Anti-inflammatory effect and high performance liquid chromatography ( HPLC ) analysis of the EtOAc extract from Panzeria alaschanica

The aerial parts of Panzeria alaschanica are used to treat pelvic inflammation and chronic pelvic inflammation. The anti-inflammatory effects of P. alaschanica have been well known in folk medicine, which suggest that extracted components from P. alaschanica could potentially treat inflammatory disease. The aim of this study was to discover the bioactive compounds of P. alaschanica. To achieve this, ethyl acetate (EtOAc) extract was extracted from the aerial parts of P. alaschanica and the antiinflammatory effects were investigated against inflammation induced by carrageenan and egg-albumin in rats. At the doses used, EtOAc extract (100, 200 and 400 mg/kg) showed significant and dosedependent anti-inflammatory effects. According to the high performance liquid chromatography (HPLC) analysis of EtOAc extract, there are two major families of compounds in the plant, namely phenylpropanoids (verbascoside, 5,6,7,4'-tetrahydroxyflavone-7-O-(6"-O-[E]-coumaroyl)-βglucopyranoside, 5,6,7,4'-tetrahydroxyflavone-7-O-(6"-O-[E]-caffeoyl)-β-glucopyranoside) and flavonoids (7,4'-dimethoxy-apigenin, isoramnetin-3-O-rutinoside, isoramnetin-3-O-glucoside, kaempferol-3-O-glucoside, hyperin), which may explain the activity.


INTRODUCTION
Inflammation is a pathophysiological response of living tissues to injuries that leads to the local accumulation of plasmatic fluid and blood cells, which involves a complex sequence of bio-chemical events closely associated to the pathogenesis of various diseases, such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, acute gout, and migraine (Vázquez et al., 2011;Kandati et al., 2012;Huang et al., 2011).Nowadays, although the synthetic anti inflammatory drugs are dominating the market, the element of toxicity from these drugs cannot be ruled out.Due to the adverse reactions of synthetic and chemical medicines that is causing gastrointestinal irritation and reappearance of symptoms after discontinuation, herbal medicines have made a comeback to improve our basic health needs.Many plants and herbs, such as ginger, turmeric and olive oil, have been shown to exhibit potent anti-inflammatory effects (Gomase et al., 2011).P. alaschanica, is a member of the Labiate family and distributed predominantly in Eerduosi of Inner Mongolia, China.P. alaschanica are one of the best-known traditional herbal medicines frequently used to treat pelvic inflammation and chronic pelvic inflammation (Li et al., 2011;Zhang et al., 2007).It is widely used in Mongolian as a substitute of the precious materia medica, Leonurus heterophyllus Sweet, which is used in treatment of edema, irregular menstruation, dysmenorrheal, amenorrhea and acute nephritis.However, there is few reported scientific study to support these claimed therapeutic and medicinal effects.During the recent years, flavonoids (Zhou and Bao, 2001;Hou et al., 2009) have been isolated from this plant, which were isolated from the ethyl acetate (EtOAc) extract of the aerial parts of P. alaschanica.
However, until recently, there have been few studies about the pharmacological effects of P. alaschanica.Recently, the anti-inflammatory effects of the EtOAc extract of P. alaschanica was studied by using eggalbumin-induced and carrageenan-induced rats.Moreover, a systematic chemical study was conducted on the EtOAc extract of P. alaschanica, which resulted in the isolation of phenylpropanoids and flavonoids (Hasan et al., 2012;Albacha et al., 2003;Meng et al., 2008;Julião et al., 2010) from the plant for the first time.This paper describes the HPLC analysis and the antiinflammatory activity of the EtOAc extract of P. alaschanica.

Plant
The aerial parts of P. alaschanica, used as experimental material, were collected in Eerduosi, Inner Mongolia of China, in July 2013 and identified by Prof. Buhebateer (Inner Mongolia University for Nationalities).A voucher (NO.20130722) was deposited in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities.

Extraction
The aerial parts of P. alaschanica (2.0 kg) was powdered and extracted by EtOAc (20 L) after extraction with 10 L chloroform (CHCl3).Evaporation of the solvent under reduced pressure resulted in the EtOAc extract (204.0 g).The dried extract was stored at 4°C before use.

Anti-inflammatory activity on carrageenan-induced paw oedema
The EtOAc extract was tested for anti-inflammatory activity on carrageenan-induced paw oedema, according to the method described by Winter et al. (1962).The animals were divided into eight groups of eight rats.The negative control group received distilled water (0.5 ml/kg, p.o.), the positive control group received luteolin (30 mg/kg, p.o.) and the test groups received the compounds at the doses of 100, 200 and 400 mg/kg p.o.The test was conducted using an electric plethysmometer 7140 (Ugo Basile, Italy).Carrageenan 2.5% (0.05 ml) was injected subcutaneously in the plantar surface of the rat's left hind paw 1 h after oral administration of the drugs to induce a progressive swelling of the paw.The paw volume, up to the tibiotarsal articulation, was measured at 0 h (before carrageenan injection) and 1, 3, 5, 7, and 12 h later.

Egg-albumin-induced inflammation in rats
In testing for the effects of the compounds against inflammation, the method described by Albacha et al. (2003), was adopted.Briefly, rats were grouped into eight (n = 8).The negative control group received distilled water (10 ml/kg, p.o.), the positive control group received luteolin (30 mg/kg, p.o.) and the test groups received the EtOAc extract at the doses of 100, 200 and mg/kg p.o.All the animals were injected with 0.1 ml of fresh eggalbumin subcutaneously into the left hind paw 30 min after the compounds and drug treatment.The volume of paw oedema of each rat was measured using a digital plethysmometer (LE 7500) prior to and 60 min after albumin injection and at every 60 to min.

Acute toxicity
For the assessment of acute toxicity, Wistar rats, male and female, were divided into groups of 10 animals.The EtOAc extract was given p.o. at the doses of 100, 500, 1000, 1500, 2000, and 3000 mg/kg from the first to seventh groups, respectively.The control group received p.o. distilled water (10 ml/kg).The mortality rate within 72 h period was determined and the LD50 was estimated according to the method described by references (Miller and Tainter, 1944).

Statistical analysis
Data were given as means ± standard error (SE); statistical analyses were done by using Student's t-test.P < 0.05 is considered significant.

Sample preparation for HPLC analysis
About 1.0 g of the dried EtOAc extract was ground into powder, accurately weighed and soaked with 25 ml water and acetonitrile solution (70 : 30, v/v) for 60 min at room temperature, then sonicated for 20 min.The solution was filtered through a 0.45 µm membrane filter before LC analysis.

Acute toxicity
No mortality was observed in groups of rats treated with the EtOAc extract of P. alaschanica.LD 50 values for the EtOAc extract is more than 3000 mg/kg.

Anti-inflammatory activity
The development of oedema induced by carrageenan corresponded to the events in the acute phase of inflammation mediated by histamine, bradykinin and prostaglandins produced under an effect of cyclooxygenase.The EtOAc extract had an antiinflammatory effect at 100, 200 and 400 mg/kg observable to 12 (p < 0.01), 7 (p < 0.01) and 4 h (p < 0.01), respectively (Figure 1A).The highest dose of EtOAc extract produced significant anti-inflammatory activity and the results were more than that of luteolin, while the activity of the medium dose is similar to luteolin.As is shown in Figure 1B, the EtOAc extract caused a significant and dose dependent inhibition of increase in paw edema.The highest dose of total flavonoids had higher anti-inflammatory effects than that of luteolin, while the activity of the lower dose is less than that of luteolin.

HPLC analysis and NMR identification of HPLC peaks
This developed HPLC assay method was applied to the analysis of the chemical constituents in the EtOAc extract obtained from P. alaschanica.The HPLC chromatogram is as shown in Figure 2, which mainly contained eight peaks and named as peaks 1 to 8. Subsequently, the peaks 1 to 8 were directly identified by the spectroscopic methods, including 1 H NMR (Table 1), 13 C NMR (Table 2) techniques, and by comparison with those reported in the literature (Hasan et al., 2012;Albacha et al., 2003;Meng et al., 2008;Julião et al., 2010).
The phenylpropanoids and flavonoids exhibited various biological properties like anti-bacterial, antiinflammatory, and antioxidant and hepatoprotective effect (Peng et al., 2003;Ghisalberti, 2000;Silva et al., 2005), which may explain the anti-inflammatory activity of the EtOAc extract.

DISCUSSION
The present experimental investigation revealed that the EtOAc extract from P. alaschanica possessed significant anti-inflammatory activity in experimental animals at a dose of 400 mg/kg.Carrageenan-induced hind paw edema is the standard experimental model of acute inflammation.Moreover, the experimental model exhibits a high degree of reproducibility (Cadirci et al., 2012).It has a biphasic effect.The first phase is due to the release of histamine and serotonin (5-HT) (0-2 h), while plateau phase is maintained by a kinin like substance (3 h) and a second accelerating phase of swelling is attributed to prostaglandins (PG) release (>4 h) (Winter et al., 1962).
The EtOAc extract from P. alaschanica produced significant inhibition of carrageenaninduced paw edema.The inhibition was similar to that of the standard drug-luteolin.There are several mediators or multi processes underlining the pathogenesis of fever.Inhibition of any of these mediators may bring about antipyresis   2002).On preliminary phytochemical screening, EtOAc extract from P. alaschanica was found to contain flavonoid compounds.Flavonoids are known to target prostaglandins, which are involved in the late phase of acute inflammation and pain perception (Morimoto et al., 1988).

Figure 1 .
Figure 1.(A) Effects of the EtOAc extract on carreageenan-induced paw oedema in rats; (B) Effects of the EtOAc extract on albumin-induced paw oedema in rats.Results are expressed as means ± SD; n = 8. *p  0.05 compared with control.**p  0.01 compared with control.***p  0.001 compared with control.

Figure 2 .
Figure 2. The HPLC-UV chromatogram of the EtOAc extract of P. Alaschanica.