Saponins of Paris polyphylla for the Improvement of Acne: Anti-Inflammatory, Antibacterial, Antioxidant and Immunomodulatory Effects

Acne is a chronic inflammatory skin disease with a recurring nature that seriously impacts patients’ quality of life. Currently, antibiotic resistance has made it less effective in treating acne. However, Paris polyphylla (P. polyphylla) is a valuable medicinal plant with a wide range of chemical components. Of these, P. polyphylla saponins modulate the effects in vivo and in vitro through antibacterial, anti-inflammatory, immunomodulatory, and antioxidant effects. Acne is primarily associated with inflammatory reactions, abnormal sebum function, micro-ecological disorders, hair follicle hyperkeratosis, and, in some patients, immune function. Therefore, the role of P. polyphylla saponins and their values in treating acne is worthy of investigation. Overall, this review first describes the distribution and characteristics of P. polyphylla and the pathogenesis of acne. Then, the potential mechanisms of P. polyphylla saponins in treating acne are listed in detail (reduction in the inflammatory response, antibacterial action, modulation of immune response and antioxidant effects, etc.). In addition, a brief description of the chemical composition of P. polyphylla saponins and its available extraction methods are described. We hope this review can serve as a quick and detailed reference for future studies on their potential acne treatment.


Introduction
Acne vulgaris is one of the most common dermatological diseases, affecting about 650 million people worldwide, with more than 85% of cases being in teenagers [1].In addition, acne brings patients a significant clinical, psychological, and economic burden.As research into acne treatment has intensified, it has been discovered that the use of antibiotic drugs may cause the development of resistance.In contrast, some medicinal plants are considered to have antibacterial, anti-inflammatory, and immune function effects.Therefore, the use of Nat medicines has become an inevitable trend.
Several studies have found that the use of active plant substances can have a beneficial effect on the treatment of acne, such as some flavonoid components, like catechins in green tea [2], licorice [3], and Chalcone A [4], which have anti-inflammatory properties.Besides, humulone and serpentine in hops [5], aloin contained in aloe vera [6], and rhubarb phenols in cassia seed [7] have shown strong antibacterial effects in the treatment of acne.Furthermore, the saponin-like substances of Shigarou have shown remarkable antiinflammatory [8], antibacterial [9].and immunity-boosting effects [10] in the treatment of acne.
Paris polyphylla belongs to the lily family and Berberidaceae.For over 2000 years, it has been used as an important Nat medicinal plant in China to treat infection, inflammation, and cancer.It was first reported in the "Shennong Materia Medica Classic" [11], and first named Chonglou in the "Dian Nan Ben Cao" [12] and later in Li Shizhen's "Compendium of Materia Medica" [13].It was listed in the "Chinensis Pharmacopoeia" for the first time in 1985 [14].Through years of cultivation and evolution, this herb has been widely distributed in Asian regions such as India [15], China [16], Bhutan, Laos, Myanmar, Nepal [17], Thailand and Vietnam (Figure 1).
Paris polyphylla belongs to the lily family and Berberidaceae.For over 2000 years, it has been used as an important Nat medicinal plant in China to treat infection, inflammation, and cancer.It was first reported in the "Shennong Materia Medica Classic" [11], and first named Chonglou in the "Dian Nan Ben Cao" [12] and later in Li Shizhen's "Compendium of Materia Medica" [13].It was listed in the "Chinensis Pharmacopoeia" for the first time in 1985 [14].Through years of cultivation and evolution, this herb has been widely distributed in Asian regions such as India [15], China [16], Bhutan, Laos, Myanmar, Nepal [17], Thailand and Vietnam (Figure 1).Traditionally, the rhizome of P. polyphylla is made into a decoction compatible with other herbal medicines.There are many kinds of prescriptions prepared in this way, such as Chongloujicaishenghua Tang [18], Lianxiuwugong Dilong Tang [19], and Qingwenjiedu soup [20], etc.They have various functions, such as heat-clearing, detoxification, and pain relief, to deal with a sore throat, snake bites, crepitant pain, and convulsions [14].
With the development of pharmacological research of P. polyphylla, the pharmacological activities, such as anti-tumor [21], antiviral [22], antibacterial [23], antifungal [24], hemostatic [25], and anti-inflammatory [26] effects have been reported and proved.Zhou et al. [21] found that Polyphyllin VII (PP VII) inhibited the differentiation of BMMs into osteoclasts by inhibiting ROS synthesis in vitro.He et al. [27] reported the anti-tumor effect of PP VII by inducing mitochondrial dysfunction and apoptosis and inhibiting PI3K/Akt and NFκB signaling pathways against lung cancer.Wang et al. [22] showed that 95% alcoholic extract of P. polyphylla could promote the release of IL-6 and kill the viruses EV71 and CVB3, thus preventing virus replication.Yang et al. [28] found that PPI could exhibit its strong anti-inflammatory ability by regulating the priming pathway of NF-κB and controlling the production of IL-8, IL-6, and TNF-α inflammatory factors.At the same time, it was found that the different types of P. polyphylla monomers (PP I, II, III, V, VI, VII and H) showed significant inhibition of the growth of Propionibacterium.acnes (P.acnes), Staphylococcus.epidermidis (S. epidermidis) and Staphylococcus.aureus (S. aureus), and their antibacterial activities were more potent than those of antibiotics [23].Traditionally, the rhizome of P. polyphylla is made into a decoction compatible with other herbal medicines.There are many kinds of prescriptions prepared in this way, such as Chongloujicaishenghua Tang [18], Lianxiuwugong Dilong Tang [19], and Qingwenjiedu soup [20], etc.They have various functions, such as heat-clearing, detoxification, and pain relief, to deal with a sore throat, snake bites, crepitant pain, and convulsions [14].
With the development of pharmacological research of P. polyphylla, the pharmacological activities, such as anti-tumor [21], antiviral [22], antibacterial [23], antifungal [24], hemostatic [25], and anti-inflammatory [26] effects have been reported and proved.Zhou et al. [21] found that Polyphyllin VII (PP VII) inhibited the differentiation of BMMs into osteoclasts by inhibiting ROS synthesis in vitro.He et al. [27] reported the anti-tumor effect of PP VII by inducing mitochondrial dysfunction and apoptosis and inhibiting PI3K/Akt and NFκB signaling pathways against lung cancer.Wang et al. [22] showed that 95% alcoholic extract of P. polyphylla could promote the release of IL-6 and kill the viruses EV71 and CVB3, thus preventing virus replication.Yang et al. [28] found that PPI could exhibit its strong anti-inflammatory ability by regulating the priming pathway of NF-κB and controlling the production of IL-8, IL-6, and TNF-α inflammatory factors.At the same time, it was found that the different types of P. polyphylla monomers (PP I, II, III, V, VI, VII and H) showed significant inhibition of the growth of Propionibacterium acnes (P.acnes), Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus), and their antibacterial activities were more potent than those of antibiotics [23].
Acne is mainly associated with the proliferation of P. acnes, inflammatory response, and abnormal sebaceous gland function [29].Therefore, early treatment of acne is mainly based on antibiotics and chemicals, which are effective in inhibiting the activity of P. acnes, reducing the production of sebum-free fatty acids and extracellular lipase [30], and decreasing the production of inflammatory factors [31].However, in recent years, studies have found that these chemical acne medications may cause mild to severe side effects, such as skin irritation caused by long-term use of benzoyl peroxide or retinoic acid, including facial skin erythema and peeling [32,33].Furthermore, treatment may run into issues with biofilm formation or antibiotic-resistant bacteria [34].Therefore, anti-acne treatment requires the development of safe and clinically effective Nat antibiotics.
Several studies have shown that the secondary metabolites (saponins [8], polysaccharides [35], flavonoids [36], etc.) obtained from P. polyphylla have a wide range of biological activities.P. polyphylla saponins are the main chemical constituents with antiinflammatory [8], antibacterial [23], and immunomodulatory [10] effects.The secondary metabolites of steroid saponins can be divided into four groups (isospirostanols, spirostanols, furostanols, and pseudo-spirostanols) which are structurally diverse and have a wide range of active effects.The literature reports that different structures of Paris saponins have different biological activities.Qin et al. [9] suggested that structural variability can significantly affect the bacterial inhibitory capacity of bacteriocin.At the same time, structural differences have different effects on the production of inflammatory mediators [8], regulation of T-cell differentiation [10], keratin regulation [37] and reduction in oxidative stress [38].As a result, this paper summarizes the role of different structures of P. polyphylla saponins in acne treatment and offers additional research ideas for acne treatment.

Distribution and Characteristics of P. polyphylla
P. polyphylla plants mainly include about 33 species and 15 varieties [39].Countries have differences due to temperature, humidity, and species differences.P. polyphylla grows widely in temperate and subtropical regions of Europe and Asia in the Northern Hemisphere, mainly in the India, China, Bhutan, Laos, Myanmar, Nepal, Japan, Thailand, Vietnam, and other countries.Among them, P. polyphylla in China includes 27 species and 15 varieties [39], of which P. polyphylla var.yunnanensis and P. polyphylla var.Chinensis are the most widely distributed and commonly used in China [14].Figure 1 shows that it is primarily found in China's Yunnan, Guizhou, and Sichuan provinces.The distribution trend declines from the Hengduan Mountains to the eastern and northeastern Sichuan, Yunnan, and Guizhou provinces [40,41].
P. polyphylla is a shade-loving angiosperm with monoecious, unbranched leaves, which are complete with flowers that bloom for up to three months in summer [42] (shown in Figure 2).It grows easily at altitudes of 1300-2500 m or in places with more than 80% canopy closure [43,44], such as bamboo forests, grassy or rocky slopes, streams, coniferous forests, shrublands, and other off-the-beaten-path deep forests or in humus-rich and well-drained soils [44].In recent years, the survival environment of P. polyphylla has been harsh, which makes the germination slow (about 7 months) [45].Therefore, many researchers have begun to cultivate it in captivity.Different growth years affect the content of effective compounds of P. polyphylla.Zhe et al. [46] found that the optimal harvesting time for P. polyphylla is 8 years of growth, considering the influence of P. polyphylla saponin content and cost factors.Therefore, P. polyphylla should be selected and harvested rationally according to the actual needs.

Pathogenesis of Acne
Acne is a chronic inflammatory skin disorder primarily affecting the face, chest, and back.Severe acne dramatically affects the patient's emotional health and quality of life.In recent years, a great deal of research has been conducted on acne treatment, in which plant extracts have been used as a popular direction in clinical treatment.Among them, P. polyphylla has strong anti-inflammatory and antimicrobial properties, and, thus, has potential anti-acne efficacy.In order to better summarize the anti-acne activities and their mechanisms of P. polyphylla, an overview of the four main pathogenic mechanisms of acne is presented, as shown in Figure 3.

Pathogenesis of Acne
Acne is a chronic inflammatory skin disorder primarily affecting the face, chest, and back.Severe acne dramatically affects the patient's emotional health and quality of life.In recent years, a great deal of research has been conducted on acne treatment, in which plant extracts have been used as a popular direction in clinical treatment.Among them, P. polyphylla has strong anti-inflammatory and antimicrobial properties, and, thus, has potential anti-acne efficacy.In order to better summarize the anti-acne activities and their mechanisms of P. polyphylla, an overview of the four main pathogenic mechanisms of acne is presented, as shown in Figure 3.

Pathogenesis of Acne
Acne is a chronic inflammatory skin disorder primarily affecting the face, chest, and back.Severe acne dramatically affects the patient's emotional health and quality of life.In recent years, a great deal of research has been conducted on acne treatment, in which plant extracts have been used as a popular direction in clinical treatment.Among them, P. polyphylla has strong anti-inflammatory and antimicrobial properties, and, thus, has potential anti-acne efficacy.In order to better summarize the anti-acne activities and their mechanisms of P. polyphylla, an overview of the four main pathogenic mechanisms of acne is presented, as shown in Figure 3.  signaling in signaling pathways by binding to TLR2, promoting the differentiation of monocytes into macrophages, and resulting in another large production of inflammatory factors, inflammatory mediators and angiogenic factors.At the same time, by promoting the differentiation of effector T cells into Th1/2/17, it inhibits the production of IL-2 and IFN-γ and promotes the production of the related factors IL-17 and IL-4, thus causing a disruption of the patient's immune system.Androgens induce lipid synthesis by upregulating PI3K/AKt/mTOR, downregulating the Wnt/β-catenin signalling pathway and thus stimulating SREBP-1 production.As acne causes the production of IL-1α, which promotes the expression of K6/K16, it promotes the proliferation and differentiation of keratinforming cells and induces the production of follicular hyperkeratosis.P. polyphylla can inhibit the production of inflammatory factors and angiogenic factors by suppressing related signaling, as well as modulating the immune system by promoting the production of IL-2 and IFN-γ and inhibiting IL-17 and IL-4.Secondly, P. polyphylla can promote Wnt/β-catenin signaling and inhibit PI3K/AKt signaling, a signaling pathway involved in lipid synthesis.Finally, P. polyphylla has an inhibitory effect on K6/K16 and regulates hair follicle hyperkeratosis.The red arrows indicate the promoting effect of P. polyphylla and the black arrows indicate the inhibiting effect of P. polyphylla.

Microbial Customization
The skin microbiota is indispensable in keratinocyte maturation and host immune regulation.S. aureus, S. epidermidis, and P. acnes (also known as Cutibacterium acnes, C. acnes) are microorganisms associated with the pathogenesis of acne [47].Among them, P. acnes is considered to be the primary pathogen causing acne [29].
Current studies have confirmed that P. acnes metabolizes short-chain fatty acids [48] (acetate, propionate, and butyrate), produces enzymes [49] (lipases, proteases, and hydrolases) and other bioactive molecules, and hydrolyzes triglycerides in sebum to free fatty acids, thus showing pro-inflammatory and keratinizing effects [50].Furthermore, P. acnes can activate NADPH oxidase 1 (Nox1) on the surface of human keratinocytes by recognizing the receptor for thrombin-activated protein (CD36), promoting the release of reactive oxygen species (ROS) and the secretion of inflammatory factors such as interleukin (IL)-8 [28].
Several studies have found that P. polyphylla is a promising medicinal plant candidate with significant inhibitory effects on multidrug resistance therapy [51].Also, P. polyphylla saponins have broad-spectrum antimicrobial effects, including acne-causing bacteria [23], such as P. acnes, S. aureus, S. epidermidis, etc.Therefore, P. polyphylla can be used as an effective medicinal plant for treating acne and reducing drug resistance.

Inflammatory Reaction
As a primary pathogen causing acne, the overgrowth of P. acnes affects humans and triggers a severe inflammatory response.The body recognizes microbial components through pattern recognition receptors (PRRS) of the innate immune system to protect the host from infection.The type of pattern recognition receptors associated with acne is the toll-like receptors (TLRs).TLRs are evolutionarily conserved transmembrane innate immune receptors involved in the first line of defense of human health and play important roles in recognizing pathogen-associated molecular patterns (PAMPs) [52].The active TLR2 is expressed in skin cells [53], such as keratinocytes, monocytes, and macrophages, and is activated by binding to Acinetobacter peptidoglycan, promoting the production of inflammatory factors (e.g., IL-8, IL-6, IL-1β, TNF-α) [54], and stimulating the differentiation of monocytes into macrophages.
Furthermore, it can activate the MAPK signaling pathway.By triggering protein phosphorylation of ERK, JNK, and P38 protein kinases [55], the expression of inflammatory proteins (iNOS, COX-2) and caspase-8 and 9 productions are promoted [56,57], thereby activating the production of NO and PGE2 inflammatory mediators.
It was found that prolonged exposure to inflammation increases the levels of inflammatory factors in hair follicles and perifollicular vessels, including human leukocyte antigen-DR (HLADR), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhe-sion molecule-1 (VCAM-1), in the perifollicular vessels [58].Expressing adhesion molecules in the vascular endothelium surrounding the sebaceous ducts causes the accumulation of lymphocytes and neutrophils in the peripheral blood, leading to an immune response and inflammation around the hair follicle [59].Acne can also induce a specific response of CD4 + T cells and promote the differentiation of T cells into Th17 and Th1 cells [60], promoting the production of inflammatory factors (such as IL-17 [61] and IL-4 [62]).Karadag et al. [62] found that serum Th1/Th2 cytokine levels were imbalanced in severe acne, with decreased levels of interferon-gamma (IFN-γ).
Due to their potent anti-inflammatory effects and ability to modulate the immune system, P. polyphylla saponins can be used as a safe and efficient phytoconstituent for treating acne.

Sebum Secretion
Excessive sebum production aggravates acne.Excess sebum leads to follicle blockage, creating an anaerobic environment that provides a suitable condition for the growth of Acinetobacter, which further interferes with the skin barrier and promotes the production of seborrheic acne.Androgens in the sebaceous glands regulate the maturation and secretion of sebaceous glands and thus stimulate the synthesis, proliferation, and differentiation of adipocytes [63].Androgen receptors bind to dihydrotestosterone (DHT) [64] by stimulating the PI3K/Akt/mTOR cascade [65], thereby activating the expression of sterol regulatory element binding protein-1 (SREBP-1) and accelerating lipid synthesis [66,67].
The PI3K/Akt signaling pathway is closely linked to acne sebum metabolism and can reduce lipogenesis (including cholesterol, triglycerides, and free fatty acids) in human sebocytes [68].Insulin-like growth factor 1 (IGF-1) regulates the PI3K/Akt/mTOR signaling pathway by binding to the insulin-like growth factor 1 receptor (IGF-1R) and decreases the transcriptional activity of the recombinant forkhead box protein O1 (FoxO1) in SZ95 sebaceous gland cells thereby inducing lipid synthesis [69].Meanwhile, IGF-1 can also promote the production of MMPs (such as MMP-2 and MMP-9) by activating the NF-κB pathway, which is linked with lipogenesis promotion [70].
The Wnt/β-catenin signaling pathway negatively regulates adipogenesis.Androgens can induce sebocyte differentiation and cause excessive sebum production by inhibiting Wnt/β-catenin signaling [71].Although there is experimental evidence that P. polyphylla saponins can regulate signaling in the PI3K/Akt [72] and Wnt/β-catenin [73] signaling pathways, the mechanism of the anti-acne action still needs further study.

Hyperkeratosis
Dyskeratosis of the basal layer and abnormal activities of keratinocytes in the ducts play parts in acne occurrence and development.Excessive keratinocyte proliferation thickens the epithelial layer of the sebaceous ducts and reduces their diameter, eventually leading to acute occlusion of the sebaceous ducts and thin-walled cystic lesions (acne).
Keratins are intermediate filament proteins that connect the cytoskeleton of keratinocytes and have an essential role in matrix rigidity sensing and downstream signaling [74].Keratins are sometimes expressed in a pairwise manner, with a type I keratin (acidic, keratins 9-20) paired with its specific type II partner (basic, keratins 1-8), for example, K6/K16 and K1/K10 [75].Generally, K1/K10 are expressed in healthy individuals, while K6/K16 are expressed in acne patients [76,77].The binding of androgens and androgen receptors on keratin-forming cells inhibits the expression of the normal K1/K10 in the basal layer [78], thereby promoting the proliferation of keratin-forming cells arranged in the hair follicle portion of the sebaceous glandular unit of the hair follicle.At the same time, keratinocytes can interact with P. acne to enter the dermis, stimulate an inflammatory cascade response that leads to impaired skin barrier function, and release pro-inflammatory factors, such as IL-1 α and TNF-α [77].The secretion of IL-1α stimulates keratin-forming cell activation and promotes acne by inducing the synthesis of keratin 6 in epidermal keratin-forming cells [79].P. polyphylla saponins have been found to regulate the expression levels of keratin and the inflammatory factors IL-1α and TNF-α [37].Thus, they can be a promising treatment for acne problems caused by hyperkeratosis.
Steroid saponins are an important research target for treating acne due to their high pharmacological activities, such as their anti-inflammatory, antibacterial, and immune system-modulating effects.Steroid saponins are glycosides consisting of a hydrophobic glycoside element (C3-linked steroidal saponins or triterpenoids) [95] and one or more hydrophilic sugar groups (glucose, galactose, pentose, or methyl pentose) [96].They are classified into four major groups by the conformation of C25, the cyclization state of the F ring, and the spiroalkane structure [97].Figure 4 describes the four groups, namely isospirostanols, spirostanols, furostanols, and pseudospirostanols [98].In addition, we analyzed and compared their structural characteristics and listed the typical monomers.

Isospirostanol Type
Most of the glycosides are diosgenin and pennogenin, containing a double bond at the 5(6) position and hydroxyl substituents at the 3β, 7β, 23β, 24β, and 27β positions, where the sugars are linked to the hydroxyl substituents to form saponins.The sugar fraction consists of D-glucose, L-rhamnose, L-arabinose, and to a lesser extent, D-xylose and L-fructose.The sugar group forms glycosides with the glycoside C3-OH and, to a lesser extent, with C1-OH, C21-OH, C23-OH, C26-OH, and C27-OH.
Currently, there is limited literature about compounds of this type for treating acne.Therefore, further studies are needed to explore the possibility of acne treatment or the causality of the potential associations.

Furostanol Type
Furostanol-type saponins are a class of F-ring cleavage compounds.The hydroxyl substituents are at the 3, 7, 17, and 26 positions and form steroidal saponins in addition to saponins linked to the sugar group at C3-OH and glucose at C26-OH to form a double sugar chain.Solvents containing -CH3 (e.g., methanol) are avoided during separation as their C22-OH is susceptible to substitution to form OCH 3 products.Plant enzymes readily metabolize their C26-OH sugar chain to close the ring with C22-OH dehydration to form the corresponding spirostanol precursor compound.Thus, the furostanol-type steroid saponins are often considered precursors of the spirostanol-type steroidal saponins [25,102].
Saponins belonging to this type include polyphyllin G (PP G No. 15 in Figure 4) and polyphyllin H (PP H, No. 16) [102].However, due to the instability of the F-ring in the furostanol-type, it is easily cleaved into a double sugar chain saponin, resulting in a lack of biological activity such as hemolysis, antibacterial action and cytotoxicity, and even difficulty in forming complexes with cholesterol [103].As a result, selecting this saponin as the active ingredient in acne treatment is complex.

Pseudospirostanol Type
The pseudospirostanols type is an F-ring open chain structure containing a fivemembered tetrahydrofuran ring.This type of component can be isolated and identified only from the stem and leaves of P. polyphylla var.yunnanensis, with a total of 13 species [104].Liu et al. [98] obtained pseudospirostanol-type compounds, including chonglouside SL-9-chonglouside SL-15 (No. 16-19; No. 21-23 in Figure 4) and abutiloside L (No. 20 in Figure 4), which have strong antibacterial effects and possibilities to be used as active antibacterial substances for the treatment of acne.

P. polyphylla Saponins Play Roles in Acne Treatment
Some key features of acne development include disturbed sebaceous gland activity resulting in excessive sebum, altered sebocyte proliferation and differentiation, dysregulation of the hormonal environment, hyperkeratinization, colonization of P. acnes, and inflammation [105,106].Therefore, the roles related to acne treatment of P. polyphylla saponins may include anti-inflammatory, antibacterial, immunomodulatory, and antioxidant effects.In addition, sebaceous gland function regulation has also been summarized (Figure 3).

Anti-Inflammatory Effects
The anti-inflammatory effects of P. polyphylla saponins have been widely reported.They demonstrate cytokine regulatory effects and a close relationship between anti-inflammation and acne treatment.The main saponins showing anti-inflammatory effects include Rhizoma Paridis total saponins and some monomers, such as PP I, PP II, PP VI, PP VII, and diosgenin.
Man et al. [8] found that P. polyphylla saponins can modulate cytokines or receptors such as VEGFD, VEGFR3, RAGE, IL-6R, IL-17BR, and CXCL16, and increase the levels of SOD and CAT enzymes [38].P. polyphylla saponins can also inhibit the abnormally active NF-κB and reduce PI3K/Akt and MAPK (including p38, ERK1/2, and JNK) phosphorylation [8].The inhibition of NO release in lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages were observed after treating Rhizoma Paridis total saponins, and there was no significant cytotoxicity [38,110].According to the structural diversity, many researchers investigated the saponin monomers' anti-inflammatory abilities in different diseases.PP I and PP VII are the most studied and widely used in current medical practice among the identified saponin monomers.
PP I is one of the saponins, showing anti-inflammatory effects and playing a part in acne treatment.Yang et al. [28] found that PP I could inhibit P. acnes-induced excessive proliferation and abnormal differentiation of HaCaT cells.It downregulated the expressions of CD36, NOX1, NLRP3, and Asc, decreased caspase-1 activity, and decreased the secretion of IL-1β and IL-8.Meanwhile, ROS production in HaCaT cells was also down-regulated.Moreover, according to Zhu et al. [37], p38 phosphorylation is inhibited by PP I in a dosedependent manner, with decreased IL-6, IL-8, TNF-α, and TLR2 expressions.Generally, the acceleration of the TLR2 pathway is correlated with increased iNOS and COX-2 expression and cytokine secretion [56].In addition to tIL-1α mentioned above, IL-1β, IL-6, IL-8, and TNF-α, IL-17 and IL-23 are key pro-inflammatory cytokines.PP I could down-regulate the expression of VEGF and IL-23 in HaCaT cells in a dose-dependent manner, thereby inhibiting the secretion of IL-17 [109].Another study also found that PP I could downregulate the expression of K16 in heat-inactivated Acinetobacter-treated HaCaT cells [37].Meanwhile, Zhu et al. [37] found that PP I contributes to the regulation of cytokine gene expression by inhibiting the PI3K/Akt/mTOR signaling pathway and NF-κB-mediated production of pro-inflammatory factors in activated macrophages, thus providing a new therapeutic target for PP I to control inflammatory acne.
Zhang et al. [112] verified the anti-inflammatory effects of PP II in vivo, using mouse and zebrafish models separately.It was found that PP II could effectively inhibit the abnormally active NF-κB pathway and reduce the production of MMP-9, thereby reducing VEGF production and decreasing the inflammatory response through anti-angiogenesis.
PP VII is an isospirostanol saponin with anti-inflammatory properties that inhibits angiogenesis, lymph angiogenesis, adhesion, inflammation, and invasiveness.In a study, PP VII inhibited NO and PGE 2 production stimulated by LPS in the model of zebrafish embryos.In the PP VII group, the biomarkers iNOS, COX-2, and MMP-9 were inhibited in LPS-induced RAW264.7 cells.NF-κB and MAPKs played parts in the research [26].Meanwhile, another study [27] proved the decreased tendencies of the protein expressions of PI3K, (p)-PI3K, Akt, p-Akt, NF-κB, and p-NF-κB after PP VII treatment.Thus, it might be concluded that PP VII can reduce inflammatory damage by affecting NF-κB, MAPKs, and PI3K/Akt pathways, which could be the targets for the subsequent treatment of inflammatory acne with PP VII.
Diosgenin plays a vital role in anti-inflammatory effects.Pro-inflammatory factors were detected in low levels after treatment with diosgenin.It was demonstrated that diosgenin inhibited IL-1β-induced NO and PGE 2 production and significantly decreased the expression of MMPs (MMP-3 and MMP-13), iNOS and COX-2 in IL-1β-stimulated human OA chondrocytes [108].Furthermore, some factors related to adhesion, such as VCAM-1 and ICAM-1, were inhibited by diosgenin [113].Choi et al. [113] found that such factors can inhibit TNF-α-induced THP-1 monocyte adhesion and decrease the expression of VCAM-1 and ICAM-1 in vivo.It was reported that the phosphorylation of p38, ERK, JNK, and Akt played parts in the diosgenin process [113].It can also decrease the production of ROS [114].
Another type of saponin named PP H was reported to have similar effects with to dexamethasone in improving bacterial-infected inflammatory skin equivalents [115].In the study, a biomimetic "interface-controlled-skin-on-chip" system was constructed.P. acnes and sodium lauryl sulfate stimulation were performed to damage the barrier function.After the treatment with PP H, a significant repair effect on the skin barrier and inhibition on the re-lease of inflammation-related cytokines were observed.Fuethermore, the effects were more prominent than those with dexamethasone.According to the study by Yang et al. [116], the in-depth mechanism may be that PP H inhibited the nuclear translocation of NF-κB P65.PP H could bind to Keap1 and activate Nrf2, thus upregulated HO-1 in LPS-induced RAW 264.7 cells, based on a molecular docking study.
Among the identified saponin monomers (PP I, PP II, PP VI, PP H, and PP VII), PP I has the strongest antibacterial effect, while PP H has a relatively weak antibacterial effect [120].
Scholars compared the antimicrobial abilities between the P. polyphylla saponins and antibiotics and found different phenomena.For example, Sun et al. [23] found that P. polyphylla saponins were slightly lower in ability than antibiotics, while in the study of Wang et al. [119], the conclusion was drawn that the MIC values for the antimicrobial activities of P. polyphylla saponins were higher than those expressed by antibiotics.
Undoubtedly, the antibacterial strength against different pathogens varies because of the different structures of P. polyphylla saponins.It was reported that the glycosyl groups and lengths of sugar chain linkages in the structure of saponins affect the antibacterial effect [9].According to Qin et al. [9], Chonglouoside SL-6, containing a trisaccharide sugar moiety at C1, exhibited the greatest most antimicrobial effect among the different saponin structures.In contrast, PP V and dioscin containing hydroxyl derivatives at C7 or C25 showed diminished activity against P. acnes.
As a result, the relationships between the chemical structure and the antibacterial capacity of P. polyphylla saponins require further investigation, potentially providing new ideas for developing acne-related drugs in the future.

Immunomodulatory Effects
The activation of immune signaling pathways can be a double-edged sword, as this is necessary to clear pathogens but harmful when too sustained or uncontrolled.For example, a recent report highlights that the persistence of inflammation in acne lesions is linked to prolonged lesions, scar formation, and loss of the sebaceous gland [121].This suggests that, apart from with anti-inflammatory treatments, moderate immunomodulation is necessary for acne.
In the study, many CD4 + T cells in acne patients differentiated into Th1 and Th17 cells, which increased IL-17 and IFN-γ [10].P. polyphylla saponins could inhibit CD4 + T cell proliferation and hinder Th1/Th17 cell differentiation [109,114,122,123], decreasing IL-4 and increasing IFN-γ production [109], thus improving the immunosuppressive function of CD4+T cells.The increase in serum interferon (IFN-γ) and IL-2 levels and the decrease in IL-4 levels were also observed, resulting in a shift in the Th1/Th2 balance towards Th1 [123].
VCAM-1 and ICAM-1 are glycoproteins on the cell surface and are members of the immunoglobulin superfamily.Stimulating vascular endothelial cells by inflammatory factors promotes the interaction of VCAM-1 and ICAM-1 with ligands, resulting in the aggregation of inflammatory macrophages, immunomodulation, platelet aggregation, and adhesion [124][125][126].The anti-angiogenesis of P. polyphylla saponins and the inhibited expression of VEGF-1 and ICAM-1 finally lead to immunomodulation.Chai et al. [127] found that P. polyphylla saponins could effectively reduce the expression of VEGF-A, VCAM-1, IL6R, IL17BR, and CXCL16 in a mouse model of lung adenocarcinoma.Furthermore, the expression levels of ICAM-1 and VCAM-1 mRNA were inhibited in response to oxidative stress [128].The reduced expression of HIF-1α and VEGF was also observed after the intervention of PP I in hypoxic laryngeal carcinoma Hep-2 cells [109].However, current studies on the mechanisms of P. polyphylla saponins in immunomodulation are mainly focused on cancer.In the future, we hope to research the relationship between P. polyphylla saponins and immunomodulatory-related mechanisms in acne pathogenesis.

Sebaceous Gland Function-Regulating Effects
Sebum is a waxy, lipid-rich biofluid excreted by the skin's sebaceous glands, and its overproduction is a known reason for acne.Increased sebum excretion is a major factor in the pathophysiology of acne.Other sebaceous gland functions include the regulation of cutaneous steroidogenesis, local androgen synthesis, and hormonal control [129].Abnormal sebaceous gland function tends to cause disturbances in lipid metabolism and increased lipid synthesis.
Although none of the literature shows direct relationships between P. polyphylla saponins and lipid synthesis, Rhizoma Paridis total saponins were found to inhibit the PI3K/Akt and MAPK (p38, Erk1/2, JNK) signaling pathway [130,131].Liu et al. [132] demonstrated that inhibition of the human sebocyte PI3K/Akt signaling pathway is associated with decreased lipogenesis (including cholesterol, triglycerides, and free fatty acids).Furthermore, the androgens can promote sebaceous gland hyperplasia by inhibiting the Wnt signaling pathway, while PP VII was reported to promote this pathway [133], which is further indicated to have the potential to reduce lipid synthesis.Therefore, further research is needed to explore the role of P. polyphylla saponins in the pathogenesis of seborrheic acne.

Follicular Hyperkeratosis Effects
Hyperkeratosis and hyperproliferation of funnel keratinocytes in acne are accompanied by overexpression of K6 and K16 [37].It was found that stimulating androgenmediated FGFR2 signaling [134] and increasing IL-1α expression [58] could increase the occurrence of acne.IL-1α subsequently activates basal keratin-forming cells by induction of K16 expression by autocrine products.Zhu et al. [37] found that K6 and K16 are detected in large amounts in the lesions of acne patients compared to the skin surface of healthy individuals.
It was reported that PP I could modulate keratin expression, inhibit IL-1α release, and suppress K16 expression in HaCaT keratinocytes treated by heat-inactivated P. acne [37].Thus, PP I may be used as a health therapy to improve seborrheic acne.

Antioxidant Effects
Excessive ROS can lead to oxidative damage to cellular lipoproteins, proteins, and DNA, thus resulting in inflammatory responses that promote acne, photooxidation, and other skin problems [135].In addition, the unsaturated fatty acids cause lipid peroxidation in lipids undergoing auto-oxidation to produce unstable hydroperoxides.The resulting hydroperoxides continue to decompose to form small molecules of compounds such as acids, aldehydes, and ketones with short carbon chains [136,137].Ayres et al. [138] found that acne patients develop lipid peroxidation in their bodies, leading to the progression of acne conditions.
Due to its complex structure, the chemical structure of P. polyphylla saponins contains phenolic hydroxyl groups, which can terminate free radical chain (•OH and O −2 ) reactions by binding to free radicals to form stable semi-keto radical structures [139].Secondly, the phenolic hydroxyl structure prevents the production of hydroxyl radicals by chelating Fe 2+ , Cu 2+ , and other trace elements in the system [140,141], thus reducing the content of reactive oxygen species.Finally, P. polyphylla saponins can reduce free radical activity by forming hydrogen bonds as a type of hydrogen donor.Comparison of the antioxidant capacity of PP VII and ascorbic acid (VC) at the same concentration has been reported to show that VC has a stronger scavenging capacity for •OH and O −2 than PP VII, but the scavenging capacity of PP VII for DPPH was stronger than that of VC at concentrations higher than 4 mg/mL [142].
Furthermore, P. polyphylla extracts can act as antioxidants with protective effects against intracellular oxidative stress.Gao et al. [143] found that PP I reduced oxidative stress by activating the SIRT3/SOD2/ROS signaling pathway.Das et al. [38] further evaluated the antioxidant activities in vitro and found that diosgenin increased the intracellular nitric oxide dismutase (NOD) and SOD.Meanwhile, P. polyphylla saponins were also proved to have the ability to resist lipid oxidation and protect DNA, thus indirectly exerting its protective effect [140] in the model of dextran-induced rat paw edema.
The clinical studies of P. polyphylla extracts on anti-acne effect were in a minority.Xu et al. [144] reported the clinical observation of a skin care product containing the extract of many leaf Paris rhizome and purslane.The acne effect was observed in the treatment of acne vulgaris.In the study of Fang Ting, a hydrogel containing P. polyphylla ex-tract for the treatment of acne was produced.The preliminary quality research and safety evaluation of that hydrogel were performed and obtained good results, but no clinical trial was designed [145].
In the study, a summary of the standard extraction techniques has been made to provide helpful information for the relevant processing industries.Figure 5 clearly shows the similarities and differences between the different extraction processes.We also described some extraction conditions of P. polyphylla saponins in Table 2. Furthermore, Table 2 outlines the extraction techniques according to the objectives and further indicates the advantages and disadvantages of the different extraction treatments to provide a basis for the later selection of extraction methods.The detailed description of these extraction methods is also shown in the Supplementary Materials attached to the manuscript.
In the study, a summary of the standard extraction techniques has been made to vide helpful information for the relevant processing industries.Figure 5 clearly show similarities and differences between the different extraction processes.We also desc some extraction conditions of P. polyphylla saponins in Table 2. Furthermore, Table 2     The ethanol concentration was 90%, the extraction pressure was 400 Mpa, the extraction time was 2 min, and the liquid-solid ratio was 40:1.

\ HPLC method
Using a 40 mL 90% ethanol aqueous solution (v/v) treatment, the system was exposed to a microwave radiation source operating at 300 W and 2450 MHz frequency and subjected to atmospheric pretreatment for 15 min of extraction.
The rates of saponin A, saponin B, saponin C, and saponin D were 1.139%, 0.539%, 0.041%, and 0.049%, respectively.The extraction was carried out by a combination of dynamic and static methods with the optimal ratio of ethanol 90%-ethyl acetate 10%, an extraction temperature of 52

Figure 1 .
Figure 1.Distribution of Paris genus.Map showing China as the center of Paris species diversity.The green line represents Hengduan Mountain, and the green arrow is a pointing illustration of the Hengduan Mountain.

Figure 1 .
Figure 1.Distribution of Paris genus.Map showing China as the center of Paris species diversity.The green line represents Hengduan Mountain, and the green arrow is a pointing illustration of the Hengduan Mountain.

Figure 3 .
Figure 3. Diagram of the pathogenesis of acne and the mechanism of action of P. polyphylla.Inflammatory response, immune regulation, sebum dysfunction, microbial colonization and follicular hyperkeratosis are the driving factors in the development of acne.Peptidoglycan from P. acnes stimulates signaling in signaling pathways by binding to TLR2, promoting the differentiation of

Figure 3 .
Figure 3. Diagram of the pathogenesis of acne and the mechanism of action of P. polyphylla.Inflammatory response, immune regulation, sebum dysfunction, microbial colonization and follicular hyperkeratosis are the driving factors in the development of acne.Peptidoglycan from P. acnes stimulates signaling in signaling pathways by binding to TLR2, promoting the differentiation of

Figure 3 .
Figure 3. Diagram of the pathogenesis of acne and the mechanism of action of P. polyphylla.Inflammatory response, immune regulation, sebum dysfunction, microbial colonization and follicular hyperkeratosis are the driving factors in the development of acne.Peptidoglycan from P. acnes stimulates
lines the extraction techniques according to the objectives and further indicates th vantages and disadvantages of the different extraction treatments to provide a bas the later selection of extraction methods.The detailed description of these extra methods is also shown in the Supplementary Materials attached to the manuscript.

Figure 5 .
Figure 5. Extraction processes of P. polyphylla saponins.The extraction process of P. polyphylla onins consists of reflux extraction (RE), ultrasonic-assisted solvent extraction (USA-SE), ultrapress-sure-assisted solvent extraction (UHPA-SE), microwave-assisted solvent extraction (MW SE), and supercritical fluid CO2 extraction (SFE-CO2).The diagram shows the different extract processes using different extraction equipment, by adjusting the reaction conditions for the ex tion of P. polyphylla saponins.

Figure 5 .
Figure 5. Extraction processes of P. polyphylla saponins.The extraction process of P. polyphylla saponins consists of reflux extraction (RE), ultrasonic-assisted solvent extraction (USA-SE), ultra-high-presssure-assisted solvent extraction (UHPA-SE), microwave-assisted solvent extraction (MWA-SE), and supercritical fluid CO 2 extraction (SFE-CO 2 ).The diagram shows the different extraction processes using different extraction equipment, by adjusting the reaction conditions for the extraction of P. polyphylla saponins.

Table 1 .
Biological activities and their mechanisms of Paris saponins.

Table 2 .
Paris extraction processes reported in literature.
•C, an extraction pressure of 315 bar, a SC-CO 2 flow rate of 39 g/min, an extractant dosage of 300 mL and an extraction time of 4 h.