Antimicrobial activity of Tinospora cordifolia: an ethnomedicinal plant

Antimicrobial activity of Tinospora cordifolia / Asian Journal of Traditional Medicines, 2012, 7(2) 

Regular articles 

Antimicrobial activity of Tinospora cordifolia: an 

ethnomedicinal plant 

Veeramuthu Duraipandiyan c , Savarimuthu Ignacimuthu a *, Kedike Balakrishna b , 

, , , 

Naif Abdullah AL-Harbi , c 

a. Division of Ethnopharmacology, Entomology Research Institute, Loyola College, 

Chennai-600 034, India 

b. Captain Srinivasa Murti Drug Research Institute for Ayurveda and Siddha, Arumbakkam, 

Chennai-600 106, Tamil Nadu, India 

c. Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies 

Abstract 

Various organic solvent extracts of Tinospora cordifolia stem were tested against some Gram-positive and Gram-negative bacteria 

and fungi. Antibacterial activity of crude extract was carried out using disc diffusion method and MIC of isolated compound was 

tested by broth microdilution method. The antifungal activity was carried out using broth microdilution method. Crude ethanolic 

extract showed good antibacterial and antifungal activity against tested microbes. A compound [(5R, 10R)-4R, 8R-Dihydroxy-2S, 

3R:15, 16-diepoxycleroda-13(16), 17, 12S, 18, 1S-dilactone] was isolated from ethanolic extract. The compound also showed good 

activity against bacteria and fungi. The lowest MIC values were seen against Bacillus subtilis (200 µg/ml), Enterococcus faecalis 

(125 µg/ml) Trichophyton simii (31.25 µg/ml), Trichophyton rubrum 57 (62.5 µg/ml) and Trichophyton rubrum 296 (62.5 µg/ml). 

The crude ethanol extract of Tinospora cordifolia and isolated compound showed activity against tested bacteria and fungi. 

Key words: antibacterial, antifungal, Tinospora cordifolia, clerodane diterpene 

1 Introduction 

India has a rich flora of medicinal plant species 

that are widely distributed throughout the country. 

They produce many unusual secondary metabolites 

which are biologically active. Despite the existence 

of a wide variety of antibacterial agents the search 

for new ones is of great importance. Microbial 

* Author to whom correspondence should be addressed. Address: 

Entomology Research Institute, Loyola College, Nungambakkam, 

Chennai 600 034, Tamil Nadu, India; Tel: +91-44-2817 8348; Fax: 

+91-44-2817 5566; E-mail: entolc@hotmail.com 

Received: 2010-12-24 Accepted: 2012-03-02 

infections are the cause of a large burden of 

diseases and bacteria are listed in the first position 

among the common microorganisms responsible 

for opportunistic diseases occurring in association 

with AIDS. Therapy of bacterial infections is a 

frequent problem due to the emergence of bacterial 

strains resistant to numerous antibiotics. In India 

catheters and lung infections caused by Gramnegative 

organisms such as Klebsiella, Pseudomonas 

and Enterococci are predominant. Nosocomial 

pneumonia is most common in the surgical intensive 

units. The search for natural products to cure disease 

represents an area of great interest in which plants 

have been the most important sources. In the Indian 

59


traditional medicine, the use of plant is a widespread 

practice among tribal and rural communities. More 

and more people in developing countries utilize 

traditional medicine for their major primary health 

care needs [1, 2] 

Natural products derived from higher plants play 

an important role as useful tools in pharmacological 

studies. With the technological advancement of 

science the isolation, identification and elucidation 

of chemical principles from natural sources have 

become much simpler and have contributed 

significantly to the development of new drugs from 

medicinal plants [3]. 

Tinospora cordifolia (Willd) Miers (Guduchi) 

belonging to Menisoermaceae (Family) is 

distributed throughout the plains of India. In folk 

and tribal medicine the whole plant and the powder 

of leaves, stem bark and roots of T. cordifolia 

are used to treat various ailments such as fever, 

jaundice, diarrhea, dysentery, general debility, 

cough, asthma, skin diseases, fracture, bites of 

poisonous insect and eye disorders [4]. In Ayurvedic 

medicine it is used for the treatment of liver and 

intestinal disorders [5]. Plant stem possess immunomodulatory 

[6], hepatoprotective [7], antipyretic 

[8], antiulcer [9] and anticancer [10] activities. 

Tinospora cordifolia has been clinically used to treat 

jaundice, rheumatoid arthritis and diabetes [11]. The 

species is rich in clerodane derived diterpinoids [12]. 

The clerodane diterpenoids comprise a large class 

of natural products with wide biological activities 

[13, 14]. The present study was undertaken to assess 

the antibacterial and antifungal activity of various 

organic and aqueous extracts of T. cordifolia stem 

and an isolated compound. 

2 Materials and methods 

2.1 Plant material 

Stems of Tinospora cordifolia were collected 

from Thandarai, Kanchipuram distict, Tamil Nadu, 

India. The plant was identified and confirmed by a 

taxonomist from the Department of Botany, Loyola 

College. Chennai and a voucher specimen (ERIC-D- 

45) is deposited at the herbarium of Entomology 

Research Institute, Loyola College, 

2.2 Preparation of crude extracts 

One kg of powder was extracted with hexane 

for a period of 48 hours. The extract was filtered 

through a Buchner funnel with Whatman number 1 

filter paper. The filtrate was evaporated to dryness 

under reduced pressure using rotary evaporator 

at 40°C. The remains of the plant material were 

extracted with ethyl acetate, ethanol, methanol 

and water sequentially in a similar manner. 

2.3 Isolation and identification of active 

compound 

Since the ethanol extract showed promising 

antibacterial and antifungal activity, it (25 g) was 

subjected to column chromatography over silica 

gel (200 g-Acme’s silica gel, 100–200 mesh) 

fractionation was followed by TLC and similar 

fractions were pooled together. The fractions were 

then subjected to antimicrobial activity and the 

active fraction was located. From the active fraction 

the pure compound was isolated and identified. 

2.4 Test organisms 

The following bacteria and fungi were used 

for the experiment. Bacteria: Bacillus subtilis 

MTCC 441, Enterococcus faecalis ATCC 29212, 

Staphylococcus aureus ATCC 25923, Escherichia 

coli ATCC 25922, Klebsiella pneumoniae 

ATCC 15380, Proteus vulgaris MTCC 1771. 

Fungi: T. rubrum MTCC 296, T. rubrum 57/01, 

T. mentagrophytes 66/01, T. simii 110/02, 

Epidermophyton floccosum 73/01, Scopulariopsis 

sp. 101/01 Aspergillus niger MTCC 1344, Botyritis 

60


cinerea, Curvularia lunata 46/0, Magnoporthe 

grisea. All the cultures were obtained from the 

Department of Microbiology, Christian Medical 

College, Vellore, Tamil Nadu, India. 

2.5 Preparation of inoculums 

Bacterial inoculum was prepared by growing 

cells in Mueller Hinton Broth (Himedia) for 24 h 

at 37°C. These cell suspensions were diluted with 

sterile MHB to provide initial cell counts of about 

10 4 CFU/ml. The filamentous fungi were grown on 

Sabouraud Dextrose Agar (SDA) slants at 28 o C for 

10 days and the spores were collected using sterile 

doubled distilled water and homogenized. Yeast was 

grown on Sabouraud Dextrose Broth (SDB) at 28 o C 

for 48 h. 

2.6 Antibacterial assay (Disc diffusion method) 

Antibacterial activity was carried out using discdiffusion 

method [16]. Petri plates were prepared 

with 20 ml of sterile Mueller Hinton Agar (MHA) 

(Hi-media, Mumbai). The test cultures were swabbed 

on the top of the solidified media and allowed to 

dry for 10 min. The tests were conducted at three 

different concentrations of the crude extract (5 mg, 

2.5 mg and 1.25 mg per disc). The loaded discs were 

placed on the surface of the medium and left for 30 

min at room temperature for compound diffusion. 

Negative control was prepared using respective 

solvent. Streptomycin (10 µg/disc) was used as 

positive control. The plates were incubated for 24 h 

at 37 o C for bacteria and 48 h at 27 o C for fungi. Zones 

of inhibition were recorded in millimeters and the 

experiment was repeated twice. 

2.7 Antifungal activity and minimum inhibitory 

concentration 

The antifungal activity and minimum 

inhibitory concentration (MIC) were performed 

according to the standard reference method [17]. 

The extracts and compound were dissolved in 

water +2% Dimethyl Sulfoxide (DMSO). The 

initial concentration of extracts was 1 mg/ml 

and for compound it was 250 µg/ml. The initial 

test concentration was serially diluted two-fold. 

Each well was inoculated with 5 µl of suspension 

containing 10 8 CFU/ml of bacteria and 10 4 spore/ 

ml of fungi, respectively. Five μl of tested broth 

was placed on the sterile MHA plates for bacteria and 

incubated at respective temperature. The antifungal 

agents Ketoconazole and Fluconazole and antibacterial 

agents Streptomycin and Ciprofloxacin were included 

in the assays as positive controls. The fungal plates 

were incubated for 24, 48 or 72 h at 27°C up to 9 days 

while bacterial plates were incubated for 24 h at 37°C. 

MIC was defined as the lowest extract concentration 

showing no visible fungal growth after incubation 

time. The MIC for bacteria was determined as the 

lowest concentration of the compound inhibiting the 

visual growth of the test cultures on the agar plate. 

3 Results and discussion 

The yield of hexane, ethyl acetate, ethanol, 

methanol and water extracts from stems of T. 

cordifolia were 15 g for hexane, 20 g for ethyl 

acetate, 25 g for ethanol, 40 g for methanol and 

5 g for water.The different solvent extracts of 

T. cordifolia were tested against selected Grampositive 

and Gram-negative pathogenic bacteria. The 

results are presented in Table 1. Hexane extract did 

not show any activity against tested bacteria for all 

tested concentrations. Ethyl acetate extract inhibited 

the growth of S. aureus (10 mm), S. typhi (9mm) 

and P. vulgaris (13 mm) at higher concentration 

of 5mg/disc. Ethanol extract showed promising 

antibacterial activity for all tested bacteria such as 

S. aureus (8 mm), B. subtilis (9mm), E. faecalis 

(9 mm), E. coli (9 mm), S. typhi (8 mm) and P. 

vulgaris (8 mm) at lowest concentrations (Table 

1). Methanol extract showed moderate activity 

against S. aureus, B. subtilis, E. faecalis, E. coli 

61


Table 1. Antibacterial activity of different solvent extracts of T. cordifolia stem 

Extracts 

Conc. 

Mg/ disc 

Disc diffusion method (inhibition zone, mm) 

S.a B.s E.f E.c S.t K.p P.v 

Hexane extract 1.25 - - - - - - 

2.5 - - - - - - - 

5 - - - - - - - 

Ethyl acetate extract 1.25 - - - - - - - 

2.5 - - - - - - - 

5 10 - - - 9 13 

Ethanol extract 1.25 8 9 9 9 8 - 8 

2.5 10 14 12 11 9 - 14 

5 12 16 15 14 12 - 16 

Methanol extract 1.25 - - - - - - - 

2.5 10 - 9 - - - 10 

5 13 10 11 11 - - 12 

Water extract 1.25 - - - - - - - 

2.5 - - - - - - - 

5 - 9 9 - - - - 

Streptomycin 10µg 12 13 12 13 nt 11 nt 

Ciprofloxacin 5 µg 20 30 16 25 nt 28 nt 

-, no activity; S.a - Staphylococcus aureus; B.s - B. subtilis; E.f- Enterococcus faecalis; E.c - Escherichia coli; K.p – Klebsiella 

pneumoniae; P.v- Proteus vulgaris; Streptomycin and Ciprofloxacin- Control antibiotics 

and P. vulgaris but did not show activity against S. 

typhi and P. vulgaris. Water extract inhibited only 

the growth of B. subtilis (9 mm) and E. faecalis 

(9 mm) at higher concentrations. The maximum 

inhibition zones were observed in ethanol extract 

against B. subtilis (16mm), E. faecalis (15 mm), E. 

coli (14 mm) and P. vulgaris (16mm). The lowest 

MIC values were recorded in ethanol extract against 

S. aureus (0.315 mg/ml), B. subtilis (0.315 mg/ml), 

E. faecalis (0.315 mg/ml) and P. vulgaris (0.315 

mg/ml) (Table 2). Antifungal activity of T. cordifolia 

stems of hexane, ethyl acetate, ethanol, methanol 

and water extracts were tested against fungi and 

the results are presented in Table 3. Hexane, ethyl 

acetate and ethanol extracts showed promising 

antifungal activity against T. rubrum 296, T. rubrum, 

T. mentagrophytes, T. simii and E. floccosum. Water 

and methanol extracts did not show antifungal 

activity. 

Antimicrobial screening results indicated that 

both antibacterial and antifungal activities were 

observed in ethanol extract of T. cordifolia. The 

active compound after bioassay guided fractionation 

was identified as 8-hydroxy arcangelisin Structure 

o f ( 5 R , 1 0 R ) - 4 R , 8 R - d i h y d r o x y - 2 S , 3 R : 1 5 , 

16-diepoxycleroda-13(16), 17, 12S, 18, 1S-dilactone 

(clerodane diterpene) (Fig. 1). The compound which 

is a clerodane diterpene has been previously reported 

by us as anticancer compound against hepatocellular 

carcinoma [15]. 

Anticancer property of this compound has been 

reported by Dhanasekaran et al [15]. Antimicrobial 

activity of this compound is given in Table 2 

and 3. The compound inhibited the growth of B. 

subtilis (200 µg/ml), E. faecalis (125 µg/ml), E. 

62


Table 2. MIC of T. cordifolia stem extracts and isolated compound against bacteria 

Microorganism 

MIC (mg/ml) 

MIC (µg/ml) 

H E Et Me W C Cfl Strep (µg/ml) 

Staphylococcus aureus >5.0 2.5 0.315 2.5 >5.0 >200 5.0 >5.0 0.315 0.625 5.0 200 5.0 >5.0 0.315 1.25 5.0 125 6.25 25 

Escherichia coli >5.0 >5.0 1.25 2.5 >5.0 200 5.0 5.0 1.25 >5.0 >5.0 >200 nt 25 

Klebsiella pneumoniae >5.0 >5.0 >5.0 >5.0 >5.0 >200 5.0 2.5 0.315 1.25 >5.0 125 nt nt 

nt, not tested; H- Hexane extract; E- Ethyl acetate extract; Et- Ethanol extract; Me- Methanol extract; W-Water extract; C- 

Isolated Compound; Strept- Streptomycin; Cfl- Ciprofloxacin (Control antibiotic for bacteria) 

Table 3: MIC of T. cordifolia stem extracts and isolated compound against fungi 

Tested organism 

Dermatophytes 

Minimum inhibitory concentration (mg/ml) 

H E Et Me W C (µg/ml) Fl (µg/ml) 

Trichophyton rubrum 296 0.125 0.250 0.125 1.0 >1.0 62.5 1.0 >1.0 62.5 25 

Trichophyton mentagrophytes 0.250 0.5 0.250 >1.0 >1.0 125 25 

Trichophyton simii 0.250 0.5 0.625 >1.0 >1.0 31.2 1.0 >1.0 125 12.5 

Scopulariopsis sp. >1.0 >1.0 >1.0 >1.0 >1.0 >200 1.0 >1.0 >1.0 >1.0 >1.0 >200 100 

Botyritis cinerea >1.0 >1.0 >1.0 >1.0 >1.0 >200 - 

Curvularia lunata >1.0 >1.0 >1.0 >1.0 >1.0 >200 1.0 >1.0 200 - 

-, no activity; H- Hexane extract; E- Ethyl acetate extract; Et- Ethanol extract; Me- Methanol extract; W- Water extract; C- 

Isolated Compound; Fluconazole Control antibiotic for fungi. 

coli (200 µg/ml) and P. vulgaris (125 µg/ml). The 

compound also inhibited the growth of T. rubrum 

57 (62.5 µg/ml), T. rubrum 296 (62.5 µg/ml), T. 

mentagrophytes (125 µg/ml), T. simii (31.2 µg/ml), 

E. floccosum (125 µg/ml) and M. grisea (200 µg/ 

ml). The compound did not show activity against 

S. aureus and S. typhi. This compound has not been 

reported previously for antimicrobial activity. The 

search for natural products to cure disease represents 

an area of great interest in which plants have 

been the most important sources. In recent years 

antimicrobial properties of Indian medicinal plants 

have been increasingly reported [18-20]. Over the 

years there have been several studies documenting 

the antibacterial properties of plants from India and 

Tamil Nadu [21-28] 

The crude extracts of T. cordifolia stem showed 

activity against bacteria and fungi. Among the 

tested extracts ethanol extract showed good activity 

against both bacteria and fungi. Aqueous, ethanol 

and chloroform crude extract of T. cordifolia 

63


The present work supports the claim in the 

traditional medicine that the plant can be used in 

the treatment of various infectious diseases as fever, 

diarrhea, dysentery, cough etc. which are of bacterial 

origin and skin diseases caused by fungi. 

stems showed antibacterial activity against E. 

coli, P. vulgaris, E. faecalis. S. typhi and Serratia 

marcesecens [29]. Dhar et al. [30] reported that 

ethanolic stem extract of T. cordifolia showed 

antiviral activity against Ranikhet diseases. The 

active compound was isolated from ethanol 

extract. Isolated compound showed activity 

against bacteria and fungi. The lowest MIC values 

were observed against E. faecalis (125 µg/ml) 

and P. vulgaris (125 µg/ml). The compound also 

showed activity against fungi; the lowest MIC 

values were seen against T. simii, T. rubrum 57 and 

T. rubrum (296). The fact that diterpenoids seem to 

be key compounds in the antimicrobial activity of 

the resinous exudates from plants has been recently 

documented with a comparative antimicrobial study 

of several plants in which the diterpenoids are the 

principal components in these extracts [31]. Zagodaspols 

et al. [32] reported that four diterpenes were 

isolated from the stem bark of Mitrephora celebica 

through bioassay-guided fractionation. These 

compounds were responsible for the antimicrobial 

activity of the plant against methicillin-resistant 

Staphylococcus aureus and Mycobacterium 

smegmatis. 

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Antimicrobial activity of Tinospora cordifolia: an ethnomedicinal plant

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