Lower-guiding Effect Of Radix Achyranthis Bidentatae In Sanmiao Wan Formula Using Acute Gouty Arthritis And Hyperuricaemia Rat Model

Sanmiao Wan(SMW) stems from Ermiao Wan, a basic prescription employed to treat gout in the clinic since Yuan dynasty, and is a famous TCM formula originally recorded in the monograph of Yi-Xue-Zheng-Zhuan(written by Yu Tuan, Qing Dynasty). SMW was used empirically to treat arthritic diseases for hundreds of years and recorded by Chinese Pharmacopoeia throughout successive dynasties, because of its good therapeutic effects of eliminating heat, excreting dampness, decreasing swelling and alleviating pain. SMW is composed of Phellodendri Chinensis Cortex(Chinese name of Huangbo, 400 g), Atractylodis Rhizome(Chinese name of Cangzhu, 600 g), and Achyranthis Bidentatae Radix(Chinese name of Niuxi, 200 g) with the compatible ratio of 2:3:1. According to the basic theories of traditional Chinese medicine, Phellodendri Chinensis Cortex served as the “Monarch drug” in SMW to treat the main disease and considered to be indispensible in the prescription. Protoberberine alkaloids such as berberine, jatrorrhizine and palmatine in Phellodendri Chinensis Cortex were proved to be the effective anti-inflammatory compounds of SMW. Atractylodis Rhizome acts as the “Minister drug” in SMW to help Phellodendri Chinensis Cortex. Essential oils such as β-eudesmol and atractylone were considered to be its active components. ABR serves as the lower-guiding drug and plays an important role in guiding the active components in SMW to the targeted tissues or organs in the lower part of the body, especially to enhance the therapeuticeffect of lower limbs. The phytoecdysones(e.g., β-ecdysterone, 25-rinokosterone) and triterpenoids(e.g., chikusetsusaponin IVa, zingibroside R1) were counted as the two main types of bioactive components of ABR. Since SMW is widely used as a therapeutic formula for gouty arthritis and hyperuricemia in clinical, acute gouty arthritis(AGA) rat model induced by monosodium urate crystal and hypoxanthine induced hyperuricemia rat model were used to in this paper, and a combination of pharmacodynamics and tissue distribution studies of alkaloids in AGA rats after multiple-dosing of SMW without or with ABR groups was used to illustrate the lower-guiding effect of ABR in SMW formula. In this study, the active alkaloids of berberine and jatrorrhizine in Phellodendri Chinensis Cortex were chosen as the model components of SMW. A sensitive and reliable ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of berberine and jatrorrhizine in rat plasma and tissues(heart, liver, spleen, lung, kidney, knee joint and ankle joint) after multiple oral administration of SMW without or with low, medium and high doses of ABR. The lower-guiding effect of ABR was investigated by combining pharmacodynamics study(knee joint swelling, synovial hyperplasia, inflammatory cell infiltration and serum uric acid levels) with the distribution studies of berberine and jatrorrhizine in the various tissues of monosodium urate crystal-induced AGA rats. The research work was illustrated in more detail bellow:Part I. Study on the Influence of Lower-guiding Effect of Achyranthis Bidentatae Radix on the Therapeutic Efficiency of Sanmiao Wan using Acute Gouty Arthritis and Hyperuricemia Rat ModelsThis study is designed to demonstrate the lower-guiding effect of ABR by making a comparason of the therapeutic effect between SMW without or with Achyranthis Bidentatae Radix(ABR) groups. Male Sprague-Dawley rats weredivided into six groups including model group, control group and SMW without or with low, medium and high doses of ABR groups, i.e., SMW(2:3:0) group(10 g/kg), SMW(2:3:1) group(12 g/kg), SMW(2:3:2) group(14 g/kg) and SMW(2:3:4) group(18 g/kg). Four drug therapy groups were orally administrated for five consecutive days, and rat model of acute gouty arthritis was established by intra-articular injection of monosodium urate crystal suspension(0.2 m L, 20 mg/m L) inside knee joint on day 2 during drug treatment slots. The therapeutic effect of SMW without or with ABR groups could be evaluated by measuring the diameter of the knee joint and pathological change in synovial lining and subsynovial tissues of AGA rats. Hyperuricaemia model induced by intraperitoneal injection of hypoxanthine(700 mg/kg) was in order to investigate the hypouricemic effect between SMW without and with ABR groups. In comparaion with control group, knee jiont swelling appeared after MSU crystal injection to rats in model group. According to the results of haematoxylin & eosin(H&E) staining, pathologic hallmarks of synovial hyperplasia, polymorphonuclear neutrophil leukocytes influx in the synovial lining and subsynovial tissues were noticeable in rat knee joints of model group after MSU injection when compared to control group. SMW(2:3:1) group, SMW(2:3:2) group and SMW(2:3:4) group have exhibited more efficiency than SMW(2:3:0) group on ameliorating knee joint swelling, inhibiting synovial hyperplasia and alleviating inflammatory cell infiltration of rats with acute gouty arthritis. What’s more, higher dose of Niuxi in SMW could control and ameliorate inflammation more significantly. Compared with the model group, treatment with SMW extracts could significantly decrease the uric acid level in hypoxanthine-treated rats. Higher dose of ABR resulted in more efficiency in reducing the uric acid levels. The results demonstrated that SMW with the lower-guiding drug ABR ameliorated the the symtoms of acute gouty arthritis rats and exhibited better anti-hyperuricemic effect. More importantly, the definite dose-response relationship of the lower-guiding drug was observed in this study. ABR is a promising guide-drug and promoter for oral joint-targeting drug delivery. The findings might also help to provide useful evidence for the clinicalapplications of this drug-guide effect.Part II. Study on the Influence of Lower-guiding Drug Achyranthis Bidentatae Radix on Tissue Distribution of the berberine and jatrorrhizine in SMW by UPLC–MS/MS.This study was designed to investigate the influence of the lower-guiding drug Achyranthis Bidentatae Radix(ABR) on plasma and tissue distribution of berberine and jatrorrhizine,the main active ingredients of Phellodendri Chinensis Cortex in Sanmiao Wan(SMW). After multiple-dosing of SMW(2:3:0)(10 g/kg), SMW(2:3:1)(12 g/kg), SMW(2:3:2)(14 g/kg), and SMW(2:3:4)(18 g/kg) groups respectively for five consecutive days, rat plasma and tissues(heart, liver, spleen, lung, kidney, knee joint and ankle) were analysed by UPLC/MS/MS. The optimal chromatographic conditions were achieved on a Shimadzu Inertsil ODS-3/C18 column(50 × 2.1 mm I.D., 2 μm). The mobile phase consisted of A(water-0.1% formic acid) and B(acetonitrile) with a linear gradient elution. The flow rate was 0.3m L/min. MS detection was conducted on a Triple Quadrupole Mass Spectrometer 5500(AB SCIEX, USA) equipped with an ESI interface in the positive ionization mode. Compared with SMW(2:3:0) group, the plasma level of berberine increased by 19.81%, 143.40%, and 681.13% in SMW(2:3:1) group, SMW(2:3:2) group and SMW(2:3:4) group. The distribution of berberine significantly decreased in heart and lung but evidently increased in liver and kidney in SMW with ABR groups. It is interesting to note that ABR increased the distribution of berberine in rat knee joint and ankle, and the berberine concentration of knee joint(targeted tissue) increased by 6.53%, 44.31%, and 212.96% in SMW(2:3:1) group, SMW(2:3:2) group and SMW(2:3:4) group. In addition, the tendency of jatrorrhizine distribution in rat plasma andtissues was similar to that of berberine.The dose-dependent lower-guiding effect of Achyranthis Bidentatae Radix was demonstrated by significantly enhancing the distribution of berberine and jatrorrhizine in lower limbs(knee and ankle) and liver, kidney belonging to lower energizer, and reducing their concentrations in heart and lung belonging to upper energizer.