Chemistry and pharmacology of the herb pair Flos Lonicerae japonicae-Forsythiae fructus

The Chinese medicine herb pair Flos Lonicerae japonicae (FLJ) and Forsythiae fructus (FF), is a representative heat-clearing (qing re) and detoxifying (jie du) combination that exhibits many pharmacological activities, including antibacterial, antiviral, antitumor, anti-inflammatory, and antioxidant effects. Extensive phytochemical studies have identified a series of bioactive compounds, such as chlorogenic acid from FLJ and forsythoside A from FF. This article provides a comprehensive review on the chemical and pharmacological principles of the traditional functions of FLJ-FF, and sheds light on further developments of this herb pair.


Introduction
Although Chinese medicine (CM) often uses multicomponent formulations and the actions of each component on multiple targets [1], the use of herb pairs-the unique clinical combination of two relatively fixed herbs-is the simplest and most fundamental form of multiherb therapy aimed at specific efficacy. The role of herb pairs has been explained by the yin and yang and five-phase theories [2], and by reference to the broader philosophical and cultural frameworks that emphasize balance between bodily functions and environmental conditions [3]. Herb pairs are simpler in composition than complete formulae but still therapeutically effective. There are several aims and principles of herbal compatibility, sometimes called the "seven relations of CM": singular application, mutual promotion, mutual assistance, mutual restraint, mutual detoxification, mutual inhibition, and mutual intoxication [4]. The principle of mutual promotion explains why herb pairs have significantly better pharmacological efficacy than individual herbs, a principle that is applied in many famous herb formulae, such as Yinqiao San [1]. Recent pharmacological investigation has clarified this mutual effect [5].
The herb pair of Flos Lonicerae japonicae (FLJ) and Forsythiae fructus (FF) has been widely used to cure febrile illness (e.g., cold and flu) at the primary stage [6]. FLJ is the flower bud of Lonicera japonica Thunb and FF is the dried fruit of Forsythia suspensa. Yinqiao San, which contains FLJ and FF with a crude weight ratio of 1:1, is used for detoxification and relieving internal heat and fever [7]. Nowadays, the various available dosage forms of FLJ-FF herb pair, such as capsules, powder, oral decoctions, and granules, are mainly indicated for cold, fever, and even upper respiratory tract infection [8]. The most familiar ones are Vc Yinqiao tablet and Shuanghuanglian oral decoction [9]. However, the mechanisms of the formulae have not been completely elucidated, and research on this combination has rarely been summarized. This article aims to provide a comprehensive and up-todate review of phytochemical and pharmacological studies of FLJ and FF.

Review
Ethnopharmacological use of the Yinqiao herb pair The FLJ-FF herb pair is described as light (qing) and floating (fu), and able to clear heat, combat swelling, and cure boils [10,11]. It has been widely used as an antipyretic, antidotal, and anti-inflammatory agent for the treatment of infections such as acute nephritis and erysipelas.

Pharmacological effects
Forsythoside A has strong antioxidant, antibacterial, and antiviral activities [14]. Forsythiaside exhibits strong antibacterial, antiviral, antioxidant, anti-inflammatory, and cyclic adenosine monophosphate phosphodiesterase inhibitory effects [15]; forsythin and rutin show a strong antioxidant effect [16]. In addition, chlorogenic acid possesses antibacterial and antiviral activities [17]. The bioactive properties of FLJ and FF are summarized below in terms of traditional functions and modern pharmacological findings.

Effects of the FLJ-FF herb pair
Studies of the synergistic action of the FLJ-FF herb pair are rare. There is little evidence for its synergistic properties, although Li et al. [18] reported a synergistic antiinflammatory effect. They established a rat model of chronic obstructive pulmonary disease and treated the animals with FLJ-FF herb pair extract (FLJ:FF, 2:3) and a single herb extract. FLJ-FF herb pair treatment improved chronic obstructive pulmonary disease pathological changes and significantly reduced interleukin-1β (IL-1β) levels in bronchoalveolar lavage fluid compared with each single herb. Duan et al. [19] evaluated anti-free radical activity of the FLJ-FF herb pair using a rat fever model. They divided Sprague-Dawley rats into different groups, treated some groups with 20 % dilute yeast suspension to create a fever model, and then tested the effectiveness of different drug combinations. The FLJ-FF herb pair showed potent free radical cleavage activity. Using microbial-plate methods, Wang et al. [20] found the FLJ-FF herb pair at the ratio of 1:6 showed the most significant inhibitory effect on Streptococcus suis 2.
One study investigated the antimicrobial activity of FLJ water and alcohol extract. The MIC and MBC values for the water extract on Staphylococcus aureus were 19.25 and 38.50 %, respectively; the MIC and MBC values for the alcohol extract on Salmonella enteritidis were 9.80 and 19.60 %, respectively, and for Staphylococcus aureus were 19.60 and 39.20 %, respectively [26]. FLJ flavonoids also showed a strong antibacterial action, especially for methicillin-resistant Staphylococcus aureus (MIC ≤5 mg/mL) [27]. These reports suggest that FLJ is a potent agent for treating various bacteria.

Detoxifying: antiviral activity
Several studies have demonstrated the antiviral activity of FF. FF aqueous extract showed antiviral activity against respiratory syncytial virus (RSV) with IC 50 50 μg/mL and CC 50 1000 μg/mL [28]. Wen et al. found that 80 % ethanol extract of FF had a significant protective effect on Madin-Darby canine kidney cells infected by the H 1 N 1 virus in a dose-dependent manner [29]. Li et al. [30] assessed the effects of forsythoside A on cell infection by avian infectious bronchitis virus; the data indicated that this compound prevented virus infection in vitro, but the mechanisms remain unclear.
Many studies have disclosed antiviral activity of FLJ, including anti-RSV, anti-HIV (human immunodeficiency virus), and anti-NDV (Newcastle disease virus) effects. Two studies used a cytopathologic effect (CPE) assay to test the antiviral activities against RSV of 44 medicinal herbs used for the treatment of respiratory tract infectious diseases in China [28,31]. FLJ showed potent antiviral activity against RSV; the IC 50 was 50.0 μg/mL and the selectivity index was more than 20.0. FLJ extract and chlorogenic acid had significant anti-cytomegalovirus activity, and the 0 % toxic dose, minimum effective concentration, and therapeutic index (TI) of these two composites for human cytomegalovirus were 3000 μg/mL, 3000 μg/mL, 1 and 100 μg/mL, 1 μg/mL, 100, respectively [32]. In in vitro tests, FLJ extract showed 104 and 72 times the TI for anti-herpes simplex virus-1 F and anti-herpes simplex-1 to acyclovir. Regarding the caviid beta-herpes virus 1, FLJ showed significant inhibition of the duplication of guinea pig cytomegalovirus at the cell level; the TI and the inhibitory duplication index were 100 and 2.61 μg/mL, respectively [33]. The anti-virus (H 9 N 2 ) and anti-avian influenza virus (least effective dose [LED] = 3.90 mg/mL, in vitro) activities of FLJ flavones have also been tested [34]. In Vero cells, three different extracts of FLJ, including volatile oil (P1), chlorogenic acids extract (P2), and flavones extract (P3), were tested for antiviral activity against the pseudorabies virus (PRV) and NDV.  16.37 %, 6.73 %, respectively. For P2 and P3, the LEDs against PRV were 0.997 and 3.097 mg/mL, respectively and against NVD, they were 0.781 and 1.563 mg/mL, respectively. These studies suggest that FLJ extracts decrease CPE lesions and neutralize viruses in a dose-dependent manner, inhibiting viruses directly and promoting cell antivirus responses [35].
Several FLJ tannins have also been investigated; 3,5-di-O-caffeoylquinic acid and methyl 3,5-di-O-caffeoylquinic acids had a strong inhibitory effect on HIV-1 reverse transcriptase (RT) and human DNA polymerase-α (HDNAPα) [36]. The IC 50 ratio of these two compounds for HIV-1 RT and HDNAP-α was 2.0 and 2.2, respectively. 3,4-di-Ocaffeoylquinic acid and methyl 3,4-di-O-caffeoylquinic acid exhibited higher inhibitory effects on HDNAP-α than on HIV-1 RT [36]. Thirteen other caffeoylquinic acids isolated from FLJ, including caffeic acid and caffeic acid methyl ester, were also found to show antiviral activities against respiratory viruses [37]. FLJ extract showed an obvious therapeutic action on mice infected with influenza A virus pneumonia [31]. The lung indexes of the FLJ group and the ribavirin group were significantly lower than in the model group, but there was no significance difference between the two treatment groups. FLJ extract reduced histopathological changes, viral duplication, and the contents of influenza virus nucleic acid compared with the model group. The tumor necrosis factor-α (TNF-α) and IL-1β expressions of the FLJ and the ribavirin groups were significantly lower than those of the model group. The FLJ chemical principles for antiviral activity were identified as chlorogenic acids, flavones, tannins, and volatile oil [31].

Detoxifying: antitumor activity
The apoptosis mechanisms induced by photodynamic therapy (PDT) in lung CH27 carcinoma cells, cultured with FLJ alcohol extract as a photosensitizer, have been explored. This extract exhibited significant photocytotoxicity in CH27 cells at a concentration range of 50-150 μg/mL, with light doses of 0.4-1.2 J/cm 2 . Apoptosis induced by PDT combined with FLJ extract was accompanied by DNA condensation, externalization of phosphatidylserine, and formation of apoptotic bodies [38]. The p38associated pathway might be involved in apoptosis induced by PDT with FLJ in CH27 cells. In another study, FLJ extract induced CH27 cell apoptosis via protein expression change and distribution of heat shock protein 27. Treatment with FLJ aqueous extract (100 μg/mL) was associated with increased stimulatory phosphorylation of c-Jun amino-terminal kinase and p38 in HepG2 cells, similar to the mitogen-activated protein kinase activation profile of protocatechuic acid [39]. This aqueous extract also decreased the viability of HepG2 cells to 50 % and triggered HepG2 cell death in a c-Jun amino-terminal kinase-dependent manner.

Heating clearing: anti-inflammatory activity
Inflammation prevents infection through production of pro-inflammatory cytokines and generation of inflammatory mediators in response to microbial products [40]. Dysregulation of inflammation has an adverse effect on the body. Although modern anti-inflammatory drugs can bring relief, new kinds of microorganisms and the emergence of drug-resistant strains have resulted in significant morbidity and mortality. In the past few decades, more attention has been focused on the anti-inflammatory effect of CM herbs, especially heat-clearing herbs [41].
Many studies have demonstrated the anti-inflammatory action of FF. FF was found to exhibit platelet-activating factor antagonistic activity and inducible nitric oxide synthase inhibitory activity [42]. An FF methanol extract and its hexane fraction showed anti-inflammatory and analgesic activity against carrageenan-induced edema, cotton pellet-induced granuloma, and acetic acid-induced vascular permeability [43]. FF extract inhibited 5-lipoxygenase and elastase with the same IC 50 values of 80 μg/mL [44]. FF ethanol extract also inhibited the secretion of the cytokine RANTES from virus-infected human bronchial epithelial cells [45]. These findings suggest that FF possesses anti-inflammatory activity through multiple target signaling pathways and multiple mechanisms of action.
Both in vivo and in vitro studies have shown that FLJ extract can inhibit various inflammatory reactions and suppress various inflammatory factors. Xu et al. [46] evaluated the anti-inflammatory property of FLJ aqueous extract in A549 cells; the extract directly inhibited both COX-1 and COX-2 activity, and IL-1-induced expression of COX-2 protein and mRNA. Kang et al. [47] examined the effect of FLJ water fraction on trypsin-induced mast cell activation. After stimulation with trypsin (100 μM), FLJ water fraction inhibited TNF-α secretion, tryptase mRNA expression, and trypsin-induced extracellular signal-regulated kinase phosphorylation in a dosedependent manner; however, it did not affect trypsin activity even at 1000 μg/mL. These studies indicate that FLJ might inhibit trypsin-induced mast cell activation through the inhibition of extracellular signal-regulated kinase phosphorylation rather than by inhibition of trypsin activity. One study evaluated the anti-inflammatory activity of n-butanol (4.2 % based on the dry weight [DW]) FJL fraction [48]. At a 400 mg/kg oral dose, it showed significant anti-inflammatory activities against arachidonic acid ear edema, croton-oil ear edema, carrageenan paw edema, and rat cotton pellet granulomatous and adjuvant-induced arthritis inflammation models in mice and rats; the inhibition rates were 27 %, 23 %, 26 %, 18 %, and 42 %, respectively and the inhibition rates for the positive drug aspirin (100 mg/kg) were 27 %, 13 %, 13 %, 0 %, and 58 %, respectively.
FLJ water extract showed an anti-inflammatory effect on proteinase activated receptor 2 (PAR2)-mediated mouse paw edema; at doses of 50, 100, and 200 mg/kg, it significantly inhibited paw thickness change and vascular permeability induced by PAR2 (inhibition rates: 41.8 %, 69.1 %, 70.9 %, and 40.2 %, 69.7 %, 68.8 %, respectively). FLJ water extracts (100 mg/kg) also significantly inhibited PAR2 agonist-induced myeloperoxidase (MPO) activity and TNF-α expression in paw tissue [49]. Tae et al. [50] used the supercritical CO 2 extraction process to obtain 1.08 % volatile oil from FLJ; pharmacological studies suggested a potent anti-inflammatory effect of the volatile oil on the ear-swelling model in mice. These reports indicate that FLJ is a safe, mild anti-inflammatory agent for treating various inflammatory disorders.

Heat clearing: antioxidant activity
Excessive reactive oxygen species result in significant damage to biological structures necessary to cellular integrity and survival. CM heat-clearing herbs are an important source of antioxidant agents. A study using a 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiment found that the CH 2 Cl 2 fraction of F. suspensa exerted the strongest scavenging activity and suggested that forsythialan A and phillygenin F are the major antioxidant constituents [51,52]. Zhang et al. [53] studied the role of forsythoside A in the elimination of reactive oxygen species and discussed the relationship between structure and activity using quantum chemical calculation. The results showed that the A and B rings in forsythoside A were active parts of its antioxidant activity, and the structure of phenolic hydroxyl groups in opposition caused higher antioxidant activity. Moreover, lianqiaoxinoside B and forsythoside H showed nearly the same antioxidant activities. These phenylethanoid glycosides have two ortho-substituting hydroxyl groups in both the caffeoyl and phenylethanoid moieties, which could be an important factor in their high antioxidant activity [54]. Lignans obtained from FF could protect human high-density lipoprotein against lipid peroxidation. In one study, they inhibited the generation of thiobarbituric acid-reactive substances in a dose-dependent manner with IC 50 values from 8.5 to 18.7 μM. Among these lignans, some exerted an inhibitory effect against the Cu 2+ -induced lipid peroxidation of high-density lipoprotein, as shown by an extended lag time prolongation at a 3.0 μM concentration [55]. The protective activity of F. suspensa against peroxynitrite (ONOO)-induced cellular damage was investigated, and its active components, phillygenin and 8-hydroxypinoresinol, were identified. These two compounds significantly reduced cell injury by 3-morpholinosydnonimine, an ONOO generator. The hydroxyl substituents of these lignans on the phenyl moieties may contribute to the antioxidant activity [56].
The antioxidant action of FLJ has been widely investigated. The FLJ ethyl acetate fraction exhibited marked scavenging/inhibitory activities with IC 50 values of 4.37, 27.58 ± 0.71, 0.47 ± 0.05, and 12.13 ± 0.79 μg/mL in the DPPH radical, total reactive oxygen species, hydroxyl radical (−OH), and peroxynitrite (ONOO − ) assays, respectively [57]. The main compounds of the ethyl acetate fraction-luteolin, caffeic acid, protocatechuic acid, and luteolin 7-O-d-glucopyranoside-also evidenced marked scavenging activities, with IC 50 values of 2.08-11.76 μM for DPPH and 1.47-6.98 μM for ONOO − [57]. The Trolox equivalent antioxidant capacity values and total phenolic content for methanolic extracts of FLJ have been demonstrated as 589.1 μmol Trolox equivalent/100 g DW and 3.63 gallic acid equivalent/100 g DW [58]. These studies suggest that FLJ is a potential natural antioxidant and beneficial chemopreventive agent. The antioxidant activity of polysaccharides with different molecule weights separated from FLJ by ultrafiltration was also studied. The reducing power of the polysaccharides had a direct correlation with antioxidant activity and concentration of certain plant extracts, and the ultrafiltration fraction had a significant inhibitory effect on superoxide radicals generated in a phenazine methosulphate/hydrogenated nicotinamide adenine dinucleotide/ nitroblue tetrazolium system. Administered to rats, crude polysaccharide extracts (50-400 mg/kg) were found to reduce lipid peroxidation malondialdehyde content, improve glutathione peroxidase and catalase activity, and significantly enhance superoxide dismutase activity in serum and tissue [59].

Limitations of this review
Few studies demonstrated a synergistic or additive effect for this herb pair. Comparable studies, using both single herbs (FLJ and FF) and the FLJ-FF herb pair, should be conducted to investigate possible synergistic or additive effects. Interdisciplinary research is needed to identify minor bioactive components using phytochemical studies, to generate reliable cell and animal models using pharmacological studies, and to elucidate underlying mechanisms using molecular biological studies.
All pharmacological studies reviewed here used in vitro or in vivo models; there was no clinical investigation of the effects of the FLJ-FF herb pair (or of single herbs). Thus, this review provides no clinical evidence for the bioactivities of FLJ and FF. In addition, some of the pharmacological targets of the FLJ-FF herb pair are still unknown.

Conclusion
The main bioactive components of FLJ and FF are flavonoids, organic acids, volatile oil, phenylethanoid glycosides, lignans, and triterpenoids. These show clear pharmacological effects, including antibacterial, antiviral, anti-inflammatory, antitumor, and antioxidant actions.