Figure 2.1Acetic Acid-induced Abdominal Writhes (1.2 per cent saline, 100 μL/10 g, i.p.) in MicePreviously Treated (60 min., p.o.) with Vehicle (water, 100 μL/10 g), Indomethacin (indo.; 10 mg/kg),or Hydroalcoholoic Extracts Obtained from Branches (HE-B) of Ixora coccinea (0.3, 1 or 2 g/kg). Thecolumns and vertical bars represent the mean±S.D. of six animals in each experimental group.Statistical significances: *p<0.05; **p<0.01.

The treatment (p.o.) with HE-L (2 g/kg) and HE-R (2 g/kg) reduced in 36 per cent (36.4±4.2 writhes) and 57 per cent (24.4±4.4), respectively, the number of cumulative writhes. The experimental group (water) presented 57.2±2.2 writhes and the treatment with the positive control, indomethacin (10 mg/kg, p.o.) inhibited 59 per cent the total number of cumulative writhes (23.4±1.3) (Figure 2.2).

Figure 2.2:Acetic Acid-induced Abdominal Writhes (1.2 per cent saline, 10 µL/g, i.p.) in Mice Previously Treated (60 min., p.o.) with Vehicle (water, 100 µL/10 g), Indomethacin (indo., 10 mg/kg), or Hydroalcoholoic Extracts Obtained from the Leaves (HE-L) and Roots (HE-R) of Ixora coccinea (2 g/kg). The columns and vertical bars represent the mean±S.D. of six animals in each experimental group. Statistical significance: *p<0.01.

The total number of acetic acid-induced abdominal writhes in the mice group treated with the vehicle (DMSO 0.5 per cent, p.o.) was 55.3±8.3 (Figure 2.3).

The administration of ME-B (2 g/kg, p.o.) reduced in 61 per cent (21.3±4.7 writhes) the total number of cumulative writhes. The group treated with indomethacin (10 mg/kg, p.o.) showed 72 per cent writhing inhibition (15.3±7.0 writhes). However, the treatment at the same dose (2 g/kg, p.o.) with ME-F did not show significant changes in the total number of cumulative writhes (52.7±8.1), when compared to the control group (Figure 2.3).

Hot-plate Assay

The treatment with HE-B (1 g/kg, p.o.) did not cause significant changes in thermal nociception, while the treatment with the positive control phentanyl (500 µg/kg, s.c.) increased substantially the thermal response time at 0 (23.2±1.8 s), 30 (15.8±2.6 s), and 60 min. (13.0±2.3 s), when compared to control group (water) (7.0±0.7, 8.5±1.1, and 7.9±1.1 s, respectively) (Figure 2.4).

Formalin Assay

In the formalin assay, the treatment with HE-B (2 g/kg, p.o.) reduced in 69 per cent (40.4±21.1 s) the inflammatory nociception (second phase), when compared to control group (130.1±30.6 s) (Figure 2.5). In similar manner, the positive control (indomethacin 10 mg/kg, p.o.) reduced in 79 per cent (27.0±12.0 s) the pain reactivity associated to the second phase. However, at the early phase of the test (first 5 min), none of the treatments showed results statistically different from control group (128.1±9.7 s) (Figure 8.5).

Figure 2.3:Acetic Acid-induced Abdominal Writhes (1.2 per cent saline, 10 µL/g, i.p.) in Mice PreviouslyTreated (60 min., p.o.) with Vehicle (DMSO 0.5 per cent), Indomethacin (indo.; 10 mg/kg), and MethanolicExtracts Obtained from the Flowers (ME-F) and Branches (ME-B) of Ixora coccinea (2 g/kg). Thecolumns and vertical bars represent the mean±S.D. of six animals in each experimental group.Statistical significance: ***p<0.001.

Figure 2.4: Thermal Antinociceptive Effect of Hydroalcoholic Extracts Obtained from the Branches(HE-B) of Ixora coccinea (q–1 g/kg p.o.), in Terms of Time Interval in which the Mouse Stands on HotPlate P55.0±1.0 ºC). Phentanyl (1–500 µg/kg s.c.) was the positive control while the negative controlgroup was treated with water (o–10 mL/g, p.o.). The results shown herein represent the mean±S.D. ofeight animals in each experimental group. Statistical significance: ***p<0.001.

Figure 2.5: Reactivity (control per cent) to Sub-plantar Application of Formalin (20 µL salineformaldehyde 1.2 per cent) in Mice Right Hind Paw Previously Treated (60 min., p.o.) with Vehicle (10µL/g), HE-B (2 g/kg), Or indomethacin (indo.; 10 mg/kg). The columns and vertical bars represent themean of phase I (0 to 5 min., neurogenic pain) and phase II (15 up 30 min., inflammatory pain)±S.D. oftwelve animals in each experimental group. Statistical significance: **p<0.01, ***p<0.001.

Rota Rod Assay

The control group (vehicle) remained on the rota rod apparatus for 60.0±0.0 s, with 1.6±0.5 falls down over a period of 1 minute. This performance was not affected by the oral administration (2 g/kg, p.o.) of HE-L, HE-B, and HE-R (60.0±0.0, 57.5±1.8, and 53.5±6.4 s, respectively) (Figure 2.6).

Figure 2.6: Evaluation of Remain Time on the Rota Rod Apparatus. Experimental groups of sixanimals were submitted to oral treatment with vehicle (water), hydroalcoholic extracts obtainedfrom the leaves (HE-L), branches (HE-B), and roots (HE-R) of Ixora coccinea (2 g/kg). The columnsand vertical bars represent mean±S.D.

Open-field Assay

The results obtained for locomotors activity and exploratory behavior of mice from open-field test corroborate those obtained from rota rod test. After treatment with HE-R, HE-B, and HE-L (2 g/kg, p.o.), the number of square crossings observed was 75.4±9.8, 67.6±6.1, and 58.4±10.6, respectively. These results do not differ from the control group result (71.7±14.1 crossings). Similar results were observed for the number of central square crossings 6.0±1.1 (HE-R), 5.1±1.1 (HE-B), 2.7±1.1 (HE-L), and 6.4±1.4 crossings (vehicle); rearing behavior 44.4±4.7 (HE-R), 40.4±4.2 (HE-G), 24.3±4.6 (HE-L), and 28.1±7.5 (vehicle); time consuming of grooming 21.3±5.8 (HE-R), 23.3±4.5 (HE-B), 19.4±5.5 (HE-L) and 12.3±2.9 s (vehicle).

Dicussion and Conclusions

The phytochemical study on the methanolic extract obtained from the flowers of Ixora coccinea (ME-F) resulted in the identification of 40 compounds, including the triterpene ursolic acid, the carbohydrate mannitol, and the steroids β-sitosterol and stigmasterol, in which this triterpene was the major compound (0.18 per cent). The triterpene lupeol was isolated from HE-L, as a mixture containing also the two terpenoids α–and β-amirin. This triterpene, previously isolated from Ixora coccinea leaves, a specimen collected in Thiruvanathapuram (India) that showed antiinflammatory and antimicotic activities (Reena et al., 1994). In this present work those isolated constituents were confirmed by spectroscopic analyzes, including NMR ¹H, ¹³C, DEPT and HMQC data). Additionally, analytical study by prospection suggested the presence of organic acids, steroids, triterpenes, anthocyanins, carbohydrates, and saponins in the hydroalcoholic extracts of flowers (HE-F), leaves (HE-L), branches (HE-B), and roots (HE-R), as well as tannins in HE-R. Comparing the GC-MS analysis of the non-polar fractions (F1-19 obtained from ME-F) and (F1-5 obtained from HE-L) to those previously results reported to Ixora coccinea roots, which was rich in palmitic, stearic, oleic and linoleic acids (Latha and Panikkar, 1998), only heavy acids, stearic and palmitic acids are present in both flowers and leaves of the specimen collected in Brazil. Those phytochemical results confirmed that Brazil climatic conditions do not affect the occurrence of the chemical constituents of Ixora coccinea, which was collected in Botanical Garden of Universidade Federal Rural do Rio de Janeiro (Brazil), been in agreement with those previously reported phytochemical data. In other hand, this present work improves its chemical data, since other compounds were cited to be isolated or detected by GC-MS analyzes, in flowers and leaves of Ixora coccinea.

Previously pharmacological results pointed out special features for a bioactive fraction obtained from the methanolic extract obtained from the flowers of Ixora coccinea Linn. (AF-ME-L) from which a non-toxic effect and antitumoral activity were detected (Latha and Panikkar, 1998). The triterpene ursolic acid was isolated from this extract and its antiviral activity was reported (Latha, 1999). Concerning to ursolic acid (3b-hydroxy-urs-12-en-28-oic acid) and its position isomer oleanolic acid (3b-hydroxy-olean-12-en-28-oic acid), belong respectively to the ursane and the oleanane groups, they share many common pharmacological properties and are included among the most notable bioactive triterpenoid compounds (Liu, 1995). During the last decades over 900 research articles have been published on ursolic acid (UA) as well as oleanolic acid (OA), reflecting tremendous interest and progress in their pharmacological importance, including in those studies UA and OA chemical modifications to make this natural products more effective and water soluble derivatives. Pharmacological research of both UA and OA, includes pre-clinical and toxicity evaluations, and human clinical uses. Among the many important pharmacological effects anticancer chemotherapies, hypolipidemic, hypoglycemic, and hepatoprotective effects; antiatherosclerotic and antidiabetogenic properties; cardiovascular protective effect; gastrointestinal transit modulating activity; antiulcer, antiinflammatory, antibacterial, and antifungal activities; additionally to immunomodulatory, and diuretic properties were confirmed, as well as their insecticidal property (Shatilo et al., 1973; Kowalewski et al., 1976; Parfentieva, 1979; Gupta et al., 1981; Vasilenko et al., 1981; 1982; Yamahara et al., 1981; Kosuge et al., 1985; Wrzeciono et al., 1985; Ma, 1986; Collins and Charles, 1987; Liu et al., 1987; Lee et al., 1988; Hao et al., 1989; Hirota et al., 1990; Huang et al., 1994; Liu et al., 1994; 1998; 1999; Liu, 1995; 2005; Sattar et al., 1995; Hsu et al., 1997; Jeong et al., 1999; Latha and Panikkar, 1999; 2001; Li et al., 1999; 2002; Mizushina et al., 2000; Tang et al., 2000; Yoshikawa et al., 2000; Marquina et al., 2001; Mix et al., 2001; Alvares et al., 2002; Murakami et al., 2004; Somova et al., 2003). Additionally, the potent inhibitory effect of ursolic acid against HIV-1 protease (with IC₅₀ values of 8.0 µM) was reported by Min et al. (1999) and confirmed by Ma et al. (2000). Kashiwada et al. (2000) confirmed antiHIV effect for its derivatives. Moreover, ursolic acid has been shown to possess antiarthritic (Iwu and Ohiri, 1980).

Both ursolic acid and oleanolic acid exist widely in plant kingdom, medicinal herbs, and also are integral part of the human diet. These two triterpenes may occur in the form of free acids or aglycones of saponins (Li et al., 1999; Marquina et al., 2001; Janicsák et al., 2006; Kowalski, 2007; Razboršek et al., 2008), which represent a source of materials for the pharmaceutical industry. The fruiting spikes of Prunella vulgaris L. (Labiatae), known as “Hsia ku Tsao” in Chinese folklore, are used as herbal remedies for human tuberculosis, jaundice, infectious hepatitis, bacillary dysentery, pleuritis with effusion, and cancer (Zurcher et al., 1954; Lee et al., 1988). The triterpenoids UA and OA among other constituents, were isolated from the whole herb. From this other herbal remedies “Khin Piah Leng” obtained from Psychotria serpens (Rubiaceae) and/or Hyptis capitata (Labiatae) a specific bioassay-directed fractionation led to the isolation and characterization of ursolic acid as one of the bioactive principle of this herbal remedies (Zurcher et al., 1954; Lee et al., 1988). Reinforcing its uses, Oguro et al. (1998) reported the inhibitory effect of oleanolic acid on 12-O-tetradecanoylphorbol-13-acetate-induced gene expression in mouse skin. Because of its biological efficacy and apparent low side effects, oleanolic acid has been patented in Japan as additive to health drinks (Okudo et al., 1990).

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