Crataegus pinnatifida: Chemical Constituents, Pharmacology, and Potential Applications

Crataegus pinnatifida (Hawthorn) is widely distributed in China and has a long history of use as a traditional medicine. The fruit of C. pinnatifida has been used for the treatment of cardiodynia, hernia, dyspepsia, postpartum blood stasis, and hemafecia and thus increasing interest in this plant has emerged in recent years. Between 1966 and 2013, numerous articles have been published on the chemical constituents, pharmacology or pharmacologic effects and toxicology of C. pinnatifida. To review the pharmacologic advances and to discuss the potential perspective for future investigation, we have summarized the main literature findings of these publications. So far, over 150 compounds including flavonoids, triterpenoids, steroids, monoterpenoids, sesquiterpenoids, lignans, hydroxycinnamic acids, organic acids and nitrogen-containing compounds have been isolated and identified from C. pinnatifida. It has been found that these constituents and extracts of C. pinnatifida have broad pharmacological effects with low toxicity on, for example, the cardiovascular, digestive, and endocrine systems, and pathogenic microorganisms, supporting the view that C. pinnatifida has favorable therapeutic effects. Thus, although C. pinnatifida has already been widely used as pharmacological therapy, due to its various active compounds, further research is warranted to develop new drugs.


Introduction
Crataegus pinnatifida, including Crataegus pinnatifida Bge. var. major N. E. Br. and C. pinnatifida Bge, is a traditional, popular Chinese medicinal herb that belongs to the Rosaceae family and is widely distributed in the north of China [1]. Modern investigations have demonstrated that C. pinnatifida has various pharmacological effects on, for example, the cardiovascular, digestive, and endocrine systems, as well as on pathogenic microorganisms [2]. To date, over 150 chemical constituents have been identified from this plant, including flavonoids, triterpenoids, steroids, lignans, organic acids, and nitrogen-containing compounds [3]. Due to its wide spectrum of biological and pharmacological effects, C. pinnatifida has a long history of use as a medicinal plant in China. In the Compendium of Materia Medica (Bencao Gangmu), a famous Traditional Chinese Medicine monograph, the earliest use of dried fruit of C. pinnatifida has been described as treatment for cardiodynia, hernia, dyspepsia, postpartum blood stasis, and hemafecia [4]. In addition to being used as a therapeutic medicine, the slightly sour fruit of C. pinnatifida is commonly used as a delicious daily food source in China [5].
In the present review we summarize the research advances on the chemical composition, pharmacology and toxicology of C. pinnatifida, which will be important for the development of new drugs and full utilization of C. pinnatifida. Additionally, we have discussed the potential perspective for future investigations of C. pinnatifida.

Chemical Composition
Research on the chemical components of C. pinnatifida started in the 1960s. Currently, over 150 compositions have been isolated and identified from C. pinnatifida, such as flavonoids, triterpenoids, steroids, monoterpenoids, sesquiterpenoids, lignans, organic acids and nitrogen-containing compounds. In this part, we describe the main chemical constituents of C. pinnatifida and their structures (Table 1). Pinnatifinoside C Leaves [12] 12 Pinnatifinoside D Leaves [12] 13 Pinnatifinoside I Leaves [12]

Triterpenoids
Since the first study of C. pinnatifida in the 1960s, triterpenoids and their derivatives have been isolated and identified. These triterpenoids are classified into tetracyclic triterpenoids and pentacyclic triterpenoids, such as ursolic acid (51)

Lipid Regulating and Anti-atherosclerosis Effects
Total flavonoids of the leaves from C. pinnatifida obviously decreased the serum levels of total cholesterol (TC) and triglyceride (TG) through gavage in high-fat/cholesterol rabbit models [50]. Total flavonoids of C. pinnatifida at a middle concentration of 50 µg/mL were able to promote the proliferation of preadipocytes but inhibit its differentiation. In addition, total flavonoids of C. pinnatifida inhibit mature adipocyte secretion of leptin and PAI-1 in a dose-dependent manner [51]. Moreover, total flavonoids of C. pinnatifida can markedly decrease the levels of total cholesterol (TC) and triglyceride (TG) in serum by controlling the gene expressions including FAS, HSL, TGH, SREBP-1c [52].
Atherosclerosis is a risk factor for coronary disease. There are a lot of theories about the pathogenesis of atherosclerosis, one of which is abnormal cholesterol levels [53]. The flavone extracted from C. pinnatifida by 70% ethanol obviously decreased the serum levels of total cholesterol (TC), triglyceride and low-density lipoprotein cholesterol (LDLC) in high-fat/cholesterol rabbit and rat models, suggesting its use to treat atherosclerosis [54].
Investigations have shown that the main antihyperlipidemic effect constituents of C. pinnatifida are hyperin and ursolic acid. Two animal models of hyperlipidemia were established in mice with 75% yolk and Triton-WR 1339 400 mg/kg (ip), respectively. The animals were administrated with hyperoside or ursolic acid extracted from C. pinnatifida in two doses. The total cholestrol (TCH), triglyceride (TG), high density lipoprotein (HDL) and superoxide dismutase (SOD) activities in serum were measured. In comparison with control groups, TCH levels in all the dosed groups were significantly decreased, while HDL and SOD activity increased; the ratio of total cholesterol/high-density lipoprotein (TC/HDL) was reduced, too. This effect could lessen damages to vascular endothelium induced by oxygen free radical (OFR) in hyperlipoidemia, thus preventing atherosclerosis [55,56]. Total flavonoids of C. pinnatifida had significant antihyperlipidemic effects and enhanced the vascular function of hyperlipidemia model rats, the mechanism of which might be relevant to the increased levels of nitric oxide (NO) in the serum and the reduction in endothelin (ET) synthesis [57,58].

Resistance to Chronic Heart Failure
In many clinical trials, C. pinnatifida extract was confirmed to be effective in the treatment of patients with chronic heart failure defined as NYHA functional class II. There were no severe side effects observed [59][60][61]. In another clinical trial, C. pinnatifida extract WS 1442, a dry extract from hawthorn leaves with flowers (dry extract ratio = 4-6.6:1, extraction solvent: ethanol 45%) standardized to 18.75% oligomeric procyanidines (OPC), was also effective in the treatment of patients with chronic stable New York Heart Association class-III heart failure. The most important constituents for the therapeutic effects of WS 1442 are OPC [62]. C. pinnatifida was capable of regulating and ameliorating cardiovascular system effects, as it enhanced myocardial contractility and expanded the coronary artery, lessened heart rhythm, myocardial oxygen consumption and peripheral resistance [63]. The mechanism of coronary artery expansion was relevant to the β-adrenergic receptor. The flavonoids of C. pinnatifida might be a new alternative botanical drug for chronic heart failure because of its good test results in pharmacological experiments.

Antihypertensive Effects
The extracts of C. pinnatifida could reduce blood pressure slowly and enduringly in mice, rabbits and cats, the mechanism of which was related to expanded peripheral vessels, and the active components were the flavanol dimers or multimers [64,65]. On compounding hypertension and hyperlipoidemia rats, extracts of C. pinnatifida at the doses of 1.5 and 2.25 g/kg/d could maintain rats' blood pressure [66].

Anti-myocardial Ischemia and Reperfusion Injury Effect
Total flavonoids isolated from the leaves of C. pinnatifida were able to reduce the degree of arrhythmia and lessen the burst size of LDH after damages in cardiocytes due to ischemia and hypoxia. Additionally, total flavonoids were able to enhance the endogenous oxygen purging system and reduce lipid peroxidation, showing it had an effect on relieving myocardial ischemia [67].

Gastrointestinal Function Regulating Effect
The alcohol extract (extracted with 60% alcohol) and the aqueous extract of C. pinnatifida had different effects on gastrointestinal function regulation. As for the alcohol extract, in the range of 2-8 mg/mL (crude drugs), the alcohol extract of charred fruits of C. pinnatifida was able to significantly reduce the contractility of rat gastric and intestine smooth muscle strips in a dose-dependent manner. [68]. In another investigation, the alcohol extract of C. pinnatifida could significantly reduce the contractility of rat gastric and intestine smooth muscle strips in the range of 5-20 mg/mL (crude drugs) in a dose-dependent manner, and the extract at the dose of 20 mg/mL (crude drugs) could inhibit the stimulation induced by acetylcholine [69]. For the aqueous extract, in the range of 5-20 mg crude drugs/mL, the aqueous extract of C. pinnatifida significantly enhanced the contractility of rat gastric and intestine smooth muscle strips in a dose-dependent manner. The extract mentioned above at the dose of 20 mg/mL could enhance the intensive contraction induced by acetylcholine and antagonize the relaxation of intestinal smooth muscle induced by atropine [70].
A common side-effect of azithromycin is gastrointestinal effects, especially for children. In a study, when injected intravenously with azithromycin, if patients (children) took hawthron slices orally, the incidence of side-effects was lower compared with the control group (p < 0.05). This study demonstrated that C. pinnatifida was able to reduce the side-effects of azithromycin on the stomach and intestine without any reported additional side-effects [71].

Digestive Enzyme Promotion Effects
C. pinnatifida contains vitamin C, vitamin B2, carotene and various organic acids, which could enhance the secretion of digestive enzymes and the enzyme activity within the stomach. Especially, amylase can enhance the activity of lipase which is able to directly help to digest fatty foods, and protease agonists from C. pinnatifida could enhance protease activity [72]. The organic acids were able to enhance mice gastrointestinal motility, and antagonize the relaxation of intestinal smooth muscle induced by atropine, though the organic acids had no effect on the stimulation of intestinal smooth muscles induced by neostigmine. This study demonstrated that it was a one-way regulation for intestinal motility [73].

Antibacterial Effects
The antibacterial effects of C. pinnatifida have been comprehensively investigated. The juice squeezed from C. pinnatifida had antibacterial effects [74]. Additionally, the extracts of C. pinnatifida could inhibit various bacilli and cocci, such as Bacteroides forsythus, Song bacillus, Smith bacillus, Proteusbacillus vulgaris, Bacillus anthraci, Corynebacterium diphtheria, Typhoid bacillus and Streptococcus hemolyticus. Skin disinfectants with extract of C. pinnatifida fruit pit as the main germicidal ingredient had preferable sterilizing effect and stability on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans [75].

Synergistic Antibacterial Effects
In vitro, the minimal inhibitory concentrations (MICs) of oxacillin, ampicillin sodium plus sulbactam sodium, ampicillin, cephazolin, and active components of C. pinnatifida were 256, 512, 512, 128, and 1,024 µg/mL against methicillin-resistant staphylococcus aureus (MRSA), respectively. However, when combined with the active components of C. pinnatifida at its sub-MIC (128 µg/mL) concentration, the MICs of the above four β-lactam antibiotics were 2, 32, 16 and 2 µg/mL respectively. The results demonstrated that these active components of C. pinnatifida had a synergistic antibacterial effect on MRSA when combined with β-lactam antibiotics [76].
In an experiment, the bacterial strains were the standard MRSA strain WHO-2 (WHO-2) and 45 clinical MRSA strains. WHO-2 possessed a high level of resistance to oxacillin (MIC = 512 mg/L) and harbors the mecA gene. The 45 clinical strains were all resistant to oxacillin (MIC > 4 mg/L) and harbor the mecA gene. (+)-Catechin (C), (−)-epicatechin gallate (ECg) and (−)-epigallocatechin (EGC) were isolated from fructus crataegi (hawthorn) guided by antibacterial sensitization activity. The combination of (+)-catechin (C) and (−)-epicatechin gallate (ECg) could enhance the activity of β-lactam antibiotics against MRSA in vitro and in vivo, which might be related to the increased accumulation of antibiotics within MRSA via suppression of important efflux pump gene expression. It was demonstrated by two-fold dilution and checkerboard methods that C (128 mg/L) combined with ECg (16 mg/L) had the greatest effect, and the combination also reduced the bacterial load in blood of septic mice challenged with a sub-lethal dose of MRSA. The mechanism is related to increased daunomycin accumulation within MRSA and down-regulated the mRNA expression of norA, norC and abcA, three important efflux pumps of MRSA [77].

Antiviral Effects
Human immunodeficiency virus (HIV) releases itself from an HIV-infected cell using serine protease, followed by attack on other cells. C. pinnatifida was found to inhibit the activity of serine protease, followed by decreasing diffusion rate of HIV in vivo [78]. Maslinic acid isolated from C. pinnatifida had a prominent effect on inhibiting the activity of HIV-1 protease. When the concentration was 17.9 mg/mL, the inhibitive rate is 100% [79]. Therefore, maslinic acid is considered as a potential candidate for novel anti-HIV therapeutics.

Anticancer and Sperm Distortion Inhibiting Effects
The anticancer effect of maslinic acid was investigated in 1989. Maslinic acid isolated from C. pinnatifida exhibited cytotoxicity on P-38 cancer cells (ED 50 = 13.0 µg/mL) [80]. Total flavonoids isolated from C. pinnatifida had no effect on normal cells, but could obviously enhance calcium concentration in tumor cells. In vitro, total flavonoids inhibited and killed Hep-2 tumor cells by calcium overload, as well as inhibited DNA biosynthesis of tumor cells [81]. Aqueous extracts of C. pinnatifida were found to inhibit sperm distortion of mice induced by cyclophosphamide, which obviously reduced the number of distorted sperm. The mechanism was related to the abundant linoleic acid and vitamin C in C. pinnatifida [82].

Immunoregulating Effects
In an experiment, the 100% decoction of C. pinnatifida was used in mice (p.o., 0.2 mL/10 mg/d, for 9 days). It was shown that the decoction could increase the weight of the immune organs of the mice (thymus and spleen), raise T lymphocyte transformation rate and T lymphocyte ANAE cell percentage, indicating the decoctum of C. pinnatifida has an obvious improving effect on the cellular immune function of the mice, which also provides an experimental basis for the clinical application [83]. The injection of C. pinnatifida (water extract and alcohol precipitate, 1 g crude drugs/mL) had the same effect as the decoction [84]. Sitosterol isolated from C. pinnatifida was able to significantly increase the leucocyte count and enhance the phagocytic activity of macrophages, and had effects on spleen and lymphocytes in mice model of immunosuppression induced by cyclophosphamide (CTX) [85]. It was confirmed that the polysaccharides extracted from C. pinnatifida were able to enhance the spleen, thymus and the phagocytic activity of macrophages, promote the formation of hemolysin and hemolysis plaque of mice [86].

Endocrine System Effects
Endocrine system imbalance is a major factor of diabetes. The regulation of the hepatic glucose output through glycogenolysis is an important target for type II diabetes therapy. Glycogenolysis is catalyzed in liver, muscle and brain by tissue specific isoforms of glycogen phosphorylase (GP). Because of its central role in glycogen metabolism, GP had been exploited as a model for structure assisted design of potent inhibitors, which might be relevant to the control of blood glucose concentrations in type II diabetes [87,88]. Maslinic acid isolated from C. pinnatifida was found to inhibit GP in moderate strength; the IC50 was 28 µmol/L. As glycogen phosphorylase inhibitors, the best effect of maslinic acid derivatives was 4 times better than maslinic acid [89]. Maslinic acid (10 µg/kg/day or 30 µg/kg/day, two weeks) was found to obviously lessen the levels of blood glucose in KK-Ay mice by lessening insulin resistance of KK-Ay mice, these results suggested that maslinic acid might be investigatedas a new drug for type II diabetes treatment [90].

Coagulation System Effects
In vitro, the extracts from the leaves of C. pinnatifida were reported to inhibit platelet aggregation of rabbit [91]. In another study, the IC50 of C. pinnatifida on platelet aggregation induced by ADP was 1.388% (g crude drugs per 100mL) [92]. In acute blood stasis rat model, total flavonoids obviously influenced the hemorheology, which decreased viscosity of plasma and hematocrit [93]. Total flavonoids isolated from the leaves of C. pinnatifida were found to inhibit thrombogenesis caused by vascular endothelial injury of artery. The mechanism is related to the enhancement of the surface charge and speeding up the fluxion of erythrocyte and soterocyte, lessening the gather and adhesion [94,95]. At low doses (between 100-500 mg/kg), C. pinnatifida water extracts was reported to inhibit platelet function significantly in Wistar albino rats. The extracts were able to change the bleeding time and the closure time, which determined by the PFA-100 and thromboxane B2 levels [96].

Antiinflammatory Effects
The inhibitory effect of ethanol extract from the leaves of C. pinnatifida on mice ear inflammation induced by dimethylbenzene was investigated [97,98]. The results showed ethanol extract of C. pinnatifida has definite antiinflammatory effects.

Antioxidant Effects
Total flavonoids of C. pinnatifida leaves was found to have a strong ability to scavenge oxygen free radicals, enhance superoxide dismutase (SOD) activities, and lessen malondialdehyde (MDA) levels. This result demonstrated that total flavonoids of C. pinnatifida leaves had good effects on protecting brain tissue, nephridial tissue, hepatic tissue and neuron by remitting oxidative stress [99][100][101][102].

Osteoporosis Inhibiting Effects
In a simulative animal model of osteoporosis induced by menopause, maslinic acid isolated from C. pinnatifida was found to inhibit osteoporosis. The mechanism was that maslinic acid was able to inhibit downstream-signal (NFκB) activities and transcription-factor (NFATcl) expressions, but it had no effects on transcriptional activities of NFATcl. Additionally, maslinic acid could also regulate the downstream-signalling (MAPK); but it had no effects on calcium flow oscillation. Therefore, the results suggested maslinic acid might be used as a new drug against osteoporosis induced by menopause [103].

Retina Protecting Effects
In an experiment, experimental rabbits were contaminated by inhaling CS 2 for 3 continuous hours on 6 consecutive days a week for a total of 3 weeks. The rabbits in the treatment group were given "haw drink compound" (water decoction with Crataegus pinnatifida, Lycium barbarum, Fructus jujubae) before contamination. After 3 weeks of the experiment, the results showed that the ultrastructures of the retinal tissues of the control group were more abnormal than those of the treatment group and the normal group. Every layer cell of the retinal in the control group showed apparent degenerative changes, but that in the treatment group was normal. This investigation demonstrated that haw drink compound could improve the tolerance to CS 2 toxicity in inducing the retinal damage of rabbits [104].

Toxicology
C. pinnatifida has been used for hundreds of years as an important traditional herbal medicine in China, as well as a daily foodstuff. However, studies of the relative systematic toxicity and safety of C. pinnatifida are lacking. So far, oral corn pollen haw liquor (an oral solution containing corn, pollen and haw) was demonstrated no have no genotoxicity effects [5]. Additionally, in order to ensure the safety of drug use, acute and long-term toxicity experiments with "semen cassiae hawthorn oat" (a capsule contains semen cassia, hawthorn and oat) were investigated. For the acute toxicity reactions, the semen cassiae hawthorn oat was orally given one-time at 10 g/kg dosages (the maximum tolerated dose in mice), and observed continuously for 14 days. For the long-term toxicity reactions, the semen cassiae hawthorn oat was orally given continuously for 8 weeks at low (1.6 g/kg) and high (2 g/kg) dosages. In the acute toxicity experiments, there were no toxic reactions or animal deaths. In the long-term toxicity experiments, there was no significant differences in the general state, weight changes, hematological indexes, biochemical indexes and organ coefficients in rats of low and high dosage group when compared with the control group. Pathologic examination did not show any structural and cellular abnormalities of each organ [105]. The above results demonstrated that C. pinnatifida is safe and non-toxic for experimental animals.

Future Perspectives and Conclusions
Medicinal plants are universally considered as important sources of new chemical substances with potential therapeutic effects. C. pinnatifida has long been used in Traditional Chinese Medicine for the treatment of cardiovascular disease, dyspepsia, infections and cancers. The flavonoids are considered to be the major bioactive constituents. C. pinnatifida has been of increasing interest in recent years, and many traditional uses have been investigated, but there is not enough systemic data about the toxicity and safety of C. pinnatifida, and few target-organ toxicity evaluations have been documented. Therefore, more investigations should be done regarding the toxicity and pharmacokinetics of C. pinnatifida.
In Traditional Chinese Medicine, C. pinnatifida is commonly used in compositions with other herbs and not used alone. Although many of the experimental results validate that C. pinnatifida exhibits significant pharmacological effects when used alone, it's important to investigate the pharmacological effects and molecular mechanisms of C. pinnatifida combined with other herbs based on modern concepts of disease pathophysiology. Furthermore, drug target-guided and bioactivity-guided isolation and purification of the chemical constituents and subsequent evaluation of the pharmacologic effects will promote the development of bioactive constituents and expand our knowledge of C. pinnatifida. Detailed investigations of the pharmacology, molecular mechanisms of action and systems biology will help to ensure which chemical constituents or multiple ingredients contribute to its pharmacological effects and help develop new effective drugs which could produce enormous benefit to society and the economy.