Journal of Ethnopharmacology 92 (2004) 291295

Anticancer and immunostimulatory compounds

from Andrographis paniculata
R. Ajaya Kumar, K. Sridevi, N. Vijaya Kumar, S. Nanduri, S. Rajagopal
Discovery Research, Dr. Reddys Laboratories, Miyapur, Hyderabad 500050, India
Received 16 April 2003; received in revised form 24 February 2004; accepted 5 March 2004

Abstract
Andrographis paniculata extract is traditionally used as a medicine to treat different diseases in India, China and Southeast Asia. In
the present study, we evaluated the anticancer and immunomodulatory activity of the methanolic extract of Andrographis paniculata in
human cancer and immune cells. The methanolic extract of Andrographis paniculata was fractionated into dichloromethane, petroleum
ether and aqueous extracts and screened for bioactivity. Our results indicate that the dichloromethane fraction of the methanolic extract
retains the active compounds contributing for both the anticancer and immunostimulatory activity. Dichloromethane fraction significantly
inhibits the proliferation of HT-29 (colon cancer) cells and augments the proliferation human peripheral blood lymphocytes (HPBLs) at low
concentrations. On further fractionation of the dichloromethane extract we could isolate three diterpene compounds, i.e. [1] andrographolide,
[2] 14-deoxyandrographolide and [3] 14-deoxy-11,12-didehydroandrographolide. Andrographolide showed anticancer activity on diverse
cancer cells representing different types of human cancers. Whereas all the three molecules showed enhanced proliferation and interleukin-2
(IL-2) induction in HPBLs.
2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Andrographis paniculata; Andrographolide; Anticancer drugs; Cytokines; Immunomodulation

1. Introduction
In recent years focus on use of non-traditional approaches
to treat diseases has been revived all over the world. The evidence collected till now shows immense potential of medicinal plants used in traditional systems (Hoareau et al., 1999).
The use of herbal extracts and nutritional supplements either
as alternative or complimentary medicine to the conventional
chemotherapy for treatment of cancer is well documented
in Ayurveda which is an alternative medical system that
has been practiced primarily in the Indian subcontinent for
5000 years (Dahanukar et al., 2000). Andrographis paniculata (Acanthaceae), also known commonly as kalmegh, is
a well known medicinal plant of Ayurveda and has been used
for centuries in Asia. About 26 different polyherbal formulations of this plant are mentioned in Ayurveda as a popular
remedy for the treatment of various disorders. Andrographis
paniculata is an annual shurb grows abundantly in India and
cultivated extensively in China and Thailand. The aerial parts
of the plant (leaves and stems) are used to extract the active
Corresponding

author. Tel.: +91-40-304-5439; fax: +91-40-304-5438.

E-mail address: Sriramrajagopal@drreddys.com (S. Rajagopal).

phytochemicals. The plant extract is known to contain diterpenes, flavonoids and stigmasterols (Siripong et al., 1992).
Extensive research of the last few decades has revealed that
the herbal extract is useful as an anti-inflammatory (Shen
et al., 2002), antiviral (Chang et al., 1991; Calabrese et al.,
2000), antithrombotic (Zhao and Fang, 1991), anticancer
(Matsuda et al., 1994), immunostimulatory (Puri et al., 1993;
See et al., 2002), hypoglycaemic (Zhang and Tan, 2000) and
hypotensive agent (Zhang and Tan, 1996).
Andrographolide, the major diterpenoid of the Andrographis paniculata extract has shown cytotoxic activity
against KB (human epidermoid carcinoma) and P388 (lymphocytic leukaemia) (Siripong et al., 1992) cells. The
methanol extract of aerial parts of Andrographis paniculata
and some of the isolated compounds showed growth inhibitory and differentiating activity on M1 (mouse myeloid
leukaemia) cells (Matsuda et al., 1994). The ethyl alcohol
extract and purified diterpene andrographolides are reported
to stimulate both antigen specific and non specific immune
responses in mice (Puri et al., 1993). However, no systematic
study has been reported addressing the immunostimulatory
activity of Andrographis paniculata extract in human immune cells. Here we report the immunomodualtory activities

0378-8741/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.03.004

292

R. Ajaya Kumar et al. / Journal of Ethnopharmacology 92 (2004) 291295

of Andrographis paniculata extract and its components

in human immune cells and anticancer activity in human
cancer cells.
2. Materials and methods
2.1. Cell lines
Human cancer cell lines SW620 and A498 were purchased
from the American Type Culture Collection, Manassas, VA,
USA. NCI/ADR-RES, U251, HT29, H522, M14, SKOV3
and DU145 were purchased from the National Cancer Institute, Bethesda, USA. All cells were maintained in RPMI
1640 supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml) and 10% foetal bovine serum. Cells were
incubated at 37 C in a humidified, 5% CO2 atmosphere.
2.2. Reagents
Streptomycin, penicillin G, foetal bovine serum, RPMI
1640, dimethyl sulphoxide (DMSO), sulphorhodamine B
(SRB), trichloroacetic acid, Tris, RNase (DNase free),
propidium iodide, sodium deoxycholate, sodium chloride, Trition X-100, aprotinin, leupeptin, PMSF and
phytohemagglutinin-A (PHA) were purchased from Sigma
Chemicals, USA. 3 H-thymidine was purchased from Amersham Pharmacia, USA.
2.3. Plant material
Fresh whole plant of Andrographis paniculatawas collected from Tirupathi, Andhrapradesh, India in August
1999. Botanical identification was done by Dr. P. Sai
Prasad Goud, taxonomist, Medicinal Plants Division, Dr.
Reddys Research Foundation (DRF) and voucher specimen
(A001/A.P/99) was deposited at the herbarium of DRF,
Hyderabad, India.
2.4. Extraction and initial fractionation
The powdered plant material (3 kg) was extracted with
methanol for 72 h (20 L 3 times). The methanolic extract was concentrated in vacuo to 10 L, diluted with water (10 L) and fractionated into petroleum ether (42.2 g),
dichloromethane (100 g) and water soluble fractions (51 g).

acetone (8:2) were found to contain andrographolide (3 g;

mp 228 C). The mixture of 14-deoxy-11,12-didehydroandrographolide (400 mg; mp 203 C) and 14-deoxyandrographolide (125 mg; mp 168 C) was separated by repeated
chromatography with mixture of chloroform and methanol.
The above three compounds were identified with the help
of spectral data and comparison with that reported in the
literature (Matsuda et al., 1994).
2.6. Cell growth assay
Cells undergoing exponential growth were seeded on
a 96-well cell culture plates at a concentration of 10 000
cells per well and incubated at 37 C in a CO2 incubator.
Twenty-four hours later cells were treated with different
concentrations of extracts or pure compounds dissolved
in DMSO to a final concentration of 0.05% in the culture
medium and incubated for 48 h. Cells were fixed by adding
ice-cold 50% trichloroacetic acid (TCA) and incubating
for 1 h at 4 C. The plates were washed with distilled water, air-dried and stained with SRB solution (0.4%, w/v, in
1% acetic acid) for 10 min at room temperature. Unbound
SRB was removed by washing thoroughly with 1% acetic
acid and the plates were air-dried. The bound SRB stain
was solubilized with 10 mM Tris buffer, and the optical
density was read on a spectrophotometric plate reader at
a single wavelength of 515 nm. At the time of drug addition, a separate reference plate for cell growth at time 0 h
(the time at which drugs were added) was also terminated
as described above. The percentage growths were calculated and the GI50 values were calculated from the growth
curves.
2.7. Isolation of peripheral blood lymphocytes
Venous blood was collected aseptically from healthy
donors in preservative free heparin tubes. The blood was
diluted 50% with phosphate buffered saline (PBS), pH 7.4
and layered onto Ficoll plus (Amersham). After centrifugation at 1500 rpm for 25 min the mononuclear cells were
collected at the interface and washed thrice with the PBS.
The cells were resuspended in RPMI 1640 medium with
10% foetal bovine serum and antibiotics.
2.8. Peripheral blood lymphocytes proliferation and IL-2
secretion

2.5. Isolation and purification of diterpenes

A portion of the dichloromethane soluble fraction (12 g)
was chromatographed on a column of silica gel (240 g)
eluted with chloroform and acetone mixture in order of increasing polarity. Fractions (250 ml each) were collected and
combined according to similar TLC pattern. Fractions 1015
of chloroformacetone (9:1) was found to be mixture (1 g) of
compounds 14-deoxy-11,12-didehydroandrographolide and
14-deoxyandrographolide. Fractions 2030 of chloroform

Proliferation of peripheral blood lymphocytes was measured as 3 H-thymidine incorporation into the newly synthesized DNA. Briefly, 1 105 cells per well in a 96-well
plate were stimulated with 5 mg/ml of PHA. After 24 h of
culturing at 37 C in a humidified CO2 incubator, different dilutions of extracts or pure compounds were added to
respective wells. Control wells received only medium containing vehicle (DMSO). The cultures were pulsed with
3 H-thymidine (0.5 Ci/well) 24 h prior to termination. After

R. Ajaya Kumar et al. / Journal of Ethnopharmacology 92 (2004) 291295

293

Fig. 1. Effect of extracts on proliferation of (A) cancer cells: HT-29 (human colon cancer) cells were incubated in complete medium with and without
different concentrations of extracts for 48 h and the percentage growth was determined by SRB method as described in Section 2 and (B) HPBLs:
PHA-stimulated HPBLs were treated with extracts for 48 h at 2.5 g/ml concentration and assayed for cellular proliferation by 3 H-thymidine incorporation
as described in Section 2.

48 h of drug addition the cells were harvested onto unifilter

plates (Packard) and incorporated thymidine was counted
using Topcount (Packard). The proliferation of PBLs was
expressed as percentage stimulation index (SI) compared to
untreated controls.
The peripheral blood lymphocytes were treated with andrographolide as described in proliferation assay. At the end
of the assay culture supernatants were collected and stored
at 70 C until use. IL-2 levels were estimated in the stored
supernatants as per manufacturers protocol using commercially available DUOSET ELISA kits from R&D systems,
USA.

3. Results and discussion

Some phytochemicals present in medicinal plants
have been reported to possess substantial antioxidant,
anti-mutagenic and anti-carcinogenic activities and are being extensively explored for their potential for treatment
and prevention of cancer (Kucuk, 2002). The well-known
compounds like paclitaxel, a diterpenoid from Taxus brevifolia, and vincristine, an alkaloid from Catharanthus roseus,
have been proved as antineoplastic drugs in clinic (Wall
and Wani, 1996; Neuss et al., 1975). The aerial parts (stems
and leaves) of the plant Andrographis paniculata have been
used traditionally as medicine to treat a broad range of diseases, including diabetes, hypertension, and cancer. Also,
Andrographis paniculata extract was reported to stimulate
both antigen specific and nonspecific immune system in
mice. However, to our knowledge the immunomodulatory
and anticancer activity of this plant extract and its components in human cells is not well defined. In the present
study, we evaluated the anticancer and immunomodulatory activity of Andrographis paniculata extract in human

cancer and immune cells to identify a novel scaffold for

further analog synthesis.
The anticancer activity of the extracts was evaluated by
incubating the HT-29 (colon cancer) cells with different
dilutions of the extracts for 48 h. As shown in Fig. 1A,
at 10 g/ml concentration the methanolic extract inhibited the proliferation HT-29 by 50%. The petroleum ether
and dichloromethane extracts inhibited the proliferation of
HT-29 cells with a GI50 value of 46 and 10 g/ml respectively. The aqueous extract did not inhibit the proliferation
of HT-29 cells. Immunomodulatory activity was assessed
by testing the effect of the extracts on HPBLs proliferation.
As shown in Fig. 1B, at a concentration of 2.5 g/ml the
methanol extract showed 18% increase in HPBLs proliferation. Among the three fractions, dichloromethane fraction
significantly enhanced the HPBLs proliferation by 52%.
The petroleum ether extract and the aqueous extract showed
18 and 4% increase of HPBLs proliferation, respectively.
These results reveal that the active compounds contributing for most of the anticancer and immunostimulatory
activity of the methanol extract are concentrated in the
dichloromethane extract.
Hence, to isolate and identify the pure active compounds
responsible for the bioactivity, we further fractionated the
dichloromethane extract and isolated three diterpene compounds, i.e. [1] andrographolide, [2] 14-deoxyandrographolide and [3] 14-deoxy-11,12-didehydroandrographolide. The
structures of these molecules were confirmed by spectral
analysis and shown in Fig. 2. Apart from these compounds
Andrographis paniculata extract is reported to contain other
constitiuents like Deoxyandrographolide 19-d-glucoside
(Chem and Xiaotion, 1982) homoandrographolide, andrographan and andrographosterin as minor constituents
(unpublished). All the three compounds were screened on
diverse cancer cells representing different types of cancers

294

R. Ajaya Kumar et al. / Journal of Ethnopharmacology 92 (2004) 291295

14
HO

14

14
O

12

HO
HO

11

HO

HO

Andrographolide

HO

HO

14-Deoxyandrographolide

12

14-Deoxy-11, 12didehydroandrographolide

Fig. 2. Structures of compounds isolated from dichloromethane extract.

and the results are summarized in Table 1. Among the three

molecules isolated andrographolide inhibited the proliferation of cancer cells with GI50 values ranging from 10 to
28 M. 14-Deoxyandrographolide showed moderate inhibition on proliferation of NCI/ADR-RES and A498 cells
and did not inhibit the proliferation of other cancer cells.
14-deoxy-11,12-didehydroandrographolide did not inhibit
the proliferation of any cancer cell line tested. These data
suggests that the anticancer activity shown by the plant extract is mainly due to the presence of andrographolide and
the other two compounds are not contributing significantly
for the anticancer activity. Our results are in agreement with
the earlier reports, where Siripong et al. (1992) demonstrated that the cytotoxic activity of andrographolide against
KB (human epidermoid carcinoma) and P388 (lymphocytic
leukaemia) cells. The growth inhibitory and differentiating
activity of the methanol extract of aerial parts of Andrographis paniculata and some of the isolated compounds
on M1 (mouse myeloid leukaemia) cells was also reported
(Matsuda et al., 1994).
In case of immunomodulatory activity, as shown in
Fig. 3A, at 1 M concentration all the three compounds
showed moderate increase in HPBLs proliferation. The andrographolide, 14-deoxy-11,12-didehydroandrographolide
and 14-deoxyandrographlide increased HPBLs proliferation
by 14, 5 and 7%, respectively. The dose response analysis of these three compounds for IL-2 induction showed that
all the three compounds could enhance the IL-2 induction in

HPBLs (Fig. 3B). Andrographolide showed more IL-2 induction when compared to 14-deoxy-11,12-didehydroandrographolide and 14-deoxyandrographlide. The difference
among the three compounds is that andrographolide at
higher concentrations showed cytotoxicity towards HPBLs, while the other two did not show any signs of toxicity. The pure compounds are less potent compared to
dichloromethane or methanolic extract in terms of their
immunomodulatory activity, which suggests that molecules
other than these diterepenes may also contribute for the immunostimulation, or the synergistic interaction among the
different components of the extract is more active. Puri et al.
(1993) also reported that Andrographis paniculata extract
stimulates both antigen specific and non-specific immune
system in mice and the whole extract is more active than
individual compounds.
From these two results it is evident that the major constituent andrographolide shows anticancer and immunostimulatory activities. The in vivo results from hollow fiber
assay conducted in immunocompetent Swiss albino mice,
demonstrated that andrographolide significantly inhibits
the cancer cell proliferation without showing any signs
of toxicity (data not shown) in mice even at high doses.
Although 14-deoxy-11,12-didehydroandrographolide and
14-deoxyandrographlide did not show in vitro anticancer activity, studies are in progress to check whether the observed
in vitro immunostimulatory activity of these compounds
will result in any objective in vivo anticancer activity

Table 1
Effect of andrographolides on growth of human cancer cells
Cell line

Cancer type

Andrographolide

14-Deoxy-11,12-didehydro andrographolide

14-Deoxy andrographolide

NCI/ADR-RES
U251
SW620
H522
M14
SKOV3
DU145
A498

Breast
CNS
Colon
Lung
Melanoma
Ovarian
Prostate
Renal

15
10
11
16
11
18
12
28

30
>100
>100
80
60
>100
>100
40

>100
>100
>100
>100
>100
>100
>100
>100

Human cancer cell lines representing different types of cancers were incubated in complete medium with and without test compounds for 48 h and the
percentage growth determined by SRB method as described in Section 2. Percentage growth of the treated cells was calculated compared to the control
untreated cells and the concentration required to inhibit the 50% growth (GI50 concentration). The numbers represent the GI50 values in micromolar
concentration.

R. Ajaya Kumar et al. / Journal of Ethnopharmacology 92 (2004) 291295

295

Fig. 3. PHA-stimulated HPBLs were treated with test compounds for 48 h at 1 M concentration and assayed for cellular proliferation. IL-2 induction
was measured at different concentrations after 48 h of treatment with test compounds as described in Section 2. (A) Proliferation by 3 H-thymidine
incorporation, (B) IL-2 levels.

indirectly through stimulation of host immune system in

terms of tumor growth inhibition, preventing the metastasis
and increasing survival time. We conclude that, owing to
its potent anticancer and immunostimulatory activities, the
diterpenoid andrographolide, can serve as a scaffold for
design and synthesis of novel, potent, non-toxic anticancer
and/or immunomodulatory molecules.

Acknowledgements
We thank Ch. Mahender, T. Srikanth and S. Siva sanjeeva
rao for their technical help in this study. We also thank Dr.
A. Venkateswarlu and R. Rajagopalan for their interest in
the present work.

References
Calabrese, C., Berman, S.H., Babish, J.G., Ma, X., Shinto, L., Dorr,
M., Wells, K., Wenner, C.A., Standish, L.J., 2000. A phase I trial
of andrographolide in HIV positive patients and normal volunteers.
Phytotherapy Research 14, 333338.
Chang, R.S., Ding, L., Chen, G.Q., Pan, Q.C., Zhao, Z.L., Smith, K.M.,
1991. Dehydroandrographolide succinic acid monoester as an inhibitor
against the human immunodeficiency virus. Proc. Soc. Exp. Biol. Med.
197, 5966.
Chem, W., Xiaotion, L., 1982. Deoxyandrographolide 19-d-glucoside
from the leaves of A. paniculata. Planta Medica 15, 245246.
Dahanukar, S.A., Kulkarnu, R.A., Rege, N.N., 2000. Pharmacology of
medicinal plants and natural products. Indian Journal of Pharmacology
32, S81S118.

Hoareau, L., Edgar, J., DaSilva, 1999. Medicinal plants: a re-emerging

health aid. Electronic Journal of Biotechnology 2, 5670.
Kucuk, O., 2002. New opportunities in chemoprevention research. Cancer
Investigation 20, 237245.
Matsuda, T., Kuroyanagi, M., Sugiyama, S., Umehara, K., Ueno, A., Nishi,
K., 1994. Cell differentiation-inducing diterpenes from Andrographis
paniculata Nees. Chemical and Pharmaceutical Bulletin, Tokyo 42,
12161225.
Neuss, N., Barnes, A.J., Huckstep, L.L., 1975. Vinca alkaloids XXXV.1.
Desacetoxyvinblastine a new minor alkaloid from Vinca rosea L.
(Catharanthus roseus G. Don). Experientia 31, 1820.
Puri, A., Saxena, R., Saxena, R.P., Saxena, K.C., Srivastava, V., Tandon,
J.S., 1993. Immunostimulant agents from Andrographis paniculata.
Journal of Natural Products 56, 995999.
See, D., Mason, S., Roshan, R., 2002. Increased tumor necrosis factor
alpha (TNF-alpha) and natural killer cell (NK) function using an
integrative approach in late stage cancers. Immunol. Invest. 31, 137
153.
Shen, Y.C., Chen, C.F., Chiou, W.F., 2002. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s)
involved in its anti-inflammatory effect. British Journal of Pharmacology 135, 399406.
Siripong, P., Kongkathip, B., Preechanukool, K., Picha, P., Tunsuwan, K.,
Taylor, W.C., 1992. Cytotoxic diterpenoid constituents from Andrographis paniculata Nees leaves. J. Sci. Soc. Thailand 18, 187194.
Wall, M.E., Wani, M.C., 1996. Camptothecin and taxol: from discovery
to clinic. Journal of Ethnopharmacology 51, 239253.
Zhang, X.F., Tan, B.K., 2000. Antihyperglycaemic and anti-oxidant properties of Andrographis paniculata in normal and diabetic rats. Clin.
Exp. Pharmacol. Physiol. 27, 358363.
Zhang, C.Y., Tan, B.K., 1996. Hypotensive activity of aqueous extract of
Andrographis paniculata in rats. Clinical Experiments in Pharmacology
and Physiology 23, 675678.
Zhao, H.Y., Fang, W.Y., 1991. Antithrombotic effects of Andrographis
paniculata Nees in preventing myocardial infarction. Chin. Med. J.
(Engl.) 104, 770775.