Studies involving in vitro and in vivo models of lipopolysaccharide-stimulated (LPS-stimulated) THP-1 cells, mouse ear-swelling, rat cotton pellet granuloma, and rat hind paw edema have confirmed that ethanol and methanol extracts from P. alkekengi calyxes exert anti-inflammatory effects. The extracts achieve these effects by inhibiting the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) [62,91]. As active ingredients isolated from P. alkekengi, physalins A, B, D, E, F, H, G, L, O, V, D1, X, VII, and I, isophysalin A, isophysalin B, and aromaphysalin B showed anti-inflammatory activity. At a concentration of 20 µM, physalins A, O, L, and G and isophysalin A inhibited the LPS-induced NO production by blocking TNF-α [47,48]. Physalins B, E, F, G, H, V, X, D1, VII, and I, isophysalin B, and aromaphysalin B reduced the levels of proinflammatory mediators NO, TNF-α, IL-6, IL-12, and interferon-γ (IFN-γ) in LPS-stimulated and IFN-γ-stimulated macrophages, RAW 264.7 cells, and 12-O-tetradecanoylphorbol-13-acetate (TPA)- and oxazolo-induced dermatitis. These effects occurred through upregulation of the signal transducer and activator of transcription 6 (STAT6) and downregulation of nuclear factor-κB (NF-κB) and the STAT1 signaling pathway [48,49,51,52,53,55,56]. The anti-inflammatory effects of four flavonoids (i.e., luteolin, apigenin, kaempferol, and quercetin) were related to inhibition of the production of NO, IL-6, IL-12, TNF-α, STAT-1, and NF-κB, the expression of C–C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and C–X–C motif chemokine ligand 1/KC (CXCL1/KC), and paw edema [57,58,59,60]. Ombuine inhibited the production of NO in LPS-damaged macrophage cells, with a half maximal inhibitory concentration (IC₅₀) value of 2.23 ± 0.19 µM [57].

Recently, in vitro experimental studies showed the anti-tumor activity of physalins in non-small cell lung cancer, human melanoma A375-S2 cells, and tumor cell lines (A549, K562). The results indicated that physalins A and B have strong anti-tumor activity and induced G2/M cell cycle arrest in non-small cell lung cancer and A375-S2 cells. The mechanism involved in this effect is related to the inhibition of Janus kinase 2 (JAK2) phosphorylation, JAK3 phosphorylation, both constitutive and induced STAT3, reactive nitrogen species (RNS), reactive oxygen species (ROS), and cyclin-dependent kinase 1/cyclin B1 (CDK1/CCNB1) complex, as well as the promotion of the p53-NADPH oxidase activator- (p53-NOXA), p38-NF-κB, and p38 mitogen-activated protein kinase/ROS (MAPK/ROS) pathways [66,67,69,70,72,73]. Physalin A also increased the content of detoxifying enzyme in HepG2 cells, induced apoptosis in HT1080 cells, and inhibited growth in prostate cancer cells (CWR22Rv1 and C42B). These effects occurred by activating the nuclear factor erythroid 2-related factor 2–antioxidant response element (Nrf2–ARE), death receptor apoptotic, JUN N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) signaling pathway; the IC₅₀ values were 20, 10.7, 14.2, and 1.9–4.3 μM, respectively [65,67,68,71]. In addition, six types of cancer cells (i.e., prostate, human HCT116 colon, human DLD-1 colon, breast, TNF-α-stimulated HeLa, and human T cell leukemia Jurkat) were treated with physalin B. The treatment inhibited the activation of TNF-α-induced NF-κB and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB pathways, whereas it promoted the activation of ERK, JNK, p38 MAPK, and P53 pathways [9,74,75,76,79]. Physalin F decreased the TOPFlash reporter activity, inhibited the effects on T-47D cells, and induced cell apoptosis via ROS-mediated mitochondrial pathways [80,81,82].

In vivo, physalins A and F clearly inhibited tumor growth by downregulating β-catenin in xenograft tumor-bearing mice [66,80]. At 10 mg/kg and 25 mg/kg, respectively, physalins B and D inhibited tumor proliferation in mice bearing sarcoma 180 tumor cells [78]. In short, the anti-tumor activity of P. alkekengi and its constituents was associated with the downregulation of JAK/STAT3, TNF-α-induced NF-κB, PMA-induced NF-κB, and phosphoinositide-3-kinase-Akt-mechanistic target of the rapamycin (PI3K/Akt/mTOR) signaling pathway. Moreover, it was linked to the upregulation of the death receptor apoptotic, p53-NOXA, p38-NF-κB, p38 MAPK/ROS, p21, and Nrf2 signaling pathway. The signaling pathways are given in Figure 5.

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 IC₅₀ values of 0.69 and 0.39 μg/mL, respectively. In vivo, physalin H dose-dependently inhibited CD4+ T cell-mediated delayed-type hypersensitivity reactions and antigen-specific T-cell response in ovalbumin-immunized mice, with IC₅₀ 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/cm² (contact treatment), and suppressed epimastigote forms of T. cruzi, with an IC₅₀ 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].

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). The minimum inhibitory concentration (MIC) values of physakengoses B, E, F, G, and H for S. aureus were 9.72 ± 2.83, 9.81 ± 1.48, 5.32 ± 1.47, 6.57 ± 0.86, and 5.78 ± 0.96 μg/mL, respectively. For B. subtilis, these values were 8.89 ± 1.63, 5.59 ± 0.85, 3.50 ± 1.49, 8.78 ± 1.67, and 3.57 ± 1.02 µg/mL, respectively. For P. aeruginosa, these values were 14.91 ± 2.56, 13.12 ± 2.42, 5.79 ± 1.15, 4.51 ± 3.02, and 3.21 ± 0.95 μg/mL, respectively [96]. Zhang et al. showed that physakengoses K, L, M, N, and O had potent antibacterial activity, with MIC values ranging from 2.16 to 12.76 mg/mL [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.

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 IC₅₀ 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.

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 IC₅₀ 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 Ca²⁺ overload in rat cardiomyocytes [107,108].

3. Summary

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.