Anti-inflammatory activity of alkanoids and triterpenoids from Trichodesma amplexicaule Roth.

Abstract

The phytochemical investigation of Trichodesma amplexicaule guided by bioassay, to isolation of triterpenoids and aliphatic phytoconstituents. To evaluate the anti-inflammatory activities of isolated phytoconstituents, models with carrageenan-induced acute arthritis and complete Freund's adjuvant (CFA)-induced chronic arthritis in rats were conducted. The investigated results showed that alkanoic acid significantly inhibited the carrageenan-induced acute arthritis. Moreover, alkane also supressed the development of chronic arthritis induced by CFA. It has been reported that alkane was the major phytoconstituent (1.03 [+ or -] 0.00135%) in in vivo studies.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Trichodesma amplexicaule; Anti-inflammatory; Triterpene; Alkane; Alkanol; Alkanoic acid

Introduction

Trichodesma amplexicaule Roth. is locally known as 'Chhota Kulpha' and naturalized in arid zone parts of Rajasthan. Traditionally it is used as an emollient and poultice (Anonymous, 1976). The roots are pounded with water and administered as a drink for children suffering from dysentery, pounded into paste used to reduce swelling of joints and leaves are used as a depurative when in cold infusions (Jain and Defilipps, 1998), for rheumatism, arthritis and as a diuretic (Chopra et al., 1956).

Trichodesma species has been examined for a variety of chemical constituents, viz., monocrotaline, supinine as pyrrolizidine alkaloids (Wassel et al., 1987; Bull et al., 1968); hexacosane, [alpha]-amyrin, lupeol (Singh, 2001), non-steroidal (Hassan et al., 1982) and fatty constituents (Hosamani, 1994). Trichodesma species have hepatotoxic (Bull et al., 1956) and antitumor (Culvenor, 1968; Kupchan et al., 1964) activities. As a part of systematic survey of botanical sources for anti-inflammatory activity, terpenoids from the aerial parts of T. amplexicaule was examined and gave reproducible activity against carrageenan-induced paw oedema and complete Freund's adjuvant-induced chronic arthritis in rats. Co-comparison of phytoconstituents in vivo and in vitro cell cultures were carried out due to continuation of our previous work (Singh and Dubey, 2001; Singh et al., 2002), which was not previously described in this plant species. So, this work was carried out in present Trichodesma species (Fig. 1).

[FIGURE 1 OMITTED]

Materials and methods

Plant material

T. amplexicaule Roth. (Boraginaceae) was collected (August, 1998) from fields of Agricultural Research Station. Durgapura, Jaipur and voucher specimens were deposited in the Herbarium, Department of Botany, University of Rajasthan, Jaipur, India, was used for present investigation (sheet no. 19440).

General experimental conditions

Melting points were determined on capillary Toshniwal melting point apparatus and are uncorrected. The spectral data were obtained on the following instruments: ir, Perkin-Elmer, 283; ms, Hewlett Packard HP 5930A; gc-ms, equipped with a HP 5933 data system, direct inlet at 70 eV; adsorbents for TLC (silica gel 60, 230-400 mesh for column chromatography and silica gel G used for TLC, Merck); TLC solvent systems: petroleum ether-benzene-alcohol (100:100:1 v/v), petroleum ether-benzene (1:1 v/v). The standards were obtained from Professor Pahup Singh, Natural Products Laboratory, Department of Chemistry, University of Rajasthan, Jaipur, India.

Extraction and characterization

Shade-dried powdered aerial parts of plant material (10.0 kg) were defatted with petroleum ether (60-80[degrees]C; 151) for 24 h, filtered and resultant residue was further Soxhlet (pressure 0.2 atm) extracted with 95% ethanol (8.01) for 36 h, filtered and concentrated (yield - 186 g). The crude ethanolic extract (160 g) was subjected to partition among petroleum ether (Fr. I, 62.12 g; 1.01), petroleum ether-benzene (2:1 v/v; Fr. II, 38.29 g; 800 ml) and benzene-hexane (2:1 v/v; Fr. III, 26.31 g; 500 ml) which were used in subsequent work. The column chromatography of Fr. I with elution by petroleum ether-benzene; 4 fractions, A-D; Fr. II with elution by benzene-hexane, 5 fractions E-I; Fr. III, elution by hexane-ethyl acetate, yielded 3 fractions, J-L, were collected (Singh et al., 2003).

Triterpene (I) and (II): A portion of fraction A-B, were combined (22.31 g) and re-chromatographed on silica gel and purified by preparative TLC with development by petroleum ether-benzene (1:1 v/v), [R.sub.f][approximately]0.26, followed by crystallization with petroleum ether-benzene (4:1 v/v), detection on TLC by Sb[Cl.sub.3], yielded I, [alpha]-amyrin (119 mg), mp 183-184[degrees]C, [C.sub.30][H.sub.50]O, [[alpha]][.sub.D] + 78.2[degrees] (CH[Cl.sub.3]), white crystals, positive to LB test. Fractions C-D (18.22 g) were further separated and purified by preparative TLC, II, lupeol (111 mg), mp 200-201[degrees]C, [C.sub.30][H.sub.50]O, [[alpha]][.sub.D] + 20[degrees] (CH[Cl.sub.3]) with solvent system: petroleum ether-benzene (1:1 v/v), detection on TLC by Sb[Cl.sub.3], [R.sub.f][approximately]0.32, positive to LB test. These isolated compounds were subjected to various physical and spectral studies and were identical with standard samples previously described (Ikan and Bergman, 1975; Nigam and Mitra, 1966; Reddy et al., 1975).

Alkane (III) and alkanol (IV): Fractions E-G (17.31 g) combined and purified by means of preparative TLC, III (123 mg), mp 56-58[degrees]C, [R.sub.f][approximately]0.38, [C.sub.26][H.sub.54], solvent system: petroleum ether-benzene-ethanol (100:100:1 v/v), crystallized with benzene-ethanol (2:1 v/v), did not gave positive test to TNM, brownish colour appeared on TLC when sprayed with 20% Sb[Cl.sub.3]. Fractions H-I, pooled (19.21 g), purified by preparative TLC, IV (247 mg), solvent system petroleum ether-benzene-ethanol (100:100:1 v/v), [R.sub.f][approximately]0.45, mp 79[degrees]C, [C.sub.26][H.sub.54]O, gave negative test to TNM, crystallized with methanol, brownish colour on TLC sprayed with Sb[Cl.sub.3]. These isolated compounds were subjected to various physical and spectral studies and were identical with their respective standards (Heilbron and Bunbury, 1953; Yamaguchi, 1970).

Alkanoic acid (V): A portion of fractions J-L, pooled (16.22 g), purified by preparative TLC (solvent mixture: heptane: benzene: ethanol, 100:100:1 v/v), yield- 313 mg, [R.sub.f][approximately]0.48, mp 68-71[degrees]C, [C.sub.26][H.sub.52][O.sub.2] crystallized with ethyl acetate-chloroform as white granules, brownish colour on TLC with Sb[Cl.sub.3] spraying reagent, did not give colour with TNM. The compound was identified by comparison (ir, nmr and ms) with an authentic sample of alkanoic acid showed the two to be indistinguishable (Ikan and Bergman, 1975; Heilbron and Bunbury, 1953; Yamaguchi, 1970).

Animals

Male wistar albino rats, 4-6 weeks old, were obtained from the Animal Centre, Jamia Hamdard University, New Delhi, India. They were housed in air conditioned room at 23 [+ or -] 2[degrees]C and fed with standard laboratory diet and tap water throughout the experiments. Rats weighing 150-200 g were used in this study.

Carrageenan-induced paw oedema in rats

This anti-inflammatory test was performed according to the method of Winter et al. (1962). Oedema in the left hind paw of rat was induced by injection 0.5 ml of 1.0% (w/v) carrageenan (Sigma St. Louis, MO) in saline into the footpad subcutaneously. The paw volume of each rat was measured before carrageenan injection and then at hourly intervals up to 10 h with Plethysmometer 7150 (UGO, Basil, Italy). The drug test groups were treated with isolated phytoconstituents (5, 10 and 20 mg/kg, body weight, s.c.) 1 h before carrageenan injection. The animals in the control group received saline only. Another group of rats was administered with indomethacin (5, 10 and 20 mg/kg, body weight, s.c.) in 1.0% CMC as a standard reference. The oedema and inhibition rate of each group were calculated as follows:

Oedema rate% = [V.sub.t] - [V.sub.0]/[V.sub.0],

Inhibition rate% = [E.sub.c] - [E.sub.t]/[E.sub.c],

where [V.sub.0] is the volume before carrageenan injection (ml); [V.sub.t] is the volume at t h after carrageen injection (ml); [E.sub.c] is the oedema rate of control group and the [E.sub.t] is the oedema rate of treated group.

Adjuvant-induced arthritis in rats

Experimental arthritis was induced in rats according to the method of Newbould (1963). The left footpad of each rat was injected with 0.5 ml of complete Freund's adjuvant (CFA, 1% suspension in olive oil; Difco). The rats in drug test groups were treated with isolated compounds (5, 10 and 20 mg/kg, body weight, p.o.) 24 h before the injection of CFA and then with daily treatment until 12 days after CFA challenge. The animals in control groups received saline only. The another group of rats was administered with indomethacin (5, 10 and 20 mg/kg, body weight, p.o.) in 1.0% CMC as a standard reference. The oedema and inhibition rate were measured with the same method as described above.

Statistical analysis

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

Results and discussion

The defatted aerial parts of T. amplexicaule were extracted with EtOH and crude ethanolic extract was subjected to silica gel column chromatography to isolate phytoconstituents I-V (I, 0.53[+ or -]0.00339%; II, 0.98[+ or -]0.00118%; III, 1.03[+ or -]0.00135; IV, 0.69[+ or -]0.00491%; V, 0.39[+ or -]0.00310%).

The rat's footpad became oedemateous soon after injection of carrageenan. The oedema rate of left footpad reached its peak at 8 h (65.2%). Administration of terpenoids significantly inhibited the development of swelling from 1 to 10 h after carrageenan injection (p<0.01, Table 1). The results, showed that alkanoic acid has the most potent anti-inflammatory effects (56.9%) at the dose of 5 mg/kg at 2 h after carrageenan injection. Indomethacin (5, 10, 20 mg/kg) showed more (66.1% at 6 h) effectiveness than isolated compounds.

According to Vineagar et al. (1987) the development of the carrageenan-induced paw oedema derives from the release of cytoplasmic enzymes and serotonin from mast cells and the increase of prostaglandin in the inflammatory area. The macrophages in carrageenan-insulated dermal tissue release much interleukin-1 to induce accumulation of polymorphic nuclear cells (PMNs) into the inflammatory area. The activated PMNs then release the lysomal enzymes and active oxygen, especially superoxide, to destroy connective tissue and induce paw swelling.

Table 2 shows the time course of oedema and inhibition rate after administration of CFA and isolated constituents. The footpad injected with CFA became swollen gradually for more than 12 days. The administration of terpenoids at the dose of 5, 10 and 20 mg/kg, significantly inhibited the development of joint swelling induced by CFA. The observed results indicated that alkane was most potent (67.7%) at the dose of 10 mg/kg against CFA-induced arthritis at 9th day. The test of short duration such as carrageenan-induced oedema in rats as well as in test of longer duration such as CFA-induced chronic arthritis in rats, the terpenoids exhibited significant anti-inflammatory effects.

Our results showed that terpenoids significantly inhibited the development of chronic joint swelling induced by CFA. Adjuvant-induced arthritis is the most frequently used chronic inflammatory model. It seems that bacterial peptidoglycan and muramyl dipeptide are responsible for its induction (Crofford and Wilder, 1993). Since the composition of bacterial adjuvant is complex and the immune response is a multiple stage process of intercellular cooperation, the mechanism is unclear (Walz et al., 1971). In Western medicine, the treatment often involves topical application of corrticosteroids which are symptomatically effective but have inherent disadvantages. In contrast, the Chinese herbal remedy containing multiple ingredients used to treat eczema (Sheehan and Atherton, 1992) is a good example of herbal approach. It may therefore, be concluded that T. amplexicaule appears to bear terpenoids affecting different mechanism relevant to inflammations arising in response to varied etiological factors. Further studies in other animal species are in progress to consolidate the inferences and seek clinical therapeutic significance for the findings.

Acknowledgements

Authors are thankful to Professor Dr. Naveen Sharma, National Institute of Ayurveda, Jaipur, for his assistance in anti-inflammatory activities and Dr. S.M. Khan, Department of Statistics, Agricultural Research Station, Durgapura, Jaipur, for his help in statistical analysis and also to Department of Biotechnology, New Delhi, for financial support.

References

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Singh, B., 2001. Scope of arid zone plants as medicinal agents. Ph.D. Thesis, University of Rajasthan, Jaipur, India.

Singh, B., Dubey, M.M., 2001. Estimation of triterpenoids from Heliotropium marifolium Koen ex Retz. I. Antimicrobial screening. Phytotherapy Res. 15, 231-234.

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B. Singh (a), P.M. Sahu (a), R.K. Lohiya (b), M.K. Sharma (a), H.L. Singh (b), S. Singh (c,*)

(a) Department of Botany, University of Rajasthan, Jaipur-302004, India

(b) Department of Chemistry, University of Rajasthan, Jaipur-302004, India

(c) Department of Entomology, Agricultural Research Station, Durgapura, Jaipur-302018, India

Received 7 April 2004; accepted 29 June 2004

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (S. Singh).

Table 1. Effects of alkanoids and triterpenoids from T. amplexicaule
Roth. and indomethacin (reference drug) on carrageenan-induced paw
oedema in rats

               Dose      Oedema rate (%)
Group          (mg/kg)*  1 h                     2 h

Control        --        39.8 [+ or -] 5.6       56.4 [+ or -] 6.5
Triterpene-I    5        29.1 [+ or -] 1.6 (a)   34.2 [+ or -] 4.4 (a)
                           (26.8)                  (39.3)
               10        26.5 [+ or -] 2.1 (a)   36.5 [+ or -] 2.9 (a)
                           (33.4)                  (35.2)
               20        21.2 [+ or -] 1.0 (a)   44.3 [+ or -] 9.2 (b)
                           (46.7)                  (21.4)
Triterpene-II   5        26.5 [+ or -] 3.5 (b)   37.2 [+ or -] 4.6 (a)
                           (33.4)                  (34.0)
               10        31.4 [+ or -] 4.3 (a)   44.8 [+ or -] 6.4 (b)
                           (21.1)                  (20.5)
               20        22.5 [+ or -] 3.4 (a)   33.5 [+ or -] 3.8 (a)
                           (43.4)                  (40.6)
Alkane          5        27.4 [+ or -] 2.9 (a)   41.3 [+ or -] 1.1 (a)
                           (31.1)                  (26.7)
               10        29.5 [+ or -] 4.9 (a)   39.3 [+ or -] 2.0 (a)
                           (25.8)                  (30.3)
               20        31.8 [+ or -] 9.2 (b)   40.3 [+ or -] 1.9 (a)
                           (20.1)                  (28.5)
Alkanol         5        29.5 [+ or -] 7.6 (a)   33.2 [+ or -] 4.7 (a)
                           (25.8)                  (41.1)
               10        33.6 [+ or -] 6.7 (a)   51.5 [+ or -] 3.0 (a)
                           (15.5)                  (8.6)
               20        30.5 [+ or -] 5.8 (a)   48.2 [+ or -] 3.9 (a)
                           (23.3)                  (14.5)
Alkanoic acid   5        19.6 [+ or -] 9.0 (b)   24.3 [+ or -] 7.6 (a)
                           (50.7)                  (56.9)
               10        20.5 [+ or -] 7.9 (a)   30.5 [+ or -] 4.8 (a)
                           (48.4)                  (45.9)
               20        18.1 [+ or -] 7.5 (a)   45.5 [+ or -] 3.9 (a)
                           (54.5)                  (19.3)
Indomethacin    5        14.3 [+ or -] 3.1 (a)   17.3 [+ or -] 1.4 (a)
                           (64.0)                  (69.3)
               10        17.2 [+ or -] 4.4 (a)   21.4 [+ or -] 6.1 (a)
                           (56.7)                  (62.0)
               20        13.0 [+ or -] 3.2 (a)   19.5 [+ or -] 5.2 (a)
                           (67.3)                  (65.4)

               Dose      Oedema rate (%)
Group          (mg/kg)*  6 h                     8 h

Control        --        63.5 [+ or -] 4.6       65.2 [+ or -] 3.9
Triterpene-I    5        56.4 [+ or -] 4.5 (a)   46.5 [+ or -] 4.4 (a)
                           (11.1)                  (28.6)
               10        54.3 [+ or -] 4.4 (a)   54.6 [+ or -] 5.4 (a)
                           (14.4)                  (16.2)
               20        39.3 [+ or -] 6.5 (a)   47.5 [+ or -] 4.5 (a)
                           (38.1)                  (27.1)
Triterpene-II   5        36.4 [+ or -] 5.6 (a)   50.5 [+ or -] 1.6 (a)
                           (42.6)                  (22.5)
               10        59.2 [+ or -] 3.0 (a)   54.4 [+ or -] 1.3 (a)
                           (6.7)                   (16.5)
               20        45.5 [+ or -] 4.2 (a)   39.6 [+ or -] 3.1 (b)
                           (28.3)                  (39.2)
Alkane          5        49.5 [+ or -] 4.2 (a)   36.5 [+ or -] 1.8 (a)
                           (22.0)                  (44.0)
               10        51.6 [+ or -] 3.9 (a)   34.2 [+ or -] 1.6 (a)
                           (18.7)                  (47.5)
               20        42.2 [+ or -] 2.6 (a)   51.4 [+ or -] 3.2 (a)
                           (33.5)                  (21.1)
Alkanol         5        46.5 [+ or -] 1.3 (a)   58.5 [+ or -] 9.1 (a)
                           (26.7)                  (10.2)
               10        54.6 [+ or -] 1.1 (b)   45.4 [+ or -] 8.2 (a)
                           (14.0)                  (30.3)
               20        49.2 [+ or -] 4.2 (a)   37.6 [+ or -] 2.8 (a)
                           (22.5)                  (42.3)
Alkanoic acid   5        33.4 [+ or -] 3.4 (a)   56.5 [+ or -] 3.1 (a)
                           (47.4)                  (13.3)
               10        47.5 [+ or -] 2.3 (a)   44.4 [+ or -] 1.0 (b)
                           (25.1)                  (31.9)
               20        39.4 [+ or -] 3.2 (b)   33.7 [+ or -] 3.4 (a)
                           (37.9)                  (48.3)
Indomethacin    5        21.5 [+ or -] 8.4 (a)   36.8 [+ or -] 4.3 (a)
                           (66.1)                  (43.5)
               10        36.4 [+ or -] 9.2 (a)   49.5 [+ or -] 3.2 (a)
                           (42.6)                  (24.0)
               20        34.5 [+ or -] 6.4 (b)   56.4 [+ or -] 4.2 (a)
                           (45.6)                  (13.4)

               Dose      Oedema rate (%)
Group          (mg/kg)*  10 h

Control        --        58.4 [+ or -] 2.1
Triterpene-I    5        44.6 [+ or -] 4.5 (a)
                           (23.3)
               10        40.8 [+ or -] 5.4 (a)
                           (29.8)
               20        50.7 [+ or -] 2.8 (a)
                           (12.8)
Triterpene-II   5        38.0 [+ or -] 8.2 (a)
                           (34.7)
               10        45.1 [+ or -] 1.6 (b)
                           (22.5)
               20        31.1 [+ or -] 1.0 (a)
                           (46.5)
Alkane          5        42.3 [+ or -] 2.8 (a)
                           (27.3)
               10        30.6 [+ or -] 3.1 (a)
                           (47.4)
               20        35.3 [+ or -] 4.2 (a)
                           (39.3)
Alkanol         5        30.4 [+ or -] 6.1 (a)
                           (47.7)
               10        35.5 [+ or -] 5.8 (b)
                           (39.0)
               20        37.8 [+ or -] 4.2 (a)
                           (35.0)
Alkanoic acid   5        48.3 [+ or -] 3.1 (a)
                           (17.0)
               10        40.0 [+ or -] 4.2 (a)
                           (31.2)
               20        33.9 [+ or -] 3.1 (a)
                           (41.7)
Indomethacin    5        30.1 [+ or -] 4.1 (b)
                           (48.2)
               10        33.4 [+ or -] 3.2 (a)
                           (42.6)
               20        31.6 [+ or -] 2.3 (a)
                           (45.7)

*mg/kg, body weight; values represent the mean [+ or -] SD of six
animals for each groups; statistically significant from control (a)
p<0.01 and (b) p<0.05 (Dunnett's 't'-test). Each value in parenthesis
indicates the percentage inhibition rate.

Table 2. Effects of alkanoids and triterpenoids from T. amplexicaule
Roth. and indomethacin (reference drug) on CFA-induced paw oedema in
rats

               Dose      Oedema rate (%)
Group          (mg/kg)*  1 day                   3 day

Control        --        38.6 [+ or -] 2.1       44.5 [+ or -] 3.9
Triterpene-I    5        19.2 [+ or -] 3.4 (a)   31.3 [+ or -] 1.1 (a)
                           (50.2)                  (29.6)
               10        20.5 [+ or -] 3.6 (a)   36.5 [+ or -] 1.2 (a)
                           (46.8)                  (17.9)
               20        24.3 [+ or -] 4.9 (b)   33.5 [+ or -] 6.5 (b)
                           (37.0)                  (24.7)
Triterpene-II   5        26.3 [+ or -] 5.6 (a)   34.8 [+ or -] 8.2 (b)
                           (31.8)                  (21.7)
               10        29.2 [+ or -] 4.2 (a)   38.3 [+ or -] 5.2 (a)
                           (24.3)                  (13.9)
               20        31.1 [+ or -] 3.1 (a)   36.9 [+ or -] 4.3 (a)
                           (19.4)                  (17.0)
Alkane          5        17.3 [+ or -] 2.8 (a)   20.4 [+ or -] 2.6 (a)
                           (55.1)                  (54.1)
               10        15.1 [+ or -] 2.2 (b)   23.5 [+ or -] 1.6 (a)
                           (60.8)                  (47.1)
               20        19.4 [+ or -] 3.3 (a)   26.4 [+ or -] 9.2 (a)
                           (49.7)                  (40.6)
Alkanol         5        22.4 [+ or -] 3.9 (a)   33.1 [+ or -] 2.7 (a)
                           (41.9)                  (25.6)
               10        25.3 [+ or -] 4.1 (a)   38.4 [+ or -] 3.4 (a)
                           (34.4)                  (13.7)
               20        28.1 [+ or -] 3.2 (a)   31.5 [+ or -] 4.8 (a)
                           (27.2)                  (29.2)
Alkanoic acid   5        30.3 [+ or -] 5.6 (a)   36.6 [+ or -] 3.9 (a)
                           (21.5)                  (17.7)
               10        23.5 [+ or -] 4.1 (a)   29.5 [+ or -] 1.8 (b)
                           (39.1)                  (33.7)
               20        32.6 [+ or -] 3.9 (a)   24.8 [+ or -] 7.3 (a)
                           (15.5)                  (44.2)
Indomethacin    5        13.3 [+ or -] 2.1 (b)   17.5 [+ or -] 4.2 (a)
                           (65.5)                  (60.4)
               10        12.2 [+ or -] 1.3 (a)   21.4 [+ or -] 3.1 (a)
                           (68.3)                  (51.9)
               20        14.4 [+ or -] 1.1 (a)   28.2 [+ or -] 1.5 (a)
                           (62.6)                  (36.6)

               Dose      Oedema rate (%)
Group          (mg/kg)*  6 day                   9 day

Control        --        59.8 [+ or -] 6.8       75.9 [+ or -] 2.8 (a)
Triterpene-I    5        38.4 [+ or -] 1.9 (a)   56.5 [+ or -] 2.8 (a)
                           (35.7)                  (25.5)
               10        29.6 [+ or -] 3.8 (a)   36.6 [+ or -] 5.3 (a)
                           (50.5)                  (51.7)
               20        47.7 [+ or -] 3.8 (a)   46.8 [+ or -] 6.2 (a)
                           (20.2)                  (38.3)
Triterpene-II   5        52.2 [+ or -] 2.5 (a)   58.4 [+ or -] 4.7 (a)
                           (12.7)                  (23.0)
               10        47.6 [+ or -] 3.4 (a)   61.4 [+ or -] 3.8 (b)
                           (20.4)                  (19.1)
               20        34.2 [+ or -] 3.3 (a)   49.5 [+ or -] 2.8 (a)
                           (42.8)                  (34.7)
Alkane          5        26.5 [+ or -] 4.6 (a)   36.4 [+ or -] 1.6 (a)
                           (55.6)                  (52.0)
               10        30.6 [+ or -] 3.3 (a)   24.5 [+ or -] 2.6 (a)
                           (48.8)                  (67.7)
               20        41.1 [+ or -] 2.2 (a)   29.2 [+ or -] 3.2 (a)
                           (31.2)                  (61.5)
Alkanol         5        48.5 [+ or -] 1.3 (a)   66.4 [+ or -] 2.4 (a)
                           (18.8)                  (12.5)
               10        43.6 [+ or -] 5.8 (a)   53.2 [+ or -] 2.1 (a)
                           (27.0)                  (29.9)
               20        33.4 [+ or -] 4.2 (a)   59.4 [+ or -] 1.9 (b)
                           (44.1)                  (21.7)
Alkanoic acid   5        50.3 [+ or -] 4.9 (a)   68.4 [+ or -] 1.1 (a)
                           (15.8)                  (9.8)
               10        37.6 [+ or -] 8.6 (a)   51.2 [+ or -] 1.1 (a)
                           (37.1)                  (32.5)
               20        34.4 [+ or -] 9.2 (a)   48.5 [+ or -] 2.8 (a)
                           (42.4)                  (36.1)
Indomethacin    5        35.5 [+ or -] 1.6 (a)   33.4 [+ or -] 3.4 (a)
                           (40.6)                  (55.9)
               10        39.6 [+ or -] 2.8 (a)   49.2 [+ or -] 4.2 (a)
                           (33.7)                  (35.1)
               20        21.5 [+ or -] 4.6 (a)   51.5 [+ or -] 8.6 (b)
                           (64.0)                  (32.1)

               Dose      Oedema rate (%)
Group          (mg/kg)*  12 day

Control        --        72.3 [+ or -] 1.9 (a)
Triterpene-I    5        61.1 [+ or -] 3.9 (a)
                           (15.4)
               10        48.2 [+ or -] 2.2 (a)
                           (33.3)
               20        64.6 [+ or -] 3.5 (a)
                           (10.6)
Triterpene-II   5        69.2 [+ or -] 7.4 (a)
                           (4.2)
               10        58.1 [+ or -] 3.2 (a)
                           (19.2)
               20        55.2 [+ or -] 2.9 (a)
                           (23.6)
Alkane          5        46.6 [+ or -] 4.6 (b)
                           (35.5)
               10        32.5 [+ or -] 3.9 (a)
                           (55.0)
               20        49.4 [+ or -] 1.0 (a)
                           (31.6)
Alkanol         5        52.3 [+ or -] 4.0 (a)
                           (27.6)
               10        68.9 [+ or -] 5.6 (a)
                           (4.7)
               20        64.5 [+ or -] 8.2 (a)
                           (10.7)
Alkanoic acid   5        59.7 [+ or -] 7.9 (a)
                           (17.4)
               10        54.2 [+ or -] 6.5 (a)
                           (25.0)
               20        59.8 [+ or -] 4.6 (a)
                           (17.2)
Indomethacin    5        55.6 [+ or -] 3.8 (a)
                           (23.0)
               10        58.2 [+ or -] 2.1 (a)
                           (19.5)
               20        61.4 [+ or -] 1.1 (a)
                           (15.0)

*mg/kg, body weight; values represent the mean [+ or -] SD of six
animals in each groups; statistically significant from control (a)
p<0.01 and (b) p<0.05 (Dunnett's 't'-test). Each value in
parenthesis indicates the percentage inhibition rate.