Natural Products from Physalis alkekengi L. var. franchetii (Mast.) Makino: A Review on Their Structural Analysis, Quality Control, Pharmacology, and Pharmacokinetics

The calyxes and fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino (P. alkekengi), a medicinal and edible plant, are frequently used as heat-clearing and detoxifying agents in thousands of Chinese medicine prescriptions. For thousands of years in China, they have been widely used in clinical practice to treat throat disease, hepatitis, and bacillary dysentery. This systematic review summarizes their structural analysis, quality control, pharmacology, and pharmacokinetics. Furthermore, the possible development trends and perspectives for future research studies on this medicinal plant are discussed. Relevant information on the calyxes and fruits of P. alkekengi was collected from electronic databases, Chinese herbal classics, and Chinese Pharmacopoeia. Moreover, information was collected from ancient documents in China. The components isolated and identified in P. alkekengi include steroids, flavonoids, phenylpropanoids, alkaloids, nucleosides, terpenoids, megastigmane, aliphatic derivatives, organic acids, coumarins, and sucrose esters. Steroids, particularly physalins and flavonoids, are the major characteristic and bioactive ingredients in P. alkekengi. According to the literature, physalins are synthesized by the mevalonate and 2-C-methyl-d-erythritol-4-phosphate pathways, and flavonoids are synthesized by the phenylpropanoid pathway. Since the chemical components and pharmacological effects of P. alkekengi are complex and varied, there are different standards for the evaluation of its quality and efficacy. In most cases, the analysis was performed using high-performance liquid chromatography coupled with ultraviolet detection. A pharmacological study showed that the crude extracts and isolated compounds from P. alkekengi had extensive in vitro and in vivo biological activities (e.g., anti-inflammatory, anti-tumor, immunosuppressive, antibacterial, anti-leishmanial, anti-asthmatic, anti-diabetic, anti-oxidative, anti-malarial, anti-Alzheimer’s disease, and vasodilatory). Moreover, the relevant anti-inflammatory and anti-tumor mechanisms were elucidated. The reported activities indicate the great pharmacological potential of P. alkekengi. Similarly, studies on the pharmacokinetics of specific compounds will also contribute to the progress of clinical research in this setting.


Introduction
P. alkekengi is a perennial plant (Figure 1a) belonging to the genus Physalis of the family Solanaceae. The calyxes and fruits of P. alkekengi (known as Jindenglong in Chinese) ( Figure 1b) are distributed in Europe and Asia. The use of the calyxes and fruits of this plant was first recorded in the prestigious monograph Shennong Bencao Jing in China [1]. Subsequently, it was included as an important traditional Chinese medicine (TCM) in the Ben Cao Gang Mu and pharmacopoeia [2]. Calyxes are green, self-expanded into an oocyst shape, slightly concave at the base, 2.5-5 cm in length, 2.5-3.5 cm in diameter, have thin In the last decades, reviews concerning research progress on the calyxes and fruits of P. alkekengi have been published, mainly focusing on the chemical components, traditional uses, toxicology, and pharmacological activities [6]; however, thus far, there are no reports on structural analysis, quality control, and pharmacokinetics. In recent years, new pharmacological activities have been discovered, and the main active ingredients in P. alkekengi are physalins and flavonoids [7]. Therefore, we herein provide a literature review on the structural analysis of physalins and flavonoids in the calyxes and fruits of P. alkekengi. We have also prepared a comprehensive and up-to-date report for the known pharmacological activities. In addition, the quality control and pharmacokinetics studies are summarized in detail. We hope that the current review will provide a theoretical basis and valuable data for future in-depth studies and the development of useful applications. In the last decades, reviews concerning research progress on the calyxes and fruits of P. alkekengi have been published, mainly focusing on the chemical components, traditional uses, toxicology, and pharmacological activities [6]; however, thus far, there are no reports on structural analysis, quality control, and pharmacokinetics. In recent years, new pharmacological activities have been discovered, and the main active ingredients in P. alkekengi are physalins and flavonoids [7]. Therefore, we herein provide a literature review on the structural analysis of physalins and flavonoids in the calyxes and fruits of P. alkekengi. We have also prepared a comprehensive and up-to-date report for the known pharmacological activities. In addition, the quality control and pharmacokinetics studies are summarized in detail. We hope that the current review will provide a theoretical basis and valuable data for future in-depth studies and the development of useful applications.

Physalins
P. alkekengi, a high-value multipurpose medicinal plant, is a rich reservoir of structurally diverse and biologically active terpenoids, termed physalins. Thus far, >70 physalin-type natural products have been isolated; most of them possess a 13,14-seco-16,24-cycloergostane skeleton, with anolides with a C-22, C-26 δ-lactone side chain or C-23, C-26 γ-lactone side chain of C28 ergostane-type steroids [8,9]. According to the bonding type of C-14, physalins can be divided into two subtypes: physalins (Type I), in which C-14 is connected to C-17 through oxygen to form an acetal bridge, and neophysalins (Type II), where C-14 is connected to C-16 and C-15/C-17 is esterified to form lactone [10].
The skeletons of physalins, through various biochemical reactions (i.e., desaturation, methylation, hydroxylation, epoxidation, cyclization, chain elongation, and glycosylation), lead to the production of various physalins [21]. As shown in Figure 3, Wu et al. [10] proposed the plausible biogenetic pathway for physalin IX, physalin V, aromaphysalin B, and physalinol A. For example, the epoxidation and hydroxylation of physalin B could produce an intermediate, which could be further ring-cleaved between C-1 and C-10, subjected to lactonization, and dehydrated to yield physalin V. Meanwhile, the cyclization of physalin B between C-11 and C-15 afforded the intermediate; subsequently, the intermediate was further epoxidized and hydrated to obtain physalin IX.

Flavonoids
Flavonoids are the second major component of P. alkekengi, with a common C6-C3-C6 tricyclic skeleton [22,23]. The main flavonoid synthetic pathway has been characterized in P. alkekengi ( Figure 4). The C6-C3-C6 carbon backbone was first synthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroylcoenzyme A, which finally enters the flavonoid synthesis pathway [24,25]. Next, 4-coumaroyl -coenzyme A combines with three molecules of malonyl-coenzyme A to yield naringenin, which is the source of all flavonoids. Chalcone synthase and chalcone isomerase are the enzymes involved in the two-step condensation [26][27][28]. Naringenin is subsequently converted to luteolin through two reactions catalyzed by flavanone 3'-hydroxylase and flavonol synthase. At the same time, the conversion of naringenin by flavanone 3hydroxylase yields dihydrokaempferol that can be hydroxylated on the 3' position of the B-ring by flavanone 3'-hydroxylase, thereby producing dihydroquercetin. The subsequent steps of dihydrokaempferol and dihydroquercetin produce kaempferol and quercetin by flavonol synthase, respectively. The last step of quercetin for the formation of stable compounds involves glycosylation by the enzyme uridine diphosphate-glucose: flavonoid-3-Oglucosyltransferase. Finally, quercetin is further converted to quercetin-3-glucoside [29,30].

Flavonoids
Flavonoids are the second major component of P. alkekengi, with a common C6-C3-C6 tricyclic skeleton [22,23]. The main flavonoid synthetic pathway has been characterized in P. alkekengi ( Figure 4). The C6-C3-C6 carbon backbone was first synthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-coenzyme A, which finally enters the flavonoid synthesis pathway [24,25]. Next, 4-coumaroyl-coenzyme A combines with three molecules of malonyl-coenzyme A to yield naringenin, which is the source of all flavonoids. Chalcone synthase and chalcone isomerase are the enzymes involved in the two-step condensation [26][27][28]. Naringenin is subsequently converted to luteolin through two reactions catalyzed by flavanone 3'-hydroxylase and flavonol synthase. At the same time, the conversion of naringenin by flavanone 3-hydroxylase yields dihydrokaempferol that can be hydroxylated on the 3' position of the B-ring by flavanone 3'-hydroxylase, thereby producing dihydroquercetin. The subsequent steps of dihydrokaempferol and dihydroquercetin produce kaempferol and quercetin by flavonol synthase, respectively. The last step of quercetin for the formation of stable compounds involves glycosylation by the enzyme uridine diphosphate-glucose: flavonoid-3-O-glucosyltransferase. Finally, quercetin is further converted to quercetin-3-glucoside [29,30].

Quality Control
The quality control of calyxes and fruits is extremely important for their use. Exten-

Quality Control
The quality control of calyxes and fruits is extremely important for their use. Extensive studies evaluated methods for the analysis of calyxes and fruits. According to the Chinese Pharmacopoeia, and based on the morphological, microscopic, and high-performance liquid chromatography (HPLC) analysis and thin-layer chromatography (TLC) identification, the minimum content of luteoloside for qualifying the calyxes and fruits of P. alkekengi is ≥0.10% [1]. However, due to the complex chemical components and diverse pharmacological activities of herbal medicines, a single quantitative marker appears to be insufficient for the assessment of quality. Currently, multiple compounds (mainly physalins, flavonoids, and polysaccharides) have been used to validate the quality of this herb by TLC, HPLC, and ultra-performance liquid chromatography-mass spectrometry (UPLC/MS) [31,32].
To obtain reliable pharmacological effects, the concentration of the chemical components of calyxes and fruits should be controlled; it is mainly determined by the season and harvesting time, as well as climatic and geographical conditions [33,34]. The effective composition of calyxes significantly changes with the growth period during harvest. A study showed that the content of physalin D in immature calyxes (0.7880 ± 0.0612%) was four-fold higher than that measured in mature calyxes (0.2028 ± 0.016%). Of note, the content of physalin D in fruits was markedly lower (immature fruits: 0.0992 ± 0.0083%; mature fruits: 0.0259 ± 0.0021%) [35]. Kranjc et al. [36] developed the first high-performance thin-layer chromatography (HPTLC) and HPTLC-MS/MS methods that can characterize physalins in crude extracts obtained from different parts of P. alkekengi at different stages of maturity. These findings indicated that only certain parts of the plant are appropriate for specific pharmaceutical applications. The HPTLC method overcame some of the drawbacks in analytical physalin profiling, providing a new approach to quality control for P. alkekengi. In addition, 31 samples of P. alkekengi collected from different habitats were analyzed using fingerprinting. The results showed that the contents of Baishan, Xinxiang, and Shenyang differed considerably [37]. Huang et al. [38] reported the fragmentation behavior of major physalins in P. alkekengi calyxes via ultra-high performance liquid chromatography (UHPLC)-quadrupole time of flight tandem mass spectrometry (QTOF-MS/MS). The content of 4,7-didehydroneophysalin B in fruits and calyxes of P. alkekengi was determined by HPLC and UPLC-MS/MS. The results showed that the contents of 4,7-didehydroneophysalin B were 2.18% (50% ethanol extract), 0.42% (70% ethanol extract) by HPLC, and 15.75-70.88 µg/g by UPLC-MS/MS. The results suggested that the content of 4,7-didehydroneophysalin B is relatively high and could be used as an index for the quality control of the medicinal material [39][40][41]. The contents of luteoloside, polysaccharides, reducing sugar, lutein, and β-carotene in samples obtained from different habitats were compared, revealing significant differences [42][43][44]. Moreover, some researchers have determined the contents of luteolin and luteoloside in pharmaceutical preparations (i.e., Physalis permviana liquid and Jinhuang yanyan tablets) [45,46]. These data provided an important theoretical basis for the harvesting of P. alkekengi, identification of physalins, and evaluation of the clinical applications of this medicinal herb. However, wide variations were observed in the contents of these compounds due to differences in the sources and time points of sample collection. Furthermore, the fruits of P. alkekengi also contained organic acids and smaller amounts of (hydroxy)cinnamoyl hexosides and amino acids [5]. The quantitative analysis of P. alkekengi is shown in Table 1. Aromatic amino acids and amino derivatives HPLC-DAD-ESI-MS Fruits 50.9-63.5 mg/kg [5] Abbreviations: APCI, atmospheric pressure chemical ionization; DAD, diode array detection; ESI, electrospray ionization interface; HPLC, high-performance liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; RP, reverse phase; TLC, thin-layer chromatography; UPLC, ultra-performance liquid chromatography; UV, ultraviolet.

Pharmacology
Pharmacological experiments showed that the various crude extracts and compounds isolated from P. alkekengi have diverse biological activities (e.g., anti-inflammatory, antitumor, immunosuppressive, anti-microbial, anti-leishmanial, anti-asthmatic, anti-diabetic, etc.). In addition, the mechanisms of action of the anti-inflammatory and anti-tumor activities were also reported. The main pharmacological activities of crude extracts and compounds are shown in Table 2.

Immunosuppressive Activity
The immunosuppressive activity of P. alkekengi mainly focused on immune cells and Trypanosoma infection. Previous studies utilizing concanavalin A (Con A)-activated spleen cells suggested that physalin B inhibited Con A-induced lymphoproliferation, mixed lymphocyte reaction (MLR), and IL-2 production [88]. Yu et al. [90] found that physalin H also significantly inhibited the proliferation of Con A-induced T cells and MLR in vitro, with IC 50 values of 0.69 and 0.39 µg/mL, respectively. In vivo, physalin H dose-dependently in-hibited CD4+ T cell-mediated delayed-type hypersensitivity reactions and antigen-specific T-cell response in ovalbumin-immunized mice, with IC 50 values of 3.61 µg/mL for 48 h and 2.75 µg/mL for 96 h. The mechanisms may be related to the modulation of T-helper 1/T-helper 2 (Th1/Th2) cytokine balance, inhibition of T cell activation, and proliferation and induction of HO-1 in T cells. Moreover, at the concentration of 40 µg, polysaccharides from fruits of P. alkekengi showed good immunosuppressive effects in mice [91]. Physalin B decreased the number of T. cruzi Dm28c and T. cruzi transmission in the gut at doses of 1 mg/mL (oral administration), 20 ng (topical application), and 57 ng/cm 2 (contact treatment), and suppressed epimastigote forms of T. cruzi, with an IC 50 value of 5.3 ± 1.9 µM [85,87]. At a concentration of 1 µg/mL, physalin B significantly increased the mortality rate (78.1%) among Rhodnius prolixus larvae infected with Trypanosoma rangeli [86]. Physalin F prevented the rejection of allogeneic heterotopic heart transplants in vivo in a concentration-dependent manner. Moreover, it inhibited the spontaneous proliferation of peripheral blood mononuclear cells in patients with human T-cell lymphotropic virus type 1-related (HTLV1-related) myelopathy at 10 µM, suggesting its potential for treatments of pathologies in the inhibition of immune responses [88,89].

Antibacterial Activity
In vitro, at the concentration of 100 µg/mL, physalin D isolated from P. alkekengi was found to be effective against Staphylococcus epidermidis (S. epidermidis), Enterococcus faecalis (E. faecalis), Staphylococcus aureus (S. aureus), and Bacillus subtilis (B. subtilis) [92]. Yang et al. [93] reported that physalins B, J, and P exhibited a good antibacterial activity against Escherichia coli (E. coli) and B. subtilis. Additionally, trichlormethane, ethanol, methanol, or aqueous extracts from P. alkekengi were also active against some Gram-positive and Gram-negative bacteria [62,[94][95][96]. Janua'rio et al. [94] found that the crude trichlormethane extract (fraction A1-29-12) inhibited the Mycobacterium tuberculosis H37RV strain at a minimum concentration of 32 µg/mL. Li et al. [95] found that the 70% ethanol extract stimulated the growth of probiotic bacteria (Lactobacillus delbrueckii) and inhibited that of pathogenic bacteria (E. coli) in a dose-dependent manner. Moreover, a study indicated that physakengoses also have potent antibacterial activity against S. aureus, B. subtilis, and Pseudomonas aeruginosa (P. aeruginosa).  [97]. However, the mechanism involved in the antibacterial activity of P. alkekengi has not been reported yet, warranting further research. The antibacterial activity is illustrated in Figure 6.

Antileishmanial Activity
Physalins exhibit potent antileishmanial activity against the cutaneous leishmaniasis [109,110]. Guimarães et al. [98] reported that physalins B and F exerted in vivo antileishmanial effects in BALB/c mice infected with Leishmania amazonensis (L. amazonensis); in vitro, they demonstrated an effect against intracellular amastigotes of Leishmania. In vitro, physalins B and F inhibited the infection of macrophages with L. amazonensis, with IC 50 values of 0.21 and 0.18 µM, respectively. Physalin F markedly reduced the lesion size and number of parasites in vivo. However, physalin D did not show this activity. This effect was associated with the inhibition of NO and proinflammatory cytokines (e.g., IL-12 and TNF-α) by physalins B and F; however, physalin D lacked immunomodulatory/anti-inflammatory activity [48,88]. Meanwhile, the results suggest that anti-inflammatory and antileishmanial activities by physalins play a role in the treatment of cutaneous leishmaniasis. Molecules 2021, 26, x FOR PEER REVIEW 16 of Figure 6. Schematic representation of antibacterial activity of P. alkekengi and its constituents.

Antileishmanial Activity
Physalins exhibit potent antileishmanial activity against the cutaneous leishmanias [109,110]. Guimara ẽs et al. [98] reported that physalins B and F exerted in vivo antileis manial effects in BALB/c mice infected with Leishmania amazonensis (L. amazonensis); vitro, they demonstrated an effect against intracellular amastigotes of Leishmania. In vitr physalins B and F inhibited the infection of macrophages with L. amazonensis, with IC values of 0.21 and 0.18 μM, respectively. Physalin F markedly reduced the lesion size an number of parasites in vivo. However, physalin D did not show this activity. This effe was associated with the inhibition of NO and proinflammatory cytokines (e.g., IL-12 an TNF-α) by physalins B and F; however, physalin D lacked immunomodulatory/anti-in flammatory activity [48,88]. Meanwhile, the results suggest that anti-inflammatory an antileishmanial activities by physalins play a role in the treatment of cutaneous leishma iasis.

Others
The anti-asthmatic activity of physalins has been increasingly reported over th years. In an in vitro study, following the oral administration of a water extract from alkekengi, the number of white blood cells and eosinophils in mice, as well as the expre sion of IL-5 and IFN-γ in lung tissue, were reduced. These findings indicated its potenc as a drug for the treatment of allergic asthma in children [99]. Moreover, some studi showed that luteolin effectively inhibited inflammation in asthmatic models [111]. Th relevant mechanisms may be related to the inhibition of iNOS/NO signaling. Thus, mo studies are required to explain the mechanisms involved in the anti-asthmatic activity the P. alkekengi extract.
Thus far, most scientific investigations on the anti-diabetic activity of P. alkekengi hav been carried out using the fruits, aerial parts, and polysaccharides obtained from the c lyxes of P. alkekengi. For the fruits and aerial parts, the ethyl acetate extract effective decreased the levels of fasting blood glucose (FBG), total cholesterol (TC), triglycerid (TG), and glycated serum protein, whereas it significantly increased those of fasting insu lin (FINS) [100,102]. Moreover, polysaccharides showed anti-hyperglycemic activity o

Antileishmanial Activity
Physalins exhibit potent antileishmanial activity against the cutaneous leishmaniasis [109,110]. Guimarães et al. [98] reported that physalins B and F exerted in vivo antileishmanial effects in BALB/c mice infected with Leishmania amazonensis (L. amazonensis); in vitro, they demonstrated an effect against intracellular amastigotes of Leishmania. In vitro, physalins B and F inhibited the infection of macrophages with L. amazonensis, with IC 50 values of 0.21 and 0.18 µM, respectively. Physalin F markedly reduced the lesion size and number of parasites in vivo. However, physalin D did not show this activity. This effect was associated with the inhibition of NO and proinflammatory cytokines (e.g., IL-12 and TNF-α) by physalins B and F; however, physalin D lacked immunomodulatory/anti-inflammatory activity [48,88]. Meanwhile, the results suggest that anti-inflammatory and antileishmanial activities by physalins play a role in the treatment of cutaneous leishmaniasis.

Others
The anti-asthmatic activity of physalins has been increasingly reported over the years. In an in vitro study, following the oral administration of a water extract from P. alkekengi, the number of white blood cells and eosinophils in mice, as well as the expression of IL-5 and IFN-γ in lung tissue, were reduced. These findings indicated its potency as a drug for the treatment of allergic asthma in children [99]. Moreover, some studies showed that luteolin effectively inhibited inflammation in asthmatic models [111]. The relevant mechanisms may be related to the inhibition of iNOS/NO signaling. Thus, more studies are required to explain the mechanisms involved in the anti-asthmatic activity of the P. alkekengi extract.
Thus far, most scientific investigations on the anti-diabetic activity of P. alkekengi have been carried out using the fruits, aerial parts, and polysaccharides obtained from the calyxes of P. alkekengi. For the fruits and aerial parts, the ethyl acetate extract effectively decreased the levels of fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), and glycated serum protein, whereas it significantly increased those of fasting insulin (FINS) [100,102]. Moreover, polysaccharides showed anti-hyperglycemic activity on alloxan-induced mice. Although research is currently at a preliminary stage, the possible mechanisms are related to the enhancement of PI3K, Akt, and glucose transporter type 4 (GLUT4) mRNA expression, as well as the inhibition of FNG and GSP expression, indicating that they are promising candidates for the development of new anti-diabetic agents [101].
The anti-ulcer and anti-Helicobacter pylori effects are newly discovered pharmacological effects of P. alkekengi. Wang et al. reported that the P. alkekengi extract showed anti-Helicobacter pylori and gastroprotective activities by reducing the intensity of gastric mucosal damage and mitigating pain sensation [63]. It was recently reported that the 70% ethanol extract of P. alkekengi treated LPS-induced acute lung injury by: (1) reducing the release of TNF-α and the accumulation of oxidation products; (2) decreasing the levels of NF-κB, phosphorylated-p38, ERK, JNK, p53, caspase 3 (CASP3), and COX-2; and (3) enhancing the translocation of Nrf2 from the cytoplasm to the nucleus [103]. It was also shown that the mechanism of P. alkekengi, which is involved in the improvement of oxidative stress damage and inflammatory response induced by acute lung injury, was related to the inhibition of NF-κB and the MAPK signaling pathway and the transduction of the apoptotic pathway, as well as the activation of the Nrf2 signaling pathway. Physalin B could be used in the treatment of dextran sulfate sodium-induced colitis in BALB/c mice by suppressing multiple inflammatory signaling pathways [50]. In addition, physalin B is effective against Alzheimer's disease through downregulation of β-amyloid (Aβ) secretion and beta-secretase 1 (BACE1) expression by activating forkhead box O1 (FoxO1) and inhibiting STAT3 phosphorylation [104]. In the diphenyl-2-picrylhydrazyl (DPPH) and thiobarbituric acid (TBA) test, physalin D showed antioxidant activity, with an IC 50 value ≥10 ± 2.1 µg/mL [92]. Physalins B, D, F, and G showed low anti-plasmodial activity; nevertheless, physalin D markedly caused parasitemia and a delay in mortality in mice infected with Plasmodium berghei [105]. Furthermore, a study demonstrated that 75% ethanol extract of calyxes and fruits of P. alkekengi significantly decreased the serum's total cholesterol and TG levels in vivo. Moreover, luteolin-7-O-β-D-glucopyranoside isolated from P. alkekengi decreased the TG levels induced by oleic acid in HepG2 cells and by high glucose in primary mouse hepatocytes, thereby exhibiting hypolipidemic activity [106]. Luteolin effectively relaxed the blood vessels and preserved the rat heart, mainly through activation of the PI3K/Akt/NO signaling pathway and enhancement of the activity of endothelial NOS, as well as amelioration of the Ca 2+ overload in rat cardiomyocytes [107,108].

Physalins
Absorption refers to the process by which the drug enters the blood circulation from the site of administration. Following the oral administration of the extract from the calyxes and fruits of P. alkekengi (0.5 g/mL) in rats, liquid chromatography with MS/MS was used to investigate the pharmacokinetic profile of physalins A, D, and L (equivalent to 2, 16, and 3 mg/mL, respectively) in plasma. The results showed similar pharmacokinetic parameters for the three physalin compounds (maximum concentration: 1.3, 1.7, and 1.3 h, respectively). The biological half-life was 2.5, 3.4, and 2.8 h; the mean residence time was 3.6, 4.9, and 4.1 h; and the area under curve was 113, 103, and 266 ng·h/mL, respectively. These data revealed that the absorption characteristics of these three physalin compounds in rats were similar. Moreover, chemical structural changes in the three compounds exerted a minimal effect on the absorption rate but a greater effect on the elimination rate [112]. This is attributed to the high degree of similarity between the chemical structures of the three physalin compounds. Another study also showed that physalins A, D, and L exhibited great similarity in the time required to reach the peak concentration (0.7, 1.2, and 0.7 h, respectively) in rat plasma. However, isophysalin B was rarely absorbed in rats due to the conversion of gastrointestinal bacteria and metabolic enzymes through a strong first-pass effect after oral administration and its low solubility in gastrointestinal fluid [113][114][115]. Pharmacokinetic studies of physalins incubated with intestinal bacterial culture showed that the concentration was significantly decreased. Furthermore, most physalins could not be detected when the reaction time was increased. These results indicated that physalins are extremely unstable in rat intestinal bacteria and have low bioavailability [115].
Distribution refers to the process by which the drug is absorbed into the blood circulation and transported to the various organs and tissues of the body. Zheng et al. [116] revealed that, after a single intragastric administration, physalin B exhibited a single-chamber model with peak concentration of 0.08 h and body clearance rate by bioavailability of 0.18 L/min/kg; the distribution decreased in the following order: C lung > C heart > C kidney > C brain > C liver > C spleen . The concentration of physalin B in the lung was >20-fold higher than that measured in all other tissues, indicating that the lung is the main target organ of physalin B. Physalin B also showed significant antitumor activity against lung cancer cell lines (IC 50 value: 1.2 µM) and became a therapeutic candidate for this disease [117]. Wu et al. [118] found that physalin D was distributed and rapidly eliminated in rats within 5 min, and the distribution characteristics in tissue decreased in the following order: C kidney > C liver > C lung > C spleen > C heart . The highest levels were recorded in the kidney, followed by the liver; however, physalin D was not detected in the brain. Therefore, kidney is the major distribution tissue for physalin D in rats, and physalin D cannot cross the blood-brain barrier. This is probably because the polarity of physalin B is lower than that of physalin D, making it easier for physalin B to cross the cell membrane than physalin D.
Metabolism is also known as biotransformation; it refers to the change in the chemical structure of the drug in the body. The main metabolic reactions of physalins in the body are phase II metabolic reactions (e.g., sulfonation, acetylation, glucuronidation, etc.). Following the oral administration of calyxes and fruits of P. alkekengi, an analytical method based on UHPLC-Q-TOF-MS/MS was applied to identify absorbed constituents and in vivo metabolites in biological fluids obtained from rats. The results identified 33 compounds in vivo: 12 and 21 compounds were predicted to be prototype components and metabolites of P. alkekengi, respectively. Lastly, sulfonation and hydroxylation were recognized as the metabolic pathways for physalin constituents [119]. Another study focused on the metabolism of physalin A in rats after oral administration. A total of 24 proposed metabolites were identified in the plasma, bile, urine, and feces. The major metabolic pathways of physalin A in the body were sulfonation, reduction, and hydroxylation. These analyses provided a framework for studying the possible metabolic pathways of other physalins and evaluating the relationship of metabolites with parent compounds in the context of the internal environment [120].
Excretion refers to the process through which the prototype of a drug or its metabolites are transported out of the body through excretory or secretory organs. A rapid and sensitive method was developed to investigate urine and feces samples collected at different exposure times after the oral administration of physalin D (25 mg/kg). The analysis showed that 12.26% of the orally administered dose of physalin D was excreted in the feces, in an unchanged form, within 72 h. The physalin D in feces was mainly excreted within 12-24 h, and the excretion ratio in feces decreased in parallel with the decreasing concentration of physalin D in the rat. Physalin D in urine was mainly excreted in the form of glucuronide and sulfate, mainly within 4-36 h, and the amount decreased with time. The excretion data of physalin D in urine and feces indicated that <14.0% of the administered dose was excreted in an unconverted form. These results revealed that physalin D was extensively and rapidly metabolized in rats after intragastric administration, leading to a short biological halflife [121]. The pharmacokinetics of physalins are shown in Table 3.  [115] Abbreviation: AUC, area under curve; CL, clearance rate; C max , maximum concentration; MRT, mean residence time; t 1/2 , biological half-life; T max , peak concentration.

Flavonoids
Flavonoids are widely distributed in the calyxes and fruits of P. alkekengi and exhibit anti-allergic, anti-inflammatory, antioxidant, and inhibitory effects on NO as the main active ingredient [122]. However, the in vivo absorption of flavonoids has been rarely investigated. Guo et al. [112] conducted a pharmacokinetic characterization of luteolin-7-O-glucopyranoside and luteolin. They found that the plasma concentrations of two flavonoids could not reach the lower limit of quantitation at most timepoints. Small amounts of compounds were detected due to the relatively low levels of flavonoids (1 and 0.4 mg/g, respectively) in extracts from the calyxes and fruits of P. alkekengi. Moreover, evidence suggested that flavonoids were usually consumed in the small intestine as a proportion of aglycone [123]. Luteolin is produced by the metabolism of luteoloside. Subsequently, it may be transported to the liver through the portal vein, where it may form a potential phase I substrate through further hydroxylation in the liver. Consequently, it produces more polar compounds through further phase I and II metabolism [124,125], such as the glucuronidation of luteolin and the hydroxylation and sulfation of other types of flavonoids [119]. Therefore, the content of prototype constituents in the body would be significantly decreased or undetectable.
Firstly, this review summarizes the structural analysis of natural products of physalins and flavonoids. Physalins are synthesized in P. alkekengi via MEV and MEP pathways, and flavonoids are synthesized via phenylpropanoid pathway. However, apart from some studies on the physalins' skeleton of natural products, there is almost no research conducted on the synthesis of specific physalins and their derivatives. The importance of the right-side (DFGH-ring) structure of physalins for biological activity is established. For example, synthesis of DFGH-ring derivatives of physalins with a hydrophobic substituent is important for the inhibitory activity [128]. Therefore, there is a need for further exploration of the synthesis of physalins and development of more clinically valuable compounds.
Secondly, a holistic quality control method that is correlated with the pharmacological effects of P. alkekengi is warranted. Current quality control methods are mainly focused on HPLC, and it is difficult to distinguish genuine products from counterfeit goods. The purpose of quality control in TCM is to monitor effective substances and their variations in the production process. By summarizing the currently available literature, we found that the contents of bioactive compounds differ significantly in samples obtained from different sources and at different collection times. Therefore, safe, high-quality, and high efficiency planting techniques for this plant should be further investigated to guide its production for TCM.
Thirdly, previous pharmacological investigations on P. alkekengi have yielded considerable evidence regarding its anti-inflammatory and anti-cancer properties and have elucidated the mechanisms of their action in vitro and in vivo. However, few studies concentrated on its immunosuppressive, anti-leishmanial, anti-asthmatic, anti-diabetic, antioxidative, anti-malarial, anti-vasodilatory, and anti-colitic effects, which warrant further exploration. Additionally, several studies have highlighted the potential of P. alkekengi as a novel therapeutic agent for the treatment of ulcers, Helicobacter pylori, LPS-induced induced acute lung injury, and Alzheimer's disease. Nevertheless, the mechanism underlying these treatment effects should be fully elucidated using current techniques.
Lastly, the absorption, distribution, metabolism, and excretion of physalins in the body are explained. These compounds are characterized by fast absorption, wide distribution, and rapid excretion. These findings indicated that physalins are extremely unstable and have low bioavailability in the intestine. Hence, they must overcome certain factors that control the sustained and stable release of drugs in the blood and improve oral bioavailability, thereby exerting good pharmacological effects. Unfortunately, however, it should be noted that few studies have investigated the pharmacokinetics of extracts and active compounds, particularly flavonoids. Consequently, further clinical application of P. alkekengi may be limited until further pharmacokinetics studies in the laboratory and clinic are performed.
In summary, P. alkekengi is an excellent, abundant, inexpensive, and edible drug. The synthesis of the main active components of P. alkekengi must be further analyzed using additional biological and chemical techniques to further expand their potential applications. In addition, the quantitative analysis of the chemical constituents of P. alkekengi should be employed for the purpose of standardization and quality control of extracts. Lastly, additional in vivo animal research and clinical trials are needed to determine whether various applications of P. alkekengi are effective and safe in a larger population.