Analgesic, anti-inflammatory and anti-ulcer properties of Thai Perilla frutescence fruit oil in animals

Abstract

Perilla frutescens fruit oil (PFO) is rich in α-linolenic acid (ALA) and exhibits biological activities. We aimed to investigate analgesic, anti-inflammatory and anti-ulcer activities of PFO and PFO-supplemented soybean milk (PFO-SM) in animal models. Analgesic activity was assessed in acetic acid-induced writhing in mice, while anti-inflammatory activity was performed in ethyl phenylpropiolate (EPP)-induced ear edema and carrageenan-induced hind paw edema in rats. Anti-ulcer effects were conducted in water immersion stress, HCl/ethanol and indomethacin-induced gastric ulcer in rats. Distinctly, PFO, containing 6.96 mg ALA and 2.61 mg LA equivalence/g, did not induce acute toxicity (LD50 > 10 mL/kg) in mice. PFO (2.5 and 5 mL/kg) and PFO-SM (0.05 mL PFO equivalence/kg) inhibited incidences of writhing (16.8, 18.0 and 32.3%, respectively) in acetic acid-induced mice. In addition, topical applications of PFO (0.1 and 1 mL/ear) significantly inhibited EPP-induced ear edema (59.3 and 65.7%, respectively) in rats, while PFO-SM slightly inhibited ear edema (25.9%). However, PFO and PFO-SM did not inhibit carrageenan-induced hind paw edema in rats. Indeed, PFO (2.5 and 5 mL/kg) significantly inhibited gastric ulcers in rats that induced by water immersion stress (92.4 and 96.6%, respectively), HCl/ethanol (74.8 and 73.3%, respectively) and indomethacin (68.8 and 88.9%, respectively), while PFO-SM did not. PFO displayed potent analgesic, anti-inflammatory and anti-ulcer properties, while PFO-SM exerted only analgesic properties. Thus, Thai PFO and its functional drink offer potential benefits in treatment of analgesic, inflammatory diseases and gastric ulcer.

Keywords: Perilla frutescence; analgesic; anti-inflammatory; anti-ulcer; fruit oil.

Figure 1. Fatty acid composition of PFO

HPLC analysis of ALA and LA in authentic standards (A) and PFO (B). The samples were derivatized with the mixture of Br-MMC and 18-crown-6 to produce a fluorogenic methyl-7-methoxycoumarin fatty acid derivative, fractionated on the C18 type column, eluted with a mobile solvent (acetonitrile:DI), and detected the fluorescence. ALA and LA in PFO concentrations were determined from their standard curves.

Figure 2. Analgesic effects of PFO, PFO-SM and ASA on acetic acid-induced writhing responses in mice

Male mice (n=6 each) were orally given DI, PFO (2.5 and 5 mL/kg), PFO-SM (0.05 mL PFO equivalence/kg) and ASA (150 mg/kg) for 30 min, then intraperitoneally injected with acetic acid (0.75%, 10 mL/kg), and recorded incidences of writhing for 15 min. Statistics was performed by one-way ANOVA test, which P<0.05 was considered significance. Numbers of writhing are expressed as mean ± SD. *P<0.05 when compared with DI. Numbers in brackets represent percentage of inhibition of the writhing.

Figure 3. Anti-inflammatory effects of PFO, PFO-SM and PBZ on EPP-induced ear edema in rats

DMSO, PBZ (1 mg/ear), PFO (0.1 and 1 mL/ear) and PFO-SM (0.01 mL PFO equivalence/ear) were topically applied to the inner and outer surfaces of rat’s ears, then induced by topical application of EPP (1 mg/20 µL/ear), and measured ear edema thickness before and after EPP application using a digital vernier caliper. Statistics was performed by one-way ANOVA test, which P<0.05 was considered significance. Data of edema thickness are expressed as mean ± SD, *P<0.05 when compared with DMSO (A). Percentage of inhibition of ear edema are shown (B).

Figure 4. Anti-inflammatory effects of PFO, PFO-SM and ASA on carrageenan-induced hind paw edema in rats

Male rats (n=6, each) were fed with DI, PFO (2.5 and 5 mL/kg), PFO-SM (0.05 mL PFO equivalence/kg) and ASA (300 mg/kg), then induced by intradermal injection of carrageenan (1%, 50 µL/paw) into the right hind paws of rats, and measured the paw edema volume using a plethysmometer. Statistics was performed by one-way ANOVA test, which P<0.05 was considered significance. Data of paw edema volume are expressed as mean ± SD, which *P<0.05 when compared with DI (A). Percentage of inhibition of paw edema are shown (B).

Figure 5. Anti-gastric ulcer effects of PFO, PFO-SM and cimetidine on water immersion, HCl/ethanol and indomethacin-induced gastric ulcer in rats

Male rats were induced with gastric ulceration by water immersion, feeding with HCl/ethanol (36.5% in ethanol, 5 mL/kg) or intraperitoneal injection of indomethacin (30 mg/kg). The rats were then treated with DI, PFO (2.5 and 5 mL/kg), PFO-SM (0.05 mL PFO equivalence/kg) and cimetidine (100 mg/kg). The rats were killed by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and measured gastric ulcer length. Statistics was performed by one-way ANOVA test, which P<0.05 was considered significant. Data of gastric ulcer length are expressed as mean ± SD. *P<0.05 when compared with DI. Percentage of inhibition of the gastric ulceration are shown in the bracket.