.

elicit HA response. On the other hand, C.


occidentalis, Z. officinale, F. vulgare, P. tuberosa,
M. dioca and H. spicatum (Zingiberaceae) which
showed little or no macrophage activation, elicited
significantly high HA titre.
The humoral immune response in terms of
plaque forming cell (PFC) counts was highest with
C. occidentalis followed by Z. officinale (160), P.
tuberosa (150), O. santnum (130), E. alba (130),
T. cordifolia (126), A. asper (126), M. dioca (125),

S. nigrum (120) and E. ailsinoides (120). The PFC


count was significant with other plants except for
C. tamala and C. intybus which like HA titre failed
to elicit PFC response over the control value.
The last column of the table shows the
medicinal uses of the plants taken up for the
present investigation (Husain et al., 1992). Plants
1, 3, 4, 5, 9, 10, 11, 14, 15 and 18 have been
used as tonics for invigorating health. Of these
plants, plant 5 also prevents ageing and prema-

ture greying of hairs. Plants 2, 3, 4, 7, 11, 12 and


17 have been used against chronic bronchitis and
diseases of chest. All these later plants with the
exclusion of plants 11 and 12 have also been
found useful against allergy-asthma. Plants 1, 3, 9
and 13 have been used in gonorrhoea and plants
1, 6, 14 and 16 against jaundice. Plants 1 and 14
have been found to be antileprotic, plant 3
antisyphlitic, plants 6 antitubercular and plant 8
antitumor. Plants 1, 2, 3, 8, 9, 10, 11, 12, 15 and

Puri et al.

16 have been found to be effective against rheumatic


disorders.
It may be recalled that plants 1 to 11 of this study
stimulate both cellular and humoral immune response of
the body, with the exception of plants 9 and 10 which
stimulate only cellular immune response. All other plants,
particularly plant 12 to 18 stimulates only humoral
immune response. Although all the ailments for which
these plants have been used may not require stimulation
of the immune response of the body, certain ailments or
condition do require immunstimulation or immunoreglutination for effective cure. As evident from the
existing literature (Atal et al., 1986; Wagner, 1984a, b),
the plants used as tonics or stimulants for invigorating
health and for treatment of chronic bronchitis, sour throat,
and jaundice of viral origin, tuberculosis, leprosy,
gonorrhoea and tumor are very much likely to require
immunostimulaton. The plants used against chronic
cases of fever may also require stimulation of immune
response. But in some cases the curative effect may also
be due to the presence of antipyretic substances in some
plants.
Allergy such as asthma (Holt and Sly, 2003; Crompton
and Mc Hardy, 1991) and rheumatism (Nuki, 1991) are
yet other disorders which develop due to alterations in
the immunological system of the body. In allergic asthma,
individuals start producing Ig E-type of antibodies instead
of the normal IgG-type, which in turn are responsible for
the allergic manifestations. Type-4 allergic manifestations
are ascribed to derangement of cell-mediated immunity
involving T-cells. In rheumatoid disorders, circulatory
antiglobulins to antibodies (rheumatoid factor) formed as
a result of immunological imbalance become attached to
the bone joints. There is also evidence of T-Cell alterations in persons genetically predisposed to rheumatism.
The immunostimulants, therefore, are most likely to be
beneficial in these disorders.
Spleen is one of the secondary lymphoid organs where
lymphocytes can interact with each other and with the
antigens since, phagocyte, macrophages, antigen presenting cells, mature T and B lymphocytes were collected
together in this organ (Ferguson, 1991a). Disorders of
spleen may accompany impairment of immune system.
Similarly, kuffer cells which have microbicidal activity
are located in liver and they may be affected in liver
disorders (Ferguson, 1991b). In view of these facts, it is
most likely that the curative effect of these plants in
spleen and probably also in liver disorders may also be
due to their immunostimulant activity.
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