Genus Periploca (Apocynaceae): A Review of Its Classification, Phytochemistry, Biological Activities and Toxicology

The genus Periploca belongs to the family Apocynaceae, which is composed of approximately ten species of plants according to incomplete statistics. Most of these plants serve as folk medicines with a long history, especially Periploca sepium and Periploca forrestii. The botanical classifications, chemical constituents, biological activities and toxicities of the genus Periploca were summarized in the literature from 1897 to early 2019. Though the botanical classification of this genus is controversial, these species are well-known to be rich sources of diverse and complex natural products—above all, cardiac steroids and C21 pregnane steroids with special structures and obvious pharmacological activities. The various crude extracts and 314 isolated metabolites from this genus have attracted much attention in intensive biological studies, indicating that they are equipped with cardiotonic, anti-inflammatory, immunosuppressive, antitumor, antimicrobial, antioxidant, insecticidal and other properties. It is noteworthy that some cardiac glycosides showed hepatotoxicity and cardiotoxicity at certain doses. Therefore, in view of the medical and agricultural value of the genus Periploca, in-depth investigations of the pharmacology in vivo, the mechanisms of biological actions, and the pharmacokinetics of the active ingredients should be carried out in the future. Moreover, in order to ensure the safety of clinical medication, the potential toxicities of cardiac glycosides or other compounds should also be paid attention. This systematic review provides an important reference base for applied research on pharmaceuticals and pesticides from this genus.


Introduction
The genus Periploca belongs to the family Apocynaceae [1] and involves approximately ten species which are diffusely distributed in temperate Asia, southern Europe and tropical Africa [2]. Many species of this genus have been historically used as folk medicines in China, especially Periploca sepium and Periploca forrestii. The root bark of P. sepium, known as "Xiangjiapi" or "Bei-wujiapi," is utilized as a traditional Chines medicine (TCM) for the treatment of rheumatoid arthritis and bone and muscle pain, which is recorded in the Chinese Pharmacopoeia (2015 version). The processing of Cortex Periplocae requires removing impurities, cleaning, slicing, and drying. The dried Cortex Periplocae (3~6 g) and other medicinal materials are decocted in water and taken for internal use [3]. The stems or the whole plant of P. forrestii, which is a well-known herb known as "Heiguteng," are widely used by the Miao nationality in China to treat many diseases, including rheumatic arthritis, traumatic injury, stomachache, dyspepsia, and amenorrhea. The usage of P. forrestii is usually decocted in water or soaked in wine for internal use, or it is smashed for external application [4]. The title genus is abundant with an array of attractive metabolites, such as steroids, oligosaccharides, terpenoids, phenylpropanoids, and flavonoids. These novel and complex metabolites and various crude extracts from the genus Periploca have attracted increasing attention to intensive biological studies, indicating that they possess significant cardiotonic, anti-inflammatory, immunosuppressive, antitumor, antimicrobial, antioxidant, insecticidal and other properties. Some cardenolide-type steroids exhibit obvious cardiotonic and antitumor effects at a certain dose. Their strong immunosuppression and insecticidal potentials provide new development prospects for pregnane glycosides. In addition, cardiac steroids were also found to have latent toxicity. In the 2000s, several researchers provided reviews of the chemical constituents and biological activities of the genus Periploca [5,6]. However, these concise reviews were expressed in Chinese. Additionally, a large amount of scientific research results on this genus has emerged in recent years. To make researchers know elaborate information on the chemical and biological activities of the genus Periploca and to make a modest contribution to the rational application of this genus, a systematic review of the genus Periploca is imperative. We covered topics including the botanical classification, phytochemistry, biological activities and toxicology of the genus Periploca through a literature survey from 1897 to early 2019. The outline of this paper and some representative plants of the genus Periploca are shown in Figure 1. decocted in water or soaked in wine for internal use, or it is smashed for external application [4]. The title genus is abundant with an array of attractive metabolites, such as steroids, oligosaccharides, terpenoids, phenylpropanoids, and flavonoids. These novel and complex metabolites and various crude extracts from the genus Periploca have attracted increasing attention to intensive biological studies, indicating that they possess significant cardiotonic, anti-inflammatory, immunosuppressive, antitumor, antimicrobial, antioxidant, insecticidal and other properties. Some cardenolide-type steroids exhibit obvious cardiotonic and antitumor effects at a certain dose. Their strong immunosuppression and insecticidal potentials provide new development prospects for pregnane glycosides. In addition, cardiac steroids were also found to have latent toxicity. In the 2000s, several researchers provided reviews of the chemical constituents and biological activities of the genus Periploca [5,6]. However, these concise reviews were expressed in Chinese. Additionally, a large amount of scientific research results on this genus has emerged in recent years. To make researchers know elaborate information on the chemical and biological activities of the genus Periploca and to make a modest contribution to the rational application of this genus, a systematic review of the genus Periploca is imperative. We covered topics including the botanical classification, phytochemistry, biological activities and toxicology of the genus Periploca through a literature survey from 1897 to early 2019. The outline of this paper and some representative plants of the genus Periploca are shown in Figure 1.

Botanical Classification of the Genus Periploca
In 1895, Schumann was the first to classify the P. species. Browicz defined the genus Periploca and recognized eleven species in 1966. According to Flora of China (1995) [2], the genus Periploca comprises about ten species, and five species are distributed in China. Additionally, in 1997, Venter [7] suggested correcting the nomenclature of Periploca to provide diagnostic characteristics for the genus and to improve classification. However, fourteen species belonging to genus Periploca were recorded by Venter, not including P. forrestii, which largely grows in southern China. Moreover, several Chinese scholars are interested in the taxonomy of this genus. Periploca omeiensis Z. Y. Zhu, which is merely distributed in Emei Mountain, China, was identified as a new species by Zhu in 1991 [8]. Furthermore, another new species, named Periploca chrysantha D. S. Yao, X. C. Chen et J. W. Ren,

Botanical Classification of the Genus Periploca
In 1895, Schumann was the first to classify the P. species. Browicz defined the genus Periploca and recognized eleven species in 1966. According to Flora of China (1995) [2], the genus Periploca comprises about ten species, and five species are distributed in China. Additionally, in 1997, Venter [7] suggested correcting the nomenclature of Periploca to provide diagnostic characteristics for the genus and to improve classification. However, fourteen species belonging to genus Periploca were recorded by Venter, not including P. forrestii, which largely grows in southern China. Moreover, several Chinese scholars are interested in the taxonomy of this genus. Periploca omeiensis Z. Y. Zhu, which is merely distributed in Emei Mountain, China, was identified as a new species by Zhu in 1991 [8]. Furthermore, another new species, named Periploca chrysantha D. S. Yao, X. C. Chen et J. W. Ren, was found in Gansu, China in 2002 and is similar to P. sepium, with a difference in the corolla [9]. However, these two new species have not been included in the flora at home and abroad to date. To our best knowledge, the botanical classification of Periploca is inconsistent at home and abroad, and the flora has not been updated in time. We did not take into account the disputes over the taxonomy of the genus Periploca and summarized all the mentioned plants of this genus which have been studied for phytochemistry and biological activities. The names and references of these thirteen studied species are shown in Table 1.

Chemical Constituents from the Genus Periploca
Phytochemical investigations of Periploca species revealed predominant metabolites, such as steroids, carbohydrates, terpenoids, phenylpropanoids, flavonoids, quinones, and aromatics. To date, three hundred and fourteen compounds (1-314) have been isolated and identified from the plants of genus Periploca.

Steroids
One hundred and forty-two steroids  were isolated from Periploca species, including forty-six cardenolides , ninety-two C 21 pregnane-type steroids , and four phytosterols (139)(140)(141)(142). Among them, approximately 80% of the C 21 pregnane-type steroids come from P. sepium, while P. forrestii is a rich source of cardenolides. Their structures, names, corresponding sources, parts of plants and references are illuminated in Table 2      89 forrestii roots [29] 108 ∆ 5 -pregnene-3β,16β,20(R)-triol P. sepium root bark [49] 109   The chemical investigation of this genus led to the discovery of cardenolides. According to their structural skeletons, forty-six cardenolides  are classified into two categories, namely periplogenin-type cardenolides  and periforgenin A-type cardenolides (41)(42)(43)(44)(45)(46). Among them, there are fourteen aglycones, sixteen monosaccharide glycosides, fourteen disaccharide glycosides, and two trisaccharide glycosides. It is worth noting that all glycosides possess a sugar chain only at the C-3 position. Additionally, every disaccharide glycoside contains a deoxysugar as the inner sugar and a moiety of glucose as the outer sugar, with a linkage mode of 1→4, while the sugar unit of monosaccharide glycosides is a deoxygenated or an oxygenated sugar.

C21 Pregnane-Type Steroids
According to the structural characteristics of the ninety-two C21 pregnane-type steroids (47-138, Table 2, Figure 3), a simple conclusion can be summarized. Up to now, all C21 steroids from Periploca species have possessed a typical pregnane unit with four unbroken rings. Most exist in the form of glycosides, with the sugar chains linked to C-3 or C-20. These saccharide chains are composed of oxygenated sugars, deoxygenated sugars, and their derivatives. Some oxygen-containing groups, such as hydroxy and methoxy groups, are mostly present at the C-3, C-14, C-16, C-17, C-20 and C-21 positions of the aglycone. Additionally, the absolute configuration at C-20 of most compounds (  The study of pharmacologically active ingredients from P. forrestii led to the discovery of periforgenin A and periforoside I (41,42), displaying a 15(14→8)abeo-(8S)-14-ketone-card skeleton characterized by a ketone at C-14 and a transformed C/D ring [28]. The structure of periforoside I was supported by X-ray diffraction in 1990. Four newly found cardiac glycosides (43)(44)(45)(46) [19,20,29,30] have different structural characteristics in the saccharide chains. The six distinct periforgenin A-type cardiac glycosides were only found in P. forrestii, which is the difference between P. forrestii and other plants of the genus.

C 21 Pregnane-Type Steroids
According to the structural characteristics of the ninety-two C 21 pregnane-type steroids (47-138, Table 2, Figure 3), a simple conclusion can be summarized. Up to now, all C 21 steroids from Periploca species have possessed a typical pregnane unit with four unbroken rings. Most exist in the form of glycosides, with the sugar chains linked to C-3 or C-20. These saccharide chains are composed of oxygenated sugars, deoxygenated sugars, and their derivatives. Some oxygen-containing groups, such as hydroxy and methoxy groups, are mostly present at the C-3, C-14, C-16, C-17, C-20 and C-21 positions of the aglycone. Additionally, the absolute configuration at C-20 of most compounds (47-81,87-92, 97-101,126-128) has been deduced to be S.
From 1967 to 1995, some Japanese researchers made great contributions to the chemical investigation of P. sepium. They isolated and identified many compounds with novel features, especially a series of pregnane glycosides. In 1987 and 1988, Itokawa and co-workers [31,36,37,41] discovered periplocosides A-K, all containing a peroxy function, from the antitumor fraction of the CHCl 3 extract from the root bark of P. sepium. During the same period, periplosides A-C were isolated by Hikino and co-workers [32]. Additionally, periplosides A and C are equipped with an orthoester group. Later, in 2008, to study the antirheumatoid arthritis effect of the genus Periploca, Zhao's group determined perperoxides A-E [33]. From further work performed by Zhao's group, it is worth noting that the peroxy function of the sugar chains of the above thirteen pregnane glycosides (periplocosides A-F, J, K and perperoxides A-E) was revised to an orthoester group according to 2D NMR spectroscopic analysis, chemical transformation, and X-ray diffraction analysis. Hence, the names of the fourteen pregnane glycosides were assigned as periplosides A-N (47-60) in 2011. The relative configurations at the spiro-quaternary carbons of the periplosides were revised according to the single-crystal X-ray structure of periploside F (52) [34,35]. With further research, in 2017, the absolute configuration of periploside C (49) was successfully established by single-crystal X-ray diffraction analysis. In addition, nine new spiro-orthoester group-containing pregnane-type steroidal glycosides, periplosides O-V, along with 3-O-formyl-periploside A (61-69), were isolated from the root bark of P. sepium [38]. To explore more about the structure-immunosuppressive activity relationship of spiroorthoester group-containing pregnanes, Zhao and co-workers performed a chemical analysis of P. chrysantha. Four new periplosides were obtained, and they were named periplosides W-Y and 3-O-formyl-periploside F (70-73) [39]. In the 1980s, chemical investigations of P. calophylla led to some pregnane glycosides with a hydroxyl group at C-14 (111)(112)(113)(114)(115). Unfortunately, the position of the sugar moiety in calocin (111) was not confirmed based on spectroscopic analysis in 1982 [54]. A new pregnane ester diglycoside and a new pregnane ester aglycone (112,113) [55]-each characterized by two hydroxyl groups that are benzoylated at C-11 and C-12-both contain the structure of 12,20-di-O-dibenzoyl drevogenin-D. Locin (114) [56] displays two β-oriented hydroxyl groups at C-12 and C-14. Calocinin (115) [57] has a special 2,6-dideoxy-L-fucose sugar unit, which was determined by spectroscopic analysis and a chemical reaction. Twenty years later, a new pregnane glycoside (116) with a β-oriented hydroxyl group at C-14 was discovered from the small branches of P. graeca [50].
It is noteworthy that the carbon side-chain at C-17 of the ten distinct compounds (123-132) is αoriented. Interestingly, these compounds all possess a β-oriented hydroxyl group at C-14, and eight are new compounds. An aglycon calogenin (123) [59] was established as containing a C-17 hydroxyethyl chain in α configuration. However, it is puzzling that the β configuration at C-17 was assigned to calogenin 3-O-β-d-digitalopyranoside-20-O-β-D-canaropyranoside (116) [50]. Plocinine (124) [60] is characterized by two cinnamoyl groups assigned to the methine protons at C-12 and C-20 of ornogenin. Compounds 125-128 are a series of 21-methoxypregnanes with a hydroxyl group in the β configuration at C-17. Similar to compounds 117-122, the pregnane glycosides 129-132 are also 21-methoxypregnane-20-one derivatives, but the remarkable difference is in the configurations at C-17. Furthermore, compounds 129 and 132 are stereoisomers at C-17 of 120 and 119, respectively. What is concerning is that compound 81 [47] was assigned the trivial name of periplocoside P in 2014, whereas 132 [61] was given the same name in 2015.
Compounds 87-96 merely have two oxygenated patterns at C-3 and C-20. In 1988, Takeya and co-workers reported that compounds 88 and 89, which were hydrolyzed with acid to yield the same aglycone (87) [49], were two new pregnane glycosides from the antitumor fraction of P. sepium. Additionally, compounds 88 and 89 both yielded 90 upon partial acid hydrolysis. Glycosides H 1 and K, isolated in 1972 and 1969, respectively [14,76], have identical relative configurations to those of compounds 89 and 90. Additionally, glycoside K is the first gregnane-type glycoside whose sugar chain links to the hydroxyl group, rather than C-3 of the aglycone. The biondianosides C,D (91,92), firstly isolated from Biondia hemsleyana (Warb.) Tsiang belonging to the same family [77], were also obtained from the genus Periploca in 2009 [52]. Periseosides C and D (93,94) are two new compounds [40]. Compound 96 [31] was established to have an R configuration at the C-20 position, which distinguishes it from compounds 87-95.
Compounds 97-106 are characterized by the inclusion of two hydroxyl groups at C-16 and C-20 of the aglycones, and the configuration of C-16 was deduced to be α. Glycoside H 2 (97) was found by Sakuma and co-workers [53] in 1980, and it showed significant potentiation of the effect of nerve growth factor. The hydrolysis of compound 97 yielded glycoside 98. Due to a hydroxyl group at the D-ring, plocoside B (100) was found to exhibit higher differentiation-inducing activity [51]. Periseoside E [40] (101), along with periseosides A-D, was obtained in 2011. The study of the small branches of P. graeca collected at Marina di Vecchiano, Italy resulted in five new pregnane glycosides (102)(103)(104)(105)(106). It is worth mentioning that 102-105 are characterized by a 6-sulfated-β-d-glucopyranosyl unit at C-16, and the occurrence of sulfated glycosides as pregnane derivatives is an unusual finding [50]. Compound 106 bears a hydroxyl group in the β configuration at C-16. Compound 107 [29] is similar to 106, with the difference between them being in the structures of the oligosaccharide moieties. Compounds 108-110 are equipped with the R configuration at C-20 and a β-oriented hydroxyl group at C-16.
It is noteworthy that the carbon side-chain at C-17 of the ten distinct compounds (123-132) is α-oriented. Interestingly, these compounds all possess a β-oriented hydroxyl group at C-14, and eight are new compounds. An aglycon calogenin (123) [59] was established as containing a C-17 hydroxyethyl chain in α configuration. However, it is puzzling that the β configuration at C-17 was assigned to calogenin 3-O-β-d-digitalopyranoside-20-O-β-d-canaropyranoside (116) [50]. Plocinine (124) [60] is characterized by two cinnamoyl groups assigned to the methine protons at C-12 and C-20 of ornogenin. Compounds 125-128 are a series of 21-methoxypregnanes with a hydroxyl group in the β configuration at C-17. Similar to compounds 117-122, the pregnane glycosides 129-132 are also 21-methoxypregnane-20-one derivatives, but the remarkable difference is in the configurations at C-17. Furthermore, compounds 129 and 132 are stereoisomers at C-17 of 120 and 119, respectively. What is concerning is that compound 81 [47] was assigned the trivial name of periplocoside P in 2014, whereas 132 [61] was given the same name in 2015.

Carbohydrates
A total of twenty-six carbohydrates have been isolated from the genus Periploca and mainly came from P. sepium and P. forrestii, eighteen of which are new compounds. Compounds 143-145 are monosaccharides, and compounds 146-168 are oligosaccharides. Carbohydrates can be considered characteristic components of Periploca species because most of them contain one or more 2,6-dideoxysugars or/and 6-deoxysugars and their derivatives. What is noticeable is that the sugar sequences of these oligosaccharides are ruled by the regularity in cardiac-and pregnane-type glycosides of the Apocynaceae (Asclepiadaceae) plants [78].
The oligosaccharides (147,148) were hydrolysis products of glycosides from P. sepium. Similarly, upon partial acid hydrolysis, compound 88 yielded 149, a deacetylation product of 147. Compounds 150-153 were isolated and elucidated as four new-type oligosaccharides in 1977, which were the first examples of oligosaccharides composed of 2,6-dideoxyaldonic lactone and 2,6-dideoxysugars [78]. Thirty years later, Zhao's group [33] engaged in the discovery of new immunosuppressive compounds from traditional Chinese medicine, which resulted in five oligosaccharides being obtained. Among them, three were new compounds, named perisaccharides A-C (154)(155)(156). In their ongoing chemical investigation of P. forrestii, four new oligosaccharides (157)(158)(159)(160) [29], characterized by diverse sugar units and an oleandro-1,5-lactone, were elucidated. In 2010, Ye and co-workers [79] reported five new oligosaccharides, perisesaccharides A-E (161)(162)(163)(164)(165), and assigned their absolute configurations. Intriguingly, they put forward that the conformation of the oleandronic acid δ-lactone should be confirmed as "boat," rather than the "chair" assessment in previous literatures, on the basis of a combination of X-ray diffraction analysis, a modified Mosher's method, CD measurements and acid hydrolysis. Additionally, the "boat" conformation of oleandronic acid δ-lactone was reported for the first time. Perisesaccharide F (166) [80], as a new natural product, was isolated from P. sepium, while another new oligosaccharide (167) [81] was discovered from P. calophylla. The structures, names, corresponding sources, parts of plants and references of these compounds are summarized in Table 3 and Figure 5. A small number of polysaccharides isolated from this genus are not listed here.

Terpenoids
There are forty-two terpenoids  composed of triterpenoids and three other types of terpenoids (169)(170)(171). According to their structural characteristics, these triterpenoids  are divided into four sub-types, namely oleanane-type triterpenes, ursane-type triterpenes, lupane-type triterpenes and cycloartane-type triterpenes. Their structures, names, corresponding sources, parts of plants and references are illuminated in Table 4 and Figure 6.

Quinones
A total of eight quinone compounds (Table 7, Figure 9) has been found in the genus Periploca, and seven of them were derived from P. forrestii. Compounds 274-279 are six emodin-type anthraquinones. An anthrone (280) was reported for the first time from P. aphylla [84]. Additionally, tanshinone IIA (281) [111] possesses a diterpenoid quinone skeleton, which was discovered from the portion of P. forrestii with anti-inflammatory activity.

Quinones
A total of eight quinone compounds (Table 7, Figure 9) has been found in the genus Periploca, and seven of them were derived from P. forrestii. Compounds 274-279 are six emodin-type anthraquinones. An anthrone (280) was reported for the first time from P. aphylla [84]. Additionally, tanshinone IIA (281) [111] possesses a diterpenoid quinone skeleton, which was discovered from the portion of P. forrestii with anti-inflammatory activity.

Quinones
A total of eight quinone compounds (Table 7, Figure 9) has been found in the genus Periploca, and seven of them were derived from P. forrestii. Compounds 274-279 are six emodin-type anthraquinones. An anthrone (280) was reported for the first time from P. aphylla [84]. Additionally, tanshinone IIA (281) [111] possesses a diterpenoid quinone skeleton, which was discovered from the portion of P. forrestii with anti-inflammatory activity.

Aromatics
Twelve aromatic acids and their derivatives 282-293 (Table 8, Figure 10) were discovered from the genus Periploca. These carboxylic acid groups could be esterified with alkanols or form ester glycosides with sugar moieties. Aromatics 294-301 are eight aromatic aldehydes and their derivatives. 4-methoxy salicylaldehyde (294) was first reported in the bark of P. graeca in 1935 [113]. Compound 301 is a new diphenylmethane [44]. Compounds 302 and 303 are two aromatic ether compounds, and a new naphthalene derivative named periplocain A (304) [108] was discovered in 2016.

Others
Two ceramide compounds 305 and 306 were obtained from the trichloromethane fraction of the 80% alcohol extract of P. forrestii for the first time in 2014 [116]. There are six aliphatic compounds (308-313) and two stereoisomeric fatty esters (312,313) [63]. Obaculactone (314) is a lactone [80]. Their structures, names, corresponding sources, parts of plants and references are showed in Table 9 and

Others
Two ceramide compounds 305 and 306 were obtained from the trichloromethane fraction of the 80% alcohol extract of P. forrestii for the first time in 2014 [116]. There are six aliphatic compounds (308-313) and two stereoisomeric fatty esters (312,313) [63]. Obaculactone (314) is a lactone [80]. Their structures, names, corresponding sources, parts of plants and references are showed in Table 9 and Figure 11. Additionally, it should be noted that some researchers have reported that the plants of genus Periploca are rich in volatile oils, whereas the volatile oil components detected online are not listed in detail in this paper.   Figure 11. Other compounds from the genus Periploca.

Biological Activities
The extracts of the genus Periploca have been investigated for a series of biological activities since the 1920s. It has been found that they show diverse and valuable activities, including cardiotonic, anti-inflammatory, immunosuppressive, antitumor, antimicrobial, antioxidant, insecticidal and other

Biological Activities
The extracts of the genus Periploca have been investigated for a series of biological activities since the 1920s. It has been found that they show diverse and valuable activities, including cardiotonic, anti-inflammatory, immunosuppressive, antitumor, antimicrobial, antioxidant, insecticidal and other properties. The biological activities of the genus Periploca, primarily interrelated species and active components are illustrated in Table 10. Table 10.
Biological activities of the genus Periploca, primarily interrelated species and active components.

Cardiotonic Effect
As early as the 1920s, periplocin (1) was detected to show digitalis-like cardiotonic capacity. Periplocin enhanced the tonus and the contractility of the cardiac muscle, as well as the tonus of the arterial muscle, whereas it led to cardiac irregularity and systolic arrest of the rat heart at excessive doses [117]. Additionally, a 10% infusion gave rise to a decrease of the frequency of the heartbeat and an increase of the tonus, arterial pressure and diuresis [118]. Through its effects on frog and rabbit hearts in situ and isolated guinea pig hearts, the total glycosides fraction extracted from the fresh stem bark of P. forrestii displayed that its cardiotonic property was analogous to that of g-strophanthin, and the average lethal dose in pigeons was 5.9 ± 1.0 mg/kg. Concerning the changes of rabbit electrocardiograms, both compounds produced the phenomena of prolonged R-R and P-R intervals, a shortened QRS interval and a flattened T wave [119]. The ethanol extract of P. calophylla, with an average lethal dose in pigeons of 31.86 ± 1.62 mg/kg, was also proven to be equipped with cardiotonic function via experiments on frog and rabbit hearts in situ, isolated guinea pig hearts, and through electrocardiograms in cats [120]. Wang's group discovered that periplocin could improve the left ventricular structure and function in rats with chronic heart failure at a dose of 8 mg/kg [121]. They inferred that the underlying mechanism of periplocin against heart failure might involve its ability to increase the expression of sarco endoplasmic reticulum Ca 2+ -ATPase mRNA, decrease the expression of phospholamban mRNA and improve the value of PLB/SERCA in chronic heart failure-model rats [122] The proliferative activity of periplocin was verified in mouse cardiac microvascular endothelial cells by Gao and co-workers [123]. Compared with periplocin (1), periplogenin (2) and xysmalogenin (25) both showed positive inotropic and negative chronotropic effects on isolated rat hearts, but their activities were weaker and failed more quickly [124].

Anti-Inflammatory and Immunosuppressive Effects
Pharmacological tests determined that the ethanol extracts, volatile oils and water extracts mainly containing polysaccharides of P. forrestii all had anti-acute inflammation properties [125][126][127]. The ethanol extract of P. forrestii displayed anti-inflammatory activity which was connected to reducing the protein expression of inducible nitric oxide synthase and cyclooxygenase-2 (COX-2); to the production of inflammation factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, nitric oxide and prostaglandin E 2 (PGE 2 ); and, mainly, to the suppression of lipopolysaccharide-mediated stimulation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases signaling pathways [128]. Additionally, the extract of P. laevigata was also able to ameliorate carrageenan-induced edema and acetic acid-induced abdominal writhing, in addition to enhancing the latency in a hot plate test [129]. In 1997, three triterpenes from P. sepium, named α-amyrin (193), α-amyrin acetate (195), and β-amyrin acetate (182), were shown to have anti-inflammatory effects in animal experiments [130]. At a dose of 20 µg/mL, Periplocin (1) could distinctly inhibit histamine release from mast cells cultured in vitro and from mast cells of sensitized rats. As such, periplocin was considered to be one of the effective anti-inflammatory ingredients of P. sepium by Gu's group [131]. It is worth noting that periplocoside A (49) showed a sufficiently preventative effect on concanavaline A-induced hepatitis by inhibiting natural killer T-derived inflammatory cytokine production, which suggested that periplocoside A possesses therapeutic potential for the treatment of human autoimmune-related hepatitis [132].
In 2005, periplocoside E (47) was first found to specifically and markedly inhibit the activation of extracellular signal-regulated kinase and Jun N-terminal kinase, while the activation of p38 was not influenced by T cells stimulated with anti-CD3, showing its potency for the treatment of T cell-mediated disorders [133]. In addition, its immunosuppressive efficacy was confirmed again by the effect of treatment with periploside A (47) on a positive selection of thymocytes from C57BL/6 mice in vitro [134]. After finding that the crude extract of P. sepium and periplocoside E had the ability to inhibit the proliferation of T cells, Zhao's group carried out a systematic chemical investigation and searched for immunosuppressive compounds, making vital contributions to this domain. Compared with the immunosuppressants rapamycin (IC 50 0.19 µM) and cyclosporin A (IC 50 0.27 µM), nine pregnane glycosides (47,(49)(50)(51)(52)(53)(54)(55)58) characterized by an orthoester group displayed significant inhibitory activities against the proliferation of T lymphocytes in vitro, with IC 50 values ranging from 0.29 to 1.97 µM. However, other pregnane glycosides without the orthoester group showed no apparent inhibitory activity [33]. Twelve spiro-orthoester group-containing periplosides (49,(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69) were evaluated for their inhibitory activities. Among them, periploside C (49), the most abundant spiro-orthoester moiety-bearing component in the root bark of P. sepium, exhibited the best selective index (SI) value at 82.5. More notable is that the length and constitution of the saccharide chain in this compound class have a crucial impact on both the suppressive activity and the SI value. Nevertheless, the substitution of a formyl group in replacement of the 4,6-dideoxy-3-O-methyl-∆ 3 -2-hexosulose function at the C-3 position of the aglycone might not affect this activity in periplosides [38]. Additionally, Zhao's group demonstrated the potential mechanism of the immunosuppressive ability of periplocoside A (periploside C, 49). Though inhibiting IL-17 production and suppressing the differentiation of Th17 cells in vitro, the oral administration of periplocoside A ameliorated experimental autoimmune encephalomyelitis [135]. The other four compounds of the same type (70)(71)(72)(73) were equipped with notable inhibitory activities against the proliferation of B and T lymphocytes, and advantageous selective index values comparable to those of cyclosporin A, which perfectly supplemented the immunosuppressive potency of the periplosides [39].
In 2006, lupane acetate (198) was found to induce the differentiation and maturation of dendritic cells, upregulate cytokines production, and enhance the immune activity of dendritic cells in vitro [136].
The ability of lupane acetate to increase the immune response of lymphocytes and macrophages from human peripheral blood might be associated with its ability to kill carcinoma cells [137]. Additionally, periplocin (1) was also discovered to protect the immune organs of tumor-bearing mice, obviously enhance the lymphocyte proliferation in mice spleens, and stimulate the production of TNF-α, IL-2 and IL-12, which indicated that it had immunoregulatory effects [138]. The polysaccharide (HGT-5A) from P. forrestii showed immunosuppressive effects on the cellular immune response and that its four ingredients played a major role [139]. However, three oligosaccharides (154)(155)(156) displayed no activity against the proliferation of T lymphocytes in vitro [33]. In 2017, by in vitro experimentation, suppressing hyaluronidase, microwave extraction combined ultrasonic pretreatment of flavonoids from P. forrestii were found to produce resistance to allergies with an IC 50 value of 1.033 mg/mL [140].
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, and extracts of P. forrestii and P. sepium showed favorable anti-arthritis efficacies. In 2004, the aqueous extract of P. sepium was found to inhibit the growth and IL-6 production of human rheumatoid arthritis-derived fibroblast-like cells in a dose-dependent manner [141]. The water-soluble components of P. forrestii could suppress the inflammatory response in collagen-induced arthritic rats through regulating the expression of the TAK-1, TAB-1, NF-κB p65, MCP-1 and CXCL-1 proteins [142]. Huang's group [143] revealed that a 60% ethanol extract of P. forrestii possessed activity against RA capacity. They inferred that the potential mechanism might be associated with the regulation of immune organ function and the levels of pro-inflammatory cytokines containing IL-1β, IL-6 and TNF-α. Liang and co-workers [144] drew a conclusion that the effect of a 50% ethanol extract of P. forrestii on RA was concerned not only with restraining the proliferation of rheumatoid arthritis synovial fibroblasts but also with the downregulation of the expression of COX-2 and PGE 2 . Through further investigation, Feng's group [145] provided new insight into the underlying mechanism of the therapeutic action in collagen-induced arthritis (CIA). By inhibiting the activation of Src kinase and the nuclear translocation of NF-κB in rats, the ethanol extract of P. forrestii was shown to be a potential candidate for the treatment of patients with RA. The anti-arthritis effect of P. forrestii was discovered to be positively correlated with the contents of total flavonoids and total saponins, and the influence of the total saponins was greater than that of the total flavonoids [146]. Sun's group [147] verified that P. forrestii saponin and periplocin (1) performed prophylactic therapeutic action against autoimmune arthritis by controlling the systemic autoimmune responses and local inflammation and bone destruction of the joints. Additionally, they deduced that the protective mechanism of P. forrestii saponin toward CIA had something to do with regulatory actions on proinflammatory factors, as well as on the crosstalk between NF-κB and c-Fos/AP-1 in vivo and in vitro [148]. Another recent biological study of the cardenolide-rich and caffeoylquinic acid-rich fractions of P. forrestii demonstrated that the inhibitory activities toward the NF-κB and MAPK signaling pathways might be involved in the antiarthritic effect based on a set of experimental results [149].

Antitumor Ability
Abundant experiments concerning the antitumor activities of P. sepium were carried out by Shan and co-workers. Above all, Shan's group explored the role of periplocin (1) in affecting human cancer cell lines in vitro from 2005 to 2017. They found that periplocin expressed a noteworthy inhibitory effect on diverse cancer cell lines-including hepatocellular carcinoma cells, colon carcinoma cells, lung carcinoma cells, and breast carcinoma cells-basically though blocking the cell cycle and inducing apoptosis [150][151][152][153]. Furthermore, the potential antitumor mechanisms of periplocin were inferred by Shan's group. Periplocin was detected to suppress the proliferation and induce apoptosis of the human hepatocarcinoma cell line SMMC-7721 via restraining Stat3 signal transduction [150]. By the downregulation of the Wnt/β-catenin signaling pathway, periplocin (0.5 µg/mL) markedly inhibited proliferation of the human colon carcinoma cell line SW840 [151]. The probable mechanism by which periplocin induced apoptosis in the human lung cancer cell line A549 was related to the downregulation of the survivin mRNA and protein [152]. In addition, periplocin had an effect on breast carcinoma MCF-7 cells with the IC 50 value of 4.88 ± 0.16 ng/mL, which was associated with the increased expression of the mRNA and protein of gene p21 WAF1/CIP1 [153]. Additionally, periplocin (1) also displayed the ability to suppress the growth of human (A549) and mouse (LL/2) lung cancer cells in vitro and in vivo by blocking the protein kinase B/extracellular signal regulated kinase signal pathway [154]. In 2013, Pan's group deduced that periplocin (1) sensitized TNF-related apoptosis-inducing ligand-resistant human hepatocellular carcinoma cells via inducing the expression of the death receptors 4 and fas-associated death domain and the downregulation several inhibitors of apoptosis, which resulted in the activation of caspases 3, 8, and 9 and led to cell apoptosis [155]. Moreover, due to the inducing of DNA double strand breaks and death-receptor mediated apoptosis in liposarcoma cells, periplocin (1) was considered to be a promising lead compound for the development of new sarcoma therapeutics by Bauer's group [156].
Additionally, periforoside E (27) showed higher cytotoxicity toward the A549 line than toward other lines. Notable is that 3β,5β-dihydroxy-14-en-card-20(22)-enolide (26) and periforoside D (45) were both inactive in terms of cytotoxic activity, indicating that the 14-hydroxy group and the C/D ring junction were responsible for this effect [19]. Moreover, the three 17α-cardiotonic steroids (38)(39)(40) showed apparently higher IC 50 values (5.4 and 7.3 µM) in vitro in the hormone-independent prostate cancer cell line PC3 than those of 17β-isomers. Additionally, in the 17α-cardenolides, the presence of sugar units of these cardenolides had nothing to do with the IC 50 values. However, a fascinating series of 17β-cardiotonic steroids (1,(3)(4)(5)24), with a 14β hydroxyl group and at least one sugar molecule, demonstrated strong antiproliferative effects with IC 50 values between 18 and 50 nM [15]. Four cardenolides (1)(2)(3)38) were tested in the human monocytic leukemia U937 and androgen-independent prostate adenocarcinoma PC3 cell lines. Periplocin (1) and periplocymarin (3) showed more effective activity toward tumor cells due to the activation of apoptotic pathways in PC3 cells and in U937 cells due to the induction of cell cycle impairment [157]. More interesting is that periplocymarin (3) was unlikely to encounter drug-drug interactions with P-glycoprotein and cytochrome P450s, suggesting that it should be taken forward for further investigations in drug development [158].
In addition to cardenolides, other types of compounds have been examined for their cytotoxic activities. Periseosides C (93) showed moderate inhibitory activity against the ConA-stimulated splenocyte proliferation in vitro [40]. Two C 21 pregnane-type steroids (52,77) and four known oligosaccharides (168,155,162,163) were tested for their cytotoxicities against the A-549 and HepG2 human cancer cell lines with IC 50 values ranging from 0.61 to 7.68 µM by Wang's group [43]. Interestingly, compound 168, a derivative of 163 with the acetyl group at C-2 of the β-D-digitalopyranosyl moiety, exhibited moderate activities, while 163 and others were not active, which implied that the presence of an acetyl group might be related to the cytotoxic activity. Besides, Shan's group discovered that baohuoside-I (255) restrained the proliferation of and arrested Eca109 human esophageal squamous carcinoma cell cycles, which might be associated with the downregulation of Cyclin B1 mRNA expression [159]. As the research continued, they reported that baohuoside-I (255), by inhibiting β-catenin-dependent signaling pathways, significantly inhibited the proliferation of the cells in a timeand dose-dependent manner with the IC 50 value of 24.8 µg/mL at 48 h and induced apoptosis of Eca109 cells in vitro and in vivo [160]. Coincidentally, lupeal acetate (198) was also found to reduce the incidence of N-nitrosomethylbenzylamine-induced esophageal tumors from 93.3% to 33.3% in rats after twenty-five weeks of treatment, which might have occurred through the activation of glycogen synthase kinase-3β expression and the inhibition of β-catenin and c-myc expression [161].
To search for potent compounds against carcinoma disease, various extracts of the genus Periploca have been studied continually. In the 1980s, Japanese scholars were the first to discover that the methanol extract of P. sepium was equipped with significant anticancer activity toward sarcoma 180 ascites in mice with ascites cancer [36,41]. The ethanol extract of P. sepium had effects on the proliferation and apoptosis of human esophageal carcinoma cells, and this action might be related to the upregulation of Rb protein expression and downregulation of the expression of apoptosis suppressor genes and the induction of apoptosis [162,163]. Additionally, the ethanol extract of P. sepium was suspended in water and extracted by ethyl acetate, and the ethyl acetate extract was capable of inducing apoptosis in human esophageal carcinoma TE-13 cells because of the downregulation of cyclin-dependent kinase 4 expression [164]. Analogously, the ethanol extract of P. forrestii stems was suspended in water and extracted sequentially with four different polar solvents. Among them, ethyl acetate extract emerged as having the strongest inhibitory property against the proliferation of K562 tumor cells, with the maximum inhibitory of 80. 71% at 25 mg/L, from which periplocin was isolated [165]. In 1995, Umehara and co-workers [51] suspended the methanol extract of P. sepium in water and extracted it with ethyl acetate. The water layer displayed differentiation-inducing activity against mouse myeloid leukemia cells. Later, the effect of the water extract of P. sepium on the differentiation of K562 tumor cells was detected, which showed that this extract could kill K562 cells but could not induce differentiation [166]. The water extract of P. sepium also inhibited the growth of gastric carcinoma BGC-823 cells and induced apoptosis, both of which which was associated with the downregulation of the expression of B-cell lymphoma-2 and surviving genes and proteins and to the upregulation of the expression of bax genes and proteins [167].
In brief, the complex mixtures and some pure chemical components from the plants of genus Periploca have presented remarkable cytotoxicity activities toward about ten different carcinoma cells, while a large proportion of investigations have focused on cytotoxic assays in vitro. For the sake of discovering antitumor lead compounds, there are still many studies worth further development.

Antimicrobial and Antioxidant Abilities
Triterpenes from this genus have been known to exhibit antibacterial properties since 2000. 3β,6α-dihydroxylup-20(29)-ene (205) showed moderate activity against Staphylococcus pyogenes [67]. Based on the antibacterial activities of oleanolic acid (172), maslinic acid acetate, β-amyrin (181) and their derivatives, Jegham's group [168] speculated the antibacterial structure-activity relationship of triterpenes. The free β-hydroxyl group at C-3 and the β-oriented carboxyl function at C-17 were closely associated with the antibacterial activities. Interestingly, the amine linked to the triterpenoid skeleton contributed to the activity against Pseudomonas aeruginosa, one of the bacteria most resistant to antibiotics and antiseptics.
The petroleum ether extract with the highest content of palmitic acid (309) from P. forrestii was detected to have antimicrobial properties [169]. Due to containing relatively high proportions of aldehydes and oxygenated monoterpenes, the essential oil from P. laevigata root bark was equipped with significant antibacterial and antifungal activities [170]. Larhsini and co-workers [171] found that the essential oil from P. laevigata leaves showed antimicrobial and antioxidant activities and had a good synergistic effect in association with conventional antibiotics. The root bark essential oil of P. sepium and 4-methoxysalicylaldehyde (294), a main component of the oil (78.8% of the total), were both elaborated to possess antimicrobial properties and moderate antioxidant activities, which indicated that the major portion of these antimicrobial and antioxidant activities was due to the presence of the aldehyde in the oil [172]. In addition, the activities of 4-methoxysalicylaldehyde (294) isolated from P. sepium oil and its six derivatives against six foodborne bacteria were evaluated by Lee's group [173]. Compound 294 (MIC 30.1-67.3 µg/mL) and 4-hydroxysalicylaldehyde (MIC 41.1-61.5 µg/mL) both inhibited the growth of the six foodborne bacteria with finer effects than did the other derivatives.
The antioxidant capacity of phenols is intimately related to their content and the number of hydroxyl groups. The methanol extract with the highest contents of phenolics and flavonoids from P. laevigata displayed the greatest antioxidant properties, followed by the water and ethyl acetate extracts [174]. Intriguingly, six lignins (236,237) were evaluated for their antioxidant abilities, and they showed no or minor antioxidant activities. Feng's group illustrated the probable structure-activity relationship. The more original hydroxyl groups of phenols are replaced by methoxyl groups, the lower the antioxidant activity is [63]. They also detected that flavanes (266-268,270) which exerted high radical-scavenging activities with IC 50 values ranging from 14.61 to 28.80 µM. Additionally, aesculitannin B (270), a flavane trimer, was able to shrink α-melanocyte-stimulating hormone-induced melanogenesis by inhibiting the expression of microphthalmia-associated transcription factor and tyrosinase in a dose-dependent manner, owing to the multiple phenolic hydroxy groups [63]. Bisflavan-3-ols 271 and 272 from the ethyl acetate fraction of P. aphylla displayed evident inhibitory potential against the enzyme lipoxygenase with IC 50 values of 19.7 and 13.5 µM. Malik and co-workers speculated that the stereochemistry of 272 was probably more conducive to the inhibition of lipoxygenase enzyme than that of 271 [84].
In the most recent three years, polysaccharides acquired from this genus have also been detected to show antioxidant and antibacterial capacities. Three crude Cortex Periplocae polysaccharides (CPPs 1-3) from the root bark of P. sepium presented evident antioxidant activities in four assays in vitro [175]. A novel polysaccharide (PLP1) from P. laevigata root bark displayed antioxidant activity in a dose-dependent manner and showed strong antibacterial activity against several Gram (+) and Gram (−) strains. Hence, it was shown to be a promising source for polysaccharides in the medical and food industries [176]. The polysaccharides isolated from P. angustifolia (PAPS) were shown to decrease the rates of lipid peroxidation and protein glycation in vitro. Furthermore, PAPS could defend human embryonic kidney cell HEK293 against oxidative stress and renal tissue against cadmium toxicity [177].
Furthermore, the antioxidant capacity of PAPS was demonstrated by the hepatoprotective potency toward cadmium chloride (CdCl 2 )-induced toxicity in rats, and it could ameliorate the alteration of liver tissue [178]. Athmouni's group [179] testified that the methanolic extract of Periploca angustifolia leaves also possessed antioxidant ability and exerted salutary effects in the treatment of Cd-induced hepatotoxicity. The methanol extract of P. hydaspidis, which is a less-studied species found in Pakistan, had the ability to adjust the levels of various parameters provoked by CCl 4 toxicity in a dose-dependent manner due to the presence of antioxidant and anti-inflammatory constituents in the methanol extract [180]. What is striking is that ZnO nanostructures with tremendous potential against multidrug-resistant E. coli, S. marcescens and E. cloacae were synthesized through reduction by P. aphylla aqueous extract without the utilization of any acid or base. In this manner, the aqueous extract of P. aphylla was proven to be a valid chelating agent [181].

Insecticidal Activity
Since 2004, extracts from the plants of genus Periploca have been found to possess favorable insecticidal activities and promising agricultural value. The ethanol extract from the root bark of P. sepium had a significant repellent effect against the oviposition of imported cabbage worms [182]. Additionally, the benzene fraction from that ethanol extract showed strong antifeeding and poisoning effects on Plutella xylostella larvae [183]. The methanol extract of P. aphylla showed activity against Leishmania infantum (IC 50 6.0 µg/mL, SI 4.0) [184]. The volatile oil from the root bark of P. sepium showed forceful contact-killing activity against Myzus avenae, and its main component was 4-methoxysalicylaldehyde (294), which also displayed antinematodal activity against Bursaphelenchus xylophilus at a minimum effective dose of 200 µg/ball [114,185]. In 2012, it was also proven that the dominating component in the oil (294) was active against Drosophila melanogaster L. and the maize weevil (Sitophilus zeamais) with the LD 50 values of 1.47 and 6.99 mg/adult, respectively [186]. In addition, the acaricidal ability of 4-methoxysalicylaldehyde (294) was greater than that of synthetic acaricides [187].
Zeng's group discovered periplocoside X (80), which induced severe time-dependent cytotoxicity in the midgut epithelial cells of the red imported fire ant and inhibited amylase activity (Solenopsis invicta). Therefore, the ant insect midgut was deduced to be a target of periplocoside X [188]. It is noteworthy that, in addition to periplocoside X (80), there are some other periplocosides capable of impacting the digestive system of insects. Hu's group has made vital contributions to the discovery of these periplocosides and has clarified the underlying mechanism. Periplocoside NW (82) [48,189,190] displayed potent stomach toxicity against some insect pests, including third-instar larvae of P. xylostella, Mythimna separata larva and Pieris rapae. The membrane system in insect midgut epithelial cells was the initial action site of periplocoside NW against insects. In 2012, periplocoside D (50) and F (52) were both detected by bioassay tests, and they showed stomach toxicity activities against third-instar larvae of M. separata with the LC 50 values of 0.39 and 0.34 mg/mL, and against third-instar larvae of P. xyllostella with the LC 50 values of 1.21 and 1.39 mg/mL, respectively [191]. The insecticidal activities against the third-instar larvae of Pseudaletia separata and P. xylostella of periplocoside P (81) were detected by an insecticidal activity bioassay.
To explore the insecticidal mechanisms of these compounds, some studies were implemented by Hu's group. Periplocoside P caused toxicity toward M. separate though activating the weakly alkaline trypsin-like protease [192] and inhibiting the activity of cytochrome P450 in the midgut. Additionally, glutathione-S-transferase might be involved in insect detoxification [193]. Through their in-depth research exploring new pesticides and their action mechanisms against insects, Hu's group found that the apical and basolateral membrane voltage (V a ) rapidly decreased in the presence of periplocoside P in a time-and dose-dependent manner, similar to the effects of Cry1Ab. They speculated that periplocoside P adjusted the transport mechanisms at the apical membrane of the midgut epithelial cells by inhibiting the V-type H + ATPase [194]. In addition, the V-type ATPase A subunit in the midgut epithelium might be regarded as the target binding site of periplocosides, which provided preliminary evidence for the mode of action of periplocosides [195]. Furthermore, aminopeptidases N and N3 of the midgut epithelium cells of M. separata larvae were extrapolated as potential targets of periplocoside E (47) by affinity chromatography [196].

Other Abilities
As early as the 1980s, glycosides K, H 1 and H 2 from P. sepium showed the potentiation of NGF-mediated nerve fiber outgrowth in organ cultures of chicken embryonic dorsal roots and sympathetic ganglia, and glycoside H 2 (97) had the best effect [53]. Later, in 2012, the alcohol extract of P. forrestii led to a good reparation of nerve injury in Pristionchus pacificus caused by thermal stimulation in the concentration range of 2-16 mg/mL, while obvious toxicity was also detected when the concentration was increased [197]. Intriguingly, the sedative and potent analgesic effects of the methanolic extract of P. aphylla at the dose of 500 mg/kg were shown in a series of animal experiments [198].
P. forrestii, a folk medicine used to cure wounds, is also equipped with wound-healing activity. Zhang's group [199] evaluated its ability through the proliferation of fibroblasts, migration and collagen production using L929 cells. The 65% ethanol eluate mainly composed of cardiac glycosides was the most effective fraction, suggesting that the cardiac glycosides may be the potential active ingredients in wound healing. In further research, periplocin (1) demonstrated its ability to significantly boost proliferation and migration and stimulate collagen production in fibroblast L929 cells. Its mechanism of promoting wound healing was intensely associated with the activation of the Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase [200]. Moreover, Abdel-Monem and co-workers [10] reported that ursolic acid played an important role in the protective effect against CCl 4 -induced injury on human hepatoma cell line (Huh7). Additionally, the antiacetylcholinesterase activities of triterpenes 199 and 198 with esterification of the alcohol function at C-3 were lower than that of lupeol (197). Ben Jannet's group speculated that the triterpenic skeleton and the free secondary alcohol function at C-3 could be responsible for the inhibition ability [87]. In addition to the abovementioned abilities, both lipoxygenase inhibitory activity [84] and antimalarial activity [201] of this genus were detected but without further studies on the mechanisms of these actions.

Toxicology
Excessive or prolonged use of P. sepium leads to toxic effects, mainly consisting of cardiotoxicity and hepatorenal toxicity. Acute toxicity tests in mice and long-term toxicity tests in rats showed that the toxicity of ethanol extract from the root bark of P. sepium was greater than that of the water extract [202,203].
The water extract from the root bark of P. sepium was dried into powder, in which the content of periplocin was 21.91 mg/g. After the water extract was injected intraperitoneally into guinea pigs, their electrocardiograms showed obvious changes, and the incidence of abnormal electrocardiograms was proportional to the dose [204]. Sun's group found that within seven days after oral administration, the water extract (0.78-15.0 g/kg) and alcohol extract (0.78-6.0 g/kg) from the root bark of P. sepium both caused obvious hepatotoxic damage in mice, which was represented by increased levels of alanine transaminase, aspartate transaminase, alkaline phosphatese and total bilirubin and a decreased content of albumin, as well as increases of the ratio of the liver to kidneys to different degrees [205]. The pathway of liver injury was inferred to be related to lipid peroxidation induced by oxidative stress [206]. Furthermore, periplocin (1) was revealed to have toxic effects at a certain dose. Deng and co-workers [207] detected that the maximum tolerable dose for the oral administration of periplocin in mice was 103 mg/kg. Periplocin was injected intraperitoneally into mice, resulting in an LD 50 value of 15.20 mg/kg. In addition, periplocin at 0.39 mg/kg brought about electrocardiogram abnormalities in half of the tested guinea pigs. Noteworthy is that the content of periplocin had a tight correlation with the acute toxicity test results of the water extract from the root bark of P. sepium, while the content of 4-methoxysalicylaldehyde (294), a quality control index of Cortex Periplocae in the Chinese Pharmacopoeia (2015 version), did not directly affect the results of acute toxicity [208].
It was demonstrated that the oral bioavailability of periplocin (1) in rats was low [209]. Gao's group [210] studied periplocin in the field of pharmacokinetics. After a single oral administration of periplocin at 50 mg/kg to rats, it was found that periplocin could not be detected at any time point, while the mean plasma profiles of its two metabolites periplocymarin (3) and periplogenin (2) were regular. Intriguingly, two metabolites participated in the pharmacokinetic process, and the main constituent in the blood was periplocymarin. In addition, periplocin could reach its peak concentration quickly after oral administration, whereas the two metabolites reached maximum concentrations more than 4.83 h after administration. It is worth noting that the tissue distributions of periplocin and two metabolites were found to include the heart, liver, spleen, lung and kidneys, but a small amount of chemical constituents were distributed in the brain [211].
To date, some toxicity studies on cardiac glycosides from the genus Peripioca have been carried out, but toxicity tests on other compounds are absent. To improve the safety of clinical therapy, comprehensive and in-depth investigations of the pharmacology and toxicity of plants of the genus Peripioca are necessary.

Conclusions
In this review, the botanical classification, chemical constituents, biological activities and toxicology studies of Periploca species were systematically summarized. There are differences in the classification of this genus at home and abroad, and some new species have not yet been included in flora. Currently, 314 diverse metabolites have been isolated from the genus Periploca, and are they are composed of steroids, carbohydrates, terpenoids, phenylpropanoids, flavonoids, quinones, aromatics, and so on. Steroids obtained from the genus Periploca account for the largest proportion. Moreover, these steroids, mainly composed of C 21 pregnane and cardiotonic steroids, should be considered the characteristic compounds of this genus. Oligosaccharides, which have a high rate of new compounds (78.3%) featuring multifarious deoxysugar and non-deoxysugar, strictly conform to the regularity of the saccharide chains in the cardenolides and C 21 pregnane glycosides of this genus. Hence, these oligosaccharides could also be regarded as representative compounds of this genus. Furthermore, Periploca species are rich in aromatic compounds and volatile oils, especially P. sepium. However, only 4-methoxy salicylaldehyde is assigned as the index component for the quality evaluation of P. sepium root bark in the Chinese Pharmacopoeia (2015 Version), which requires its content to be not less than 0.20% [3]. Other components characterized by a high content, favorable pharmacological activity or potential toxicity should perhaps also be used as legal quality evaluation indicators.
In regard to biological activities, the genus Periploca has received increasing attention over the last fifteen years. The relationships between various activities and their main related plant sources and active ingredients are concisely concluded. It is worth noting that periplocin is a research hotspot and has been demonstrated to possess various activities, such as cardiotonic, antitumor, immunosuppressive and wound-healing effects. However, periplocin has hepatotoxicity and cardiotoxicity at certain doses, suggesting that further pharmacological, toxicological and pharmacokinetics studies on this active compound are necessary. In addition, a series of periplosides with fascinating immunosuppressive and insecticidal properties should be another research focus. In view of periplosides' available immunosuppressive effect in vitro, detailed animal experiments in vivo and the structure-activity relationships of the periplosides should also be studied to identify new immunosuppressants for the treatment of RA. More importantly, periplosides may be promising insecticidal alternatives, which is of great value to agriculture. Thus far, most experiments of biological activities of the genus Periploca have been conducted in vitro. Taking into account the important medical and agricultural value of this genus, intensive studies on the bioactivity-guided chemical isolation, pharmacological activities in vivo, and underlying mechanisms of the biological activities of this genus should be further performed.