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International Journal of Pharmacological Research www.ssjournals.com
ISSN: 2277-3312 Journal DOI:10.7439/ijpr
IJPR Volume 6 Issue 01 (2016) 29
Immunomodulation Potential of Andrographis Paniculata and
Tinospora Cordifolia Methanolic Extracts in Combination Forms
Yadav Ra*, Yadav Na and Kharya M Db
a*Department of Pharmacy, SRMS, College of Engg. and Tech., Bareilly, U.P.
bDepartment of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar, M.P.
Corresponding author*
Rajesh Yadav
Department of Pharmacy, SRMS, College of Engg.and Tech., Bareilly, U.P.
E-mail: raj_ishu78@rediffmail.com
1. Introduction
Indian traditional systems of medicines like Ayurveda and Siddha have the potential to the body’s natural
resistance to disease [1]. Recent studies with plants have revealed many compounds with potent antioxidant,
antineoplastic, antiulcer, anti-inflammatory and immunostimulating potential [2]. It is believed that the immunomodulatory
drugs promote positive health and maintain organic resistance against infections by establishing body’s equilibrium and
conditioning the body tissues [3]. The restorative and rejuvenating power of herbal remedies might be due to their action
on the immune system and thereby responsible for the protection of the organism from extraneous substances and
maintaining the homeostasis. Beyond this pharmaceutical approach to plants, there is a wide tendency to utilize herbal
products to supplement the diet, mainly with the intention of improving the quality of life and preventing the diseases of
elderly people [1]. India has been identified as a major resourceful area in the traditional and alternative medicines
globally. Several plant products are known to exhibit immense medicinal value against human diseases. The plants use as
an immunomodulator namely Andrographis paniculata and Tinospora cordifolia. They have good impact in the
treatment and management of HIV-AIDS because these plant not only treats disease but also enhance the body
vitality and immunity. The humoral and cell-mediated immune response was observed through delayed type
hypersensitivity (DTH) model.
Andrographis paniculata, is an herbaceous plant of family Acanthaceae, also called King of Natural Bitters, is a
traditional India, Southeast Asian and Chinese herb, used for centuries in Ayurvedic medicine. Since ancient times, A.
paniculata is being used in Ayurvedic and traditional Siddha systems as well as in tribal medicine in India and some other
countries for multiple clinical applications. It has three major spheres of influence, first by offering extensive immune
system support, second to protect the liver and third to strengthen the cardiovascular system [4]. The herb has been revered
for treating infectious diseases and is highly regarded for preventating many diseases, due to its powerful immune
strengthening benefits. The global flu epidemic of 1918 was one of the most devastating infectious outbreaks in World
Abstract
Objective: This work mainly focuses on the immunomodulatory effect of different drug combinations (DC-I to DC-V)
of methanolic extracts of aerial parts of Andrographis paniculata (AP) and mature stems of Tinospora cordifolia (TC).
Materials and Methods: The coarse powder (40-mesh) of shade dried aerial parts of AP and mature stems of TC
(500g each) was subjected separately to successive extraction with 1000ml each of petroleum ether (60-80oC) followed
by methanol. The doses of DC-I to DC-V were selected and were administered orally at doses of 200 mg/kg body weight
to Albino Wistar rats and compared with the control group and standard drug i.e. cyclophosphamide (100mg/kg)
respectively.
Results: Results suggest that although all the five tested combinations (DC-I to DC-V) prepared using different
proportion of methanol extracts of A. paniculata and T. cordifolia (100:Zero, 75:25, 50:50, 25:75 and Zero:100) showed
a significant increase in macrophase count, neutrophil adhesion(NA), haemagglutinating antibody (HA) titre and
delayed type hypersensitivity (DTH) response. In rats immunized with sheep RBC, DC-I to DC-V enhanced the
humoral antibody response to the antigen and significantly potentiated the cellular immunity by facilitating the
footpad thickness response to sheep RBC in sensitized rats on chosen experimental models.
Conclusion: The values of macrophase, NA, HA, DTH and cellular responses of combination DC-II at a 200 mg/kg
body weight were statistically significant as compared to other combinations. Combination DC-II exhibited best
immunostimulant potential, indicating that it has promising immunomodulatory activity, as proposed from commercial
point of view.
Keywords: Andrographis paniculata, Humoral and cellular responses, Immunostimulant, Methanol extract, Sheep RBC,
Tinospora cordifolia.
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 30
History. No country escaped its on-slaught except India where Andrographis paniculata, an amazing herb was credited
with stopping the spread of the deadly virus. Its most active and major constituent-Andrographolide is a Lactone ring in
basic diterpene glycoside molecules [5]. Andrographis paniculata is also reported to possess anti-inflammatory [6], Anti-
Oxidation [7], Anti-hepatotoxicity [8], Anti-hyperglycemic effect [9], Anti-infection [10], Anti-cancer [11], Anti-
atherosclerosis activity [12].
Tinospora cordifolia is a large extensively spreading, perennial climber belonging to the family Menispermaceae.
It is widely distributed throughout tropical and subtropical India. In Hindi, the plant is commonly known as Giloya, Giloe
or Amrita. Giloya is a Hindu mythological term that refers to the heavenly elixir which has saved celestial beings from old
age and kept them eternally young. Guduchi, the Sanskrit name means one which protects the entire body [13]. The active
adaptogenic constituents are diterpene compounds including tinosporone, tinosporic acid, cordifolisides A to E, syringen,
the yellow alkaloid, berberine, giloin, crude giloininand, a glucosidal bitter principle as well as polysaccharides, including
arabinogalactan polysaccharide [7-8]. It shows significant bactericidal activities. It improves bacterial clearance as well as
improves phagocytic and intracellular bactericidal capacities of neutrophils. It also stimulates macrophage action. As a
result it stimulates immune system of body [15]. In Ayurveda also called as Amrita, it is used as “rasayana” which has
powerful immunostimulant activity [16]. Charaka described rasayana as antiaging, which increased the life span, promoted
intelligence, improved memory and ensured freedom from diseases, indicating immunostimulant effect [17]. T. cordifolia
is used to strengthen the immune system of the body by keeping the function of its various organs in harmony. It has great
potency to alleviate impurity of body organs. T. cordifolia in Vedic age was considered as one of the most rejuvenating
herbs working well on the entire seven Dhatus (the constituent elements of the body) keeping the bodies free from all
types of illness [18]. Scientists realize that the effective life span of any antibiotic or synthetic molecule is limited so new
sources especially plant sources need to be investigated. Therefore, in the today’s world of modern medicine T. cordifolia
is rightly called as “The Magical Rejuvenating Herb”. Categorized as “rasayana” in traditional Indian System of
Medicine it is used as general tonic because of its anti-inflammatory, anti-arthritic, anti-allergic, anti-malarial and
immunomodulatory properties [19-21]. Its general adaptogenic and immunomodulatory activity was implicated in
fighting infections.
Chemotherapeutic agents of today have mainly immunosuppressive activity and most of them are cytotoxic and
exerts a variety of side effects. Then metabolism and clinical safety has not been clearly established. This has given rise to
stimulation in the research for locating natural resources showing immunomodulatory activity. Presently due to variety of
reasons, cases of immunity impairment and its associated problems are increasing. Unfortunately existing therapy does not
provide any cure to handle such situation.
However Andrographis paniculata and Tinospora cordifolia have been described as over all rejuvenator in
Ayurveda with its modifying effects on immunity, may provide a radical cure in such complications. It was therefore
decided to investigate these plants the renowned drugs of Ayurveda for developing an immunomodulatory potential.
Although the literature revealed that A. paniculata and T. cordifolia possess good level of immunostimulant
property. And based on the studies, these drugs have also been suggested for their clinical use [8-10], however as no study
is available on immunomodulation where both of these drugs i.e. A. paniculata and T. cordifolia have been used together
as combination.
The studies also revealed that although the drugs possess immunomodulatory activities, their mode of action can
be different. Further, there are growing evidences that drugs capable of modulating single pathway or target are of limited
value as immune related therapy and therefore system biology aiming at multi-valuable approaches are now gaining more
interest/important. Keeping these facts in mind, it was thought to evaluate the immunomodulation potential of these two
highly valued drugs in their combined form. And hence the study was planned accordingly.
The extracts from A. paniculata and T. cordifolia were subjected to evaluate the bio-actives following to
preliminary phytochemical screening of extracts. After performing different chemical tests in each extracts of A.
paniculata and T. cordifolia, it was found that the bio-actives were present in methanolic extracts of A. paniculata and T.
cordifolia. Therefore, the petroleum ether extracts was not used and only the methanolic extracts were subjected to further
studies. The five Drug combinations (DC-I to DC-V) prepared from methanol extracts of A. paniculata (MEAP) and T.
cordifolia (METC) and evaluated for their immunomodulatory activity studies by using different animal models like
Modulation of Macrophage Function, Humoral response in normal and cyclophosphamide induced immunodeficient rats,
Neutrophil Adhesion (NA) Test, Haemagglutinating antibody (HA) Titre, Delayed type hypersensitivity (DTH) response in
rats and compared with the control group and standard drug i.e. cyclophosphamide (100mg/kg) respectively. Therefore,
present investigation is aimed at studying the immunomodulatory potential of DC-I to DC-V in order to justify the
claims that the andrographolide, berberine present in the extracts of AP and TC may be responsible for its
immunomodulatory potential.
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 31
2. Materials and Methods
2.1 Plant material:
The Aerial parts of Andrographis paniculata and selected mature stems of the Tinospora cordifolia were
collected from medicinal plant garden of Department of Pharmaceutical Sciences, University Campus, Dr. H.S. Gour
Vishwavidyalaya, Sagar (M.P.).The plant material was identified and authenticated taxonomically at the Botany
Department of Dr. H.S Gour University, Sagar M.P. (Ref no-1417 and 1418, respectively, dated- 28.01.2011). A voucher
specimen of the collected sample was deposited in the Departmental herbarium for future reference.
2.2Preparation of extracts:
The collected plant materials were washed with water to make them free from any dust or foreign matter. A.
paniculata (aerial parts) was dried as such in open shade whereas for convenience of drying T. cordifolia (mature stems)
were cut into small pieces, crushed and dried in open shade. After air drying the plant materials were powdered (40 mesh
size). 500g of each powder was separately extracted by soxhlet successively with 1000ml each of petroleum ether (60-
80oC) followed by methanol. The appearance of colorless solvent in the siphon tube was taken as the end point of
extraction. The successive extracts were separately filtered and concentrated at reduced temperature on a rotary
evaporator and dried over a desiccator at room temperature to obtain total extracts i.e. petroleum ether and methanol
extracts of A. paniculata and Tinospora cordifolia. After completion of extraction, extracts were weighted (w/w);
percentage yield was calculated and abbreviated separately. The biologically potent methanol extract was prepared for
herbal tablet formulation.
2.3 Preparation of Drug combinations from methanol extracts:
The studies also revealed that although the drugs possess immunomodulatory activities, their mode of action can
be different. Further, there are growing evidences that drugs capable of modulating single pathway or target are of limited
value as immune related therapy and therefore system biology aiming at multi-valuable approaches are now gaining more
interest/important. Keeping these facts in mind, it was thought to evaluate the immunomodulation potential of these two
highly valued drugs in their combined form.
2.4Animal selection:
Swiss Albino mice of either sex (20-25gms.) and Wistar albino rats of either sex (200-250gms.) were selected for
carrying out Pharmacological activity. Animals were housed at room temperature (23±20C) with 12h light and 12h dark
cycle and relative humidity (55±10%) and were given water (ad libitum) and were fed with rat pellet feed. Experiments on
animals were conducted after getting approval from Institutional Animal Ethics Committee Dr. H.S. Gour Central
University, Sagar, M.P. (Registration No. 379/01/ab/CPCSEA) which is registered with Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA), Government of India.
2.5Acute toxicity studies:
The acute oral toxicity studies and selection of dose was done as per guidelines of Organization for
Economic Co-operation and Development (OECD), draft guidelines 423 received from Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment,
Government of India. Healthy albino mice of either sex were used for acute toxicity study to determine LD50 of
investigating five different Drug combinations (DC-I to DC-V) of A. paniculata and T. cordifolia
The animals were randomly selected, marked to permit individual identification and kept in the cages for 7 days
prior to dosing to allow for acclimatization to the laboratory conditions.
The test substances, Drug Combinations (DC-I to DC-V) of AP and TC were administered in a single dose by
gavage. Three mice (n=3) were used in each category and starting dose lied in the range of 5-5000mg/kg body weight
(OECD guideline 423). Dose ranging between 5, 300, 2000 and 5000mg/kg of body wt. of extract combinations were
administered stepwise to the mice according to their weights. After the treatment, mice were observed individually once
during the first 30 minutes, and then periodically during the first 24 hrs. There was no mortality till the dose of 2000mg/kg
body weight in extract combinations. Considering this dose of 2000mg/kg body weight, 1/10th of this dose i.e. 200mg/kg
body weight was taken as effective dose for five extract drug combinations (DC-I to DC-V) from methanol extracts of A.
paniculata (MEAP) and T. cordifolia (METC) for carrying out their immunomodulatory screening.
2.6 Models for Immunomodulatory Screening of different Extract Combinations of AP and TC
2.6.1 Dose selection and Preparation for study:
After carrying out acute toxicity studies, it was observed that lethal dose for DC-I to DC-V combination was
2000mg/kg body weight and hence 1/10th of the dose i.e. 200mg/kg body weight was taken as effective dose for different
Drug combinations (DC-I to DC-V) of extracts for performing the immunomodulatory screening.
2.6.2 Standard Drugs, Chemicals and reagents
The drugs, chemicals, and reagents procured from S.D. Fine Chemicals, (Mumbai, India) were of analytical
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 32
grade. Cyclophosphamide (German Remedies Limited, Kundaim Industrial Estate, Ponda, Goa) was used to produce
immunosuppression in rats (Dose- 100mg/kg body weight). Sheep Red Blood Cells (SRBCs) at dose of 0.5ml were
injected intraperitoneally for immunization and challenge to the rats. Alsever’s solution- for collection of blood
from sheep and Phosphate Buffer saline (PBS -7.2pH)- for collection of peritoneal fluid form mice were used.
2.6.3Sheep Red Blood Cells (SRBCs)
Blood from healthy Sheep was collected from local butcher house in Alsever’s solution. It was washed three
times with pyrogen free 0.9% normal saline and centrifuged at 3000 rpm for 5min. Supernatant was discarded. The settled
SRBC was then suspended in normal saline and total SRBC counted using Neubauer chamber and RBC of this suspension
was adjusted to a concentration of 5×109 SRBC (0.5ml) and injected intraperitoneally for immunization and challenge
[26].
2.6.4Preparation of Alsever’s solution
Formula:
Citric acid
0.055gm
Sodium citrate
0.8gm
Glucose
2.05gm
Sodium chloride
0.42gm
Distilled water to make volume up to
100 ml
All the above ingredients were weighed and dissolved in distilled water and the volume was made up to 100ml.
The solution was stored in refrigerator [27].
2.6.5Preparation of Phosphate Buffer saline, (PBS) (7.2 pH)
Take 50ml of 0.2M of Pot. di hydrogen phosphate (KH2PO4), [27.218gms. KH2PO4 in 1000ml. water] was taken
in 200ml. volumetric flask and 34.7ml. of 0.2M Sod. hydroxide was added and volume was made up to 200ml [28].
2.6.6Statistical Analysis of Experimental Data
All the experimental data for statistical analysis were presented as mean± SEM (n=4 in each group). Results
obtained were statistically analyzed by using One-way ANOVA followed by Dunnett’s comparision test. Statistical
significance on comparison with control group was indicated by *mark, where *P<0.05 was considered as significant
value.
2.6.7 Modulation of Macrophage Function
Albino male mice (20-25g) of either sex were housed under standard laboratory conditions prior to experimentation.
They were fasted for a period of 24hrs. Allowing free access to drinking water, prior to p.o. drug administration.
Group-1: (control, n=4) received only clean tap water.
Group-2 to 6: (received Drug combination samples DC-I to DC-V, n=4) @ 200mg/kg body weight.
Peritoneal macrophage was isolated from the treated mice (n=4) on day 5th, 10th and 15th consecutively and also
from control mice. Peritoneal fluid was collected in Phosphate buffer saline (PBS, ph 7.2) and the macrophage count was
done [29].
2.6.8 Humoral response in normal and cyclophosphamide induced immunodeficient rats
The depression in immune system associated with cancer, surgery, infection and certain drugs is characterized by
the reduction in the number and function of neutrophils and macrophages as well as in intracellular bactericidal capacity of
these cells. Cyclophosphamide is converted in the organism from a non-reactive to a highly reactive form. This alkylating
agent, which is inactive in vitro, is activated in vivo by enzymatic cleavage of the phosphamide group, which releases the
active portion of the compound once the molecule is split. The alkylating agents are thought to act by combining directly
with certain intracellular molecules, particularly nucleic acid and proteins. It blocks the conversion of a precursor
population (possibly small lymphocytes) to lyphoblasts and the thymus dependent lymphocytes are preferentially affected
by cyclophosphamide.
Treatment schedule:
In this procedure male albino rats (200-250g) were divided into three groups.
Group- A: (–ve control, n=4) was administered tap water from day (-9) to day (+5) and on day (+2) 100mg/kg body weight
of cyclophosphamide was administered orally in addition to water.
Group-B: (+ve control, n=4) was administered tap water from day (-9) to day (+5).
Group-C1 to C5: (Drug combination samples DC-I to DC-V, n=4) was administered 200mg/kg body weight of DC-I to
DC-V once a day orally from day (-9) to day (+5) and on day (+2) 100mg/kg body weight of cyclophosphamide was
administered orally in addition to DC-I to DC-V treatment.
On day 0, rats in all groups were immunized (ip) with 0.5ml/100g body weight with SRBCs.
On day (+6), blood was collected from each rat and serum separated.
The value of highest serum dilution carrying visible hemagglutination was taken as the antibody titre[30].
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 33
Anti-SRBC-hemagglutination antibody titre in all drug combinations was found better (7.34±3.22 to 6.19±2.03)
than positive control (4.32±2.64) but with the Drug combination (DC-II) treated cyclophosphamide induced rats it was
found to be maximum (9.49±3.74) as compared to cyclophosphamide induced control rats (1.96±0.18).
2.6.9 Immunostimulant Activity of Drug Combination (DC-I TO DC-V) was performed on the following:
Immunostimulants, also known as immunostimulators, are substances (plants and nutrients) that stimulate the
immune system by inducing activation or increasing activity of any of its components. Immunostimulant therapy may be
beneficial for patients under a variety of settings that include prevention and treatment of various infectious diseases. It is
important to know the appropriate use of such treatments so that the ideal immunostimulant preparation is selected for
each individual patient. An ideal situation when a host is exposed to pathogen challenge (e.g. bacteria or virus) is to have
optimal immunity that protects the host from disease. In many cases specific therapy in the form of antibacterial,
antiprotozoal, antiparasitic or antifungal therapy will work in combination with the immune system to aid with pathogen
clearance. In some instances, the addition of an immunostimulant will aid in “boosting” the immune response [31].
(a) Neutrophil Adhesion (NA) Test
(b) Hemagglutinating Antibody (HA) Titre
(c) Delayed Type Hypersensitivity (DTH) response in rats
Treatment: Albino Wistar male rats (200-250g) were used for the study. Animals were housed properly under standard
conditions of temperature (23±2°C), 12:12h light/dark cycles and fed with standard pellet diet and water ad libitum. Fresh
SRBC in Alsever’s solution were obtained from authentic source. The animals were divided into six groups consisting of
four animals per group. A group of four untreated rats was taken as control (Group 1). The Drug combinations (DC-I to
DC-V) were fed orally for 14 days at a dose of 200mg/kg body weight (Group 2-6) for assessment of immunomodulatory
effect. On 14th day, all groups of rats were challenged with SRBCs (0.5ml/100g body weight I.P.).
(a) Neutrophil Adhesion (NA) test
On 14th day of Drug combinations treatment, blood samples were collected (before challenge) by puncturing the
tail-vein into heparanized vials and analyzed for total leukocyte counts (TLC) and differential leukocyte counts (DLC).
After initial counts, blood samples were incubated with 80mg/ml of nylon fibers for 15min. at 370C. The incubated blood
samples were again analyzed for TLC and DLC. The product of TLC and percentage neutrophil gives Neutrophil Index
(NI) of the treated and untreated blood samples and the difference was taken as index of neutrophil adhesion (NA)[32].
Percent neutrophil adhesion was calculated as below:-
NIu –NIt
Neutrophil adhesion (NA) (%) = X 100.
NIu
Where NIu = Neutrophil index of untreated blood sample.
NIt = Neutrophil index of treated blood sample.
(b) Hemagglutinating Antibody (HA) titre
Rats of groups DC-I to DC-V were pretreated with Drug combinations for 14 days and each rat was immunized
with SRBC (0.5ml/100g body weight I.P.), including control rats. The animals were treated with drug combinations for 14
more days and blood samples were collected from each rat on day 15 for HA titre. The titre was determined by
titrating serum dilutions with SRBCs. The micro titre plates were incubated at room temperature for 2 hours and examined
visually for agglutination. The highest number of dilution of serum showing hemagglutination was expressed as HA
titre[33].
(c) Delayed Type Hypersensitivity (DTH) response
DTH response was determined by the significant decrease or increase in paw volume. All the six groups of
SRBCs immunized rats were challenged by subcutaneous administration of SRBCs 0.5ml in right hind foot pad on
28th day and 0.2ml of 0.9% normal saline was similarly injected into left hind foot pad as control. The cell mediated
immune response was measured at 24h after SRBCs challenged on 28th day in terms of increase in paw volume
(plethysmometrically). The DTH response was expressed as the mean percent increase in paw volume between the
right foot pad injected with SRBCs and left foot pad injected with normal saline[34].
3. Results
Immunomodulators are biologic response modifying compounds that affect the immune response in either a
positive or negative fashion, where it results in an enhancement of immune reaction; it is named as immunostimulation
which implies stimulation of non-specific system i.e. stimulation of the function and efficiency of granulocytes,
macrophages and certain T-lymphocytes [37]. Immunosupression implies mainly to reduce resistance against infections,
stress and may be because of environmental or chemotherapeutic factors. Immunostimulation and immunosuppression
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 34
both need to be tackled in order to regulate the normal immunological functioning. Hence, immunostimulating agents and
immunosuppressing agents have their own standing. A number of disorders can be treated by biologic response modifiers;
these include immunodeficiency diseases and autoimmune disorders. These drugs may work on cellular or humoral
immune systems or both [38-39].
There are several herbal preparations used in the indigenous system of medicinewhich can enhance the body’s im
mune status. The drugs namely A. paniculata and T. cordifolia were collected and authenticated. The drugs were cut into
pieces and powdered to a coarse consistency, which were subjected to extraction (soxhlet) by using petroleum ether (60-
80) and methanol in succession as solvents. The extracts obtained were concentrated and dried in desiccators (Table 1 &
2). Table 1: Extract detail of A. paniculata and T. cordifolia
Drug
Extract
A. paniculata
(Aerial parts)
Petroleum ether(60-80ºC) extract
Methanol extract
T. cordifolia
(Stems)
Petroleum ether (60-80ºC) extract
Methanol extract
Table 2: Yield of extraction derived extracts of A. paniculata and T. cordifolia
Plant material
Extracted material
Extract
Nature
Color
%Yield (w/w)
A. paniculata
(Aerial parts)
Petroleum ether
(60-80°C) Extract
Solid
Light -brown
1.06
Methanol Extract
Solid
Dark brown
12.36
T. cordifolia (stems)
Petroleum ether
(60-80°C) Extract
Solid
Light brown
2.31
Methanol Extract
Semi solid
Light brown
11.58
All the individual dried extracts were checked for their active ingredients by proximate chemical analysis. For the
design and preparation of different Drug Combinations (DC-I to DC-V), the individual dried extracts of Andrographis
paniculata and Tinospora cordifolia were mixed in a requisite amount and 2% Tween 80 suspension was added to the
each dried Drug combination in distilled water (100ml) as a suspending agent (Table 3).
Table 3: Drug combinations of methanol extracts of A. paniculata and T. cordifolia
S.No.
Drug Combination
Quantity of (MEAP)
Quantity of (METC)
1
DC-I
100%
Zero
2
DC-II
75%
25%
3
DC-III
50%
50%
4
DC-IV
25%
75%
5
DC-V
Zero
100%
Note: The quantity of each Extract Combination is 5g.
The prepared Drug Combinations (DC-I to DC-V) were subjected to toxicity studies followed by dose selection
was carried out as per guidelines of Organization for Economic Co-operation and Development (OECD), Draft guidelines
423 received from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA),
Ministry of Social Justice and Empowerment, Government of India. During acute oral toxicity study, animals were
observed for their Behavioural, Neurological and Autonomic profile. For acute oral toxicity studies of Five Drug
Combinations (DC-I to DC-V) of methanol extracts of A. paniculata and T. cordifolia were administered in a single dose
by gavage. Three animals were used in each category and starting dose lied in the range of 5-5000 mg /kg body weight
(OECD guideline 423). 1/10thof the lethal dose was used as effective dose for immunomodulatory activity screening. It
was observed that administration of single dose of different Drug combinations of methanol extract of A. paniculata herb
and methanol extract of stem of T. cordifolia at dose of 200mg/kg, orally did not have any deleterious effects (Table 4).
Table 4: Acute toxicity study for LD50 determination of Drug Combinations (DC-I to DC- V) of methanol extracts
from A. paniculata and T. cordifolia
S.No.
Drug Combination
LD50 Cut-off
Vehicle
1
DC-I (100%)
2000 mg/kg
Tween-80
2
DC-II (75-25%)
2000 mg/kg
Tween-80
3
DC-III (50-50%)
2000 mg/kg
Tween-80
4
DC-IV (25-75%)
2000 mg/kg
Tween-80
5
DC-V (100%)
2000 mg/kg
Tween-80
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 35
Immunomodulatory agents of plant and animal origin increase the immune responsiveness of the body against
pathogens by activating primarily the non-specific immune system i.e. stimulation of the function and efficiency of the
macrophages and other complements. However, these drugs need to be subjected to systematic scientific studies to
substantiate the therapeutic claims made with regard to their utility. The time dependent effect of different Drug
Combinations (DC-I to DC-V) on morphometric functional changes in mice (peritoneal macrophages) were evaluated
where Drug combination (DC-II) treated (200mg/kg b. wt. p.o.) animals showed a very significant increase in the
macrophage count and the maximum number of macrophage cells (8,233±176.38, 9,243±208.17 and 9,891±218.58) were
found to be on the 5th, 10th and 15th days respectively of extract administration as compared to control (3,233±497.77,
3,600±461.88 and 3,233±523.87).
The effects of Drug combination (DC-II) at dose 200mg/kg b.wt. p.o. of AP and TC treatment on morphometric
and functional changes of macrophages in mice showed very significant increase (p<0.01) in the number of macrophage
cells on the 5th, 10th and 15th days of drug administration. Thus it significantly activated macrophages and enhanced their
function as compared to control, suggesting that (DC-II) combination possess potential immunostimulant effect (Table 5,
Figure 1).
Table 5: Effect of Drug Combinations (DC-I to DC-V) on morphometric and functional changes of macrophage in
male albino mice
Macrophage count(Cell/mm3)
Days after Drug Combinations (DC-I to DC-V) (200mg/kg. P.O.)
Day 5th
Day 10th
Day 15th
Control Group
3,233±497.77
3,600±461.88
3,233±523.87
DC-I Group
6,033±185.59*
6,766±352.77*
7,600±305.5*
DC-II Group
8,233±176.38**
9,243±208.17**
9,891±218.58**
DC-III Group
7,366±233.33*
8,000±57.73*
8,533±233.33*
DC-IV Group
5,266±371.18
5,733±348.01
6,433±284.80*
DC-V Group
5,966±120.19
6,500±360.56*
7,333±240.37*
Values are expressed as mean±SEM, (n = 4), Comparison of Group I (Control) was made with all groups.
**p<0.01 Very Significant compared to control group (ANOVA followed by Dunnett’s test). *p<0.05 Significant compared
to control group (ANOVA followed by Dunnett’s test).
Figure 1: Effect of Drug Combinations (DC-I to DC-V) on morphometric and functional changes of macrophage in
male albino mice.
Cyclophosphamide is modified nitrogen mustard which is converted in the organism from a non-reactive to a
highly reacting form. Alkylation agents are thought to act by combining directly with certain intracellular molecules,
particularly nucleic acid and proteins. Thus such molecules are denatured by formation of intracellular cells. It blocks the
conversion of a precursor population (possibly small lymphocytes) to lyphoblasts. The thymus dependent lymphocytes are
preferentially affected by cyclophosphamide.
0
2000
4000
6000
8000
10000
12000
Control Group DC-I Group DC-II Group DC--III Group DC-IV Group DC-V Group
Cell/mm3
Days after Drug Combinations
5 Days after Drug Combinations (DC-I-V) (200mg/kg. P.O.)
10 Days after Drug Combinations (DC-I-V) (200mg/kg. P.O.)
15 Days after Drug Combinations (DC-I-V) (200mg/kg. P.O.)
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 36
The findings of present studies on cyclophosphamide induced immunosuppression model reveals that methanol
extracts of both the chosen drugs i.e. A. paniculata and T. cordifolia on Anti-SRBC-hemagglutination antibody titre in the
Drug combination-DC-II treated cyclophosphamide induced rats was found to be (9.49±3.74) as compared to
cyclophosphamide induced control rats (1.96±0.18). The Anti-SRBC-hemagglutination antibody titre in the control rats
was (4.32±2.64). The suppressive effect of cyclophosphamide was protected by animals treated with Drug combination-
DC-I to DC-V and result revealed that administration of Drug combinations of AP and TC could stimulate the haemopoetic
system. It shows that the Drug combination-DC-II very significantly (P<0.01) protected cyclophosphamide induced
humoral immunosuppression in rats as compared to control groups. This suggested a significant potentiating action of
Drug combination (DC-II) on humoral immunity, as plant extracts significantly protected cyclophosphamide induced
humoral immunosuppression in rats (Table 6, Figure 2).
Table 6: Effect of Drug combinations (DC-I to DC-V) on hemagglutination Antibody titre in Normal and
Cyclophosphamide (CYMP, 100mg/kg) induced immunodeficient rats
Group
Treatment
Value
A
–ve Control (SRBC + CYMP)
1.96±0.18
B
+ve Control (SRBC)
4.32±2.64
C1
Drug combination (DC-I+SRBC+CYMP)
7.34±3.22*
C2
Drug combination (DC-II+SRBC+CYMP)
9.49±3.74**
C3
Drug combination (DC-III+SRBC+CYMP)
7.32±2.89*
C4
Drug combination (DC-IV+SRBC+CYMP)
6.80±2.21*
C5
Drug combination (DC-V+SRBC+CYMP)
6.19±2.03*
Values are expressed as mean±SEM, (n = 4), Comparison of Group I (Control) was made with all groups.
**p<0.01Very Significant compared to control group (ANOVA followed by Dunnett’s test). *p<0.05 Significant compared
to control group (ANOVA followed by Dunnett’s test).
Figure 2: Effect of Drug combinations (DC-I to DC-V) on hemagglutination Antibody titre in Normal and
Cyclophosphamide (CYMP, 100mg/kg) induced immunodeficient rats.
Neutrophil Adhesion Test is an indicative of the marginalization of phagocytic cells in the blood vessels, i.e. an
indication of immunostimulation. As per the present findings on neutrophil adhesion, the % neutrophil adhesion in control
group animals was 32.12, whereas in Drug combinations (DC-I,III,IV,V) treated group animals, at dose of 200mg/kg body
weight, it was 39.84, 38.30, 37.50 and 35.91 respectively, while for Drug combination (DC-II) treated group animals at
dose of 200mg/kg body weight, it was 54.76. The treatments showed very significant (P<0.01) increase at a dose of
200mg/kg body weight in Drug combination-DC-II treated group animals as compared to control, proving the
immunostimulant action of the Drug combination (DC-II) of methanol extracts of AP and TC (Table 7, Figure 3 ).
0
2
4
6
8
10
12
A (–ve
Control) B (+ve
Control) C1 (DC-I) C2 (DC-II) C3 (DC-III) C4 (DC-IV) C5 (DC-V)
Group
Hemagglutination antibody titre
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 37
Table 7: Effect of Drug combinations (DC-I to DC-V) on Neutrophil Adhesion in rats
S. No.
Group
TLC(103/mm3) (A)
Neutrophil % (B)
Neutrophil Index
(A X B) = (C)
Neutrophil
Adhesion (%)
UB
FTB
UB
FTB
UB
FTB
1
Control
3.7±0.05
2.7±0.05
38.3±0.66
35.6±0.66
141.7±2.42
96.2±0.87
32.12
2
DC-I
3.7±0.02
2.7±0.04
38.3±0.33
31.6±0.66
141.8±2.51
85.3±0.36
39.84*
3
DC-II
4.6±0.06
3.1±0.05
48.6±0.33
32.6±0.57
223.5±2.81
101.1±1.52
54.76**
4
DC-III
5.7±0.05
4.8±0.33
51.3±0.57
37.6±0.57
292.4±1.87
180.4±2.21
38.30*
5
DC-IV
3.9±0.05
2.9±0.04
39.6±0.33
33.3±0.57
154.4±1.07
96.5±0.66
37.50*
6
DC-V
5.1±0.05
4.2±0.05
52.3±0.33
40.6±0.33
268.7±1.32
172.2±2.31
35.91*
Values are expressed as mean±SEM, (n = 4), Comparison of Group I (Control) was made with all groups.
**p<0.01Very Significant compared to control group (ANOVA followed by Dunnett’s test). *p<0.05 Significant compared
to control group (ANOVA followed by Dunnett’s test).
Figure 3: Effect of Drug combinations (DC-I to DC-V) on Neutrophil Adhesion in rats
Hemagglutination reaction: The antigen antibody reaction results in agglutination. The relative strength of an
antibody titer is defined as the reciprocal of the highest dilution which is still capable of causing visible agglutination. The
augmentation of humoral response by DC-I to DC-V, as evidenced by an enhancement of antibody responsiveness to
SRBC in rats, indicated the enhanced responsiveness of macrophages and B lymphocytes subsets involved in antibody
synthesis. Agglutination tests can be used to measure the level of antibodies to particulate antigens. In this test, serum
containing antibodies was collected from animals of each group and serial dilutions were done in microtiter plate. For
present study on Hemagglutinating Antibody (HA) titer, the animlas were treated with different Drug combinations (DC-I
to DC-V) for 14 more days and blood samples were analyzed from each rat on day 15 for HA titre where values were
very significantly (P<0.01) increased at the dose of 200mg/kg body weight for Drug combination (DC-II) (8.59±0.221)
as compared to control (3.58±0.077), suggesting that 14 days pretreatment of drug combination-II of AP and TC was
capable to enhance responsiveness of macrophages and lymphocytes inbolved in antibody synthesis and showed possible
immunostimulant action of the Drug combination-DC-II of methanol extracts of AP and TC (Table 8, Figure 4).
Table 8: Effect of Drug combinations (DC-I to DC-V) on HA Titre to antigenic challenge by Sheep RBCs in rats
Group
H A Titre
Control
3.58±0.077
DC-I
6.90±0.233*
DC-II
8.59±0.221**
DC-III
6.42±0.231*
DC-IV
5.90±0.211*
DC-V
5.20±0.201*
Values are expressed as mean± SEM, (n = 4), Comparison of Group I (Control) was made with all groups.
**p<0.01Very Significant compared to control group (ANOVA followed by Dunnett’s test). *p<0.05 Significant compared
to control group (ANOVA followed by Dunnett’s test).
0
40
80
120
160
200
240
280
320
Control DC-I DC-II DC-III DC-IV DC-V
Neutrophil Index (UB) Neutrophil Index (FTB) Neutrophil Adhesion (%)
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 38
Figure 4: Effect of Drug combinations (DC-I to DC-V) on HA Titre to antigenic challenge by Sheep RBCs in rats
In the present investigation, DTH reaction was used to study the effect of Drug combinations (DC-I to DC-V) on
cell mediated immunity, using SRBC as an antigen. On 28th day after 24h of challenge in the control group animals, paw
edema was 2.03±0.141while in Drug combinations (DC-I to DC-V) treated group animals at dose of 200mg/kg body
weight, it was 5.32±0.124, 8.82±0.228, 6.83±0.233, 6.12±0.201and 4.76±0.217respectively . Therefore, the peak edema
after 24h of challenge was the evaluating parameter. The results with Drug combination (DC-II) of AP and TC (200mg/kg
body weight) were statistically very significant (P<0.01) with regard to increase in paw volume compared to control
treatment. Thus, the results obtained with Drug combination (DC-II) treatment concluded that the DC-II is able to
stimulate the macrophages function to stimulate T cells for the hypersensitivity reaction in the immunized rats (Table 9,
Figure 5 ).
Table 9: Effect of Drug combinations (DC-I to DC-V) on DTH response to antigenic challenge by Sheep RBCs in
rats
Group
DTH response (%increase in paw volume)
Control
2.03±0.141
DC-I
5.32±0.124*
DC-II
8.82±0.228**
DC-III
6.83±0.233*
DC-IV
6.12±0.201*
DC-V
4.76±0.217*
Values are expressed as mean±SEM, (n = 4), Comparison of Group I (Control) was made with all groups.
**p<0.01Very Significant compared to control group (ANOVA followed by Dunnett’s test). *p<0.05 Significant compared
to control group (ANOVA followed by Dunnett’s test).
Figure 5: Effect of Drug combinations (DC-I to DC-V) on DTH response to antigenic challenge by Sheep RBCs in
rats
0
1
2
3
4
5
6
7
8
9
10
Control DC-I DC-II DC-III DC-IV DC-V
Group
H. A. Titre
0
1
2
3
4
5
6
7
8
9
10
Control DC-I DC-II DC-III DC-IV DC-V
Group
DTH response (% increase in paw volume)
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 39
Increase in DTH response indicated that DC-II of AP and TC has a stimulatory effect on lymphocytes and
accessory cell types required for the expression of the reaction i.e. cell mediated immunity [40].
4. Discussion
The literature revealed that although when used individually the Andrographis paniculata and
Tinospora cordifolia exhibited immunomodulatory activities, however their immunomodulation potential, if
used in combination, was yet to be evaluated.
A good number of herbal drugs are known to possess immunomodulatory properties and generally act by
stimulating both specific and nonspecific immunity. Many plants used in traditional medicine also have
immunomodulating activities. Some of these stimulate both humoral and cell-mediated immunity, while others activate
only the cellular components of the immune system. Some of these plants also suppress both humoral and cell-mediated
immunity [35]. The plants, Andrographis paniculata (Kalmegh) and Tinospora cordifolia (Giloya) are becoming
increasingly popular for a variety of diseases and infective conditions, primarily influencing the host defence mechanism.
Immunity, both cell (Cellular) and antibody mediated (Humoral) are triggered by antigens. In Cellular responses, CD8+ T
cells proliferate into “killer” T cells and directly attack the invading antigen while in humoral responses, B cells transform
into plasma cells which synthesize and secrete specific proteins called antibodies or immunoglobulins. Antibodies bind to
and inactivate a particular antigen. A cell-mediated immune response begins with activation of a small number of T cells
(Lymphocytes) by a particular antigen. Once a T cell is activated, it can undergo proliferation and differentiation into a
clone of cells, a population of identical cells that can recognize the same antigen and carry out some aspect of the
immune attack. The body contains not only millions of different T cells but also millions of different B cells, each capable
of responding to a specific antigen. Whereas cytotoxic T cells leave lymphatic tissue to seek out and destroy a foreign
antigen. In the presence of a foreign antigen, specific B cells in lymph nodes, the spleen or lymphatic tissue in the
gastrointestinal tract become activated. They differentiate into plasma cells that secrete specific antibodies, which then
circulate in the lymph and blood to reach the site of invasion [36].
5. Conclusion
For catering the need of the hour for management of immune responses, the present study was designed to
evaluate the combined immunomodulation potential of both these highly valued drugs. The present study was focused on
their methanol extracts as they were found to possess important bio-actives with proven immunomodulatory activities.
Subsequent in-depth screening, revealed better degree of
immunomodulatory
activity when methanol extracts of both of
these drugs were mixed/combined together. Although all the five tested combinations (DC-I to DC-V) prepared using
different proportion of methanol extracts of A. paniculata and T. cordifolia (100:Zero, 75:25, 50:50, 25:75 and Zero:100)
revealed to modulate good level of activity on chosen experimental models, the combination DC-II showed best
immunomodulatory activity. After arriving to a conclusion that combination DC-II exhibited best immunostimulant
potential, as proposed, from commercial point of view so-that the formulation can be explored and exploited for the
benefit of the people suffering from immunity related disorders.
The overall present studies revealed that methanol extracts of both the chosen drugs i.e. A. paniculata and T.
cordifolia reported for possessing immunomodulatory activities, when used in combinations showed higher level of
activity with broader level of spectrum profile.
References
[1] Sagrawat H, Khan Y. Immunomodulatory plants: A phytopharmacological. Pharmacog Revi. 2007; 1: 248-260.
[2] Wanger H. Plant derived natural products with immunostimulatory activity: recent advances. Pure Appl Chemistry
1990; 62(7): 1217-1222.
[3] Dasgupta PSC, Gomes A. Immunopotentiating activity of immune-21: A poly herbal product. Ind J Pharmacol 1998;
30:163-168.
[4] Maffei M. Dietary Supplements of Plant Origin-Nutrition and Health Approach. Taylor and Francis, E-Library; 2003:
18.
[5] Winter. Inter., Herbal Association; 2009: 8-18.
[6] Shen YC, Chen CF, Chiou WF. Andrographolide prevents oxygen radical production by human neutrophils: possible
mechanism(s) involved in its anti-inflammatory effect. Br J Pharmacol 2002; 135 (2): 399-406.
[7] Kapil A, Koul IB, Banerjee SK, Gupta BD. Antihepatotoxic effects of major diterpenoid constituents of Andrographis
paniculata. Biochem Pharmacol 1993; 46 (1): 182-185.
[8] Zhang XF, Tan BK. Antihyperglycaemic and anti-oxidant properties of Andrographis paniculata in normal and
diabetic rats. Clin Exp Pharmacol Physiol 2000; 27 (5-6):358-363.
Yadav R. et al Research Article
IJPR Volume 6 Issue 01 (2016) 40
[9] Sheeja K, Kuttan G. Activation of cytotoxic T lymphocyte responses and attenuation of tumor growth in vivo by
Andrographis paniculata extract and andrographolide. Immunopharmacol Immunotoxicol 2007b; 29 (1): 81-93.
[10] Zhang CY, Tan BKH. Hypotensive activity of aqueous extract of Andrographis paniculata in rats. Clin Exp
Pharmacol Physiol 1996; 23: 675-678.
[11] Nadkarni KM. Indian Materia Medica. Bombay: Popular Prakashan; 1976: 498.
[12] Thatte U M, Chhabria S, Karandikar S M, Dahanukar SA. Protective effects of Indian medical plants against
cyclophosphamide neutropenia. J of Post Grad Medicine 1987; 33: 185-188.
[13] Kulichenko LL, Kireyeva LV, Malyshkina EN. A randomized, controlled study of Kan Jang versus amantadine in the
treatment of influenza in Volgograd. J Herb Pharmcother 2003; (3): 77-92.
[14] Kapil A, Sharma S. Immunopotentiating compounds from Tinospora cordifolia. J Ethnopharmacol 1997; 58: 89-95.
[15] Singh RP, Banerjee S, Kumar PV, Raveesha KA, Rao AR. Tinospora cordifolia induces enzymes of carcinogen/drug
metabolism and antioxidant system, and inhibits lipid peroxidation in mice. Phytomedicine 2006; 13(1-2): 74.
[16] Chopra A, Doiphode V. Ayurvedic medicine. Core concept, therapeutic principles, and current relevance. Med Chin
North Am 2002; 86; 75-89.
[17] Rege NN, Nazareth HM, Bapat RD, Dhanukar SA. Modulation of immunosuppression in obstructive jaundice by
Tinospora cordifolia. Ind J Med Res 1989; 90: 478-83.
[18] Gabhe SY, Tatke PA, Khan TA. Evaluations of the immunomodulatory activity of the methanol extract of Ficus
benghalensis roots in rats. Ind J Pharmacol 2006; 38(4): 271-275.
[19] Thakur M, Bhargava S, Dixit V K. Immunomodulatory activity of. Chlorophytum borivilianum Sant. eCAM
doi:10.1093/eCAM/nel., 2006; 1094.
[20] Savadi RV, Yadav R, Yadav N. Study on immunomodulatory activity of ethanolic extract of Spilanthes acmella Murr.
leaves. Ind J of Nat Prod Resources 2010; 1(2): 204-207.
[21] Ghosal S, Baumic S, Chattopadhayay S. Leishmanicidal Compounds from the Fruits of Piper longum. Phytotherpy
Research 1995; 302-308.
[22] Rao CS, Raju C, Gopumadhavan S, Chauhan BL, Kulkarni RD, Mitra SK. Immunotherapeutic modification by an
ayurvedic formulation Septilin. Ind J of Experimental Biol 1994; 32: 553-561.
[23] Kumar S, Gupta P, Sharma S, Kumar D. A review on immunostimulatory plants. J of Chinese Integrative Medicine
2011; 9(2): 117–128.
[24] Postage J R, Norris J R, Ribbons DW. Methods in microbiology. London: Academic press; 1969: 1, 611.
[25] Wilkonson P C. Neutrophil Adhesion Test. In, Vane J K, Ferreria SH (ed). Hand Book of Experimental Pharmacology.
Berlin: Vol. I, Springer-Verlag, p. 109; 1978.
[26] Mitra SS. Immunomodulatory effect of IM-133. Phytother Research 1999; 13: 341-343.
[27] Claman HN. The biology of the immune response. J Amer Med Assoc 1987; 258(20): 3007.
[28] Thatte U M, Chhabria S, Karandikar S M, Dahanukar SA. Protective effects of Indian medical plants against
cyclophosphamide neutropenia. J of Post Grad Medicine 1987; 33: 185-188.
[29] Vasudevan DM, Sreekumari S. Text book of biochemistry for medical students. New Delhi: Jaypee brother’s medical;
1995: 194.
[30] Hennessey LR, Baker JR. Basic and Clinical immunology. In: Stites DP editor. Immunolmodulators. New Jersey:
8th ed., Lange International, p. 781-783; 1994.
[31] Wireda D, Reasor MJ. Immunomodulating drug. In: Craig CR, Stitzel RE editors. Modern Pharmacology. London:
3rd ed., Littel, Brown and co, p, 821, 832-835; 1986.
[32] Turner MA. Screening Methods in Pharmacology. Academic Press New York; 1965: 26.
[33] Gupta RS Sharma A. Antifertility effect of Tinospora cordifolia (Willd.) stem extract in male rats. Indian J Exp Biol
2004; 41: 885-889.
[34] Ho YT, Yang JS, Lu CC, Chiang JH, Li TC, Lin JJ, Lai KC, Liao CL, Lin JG, Chung JG. Berberine
inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model. Phytomed 2009;
16(9): 887-890.
[35] Stephen H, Comac L. Miracle herbs: How herbs combine with modern medicine to treat cancer, he art disease, AIDS
and more. Kensington Publishing Corporation, New York; 2000.
[36] Kalikar MV, Thawani VR, Varadpande UK, Sontakke SD, Singh RP, Khiyani RK. Immunomodulatory effect of
Tinospora cordifolia extract in human immunodeficiency virus positive patients. Indian J Pharmacol 1998;
40(3):107-110.
[37] Puri A, Saxena R, Saxena R P, Saxena K C. Immunostimulant agents from Andrographis paniculata. J Nat Prod
1993; 56: 995–999.
[38] Reddy MK, Reddy MVB, Gunasekar D, Murthy MM, Caux C, Bodo B. A flavone and an unusual 23-carbon terpenoid
from Andrographis paniculata. Phytochem 2003; 62: 1271-1275.
[39] Matsuda T, Kuroyanagi M, Sugiyama S, Umehara K, Ueno A, Nishi K. Cell differentiation-inducing diterpenes from
Andrographis paniculata Nees. Chem Pharm Bull 1994; 42 (6): 1216-1225.
[40] Kiem PV, Minh CV, Dat NT, Kinh LV, Hang DT, Nam NH, Cuong NX, Huong HT, Lau TV. Aporphine
alkaloids, clerodane diterpenes, and other constituents from Tinospora cordifolia. Fitoterapia 2010; 81(6): 485-
489.
