African Journal of Biotechnology Vol. 7 (2), pp.

081-085, 18 January, 2008

Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2008 Academic Journals

Full Length Research Paper

Enhanced Phagocytosis and Antibody Production by

Tinospora cordifolia - A new dimension in
Immunomodulation
M. S. Ranjith1*, A.J.A. Ranjitsingh2, S. Gokul Shankar3, G. S. Vijayalaksmi5, K. Deepa5 and
Harcharan Singh Sidhu1
1
Microbiology Unit, AIMST University, Kedah, Malaysia.
2
Department of Advanced Zoology and Biotechnology, Sri Paramakalyani College, Alwarkurichi, India.
3
R and D Center, Cholayil Private Limited, Chennai, India.
4
Sri Paramakalyani Center for Environmental Sciences, M.S. University, Tirunelveli, India.
5
Anatomy Unit, AIMST University, Kedah, Malaysia.
Accepted 18 December, 2007

Tinospora cordifolia (guduchi) is a widely used shrub in ayurvedic systems of medicine known to
possess immunomodulatory properties. In the present study the aqueous extract of T. cordifolia was
found to enhance phagocytosis in vitro. The aqueous and ethanolic extracts also induced an increase
in antibody production in vivo.

Key words: Immunomodulation, phagocytosis assay, Tinospora cordifolia.

INTRODUCTION

One of the most important components of immune ability of macrophages and increase the antibody pro-
system in the initial stages of the defense is phagocytosis duction by B cells have been well documented by several
and it is an innate response. Phagocytic cells include workers (Chopra et al., 1956; Kirtikar and Basu 1975;
neutrophils, eosinophils, macrophages and monocytes Nadkarni et al., 1976; Chopra et al., 1982; Zhao et al.,
that recognize foreign substances and invading microor- 1991). In the system of alternative medicine several
ganisms. These cells engulf and destroy the foreign plants are reported to have immune boosting property.
substances with their intracellular killing mechanisms. Among them, Tinospora cordifolia locally known as
When the innate immune response fails the next level of guduchi is of great interest for several researchers (Rege
defense is provided by B cells (antibody mediated et al., 1993; Thatte et al., 1994; Gunabakshi et al., 1998).
immune response) and T cells (cell mediated immune The present study was aimed at evaluating the effect of
response). Any means by which these defense systems the above plant in phagocyte mediated immunity and
can be catalyzed/enhanced will prove to boost the overall antibody production through in vitro and in vivo studies.
immune response and well being of the host (Gottlieb et
al., 1987).
MATERIALS AND METHODS
Since ancient times, people have used plants for their
healing, preventive, curative, rejuvenative and immuno- Collection of plant
modulating properties (Swerdlow, 2000). In the recent
years, this ancient knowledge has gained global accept- T. cordifolia was locally collected and got authenticated at the R
and D Center, Cholayil Private Limited, Chennai, India.
ance. Use of plant compounds to enhance the phagocytic
Extraction procedure

Non-destructive cold process extraction was employed using stem

*Corresponding author. E-mail: [email protected]. Phone: powder of T. cordifolia with ethanol, ethyl acetate and chloroform.
+6 016 4083740. For aqueous extraction, heat distillation process was used. The
082 Afr. J. Biotechnol.

crushed semi-dried stem powder of T. cordifolia was soaked sepa- Appropriate controls were maintained.
rately in ethanol, ethyl acetate and chloroform in a ratio of 1:5 Two hundred macrophages were scanned from each of the test
solute versus solvent in a conical flask. The entire set up was kept and control smears. The cells with ingested candida were counted.
at room temperature for 24 h with intermittent shaking. After 24 h, This was expressed as percentage phagocytosis. While counting
the mixture was filtered through Whattman No.1 filter paper and the these macrophages with phagocytosed candida, a record of all
filtrate was dried to evaporate the solvent. The extract settled at the intracellular forms of candida was maintained. Three forms were
bottom was used for the experiment at varying concentration. The observed; (a) yeast cells with homogenous blue cytoplasm, (b)
concentrated extracts were dissolved in dimethyl sulfoxide (DMSO) yeast cells with partially or completely decolorized cytoplasm (ghost
and used for further tests. In the case of aqueous extraction the cells), and (c) filamentous forms which consist of blue staining yeast
stem powder was boiled in water at 1:5 ratio at 100°C for 30 min cell with one (rarely two) bud, denoting germination within the
(Sonia et al., 2000). After 30 min the mixture was filtered and the macrophage. With the given incubation length, the filamentous
filtrate was stored in a refrigerator until use. forms constituted less than 8% of total intracellular forms. The
"ghost cells" were considered as killed/digested organisms by
macrophages. The intracellular killing capacity (ICK) was expressed
Phagocytosis assay as the number of ghost cells present per 100 phagocytosed
candidae (Rege and Dahanukar, 1993).
Preparation of macrophage suspension

Peritoneal exudate was collected from the peritoneal cavity of Antibody production
anaesthetized rats (Vanfurth et al., 1978). The fluid was transferred
to a sterile test tube and centrifuged at 500 rpm for 10 min. After Wistar rats (150 - 200 g) were used for the experiment. Animals
removing the supernatant, the pellet was resuspended in 1 ml of were housed under standard conditions of temperature (23±1°C),
cold distilled water along with 1 ml of minimum essential medium 12 h light/dark cycle and fed with standard pellet diet (Gold Muhor,
(MEM; Hi-media) to destroy the neutrophils. This suspension was Lipton India Ltd.) and water ad libitum. The animals were divided
centrifuged again for 10 min, the supernatant was removed and the into 4 groups consisting of six animals in each group was treated
pellet was resuspended in 2 ml of MEM. Total and differential separately with 10 mg/kg body weight of each of the four extracts
counts of the peritoneal exudate cells were carried out. Recovery of chosen for the study. Two animals were maintained as control. The
macrophages was approximately 85 - 90%. Viability of cells was extracts at above concentrations were prepared in 5 ml of water
determined using a dye exclusion technique and was found to be and were fed orally to each animal for 14 days.
more than 96%. The final concentration was adjusted to 105 cells After 14 days of treatment the Sheep Red Blood Cells (SRBC)
per ml (Cline, 1973). (collected in Alsever's solution, were washed three times in large
volumes of pyrogen free 0.9% normal saline) at a concentration of
0.5 x 109 cells/ml was injected. The antibody produced against
Preparation of yeast cell suspension SRBC in each extract treated group and control was estimated by
micro-agglutination test. An equal volume of 0.6% washed SRBC
Candida albicans used in the present study is a clinical isolate from was added to doubling dilutions of sera made in microtiter trays,
patient with acute vaginal candidiasis (Obtained from Department of and the pattern of agglutination was read after the trays had stood
Microbiology, The New College, Chennai). This strain was grown in at room temperature overnight. For the titration of 2-mercapto-
the medium for growth of yeast phase (MGYP). This medium is ethanol (2-ME) – resistant hemagglutinin, sera were incubated with
composed of 0.3% malt extract, 1% glucose, 0.3% yeast extract equal volumes of 0.1 M 2-ME in phosphate-buffered saline at 37°C
and 0.5% peptone adjusted to pH 6.4 - 6.8. After incubation in this for 1 h before the addition of SRBC.
medium for 24 h at room temperature (28°C - 32°C), the organisms
entered the yeast phase. The cells were washed twice in 0.9%
saline. The yeast characteristics of C. albicans and their viability RESULTS AND DISCUSSION
were checked and verified with a haemocytometer. For this pur-
pose, 0.5 ml of 0.3% trypan blue and 0.1% eosin (3:1) were added Aqueous extract of T. cordifolia exhibited boosting of
to 0.5 ml of the washed cell suspension, mixed well for 10 min and
the hemocytometer chamber was filled with this mixture. The yeast
phagocytic ability of macrophage in vitro at 5 µg/ml. The
cells looked as single spherical cells or attached doublets; the above extract did not show any activity at 1 µg/ml and the
filamentous forms were absent and 98% of yeast cells were viable, activity was constant above 5 up to 20 µg/ml. Ethanolic,
as shown by their ability to exclude the dye (Lehrer et al., 1975). chloroform and ethyl acetate did not show any activity up
The concentration was then adjusted to 108 yeast cells/ml (Cline, to 20 µg/ml and was found to be comparable to the
1973).
control (Table 1). Treatment with the aqueous and
ethanolic extracts of T. cordifolia at 10 mg/kg body weight
Assay system for 14 days significantly increased the antibody produc-
tion against SRBC in animals when compared to control.
The assay system consisted of 0.1 ml of C. albicans suspension,
0.3 ml of macrophage suspension, 0.3 ml of MEM and 0.1 ml of
Other extracts of the above plant did not show activity
pooled serum obtained from normal volunteers with blood group (Figures 1 and 2).
AB. These quantities were selected to facilitate optimal pelleting of Medicinal plants and their compounds have a special
organisms with macrophages. The tubes containing the assay place in the maintenance of health and well-being of
system were incubated at 37°C in a carbon dioxide environment for humans as well as used as therapeutic purposes
90 min with the different solvent extracts and aqueous preparation (Gerzon, 1980; Jardine, 1980; Kinghorn and Balandrin
of the plant at different concentrations; 1, 5, 10, 15 and 20 µg/ml.
Smears were prepared using the cytocentrifuge, stained with
1993; Wall and Wani 1993; Cragg et al., 1997). T.
Giemsa and examined by light microscopy under oil-immersion. cordifolia has been extensively used in the treatment of
 Ranjith et al. 083

Table 1. Effect of Tinospora cordifolia in boosting phagocytosis against Candida albicans.

Concentration (µg/ml)/the ratio of phagocyte vs. ingested cells (mean ± SD)

Extract 1 5 10 15 20 Control
Aqueous 1:5 ± 0.06 1:18 ± 0.8 1:19 ± 0.9 1:17 ± 1 1:20 ± 0.8
Ethanol 1:4 ± 0.4 1:6 ± 0.5 1:5 ± 0.5 1:8 ± 0.6 1:5 ± 0.9
1:6 ± 0.4
Ethyl acetate 1:5 ± 0.06 1:7 ± 0.08 1:7 ± 0.07 1:6 ± 0.04 1:5 ± 0.05
Chloroform 1:4 ± 0.06 1:6 ± 0.07 1:4 ± 0.06 1:5 ± 0.05 1:7 ± 0.05

Figure 1. Effect of aqueous and ethanolic extracts of T. cordifolia in antibody production.

various types of haematological, hepatic, neurological, al., 1999; Dikshit et al., 2000; Manjrekar et al., 2000). It
diabetic and inflammatory conditions (Bhavmishra, 2002). has been also observed that extracts of several medicinal
The immunomodulatory and anti-inflammatory property of plants stimulate the macrophages (Broker and Bhatt
T.cordifolia has been well documented (Tripathi et al., 1953, Atal et al., 1986, Thatte et al., 1994). Interestingly
1997; Bishayi et al., 2002; Kasture et al., 2001; in our study, none of the other extracts of the same plant
Subramanian et al., 2002). The present study was aimed showed above activity. We presume that active com-
at the scientific validation of the immunomodulatory pounds responsible for enhancing phagocytosis in T.
properties of T. cordifolia. cordifolia may be highly polar in nature and hence
In our study, we found that the aqueous extract of T. present in the aqueous extract. Previous authors have
cordifolia was effective in boosting phagocyte mediated reported similar findings that most of the therapeutic
immune response in vitro. The extract at a concentration compounds in plants are polar in nature and require polar
of 5 µg/ml showed 200% increase in phagocytic ability of solvents for their extraction. However, the ethanolic
macrophages as compared to control. T. cordifolia is extract failed to record significant activity in our present
reported to benefit the immune system in a variety of study. The active principle in T. cordifolia includes
ways (Rege et al., 1993; Nagarkatti et al., 1994; Kapil alkaloids, glycosides, diterpinoid lactones, steroids, ses-
and Sharma 1997). quiterpenoids, phenolics and aliphatic compounds that
The alcoholic and aqueous extracts of T. cordifolia may be responsible for the activity (Singh et al., 2003).
have been tested successfully for immunomodulatory To understand whether this plant possesses any other
properties (Thatte et al., 1987; Dahanukar et al., 1988; immunomodulatory properties other than phagocytosis
Thatte and Dahanukar 1989; Rege et al., 1989; Rege et we studied its effect on humoral antibody production. The
084 Afr. J. Biotechnol.

Figure 2. Effect of chloroform and ethyl acetate extracts of T. cordifolia in

antibody production.

study reveals that aqueous and ethanolic extracts of T. Bishayi B, Roychowdhury S, Ghosh S, Sengupta M (2002).
Hepatoprotective and immunomodulatory properties of Tinospora
cordifolia enhanced antibody production when SRBC was
cordifolia in CCl4 intoxicated mature albino rats. J. Toxicol. Sci. 27:
used as an antigen. In our present study, we have 139-146.
revalidated the immunomodulatory properties of the Broker R, Bhatt JV (1953). Symposium on antibacterial substances from
extracts of this plant. soil, plants and other sources. XV. Phagocytic coefficient as a
measure for evaluating plant antibiotics. Indian J. Pharm. 15: 309-
Boosting of macrophage mediated innate (non- 310.
specific) immunity coupled with increased antibody Chopra RN, Chopra LC, Handa KD, Kapur LD (eds) (1982). Indigenous
production (acquired specific immunity) using a safe plant Drugs of India. 2nd ed. Kolkata: M/S Dhar VN & Sons.
based compound could emerge as an alternate way in Chopra RN, Nayar SL, Chopra IC (eds) (1956). Glossary of Indian
Medicinal plants. New Delhi: CSIR.
preventing various diseases per se. The present study Cline MJ (1973). A new white cell test which measures individual
reveals the multifaceted immunomodulatory potential of phagocyte function in mixed leucocyte population. J. Lab. Clin. Med.,
T. cordifolia. In this era of immunosuppression, steroid 81: 3111-3116.
treatment, organ transplantation, HIV/AIDS, chemothe- Cragg GM, Newman DJ, Snader KM (1997). Natural products in drug
discovery and development. J. Nat. Prod., 60: 52-60.
rapy, etc, there is an inevitable need to judiciously exploit
Dahanukar SA, Thatte UM, Pai N, More PB, Karandikar SM, (1988).
and utilize the immunomodulatory properties of the Immunotherapeutic modification by Tinospora cordifolia of abdominal
unique medicinal plants like T. cordifolia in modern sepsis induced by caecal ligation in rats. Indian J. Gastroenterol. 7:
medicine. 21-23.
Dikshit V, Damre AS, Kulkarni KR, Gokhale A, Saraf MN (2000).
Preliminary screening of imunocin for immunomodulatory activity.
Indian J. Pharm. Sci. 62: 257.
REFERENCES Gerzon K (1980). Anticancer Agents Based on Natural Product Models,
ed. by Cassady JM, Douros JD, Academic Press, New York, pp.
Atal CK, Sharma ML, Kaul A, Khajuria A (1986). Immunomodulating 271-317.
agents of plant origin. I: Preliminary screening. J Ethnopharmacol. Gottlieb AA, Gottlieb MS, Scholes VE (1987). Reconstitution of immune
18: 133-141. function in AIDS/ARC. Medicine from plant sources. Concepts
Bhavmishra (1929). Sarth Bhavaprakasha, Raghuwanshi Prakashan 1 Immunopathol., 4: 261-274.
Edition, p. 109. Gunabakshi DN, Sensarma P, Pal DC (1998). Medicinal Plants of India,
 Ranjith et al. 085

Pilgrims Publishing. Subramanian M, Chintalwar GJ, Chattopadhyay S (2002). Antioxidant

Jardine I (1980). Anticancer Agents Based on Natural Product Models, properties of a Tinospora cordifolia polysaccharide against iron-
ed. by Cassady JM, Douros JD, Academic Press, New York, pp. mediated lipid damage and gamma-ray induced protein damage.
319-351. Redox Rep. 7: 137-143.
Kapil A, Sharma S (1997). Immunopotentiating compounds from Swerdlow J (2000). Nature’s Medicine. Plants That Heal, National
Tinospora cordifolia . J. Ethnopharmacol. 58: 89-95. Geographic Society.
Kasture SB, Kasture VS, Chopde CT (2001). Anti-inflammatory activity Thatte UM, Chhabria S, Karandikar SM, Dahanukar SA (1987).
of Rubia cordifolia roots. J. Nat. Remedies 1(2): 111-115. Immunotherapeutic modification of E.coli induced abdominal sepsis
Kinghorn AD, Balandrin MF (1993). Human Medical Agents from Plants, and mortality in mice by Indian medicinal plants. Indian Drugs 25: 95-
American Chemical Society Symposium Series 534. American 97.
Chemical Society, Washington, DC, pp. 80-95. Thatte UM, Dahanukar SA. (1989). Immunotherapeutic modification of
Kirtikar KR, Basu BD (eds) (1975). Indian Medicinal Plants, Vol 1. 2nd experimental infections by Indian medicinal plants. Phytother Res. 3:
ed. New Connaught Place, Dehra Dun: M/S Bishen Singh, Mahendra 43-49.
Pal Singh. Thatte UM, Rao SG, Dahanukar SA (1994). Tinospora cordifolia
Lehrer RI, Ladra KM, Hake RB (1975). Non-oxidative fungicidal induces colony stimulating activity in serum. J. Postgrad. Med. 40:
mechanisms of mammalian granulocytes. Demonstration of 202-203.
components with candidicidal activity in human, rabbit and guinea Tripathi YB, Sharma M, Manickam M (1997). Rubiadin: A new
pig leukocytes. Infect. Immun., 11: 1226-1234. antioxidant from Rubia cardifolia. Indian J. Biochem. Biophys. 34(3):
Manjrekar PN, Jolly CI, Narayanan S (2000). Comparative studies of 302-306.
the immunomodulatory activity of Tinospora cordifolia and Tinospora Vanfurth R, Theda L, Vanzwet Leijh PCJ (1978). In vitro determination
sinensis. Fitoterapia 71: 254-257. of phagocytosis and intracellular killing by polymorphonuclear and
Nadkarni KM, Nadkarni AK (eds) (1976). Indian Materia Medica, Vol 1. mononuclear phagocytes. In: Weir DM, editor. Handbook of
3rd ed. Mumbai: M/S Popular Prakasan Pvt. Ltd. Experimental Immunology, Vol 2. Oxford, Blackwell Scientific
Nagarkatti DS, Rege NN, Desai NK, Dahanukar SA (1994). Modulation Publications, 32: 1-19.
of Kupffer cell activity by Tinospora cordifolia in liver damage. J. Wall ME, Wani MC (1993). Human Medicinal Agents from Plants, ed.
Postgrad. Med. 40: 65-67. by Kinghorn AD, Balandrin MF, American Chemical Society
Rege NN, Bapat RD, Koti R, Desai NK, Dahanukar S (1993). Symposium Series 534: 149-169.
Immunotherapy with Tinospora cordifolia: A new lead in the Zhao TF, Wang X, Rimando AM, Che C (1991). Folkloric medicinal
management of obstructive jaundice. Indian J. Gastroenterol. 12: 5- plants: Tinospora sagittata var. cravaniana and Mahonia bealei.
8. Plant. Med. 57: 505.
Rege NN, Dahanukar SA (1993). Quantitation of microbicidal activity of
mononuclear phagocytes: an in vitro technique. J. Postgrad. Med.
39: 22-25.
Rege NN, Nazareth HM, Bapat RD, Dahanukar SA (1989). Modulation
of immunosuppression in obstructive jaundice by Tinospora
cordifolia. Indian J. Med. Res. 90: 478-483.
Rege NN, Thatte UM, Dahanukar SA (1999). Adaptogenic properties of
six rasayana herbs used in Ayurvedic medicine. Phytother. Res.13:
275-91.
Singh SS, Pandey SC, Srivastava S, Gupta VS, Patro B, Ghosh AC
(2003). Chemistry and medicinal properties of Tinospora cordifolia
(guduchi) Indian J. Pharmacol. 35: 83-91. Educational Forum.
Sonia RN, Igna´cio Jose´ Luiz P, Ferreira MBA, Claire FK (2001). Nitric
oxide production by murine peritoneal macrophages in vitro and in
vivo treated with Phyllanthus tenellus extracts. J. Ethnopharmacol.
74: 181-187.