Ziziphus jujuba Mill., a plant used as medicinal food: a review of its phytochemistry, pharmacology, quality control and future research

Abstract

Jujubae Fructus (ZJF) [called Dazao (大枣) in Chinese], the fruit of Ziziphus jujuba Mill. (ZJ), is utilized as a food and traditional medicine in China. In TCM use, ZJF is traditionally used to treat and nourish the stomach, tonify the spleen, and nourish the blood, as well as for overall nourishing and strength. According to the available literature from 1974 to March 2019, more than 278 compounds have been isolated and identified from ZJ. Local books, papers and dissertations were also searched. The aim of this review was to examine this plant’s traditional uses, botany, phytochemistry, pharmacological effects, toxicity, pharmacokinetics, quality control and economically important uses. In vivo and in vitro scientific investigations have initially confirmed its pharmacological potential by showing anti-inflammatory, antioxidant, antimicrobial, gastrointestinal protective, cardiovascular, neuroprotective, anticancer, anti-HIV, sedative-hypnotic and anxiolytic effects. Bioactive metabolites belonging to different classes are responsible for these activities, including triterpenoid acids, saponins, cyclopeptide alkaloids, flavonoids and neo-lignans, which are considered the characteristic and active components of ZJ. The TCM use of ZJF, including tonifying and replenishing the middle Qi and nourishing the blood to tranquilize, is based on its gastrointestinal protective, cardiovascular, neuroprotective, sedative-hypnotic and anxiolytic properties. Its detoxification effects are attributed to its anti-inflammatory, antiviral, anticancer and antibacterial activities. Moreover, the TCM characteristics of ZJF (sweet flavour; warm nature; and spleen, stomach, and heart meridian effects) support its traditional uses and pharmacological effects. We encourage more studies to further clarify the relationship between modern applications and traditional uses in the future. Furthermore, no one has studied ZJ blossoms, and researchers should allocate more time to the study of ZJ blossoms. Additionally, unsolved problems include the scientific principle of the Chinese material medica processing [CMMP (中药炮制) in Chinese] of ZJF, the molecular mechanisms of the biological activity of ZJ and its other medicinal parts, the overall pharmacokinetics rather than single molecule pharmacokinetics, the efficacy and the toxicology. All of the unsolved problems noted above require further study.

Introduction

Ziziphus jujuba Mill. (ZJ) (Family Rhamnaceae) is a small tree, or less commonly, a shrub; the fruits of ZJ are notably used in TCM “Jujubae Fructus” (ZJF) (大枣/红枣 in Chinese). The characteristics of ZJF in TCM are summarized as sweet in flavour, with a warm nature and good spleen, stomach, and heart meridian distributions (Pharmacopoeia Commission of PRC 2015). According to Chen and Zhang (2014), these characteristics are similar to the characterization of anti-inflammatory agents in TCM. ZJF is used to nourish the stomach, fortify the spleen, and replenish blood, as well as for overall enriching and tonifying and to improve one’s health (Editorial Board of Flora of China 1982). In Shennong’s Classic of Materia Medica, ZJF is considered an effective drug for treating heart and abdominal pathogenic Qi, tonifying the spleen and stomach, as well as being beneficial for the twelve meridians, balancing stomach Qi, unblocking the nine orifices, tonifying a shortage of Qi and fluid-humour, tonifying an insufficiency of middle Qi, tranquilizing, heaviness of the limbs, and harmonizing the hundred medicinals. ZJF extracts are used to treat fullness in the chest and ribs or chronic hepatitis in Japan (Kubota et al. 2009). Five forms of ZJ are available, including ZJF, Ziziphus jujuba leaves (ZJL), Ziziphus jujuba core (ZJC), Ziziphus jujuba bark (ZJB) and Ziziphus jujuba roots (ZJR) (Fig. 1) (Wang 2014), although ZJF is most frequently used in TCM prescriptions (Liu et al. 2017d). The main production areas of ZJF are Hebei, Henan, Shandong, Shaanxi and other places in China [cultivated in Asia, Africa, Europe, and North and South America] (Editorial Board of Flora of China 2007). Sixty-six Chinese medicinal preparations containing ZJF are listed in the 2015 edition of the Chinese Pharmacopoeia, such as Anshen Bunao liquid (安神补脑液 in Chinese), in which the function of ZJF is to tonify Qi, nourish the blood and tranquilize; Xiangsha Yangwei pills (香砂养胃丸 in Chinese), in which the function of ZJF is to warm the middle to harmonize the stomach; Jianpi Shenxue tablets (健脾生血片 in Chinese), in which the function of ZJF is to fortify the spleen to harmonize the stomach and nourish the blood to tranquilize, etc. (Pharmacopoeia Commission of PRC 2015).

Fig. 1

Whole ZJ plant (A); the leaves and flowers of ZJ (B); the fruits of ZJ (C); the stem bark of ZJ(D); the cores of ZJ (E); the roots of ZJ (F)

Modern studies have shown that ZJF has anti-inflammatory (Goyal et al. 2011), antioxidant (Ko et al. 2008), anticancer (Lee et al. 2003), and anti-HIV effects (Fujioka et al. 1994), in addition to serving as a sweetness inhibitor (Suttisri et al. 1995), and other functions. Although only ZJF is used in TCM, some studies have reported the pharmacological effects and phytochemistry of Ziziphus jujuba leaves (ZJL) (Yoshikawa et al. 1991, 1992), Ziziphus jujuba roots (ZJR) (Kang et al. 2015, 2016, 2017), Ziziphus jujuba stem bark (ZJSB) (Han et al. 1989) and Ziziphus jujuba seeds (ZJS) (Choi et al. 2011). Although approximately 278 components have been separated and identified from ZJ, the mechanisms and material basis of its efficacy are not still clear. Therefore, it is necessary for us to present a systematic overview of ZJ and discuss possible future research directions.

Traditional uses

While different from clinical studies, ethnopharmacological fieldwork is also focused on understanding the medical use of substances (Heinrich et al. 2018). In classical Chinese herbal medicine, ZJF is considered an effective drug to treat heart and abdominal pathogenic Qi; tonify the spleen and stomach; balance stomach Qi; tonify a shortage of Qi, fluid and humour; tonify insufficiency of middle Qi; tranquilize; harmonize the nature of medicinals (Shennong’s Classic of Materia Medica); moisten the lungs and heart; suppress cough; tonify the five viscera; treat consumptive disease; eliminate intestinal and stomach pathogenic Qi (Rihuazi Materia Medica); tonify fluids and humour; nourish spleen Qi; strengthen will (Wupu Materia Medica; Dietetic Materia Medica), harmonize nutrients and defenses; soothe yin-blood; clear the twelve meridians; unblock the nine orifices (Shennong’s Classic of Materia Medica); maintain beauty and youth (Wupu Materia Medica); warm and tonify the spleen and stomach (Compendium of Materia Medica); and reduce the toxicity of Caowu (Aconiti kusnezoffii radix), Fuzi (Aconiti lateralis radix praeparata), and Chuanwu (Aconiti radix) (Variorum of the Classic of Materia Medica). There are some differences in the records of traditional usage of ZJF in different classical works associated with herbal medicine that need to be further verified in clinical practice. According to many ancient books, ZJF has considerable effects for tonifying and replenishing the middle Qi and nourishing the blood to calm. Furthermore, ZJF has been used for spleen deficiency, decreased appetite, loose stool, fatigue and lack of strength, blood deficiency and sallow complexion, women’s hysteria, and restlessness of mind and will (Huang 2002). In the Chinese Pharmacopoeia (Pharmacopoeia Commission of PRC 2015), preparations containing ZJF are mainly used for tonifying and replenishing the middle Qi and nourishing the blood to tranquilize. In traditional use, ZJF is directly eaten or combined with other drugs to prepare decoction for oral use.

In addition, ZJL, ZJC, ZJB and ZJR are also widely used in Chinese folk traditional medicine. ZJL is characterized as having a sweet flavour and a warm nature. It is mainly used for detoxifying and heat-clearing. It is effective for treating fevers in children, sores and furuncles, hot prickly heat, athlete’s foot, and burns and scalds (using a decoction with water for oral administration or douching). ZJC is characterized as being bitter in flavour with a flat nature and is useful for liver and kidney meridian distributions in TCM; it has also been described as useful in treatment for detoxifying and promoting wound healing. Additionally, it is suitable for the treatment of chancre sores and ulcerative gingivitis; ZJC may be used for external application after grinding it into powder after carbonizing it through burning. ZJB is bitter with an acerbic flavour and a warm nature; it is effective for lung and large intestine meridian distributions. It is used for astringing the intestines and improving diarrhoea, suppressing cough and stopping bleeding, and it has been noted to be suitable for diarrhoea, dysentery, cough, flooding and spotting; 6–9 g of the ground powder is decocted with water for oral administration or 1.5–3 g of powder are taken with water. For uses such as bleeding due to traumatic injury and burns, appropriate amounts should be used externally use through decocting with water for douching or grinding it into powder and applying it directly. ZJR is sweet in flavour with a warm nature and is effective for liver, spleen, and kidney meridian distributions. It is used for regulating menstruation to stop bleeding, dispelling wind to relieve pain, and tonifying the spleen to improve diarrhoea. It is suitable for menstrual irregularities, infertility, flooding and spotting, haematemesis, stomach pain, impediment pain, spleen deficiency and diarrhoea (by decocting 10–30 g with water for oral administration), rubella, and erysipelas (for external use with an appropriate amount. or by decocting with water for douching) (Wang 2014). In sum, all parts of ZJ are useful (Fig. 2), and the literature related to any portion of ZJ needs to be further organized and studied to provide a basis for clinical use and for inclusion in the Chinese Pharmacopoeia.

Fig. 2

Traditional uses of different parts of ZJ

Different parts of ZJ have different effects, which is directly related to the contents of their different chemical components; both ZJF and ZJR fortify the spleen, both ZJL and ZJC have detoxicating effects, and both ZJB and ZJR are effective at stopping bleeding, which may be related to some chemical composition shared between them (Table S1).

Botany

ZJ (syn. Z. Mauritania) is a common species in the genus Ziziphus of Dicotyledoneae of Angiospermae that belongs to the plant family Rhamnaceae (Rodríguez Villanueva and Rodríguez Villanueva 2017). There are approximately 170 species of genus Ziziphus in the world, and 12 species of genus Ziziphus are cultivated for their important economic and medicinal value (Li 2015). At present, there are more than 750 varieties of ZJ in China (Liu 2008).

Ziziphus jujuba Mill. (ZJ) is a deciduous tree native to China that grows approximately 10 metres high (Fig. 1). It has brown or grey-brown bark, and its branchlets can have 2 stipular spines; the spines are long, erect, and stout, whereas the short spines recurved. The stipular spines are slender and caducous. The petioles are glabrous or sparsely puberulent. The leaf blades are abaxially pale green, adaxially dark green, ovate, and papery. The flowers are yellow-green, bisexual, 5-merous, and glabrous with a 2–3 mm pedicel. The sepals are ovate-triangular, oblong or narrowly ovoid and are drupe red at maturity before turning red–purple; the petals are obovate, the disk is orbicular, and the seeds are compressed-orbicular (Editorial Board of Flora of China 2007).

ZJ grows on high mountains, hills, clear and dry slopes and plains and is also widely planted below 1700 m above sea level. ZJF is the fruit of ZJ (Fig. 1); it is collected when it matures in autumn, washed, and dried in the sun (Pharmacopoeia Commission of PRC 2015).

Phytochemistry

There are approximately 170 species of genus Ziziphus in the world. The main components of genus Ziziphus are triterpenoid acids, alkaloids, saponins, flavonoids and their glycosides (Che and Zhang 2011).

Triterpenoid acids, saponins, alkaloids, flavonoids and simple phenols are the dominant pharmacologically active compounds of ZJ. ZJF is also a good source of medium-chain fatty acids and β-carotene (Guil-Guerrero et al. 2004), and the chemical composition of ZJF varies between varieties, different cultivation areas and different water and fertilizer management (Jiao and Liu 2018).

According to the available literature, approximately 278 compounds have been isolated from ZJ, including 55 triterpenoid acids (1–55) (Fig. 3), 26 saponins (56–81) (Fig. 4), 37 alkaloids (82–118) (Fig. 5), 37 flavonoids (119–155) (Fig. 6), 11 sterols (156–166) (Fig. 7), 14 simple phenols (167–180) (Fig. 8), 10 glycosides (181–190) (Fig. 9), 9 nucleosides and nucleobases (191–199) (Fig. 10), 29 amino acids (200–228) (Fig. S1), 7 vitamins (229–235) (Fig. S2), 2 amides (236–237) (Fig. S3), 28 fatty acid derivatives (238–265) (Fig. S4), 5 saccharides (266–270) (Fig. S5), and 8 other compounds (271–278) (Fig. S6). While there have been no components isolated from ZJ flowers, 69 components (23, 25, 28, 56–81, 119–121, 124–126, 128, 133, 144, 148, 151, 153, 156–157, 160–165, 167, 169, 173, 179, 189, 240–243, 245, 251–255, 260–261, and 277–278) were isolated from ZJL, 73 constituents (1, 7, 134, 137–143, 145–147, 149, 156–157, 160–161, 163–174, 186–188, 200–210, 212–221, 223–225, 227, 240–245, 250, 252–254, and 262–266) were isolated from ZJS, and 42 components (1, 2, 4, 11–16, 25–27, 29–49, 83, 90, 92 and 96–101) were isolated from ZJR. Moreover, 30 components were isolated from ZJB, including 8 (1, 8, 23, 25, 27, 105, 108, and 118) from the root bark and 22 (83–91, 93–95, 102–107, 109–111, and 155) from the stem bark. Furthermore, 150 components (1–6, 9–10, 17–28, 50–55, 65, 82, 112–117, 119, 121–127, 129–132, 135–136, 148, 150–152, 154, 156–159, 167–185, 190–249, 255–259, 267–274, and 276) were isolated from ZJF. Confusingly, there are multiple terms used for the alkaloids isolated. For example, Daechuine-S1 (104) is also called Frangufoline, Daechuine-S2 (105) is also known as Frangulanine, and Daechuine-S4 (106) is also named Franganine, and so on. The corresponding names, pharmaceutical effects, parts of the plant, quantity (total), and place of origin of the compounds from ZJ are systematically listed in Table S1. The Table S1 also includes data concerning the source of the respective compounds, their concentrations, etc. The contents of polysaccharides, flavonoids and nucleotides in ZJF could be affected by its drying process and maturity (Chen et al. 2013). In addition, the amount of bioactive substances and antioxidant capacities vary among different ZJ cultivars (Kou et al. 2015). The contents of the chemical components (fatty acids, phenols, α-tocopherols and β-carotene composition) also differed between different parts (fruit and leaves) of ZJ (San and Yildirim 2010).

Fig. 3

The triterpenoid acids isolated from ZJ

Fig. 4

The saponins isolated from ZJ

Fig. 5

The alkaloids isolated from ZJ

Fig. 6

The flavonoids isolated from ZJ

Fig. 7

The sterols isolated from ZJ

Fig. 8

The simple phenols isolated from ZJ

Fig. 9

The glycosides isolated from ZJ

Fig. 10

The nucleosides and nucleobases isolated from ZJ

Triterpenoid acids

Fifty-five triterpenoid acids (1–55) (Fig. 3) were isolated and identified from ZJ (Yagi et al. 1978a, b; Kundu et al. 1989; Bai et al. 1992; Su et al. 2002; Lee et al. 2003, 2004; Mishra et al. 2007; Niu 2008; Guo 2009; Guo et al. 2009b, c; Fujiwara et al. 2011; Guo et al. 2011a, b; Bai et al. 2016; Kang et al. 2016, 2017; Masullo et al. 2019), some of which were reported to exhibit anticancer and anti-HIV properties. The mechanisms of action of the triterpenoids against cancer (Qiao et al. 2014) and HIV are worthy of further investigation. To date, 17 lupane-type triterpenoid acids (1–17), 5 oleanane-type triterpenoid acids (18–22), 27 ceanothane-type triterpenoid acids (23–49) and 6 ursane-type triterpenoid acids (50–55) have been isolated. These triterpenoid acids are pentacyclic triterpenoids, and among the triterpenoid acids, compounds 1–6, 9–10, 17–28, and 50–55 were isolated from ZJF; compounds 1, 2, 4, 11–16, 25–27, and 29–49 were isolated from ZJR; compounds 1, 8, 23, 25, and 27 were isolated from ZJ root bark; and compounds 1, 7, 23, 25, and 28 were isolated from the ZJ leaves and seeds.

Triterpenoid acids are one of the most important components of ZJ and are expected to be the main active compounds responsible for the pharmacologic activity of ZJ activities in its various applications. The triterpenoids oleanolic acid and betulinic acid are the main active ingredients in ZJF, and they have therefore been selected as markers for evaluating the quality of ZJF and its related preparations (Pharmacopoeia Commission of PRC 2015).

Saponins

Twenty-six saponins (56–81) (Fig. 4) were isolated from fresh ZJL (Yoshikawa et al. 1991, 1992) and dry ZJ leaves (Masullo et al. 2019), and jujuboside B (65) was also isolated from ZJF (Niu 2008). These saponins are mainly dammarane type triterpene saponins, and their mother structures can generally be divided into three main types, all of which include a glycosyl unit linked to C-3 and C-20. These glycosyl units are mainly d-glucose, d-galactose, l-6-deoxy-talose, l-rhamnose, l-arabinose, d-xylose and acetyl rhamnose (Guo 2009). Compounds 56–66 showed sweet-reducing activities (Yoshikawa et al. 1991, 1992) and can be used as raw materials for sweetness inhibitor products (Liu et al. 2015).

Alkaloids

Thirty-seven alkaloids (82–118) (Fig. 5) have isolated from ZJ (Otsuka et al. 1974; Tschesche et al. 1976; Han et al. 1987, 1989; Khokhar et al. 1994; Tripathi et al. 2001; Kang et al. 2015; Bai et al. 2016), mostly composed of cyclopeptides and aporphine alkaloids and mainly distributed in the root and stem bark. Since the 1980s, thirteen- and fourteen-membered ring type cyclopeptide alkaloid skeletons have been found in ZJ. Alkaloids (82–118) can also be used as chemical taxonomic representatives and markers for the genus Ziziphus (Che and Zhang 2011).

Flavonoids

To date, 37 flavonoids (119–155) (Fig. 6) have been isolated and identified from ZJ. Twelve of these flavonols (119, 121–127, and 129–132) have been isolated from ZJ fruit (Niu 2008; Guo 2009; Guo et al. 2009c; Pawlowska et al. 2009; Wang et al. 2010; Choi et al. 2011, 2012; Gao et al. 2012a, b; Bai et al. 2016; Elaloui et al. 2016; Cui et al. 2017; Pu et al. 2018), while 9 flavonols (119–121, 124–126, 128, and 132–133) are present in ZJL (Cui et al. 2017; Masullo et al. 2019) and one (117) is present in ZJS (Alam et al. 2017). Two flavanones (135–136) were first isolated from ZJF in 1981 (Okamura et al. 1981). Eleven flavones (137–143, 145–147, and 149) have been isolated from ZJS (Fu et al. 2016; Choi et al. 2011) and 2 flavones (144 and 148) have been found in ZJL and ZJF (Elaloui et al. 2016; Masullo et al. 2019). Four flavan-3-ols (150–152 and 154) have been isolated from ZJF (Choi et al. 2011, 2012; Gao et al. 2012a, b; Pu et al. 2018), and 3 flavan-3-ols (151, 153 and 155) are present in ZJL (Cui et al. 2017) and ZJB (Malik et al. 2002). Cheng et al. reported the presence of sedating flavonoids such as spinosin (137) and swertish (146) (Cheng et al. 2000). Flavonoids can be isolated from most natural plants and therefore cannot be used as chemical markers for the genus Ziziphus.

Sterols

Eleven sterols (156–166) (Fig. 7) have been reported in ZJ. Two compounds (156–157) were isolated from ZJ fruit, leaves and seeds (Guo 2009; Guo et al. 2009c; Aloui et al. 2012; Liu et al. 2015; Elaloui et al. 2016). β-daucosterol (158) and 3β, 6β-stigmast-4-en-3,6-diol (159) were isolated from ZJF in 2008 and 2009 (Niu 2008; Guo 2009; Guo et al. 2009c). Five compounds (160–161 and 163–165) have been isolated from ZJL and ZJS (Aloui et al. 2012; Elaloui et al. 2016), while stigmastanol (162) and cholesterol (166) were only isolated from ZJL (Elaloui et al. 2016) and ZJS (Aloui et al. 2012), respectively.

Simple phenols

Fourteen simple phenols (167–180) (Fig. 8) have been isolated from ZJ and identified (Niu 2008; Wang et al. 2010; Zhang, et al. 2010; Wang et al. 2011; Gao et al. 2012a, b; Bai et al. 2016; Cui et al. 2017; Pu, et al. 2018), including 5 hydroxycinnamic acids (167–171), 6 benzoic acids (172–177), 2 benzaldehydes (178–179) and 1 trihydroxybenzene (180). Among the simple phenols, compounds 167–180 were isolated from ZJ fruit, compounds 167–174 were isolated from ZJ seeds, and compounds 167, 169, 173, and 179 were isolated from ZJ leaves.

ZJF’s antioxidant capacity is closely related the concentration of efficient oxygen- free radical scavengers, such as vitamin C and phenolic compounds (Jiao and Liu 2018), which vary by the variety of ZJF and the source of the fresh ZJF. Vitamin C content is generally higher than 200 mg/100 g FW, with most samples containing between 300 and 500 mg/100 g FW; the highest level of vitamin C found has been 600 mg/100 g FW. The total phenol content ranges between 0.558 and 2.520 mg GAE/g FW, and while these compounds provide the material basis for the antioxidant effects of ZJF, both vitamin C and phenol concentrations are decreased after ZJF is dried (Jiao and Liu 2018).

Glycosides

Ten glycosides (181–190) (Fig. 9) were separated and identified from ZJ (Okamura et al. 1981; Niu 2008; Alam et al. 2017; Cui et al. 2017; Pu et al. 2018), including 6 compounds (181–185 and 190) isolated from ZJ fruit, 3 compounds (186–188) isolated from ZJ seeds, and compound 189, which was isolated from ZJ leaves.

Nucleosides and nucleobases

Nine nucleosides and nucleobases (191–199) (Fig. 10) were isolated and identified from ZJ (Guo et al. 2010b), all of which were from the ZJ fruit. The cAMP contents were much higher in the mature flesh of ZJ (38.05 nmol/g fw) than in the other tested materials from 14 types of horticultural plants and was the highest among the higher plant fruits. The highest cAMP content found in the test was 302.50 nmol/g fw. from Muzao of Shanxi, which was also the highest among the higher plants (Liu and Wang 1991).

Amino acids

Twenty-nine amino acids (200–228) (Fig. S1) were isolated and identified from ZJ (Choi et al. 2011, 2012; Pu et al. 2018). All of the compounds were found in the ZJ fruit, and 25 compounds (200–210, 212–221, 223–225 and 227) were isolated from ZJ seeds. These amino acids include eight amino acids that are essential for the human body, including arginine and histidine, which cannot be synthesized by children.

Vitamins

All of the compounds (229–235) (Fig. S2) were isolated from ZJ fruit (Gao et al. 2012a, b; Liu et al. 2015). ZJF is known as a “natural vitamin pill” and is rich in vitamins A, B, and C. For example, concentrations of vitamin C in fresh jujube fruit are up to 400–600 mg/100 g (Jiao and Liu 2018), which is 70–80 times that of an apple (Liu et al. 2015).

Amides

Two compounds (236–237) (Fig. S3) were isolated from ZJ fruit (Guo 2009; Guo et al. 2009c).

Fatty acid derivatives

Twenty-eight fatty acid derivatives (238–265) (Fig. S4) were isolated and identified from ZJ (Bai et al. 1992; Su et al. 2002; Guo 2009; Guo et al. 2009c, 2011a; Aloui et al. 2012; Liu et al. 2015; Bai et al. 2016; Elaloui et al. 2016; Cui et al. 2017), including 17 compounds (238–249 and 255–259) that were separated from ZJ fruit, 14 compounds (240–245, 250, 252–254, and 262–265) isolated from ZJ seeds, and 12 compounds (240–243, 245, 251–255, 260–261) isolated from ZJ leaves.

Saccharides

Five compounds (266–270) (Fig. S5) were isolated from ZJ (Guo 2009; Guo et al. 2009c; Gao et al. 2012a, b; Alam et al. 2017; Pu et al. 2018), including 1 compound (266) that was isolated from ZJ seeds and 4 compounds (267–270) isolated from ZJ fruit.

Others

Eight other compounds (271–278) (Fig. S6) were also isolated and identified (Okamura et al. 1981; Fukuyama et al. 1986; Heo et al. 2003; Niu 2008; Guo 2009; Guo et al. 2009b; Gao et al. 2012b; Elaloui et al. 2016; Pu et al. 2018; Masullo et al. 2019).

Pharmacological effects

The biological activities of ZJ and its components have long been studied in vitro and using animal models (in vivo) (Huang et al. 2007; Vahedi et al. 2008; Li et al. 2011c; Gao et al. 2013; Ding et al. 2016; Rodríguez Villanueva and Rodríguez Villanueva 2017), but there is no evidence from human interventional and epidemiological studies. In these two respects, more research should be performed. Multiple pharmacological effects have been associated with ZJ and compounds isolated from ZJ in animals and cells, as described below.

Anti-inflammatory activity

The control of inflammation is very important to improving health and vitality, as inflammation can aggravate arthritis, diabetes, etc. (Ignat et al. 2011).

ZJF potentially plays a protective role by attenuating NOS activity in anti-acute and chronic inflammation in Wistar albino rats. In one study, one of three doses (100, 200 and 400 mg/kg) of 60% ethanol extract of ZJF or indomethacin (10 mg/kg) were administered orally one hour before an injection of carrageenan (Goyal et al. 2011). Pretreatment with ZJF extract showed marked dose-dependent attenuation in oedema compared to control in the acute study, and ZJF extract significantly decreased granuloma tissue formation compared to control in the chronic study. This study therefore provides the ethnopharmacological basis for the use of ZJF as an anti-inflammatory agent.

In Shizao Tang (Euphorbia kansui, Euphorbia fischeriana, Daphne genkwa, ZJF) from Shanghanlun (Treatise on Cold Damage Diseases), ZJF is used in traditional Chinese formulas as an antidote to relieve the drastic inflammatory response due to Euphorbia species. Yu et al. determined that the triterpene acid fraction was the most active part of ZJF through the inhibition of inflammatory cells activated by Euphorbia kansui and a phorbol ester (prostratin) isolated from Euphorbia fischeriana (Yu et al. 2012). The triterpene acid fraction of ZJF might be helpful in attenuating the irritant action of Euphorbiaceae plants and protecting the gastrointestinal tissue from potent inflammatory injury, further confirming the rationality of the medicines (Euphorbia kansui, Euphorbia fischeriana, Daphne genkwa, ZJF) of Shizao Tang.

The presence of a ketone at the C-3 of oleanonic acid (19) implies an increase in inhibition of in vivo inflammatory processes and models related to 5-lipoxygenase activity. Oleanonic acid (19), with an IC₅₀ of 17 μM, reduced leukotriene B4 production from rat peritoneal leukocytes and was more active against in vitro leukotriene formation and dermatitis due to TPA (Giner-Larza et al. 2001). Its efficacy and effectiveness in inflammatory cells, however, requires further study.

Maslinic acid (20) (purity: 94.7%) was orally administered to CAIA mice daily at a dose of 200 mg/kg of body weight for 11 days starting on day 1, and the daily intake of maslinic acid for 12 weeks prevented and alleviated arthritis. The preventive effects of 20 on arthritis are attributed to the promotion of tissue formation and inhibition of synovium inflammation through toll-like receptor signal inactivation and downregulation of leukotrienes by the glucocorticoid receptor (Shimazu et al. 2018). Further studies, such as the use of localized cells for time-course analyses, will provide more detailed insights into the indirect and direct antiarthritis effects of 20.

Compounds 137–138, 140–143, and 149 showed moderate inhibitory activities against COX-1 and COX-2 enzymes (Table 1). SC560 and NS398 were used as positive controls for COX-1 (IC₅₀ 0.02 μM) and COX-2 (IC₅₀ 0.22 μM), although no COX-2 selectivity was evident for any of the active compounds (Fu et al. 2016).

Table 1 Inhibitory effects of compounds 137–138, 140–143, 149 against COX-1 and COX-2

Antioxidant activity

Ko et al. reported full and detailed descriptions of 70 antioxidant Korean medicinal plants using three oxidation reactions [luminol/Fenton reagent, 2,7-dichlorodihydro- fluorescein (DCHF)/Fenton reagent and DCHF/peroxynitrite] (Ko et al. 2008) and confirmed the in vitro antioxidant activity of ZJF. The antioxidant activity of ZJF was lower compared to other plants, and while this study is not suitable to evaluate the antioxidant activity of ZJF through pure redox-chemical experiments, it can serve as a reference.

Scientists have found that the antioxidant ability (scavenging effect on the DPPH radical) of ZJF was related to the variety of ZJF (Li et al. 2005). It was also found that the peel from all the varieties had the highest antioxidant capacity using the DPPH and FRAP assays, which was reflected by the high total concentrations of phenols (protocatechuic, caffeic, chlorogenic and gallic acids), flavonoids, and anthocyanins in their peels (Zhang et al. 2010). The free, esterified, glycosylated, and insoluble binding forms of 8 phenolic acids found in the peel, pulp, and seed of ZJF were studied using HPLC-ECD. The glycoside and insoluble-binding phenolic acid fractions in the ZJF pulp displayed the highest total phenolic contents and the strongest antioxidant effects determined by DPPH and FRAP assays (Wang et al. 2011).

Traumatic acid (255) shows stimulatory and antioxidant effects on collagen biosynthesis and can be used to treat many skin diseases associated with oxidative stress and collagen biosynthesis disorders (Jabłońska-Trypuć et al. 2016), which corresponds with the traditional use of ZJF for maintaining beauty and youth and confirming the Chinese proverb, “three jujubes a day, youth never grows old” due to the link between antioxidation and anti-aging (Sohal and Orr 2012; Koltover 2017). However, the molecular mechanisms of the collagen content changes in the cells remains unclear and requires further study.

ZJF and ZJF extract are potential sources of natural antioxidants in the food industry, though the basic metabolic functions of ROS should be investigated. In fact, removal of excess ROS can disrupt cellular signalling pathways and increase the risk for chronic diseases (Finley et al. 2011), highlighting the need for further scientific research.

One neutral polysaccharide fraction and three acidic polysaccharide fractions were separated from a hot water extraction of ZJF by Chang et al., and the average molecular weight of the fractions ranged from 40,566 to 129,518 Da. Four polysaccharide fractions (12.33–19.30 μM) were found to be more effective in scavenging superoxide anions than hydroxyl radicals, while the chelating effect of acidic polysaccharides on ferrous ion was more pronounced. Data on the antioxidant activity of different polysaccharide fractions from ZJF are significantly different at P < 0.05 (Chang et al. 2010).

Antioxidant effects of solvent extracts from the pulp, leaf, and seed of ZJ via sonication were investigated by Kim and Son. For ABTS radical scavenging activity analysis at a concentration of 1 mg/mL, 70% ethanol ZJS (94.76 ± 0.23%) and 80% methanol ZJL (95.46 ± 0.14%) extracts were observed to have higher activity than the controls, such as vitamin C (89.27 ± 0.12%), vitamin E (88.53 ± 0.12%), BHA (89.60 ± 0.00%), and BHT (84.07 ± 0.50%). Pulp and ZJS extracts at concentrations of 0.1–1 mg/mL showed SOD-like activity of 24.46–34.84%, and the 80% methanol ZJL extract demonstrated the highest activity (43.66 ± 0.37%) among the samples. The results showed that ZJ pulp and ZJS have excellent antioxidants activity, and the antioxidant effects of ZJL were found to be even higher than those of vitamins E and C (Kim and Son 2011), which provides a basis for the development of ZJL-related products.

However, it is important to note that pure redox-chemistry studies have no pharmacological relevance (Gafner 2018). In animal or human studies, there is little evidence that antioxidant activity observed in vitro has an impact on health. Pure redox-chemistry tests that prove the in vitro antioxidant effects of compounds isolated from ZJ and the different medicinal uses of ZJ are insufficient, but they can be used as a pre-test to provide some references or directions for further study of the antioxidant activity of ZJ.

Antimicrobial activity

ZJR ethanol extracts (1 and 2 mg/mL) had obvious inhibitory effects on fungi, including Candida tropicalis, C. albicans, Malassezia furfur (strains 1374 and 1765), Aspergillus niger, and A. flavus when compared with nystatin as the standard. (Sarfaraz et al. 2002). It is not clear, however, which components of ZJR play an antimicrobial role. Additionally, ZJRB extracts demonstrated antibacterial effects against 20 different bacteria (Elmahi et al. 1997). Ceanothic acid (25) showed growth inhibitory activities against Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis and Actinomyces viscosus, with MICs of 513.68, 127.39, 127.39 and 86.30 μM, respectively (Li et al. 1997). However, the MIC values of the positive control sanguinarine were 45.13, 6.02, 6.02 and 45.13 μM, respectively. The efficacy of compound 25 is lower than that of positive drugs, and it has little potential to become a lead antimicrobial compound.

According to microdilution antifungal susceptibility testing, magnoflorine (116) demonstrated high growth inhibitory effects when tested with Candida strains, with a MIC of 146.02 μM. Cytotoxicity testing demonstrated that it has no toxicity to HaCaT cells, even at treatments doses of 584.08 μM. Therefore, 116 is a good candidate lead compound for new antifungal agents (Kim et al. 2018).

Gastrointestinal protective activity

In hamster models, ZJF extracts (771 g kg⁻¹ of water-soluble carbohydrate concentrates at 5.0 and 15 g kg⁻¹ of diet), including hemicellulose, pectin polysaccharide, glucose, and fructose were observed to be effective in maintaining intestinal health by reducing intestinal mucosal exposure to toxic ammonia and other harmful substances (Huang et al. 2008). ZJ polysaccharides have also been shown to improve intestinal oxidative damage induced by ischaemia–reperfusion in rabbits. ZJ polysaccharides [fructose (21.6%), glucose (23%), mannose (12.9%) and xylose (31.3%)] have antioxidant activities that may contribute to this observed activity (Wang 2011), which is consistent with the traditional usage of ZJF for the role of tonifying the spleen and stomach.

Maslinic acid (20) (21.16 and 211.55 μM, inhibits H⁺ and K-ATPase activity) and ursolic acid (52) (218.98 μM, favours the gastric mucus barrier) demonstrate gastroprotective activity via different mechanisms of action (Da Rosa et al. 2017), which is consistent with the traditional Chinese medicine theory that ZJF can be used for gastric diseases. Maslinic acid (20) inhibits H⁺, K⁺-ATPase activity, whereas ursolic acid (52) affects the gastric mucus barrier. Maslinic acid (20) was absorbed with a peak plasmatic concentration at 30 min and oral bioavailability of 6.25% (Juan and Planas 2016). The bioavailability of ursolic acid (52) by oral administration is low since it is absorbed by the intestine through passive diffusion, although ursolic acid (52) dispersion preparations can increase the solubility and bioavailability (Zhang and Shen 2018). The components of ZJF involved in gastrointestinal protection are not limited to maslinic acid and ursolic acid, as other components may improve their bioavailability through solubilization or other mechanisms to allow them to deposit in their intact form to play an active role in vivo (Yin et al. 2012).

Cardiovascular activity

Betulinic acid (1) (at 10 μM) can upregulate endothelial NO synthase (eNOS) expression and downregulate NADPH oxidase expression in human endothelial cells, and thus has therapeutic potential in cardiovascular disease (Steinkamp-Fenske et al. 2007); betulinic acid (1) has also been shown to act as a TGR5 agonist (Genet et al. 2010; Lo et al. 2016).

NF-κB is an attractive target for cardiac hypertrophy. In abdominal aortic constriction rats, oleanonic acid (19) (15 or 45 mg/kg/day, oral gavage) markedly reduced left ventricular wall thickness and heart size in a dose-dependent manner. Further studies have shown that 19 can effectively improve cardiac hypertrophy by inhibiting the PKCζ-NF-κB signalling pathway (Gao et al. 2018). However, whether 19 inhibits the activation of the PKCζ-NF-κB pathway by reducing the activity of 5-lipoxygenase requires further study.

Beginning 1 day after surgery (sham surgery or aortic banding), all of the C57 mice were administered maslinic acid (20) (20 mg/kg) or vehicle orally for the following 4 weeks, and 20 was found to protect against pressure overload-induced cardiac fibrosis and cardiac hypertrophy. Further studies have shown that 20 can inhibit the activation of the ERK and AKT signalling pathways and reduce cardiomyocyte hypertrophy in vitro, making it a potential treatment option for cardiac hypertrophy (Liu et al. 2018e). However, the differences between the ERK and AKT signalling pathways in cardiac myocyte function and survival require further elucidation.

The prevention of foam cell formation is considered one of the main mechanisms of preventing atherosclerosis. Triterpenoids such as oleanonic acid (19) and pomonic acid (53) from ZJF, which contain a carboxylic acid at C-28, play an important role in inhibiting foam cell formation in human macrophages. Therefore, triterpenoids may be useful for preventing atherosclerosis (Fujiwara et al. 2011).

Jujuboside B (65) (IC₅₀ = 92.1 μM) inhibited collagen (2 mg/mL)-induced platelet aggregation (aspirin as control, IC₅₀ = 130.5 μM). Furthermore, 65 (IC₅₀ = 201.5 μM) dose-dependently inhibited thrombin (0.4 U/mL)-induced platelet aggregation (aspirin as control, IC₅₀ = 1810.5 μM). Additionally, treatment with 65 (100 mg/kg) increased protection against acute thromboembolism in mice (~ 63% protection) (aspirin as control, 50 mg/kg) when 65 and aspirin were orally administered to mice. Effective inhibition of platelet aggregation has also been demonstrated by 65 both in vivo and in vitro, and 65 is therefore considered effective for the treatment and prevention of cardiovascular diseases connected to platelet hyperaggregation (Seo et al. 2012). Further research is needed to determine the appropriate systemic concentrations of 65 in order to minimize its side effects (extension of bleeding time) and maximize its protective effects (protection against thrombosis).

A neo-lignan (278) isolated from ZJL has been shown to increase the release of endogenous prostaglandin I₂ from the rat aorta by up to 25.3% at 7.76 μM (Fukuyama et al. 1986), and it too could be considered for use in the treatment of cardiovascular diseases (Zhang 2008a).

To study the enhancing effect of ZJF on haematopoietic function in cultured Hep3B human hepatocellular carcinoma cells, Chen et al. studied its effects on the expression of erythropoietin. Application of chemically standardized ZJF water extract (0.75–3.0 mg/mL) stimulated the expression of erythropoietin in a dose- dependent manner, with the highest response found to be an ~ 100% increase (Chen et al. 2014b). These results confirmed the haematopoietic function of ZJF in the regulation of the expression of erythropoietin in liver cells. In addition, a study by Cheng et al. showed that gavage with the bond phenols from jujube peel (JPBP) or the free phenols from jujube peel (JPFP) [(approximately 2 mL, equal to 300 mg/kg rat)/d] could reduce aluminium toxicity and prevent ISO-induced myocardial injury in rats (Cheng et al. 2012). While it would have been better if the phytochemical analyses of the chemically standardized ZJF water extract, JPFP and JPBP were evaluated in the above studies, the relationship between the structure of the active components and the pharmacological activities of ZJF have also not been investigated.

The research results discussed above are consistent with the effects of ZJF as having a sweet flavour and a warm nature, and the traditional use of ZJF for blood-nourishing and detoxification functions.

Neuroprotective effects

As for ZJF water extract, the treatment of 72 h at several different concentrations (0.75, 1.5, and 3.0 mg/mL) induced the expressions of NF200, NF160, and NF68, with the highest induction by 100, 150, and ~ 150%, respectively [GAPDH served as loading control; NGF (50 ng/mL) served as the positive control]. The expressions of NFs in ZJF-treated cultures demonstrated a dose-dependent increase. The results support the use of ZJF as a food supplement for the prevention of neurodegenerative diseases in which neurotrophin deficiency is involved (Chen et al. 2014c). However, the mechanism of neuroprotective effects of aqueous extracts of ZJF needs further study. In addition, ZJF has been proved to play a role in antioxidant enzymes in cultured astrocytes and regulating expressions of neurotrophic factors (Chen et al. 2014d), which are consistent with the traditional use of ZJF to calm the nerves.

Qian et al. employed a time window study with intracerebroventricular (i.c.v.) administration of maslinic acid (20) to investigate its synergistic effects. The presence of 20 prolonged the therapeutic time window for MK-801 from one to 3 h. Compared with the single treatment group or the vehicle, when 20 (0.85 μM) was given 15 min before middle cerebral artery occlusion followed by MK-801 (0.25 mg/kg) administration 1 h after the artery occlusion the combination therapy demonstrated synergistic effects on infarct volume. The synergistic effect of co-treatment with 20 and MK-801 on neuroprotection might be related to the improvement in glial function, particularly the increased GLT-1 expression. The combination of 20 and MK-801 may prove to be a potential treatment strategy for acute ischaemic stroke (Qian et al. 2016), although 20 alone had minimal effects on glial function under normal physiological conditions.

Vitexin (147), which helps to increase neuroprotective factors and pathways and counteract targets that induce neurodegeneration, such as neuroinflammation (30–60 mg/kg vitexin was administered intraperitoneally to young rats); motor impairment (10–100 μM vitexin was administered in Parkinson disease simulation models); reduced cognition (5, 15, or 30 μM vitexin was added to the RAW 264.7 cell line), and redox imbalances and/or abnormal protein aggregation. These results provide strong support for the scientific exploration of vitexin for these pathologies (Lima et al. 2018).

Sedative-hypnotic and anxiolytic effects

The leaves and seeds from many Ziziphus species have been shown to promote sleep and depress the activity of the central nervous system, but these functions were not related to muscle relaxant or anticonvulsant activities (Peng et al. 2000). Lin et al. reported the anti-anxiety effects of multi-herbal medicines containing ZJS extract in mice (Lin et al. 2003).

Spinosin (137) and swertish (146) have significant sedative effects. Compared with the control group, oral administration of 137 (0.04 mmol/kg) and 146 (0.04 mmol/kg) prolonged sleeping time by 29 ± 31% compared to that induced by pentobarbital. The sedative effects of 146 have also been studied following the acid hydrolysis of 137 (Cheng et al. 2000).

Yang et al. studied the sedative and hypnotic activities of oleamide (276) in mice, and found that it dose-dependently inhibited motion activity in mice in doses ranging from 43.7 to 175.0 mg/kg by intraperitoneal injection. The positive control in this study was 2.5 mg/kg diazepam. Moreover, 276 promoted the hypnotic effects induced by sodium pentobarbital; these results provide further evidence for the sedative and hypnotic activities of 276 (Yang et al. 1999) and are consistent with the sedative effects targeted in the traditional usage of ZJF.

Unfortunately, few studies have focused on the material basis of the sedative effects of ZJF, and it is therefore necessary to strengthen the research in this respect in the future.

Hepatoprotective activity

Ent-epicatechinoceanothic acid A (47) showed antiproliferative effects towards the rat hepatic stellate cell line HSC-T6 with an IC₅₀ value of 43.5 μM (95% CI 28.6–66.2 μM), while (-)-epigallocatechin gallate, which was used as the positive control, exhibited an IC₅₀ value of 31.6 μM (95% CI 24.1–41.4 μM) (Kang et al. 2017). This finding indicates that 47 exhibited hepatoprotective effects in vitro, although the mechanism of these effects has not yet been elucidated.

ZJF has been shown to effectively prevent liver injury, mainly through downregulation of inflammatory responses and oxidative stress (Shen et al. 2009). Treatment with polysaccharides from ZJF (PZJF) (400 mg/kg, i.g.) significantly (P < 0.01) reduced the activities of CCl₄-elevated aspartate aminotransferase (AST), lactic dehydrogenase (LDH) and alanine aminotransferase (ALT) in serum and hepatic malondialdehyde (MDA) levels in mice. The results are indicators of hepatic injury induced in the mice by CCl₄, as shown in the significant increase in serum activities of ALT and AST from 26.0 ± 7.7 to 25.0 ± 6.5 IU/L in the untreated normal group to 46.6 ± 9.0 and 59.3 ± 11.9 IU/L in the CCl₄ group (P < 0.01), respectively. The CCl₄-induced hepatic injury was further confirmed by the increased in LDH from 166.0 ± 46.5 U/L to 265.6 ± 44.4 U/L (P < 0.01). At a dosage of 400 mg/kg BW, the AST, LDH and ALT decreased to 36.9 ± 9.6 IU/L, 183.2 ± 20.3 U/L, and 30.2 ± 5.6 IU/L, respectively, which were close to the levels obtained by the positive agent, biphenyldicarboxylate pills (BP) (400 mg/kg BW), with which the values were 25.0 ± 6.1 IU/L, 157.2 ± 49.2 U/L and 26.8 ± 6.2 IU/L, respectively. A prominent elevation in the MDA level and a significant reduction in the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels in the CCl₄-intoxicated group were clearly observed in comparison with the untreated normal group (P < 0.01). Interestingly, after the administration of PZJF at a dose of 400 mg/kg BW, there were significant increases in GSH-Px and SOD levels and a conspicuous reduction in MDA content from 251.6 ± 84.9 U/gprot, 66.1 ± 10.4 U/mgprot and 49.4 ± 5.4 nmol/mgprot in the CCl₄-intoxicated group to 532.2 ± 150.6 U/gprot, 192.5 ± 33.2 U/mgprot and 39.4 ± 4.2 nmol/mgprot (P < 0.01), respectively. These findings were very close to the 581.7 ± 116.6 U/gprot, 203.7 ± 33.2 U/mgprot and 37.6 ± 3.7 nmol/mgprot of the normal group (P > 0.05) and the 581.7 ± 116.6 U/gprot, 203.7 ± 33.2 U/mgprot and 37.6 ± 3.7 nmol/mgprot values of the positive drug BP (400 mg/kg BW, P > 0.05), respectively. Mice treated with PZJF exhibited a better antioxidant system profile and hepatosomatic index, with normal GSH-Px and SOD activities in the liver (Wang et al. 2012), which suggested that ZJF exerts hepatoprotective effects in vivo.

Wang et al. found that maslinic acid (20) (12.5, 25, 50 mg/kg, i.p.) dose- dependently increased the expression of HO-1 and Nrf2 and contributes to the protection against LPS/D-gal-induced liver injury by inhibiting the NF-κB and activating the Nrf2 signalling pathways (Wang et al. 2018b).

Anticancer and cytotoxic pro-apoptotic effects

Anticancer effect

The antitumour mechanisms of Jujuboside B (65) in vivo and in vitro have been investigated by Xu et al. HCT 116 cells were subcutaneously implanted into the right flank of each nude mouse. When the tumour size reached 60 mm³, the mice were treated intraperitoneally with 65 (40 mg/kg) three times per week for 5 weeks. Compared with the control group, the group treated with 65 showed tumour growth inhibition of approximately 60% (*P < 0.05 vs the untreated control group). No obvious change in body weight or toxicity were observed during the experimental period, and these results showed that 65 suppressed tumour growth in vivo. Further in vitro and other experimental results demonstrated that 65 induced protective autophagy to delay extrinsic pathway-mediated apoptosis (Xu et al. 2014).

Kangsamaksin et al. studied the effects of lupeol (8) and stigmasterol (157) on tumour and their anticancer activities in vivo and endothelial cells in vitro. In vivo, single or combined use of 8 (10 mg/kg) and 157 (10 mg/kg) via oral gavage disrupted tumour angiogenesis and reduced the growth of cholangiocarcinoma tumour xenografts in mice. Further in vitro and other experimental results have demonstrated that 8 and 157 are attractive candidates for anticancer treatment focused on cholangiocarcinoma tumours by targeting tumour endothelial cells and exerting anti-inflammatory activity (Kangsamaksin et al. 2017), although these findings require further verification in clinical trials.

Cytotoxic pro-apoptotic effect

The cytotoxicities of lupane-type triterpenoic acids (1, 3, 5, and 6) against A549, B16(F-10), K562, LOX-IMVI, PC-3 and SK-MEL-2 cancer cell lines (Table 2) were studied in vitro using the sulforhodamine B (SRB) method with adriamycin serving as the positive control. These results showed that the coumaroyl moiety at the C-3 position in lupane-type triterpenes may play an important role in enhancing the cytotoxic activity (Lee et al. 2003), although the mechanism of action of 3 and 5 requires further study.

Table 2 Cytotoxicity of compounds 1, 3, 5 and 6 isolated from ZJF against tumour cell lines^a

A large amount of in vitro evidence shows that betulinic acid (1) is effective against cervical, ovarian, human melanoma, and non-small-cell and small-cell lung cancers as well as head and neck carcinomas (Eiznhamer and Xu 2004). Betulinic acid demonstrated selective toxicity against cultured human melanoma cells (Kim et al. 1998) and induced apoptosis (Kim et al. 1998; Liu et al. 2004) in sensitive cells in a CD95- and p53-independent fashion (Eiznhamer and Xu 2004).

The induction of apoptosis is one mechanism for the anticancer activities of ZJF extracts in different cell lines (Vahedi et al. 2008). Huang et al. found that the ZJF extracts decreased the activity of the human hepatoma cells (HepG2) (Huang et al. 2007). Triterpenic acids from ZJ have been shown to inhibit the growth of MCF-7 and SKBR3 breast cancer cell lines and induce their apoptosis (Plastina et al. 2012). Choi et al. found that ZJF extracts from eight growth stages (S1–8) had dose-dependent inhibitory effects on HeLa cervical cancer cells. However, the inhibitory effects on A549 lung cancer and Hel299 normal lung cells decreased with fruit maturation and were closely related to the flavonoid contents and fruit antioxidant activities (Choi et al. 2012). In vitro studies showed that deproteinized polysaccharides from ZJF inhibited melanoma cells at the G2/M phase of the cell cycle accompanied by the formation of apoptotic bodies and increases in caspase-9 and caspase-3 activities (Hung et al. 2012).

Four isomers of coumaroyl alphitolic acid (3, 4, 5, and 16) showed strong apoptotic cell death-inducing activities in a concentration-dependent manner with IC₅₀ values of approximately 9–12 μM in human cancer cell lines [IC₅₀ values (μM) (A549: 3 (9.4 ± 1.2), 4 (12.0 ± 1.4), 5 (10.5 ± 1.6), and 16 (12.1 ± 1.8); MDA-MB-231: 3 (10.5 ± 1.1), 4 (11.4 ± 1.3), 5 (11.5 ± 1.8), and 16 (12.5 ± 2.2); and PC-3: 3 (10.1 ± 1.5), 4 (11.7 ± 1.7), 5 (10.9 ± 1.9), and 16 (12.1 ± 2.1)]. Compound 3 can induce apoptotic cell death in these cancer cells by increasing mitochondrial ROS production and the subsequent activation of p38 MAPK. These results provide a reasonable basis for using Ziziphus extracts to treat cancer in traditional oriental medicine (Shin et al. 2018). However, it is necessary to further clarify the exact mechanism by which 3 or other isomers of coumaroyl alphitolic acid increase the production of mitochondrial ROS in order to better understand the anticancer activities of the isomers of p-coumaroyl alphitolic acid from ZJF.

Pentacyclic triterpenes that possess a carboxylic acid functional group at C-28, including betulonic acid (6), oleanonic acid (19), ursonic acid (51), and ursolic acid (52), had significant cytotoxic effects against three human cancer cell lines (HT29 colorectal carcinoma cells, HONE-1 nasopharyngeal carcinoma, and KB oral epidermoid carcinoma) and resulted in IC₅₀ values ranging from 4.0 to 8.8 μM (Chiang et al. 2005). However, the mechanism of action of this effect has not been clarified and requires further study.

2α-hydroxyursolic acid (50) at the concentrations of 15, 20, and 25 μM showed anticancer activities by inhibiting MDA-MB-231 human breast cancer cell proliferation in a dose-dependent manner (P < 0.05) and inducing apoptosis by regulating the p38/MAPK signalling pathway. The EC₅₀ was 19.82 μM, suggesting a specific anti-proliferative activity of 50 towards MDA-MB-231 human breast cancer cells (Jiang et al. 2016).

The anticancer characteristics of three anticancer biomarkers (8, 52, and 156) were explored by Alam et al. in vitro against HepG2 and MCF-7 cell lines using the MTT assay, and an effective HPTLC method was developed for the simultaneous analysis of these three compounds [8, 52, and 156 (7.47, 5.50 and 11.85 μg/mg, respectively)] in different species. The results also supported their strong anticancer effects (Alam et al. 2018). However, the anticancer effects and mechanisms of these compounds in vivo still require further study.

Sweetness inhibitors

Extracts from ZJL inhibited sweet taste sensations in flies, hamsters and rats. Anti-sweet substances separated from ZJ included jujubasaponin II (57), III (58), IV (59), V (60) and VI (61) from ZJL; jujuboside B (65) from ZJL and ZJF; and zizyphus saponins I-III (62–64) from dried ZJL. Compounds 57, 58 and 66, the only three compounds with acyl groups, were up to four times more active at inhibiting sucrose sweet tastes than the other anti-sweet components, which could be used to reduce obesity in overweight or diabetic patients (Suttisri et al. 1995). Ziziphin (66) extracted from ZJL inhibited the sweetness induced by aspartame, d-glucose, d-fructose, glycine, naringin dihydrochalcone, sodium saccharin and steviosides (Kurihara et al. 1988). However, it did not inhibit hydrochloric acid-induced acidity or quinine-associated bitterness, suggesting that 66 has a strong specificity for sweetness (Kurihara. 1992). While 66 was also found to suppress human sweetness receptors (Smith and Halpern 1983), the mechanism of action of 66 is considered to be due to changing the taste. Anti-sweetness potencies of these compounds are expressed as relative anti-sweetness potencies on a molar comparison basis to gymnemic acid I, and their anti-sweetness potencies are 0.5 (57–58, 66), 0.25 (59–61, 64–65) and 0.125 (62–63), respectively (Suttisri et al. 1995).

Immunostimulant activity

ZJL might stimulate the intracellular killing capacity and chemotactic and phagocytic activities of human neutrophils at 0.005–0.050 mg/mL, although it did not show any significant effect at a concentration of 0.100 mg/mL (Ganachari et al. 2004). The ZJL water-alcohol extract stimulates the cell-mediated immune system by increasing the phagocytic functions of neutrophils. It is logical to suggest that this preparation may be useful as an adjuvant in several immunosuppressed clinical conditions, although further in-depth study is necessary to determine which components of ZJL play this role.

Zhao et al. found that crude ZJJ significantly increased the spleen and thymus indices in mice and enhanced the proliferation of peritoneal macrophages and spleen cells. Ju-B-2 pectic polysaccharides from ZJJ fruits had a dramatic (P < 0.01) effect in promoting splenocyte proliferation at a higher dose (> 0.030 mg/mL), while Ju-B-3 pectic polysaccharides from ZJJ fruits did not demonstrate any proliferation effects compared to the control group. Based on their structures, rhamnuronic acid and its side chains were considered to be the main factors causing stimulating immune responses (Zhao et al. 2006), although the structure–activity relationship of these polysaccharides needs further study.

Chen et al. investigated the roles of ZJF on the expressions of pro-inflammatory cytokines in cultured macrophages. Application of various chemically standardized concentrations of ZJF water extract (0–3.0 mg/mL) for 24 h stimulated the transcriptional expression of TNF-α, IL-1β, and IL-6 in cultured RAW 264.7 macrophages. In contrast, pretreatment with ZJF water extract suppressed the expression of IL-6 and IL-1β, but not TNF-α in LPS-stimulated macrophages. The IL-6 and IL-1β cytokines in LPS-induced macrophages were suppressed by ZJF water extract in both protein and mRNA levels. In parallel, the inhibition by ZJF water extract on the transcriptional activity of NF-κB was revealed in LPS-induced macrophages. These results confirmed that ZJF served bidirectional immune- modulatory roles by regulating the expressions of pro-inflammatory cytokines in macrophages (Chen et al. 2014a). However, which components in the aqueous extract of ZJF play a double regulatory role is an important direction for future research.

Lai et al. injected maslinic acid (20) i.p. at different doses (0, 8, 16 and 32 mg/kg) into leukaemia mice for 2 weeks. Monocyte (at 32 mg/kg treatment) and T cell (at 16 mg/kg treatment) markers were increased by 20, but B-cell markers (at 8 mg/kg treatment) were reduced. Moreover, at the 32 mg/kg dose, 20 increased macrophage-induced phagocytosis by peripheral blood mononuclear cells in the peritoneal cavity and increased NK cell activities at a target cell: splenocyte ratio of 25:1; however, B-cell and T-cell proliferation were not affected, the detailed mechanism behind this has yet to be clarified. The results showed that 20 increased immune responses by enhancing NK cell effects and macrophage phagocytosis in leukaemic mice (Lai et al. 2019), which is consistent with the traditional use of ZJF for invigorating the spleen.

Wound-healing activity

In their book on herbal drugs, Ansari et al. mentioned ZJR use in wound healing (Ansari et al. 2006). Very recently, Chopda, M.Z. confirmed the wound healing activity of ZJR in an experimental rat model using a topical ointment form of 5 mg/mL and 10 mg/mL concentrations by topical application (Chopda 2009), which confirms the view of Ansari et al. (Ansari et al. 2006). Additionally, at the 1 μM dose, squalene (275) from ZJL can promote wound healing by promoting macrophage responses during inflammation, meaning 275 was useful at the resolution stage of wound healing. These results are consistent with the traditional Chinese medicine treatment for traumatic haemorrhage. Compound 275 is currently only separated from ZJ leaves, not from ZJ root bark and bark, and its mechanism needs further study (Sánchez-Quesada et al. 2018).

Additionally, local application of adelmidrol + trans-traumatic acid (255) plays an important role in the closure and healing of diabetic wounds in a streptozotocin- induced diabetic mouse model (Siracusa et al. 2018), which is consistent with the traditional use of ZJ for wound healing. However, the relationship between the activity of ZJ on wound healing and compound 255 requires further research.

Lupeol (8) has many pharmacological effects, such as antioxidant, anti-inflammatory, anti-mutagenic, and anti-diabetic effects. Pereira Beserra et al. studied the effects of 8 (0.23, 2.34, 23.43, and 46.87 μM) in wound healing assays as well as its signal transduction mechanism in vitro in human neonatal prepuce keratinocytes and fibroblasts. The results demonstrated that 8 has therapeutic potential in promoting wound healing (Pereira Beserra et al. 2018), although further in vivo and clinical studies are needed to explore these effects and to develop 8 as a therapeutic agent for the treatment of skin wounds.

Chlorogenic acid (171) or PBS was applied directly to the wound area twice a day, and at 13 days post-infection, the wound healing rate of the 171-treated groups reached 72.35% ± 2.86 (1/16MIC) or 83.85% ± 4.82 (1/2MIC), while the control group treated with PBS demonstrated wound closure of 53.53% ± 6.58. On the 5th and 7th days after infection, the high-dose-treated group had a significant decrease in bacterial number. In a mouse wound model, the wound healing speed of the 171-treated groups was faster and the number of bacteria in the wound area was also reduced, suggesting that 171 has the potential to be used as a wound healing agent (Wang et al. 2018a) and is expected to be developed as a drug for wound healing.

Anti-HIV activity

Betulinic acid (1) inhibited HIV replication in H9 lymphocytes with an EC₅₀ value of 1.4 µM and inhibited the growth of uninfected H9 cells with an IC₅₀ value of 13 µM. Betulinic acid has a lupane skeleton and was the first identified triterpene to show anti-HIV effects. Platanic acid and 1 are structurally interrelated, except that the isopropenyl group in 1 is replaced with an acetyl group in platanic acid. The C-19 substituents, the C-17 carboxylic acid group and the C-3 hydroxy group contribute to the enhanced anti-HIV effects (Fujioka et al. 1994). The mechanism of the anti-HIV activity of compound 1 requires further study.

PA-457 (as a positive control, IC₅₀: 15.2 µM) and epiceanothic acid (26) were detected in MT-4 cells acutely infected with HIV-1_NL4–3, and compound 26 showed only moderate anti-HIV effects with an IC₅₀ value of 38.9 µM and a TI of 2.49 (Zhang et al. 2011). The mechanism of compound 26 against HIV and whether it can be used as an anti-HIV drug or lead compound needs further study.

An in silico wafer virtual screening method was used to identify lead compounds from a natural compound library or database to be used as an HIV-1 protease inhibitor. HIV-1 protease was examined using a virtual screening against the Indonesian Herbal Database with AutoDock. The results found that ursonic acid (51) had anti-HIV effects (Yanuar et al. 2014), although whether 51 truly exerts anti-HIV effects needs to be studied both in vivo and in vitro.

Anti-asthmatic activity

The anti-asthmatic activities of the ethanolic extracts of ZJF (EZJF) and Jujuboside B (65) were evaluated by various screening methods. The results showed that EZJF and 65 significantly inhibited milk-induced eosinophilia and leucocytosis, clonidine- induced catalepsy, passive paw anaphylaxis, and clonidine-induced mast cell degranulation. After 65 pretreatment (33, 66, and 132 mg/kg, p.o.) in mice, the number of inflammatory cells in BAL fluid was significantly reduced and the degree of pulmonary inflammation was alleviated. High TH2 cytokine expression levels were observed in lung homogenates and BAL fluid, whereas serum levels were markedly decreased. Therefore, EZJF and 65 displayed strong anti-asthmatic effects and have a potential role in the treatment of asthma (Ninave and Patil 2018). This finding is consistent with the traditional use of ZJF for its lung-moistening effects, although the results of this study were weakened because the two researchers (Ninave and Patil 2018) did not perform mass spectrometric analysis of Jujuboside B (65) to determine the exact molecular weight.

Others

The other pharmacological effects of ZJF can be found in the supplementary materials (Pharmacological effects S1-S13).

Some of these pharmacological studies prove the traditional usage of ZJ, and some are the development of the traditional usage of ZJ. These results can be considered conducive to the rational use of ZJ and the development of a local economy.

Toxicity

Thus far, no report has been published on the toxicity of ZJF. The recommended daily dose of ZJF is 6–15 g (Pharmacopoeia Commission of PRC 2015), and relevant reports have shown that the ethanolic extract from Zizyphus sativa Gaertn (Syn. = Zizyphus jujuba Mill.) fruit (prepared by extraction of ZJF along with the seeds with 95% ethanol) resulted in no acute or chronic toxicity in Swiss albino mice. In an acute toxicity test, animals that received three doses of 0.5 g/kg, 1 g/kg and 3 g/kg body weight (p.o.) were observed every 24 h and treated by chronic treatment and dose (100 mg/kg body weight extract) for 3 months. In addition to examining the effects on average body weight and the weight of important organs, external visceral toxicity, morphological changes, spermatogenic dysfunction, and haematological changes were also recorded. The crude drug had no obvious toxic effects (Shah et al. 1989). Administration of ZJF extract (prepared by extraction of ZJF with 60% ethanol using Soxhlet extraction) did not cause any behavioural changes, toxic symptoms or mortality, even at high doses (2 g/kg, p.o.) in Wistar albino rats. However, the toxicity of ZJF aqueous extracts has not yet been studied. Long-term traditional clinical experience shows that it is used with caution in cases of food stagnation or damp-phlegm because it can help induce distention in the middle energizer and the dampness can produce heat (Zhang 2008b). It is also used in some prescriptions to reduce the toxicity of various herbs and protect the stomach Qi. For example, Jingdaji (Euphorbiae pekinensis radix), Gansui (Kansui radix), and Yuanhua (Genkwa flos) in Shizao Tang from Shanghanlun (Treatise on Cold Damage Diseases) are used in conjunction with ZJF to alleviate toxicity and the violent effects associated with three herbs to protect the stomach Qi. Additionally, it is also compatible with Shengjiang (Zingiberis rhizoma recens) to enhance the coordination of the spleen and stomach (Zhang 2008b) and harmonize the nutrient and defence (Huang 2002).

Pharmacokinetics

The Chinese Pharmacopoeia controls the quality of ZJF using betulinic acid (1) and oleanolic acid (18). The pharmacokinetics of these two compounds have been well studied (Udeani et al. 1999; Cheng et al. 2003; Song et al. 2006; Jeong et al. 2007).

The extent of binding of 1 to plasma proteins in dog, mouse and rat plasma was determined by LC/MS. The results showed that in dog or rat plasma, 1 was 99.99% bound to serum proteins at 25 and 15 µg/mL and that 1 was also 99.97% bound at 5 µg/mL (Cheng et al. 2003).

Udeani et al. examined CD-1 rats at designated times after a single intraperitoneal (IP) dose of 1 at 250 or 500 mg/kg. The results demonstrated that after 500 and 250 mg/kg 1 IP, serum concentrations reached peaks at 0.23 and 0.15 h, respectively. The distribution of 1 in the tissues and organs of the mice was studied, and compound 1 was found to be mostly distributed in the perirenal fat and less in the heart and brain. After administering different doses, although the elimination phase remained basically the same, the absorption rate was obviously different (Udeani et al. 1999).

Song et al. studied the pharmacokinetics of oleanolic acid (18) in 18 healthy male Chinese volunteers. The concentration–time curves in plasma following oral administration of 40 mg oleanolic acid (18) for most of the 18 subjects were consistent with the single compartment model. The mean values of C_max, T_max, AUC_0-48, AUC_0-∞, t_1/2, CL/F, and V/F of 18 after p.o. administration of a single 40 mg dose were 12.12 ± 6.84 ng/mL, 5.2 ± 2.9 h, 114.34 ± 74.87 ng h/mL, 124.29 ± 106.77 ng h/mL, 8.73 ± 6.11 h, 555.3 ± 347.7 L/h, and 3371.1 ± 1990.1 L, respectively. These results showed that V_d was 2.5 times larger than the average total plasma volume in a man, potentially because 18 was distributed to a great extent out of the blood or amassed in a specific tissue (Song et al. 2006).

Jeong et al. studied the absorption and metabolism of 18 by using a Caco-2 cell permeation and rat liver microsome model and found that 18 underwent transmembrane transport mediated by passive transport. The osmotic coefficient showed that its intestinal absorption was poor, and 18 could be metabolized by liver microsomes but was not stable. The rats were administered different doses (0.5, 1 and 2 mg/kg doses) of 18, and the pharmacokinetic parameters of 18 showed good linear-dose pharmacokinetics as evidenced by the unaltered CL (28.6–33.0 mL/min/kg), V_ss (437–583 mL/kg), dose-normalized AUC (16.0–17.9 mg min/mL based on 1 mg/kg) and t_1/2 (41.9–52.7 min), but the oral bioavailability of 18 was very low, which may be closely related to its poor intestinal absorption and extensive metabolic clearance in the liver (Jeong et al. 2007).

Many studies have shown that the lipid solubility of 18 is strong and that its water solubility is very low because of the specific parent nucleus present in soap. The structure of pentacyclic triterpenoid saponins distributes these compounds widely in lipophilic tissues, but their bioavailability is low; thus, the clinical applications of existing preparations are greatly limited (Cheng and Xiong 2008).

Unfortunately, pharmacokinetic studies of whole ZJF plants and some compounds have not been reported; thus, this is an important research direction for future studies.

Quality control

To control the quality of ZJF, the Chinese Pharmacopoeia suggests examining the plant origin and performing morphological, microscopic and TLC identification in addition to determining the total ash and aflatoxin. Qualified ZJF samples should contain betulinic acid (1) and oleanolic acid (18) (Pharmacopoeia Commission of PRC 2015) (Fig. 11), although the presence of 1 and 18 alone seems insufficient to assess the quality of ZJF. Recently, various bioactive components in ZJF were examined using TLC, HPLC–DAD, HPLC-ELSD-MS, RP-HPLC, UPLCDAD-MS, HPLC, MS and NMR methods, such as betulinic acid (1), alphitolic acid (2), oleanolic acid (18), zizyberanalic acid(colubrinic acid) (23), zizyberanal acid (24), ceanothic acid (25), epiceanothic acid (26), zizyberenalic acid (27), ceanothenic acid (28), ursonic acid (51), ursolic acid (52), zizyberanone (271), nucleosides, nucleobases, etc. (Lee et al. 2004; Guo et al. 2009a, b, 2010a, b). Although scholars at home and abroad have performed much research on its chemical constituents, the water-soluble components with high polarity (Ji et al. 2017) require relatively highly technical experimental conditions for separation and purification, and the current research is not yet sufficiently detailed.

Fig. 11

Thin layer chromatogram of ZJF

To date, the research on pharmacological activities and quality control of the water-soluble components of ZJF is relatively weak, and the research in this area should therefore be further strengthened. At present, betulinic acid (1) and oleanolic acid (18) are used for ZJF quality control (Pharmacopoeia Commission of PRC 2015) in the current Pharmacopoeia. It is not sufficient for comprehensive quality control of ZJF with betulinic acid (1) and oleanolic acid (18) because betulinic acid (1) and oleanolic acid (18) are insoluble in water, while ZJF is mostly used as a decoction (water extract) in clinical treatment.

Therefore, it is necessary to find other effective components and establish quality control standards corresponding to their efficacy. If necessary, further study of the ZJF fingerprint will provide stronger quality assurance for ZJF quality control (Liu et al. 2015).

Economically important use

The diversity of plant usages provides opportunities for the development of new food and/or pharmaceutical products (Yao et al. 2018). Zizyphus jujuba has medicinal value throughout the body. Its fruit is rich in nutrients and chemical components and has a wide range of pharmacological effects. Its important dietary and therapeutic ingredients are at the top of the list. The saccharides are the most concentrated of the nutrients in ZJF, as the content of soluble saccharides can reach approximately 30% in mature fresh ZJF, and the content of the dry ZJF can reach 60–70% or even higher, which is much higher than that of other fruits. The vitamin C in fresh ZJF can reach more than 600 mg/100 g FW (Jiao and Liu 2018), and either fresh or dried fruits can be used. In addition to being a common fruit, it is also an important festival product, TCM, and food product that can be deep-processed into products and functional foods, such as jujube formula granules (Liu et al. 2017a), dietary fibre biscuits (Liu et al. 2017f), jujube polysaccharide capsules (Liu et al. 2016a), edible red pigments (Liu et al. 2016b), jujube brown sugar ginger tea (Liu et al. 2018a), jujube yogurt (Liu et al. 2018b), etc. The jujube core can be converted into furfural (Liu et al. 2017b), activated carbon (Liu et al. 2017c), and other products. Jujube leaves can be used to produce sweetener inhibitors (Liu et al. 2017e) and chlorophyll (Liu et al. 2017g). Li et al. closely studied the nutritional composition of five cultivars of Chinese jujube (Li et al. 2007) and polysaccharides from ZJF (Li et al. 2011a, b, 2013), which laid the foundation for the further development of functional foods from ZJF. Siriamornpun et al. offered practical information about how best to use the bioactive compounds and health implications of the green ZJF and ripe ZJF as potential sources of nutritive and functional applications (Siriamornpun et al. 2015). ZJ is the main cash crop of farmers in Xinjiang, Shaanxi, Shanxi, Henan and other provinces. At present, China has 99% of the world’s jujube resources and accounts for nearly 100% of the international trade in jujube products. The total annual output value of the jujube industry is more than 20 billion Yuan. In many key jujube producing counties, the income of the jujube industry accounts for 40% of farmers’ income, reaching as high as 80% in some counties (Liu 2008). This is also a way for farmers to escape poverty and become rich. It is worth noting that ZJ is an entomophilic plant pollinated by flies, ants, bees, and other insects, and the unabi fly in particular is a pest of the culture fruits (Gusakova et al. 1999). More research is necessary to determine how to optimize the relationship between the yield of ZJF and these insects to maximize the benefits to fruit farmers.

Conclusions and future prospects

ZJ is a well-known plant worldwide, and its fruit (ZJF) is listed in the Chinese Pharmacopoeia. ZJF plays an important role in both the food industry and health care. ZJF includes more triterpenoid acids, cyclopeptide alkaloids and flavonoids than the other plant parts, which may explain why ZJF is more frequently used in TCM prescriptions than other plant parts. Currently, according to the available literature, approximately 278 compounds have been isolated and identified from ZJ. It mainly contains triterpenoid acids, saponins, alkaloids, flavonoids and simple phenols, which provide the material basis for ZJ to have obvious antioxidation, anti-inflammation, antibacterial, anti-anxiety, anticancer, sweet inhibitor, anti-HIV and other pharmacological effects. Pharmacological studies in vitro and in vivo have increasingly confirmed the traditional uses of ZJF as tonifying and replenishing the middle Qi and nourishing the blood to tranquilize.

We showed preliminary evidence of a relationship between modern pharmaceutical studies and traditional uses. The strong gastrointestinal protective, cardiovascular, neuroprotective, sedative-hypnotic and anxiolytic effects correspond to ZJF’s TCM characteristics (sweet flavour, warm nature and spleen, and stomach and heart meridians). Moreover, the remarkable sedative-hypnotic, anxiolytic, antioxidant and anti-inflammatory capacities of ZJF contribute to its neuroprotective and anticancer activities. However, in studies, some experiments lacked controlled clinical trials, so it is impossible to draw unequivocal conclusions about the effects of these compounds in humans.

Not all the available information contributes to traditional evidence-based usages, and there is not even enough evidence to make it a registered evidence-based drug because of lack of the best basis for in vivo experiments and clinical research. If ZJF is to become an evidence-based drug, in vivo experiments and clinical studies should be conducted.

In future studies, we encourage more clinical studies and in vivo experiments to further clarify the relationship between modern applications and traditional uses of this plant.

Nevertheless, there are still some questions that have not been clarified and that require further study by researchers to promote further scientific and clinical research.

First, the traditional actions of ZJF to tonify and replenish the middle Qi and nourish the blood to tranquilize require more modern pharmacological studies to elucidate their intrinsic mechanisms.

Second, previously reported pharmacological studies mostly focus on a limited number of ingredients, and only a few studies have focused on holistic pharmacodynamics. Therefore, we need to answer whether these identified compounds can achieve the equivalent effect of ZJF, and, if not, to what extent. Otherwise, more bioactive ingredients, especially Qi-enriching, spleen-invigorating, blood-nourishing and sedative substances should be identified through chemical standardization and bioactivity-guided separation strategies for bioactive compounds, and their mechanism of action is still unclear and needs further study.

Third, no recent studies have investigated the clinical differences between ZJF, ZJB, ZJC, ZJL, and ZJR. These should be one of the main research directions for future studies.

Fourth, though the fruit has been traditionally used as a TCM, some researchers have studied the phytochemicals of other plant parts (seeds, barks, and leaves, but not in flowers) and revealed their pharmacological effects. Therefore, it is necessary to compare the chemical constituents from different parts and their corresponding pharmacological effects. Furthermore, no one has studied ZJ blossoms, and exploratory research should therefore focus on ZJ blossoms.

Fifth, ZJF is commonly combined with other TCMs [such as Gancao (Glycyrrhizae radix et rhizoma), Ejiao (Asini corii colla), Tinglizi (Descurainiae semen lepidii semen), and Danggui (Angelicae sinensis radix)] in conventional therapies. The interactions between ZJF and other TCMs as well as their underlying mechanisms need further study (Gao and Zhong 2006).

Sixth, there are currently no reports focused on ZJF toxicity. However, we are encouraged by the evaluation of side effects or toxicity associated with ZJB, ZJC, ZJL, and ZJR in in vitro, in vivo and clinical studies.

Seventh, ZJF has been stir-baked to yield yellow, brown, and charcoal colours; steamed; and stir-fried with wine, vinegar, etc. during its traditional processing methods (Liu et al. 2018d). The scientific principles and mechanisms of CMMP associated with these changes are not clear, and therefore require further study.

Eighth, in terms of the number and depth of research of papers published at home and abroad, there are still very few works focused on ZJF storage, preservation and processing, which is a weakness in the ZJF scientific research.

Ninth, there are some differences in the records of traditional usage of ZJF in different classical works associated with herbal medicine that need to be further verified in clinical practice.

Tenth, the volatile components in ZJF are relatively complex. Different processing methods have greater impacts on the volatile components in ZJF, which may create changes in its efficacy, and this aspect requires further study (Lu et al. 2013; Wang et al. 2014).

We believe that if we can answer the above questions, we will be one step closer to understanding the roles and characteristics of ZJ and its components.