Chemoprofile and bioactivities of Taverniera cuneifolia (Roth) Arn.: a wild relative and possible substitute of Glycyrrhiza glabra L.

Introduction

The genus Taverniera belonging to the family of Fabaceae, includes twelve species and is endemic to the Northeast African and Southwestern Asian countries (Naik, 1998; Stadler et al., 1994). Literature available on this plant is scarce, except for Taverniera abbysinica, which is used as a 'drug for sudden illness' in the African subcontinent (Stadler et al., 1994). Taverniera cuneifolia (Roth) Arn., is often referred as Indian licorice as its roots are sweet and taste very similar to that of Glycyrrhiza glabra L., popularly known as commercial licorice (Zone, 2005). The roots of G. glabra are very widely used in traditional systems of medicines all over the world (Grieve, 1992). G. glabra is rich in bioactivities like antiviral, anticancer, anti-ulcer, anti-diabetic, anti-inflammatory, anti-oxidant, anti-thrombic, anti-malarial, anti-fungal, anti-bacterial, estrogenic, immuno-stimulant, anti-allergenic and expectorant activities (Olukoga and Donaldson, 2000; Baltina, 2003; Sasaki et al., 2003, Cinatl et al., 2003, Rastogi and Mehrotra,1989). The commercial licorice has a huge demand in the Indian system of medicine, Ayurveda and majority of the requirement of the Ayurvedic drug industry in India is met through import from Afghanistan and Pakistan (Rastogi and Mehrotra, 1989). A number of plants often referred as Indian licorices, could be potential alternatives to G. glabra. Not many studies are available on the scientific validity of indigenous alternatives or wild relatives of G. glabra. In this communication, we are presenting the chemoprofile and bioactivities of the root extracts of T. cuneifolia and it is compared with that of G. glabra.

Materials and methods

Roots of T. cuneifolia were collected from Osmana-bad district of the Maharashtra state, India. Dried roots and runner pieces of G. glabra L. were obtained from local stores. Twenty grams of shade dried and powdered plant materials were extracted in 200ml of 70% ethanol using a soxhlet extractor. The extracts were then filtered and evaporated to yield brown residues. The residues suspended in distilled dimethyl sulphoxide (DMSO) was used as crude extracts. Plant materials were also extracted sequentially using solvents with increasing polarity for 6-8 h. Flavonoids and Coumarins were extracted by refluxing 1-g root powder in 5 ml of methanol at 60 [degrees]C in a water bath for 30min. Extracts were filtered, concentrated and re-suspended in 1 ml of methanol. Saponin extract residues were re-suspended in chloroform/methanol (1:1).

HPTLC analysis

Precoated TLC plates (Silica Gel 60 F254) of 0.2 mm thickness and 20x20 cm size were purchased from Merck KGaA, Germany. Standard Glycyrrhizin was purchased from Sigma (USA) and 1 mg/ml stock solution was prepared in ethanol. Ten microliter of the crude extracts and standard glycyrrhizin (5[micro]l] were loaded as 10-mm streak on HPTLC plates at 10-mm distance between two streaks, using a Linomat IV an automatic spotter (Camag, Pvt. Ltd., Switzerland). Plates were allowed to dry for few minutes and developed using n-butanol: acetic acid: water (7:1:2) as a solvent system. Plates were dried, observed under UV 254 and 366 nm and scanned using a Camag Scanner III (Switzerland). UV Spectra, Rf value, % AUC and [lambda] max of each chromatophores were documented. Plates were derivatized using specific detection reagents (e.g. for glycyrrhizin, anisaldehyde: sulfuric acid reagent) and observed visually as well as under UV at 254 and 366 nm. All the extractions, chromatographic separations and analysis were done as per Wagner and Bladt (1996).

Anti-inflammatory assay

Wistar rats having an average body weight of 150-200 g were orally fed with ethanol/chloroform/petroleum ether extracts of T. cuneifolia, for 3 days prior to the injection of Carrageenan with doses of 250 mg and 500 mg/kg body weight. The animals were divided into eight groups of six each. Group I served as control, received distilled water, Group II and III received ethanol extract, Group IV and V received chloroform, Group VI and VII received petroleum extract. Group VIII served as positive control and received Na-Diclofenac, 9 mg/Kg (Cipla Pvt. Ltd., India). Drug was administrated orally, daily as a single dose. After pre-treating the animals for 3 days, on the day 4, 0.1-ml Carrageenan (10%) in distilled water was injected subcutaneously into the plantar plexus of the hind paw (Winter et al., 1962). Before injection, initial paw volume was recorded, plethysmographically. After injecting carrageenan, paw volume was recorded at 2, 4, 6 h and thereafter everyday for the next 5 days. Na-Diclofenac treatment was continued for the next 5 days. Statistical analysis was done by Students t-test.

Cytotoxicity testing

Cytotoxicity of crude extracts were determined in MT-2 cell line (T lymphoid cell line from NIH AIDS Research and Reference Programme, USA). Extract concentrations ranging from 0.24 to 500[[micro].g]/ml were prepared in 96 well flat-bottom tissue culture plates, using RPMI 1640 medium (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) containing 10% FCS (Moregate, Australia). To each well, washed, 2 x [10.sup.4] MT-2 cells were added. The plates were incubated for 5 days at 37[degrees]C. On the fifth day, plates were examined microscopically for cytotoxic effect and the cell viability was determined by trypan blue dye (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) exclusion method (Sasaki et al., 2003; Hu and Hsiung, 1989; Nokta and Pollard, 1992).

Virus titration

Seven serial four-fold dilutions of virus stock (ranging from 1:16 to 1: 65,536) were prepared. MT-2 cells were added to each well and the plates were incubated at 37[degrees]C. After overnight adsorption, cells were washed and incubated at 37[degrees]C for 7 days. At the end of incubation period, culture supernatant was tested for the presence of p24 antigen using a commercial ELISA kit (Coulter Inc., USA). The [TCID.sub.50]/ml was calculated by Spearman-Karber method (Sasaki et al., 2003; Hu and Hsiung, 1989; Nokta and Pollard, 1992).

Anti-HIV activity

Using MT-2 cell line and HIV-1 III B strain, sub toxic concentrations of the extracts were tested for its ability to inactivate cell free HIV and to inhibit HIV replication (Sasaki et al., 2003; Hu and Hsiung, 1989; Nokta and Pollard, 1992).Inhibition of HIV-1 III B growth was monitored by Inhibition of cytopathic effect (CPE) and reduction in HIV-1 p24 antigen (Sasaki et al., 2003; Hu and Hsiung, 1989; Nokta and Pollard, 1992). Both the methods used for anti-HIV testing of T. cuneifolia extract have been standardized using known HIV inhibitors like AZT and nonoxynol -9 (Cipla Pvt. Ltd., India).

(a) Viral binding inhibition assay: Sub toxic concentrations of the extracts, i.e. 62.5, 31.25 and 15.6 [micro]g/ml were mixed with 100 [micro].1 of pre-titrated HIV-1 III B virus (100 [TCID sub.50]/ml in separate tubes. The virus-compound mixture was incubated for l h at 37 [degrees] C. The MT-2 cells were washed using RPMI + 2% FCS and 200 [micro] of 0.1 x [10.sup.6] cells were incubated for 2h at 37 [degrees] c. After the last wash, supernatant was tested for p24 antigen (0 PID--post infection day). The pellet was resuspended in growth medium and transferred to 24 well plates containing sub toxic concentrations of the extract. The plates were incubated at 37[degrees]C for 5 days. On the fifth day, plates were read microscopically for inhibition of CPE. The supernatants (5 PID) were tested for p24 antigen. Cell control, virus control and nonoxynol-9 controls were maintained. All the incubations were done in a [CO.sub.2] incubator (Nokta and Pollard, 1992).

(b)Viral replication inhibition assay: MT-2 cells (0.1 x [10.sup.6] cells/ml) were infected with HIV-1 III B Virus ([100TCID.sub.50]/ml) and incubated for 2h at 37[degrees]C. After incubation, cells were washed thrice using RPMI + 2% FCS to remove residual virus. From the last wash, supernatant was tested for baseline p24 antigen levels (0 PID). The pellet was re-suspended in growth medium (RPMI+10% FCS). The cell suspension was transferred to 24 well tissue culture plates. Sub toxic concentrations of the extract, 62.5, 31.25 and 15.6 [mu]/ml were then added to each well and incubated at 37[degrees]C for 5 days. On day 5, the plates were examined micro-scopically for CPE and the supernatant (5 PID) was tested for p24 antigen. Cell control, virus control and AZT controls were also maintained (Nokta and Pollard, 1992).

Anti-tumor activity

Discs of 0.5-cm height and 1.5-cm diameter were collected from the core of surface sterilized potato tubers and transferred into wells of 1.5-cm diameter made in water agar (1.5%) plates. 0.5ml of extracts (2mg/ml) in test tubes were mixed with 1.5 ml distilled water and 2 ml of 48-h old broth culture of Agrobacterium tumefaciens (approximately 5 x [10.sup.9] cells/ml). 50 [mu] of this suspension was used for inoculation of discs. Mixture without extract was used as control. All the plates were incubated at 29[+ or -]1[degrees]C and the tumors developed on the discs were counted on the twelfth day (McLaughlin, 1991).

Protective effect of extracts against mutagen induced toxicity

The protective effects of the extracts were tested against ethyl methane sulfonate (EMS) (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) induced toxicity in Salmonella typhimurium NCIM 2501. The dose of EMS was determined by testing various concentrations and it was found that 1 [micro] EMS killed about 75% of inoculum and thus it was used at 1 [micro]l/plate concentration. 50 [micro]l of 24-h old broth culture of S. typhimurium was mixed with 50 [micro]l of phosphate buffer and various concentrations of the extracts (0, 2, 4, 6 and 8 mg in separate tubes). 1 [micro]l of EMS were added to these tubes aseptically and incubated for 30min at 37[degrees]C. After incubation, 2 ml of molten top agar was added to each tube and poured into nutrient agar petriplates and incubated for 24 h at 37 [+ or -] 1 [degrees]C Surviving colonies were counted and percent of survival was calculated by comparing with the control (Maron and Ames, 1983).

Inhibition of serum induced germ tube formation in Candida albicans

(ATCC 10231) was grown in yeast extract peptone dextrose (YPD) broth (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) for 24h at 28[+ or -] 1[degrees]C in a shaking incubator. 5.0 x [10.sup.6] cells/ml from this culture was used as inoculum for 1 ml of 25% human serum (Odds, 1988). Various concentrations of extracts were added aseptically in separate vials and incubated at 37[degrees]C for 90min. After incubation, a drop of the culture was observed under a microscope for germ tube formation and compared with the control (without drug) (Odds, 1988).

Results

Chemoprofile of T. cuneifolia and G. glabra

The crude extract of the roots of T. cuneifolia was resolved into twelve chromatophores, while that of G. glabra yielded eight, corresponding to the Rf values 0.31-0.90and 0.07-0.90, respectively. Standard glycyrrhizin exhibited an Rf value of 0.31 and characteristic spectrum at 200 nm. The chromatophores present in T. cuneifolia extract at the Rf 0.33 and in G. glabra at the Rf value 0.32 showed quenching like that of standard glycyrrhizin, under UV 254 nm. After derivatization with anisaldehyde: sulfuric acid reagent, pink-violet spots were developed at Rf 0.31 in standard glycyrrhizin lane and at Rf 0.32, 0.33 in G. glabra and T. cuneifolia lanes under visible light (Fig. 1B). Under UV, 366nm yellow green fluorescent zones were observed in standard glycyrrhizin as well as at the corresponding spots of G. glabra and T. cuneifolia lanes (Fig. 1A). Spectra of standard glycyrrhizin showed perfect matching with that of corresponding spots in G. glabra and T. cuneifolia lanes. In addition to glycyrrhizin, three more chromatophores (E-1-E-3) in crude extracts of both the plants were found similar, based on their absorption maxima ([lambda] max) and spectra. The percentage area under the concentration curve (% AUC) of glycyrrhizin in G. glabra extract was 15.88 and in T. cuneifolia 13.20. Comparison of the UV spectra of similar chromatophores of the different extracts of T. cuneifolia and G. glabra showed perfect overlapping of at least eighteen chromatophores (Table 1).

Anti-inflammatory activity

Na-diclofenac exhibited 11% inhibition in edema volume 3h after carrageenan injection and 79% on the fifth day. Ethanol extract of T. cuneifolia caused 22% inhibition after 6h and 74% on fifth day at 500-mg/kg-body weight. At same concentration. Chloroform fraction showed 19% inhibition, 3h after carrageenan injection and 50% on the fifth day. Maximum inhibition caused by the Chloroform extract was 60% on the fourth day. Petroleum ether fraction failed to inhibit carrageenan induced paw edema (Table 2 and Fig. 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Anti-HIV activity

T. cuneifolia extract exhibited marked toxic effects on MT-2 cells. 50% cytotoxicity was noted at 62.5 [micro]g/ml concentration. The sub toxic concentration of the extract was found to be 31.25 [micro]g/ml Crude extract of T. cuneifolia failed to demonstrate anti-HIV activity at concentrations, 62.5, 31.25 and 15.6 [micro]g/ml (Table 3).

Anti-tumor activity

Fifty-percentage inhibition of tumor formation was found at 0.250mg/ml concentration of T. cuneifolia extract while G. glabra extract caused 59% inhibition (Table 3). The extracts of T. cuneifolia and G. glabra showed no effect on the in vitro growth of A. tumefaciens up to the 10mg/plate concentration (data not shown).

Protective activity of extracts against mutagen induced toxicity

Percentage survival of S. typhimurium cells improved with increase in concentration of the extract, i.e. 52%, 60% and 75% at 2, 4 and 6mg/plate concentration, respectively. A maximum of 75% survival was found at 6-mg/plate concentration of T. cuneifolia crude extract. Further increase in concentration reduced the percentage survival to 51%. G. glabra extract possessed better protective activity than T. cuneifolia. G. glabra extract protected 97% cells against EMS toxicity at 8 mg/disc concentration (Table 4).

Table 1. Analysis of similar chromatophores from extracts of T
cuneifolia (Roth) Arn., and G. glabra L

Extract     Solvent system                              Rf *
                                                        Chromatophores

Crude       n-Butanol: acetic acid: [H.sub.2]0 (7:1:2)  E-l
                                                        E-2
                                                        E-3
                                                         GL    0.31(100)

Saponins    Chloroform:methanol (95:5)                  S-l
                                                        S-2
                                                        S-3
Flavonoids  Ethylacetate: formic acid: acetic acid:     F-l
                                                        F-2

Coumarins   Toluene: diethyl ether (1:1)                C-l
                                                        C-2
                                                        C-3

T.cuneifolia  G. glabra     [lambda] max

0.13 (3.83)   0.12(1.38)    190
0.20 (10.04)  0.22 (5.63)   190
0.27 (19.54)  0.27 (6.33)   257,252
0.33 (13.20)  0.32(15.88)   257,190
0.31 (13.50)  0.33 (11.37)  305
0.38 (07.15)  0.38 (04.52)  297
0.97 (24.92)  0.97 (07.55)  199
0.60 (02.39)  0.57 (03.53)  319
0.98 (86.04)  0.99 (22.62)  202
0.30 (03.79)  0.29 (12.54)  320, 328
0.35(03.17)   0.37 (16.78)  331, 326
0.71 (57.10)  0.68 (12.31)  400

* values in parenthesis show % AUC in extract, GL--standard glycyrrhizin
(E--chromatophores from ethanol extract (crude)) ,S--chromatophores from
Saponin extract, F--chromatophores from flavonoid extract,
C--chromatophroes from Coumarin extract).

Table 2. Effect of extracts of T. cuneifolia, on Carrageenan induced
paw edema in Wistar rats


Dose (mg/kg                    Edema volume (a) (mm)

                       Initial              3h

Control (no extract)   29.5 [+ or -] 1.87              65+1.41

EE 250                   29 [+ or -] 2.36   65.5 [+ or -] 2.34

   500                 29.3 [+ or -] 2.80  57.50 [+ or -] 3.27

CE 250                29.16 [+ or -] 2.31  62.33 [+ or -] 3.38

   500                27.67 [+ or -] 1.36  56.83 [+ or -] 1.94

PE 250                   29 [+ or -] 1.41  64.83 [+ or -] 1.60

   500                   29 [+ or -] 2.09     68 [+ or -] 2.28

Diclofenac            27.33 [+ or -] 1.75  59.16 [+ or -] 3.06
(9 mg/kg
body weight))

Dose (mg/kg                    Edema volume (a) (mm)

                       4h                   6h

Control (no extract)   65.5 [+ or -] 1.87   64.5 [+ or -] 1.51

EE 250                   67 [+ or -] 3.22   66.5 [+ or -] 3.20

   500                57.33 [+ or -] 4.27     56 [+ or -] 4.04

CE 250                    63 [+ or -] 2.0  62.55 [+ or -] 1.87

   500                57.33 [+ or -] 2.42   52.8 [+ or -] 1.86

PE 250                   66 [+ or -] 1.41  65.33 [+ or -] 1.63

   500                 66.5 [+ or -] 1.22  63.16 [+ or -] 0.98

Diclofenac            56.83 [+ or -] 2.78   54.5 [+ or -] 2.25
(9 mg/kg
body weight))

Dose (mg/kg                    Edema volume (a) (mm)

                       24 h                48 h

Control (no extract)   60.8 [+ or -] 1.32  55.67 [+ or -] 1.36

EE 250                 64.5 [+ or -] 3.20   60.5 [+ or -] 3.20

   500                52.33 [+ or -] 4.32  47.16 [+ or -] 3.76

CE 250                59.33 [+ or -] 1.36  56.67 [+ or -] 1.75

   500                   49 [+ or -] 1.60  43.83 [+ or -] 1.54

PE 250                61.33 [+ or -] 1.21   57.5 [+ or -] 1.76

   500                58.33 [+ or -] 1.63     54 [+ or -] 1.54

Diclofenac            49.16 [+ or -] 3.37  44.67 [+ or -] 2.50
(9 mg/kg
body weight))

Dose (mg/kg                    Edema volume (a) (mm)

                      72 h                  96 h

Control (no extract)  49.83 [+ or -] 0.75   46.33 [+ or -] 1.21

EE 250                55.67 [+ or -] 2.87   49.83 [+ or -] 2.78

   500                   42 [+ or -] 2.75      37 [+ or -] 2.09

CE 250                 53.5 [+ or -] 2.16    50.5 [+ or -] 1.87

  500                    39 [+ or -] 1.16   34.33 [+ or -] 1.26

PE 250                 54.5 [+ or -] 1.37   49.67 [+ or -] 1.86

  500                 48.67 [+ or -] 1.21      44 [+ or -] 0.89

Diclofenac                     38.66+1.75  33.16 [+ or -] +2.04
(9 mg/kg
body weight))

Dose (mg/kg            Edema volume (a) (mm)

                         120 h

Control (no extract)     42.67 [+ or -] 1.03

EE 250                   44.67 [+ or -] 2.16

   500                  32.8 * [+ or -] 1.72

CE 250                   47.16 [+ or -] 1.32

   500                34.33 ** [+ or -] 1.63

PE 250                    45.5 [+ or -] 1.97

   500                    39.5 [+ or -] 1.64

Diclofenac             30.16 * [+ or -] 1.47
(9 mg/kg
body weight))

* P values <0.01; ** <0.05.
(a) Edema volume was measured plethysmographically,
EE--ethanol extract, CE--chloroform extract,
PE--petroleum ether extract.

Inhibition of serum induced germ tube formation in C. albicans

T. cuneifolia curde extract inhibited serum induced germ tube formation. 97% germ tube formation was observed even in presence of the extract (Table 5). Both the extracts did not affect C. albicans yeast phase growth up to 10-mg/ml concentration (data not shown).

Discussion

Chromatograhic and spectral analysis of extracts of both the plants exhibited considerable similarity in chemoprofile. The similar chromatophroes included the sweetening principle, glycyrrhizin. T. cuneifolia contained 13.20% glycyrrhizin in its roots compared to 15.88% in G. glabra. T. cuneifolia possessed considerable in vivo anti-inflammatory activity and it was found to be associated with ethanol and chloroform soluble fractions (Table 2, Fig. 2). Terpenoids and flavonoids (which are reported to have in vivo anti-inflammatory activity (Baltina et al., 2003; Finney and Somers, 1959). T. cuneifolia extract was cytotoxic to MT-2 cell line at [greater than or equal to]62.5 [micro]g/ml conentrations (Fig. 3). G. glabra curde extracts, its fractions and some of the constituents like glycyrrhizin 18[alpha]-glycyrrhetinic acid 18[beta]-glycyrrhetinic acid, licocoumarone are reported to possess cytotoxic activity (Chung et al., 2001). Mechanism of cytotoxic activity of these compounds is not very clear but these compounds induce apoptosis in human cell lines and thus are considerrd to be potent anticancer agents (Watanbe et at., 2002).

G. glabra extracts, glycyrrhizin and its derivatives are reported to inhibit growth of viruses like HIV, SARS, Hepatitis B and C, Influenza through the potentiation of immune response, inhibition of reverse transcriptase and induction of interferon production (Baltina, 2003; Sasaki et al., 2003; Cinatl et al., 2003). Glycyrrhizin induced [beta]-chemokines like [CCl.sub.3], [CCl.sub.4] and [CCl.sub.5] compete with virus for binding to co-receptor CCR5 and thus affect the entry of HIV (Sasaki et al., 2003). In our study T. cuneifolia crude extract even though contains glycyrrhizin did not show anti-HIV activity. A detailed study exploring the status of immune response, chemokine production, which is beyond the scope of this study, may reveal the probable reasons for the observed lack of anti-HIV activity.

T. cuneifolia extract caused 50% inhibition of Agrobacterium induced tumors while the inhibition by G. glabra extract was 59% (Table 3). This is the first report of inhibition of plant tumors by the extracts of these plants. However in different model systems scientists have reported inhibition of tumors by extracts and constituents like glycyrrhizin 18-[alpha] glycyrrhetinic acid, isoliquiritigenin, glabridin, licocoumarone, etc. of G. glabra (Grieve, 1992; Baltina, 2003; Rastogi and Mehrotra, 1989; Olukoga and Donaldson, 2000).

Table 3. Inhibition of A. tumefaciens induced tumors in
potato discs by crude extracts of T. cuneifolia and G. glabra

Crude        Number of    Tumor      Inhibition
extract      tumors/disc  induction  of tumor
(0.5 mg/m1)               (%)        induction
                                     (%)

Control (no  22[+ or -]2.0  100        00
extract)
G. glabra    09[+ or -]1.0  41         59
T.           11[+ or -]1.0  50         50
Table 4. Protective effect of the ethanol extract of T cuneifolia
and G. glabra against EMS toxicity in S. typhimurium

Extracts    Extract     Number of colonies/plate      Percentage
           (mg/plate)                                 survival

                        Control (no     EMS(1
                        EMS)            [micro]1/m1)

Control     0          288[+ or -]21.0  64[+ or -]11.0    22
T           2          242[+ or -]21.0  125[+ or -]14.0   52

cuneifolia  4          272[+ or -]26.0  163[+ or -]17.0   60
            6          300[+ or -]29.0  224[+ or -]21.0   75
            8          312[+ or -]32.0  159[+ or -]15.0   51

G. glabra   2          333[+ or -]27.0  104[+ or -]14.0   31
4                      299[+ or -]25.0  116[+ or -]18.0   39
6                      137[+ or -]11.0  64[+ or -]9.0     47
8                      240[+ or -]24.0  233[+ or -]22.0   97

[+ or -] Standard deviation

Both the plant extracts protected S typhimurium cells from EMS induced toxicity (Table 4) 6 mg/plate concentration was found to be optimum, showing 75% survival compared to that of 22% in control plates without (Table 4) G glabra crude extract caused97% survival at 8mg/plate concentration (Table 4) This is the first report for T. cuneifolia G. glabra crude extract and components like glycyrrhizin 18-[alpha] and 18-[beta] glycyrrhetinic acid, and a flavonoid, glabrene found in the leaves are reported to show considerable antimutagenic as well as desmutagenic activity (Zani et al., 1993). Formation of germ tubes is an important step in the establishment of infection by the human fungal pathogen, C. albicans (Odds, 1988). The extract of T. cuneifolia inhibited germ tube formation in C. albicans. In conclusion, both T cuneifolia and G glabra extracts possessed similar chemoprofile. The similarity is also reflected in the bioactivities. Results of this study indicate that T. cuneifolia could be a good source for phytochemicals with useful bioactivities in parts of the world where G. glabra is not cultivated or not available.

[FIGURE 3 OMITTED]

Table 5 Effect of crude extracts of T. cuneifolia and
G glabra on germ tube formation by c . albicans

Extract (1[micro]g/m1)  % Induction of  % Induction
                        germ of         of germ tube

No extract              100             0
T. cuneifolia           15              85
G. glabra               97              3

Acknowledgements

Science and Technology Cell, Government of Maharashtra, Mumbai (India) is thanked for the financial assistance. We thank Dr. Krishnammacharylu, School of Earth Science, SRTM University for helpful discussion on the statistical analysis of the data presented in this paper.

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Gajanan B. Zore (a), Umakanth B. Winston (b), Babasaheb S. Surwase (a), Nisha S. Meshram(c), V.D. Sangle(d), Smita S. Kulkarni(b), S. Mohan Karuppayil(a) *

(a) School of Life Sciences SRTM University, Nanded, Maharashtra, India

(b) National AIDS Research Insitute, Pune, Maharashtra, India

(c) Science and Technology Cell, Government of Maharashtra, Mumbai, Maharashtra, India

(d) Glenmark Pharmaceuticals Ltd., Mumbai, Maharashtra, India

Received 26 September 2006L: accepted 12 December 2006