Physalis alkekengi L. var. franchetii (Mast.) Makino: An ethnomedical, phytochemical and pharmacological review

Physalis alkekengi L. var. franchetii (Mast.) Makino: An ethnomedical, phytochemical and pharmacological review

Author’s Accepted Manuscript Physalis alkekengi L. var. franchetii (Mast.) Makino: an ethnomedical, phytochemical and pharmacological review Ai-Ling L...

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Author’s Accepted Manuscript Physalis alkekengi L. var. franchetii (Mast.) Makino: an ethnomedical, phytochemical and pharmacological review Ai-Ling Li, Bang-Jiao Chen, Guo-Hui Li, MingXing Zhou, Yan-Ru Li, Dong-Mei Ren, HongXiang Lou, Xiao-Ning Wang, Tao Shen www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(17)31342-9 http://dx.doi.org/10.1016/j.jep.2017.08.022 JEP10992

To appear in: Journal of Ethnopharmacology Received date: 5 April 2017 Revised date: 14 August 2017 Accepted date: 18 August 2017 Cite this article as: Ai-Ling Li, Bang-Jiao Chen, Guo-Hui Li, Ming-Xing Zhou, Yan-Ru Li, Dong-Mei Ren, Hong-Xiang Lou, Xiao-Ning Wang and Tao Shen, Physalis alkekengi L. var. franchetii (Mast.) Makino: an ethnomedical, phytochemical and pharmacological review, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.08.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Physalis alkekengi L. var. franchetii (Mast.) Makino: an ethnomedical, phytochemical and pharmacological review Ai-Ling Li1, Bang-Jiao Chen2, Guo-Hui Li3, Ming-Xing Zhou1, Yan-Ru Li1, Dong-Mei Ren1, Hong-Xiang Lou1, Xiao-Ning Wang*,1, Tao Shen*,1 1

Key Lab of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University, Jinan, P. R. China;

2

Department of Pharmacy, The Third Hospital of Jinan, Jinan, P. R. China

3

Department of Pharmacy, Jinan Maternity and Child Care Hospital, Jinan, P. R. China

* Corresponding author at: School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xi Road, Jinan 250012, P. R. China; Tel.: 86-531-88382028; fax: 86-531-88382548; E-mail address: [email protected] (X.-N. Wang); [email protected] (T. Shen)

Abstract Ethnopharmacological relevance: The calyxes and fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino (Physalis Calyx seu Fructus), have been widely used in traditional and indigenous Chinese medicines for the therapy of cough, excessive phlegm, pharyngitis, sore throat, dysuria, pemphigus, eczema, and jaundice with a long history. Aim of the review: The present review aims to achieve a comprehensive and up-to-date investigation in ethnomedical uses, phytochemistry, pharmacology, and toxicity of P. alkekengi var. franchetii, particularly its calyxes and fruits. Through analysis of these findings, evidences supporting their applications in ethnomedicines are illustrated. Possible perspectives and opportunities for the future research are analyzed to highlight the gaps in our knowledge that deserves further investigation. Material and Methods: Information on P. alkekengi var. franchetii was collected via electronic search of major scientific databases (e.g. Web of Science, SciFinder, Google Scholar, Pubmed, Elsevier, SpringerLink, Wiley online and China Knowledge Resource Integrated) for publications on this medicinal plant. Information was also obtained from local classic herbal literature on ethnopharmacology. 1

Results: About 124 chemical ingredients have been characterized from different parts of this plant. Steroids (particularly physalins) and flavonoids are the major characteristic and bioactive constituents. The crude extracts and the isolated compounds have demonstrated various in vitro and in vivo pharmacological functions, such as anti-inflammation, inhibition of tumor cell proliferation, antimicrobial activity, diuretic effect, anti-diabetes, anti-asthma, immunomodulation, and anti-oxidation. Conclusions: P. alkekengi var. franchetii is an important medicinal plant for the ethnomedical therapy of microbial infection, inflammation, and respiratory diseases (e.g. cough, excessive phlegm, pharyngitis). Phytochemical and pharmacological investigations of this plant definitely increased in the past half century. The chemical profiles, including ingredients and structures, have been adequately verified. Modern pharmacological studies supported its uses in the traditional and folk medicines, however, the molecular mechanisms of purified compounds remained unclear and were worth of further exploration. Therefore, the researchers should be paid more attention to a better utilization of this plant.

Graphical Abstract

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Keywords: Physalis alkekengi var. franchetii, Phytochemistry, Pharmacology, Ethnomedical uses

Chemical compounds studied in this article: calystegin B1 (PubChem CID: 164245) calystegin B2 (PubChem CID: 443000) chlorogenic acid (PubChem CID: 1794427) ferulic acid (PubChem CID: 445858) isophysalin A (PubChem CID: 101575890) luteolin (PubChem CID: 5280445) luteolin-7-O-β-D-glucopyranoside (PubChem CID: 5280637) physalin A (PubChem CID: 44577487) physalin B (PubChem CID: 101650337) physalin D (PubChem CID: 431071) physalin F (PubChem CID: 101528280) physalin L (PubChem CID: 101637227)

Abbreviation ERK, extracellular signal-regulated kinase; EtOAc, ethyl acetate; EtOH, ethyl alcohol; COX-2, cyclooxygenase-2; IL, interleukin; INF-γ, interferon γ; JAK, Janus kinases; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MIC, minimum inhibitory concentration; MIR-2, inflammatory protein-2; MMP, matrix metalloproteinase; NF-κB, nuclear factor κB; NO, nitric oxide; iNOS, inducible nitric oxide synthase; PGE2, prostaglandin E2; ROS, reactive oxygen species; STAT3, the signal transducers and activators of transcription; TNF-??, tumor necrosis factor ??; WSP, water soluble polysaccharide.

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1. Introduction Physalis Calyx seu Fructus, named as ‘Jin-Deng-Long’ (锦灯笼) in Chinese, is the calyxes and fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino, and has a long history of the treatment of human diseases in China. Its medical values have been recorded in many Chinese medical documents, exemplified by ‘Shennong’s Classic of Materia Medica’ (Shen Nong Ben Cao Jing, 神农本草经), and ‘Compendium of Materia Medica’ (Ben Cao Gang Mu, 本草纲目). According to traditional Chinese medicine (TCM) theory, it is commonly used for the treatment of cough, excessive phlegm, pharyngitis, sore throat, dysuria, pemphigus, and eczema (Pharmacopoeia Commission of PRC, 2015). Since the wide applications of P. alkekengi var. franchetii in indigenous medicines, plenty of investigations on the phytochemical and pharmacological aspects of this plant have been developed, and given rise to many interesting and attractive results. Chemical constituents covering steroids, flavonoids, phenylpropanoids, and alkaloids, have been isolated from different parts of the plant. Crude extracts and isolated ingredients of this plant demonstrated various pharmacological effects, such as anti-inflammation, inhibition of tumor cell proliferation, antimicrobial, diuretic effect, anti-diabetes, anti-asthma, immunomodulation, and anti-oxidation, verified by the bioassay in vitro and in vivo (Gao et al., 2014; Guo et al., 2012). Although several concise reviews concerning the phytochemical and biological aspects have been published, these reviews were composed in Chinese and not comprehensive. Different from above literatures, the present review provides a comprehensive and up-to-date survey on ethnomedical uses, phytochemistry, pharmacology, and toxicology of this plant. More importantly, correlations of ethnomedical uses, pharmacology, toxicology, and phytochemical aspects have been discussed on the basis of the research findings in these fields. Besides, the major achievements, shortcomings, as well as the possible perspectives and trends for future studies of the calyxes and fruits of P. alkekengi var. franchetii have also been put forward.

2. Botanical characterization and distribution Five synonyms are known for Physalis alkekengi var. franchetii (Solanaceae), including ‘Physalis franchetii Mast., Physalis franchetii var. bunyardii Makino, Physalis glabripes Pojark., Physalis praetermissa Pojark., and Physalis szechuanica Pojark.’ (The plant List, 2017). P. alkekengi var. franchetii is a perennial herb growing to the height of 40 – 80 cm. The stem is

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little branched, nodes sometimes inflated, and pubescent. Leaf is blade narrowly to broadly ovate, 5 – 15 cm long, 2 – 8 cm broad, glabrescent and sometimes ciliate, base oblique, cuneate, margin entire or coarsely dentate, sometimes with salient, unequal deltate lobes, and apex acuminate. Pedicel is 0.6 – 1.6 cm in length, glabrescent, puberulent or densely and persistently villous. Fruiting calyx is red, ovate, rounded, 2.5 – 4 × 2 – 3.5 cm, subleathery, invaginated at base, and glabrescent. Fruiting pedicel is 2 – 3 cm in length. Berry is shiny, orange-red, globose, and 1 – 1.5 cm in diameter. The seeds are ca. 2 mm in diameter, pale yellow, and reniform. Pictures of P. alkekengi var. franchetii have been shown in figure 1. It is widely distributed in Asia and Europe. In China, it mainly grows in Gansu, Shanxi, Henan, Hubei, Sichuan, Guizhou, and Yunnan provinces (Editorial Board of Flora of China, 1978; Zhang et al, 1994).

3. Ethnomedical uses and preparations Ethnomedical uses of P. alkekengi var. franchetii date back to over 2000 years ago. Its medical values were firstly recorded in the Shennong’s Classic of Materia Medica’ (Shen Nong Ben Cao Jing, 神 农本草经) arisen in the period of the Warring States, the Qin and Han Dynasties (B. C. 475 – A. D. 220). In this TCM monograph, P. alkekengi var. franchetii was classified into a ‘medium grade’ drug with undefined toxicity, and was described as an agent of treating dysuria and dystocia (Gu and Yang, 2007). Subsequently, its medical uses were documented in plenty of well-known TCM classics, such as Shen Nong Ben Cao Jing Ji Zhu (神农本草经集注, A. D. 480), Xin Xiu Ben Cao (新修本草, A. D. 659), Dian Nan Ben Cao (滇南本草, A. D. 1436), Compendium of Materia Medica (Ben Cao Gang Mu, 本草纲目, A. D. 1578), and Chinese Pharmacopoeia (2015 Edition). Based on the descriptions in these TCM monographs and traditional applications by local residents in folk medicines, it is concluded that the calyxes and fruits of P. alkekengi var. franchetii are externally and/or internally used to treat cough, excessive phlegm, pharyngitis, sore throat, dysuria, dermatosis, dystocia, jaundice, hemorrhoids, and bronchocephalitis. The clinical dosages for adults suggested by the TCM monographs and Chinese Pharmacopoeia are 5 – 9 g/day for internal use, and reasonable amount for external application. The usages of this plant have been summarized as follows. Infusion tea of the calyxes of P. alkekengi var. franchetii was able to cure cough, excessive phlegm, sore throats and laryngeal cancer (Liaoning College of Traditional Chinese Medicine, 1973). The dry powder of fruits was used for the therapy of bronchocephalitis (Ye, 1953). Administration of its fruits could relieve the infant jaundice (Tao and Shang, 1987). The aerial parts of P. alkekengi var. franchetii were externally used for the therapy of

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hemorrhoids. The fresh jam and powders of dry fruits were applied externally for the treatment of dermatosis, such as felon, pemphigus, and eczema (Guiyang Health Bureau, 1959; Li, 1978). In Turkish folk medicine, the plant was used as diuretic, antipyretic, and sedative agents (Baytop, 1999). It has also been adopted for the treatment of cough, urinary problem, middle ear infection, and sore throats in Eastern Asia and European folk medicines (Hong et al., 2015; Kim et al., 1997). Beside these ethnomedical applications in the form of single medicine, the calyxes and fruits of P. alkekengi var. franchetii (Physalis Calyx seu Fructus) were commonly used in multi-component preparations to improve their therapeutic efficacy because of the TCM’s synergic effects, such as Jin Deng Shan Gen Decoction (金灯山根汤). Moreover, the calyxes and fruits of P. alkekengi var. franchetii were developed into modern pharmaceutics preparation [e.g. Ju Hong Hua Tan Pill (橘红化痰丸)]. The traditional uses of the calyxes and fruits of P. alkekengi var. franchetii in the form of compound and modern pharmaceutical preparations have been summarized in Table 1. The names of crude drugs in Table 1 were established on the basis of Chinese Pharmacopoeia (2015 Edition).

4. Phytochemistry Phytochemical investigations of P. alkekengi var. franchetii date back to the year of 1965 (Yamaguchi and Nishimoto, 1965). The information of the isolated constituents has been briefly provided by two Chinese reviews (Gao et al., 2014; Guo et al., 2012). Up to date, approximately 124 ingredients covering steroids, flavonoids, phenylpropanoids, and alkaloids, have been isolated from different parts of this plant (Table 2). Among them, steroids (mostly physalins) and flavonoids have been regarded to be the characteristic and principal bioactive substances of P. alkekengi var. franchetii.

4.1 Steroids About fifty-eight steroids have been reported from the calyxes, fruits and aerial parts of P. alkekengi var. franchetii. Among them, a group of steroids bearing 13, 14-seco-16, 24-cycloergostane skeletons, named as physalins, are the predominate steroids in this plant. Physalin A (1), isolated from the leaves of P. alkekengi var. franchetii in 1969, was the first member of this group (Matsuura et al., 1969). Subsequently, a series of physalins with complex and diverse structures were reported from the genus Physalis. Up to now, phytochemical investigations of P. alkekengi var. franchetii lead to the isolation of fifty physalins (1–49 and 53), and most of which are firstly discovered structures (Table 2 and Figure 2). The structural diversity of physalins is produced by cyclization, changes in the degree of

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unsaturation, and the variations in the substitution pattern of rings. For example, physalins F (12), J (15), and III (34) contain 5, 6-epoxy moiety (Qiu et al., 2008c; Yang et al., 2016; Xu et al., 2013). Physalins K (16) and Q (22) are 2α, 5α-epidioxy derivatives (Makino et al., 1995a). An additional bond between C-11 and C-16 exists in the structure of physalin R (23) (Makino et al., 1995b). Physalin S (25) possesses a 6β-hydroxy-3, 5-cyclo steroidal skeleton which might be produced by an acid catalyzed rearrangement reaction of 3-hydroxy-△5 physalin B (Makino et al., 1995b). Physalins W (27), X (28), Y (30), and Z (31), as well as 3-O-methylphysalin X (29), and isophysalin G (43) are 3-hydroxyl and/or 3-methoxyl substituted physalins, while physalin C (3) and 5α, 6β-dihydroxyphysalin R (24) are ingredients with 5,6-dihydroxyl groups (Li et al., 2012).These physalins have been determined to be the bioactive ingredients contributing to the anti-inflammatory, anti-tumor and antimicrobial functions of P. alkekengi var. franchetii.. Furthermore, ingredients 50–52 sharing polyoxygenated ergostane structures (named as withanolides), and ingredients 54–58 possessing stigmastane skeletons have been isolated from the fruits, calyxes and aerial parts of this plant (Li et al., 2014; Sharma et al., 1974; Liang and Cai, 2007).

4.2 Flavonoids Nineteen flavonoids have been isolated from the calyxes of P. alkekengi var. franchetii in the form of flavones and flavonols (Table 2 and Figure 3). Theisolated flavonoids include luteolin and their glycosides (59–63), quercetin derivatives (64–68), and kaempferol glycosides (69–70), which are widely-distributed in the medical plants (Xing and Jiang, 2013; Cai et al., 2009; Han et al., 2015; Lin and Wang, 2011; Qiu et al., 2007; Qiu et al., 2008c; Xu et al., 2009). Moreover, four flavone analogues, apigenin-O-β-D-glucopyranoside (71), diosmetin-O-β-D-di-glucopyranoside (72), chrysoeriol and its glucopyanoside (76 and 77), as well as three flavonol analogues, 3’,4’-dimethoxymyricetin (73), ombuine (74), and 5,4’,5’-trihydroxy-7,3’-dimethoxy-flavonol (75), were obtained from the calyxes of this plant (Shu et al., 2014; Cai et al., 2009; Qiu et al., 2008c; Xu et al., 2009; Chen et al., 2007b). These isolated flavonoids have been best known for their beneficial biological functions, including anti-oxidation, anti-inflammation, and inhibition of tumor proliferation (Birt et al., 2001; Nijveldt et al., 2001; Yao et al., 2004).

4.3 Phenylpropanoids Eight phenylpropanoids, consisting of four phenylpropionic acid derivatives and four lignans, have been purified from the calyxes of P. alkekengi var. franchetii (Table 2 and Figure 4). The isolated 7

phenylpropionic acid derivatives are well-known chemical ingredients, covering ferulic acid (78), 3-caffeoylquinic acid methyl ester (79), chlorogenic acid (80), and syringalide (81), which demonstrate evident antibacterial, antiviral and anti-inflammatory properties (Li et al., 2002; Chen et al., 2014; Shu et al., 2014). All of the isolated lignans possess the tetrahydrofuran-type skeleton and exist in the form of

glycosides,

comprising

syringaresinol

glycosides

(82

and

85),

(+)-pinoresinol-O-β-D-di-glucopyranoside (83), and (+)-medioresinol-O-β-D-di-glucopyranoside (84) (Shu et al., 2014; Chen et al., 2014).

4.4 N-containing compounds Approximately thirteen N-containing ingredients have been discovered, covering alkaloids, nucleosides and peptides (Table 2 and Figure 4). The alkaloids mainly occur in the root of P. alkekengi var. franchetii. Five calystegins (88–92), 1β-amino-2α, 3β, 5β-trihydroxycycloheptane (93), 3α-tigloyloxytropane (94), and phygrine (95) have been isolated from the root of this plant (Asano et al., 1996; Yamaguchi and Nishimoto, 1965; Basey et al., 1992). Two cinnamamide derivatives, N-transferuloyltyramine (86) and N-p-coumaroyltyramine (87), were found in the calyxes of P. alkekengi var. franchetii (Chen et al., 2014). Two nucleosides adenine (96) and adenosine (97), as well as one peptide cyclo(tyrosine-amidocaproic)-bipeptide (98) were obtained from the calyxes of this plant (Chen et al., 2014; Cai et al., 2009).

4.5 Miscellaneous constituents Besides above mentioned ingredients, terpenoids (99 and 100) (Xing and Jiang, 2013), megastigmanes (101–106) (Qiu et al., 2008b; Qiu et al., 2008d), aliphatic derivatives (107–109) (Chen et al., 2014), organic acids (110–112) (Chen et al., 2007b; Cai et al., 2009), coumarin (113) (Shu et al., 2014), phenethyl alcohol (114) (Xing and Jiang, 2013), sucrose esters (115–124)(Zhang et al., 2016), and polysaccharides (Shang et al., 2011; Tong et al., 2008) have been identified from different parts of P. alkekengi var. franchetii (Table 2 and Figure 5).

5. Pharmacology Since the flavonoids (59–77) and phenylpropanoids (78–81) are widely distributed in plant kingdom and demonstrate diverse biological functions, their phytochemical and pharmacological aspects have been comprehensively summarized (Agrawal, 2011; Han et al., 2007; Harborne, 2013; Nijveldt et al., 2001; Srinivasan et al., 2007; Zhao and Moghadasian, 2008). In the present review, we 8

focused on the pharmacological effects related to the ethnomedical uses of P. alkekengi var. franchetii, to establish the correlations of ethnomedical uses, phytochemistry, and pharmacology.

5.1 Anti-inflammatory activity A 50% EtOH fraction of P. alkekengi var. franchetii, mainly consisting of flavonoids (60, 71 and 72) and physalins (4, 12, 15, 20 and 21), evidently decreased the levels of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), and interleukin 6 (IL-6), and downregulated the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and NF-κB in lipopolysaccharide (LPS)- stimulated THP-1 cells at concentrations ranging from 25 to 100 μg/mL (Shu et al., 2016). Furthermore, treatment with the 50% EtOH fraction (200 mg/kg) significantly reduced ear edema in xylene-induced acute rat ear edema model, and inhibited granulomatous tissue formation in cotton pellet-induced rat model. Similarly, the methanol extract of P. alkekengi var. franchetii (20–60 μg/mL) was capable of decreasing the levels of NO, IL-6, and TNF-α, downregulating the MMP-9 expression, and inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of AP-1 in LPS-induced RAW 264.7 macrophages (Hong et al., 2015). The anti-inflammatory effect was confirmed by ovalbumin-induced mice asthma model. Treatment with the methanol extract of P. alkekengi var. franchetii at a dose of 30 mg/kg definitely decreased inflammatory cell counts and the productions of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluids. The expressions of iNOS, matrix metalloproteinase-9 (MMP-9), and mucus production in lung tissue have also been inhibited. Moniruzzaman et al (2016) separated the MeOH extract of P. alkekengi var. franchetii into n-hexane, chloroform, ethyl acetate (EtOAc) and butanol fractions, and found that the EtOAc and butanol fractions dose-dependently inhibited the productions of inflammaotry mediators (e.g. NO, TNF-α, IL-6) in LPS-induced BV microglial cells. The EtOAc fraction was the potent one with approximate inhibition rates of 100%, 60%, 65% for NO, TNF-α and IL-6, respectively, at a dose of 100 μg/mL, and therefore was subjected to the further research. Research on mechanism indicated that the anti-inflammatory effect of the EtOAc fraction was associated with inhibition of NF-κB, as well as disrupitons of Akt and MAPK signaling pathways. In the studies in vivo, the EtOAc fraction of P. alkekengi var. franchetii at doses of 100 and 200 mg/kg significantly inhibited acetic acid-induced writhing, formalin-induced licking time and edema in mice (Moniruzzaman et al., 2016). The powder of the calyxes of P. alkekengi var. franchetii and sesame oil were mixed together to prepared an oil preparation of P. alkekengi var. franchetii, and evaluated for its anti-eczema activity using 2, 4-dinitrochlorobenzene induced rat acute atopic dermatitis model and guinea pig chronic 9

atopic dermatitis model in vivo (Miao et al., 2014). External applications of P. alkekengi var. franchetii oil at a dose of 0.36 g evidently improved ear swelling and pathological changes in these two models in vivo. This result supported the traditional use of P. alkekengi var. franchetii for the remedy of eczema and atopic dermatitis. Ingredients isolated from P. alkekengi var. franchetii were investigated for their anti-inflammatory effect in vitro and in vivo. Luteolin (59) and luteolin-7-O-β-D-glucopyranoside (60) are representatives of P. alkekengi var. franchetii flavonoids with diverse biological activities. Luteolin (59) dose-dependently inhibited the expression and production of NO, PGE2, iNOS, COX-2, TNF-α, and IL-6 in LPS-stimulated rat macrophage 264.7 cells at doses of 5, 10, and 25 μM (Chen et al., 2007a). Its anti-inflammatory effect was associated with blocking NF-κB and AP-1 activation. The anti-inflammatory property of 59 has been tested using rat inflammation models in vivo (Li et al., 2007). Oral administration of 59 at doses of 10 and 50 mg/kg significantly alleviated the paw edema in carrageenan-induced acute rat inflammation model. 59 also inhibited cotton pellet-induced granuloma formation at 1, 10 and 50 mg/kg in chronic rat inflammation model. Its anti-inflammatory effect was comparable to the positive control indomethacin (50 mg/kg) (Li et al., 2007). Similar anti-inflammatory functions of structurally related flavonoids, such as apigenin, quercetin, kaempferol and their analogues, have been determined by bioassay in vitro and in vivo (Funakoshi-Tago et al., 2011; Hämäläinen et al., 2007; Kim et al., 2004). Therefore, the ethnomedical applications of P. alkekengi var. franchetii on inflammation-related diseases are partly attributed to these flavonoids (59–77). In addition, ferulic acid (78) at 50 μM inhibited the respiratory syncytial virus and LPS-induced productions of macrophage inflammatory protein-2 (MIR-2) in RAW 264.7 cells (Sakai et al., 1999; Sakai et al., 1997). Besides flavonoids, physalin is the other dominant group of anti-inflammatory substances in this ethnomedical plant. Physalins A (1), G (13), L (17), O (20), and isophysalin A (39) inhibited the LPS-induced NO production with inhibition rates of 90.33%, 77.42%, 70.97%, 87.10% and 83.88% at 20 μM in RAW 264.7 cells (Ji et al., 2012). Hydrocortisone was used as a positive control, and displayed an inhibition rate of 87.22% at 20 μM. Chen et al. (2012) have also tested the anti-inflammatory activity of 1 , and suggested that 1 inhibited the NO formation assay in LPS-induced RAW 264.7 cells, with an IC50 value of 2.57 μM (Chen et al., 2012). They have also investigated the synergistic anti-inflammatory effect of steroid and flavonoid. The results indicated that combinational applications of 1 and 59, as well as 1 and 60 demonstrated the synergistic functions on inhibiting NO production, decreasing TNF-α level, and downregulating iNOS protein expression in LPS-induced RAW 264.7 macrophages 10

(Chen et al., 2012). Anti-inflammatory effect of physalin F (12) was investigated using a collagen-induced DBA/1 mice arthritis model (Brustolim et al., 2010). Oral administration with physalin F (12) at a dose of 20 mg/kg markedly decreased the paw edema in 20 days after treatment, while the positive control dexamethasone (2.5 mg/kg) inhibited the paw edema in 10 days after treatment. Pinto et al (2010) evaluated the anti-inflammatory effect of physalin E (11) using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced acute dermatitis mice ear model and oxazolone-induced chronic mice ear model. Treatment of mice ear with 11 (0.125–0.5 mg/ear) evidently alleviated ear edema, blocked the production of pro-inflammatory cytokines (e.g. TNF-α and IFN-γ), and inhibited myeloperoxidase activity. In this study, pretreatment with mifepristone, a glucocorticoid receptor antagonist, completely inhibited the anti-inflammatory effect of physalins B (4), E (11) and F (12). 4 and 12 have been investigated for their anti-inflammatory effect using intestinal ischaemia and reperfusion (I/R) inflammation model in mice (Vieira et al., 2005). Physalins B (4) and F (12) at a dose of 20 mg/kg, as well as the positive control dexamethasone (10 mg/kg), markedly prevented intestinal neutrophil influx and haemorrhage, decreased intestinal vascular permeability, and suppressed the increase of TNF-α. Similar with the previously reported results (Pinto et al., 2010), the anti-inflammatory effects of physalins B (4) and F (12) could be reversed by the corticoid receptor antagonist RU486. Above two documents suggested that the anti-inflammatory effects of physalins, exemplified by 4 and 12, might be attributed to the activation of glucocorticoid receptors. Ozawa et al. (2013) reported that physalins with 5β, 6β-epoxy moiety and C5−C6 olefin functionality in ring B inhibited NF-κB signaling pathway, but had different modes of action in inhibiting NF-κB activation. Further study indicated that 5β, 6β-epoxy substituted physalins disturbed NF-κB activation through inhibiting phosphorylation and degradation of IκBα, however, physalins with C5−C6 olefinic functionality inhibited NF-κB via obstructing nuclear translocation and DNA binding of RelA/p50 protein dimer (Ozawa et al., 2013). A ‘Jin Deng Long Injection’, prepared from the aqueous of the calyxes of P. alkekengi var. franchetii by Beijing Hospital of Traditional Chinese Medicine, has been clinically evaluated for its therapeutic effect against of children’s upper respiratory tract inflammation, such as suppurative tonsillitis, herpetic pharyngitis (Li et al., 1986). In this clinical survey containing 191 cases, after intramuscular administration of ‘Jin Deng Long Injection’ equivalent to 5.4–8.0 g of the calyxes of P. alkekengi var. franchetii, for children older than 5, and half of the dose for children less than 5, the symptoms of fever, sore throat, and congestion of throat for patients disappeared or improved within 2−4 days, with a cure rate approximately at 95%. Subsequently, a phytochemical investigation was 11

carried out on this injection, and verified physalin B (4) as the main bioactive substance (He and Wang, 1986). The anti-inflammatory functions of the calyx and fruit extracts of P. alkekengi var. franchetii have been sufficiently investigated using diverse in vitro and in vivo models, and the clinical study. The bioactive doses of the extract of this plant are 20–100 μg/mL for cell-based assay (Shu et al., 2016; Hong et al., 2015), and 10–200 mg/kg for different mice models (Moniruzzaman et al., 2016). The clinically effective dose is 5.4 – 8.0 g, which is consistent with suggested dose (5–9 g) in Chinese Pharmacopia (Li et al., 1986). The anti-inflammatory effects of P. alkekengi var. franchetii and its constituents were exerted by inhibiting NF-κB and AP-1 pathways, and downregulating inflammatory mediators (e.g. PGE2, ILs, TNF-α), and some key enzymes (e.g. COX2, iNOS) involved in inflammatory response (Figure 6). All of these data definitely supported the ethnomedical uses of this plant for the therapy of inflammation-related diseases, such as cough, excessive phlegm, sore throat, and eczema. Phyaslins (e.g. 1, 4, and 11) and flavonoids are the ingredients sustaining the anti-inflammatory application of P. alkekengi var. franchetii.

5.2 Antimicrobial activity Chlorogenic acid (80) demonstrated antibacterial activities against Staphylococcus aureus, Streptococcus pneumoniae, Bacillus subtilis, Escherichia coli, Shigella dysenteriae, and Salmonella Typhimurium, with MIC values of 20–80 μg/mL (Lou et al., 2011). 80 also inhibited the proliferation of influenza virus in MCDK cells, and adenovirus and respiratory syncytial virus in HeLa cells in vitro (Li et al., 2005). Intraperitoneal administration of 80 at doses of 5 and 10 mg/kg evidently decreased the mortality and lung index of influenza virus-infected mice. Physakengoses B (116) and E–H (119–122) demonstrated antibacterial activities against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli with MIC values ranging from 3.5 to 14.9 μg/mL (Zhang et al., 2016). Physalins B (4), D (9), F (12), and G (13) displayed antimalarial effect against Plasmodium falciparum in vitro, with IC50 values ranging from 2.2 to 55 μM (Sá et al., 2011).Among them, physalin F (12) was the most potent ingredient (IC50 = 2.2 μM and LC50 = 13.3 μM), while physalin D (9) was the weakest (IC50 = 55 μM and LC50 = 570 μM). Then, 9 and 12 were subjected to the Plasmodium berghei-infected mice model to confirm its bioassay in vivo. Interestingly, treatment of P. berghei-infected mice with 12 (50 and 100 mg/kg) increased parasitemia levels and mortality, however, treatment with 9 significantly reduced parasitemia and mortality in P. berghei-infected mice. The author mentioned that this unexpected result might be caused by the potent immunosuppressive 12

activity of 9, which destroyed the antimalarial immune response in infected animals. Through analyzing the characteristics of the tested microorganism in these studies, it was concluded that the purified constituents were active in inhibition of bacteria, influenza virus and malaria (Li et al., 2005; Sá et al., 2011; Zhang et al., 2016; Lou et al., 2011). These antimicrobial results, together with the anti-inflammatory property, definitely supported the traditional use of P. alkekengi var. franchetii for the therapy of bacterium and/or virus infection related diseases, such as excessive phlegm, sore throat, diarrhea, gastritis and turbid urine.

5.3 Inhibition of tumor cell proliferation Antiproliferative functions of the crude extracts and purified ingredients of the calyxes and fruits of P. alkekengi var. franchetii on tumor cells, especially the anti-tumor effects of physalins, are hot topic in the field of pharmacological aspects of this ethnomedical plant. The aqueous extract of the calyxes of P. alkekengi var. franchetii dose-dependently inhibited the proliferation of human lung cancer SPC-A-1 cells with a maximum inhibition rate of 79.9% at 25 mg/mL (Xin et al., 2010). Physalin A (1) dose-dependently inhibited the growth of human lung cancer H292, H460, H358, H1975, and A549 cells with an IC50 values ranging from 5 to 30 μM (Kang et al., 2016; Zhu et al., 2016). Mechanic study indicated that 1 was able to induce the cell apoptosis, increase the intracellular ROS level, arrest cells in G2/M phase, and inhibit phosphorylations of STAT3 and JAKs. He et al (2013a, b) found that 1 at 10 and 15 μM induced apoptosis and autophagy in human fibrosarcoma HT1080 cells and human melanoma A375-S2 cells. In HT1080 cells, physalin A (1) at 10 μM induced apoptosis through up-regulating the expressions of caspases 3 and 8, and no significant changes in caspase 9, Bid, Bax and Bcl-2, were observed (He et al., 2013b). In A375-S2 cells, apoptosis-related proteins, including caspases 3, 8, 9, Bid, Bax and Bcl-2 were activated by 1 at 15 μM (He et al., 2013a). Furthermore, physalin A (1)-induced apoptosis was associated with ROS generation and activation of P53-Noxa pathway in A375-S2 cells. Treatment of cells with ROS scavengers N-acetylcysteine, glutathione (GSH), p53 inhibitor pifithrin-α and Noxa-siRNA completely inhibited the cell apoptosis. Importantly, 1 did not demonstrated cytotoxicity on human normal peripheral blood mononuclear cells under the active concentrations on inhibitions of fibrosarcoma and melanoma cells. These results suggested that anti-proliferative effect of 1 was regulated by p53-Noxa-mediated ROS generation, the JAK2/3-STAT3 and p38 MAPK/ROS pathways. Physalin B (4) was investigated for its antiproliferative effects against human melanoma cells (A375 and A 2058), rat cardiac myoblast H9c2 cells, human normal aorta smooth muscle T/G HA-VSMC cells, and human skin fibroblast CCD-966SK 13

cells (Hsu et al., 2012). The results indicated that 4 was particularly active against human melanoma A375 and A 2058 cells (IC50 < 4.6 μg/mL), and had low cytotoxicities against H9c2, T/G HA-VSMC, and CCD-966SK cells. Further investigation revealed that 4 at a dose of 3 μg/mL induced apoptosis via activation of Noxa, caspase 3 and Bax in A375 cells, a similar mechanism with 1. Vandenberghe et al (2008) have verified Noxa-associated apoptosis of 4, and also found that 4 was an inhibitor of the ubiquitin-proteasome pathway. With the exception of physalins, flavonoids and phenylpropanoids possessed antiproliferative activities against tumor cells. Chlorogenic acid (80) dose-dependently inhibited the proteolytic activity of MMP-9 with an IC50 value of about 50 nM in human hepatocellular carcinoma Hep3B cells, suggesting the potential of 80 as an anti-metastasis agent (Jin et al., 2005). Ferulic acid (78) at concentrations of 250 and 500 ppm was capable of inhibiting formation of aberrant crypt foci, and incidences of colonic carcinomas in azoxymethane-induced colon carcinogenesis in mice (Kawabata et al., 2000). Further research indicated that the antiproliferative effect of 78 was caused by activations of phase II detoxifying enzymes, glutathione S-transferase and quinone reductase in liver and colon of mice. Treatment of mice with 78 and 80 at a dose of 10 μmol per mouse evidently inhibited the formation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumors, with the inhibition rates of 35% and 60%, respectively (Huang et al., 1988). The anti-tumor properties of flavonoids, including luteolin (59), quercetin, and their analogues, have been extensively investigated (Lin et al., 2008; Murakami et al., 2008). These flavonoids could induce cancer cell apoptosis, arrest cancer cell cycle, and inhibit metastasis and angiogenesis through acting with multiple targets, such as p53, phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, NF-κB, Nrf2, X-linked inhibitor of apoptosis protein, MAPK, and extracellular signal-regulated kinase (ERK). Taken together, physalins, flavonoids, and phenylpropanoids are the main anti-tumor substances. As a group of representative constituents in genus Physalis, physalins A (1) and B (4) have received great interest, and have been verified for their anti-tumor mechanism on the cell-based assay (He et al., 2013b; Vandenberghe et al., 2008)(Figure 6). These results suggested that 1 and 4 might be promising therapeutic agents for cancer, especially for the treatment of melanoma. However, limited by the amount of physalins purified from the plant, effectiveness on inhibition of tumor proliferation has not been confirmed by the assay in vivo. The induction of phase II detoxifying enzymes and antioxidant enzymes by ferulic acid (78) implied the potential of 78 as a chemopreventive agent (Huang et al., 1988).

14

5.4 Anti-diabetes The steroidal fraction of the calyxes of P. alkekengi var. franchetii was purified by partition and column chromatography, and then evaluated for its hypoglycemic activity (Liu et al., 2010). Oral administration of the steroidal fraction at a dose of 150 mg/kg a day significantly decreased the blood glucose level, reduced water intake, and increased the body weight of diabetic mice induced by alloxan, demonstrating equal potency with the clinical anti-diabetic traditional medicine ‘Xiao Ke Pill’. These results suggested that the steroidal fraction of fruits or calyxes could be used to treat hyperglycaemia. A polysaccharide isolated from the fruits of P. alkekengi var. franchetii demonstrated anti-diabetic potency (Tong et al., 2008). Oral administration of this polysaccharide (50 and 100 mg/kg) evidently reduced blood glucose level and water intake, and increase the body weight in alloxan-induced diabetic mice model. Calystegins (88 – 92) are a group of structure analogues of glucose and galactose, and thus they can block process of carbohydrate metabolism through competitive inhibitions of key glucosidases and galactosidases. Calystegin B2 (91) has been found to be a potent competitive inhibitor of almond β-glucosidase (Ki = 1.2 μM) and coffee bean α-galactosidase (Ki = 0.86 μM) (Asano et al., 1995). Calystegin B1 (90) has been verified to be potent competitive inhibitors of almond β-glucosidase (Ki = 1.9 μM) and bovine liver β-galactosidase (Ki = 1.6 μM), but inactive against α-galactosidase. Beside these pure ingredients, ferulic acid (78) and chlorogenic acid (80) have been comprehensively investigated for their therapeutic effects on diabetes mellitus, such as diabetic complication using diverse diabetic models in vivo. These two ingredients could decrease the blood glucose level, stimulate insulin secretion, improve glucose tolerance and insulin resistance, lower serum and hepatic CG and TG levels, inhibit fat absorption, and promote fat metabolism in vivo (Mancuso and Santangelo, 2014; Meng et al., 2013). Furthermore, clinical studies indicated that daily consumption of 78 and 80 rich food would reduce the risk of type 2 diabetes mellitus (Huxley et al., 2009).

5.5 Anti-asthma Aqueous extract of the calyxes of P. alkekengi var. franchetii was evaluated for its anti-asthmatic function using ovalbumina-induced mice asthma model (Bao, 2008). Treatment with P. alkekengi var. franchetii calyxes (0.5 g per mouse for six weeks) significantly decreased the levels of white blood cell and eosinophil, and inhibited the expression of IL-5 and IFN-γ in the lung tissue, comparable to the clinically used TCM, ‘Xiao Qing Long Decoction’. The anti-asthma effect might be partly attributed to 15

the structural similarity of physalins to the clinically used steroid hormone, as well as its potent anti-inflammatory effect, for instance, inhibition of the pro-inflammatory cytokines production (see section 5.1). This data implied the potential of P. alkekengi var. franchetii as anti-asthma agent.

5.6 Diuretic effect Wu et al (2012) reported that intragastric administration of the EtOH extract of calyxes of P. alkekengi var. franchetii was capable of promoting the excretion of urine in mice. As a positive control, hydrochlorothiazide (10 mg/kg) demonstrated a potent and persistent diuretic effect until the fifth hour of treatment. While the EtOH extract (200 mg/kg) displayed equal diuretic effect at the second hour of extract administration, but had no effect on the excretion of urine at the fifth hour. It implied that the diuretic effect of P. alkekengi var. franchetii was short-acting compared with that of hydrochlorothiazide. This data supported the traditional uses of P. alkekengi var. franchetii for the treatment of chronic nephritis and urinary calculus.

5.7 Vasodilative effect The aqueous extract of calyxes of P. alkekengi var. franchetii was evaluated for its function on arterial vasodilatation by the rat aortic ring assay (Liu et al., 2008). The data indicated that aqueous extract of P. alkekengi var. franchetii, equal to 32 mg/mL of the calyxes, was capable of relieving phenylephrine- and KCl- induced vasoconstriction, and this vasodilative effect could not be blocked by nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester. Liu et al (2008) further investigated the mechanism of P. alkekengi var. franchetii on smooth muscle contraction, and found that the P. alkekengi var. franchetii’s vasodilatation was not disturbed by K+ channel inhibitors, tetrathylamonium and 4-aminopyrimide. Collectively, the aqueous extract of P. alkekengi var. franchetii had an endothelium-independent vasodilatation which might be related to the inhibitions of calcium influx and protein kinase C (PKC) signaling pathway (Figure 6).

5.8 Miscellaneous bioactivities The EtOAc, BuOH and aqueous extracts of the calyxes of P. alkekengi var. franchetii demonstrated potent DPPH radical scavenging activities with the clearance rates of approximately 100% at a concentration of 2 mg/mL, which were comparable to the positive control butylated hydroxytoluene (Wang et al., 2010). A crude polysaccharide from the fruits and calyxes of P. alkekengi var. franchetii displayed high potency on DPPH, OH, and superoxide anion-scavenging activities with scavenging rates 16

of 53.3%, 80.5%, and 68.3% (Ge et al., 2009). Gong et al (2002) have evaluated the analgesic effect of P. alkekengi var. franchetii using acetic acid-induced writhes assay, the hot plate assay, and electrical stimulation vocalization test. The results indicated that administration of the aqueous extracts, which were equal to 500 and 800 mg/kg of the calyxes, evidently inhibited the writhing reaction, prolonged the latency of licking paw and raise the pain threshold in tail stimulation-vocalization test in mice. The analgesic effect was reversed by naloxone, suggesting that this analgesic activity was exerted by an interaction with brain opioid mechanism (Chamberlain and Klein, 1994). Intestinal microflora imbalance and deficiency of intestinal probiotic bacterium give rise to abdominal pain, diarrhea, and astriction. It was found that total physalin extract of P. alkekengi var. franchetii promoted the growth of Lactobacillus delbrueckii and inhibited the growth of Escherichia coli at concentrations of 0.78–1.56 mg/mL in vitro (Li et al., 2012). Subsequently, two studies in vivo indicated that intragastrically administration of the aqueous extract of calyxes of P. alkekengi var. franchetii (20–80 mg/kg) counteracted levofloxacin-induced decrease of Bacillus acidilactici, Lactobacillus bacteria, and increased the diversity of bacterium composition in mice intestine (Li et al., 2013; Wang et al., 2014). Regulation of intestinal microflora of P. alkekengi var. franchetii might be related to flavonoid glycoside 60 and physalins 2, 3, 9, 15, 21, 24 and 26. Water soluble polysaccharide (WSP) purified from the stem of P. alkekengi var. franchetii was subjected to an assay for the production of ovalbumin-specific antibody (Shang et al., 2011). The data indicated that WSP at a dose of 10 mg/kg significantly enhanced ovalbumin-specific antibody titers (IgG, IgG1, IgG2b) in ICR mice serum, and suggested the potential application of WSP as an adjuvant for improving the efficacy of vaccine. The adjuvant effect of polysaccharide from P. alkekengi var. franchetii has also been verified by Yang et al (2014). Physalins B (4), F (12) and G (13) inhibited the concanavalin-activated splenocyte proliferation (Soares et al., 2006). Physalin B (4) at a concentration of 2 μg/mL produced a 100% inhibition of cell proliferation in mixed lymphocyte reaction. More importantly, administration of physalins B (4), F (12) and G (13) at a dose of 1 mg/day/mouse prevented the rejection of allogeneic heterotopic heart transplant in mice. These data indicated the potential application of physalins as immunosuppressive agents.

6.

Toxicology An acute toxicity study in vivo indicated that no adverse effects on the general behavior or 17

appearance of the mice appeared after oral administration of the 50% EtOH extract of P. alkekengi var. franchetii at a dose of 12.8 g/kg in ICR mice (Shu et al., 2016). Similarly, Zhang (2008) reported that oral administration 20 g/kg of the EtOH extract of P. alkekengi var. franchetii did not cause any toxic effect in ICR mice. However, Wang et al. (2004) reported that oral administration of the extract from calyxes of P. alkekengi var. franchetii at doses of 0.75 and 1.5 g/kg a day gave rise to the death of rat in adrenaline- and alloxan- induced rat diabetic models. The toxicity of the calyxes might be produced by the non-selective cytotoxicity of physalins. These contradictory data might be caused by different extraction method, origin of materials and in vivo models, as well as an error in reporting the experiments. The results also implied that diabetic patients might be sensitive to the cytotoxicity of physalins and the calyxes of this plant. In addition, intraperitoneal administration of the aqueous extract of P. alkekengi var. franchetii fruits to female rats disorganized the estrus cycle, produced a 100% diestrus, and a 96% decrease in the number of pups born (Vessal et al., 1991).

7.

Conclusions P. alkekengi var. franchetii is a medicinal plant that has been long used in TCM and folk medicines.

The present review comprehensively summarizes the ethnomedical uses, phytochemical, pharmacological, and toxicological aspects of this medicinal plant, especially the fruits and calyxes of P. alkekengi var. franchetii. Phytochemical investigations of this plant have focused on the fruits and calyxes because they are the medicinal tissues in TCM and folk medicines, while other tissues (e.g. leaf, stem, and root) have received rather less attention. Physalins and flavonoids are regard to be major ingredients of this plant, and might be typical chemotaxonomic markers for identification of P. alkekengi var. franchetii from the phytochemical point of view. In Chinese Pharmacopoeia (2015 Edition), physalin L (17) and luteolin-7-O-β-D-glucopyranoside (60) have been documented as the standard for the identification and quality control of Physalis Calyx seu Fructus (Pharmacopoeia Commission of PRC, 2015). Moreover, these two types of structures were predominantly discovered in the fruits and calyxes, and only seven physalins (6, 7, 16, 22, and 26-28) have been isolated from leaf, stem, and aerial parts of this plant. Pharmacological studies on crude extracts and chemical ingredients provided pragmatic evidences for its traditional uses, and have revealed this plant to be a valuable source for therapeutic molecules. Regarding the chemical ingredients contributed to medicinal values, we have concluded that: (i) physalins, flavonoids and phenylpropanoids contribute to the majority of pharmacological functions of P. alkekengi var. franchetii; (ii) physalins are responsible for anti-inflammatory, antimicrobial, 18

anti-cancer and immunosuppressive activities; flavonoids for anti-diabetic, anti-inflammatory and anti-cancer activities; phenylpropanoid (e.g. chlorogenic acid (80) and ferulic acid (78)] for anti-diabetic, antimicrobial and anti-cancer effects; alkaloids (e.g. calystegins) for anti-diabetic effect. These different chemical ingredients might have synergetic effect (Chen et al., 2012). Furthermore, these pharmacological results furnished documents for establishing the correlations between ethnomedical uses and biological functions: ethnomedical uses for cough, pharyngitis, excessive phlegm, sore throat were related to anti-inflammatory and antimicrobial activities; dysuria to diuretic effect; pemphigus to anti-inflammatory and immunosuppressive effect; laryngeal cancer to inhibitory effect on tumor cell proliferation; eczema to anti-inflammatory effect; bronchocephalitis and felon to antimicrobial activity. Besides these pharmacological activities related to ethnomedical uses, P. alkekengi var. franchetii may possess a variety of potential biological activities since the existences of flavonoids and phenylpropanoids.

Cumulative

evidences

have

verified

antithrombotic,

antimutagenic,

chemopreventive, antiviral, antioxidant, neuroprotective, and cardioprotective effects of flavonoids (Agrawal, 2011; Nijveldt et al., 2001; Havsteen, 2002; Harborne, 2013), and have approved antioxidant, hepatoprotective, neuroprotective, antithrombotic, anti-atherogenic, chemopreventive, and antiviral effects of phenylpropanoids (Srinivasan et al., 2007; Ou and Kwok, 2004; Kwon et al, 2010; Wang et al., 2009; Xu et al., 2012). Thus, the calyxes and fruits of P. alkekengi var. franchetii have the potential of protecting human against virus-infection, neurodegenerative diseases, cardiovascular disease, and cancer. The data on the toxicological aspects were inconsistent. These contradictory results might be caused by different origin and fractions of plant materials adopted in the bioassay (Shu et al., 2016; Wang et al., 2004; Zhang, 2008). Through detailed analysis of the experimental information (e.g. extract preparation, the model used), it has been concluded that treatment of mice with P. alkekengi var. franchetii extract at a dose of 12.8 g/kg or even 20 g/kg would not give rise to toxicity in mice model, suggesting a low toxicity of this plant (Shu et al., 2016; Zhang, 2008). The reproductive system toxicity of P. alkekengi var. franchetii might exist because of its suppression of diestrus and pups born (Vessal et al., 1991). Watchfully, the fruits and calyxes could not be used by pregnant woman in traditional Chinese medicines since their oxytoic effect. These results implied the harmful effect of P. alkekengi var. franchetii on female reproductive system.

8. Future perspectives 19

Although there has been considerable progress with regards to the phytochemistry and pharmacology of P. alkekengi var. franchetii, important areas remain to be explored for a better understanding of its pharmacological effects and clinical efficacy. (i) Physalins and flavonoids have been verified as the dominant bioactive substances; however, their mechanisms of action, for example, inflammation-related diseases and tumor, remain unclear and should be investigated using modern of pharmacological techniques. (ii) Plenty of ethnomedical uses and pharmacological functions have only been validated by cell-based bioassay or evaluated using the unrepresentative animal modes. For instance, antimicrobial activity and inhibition on cancer proliferation of crude extracts and ingredients have been only evaluated by bioassay in vitro; their anti-inflammatory effects have been mainly verified using classic xylene-, albumen-, and cotton pellet-induced rat inflammatory models, which might not precisely reveal the therapeutic effect on respiratory inflammation-related disease, such as cough, excessive phlegm, sore throat. Thus, representative and appropriate animal models should be adopted to evaluate their efficiency on diseases, and to support the application in ethnomedicines. Moreover, the pharmacokinetic profiles of active ingredients (e.g. physalins) should be noted. (iii) Toxicological studies were limited and have not given a solid data. Thus, extensive toxicity and safety evaluation should be conducted to confirm its side effects, acute and chronic toxicities. (iv) Based upon the ethnomedical uses, chemical ingredients and pharmacological functions of P. alkekengi var. franchetii, future studies on finding new lead compounds and developing crude extracts and ingredients into new drugs and dietary supplements are significant work. Throughout our literature review, we observed that phytochemical and pharmacological investigations supported the ethnomedical application of P. alkekengi var. franchetii in TCM and folk medicines. The effectiveness of crude extracts and purified ingredients for the therapy of diseases provided possibilities of discovering lead compounds for drug research and development, and also supported the clinical application of P. alkekengi var. franchetii in modern medicine.

Acknowledgments The authors wish to acknowledge the financial supports from NNSFs of China (31470419 and 81673558), NSF of Shandong (ZR2014HM019), and Young Scholars Program of Shandong University (2015WLJH50).

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Decoction

Decoction

Zi yin decoction

Treating chronic nephritis

Treating urinary calculus

Treating Behcet syndrome

huo

yan

Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Flos Lonicerae Japonicae (Lonicera japonica Thunb.), Herba Taraxaci (Taraxacum mongolicum Hand-Mazz.), Herba Corydalis Bunge (Corydalis bungeana Turcz.), Radix Trichosanthis (Trichosanthes kirilowii Maxim.), Bulbus Fritillariae Thunbergii (Fritillaria thunbergii Miq), Herba Menthae (Mentha haplocalyx Briq.), Fructus Arctii (Arctium lappa L.), Radix Scrophulariae (Scrophularia ningpoensis Hemsl.), and Radix Rehmanniae (Rehmannia glutinosa Libosch.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Flos Lonicerae Japonicae (Lonicera japonica Thunb.), Fructus Forsythiae (Forsythia suspensa (Thnub.)Vahl), Radix et Rhizoma Sophorae Tonkinensis (Sophora tonkinensis Gagnep.), Fructus Gardeniae (Gardenia jasminoides Ellis), Radix Isatidis (Isatis indigotica Fort.), Radix Achyranthis Bidentatae (Achyranthes bidentata Bl.), Indigo Naturalis (Isatis indigotica Fort.), Herba Menthae (Mentha haplocalyx Briq.), Radix Scrophulariae (Scrophularia ningpoensis Hemsl.), and Radix et Rhizoma Glycyrrhizae (Glycyrrhiz uralensis Fisch.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Fructus Chaenomelis (Chaenomeles speciosa (Sweet) Nakai), Fructus Jujubae (Ziziphus jujube Mill.), and Herba Plantaginis (Plantago asiatica L.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Radix et Rhizoma Gentianae (Gentiana scabra Bge.), Rhizome of Smilax ocreafa A. D, Roots of Cinnamomum camphora (L.) Presl., and Herba Plantaginis (Plantago asiatica L.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Radix Adenophorae (Adenophora stricta Miq.), Radix Glehniae (Glehnia littoralis Fr. Schmidtex Miq.), Radix Scrophulariae (Scrophularia ningpoensis Hemsl.), Cortex Moutan (Paeonia suffruticosa Andr.), Caulis Dendrobii (Dendrobium nobile Lindl.), Fructus Corni (Cornus officinalis Sieb.et Zucc.), Fructus Lycii (Lycium barbarum L.), and Radix Astragali (Astragalus membranaceus (Fisch.) Bge)

Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Fructus Arctii (Arctium lappa L.), and Radix et Rhizoma Glycyrrhizae (Glycyrrhiz uralensis Fisch.) Physalis Calyx seu Fructus (P. alkekengi var. francheti), Fructus Cannarii (Canarium album Raeusch.), Radix et Rhizoma Sophorae Tonkinensis (Sophora tonkinensis Gagnep.), Semen Sterculiae Lychnophorae (Sterculia lychnophora Hance), Radix Trichosanthis (Trichosanthes kirilowii Maxim.), Radix Ophiopogonis (Ophiopogon japonicas (Thunb.) Ker-Gawl.), and Fructus Chebulae (Terminalia chebula Retz.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Radix Platycodonis (Platycodon grandiflorum (Jacq.) A DC.), Semen Armeniacae Amarum (Prunus armeniaca L.), Radix Peucedani (Peucedanum praeruptorum Dunn), and Radix et Rhizoma Glycyrrhizae (Glycyrrhiz uralensis Fisch.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Semen Armeniacae Amarum (Prunus armeniaca L.), and Radix Scrophulariae (Scrophularia ningpoensis Hemsl.)

Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Radix et Rhizoma Sophorae Tonkinensis (Sophora tonkinensis Gagnep.), Fructus Arctii (Arctium lappa L.), Radix Platycodonis (Platycodon grandiflorum (Jacq.) A DC.), Rhizoma Belamcandae (Belamcanda chinensis (L.)DC.), and Radix et Rhizoma Glycyrrhizae (Glycyrrhiz uralensis Fisch.) Physalis Calyx seu Fructus (P. alkekengi var. franchetii), and Borneolum Syntheticum

Composition a Crude drug names (Latin names of original plants)

Health

(Guiyang Health Bureau, 1959) (Zhao, 2006)

Folk recipe

(Ni and Ge, 2004)

(Writing Group of Shandong Zhong Cao Yao Shou Ce, 1970) (Health Bureau of Shanxi Revolutionary Committee, 1972) (Hu, 1997)

(Beijing Public Bureau, 1961)

(Guiyang Health Bureau, 1959) (Jiang, 2001)

(Wei, 1992)

References

+

Treating phlegm

cough

and

27

Ju hong hua tan pill Physalis Calyx seu Fructus (P. alkekengi var. franchetii), Exocarpium Citri Rubrum (Citrus reticulata Blanco), Bulbus Fritillariae CFDAb [Approved by China Cirrhosae (Fritillaria cirrhosa D. Don), Semen Armeniacae Amarum (Prunus armeniaca L. var. ansu Maxim), Pericarpium Papaveris Food and Drug (Papaver somniferum L.), Fructus Schisandrae Chinensis (Schisandra chfnensis (Turcz.) Baill.), Alumen, and Radix et Rhizoma Administration (CFDA)] Glycyrrhizae (Glycyrrhiz uralensis Fisch.) a The crude drug names in column 3 were proved based on the Chinese Pharmacopoeia (2015 Edition) and the Latin names of the original plants were identified with the web of www.theplantlist.org. b Cited from the website of China Food and Drug Administration (http://www.sda.com.cn)

excessive

Qing e decoction

Treating acute tonsillitis

jiang

Jie du shuang decoction

Treating peritonsillar abscess

excessive

Decoction

and

Treating phlegm

cough

Decoction

Qing guo ointment

Decoction

Pulvis

Preparation name / dosage form Jin deng shan gen decoction

Treating acute bronchitis

Treating acute pharyngitis, sore throat, acute tonsillitis, periamygdalitis, acute epiglottitis Treating pharyngitis and sore throat

Traditional uses

Table 1 Traditional uses of the calyxes and fruits of P. alkekengi var. franchetii in China

Table 2 Chemical ingredients isolated from the plant of Physalis alkekengi var. franchetii Classification Steroids

No 1

Chemical ingredient Physalin A

Part of plant Calyx

2 3 4

3β-Hydroxy-2-hydrophysalin A 5α,6β-Dihydroxy-25,27-dihydro-7-deoxyphysalin A Physalin B

Calyx Calyx Calyx

5 6 7

4,7-Dehydrophysalin B 3α-Methoxy-2,3-dihydro-4,7-didehydrophysalin B 3β-Methoxy-2,3-dihydro-4,7-didehydrophysalin B

Calyx Stem and leaf Stem and leaf , calyx

8

Physalin C

Calyx

9

Physalin D

Calyx

10 11 12

Physalin D1 Physalin E Physalin F

Fruit Calyx Calyx

13 14 15 16 17

Physalin G Physalin I Physalin J Physalin K Physalin L

Calyx Calyx Calyx Leaf Calyx

18 19 20

Physalin M Physalin N Physalin O

Calyx Calyx Calyx

21

Physalin P

Calyx, fruit

22 23 24 25 26 27 28

Physalin Q Physalin R 5α, 6β-Dihydroxyphysalin R Physalin S Physalin T Physalin W Physalin X

Leaf Calyx Calyx Fruit Aerial parts Aerial parts Aerial parts

29 30 31

3-O-Methylphysalin X Physalin Y Physalin Z

Calyx Calyx Calyx

32 33 34 35 36 37 38 39 40

Physalin I Physalin II Physalin III Physalin IV Physalin V Physalin VI Physalin VII Isophysalin A Isophysalin B

Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx

41 42

7β-Methoxylisophysalin B 7β-Methoxylisophysalin C

Calyx Calyx

28

Ref (Xing and Jiang, 2013; Li and Li, 2002; Lin and Wang, 2011; Xu et al., 2009; Li et al., 2014; Qiu et al., 2008c; Yang et al., 2016; Chen et al., 2007b; Matsuura et al., 1969; Sunayama et al., 1993) (Li et al., 2012) (Li et al., 2012) (Xing and Jiang, 2013; Li and Li, 2002; Xu et al., 2009; Han et al., 2015; Li et al., 2014; Qiu et al., 2008c; Yang et al., 2016; Chen et al., 2007b) (Yang et al., 2016) (Qiu et al., 2008a) (Qiu et al., 2008a; Yang et al., 2016; Han et al., 2015) (Kawai and Matsuura, 1970; Xu et al., 2013; Yang et al., 2016) (Li et al., 2010; Cai et al., 2009; Lin and Wang, 2011; Li et al., 2014; Qiu et al., 2008c; Yang et al., 2016; Chen et al., 2007b) (Li et al., 2014) (Qiu et al., 2008c) (Xing and Jiang, 2013; Qiu et al., 2008c; Yang et al., 2016) (Yang et al., 2016; Chen et al., 2007b) (Xu et al., 2013; Yang et al., 2016) (Yang et al., 2016) (Makino et al., 1995a) (Li et al., 2002; Lin and Wang, 2011; Xu et al., 2009; Kawai et al., 1987; Li et al., 2014; Qiu et al., 2008c; Yang et al., 2016) (Li et al., 2002; Xu et al., 2009; Yang et al., 2016) (Kawai et al., 1992; Yang et al., 2016) (Lin and Wang, 2011; Xu et al., 2009; Qiu et al., 2008c; Kawai et al., 1992; Yang et al., 2016) (Li et al., 2010; Liang and Cai, 2007; Cai et al., 2009; Xu et al., 2013; Yang et al., 2016; Zhang et al., 2009; Chen et al., 2007b) (Makino et al., 1995a) (Li and Li, 2002; Makino et al., 1995b) (Li et al., 2012) (Makino et al., 1995b) (Kawai et al., 2001) (Chen et al., 2007c; Qiu et al., 2008c) (Chen et al., 2007c; Li et al., 2014; Qiu et al., 2008c) (Xu et al., 2013) (Qiu et al., 2008c) (Qiu et al., 2008c; Xu et al., 2013; Yang et al., 2016) (Qiu et al., 2008c) (Qiu et al., 2008c) (Xu et al., 2013) (Xu et al., 2013) (Yang et al., 2016) (Yang et al., 2016) (Yang et al., 2016) (Yang et al., 2016) (Yang et al., 2016; Han et al., 2015; Sunayama et al., 1993) (Yang et al., 2016) (Yang et al., 2016)

43 44 45

Isophysalin G Isophysalin I 25,27-Dihydro-4,7-dedehydro-7-deoxyneophysalin A 5α-Hydroxy-25,27-dihydro-4,7-didehydro-7-deoxyn eophysalin A 4,7-Didehydroneophysalin B

Calyx Calyx Calyx

Calyx Calyx Fruit Fruit Fruit Calyx

54 55 56 57 58

Alkekengilin A Alkekengilin B Alkekenginin A Alkekenginin B Philadelphicalactone A 16,24-Cyclo-13,14-secoergosta-2-ene-18,26-dioica cid-14:17,14:27-diepoxy-11β,13,20,22-tetrahydrox y-5α-methoxy-1,15-dioxo-γ-lactoneδ-lactone Physanol A Physanol B β-Sitosterol Daucosterol Saringosterol

(Xing and Jiang, 2013; Li et al., 2010; Liang and Cai, 2007; Cai et al., 2009; Li et al., 2014; Qiu et al., 2008c; Sunayama et al., 1993; Zhang et al., 2009) (Li et al., 2010) (Li et al., 2010) (Li et al., 2014) (Li et al., 2014) (Li et al., 2014) (Yang et al., 2016)

Aerial part Aerial part Calyx Calyx Calyx

(Sharma et al., 1974) (Sharma et al., 1974) (Liang and Cai, 2007) (Liang and Cai, 2007) (Chen et al., 2007b)

59

Luteolin

Calyx

60

Luteolin-7-O-β-D-glucopyranoside

Calyx

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

Luteolin-4’-O-β-D-glucopyranoside Luteolin-7,4'-di-O-β-D-glucopyranoside Luteolin-7,3’-di-O-β-D-glucopyranoside 3’,7-Dimethylquercetin 3’,4’,7-Trimethylquercetin 3’,4’-Dimethyl quercetin Quercetin-3-O-β-D-glucopyranoside Quercetin-3,7-di-O-β-D-glucopyranoside Kaempferol-3-O-β-D-Glucose 3,7-Di-O-α-L-rhamnopyransoyl kaempferol Apigenin-O-β-D-glucopyranoside Diosmetin-O-β-D-glucopyranoside 3’,4’-Dimethoxymyricetin Ombuine 5,4’,5’-Trihydroxy-7,3’-dimethoxy-flavonol Chrysoeriol Chrysoeriol-O-β-D-glucopyanoside

Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx

(Cai et al., 2009; Lin and Wang, 2011; Qiu et al., 2008c; Xu et al., 2009) (Cai et al., 2009; Lin and Wang, 2011; Qiu et al., 2007; Qiu et al., 2008c; Xu et al., 2009) (Qiu et al., 2007; Qiu et al., 2008c) (Qiu et al., 2008c) (Qiu et al., 2007; Qiu et al., 2008c) (Han et al., 2015) (Han et al., 2015) (Xing and Jiang, 2013) (Qiu et al., 2007; Qiu et al., 2008c) (Qiu et al., 2007; Qiu et al., 2008c) (Xing and Jiang, 2013) (Xing and Jiang, 2013) (Shu et al., 2014) (Shu et al., 2014) (Cai et al., 2009) (Qiu et al., 2008c; Xu et al., 2009) (Xu et al., 2009) (Chen et al., 2007b) (Shu et al., 2014)

Phenylpropanoid

78 79 80 81 82 83 84 85

Ferulic acid 3-Caffeoylquinic acid methyl ester Chlorogenic acid Syringalide (+)-Syringaresinol-O-β-D-di-glucopyranoside (+)-Pinoresinol-O-β-D-di-glucopyranoside (+)-Medioresinol-O-β-D-di-glucopyranoside Syringaresinol-4’-O-β-D-glucopyranoside

Calyx Calyx Calyx Calyx Calyx Calyx Calyx Calyx

(Li et al., 2002) (Chen et al., 2014) (Chen et al., 2014) (Shu et al., 2014) (Shu et al., 2014) (Shu et al., 2014) (Shu et al., 2014) (Chen et al., 2014)

Alkaloids

86 87 88 89 90 91

N-Transferuloyltyramine N-p-Coumaroyltyramine Calystegin A3 Calystegin A5 Calystegin B1 Calystegin B2

Calyx Calyx Roots Roots Roots Roots

(Chen et al., 2014) (Chen et al., 2014) (Asano et al., 1996) (Asano et al., 1996) (Asano et al., 1996) (Asano et al., 1996)

46 47

48 49 50 51 52 53

flavnoids

29

Calyx Calyx, Fruit

(Qiu et al., 2008c) (Yang et al., 2016) (Sunayama et al., 1993; Xu et al., 2013; Yang et al., 2016; Zhang et al., 2009) (Li et al., 2012; Zhang et al., 2009)

Nucleosides

Terpenoids

Megastigmane

92 93 94 95 96 97 98

Calystegin B3 1β-Amino-2α,3β,5β-trihydroxycycloheptane 3α-Tigloyloxytrropane Phygrine Adenine Adenosine Cyclo(tyrosine-amidocaproic)-bipeptid

Roots Roots Root Root and aerial part Calyx Calyx Calyx

(Asano et al., 1996) (Asano et al., 1996) (Yamaguchi and Nishimoto, 1965) (Basey et al., 1992) (Chen et al., 2014) (Chen et al., 2014) (Cai et al., 2009)

99 100

Oleanolic acid Neryl-1-O-β-D-glucopyranosyl-(1/2) O-[a-L-arabinopyranosyl-(1/6)]-O-βglucopyranoside

Calyx Calyx

(Xing and Jiang, 2013) (Chen et al., 2014)

(6S,9R)-Roseoside (6S,9S)-Roseoside (6R,9S)-3-Oxo-α-ionol-β-D-glucopyranoside Citroside A (6R,9S)-3-Oxo-α-ionyl-9-O-β-D-glucopyranosyl-(1'' →6')-β-D-glucopyranoside (6S,9S)-3-Oxo-α-ionyl-9-O-β-D-glucopyranosyl-(1'' →6')-β-D-glucopyranoside

Stem and leaf Stem and leaf Stem and leaf Stem and leaf Stem and leaf

(Qiu et al., 2008b; Qiu et al., 2008d) (Qiu et al., 2008b; Qiu et al., 2008d) (Qiu et al., 2008b; Qiu et al., 2008d) (Qiu et al., 2008b; Qiu et al., 2008d) (Qiu et al., 2008d)

Stem and leaf

(Qiu et al., 2008d)

107

(Z)-9,10,11-trihydroxy-12-octadecenoic acid

Calyx

(Xing and Jiang, 2013)

108 109

N-pen-tadecanoic acid Tetra-cosanic acid

Calyx Calyx

(Xing and Jiang, 2013) (Xing and Jiang, 2013)

110 111 112 113

Syringic acid 1,5-Dimethyl citrate 5-Hydroxymethylfuroic acid Scopoleti-O-β-D-di-glucopyranoside

Calyx Calyx Calyx Calyx

(Chen et al., 2007b) (Cai et al., 2009) (Cai et al., 2009) (Shu et al., 2014)

114 115 116 117 118 119 120 121 122 123 124

3,4-Dihydroxyphenethyl alcohol Physakengose A Physakengose B Physakengose C Physakengose D Physakengose E Physakengose F Physakengose J Physakengose H Physakengose I Physakengose J

Calyx Aerial part Aerial part Aerial part Aerial part Aerial part Aerial part Aerial part Aerial part Aerial part Aerial part

(Xing and Jiang, 2013) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016) (Zhang et al., 2016)

101 102 103 104 105 106

Aliphatic derivatives

Organic acid

Coumarin

Sucrose esters

– -

D

30

Figure 1 Pictures of Physalis alkekengi var. franchetii. (A) the whole plant; (B) fruit and calyx; (C) Physalis Calyx seu Fructus, medicinal materials used in TCM

31

Figure 2 Steroids isolated from P. alkekengi var. franchetii

32

Figure 3 Flavonoids isolated from P. alkekengi var. franchetii

Figure 4 Phenylpropanoids and N-containing compounds isolated from P. alkekengi var. franchetii

33

Figure 5 Miscellaneous constituents isolated from P. alkekengi var. franchetii

Figure 6 Mechanism of pharmacological activities that are related to the ethnomedical uses of P. alkekengi var. franchetii

34