| Despite the progress that has been made in the development of pain therapies in the past decades, there is still the need for new analgesic agents with confirmed efficacy and minimum side effects for the treatment of various painful conditions, especially chronic painful diseases. Natural products are one of the major sources of new drugs. Some clinically used analgesic agents, such as aspirin and morphine, were developed from natural products. In the search for novel analgesics with better new efficacy and safety,Bauhinia glauca subsp. hupehana has been chosen as research object in our study according to its traditional use in the treatment of painful conditions.B. glauca was considered being warm in property, bitter with taste and non-toxic in traditional Chinese medicine. It is a member of the genus Bauhinia, which belongs to the family Leguminosae. It is widely grown in the south and southwest of China. In folklore medicine, the leaves, stems and roots of B. glauca were used in the treatment of inflammatory and painful conditions, such as backache, testicular pain,hemostasis swelling pain and rheumatic arthritis. Although B. glauca appeared to be a useful pain–relief herb in traditional medicine for hundreds of years, there have been few reports on the antinociceptive activity of this plant and the mechanisms of its analgesic action. To explore and verify its medicinal value, experiments have been carried out in this study to investigate the analgesic and anti-inflammatory activities of the 85% ethanolic extract of B. glauca(BHE). The major chemical constituents of BHE have been isolated and identified and some of the isolated compounds were evaluated for their biological activity.1. Analgesic and anti-inflammatory activities of BHEAnalgesic activity The antinociceptive activity of BHE was assessed in mice using chemically and heat–induced pain models, such as the acetic acid–induced writhing, hot plate, tail–flick and glutamate tests. The acetic acid induced abdominal constriction test revealed that treatment with BHE could significantly decrease the abdominal constriction response at doses of 100 and 200 mg/kg(p.o.), showing by the mean number of writhings, and the inhibition of writhing was dose–dependent. The tail–flick test showed that BHE could significantly prolong the latency of nociceptive response of the mouse’s tail under infrared radiation(IR) compared with the control group at doses of 100 and 200 mg/kg(p.o.). This result suggested that BHE has central antinociceptive activity and the effect was dose–dependent. The hot plate test demonstrated that BHE could significantly prolong the duration of the mouse’s feet on the hot plate at the doses of 100 and 200 mg/kg. This result confirmed the central effect of BHE. The glutamate–induced nociception test showed that BHE(100 and 200 mg/kg)dose–dependently produced significant inhibition of the glutamate–induced nociception.The antinociceptive activities of some fractions of BHE have also been evaluated by using the acetic acid–induced writhing and tail–flick tests. The both tests showed that the ethyl acetate and n-butanol fractions had significant analgesic activity at the dose of 200 mg/kg(p.o.). The ethyl acetate and n-butanol fractions were thus the main active fractions responsible for the analgesic activity of BHE. The antinociceptive effects of these tow fractions involved probably the central nerve system.Possible mechanism of the antinociceptive effect The mechanism of antinociceptive effect of BHE was evaluated in mice to observe the involvement of the opioid system, the cyclic guanosine monophosphate(c GMP) pathway and the ATP–sensitive K+channel pathway. For the assessment of the involvement of the opioid system in the analgesic activity of BHE, the non–selective opioid receptor antagonist naltrexone(5 mg/kg, s.c.) was administered. The data obtained in the hot plate test showed that naltrexone was not able to reverse the antinociceptive effect produced by BHE, showing that the antinociceptive effect of BHE was not due to the opioid receptor.The current study also investigated the involvement of the c GMP pathway in theantinociceptive activity of BHE. To evaluate the involvement of c GMP in the analgesic activity of BHE, methylene blue(MB), a guanylyl cyclase inhibitor, was administered prior to inducing nociception with an intraperitoneal injection of acetic acid. This result illustrated that methylene blue significantly decreased the nociception caused by acetic acid and also enhanced the antinociceptive effect of BHE, suggesting that the antinociceptive activity of BHE may involve the c GMP pathway. It has been also observed that the antinociceptive effect of BHE may involve the participation of the ATP–sensitive K+channel pathway because glibenclamide, an ATP–sensitive K+channel antagonist, could partially reverse the antinociceptive activity of BHE. The glutamate test showed also an association between the antinociceptive activity of BHE and the glutamatergic system.Acute toxicity The acute toxicity of BHE was tested according to the up–and–down method. BHE was found to be completely safe up to a dose of 2 g/kg(p.o.), and no gross effects or mortality were observed during 24 h for any animal. Therefore, the LD50(lethal dose, 50%) value for BHE in mice was estimated to be higher than 2 g/kg(p.o.).Because the dose used in the acute toxicity test was 20–fold higher than the effective dose used in the other experiments(100 mg/kg, p.o.), it is assumed that the doses given to the mice in this study(50, 100, and 200 mg/kg, p.o.) were safe.Anti-inflammatory activity The anti-inflammatory activity of BHE was assessed in mice using xylene-induced mice ear oedema and carrageen induced rat paw oedema tests. In the xylene-induced mice ear oedema and carrageen induced rat paw oedema tests, BHE exhibited significant anti-inflammatory activity at doses of 100 and 200 mg/kg(i.p.). The anti-inflammatory activity of BHE was dose–dependent2. Phytochemical constituents of BHEThe phytochemical constituents of BHE have been isolated in several steps, including extracting the herbal material under reflux, liquid-liquid partition and different types of column chromatography. The structures of the isolated compounds were identified by NMR, UV and MS methods. Twenty-two isolated compounds included flavonoids, phenolic acids, alkaloids, lignans and small molecular compoundsand the majority of them were flavonoids. They were identified to be gallic acid, luteolin,-sitosterol, 5, 7-Dihydroxychromone, umbelliferone, anisaldehyde, cinnamic acid, quercetin, fisetin, farrerol, peperomin B, eriodictyol, 3’, 4’, 7-trimethoxyflavone,ethyl gallate,(-)-epicatechin,(E)-Ethyl 3-(4’-phenmethyl) acrylate,3’,4’,7-Trihydroxyflavone, N-(2-hydroxyethyl)-N-methylcinnamamide,4’-hydroxy-7-methoxyflavonone, garbanzol, syzalterin and tupichinol E, respectively.Among them, peperomin B and N-(2-hydroxyethyl)-N-methylcinnamamide were isolated from the genus Bauhinia for the first time.3. Analgesic and anti-inflammatory activities of isolated compoundsThe compounds gallic acid, peperomin B and farrerol were tested for analgesic activity. In the acetic acid induced abdominal constriction test, peperomin B could significantly decrease the abdominal constriction response at the dose of 50 mg/kg(i.p.) and the inhibition rate was 54.89%. Gallic acid and farrerol have not shown analgesic activity. Peperomin B showed negative result in the tail–flick test, indicating that its antinociceptive effect was peripheral but not central activity. The anti-inflammatory activity of peperomin B was investigated by the xylene-induced mice ear oedema test,in which it significantly decreased the ear oedema. According to the literatures,peperomin B elicits anti-inflammatory effect through the mechanism of inhibiting the NF- B signaling pathway activated by TNF- and IL-1. |
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