Anti-inflammatory and antimicrobial activities of triterpenoids from Strobilanthes callosus Nees. (Short Communication).

B. Singh (1), P.M. Sahu (1) and M.K. Sharma (1)

Summary

The anti-inflammatory and antimicrobial activities of the 95% ethanol extract, benzene fraction and isolated triterpenoids of Strobilanthes callosus were investigated. In the carrageenan-induced paw edema inflammation model, the taraxerol showed a high reduction of edema, but the antimicrobial effect observed was lower at the two doses employed. These results confirm the use of this plant in folk medicine as an anti-inflammatory and antimicrobial herbal drug.

Key words: Strobilanthes callosus, triterpenoids, anti-inflammatory and antimicrobial activities

* Introduction

Strobilanthes callosus Nees. (syn. Carvia callosa; family-Acanthaceae) is known locally as Karvi', and its stem bark is used as an emollient but in formulations for painful and ineffectual attempts to urinate or defecate (Jam and Defilipps, 1991), is used externally for mumps and flowers, and used as a vulnerary (Nadkarni, 1954). Lilkewise, pounded leaves are rubbed on to the body during the cold period of an intermittent fever and used as a poultice to treat ague in children to alleviate coughing (Kirtikar and Basu, 1935; Chopra et al., 1956), as an astringent and diuretic, and to treat arthritis (Anonymous, 1954). The ethanolic extract of S. heyneanus showed GOT and GPT activities (Nayar et al., 1988), along with other activities such as analgesic, anti-inflammatory and anti-immunosuppressant (Ravishankar et al., 1987a). The petroleum ether and aqueous extracts of roots of S. heyneanus were screened for CNS and depressant activity (Ravishankar, 1987b).

The roots contain lupeol (Burkill, 1935) and have been examined by influorescence for volatile oil-borucol, sitosterol, hexacosane, campesterol and others (Ilyas et al., 1979; Rastogi and Mehrotra, 1979). The quinazolinone alkaloid indirubin showed anticancer and hypotensive action (Li et al., 1993). Li et al. (1997) formulated a Chinese drug 'Shengma' through the combination of Serratulla and Strobilanthes, but there are no literature reports of anti-inflammatory and antimicrobial screening of S. callosus; hence, this study was carried Out.

* Materials and Methods

Plant material

S. callosus Nees. used for the present investigation was collected from the Mt. Abu hills of Rajasthan in August, 1999, and authenticated by the Herbarium of the Department of Botany, University of Rajasthan, Jaipur (specimen no. 20041).

Extraction and characterization

Shade-dried and powdered aerial parts of the plant material (5.0 kg) were defatted using petroleum ether (60-80 [degrees]C) for 24 h, filtered, and the resultant residue further Soxhlet extracted with 95% ethanol for 36 h, filtered, and evaporated to dryness (yield 3.48%). The concentrated ethanolic extract was fractionated sequentially using petroleum ether (41.23%), benzene (39.52%), chloroform (8.69%) and residue.

The benzene-soluble fraction was re-dissolved and applied on thin layer chromatography plates (silica gel G; hexane:acetone; 8:2; Fazli and Hardman, 1968). The developed plates were sprayed wtih 20% [SbCl.sub.3] (in [CHCl.sub.3], Heftmann, 1975) and heated in an oven (120 [degrees]C) for 10 mm. Six positive spots were observed.

Preparative tic of the benzene fraction was carried out, along with reference standards (lupeol, sitosterol, campesterol, [alpha]-amyrin acetate, taraxerol and taraxasterol etc.) developed in the above-mentioned organic solvent system. Later, these chromatograms were exposed to [I.sub.2] vapors, marked, evaporated and the spots scraped separately with sorbent and eluted with chloroform and concentrated. Each of the compounds was crystallized using methanol:acetone (1:2; Kaul et al., 1967). The crastals were dried in vacuo and subjected to various physical and spectral studies for characterization (Heilbron and Bundury, 1953; Yamaguchi, 1970).

Animals

Male wistar albino rats, 4-6 weeks old, 150-200 g each, were obtained from the Animal Centre, Hamdard University, New Delhi, India. They were housed in an airconditioned room at 223 and fed with standard laboratory diet and tap water throughout the experiments.

Carrageenan-induced paw edema

Anti-inflammatory activity was measured according to the method of Winter et al. (1962). Edema in the left hind paw of the rat was induced by subcutaneous injection of 0.05 ml of 1.0% (w/v) carrageenan (Sigma, St. Louis, MO) in saline into the footpad. The paw volume of each rat was measured before carrageenan injection and then at hourly intervals up to 10 times using a plethysmometer 7150 (UGO, Basil, Italy). The drug test groups were treated with ethanolic extract, [alpha]amyrin acetate, taraxerol and taraxasterol (10 and 20 mg/kg body wt., s.c.) before carrageenan injection. The animals in the control group received saline only. Another group of rats was administered indomethacin (10 and 20 mg/kg body wt. s.c.) in 1.0% CMC as standard reference. The edema and inhibition rate of each group were calculated.

Statistical analysis

Data were expressed as mean [+ or -] s.e.m., assessed statistically by analysis of variance (ANOVA). The difference between drug-treated groups and the control group was evaluated using Dunnett's 't' test. P < 0.01 was considered statistically significant.

Sources of test microbes

The pure cultures of bacteria, Escherichia coli (ATCC 5922), Staphylococcus aureus (ATCC 25923), Enterobacter cloacae (ATCC 25924), Klebsiella pneumoniae (ATCC 10031) and Bacillus thuringiensis (Kurastaki), were obtained from S.M.S. Medical College, Jaipur and maintained on a nutrient broth medium by incubating at 37 [degrees]C for 48 h. Fungi, Aspergillus niger A. flavus, Rhizoctonia phaseoli and Penicillium chrysogenum (from the Seed Pathology Laboratory, Department of Botany, University of Rajasthan, Jaipur) were grown on a potato dextrose agar (PDA) medium and incubated at 27 [degrees]C for 48 h (Jain and Singh, 1998).

Antimicrobial assay

Antibacterial and antifungal activities were screened by the disc-diffusion method (Gould and Bowie, 1952). The filter paper discs (Whatman No. 1, 6-mm) saturated with sequential extracts (8 mg/disc), isolated compounds (1 mg/disc) and reference antibiotics (gentamycin - 1 mg/disc for bacteria and mycostatin - 1 mg/disc for fungi). The inhibition zone (s) were recorded and the activity index (AI) calculated by comparison with respective compounds (AI = Inhibition zone of test sample/Inhibition zone of the standard).

* Result and Discussion

The benzene extract of S. callosus contains a number of triterpenoids. These compounds were separated by preparative tlc and identified on the basis of [R.sub.f], mp, spectral data, and comparison with the authentic markers lupeol (0.31%), [beta]-sitosterol (0.79%), campesterol (0.99%), [alpha]-amyrin acetate (0.82%), taraxerol (0.69%) and taraxasterol (0.52%).

Table 1 shows the edema and inhibition rates after administration of carrageenan, ethanolic crude extract and three isolated compounds of S. callosus. The footpad injected with carrageenan became edemateous for 10 h. Administration of the ethanolic extract (100, 200 mg/kg body wt. dose s.c.) and others (10, 20 mg/kg body wt. s.c.) showed a promotion of the inhibition rate. The anti-inflammatory activity of taraxerol was apparent as early as 1 h after carrageenan injection and was maintained until the experiment was terminated at 10 h. The anti-inflammatory effect of taraxerol (48.61%) is similar to that of indomethacin (48.7%).

In this study we showed that in control animals, the subcutaneous injection of carrageenan produced a local edema within 30 min that increased progressively to reach its maximal intensity 7 h after injection of the phlogistic agent. A pre-treatment with extract and isolated triterpenoids of Strobilanthes in a dose equivalent to 100,200 mg/kg body wt. for extract and 10,20 mg/kg body wt. for isolated taraxerol significantly reduced the edema 1 h after carrageenan injection.

Conclusions

Nevertheless, a dose of extract (400 mg/kg body wt.) equivalent to 50 mg/kg body wt. of taraxerol shows less inhibition (16.6%) and no effect is observed until 3 h. This dose causes strong diarrhea in the animals during the 3 h. The observed diarrhea reflects the toxic range. No conclusion can be drawn on dose dependency, when malabsorption may occur and bioavailability cannot be ensured. The other three triterpenoids could not be screened, but these had been screened in our previous

study.

The observed results indicated that the benzene fraction showed higher activity against S. aureaus (IZ-14.00; AI-0.49) and A. flavus (IZ-9.00; AI-0.67) at the dose of 8 mg/disc but were less active at the dose of 4 mg/disc (IZ-0.9; AI-0.36). The activity index was calculated on the basis of comparison with standard (gentamycin for bacteria and mycostain for fungi) and isolated compounds. Among the isolated triterpenoids, taraxerol exhibited maximum activity against K. pneumoniae (Table 2). Sitosterol and [beta]-amyrin acetate showed better activity against E. ccli, K. pneumoniae (Jain and Singh, 1999) and P. chtysogenum (Singh and Dubey, 2001). Inhibition zones were recorded in thriplicate along with the standard deviation.

Based on the results of this study, we came to the conclusion that the ethanolic extract of aerial parts of S. callosus does have pronounced anti-inflammatory activity when used subsutaneously (Calvo et al., 1998). The antimicrobial activity was found to be associated with the benzene fraction of this plant material. The benzene fraction and taraxerol from this plant species demonstrated greater activity against selected microorganisms. These conclusions provide a scientific basis for the utilization of the plant in Indian folk medicine for the treatment of variety of subcutaneous inflammatory and microbial conditions.

Table 1

Anti-inflammatory activity of triterpenoids from S. callosus Nees. on
carrageenan- induced paw edema.

Extract/compound   Dose (mg/kg) *   Edema rate (%) after injection
                                   1 h

Control             -              41.53 [+ or -] 3.62
Ethanolic extract  100             37.16 [+ or -] 1.16 (a) (10.5)
                   200             34.18 [+ or -] 1.25 (b) (17.6)
[alpha]-amyrin      10             31.35 [+ or -] 2.68 (a) (24.5)
                    20             28.25 [+ or -] 3-52 (a) (31.9)
Taraxerol           10             24.16 [+ or -] 4.61 (a) (41.8)
                    20             21.34 [+ or -] 3.33 (a) (48.6)
Taraxasterol        10             27.64 [+ or -] 2.76 (a) (33.4)
                    20             26.54 [+ or -] 6.54 (a) (36.0)
Indomethacin        10             17.16 [+ or -] 3.69 (a) (58.6)
                    20             14.28 [+ or -] 4.38 (a) (65.6)

Extract/compound    Edema rate (%) after injection
                   3 h

Control            54.28 [+ or -] 2.31
Ethanolic extract  43.56 [+ or -] 3.56 (a) (19.7)
                   48.64 [+ or -] 4.26 (b) (10.3)
[alpha]-amyrin     38.45 [+ or -] 3.42 (a) (29.7)
                   36.54 [+ or -] 4.25 (b) (32.6)
Taraxerol          31.35 [+ or -] 3.35 (a) (42.2)
                   29.61 [+ or -] 2.21 (a) (45.4)
Taraxasterol       39.65 [+ or -] 5.69 (a) (27.1)
                   43.64 [+ or -] 4.56 (a) (19.6)
Indomethacin       21.56 [+ or -] 1.19 (a) (60.2)
                   19.61 [+ or -] 2.68 (a) (63.8)

Extract/compound    Edema rate (%) after injection
                   5 h

Control            62.81 [+ or -] 4.13
Ethanolic extract  52.61 [+ or -] 5.61 (a) (16.2)
                   54.71 [+ or -] 6.34 (a) (12.8)
[alpha]-amyrin     52.61 [+ or -] 5.61 (a) (16.2)
                   41.45 [+ or -] 3.69 (a) (34.0)
Taraxerol          36.40 [+ or -] 4.11 (a) (42.0)
                   39.43 [+ or -] 6.16 (a) (37.2)
Taraxasterol       45.59 [+ or -] 4.56 (a) (27.4)
                   48.61 [+ or -] 5.54 (a) (22.6)
Indomethacin       27.16 [+ or -] 5.38 (a) (56.7)
                   32.16 [+ or -] 4.35 (a) (48.7)

Extract/compound    Edema rate (%) after injection
                   7 h

Control            79.33 [+ or -] 1.19
Ethanolic extract  58.54 [+ or -] 2.63 (a) (26.2)
                   63.61 [+ or -] 1.13 (a) (19.8)
[alpha]-amyrin     58.54 [+ or -] 2.63 (a) (26.2)
                   54.65 [+ or -] 4.65 (a) (3.11)
Taraxerol          43.54 [+ or -] 5.56 (a) (45.1)
                   49.66 [+ or -] 6.69 (a) (37.4)
Taraxasterol       56.56 [+ or -] 2.19 (a) (28.7)
                   60.61 [+ or -] 2.89 (a) (23.5)
Indomethacin       31.34 [+ or -] 4.56 (a) (60.4)
                   34.56 [+ or -] 3.36 (a) (56.4)

Extract/compound    Edema rate (%) after injection
                   10 h

Control            76.24 [+ or -] 2.83
Ethanolic extract  62.66 [+ or -] 6.56 (a) (17.9)
                   59.61 [+ or -] 4.41 (a) (21.9)
[alpha]-amyrin     62.66 [+ or -] 6.56 (a) (17.9)
                   59.16 [+ or -] 3.34 (a) (22.5)
Taraxerol          40.41 [+ or -] 4.46 (a) (47.0)
                   46.51 [+ or -] 5.61     (39.0)
Taraxasterol       52.56 [+ or -] 6.61 (a) (29.8)
                   62.76 [+ or -] 7.70 (a) (19.7)
Indomethacin       42.61 [+ or -] 3.36 (a) (44.1)
                   48.98 [+ or -] 4.16 (a) (35.7)

* mg/kg body weight

Values represent the mean [+ or -] s.e.m. of six animals for each group.
Statistically significant from control: (a) P <0.01 and (b) P <0.05
(Dunnett's 't' test). Each value in parentheses indicates the percentage
inhibition rate.
Table 2

Antimicrobial activity of sub-fractions and isolated triterpenoids from
Strobilanthes callosus Nees.

Microorganisms                       Active sub-fractions
                            PE                   [C.sub.6][H.sub.6]

Bacteria
E. Coli            IZ *     10.00 [+ or -] .122  12.00 [+ or -] .218
                   AI **     0.41                 0.48
S. aureus          IZ       13.00 [+ or -] .241  14.00 [+ or -] .223
                   AI        0.43                 0.49
C. cloacae         IZ        8.00 [+ or -] .101  19.00 [+ or -] .112
                   AI        0.25                 0.28
E. penumoniae      IZ       12.00 [+ or -] .152  14.00 [+ or -] .211
                   AI        0.46                 0.54
E. thuringiensis   IZ       11.00 [+ or -] .228  15.00 [+ or -] .228
                   AI        0.58                 0.75

Fungi
A. niger           IZ        7.00 [+ or -] .087   8.00 [+ or -] .217
                   AI        0.40                 0.45
A. flavus          IZ        6.00 [+ or -] .079   9.00 [+ or -] .116
                   AI        0.45                 0.67
R. phaseoli        IZ        7.00 [+ or -] .110   6.00 [+ or -] .071
                   AI        0.55                 0.47
P. chrysogenum     IZ        8.00 [+ or -] .077   9.00 [+ or -] 0.92
                   AI        0.52                 0.58

Microorganisms    Active sub-fractions    Isolated compounds
                  [CHCl.sub.2]         I

Bacteria
E. Coli            8.00 [+ or -] .132  16.00 [+ or -] .416
                   0.32                 0.65
S. aureus          9.00 [+ or -] .157  18.00 [+ or -] .479
                   0.30                 0.62
C. cloacae         8.00 [+ or -] .167  14.00 [+ or -] .327
                   0.26                 0.45
E. penumoniae     10.00 [+ or -] .173  12.00 [+ or -] .289
                   0.39                 0.48
E. thuringiensis  11.00 [+ or -] .216  16.00 [+ or -] .322
                   0.55                 0.80

Fungi
A. niger           6.00 [+ or -] .069   9.00 [+ or -] .178
                   0.37                 0.52
A. flavus          7.00 [+ or -] .092   8.00 [+ or -] .163
                   0.52                 0.59
R. phaseoli        6.00 [+ or -] .066   9.00 [+ or -] .217
                   0.51                 0.71
P. chrysogenum     7.00 [+ or -] 0.94   8.00 [+ or -] .185
                   0.45                 0.51

Microorganisms                Isolated compounds
                  II                   III

Bacteria
E. Coli           14.00 [+ or -] .423  13.00 [+ or -] .398
                   0.58                 0.53
S. aureus         15.00 [+ or -] .418  16.00 [+ or -] .410
                   0.52                 0.54
C. cloacae        12.00 [+ or -] .379  16.00 [+ or -] .451
                   0.38                 0.52
E. penumoniae     13.00 [+ or -] .298  14.00 [+ or -] .436
                   0.52                 0.56
E. thuringiensis  12.00 [+ or -] .179  10.00 [+ or -] .476
                   0.63                 0.53

Fungi
A. niger          10.00 [+ or -] .198   8.00 [+ or -] .380
                   0.57                 0.45
A. flavus          9.00 [+ or -] .116  10.00 [+ or -] .462
                   0.66                 0.74
R. phaseoli        7.00 [+ or -] .108   8.00 [+ or -] .443
                   0.58                 0.63
P. chrysogenum    10.00 [+ or -] .176   9.00 [+ or -] .489
                   0.63                 0.58

Microorganisms                Isolated compounds
                  IV                   V

Bacteria
E. Coli           17.00 [+ or -] .486  16.00 [+ or -] .431
                   0.71                 0.67
S. aureus         14.00 [+ or -] .163  12.00 [+ or -] .216
                   0.49                 0.42
C. cloacae        11.00 [+ or -] .112  13.00 [+ or -] .223
                   0.34                 0.42
E. penumoniae     16.00 [+ or -] .321  18.00 [+ or -] .498
                   0.62                 0.71
E. thuringiensis  15.00 [+ or -] .228  16.00 [+ or -] .473
                   0.76                 0.81

Fungi
A. niger          10.00 [+ or -] .107   9.09 [+ or -] .102
                   0.56                 0.51
A. flavus          9.00 [+ or -] 0.88   8.00 [+ or -] .092
                   0.67                 0.60
R. phaseoli       10.00 [+ or -] 0.67  11.00 [+ or -] 0.60
                   0.80                 0.86
P. chrysogenum    11.00 [+ or -] .123   9.00 [+ or -] .193
                   0.69                 0.57

Microorganisms     Isolated compounds
                  VI

Bacteria
E. Coli           14.00 [+ or -] .329
                   0.59
S. aureus         17.00 [+ or -] .421
                   0.57
C. cloacae        15.00 [+ or -] .399
                   0.47
E. penumoniae     11.00 [+ or -] .267
                   0.43
E. thuringiensis  13.00 [+ or -] .283
                   0.66

Fungi
A. niger          10.00 [+ or -] .176
                   0.56
A. flavus         10.00 [+ or -] .126
                   0.74
R. phaseoli        9.00 [+ or -] 0.74
                   0.72
P. chrysogenum     8.00 [+ or -] .096
                   0.51

* Inhibition zone (in mm) includes the disc diameter (6 mm); ** Activity
index = Inhibition zone of test sample/Inhibition zone of standard

Abbreviations used: PE - petroleum ether; [C.sub.6][H.sub.6] - benzene;
[CHCl.sub.3] - chloroform

Isolated compounds: I - lupeol; II - sitosterol; III - campesterol IV -
[alpha]-amyrin acetate; V - taraxerol; VI - taraxasterol.

All values are mean [+ or -] SD of triplicate readings.

Acknowledgements

The authors are thankful to the Department of Biotechnology, New Delhi, India, for financial support.

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B. Singh (1)

P. M. Sahu (1)

M. K. Sharma (1)

(1) Department of Botany, University of Rajasthan, Jaipur, India

* Address

Manoj Kumar Sharma, Department of Botany, University of Rajasthan, Jaipur-302004, India

Fax: ++91-141-624642;

e-mail: priyankasharma@ id.eth.net