Fatty acids are associated with strong anti-inflammatory and antioxidant effects (Lin et al., 2017). Sesame oil obtained from Sesamum indicum L. (Pedaliaceae) seeds contains 45% of linoleic acid, 40% of oleic acid, 10% of palmitic acid, and 5% of stearic acid (Moore et al., 2020).

Linoleic acid 18:2 (ω-6) is a common polyunsaturated fatty acid present in plant oils. It is considered as an essential omega-6 fatty acid, a precursor of arachidonic acid 20:4 (n–6), prostaglandins, and leukotrienes well implicated in the inflammation process (Moore et al., 2020).

Oleic acid is a monounsaturated fatty acid called omega-9. It can be used to enhance the topical delivery of drugs and improve the trans-epithelial distribution of compounds present in the mixtures. Oleic acid could, however, cause some form of toxicity to the skin cells. This has been studied on keratinocytes even at low concentrations. In contrast, when oleic acid was applied to reconstructed human epidermis or injured skins, no alteration in morphology was observed. This is related to the modulation of the thickness of the stratum corneum, the outermost layer of the epidermis, in the control of oleic acid-induced toxicity. Low amounts of oleic acid are sufficient to cause modulation of local cytokines production without affecting skin morphology while inducing skin irritation in vivo (Boelsma et al., 1996). In addition, palmitic and stearic acids also have effects on the wound healing process since they can modulate cell migration, proliferation, and the production of inflammatory mediators (Silva et al., 2018).

To conclude about fatty acids content, oils with high content of linoleic acid 18:2 (n−6) (such as sunflower oil, Helianthus sp., and sesame oil) are more favorable for treating skin disorders than those with high content of oleic acid 18:1 (n−9) (such as olive oil, Olea europaea) (Moore et al., 2020). Due to its important presence in Kampo ointments, sesame oil plays a decisive role. It influences the extraction of herbs and has a therapeutic emollient role.

Several physicochemical steps will affect the molecular structure of sesame oil due to exposure to high temperatures (at 200°C for 10 min for Chuōkō to 1 h for Shiunkō at 220°C–230°C). One of the main transformations is the polymerization step mentioned in the Japanese reference books for Kampo formulas (Japan Pharmaceutical Manufacturers Association, 2005).

The polymerization process results in the formation of non-volatile polar compounds, triacylglycerol dimers, and polymers. Also, hydrolysis and oxidation influence the properties of the oil (Choe and Min, 2007). Dimerization and polymerization in heated oils are radical reactions that produce dimers and polymers, respectively (Khor et al., 2019). The smoke point reflects the breakdown of fats into glycerol and free fatty acids (Ramroudi et al., 2022). It depends on the free fatty acids content and the refining process used. The smoke point of sesame oil is between 184°C and 242°C, depending on the study (Ramroudi et al., 2022). The more unsaturated the vegetable oil is, the more carbon–carbon bonds it contains, and the quicker the oil will polymerize.

Thus, the polymerization of oleic acid leads to the production of acyclic polymers which will probably influence the extraction of metabolites from herbs. Linoleic acid is more easily polymerized at high temperatures than oleic acid (Choe and Min, 2007). As a result, sesame oil is subject to these changes. The interesting polymerization process, by changing the polarity of the oil, will probably influence the extraction of the crude drugs.

Beeswax has been used since ancient times for its antimicrobial properties in European and Asian traditional medicines (Cornara et al., 2017). It includes a mixture of more than 300 components such as hydrocarbons (chain length of 27–33 carbons), free fatty acids, fatty acid alcohols and esters, and exogenous substances such as propolis or pollen.

Several studies carried out on the hive products proved an antibacterial effect with an important intensity for propolis and a lighter intensity for beeswax although significant (Fratini et al., 2016). Beeswax showed in vitro antimicrobic effectiveness against some infectious germs tested with zone of the inhibition assay using the agar diffusion method. Among the germs tested favorably in the higher zones of inhibition, we find Candida albicans, Staphylococcus aureus, and Bacillus subtilis (Ghanem and Arabia, 2011). Also, the extracts of beeswax in methanol or ethanol have been evaluated using the zone of inhibition of the development of microorganisms. The results showed a significant inhibition zone for the beeswax methanolic extract on S. enterica and C. tropicalis. For 70% methanol extract, the most sensitive germs were E. coli, C. albican, C. tropicalis, and Aspergillus niger (Kacániová et al., 2012). Research works hold in vitro also demonstrated that the inhibitory effect is enhanced synergistically with other natural products such as propolis or olive oil (Fratini et al., 2016). Therefore, it can be assumed that it would be the same with sesame oil and herbs used for Kampo ointments. Concerning the mechanism of action and specific properties of beeswax, its antibacterial mode of action is still however poorly studied (Fratini et al., 2016). Moreover, beeswax is a difficult to standardize product and very sensitive to biotic and abiotic factors during its production by bees.

Topical application of preparations containing beeswax forms a protective barrier against several external factors by forming a film on the skin surface. Main compounds such as squalene (C₃₀H₅₀; 410.3912), 10-hydroxy-trans-2-decenoic acid (named queen bee acid; C₁₀H₁₈O₃; 186.1256), and flavones such as chrysin (C₁₅H₁₀O₄; 254.0579) and apigenin (C₁₅H₁₀O₅; 270.0528) are influential for wound care, atopic dermatitis, or psoriasis. Also, some compounds such as sterols reduce the water loss on skin wounds and are beneficial for treating atopic dermatitis. The presence of squalene, omega-hydroxy amino acids such as queen bee acid, and specific flavones such as chrysin are responsible for the antiseptic activity. Additionally, it contains α-carotene (C₄₀H₅₆; 536.8726), source of retinol (C₂₀H₃₀O; 286.2297), which delays collagen degradation and stimulates mitotic division in the epidermis and as a result wound healing (Kurek-Górecka et al., 2020).

The Shiunkō formula contains a small quantity of lard (1.3% mass). It is therefore only used in high-temperature extraction protocols. However, this ingredient should be taken into account, especially during the extraction process, while changing the fatty acids composition a little. Lard does not contain trans-unsaturated fatty acids but triglycerides with a high rate of saturated fatty acids such as oleic (C₁₈H₃₄O₂; 282.2559), palmitic (C₁₆H₃₂O₂; 256.2402), stearic (C₁₈H₃₆O₂; 284.2715), and linoleic acids (C₁₈H₃₂O₂; 280.2402).

Only regarding the therapeutic properties of lard, some studies have investigated the impact of skin applications of animal fat blends on atopic dermatitis lesions. Results showed that the lard mixture significantly ameliorated the macroscopic and microscopic signs and reduced skin thickness and mastocyte incorporation in mice skin lesions. The mixture also had an anti-allergic effect by reducing the involvement of interleukins and E-type immunoglobulins (Lee et al., 2020).

In vivo, use of a lard-based ointment on inflamed rat feet reduced paws inflammation. In addition, antioxidant enzymes and inflammatory markers of circulating neutrophils were reduced. A beneficial action of lard is induced by several components such as 5-dodecanolide (C₁₂H₂₂O₂; 198.1620), oleamide (C₁₈H₃₅NO; 281.2719), and resolvin D1 (C₂₂H₃₂O₅; 376.2250). This anti-inflammatory effect occurs via neutrophils and peripheral mononuclear cells stimulated by lipopolysaccharides, decreasing the production of TNFα and the activity of pro-inflammatory enzymes (Capó et al., 2021). These favorable actions suggest that the addition of lard to the Shiunkō formula is potentially related to therapeutic activity of interest for skin wound healing and chronic wound treatments. Nevertheless, the low concentration of lard raises questions about its real usefulness as an excipient in the Shiunkō formula, especially as it is not present in the other two topical formulas.

According to the Japanese Pharmacopeia, turmeric, ukon in Japanese (ウコン; 鬱金), is the rhizome of Curcuma longa L. (Zingiberaceae) (The society of Japanese pharmacopeia, 2016; WFO, 2022). It can be prepared with or without cork layers, usually with a blanching step (The society of Japanese pharmacopeia, 2016). Originally from India, the plant arrived in Japan a few centuries ago and started to be cultivated in southern prefectures such as Kyushu or Okinawa. Turmeric is the dried rhizome harvested after the flowering period in summer. The rhizome contains not less than 1.0% and not more than 5.0% of total curcuminoids (The society of Japanese pharmacopeia, 2016). This group includes curcumin (C₂₁H₂₀O₆; 368.1260), demethoxycurcumin (C₂₀H₁₈O₅; 338.1154), and bisdemethoxycurcumin (C₁₉H₁₆O₄; 308.1049). Rhizome also contains sesquiterpenes such as caryophyllene (C₁₅H₂₄; 204.1878), monoterpenes such as eucalyptol (C₁₀H₁₈O; 154.1358), steroids and lignans (Keio University Kampo Department).

The therapeutic properties of turmeric are well known for topical wound treatments. Curcumin is an asset for remedies containing it, as this molecule acts on the different phases of the wound healing process, from the inflammatory phase to re-epithelialization (Vollono et al., 2019). Studies involving murine models to qualify the wound healing properties were conducted on induced wounds, severe burns, or diabetic ulcers. For burns, a strong influence on the hydroxyproline levels in the skin of the mice treated with turmeric has been proven. Improved collagen deposition, angiogenesis, granulation tissue formation, and epithelialization were demonstrated in vitro (Kulac et al., 2013). The same results have been observed on the murine model for induced wounds with a better maturation and cross linking of collagen, increased stability of acid-soluble collagen, aldehyde content, shrinkage temperature, and tensile strength (Panchatcharam et al., 2006). Topical applications of curcumin-enriched remedies also accelerate wound healing in mice by regulating the levels of various cytokines (Yen et al., 2018). Regarding diabetic ulcers, curcumin application increased wound healing and decreased the expression of inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and matrix metallopeptidase 9 (MMP-9). Curcumin also increased the levels of anti-inflammatory modulators such as IL-10, superoxide dismutase, catalase, and glutathione peroxidase. The curcumin application provided granulation tissue improvement with fibroblast proliferation, collagen deposition, and effective epithelialization (Kant et al., 2014). These interesting properties have been the subject of other studies, notably on the effectiveness of gel forms for the topical application of curcumin-based medicines (Mohanty and Sahoo, 2017).

In a nutshell, curcumin acts on the key phases of the healing process such as inflammation (inhibiting the activity of NF- κB, TNF-α, and IL-1 cytokines, action on ROS, and a dose-dependent antioxidant effect), proliferation (increased fibroblast migration, granulation, collagen deposition, and re-epithelialization), and remodeling (increased production of transforming growth factor-β (TGF) and fibroblast proliferation) (Akbik et al., 2014). All these interesting properties for wound healing need to be considered in Kampo ointments as curcumin is a lipophilic polyphenol substance.

Turmeric also includes terpene-class compounds such as sesquiterpene ketones: ar-turmerone (C₁₅H₂₀O; 216.1514), α-turmerone (C₁₅H₂₂O; 218.1671), and (+)-β-turmerone (C₁₅H₂₂O; 218.1671). Monoterpenes such as α-phellandrene are also found (Avanço et al., 2017). These ketones, as secondary metabolites, are of great interest because of their bacteriostatic and antibacterial properties. Such properties are obviously recognized in the management of chronic wounds where recurrent infections and bacterial resistance are issues. Moreover, a study carried out on turmeric essential oil in vitro showed antioxidant, antifungal, and antimycotoxigenic actions on Fusarium verticillioides cultures (Avanço et al., 2017). Finally, curcumin has a rather complex biological action on dermatological cell lines with ambivalence regarding apoptosis and wound healing since cell death can be induced while favorable biological effects on wound healing are clearly demonstrated (Mehta et al., 2016). Additionally, studies on the biological effects of Kampo extracts on wound healing should include not only the evaluation of cell proliferation and migration but also the antibacterial component, which is strongly involved in chronic wound management.

The Amur cork tree, Phellodendron sp., is named kihada in Japanese (キハダ; 黄蘗). Its bark, named ōbaku (オウバク; 黃柏), is largely used and studied in Asia and is also one of the 50 fundamental herbs known as huáng bǎi (黄柏) in Chinese (Ergil et al., 2002). According to the Japanese Pharmacopeia, the bark comes from Phellodendron amurense Rupr. or P. chinense C.K.Schneid. (Rutaceae), with the periderm removed, revealing a characteristic yellow color (The society of Japanese pharmacopeia, 2016; WFO, 2022). The bark has been traditionally used for its digestive properties for oral intakes and as an anti-inflammatory and antibacterial remedy for topical uses (Li et al., 2006). Amur cork trees are at least 12 years old when the bark is collected during the summer. The inner bark contains at least 1.2% of berberine (C₂₀H₁₈NO₄ ⁺; 336.1236) (The society of Japanese pharmacopeia, 2016). It is a hydrophobic alkaloid isoquinoline of the protoberberine class, a quaternary ammonium salt, and a hydrophilic yellow pigment (Leitao da-Cunha et al., 2005; Patel, 2021).

While berberine is the most distinctive compound, jatrorrhizine (C₂₀H₂₀NO₄ ⁺; 338.1392), palmatine (C₂₁H₂₂NO₄ ⁺; 352.1549), and coptisine (C₁₉H₁₄NO₄ ⁺; 320.0923) are some of the other typical secondary metabolites (Kamimura et al., 2019). The bark contains bitter taste elements such as obacunone (C₂₆H₃₀O₇; 454.1991), limonin (also known as obaculactone C₂₆H₃₀O₈; 470.1941), and phytosteroids such as β-sitosterol (C₂₉H₅₀O; 414.3862) or campesterol (C₂₈H₄₈O; 400.3705) (Keio University Kampo Department). In a recent study, the therapeutic action of berberine was evaluated using both in vitro and in vivo models, first, in a cell model with hyperglycemic treatment and, second, in diabetic rats’ wounds infected via streptozotocin induction. These experiments clarified the action of berberine on thioredoxin reductases (TrxR1), a key enzyme in redox homeostasis. This was carried out by suppressing c-Jun N-terminal kinase (JNK) signaling, thereby inhibiting oxidative stress and apoptosis. By taking this action, better cell proliferation was observed. Accelerated wound healing was also correlated with a decrease in the action of MMP-9 and regulation of TGF-β1 and tissue inhibitors of MMP-1 (TIMP1). In vitro, treatments with berberine caused an acceleration of wound healing and an improved synthesis of the extracellular matrix. In vitro, there was also an inhibition of cell damage induced by hyperglycemic treatments. In vivo, for diabetic rats, topical treatment enriched with berberine led to an acceleration of wound healing by stimulation of the TrxR1 enzyme (Zhou et al., 2021). For palmatine, therapeutic actions with the modulation of inflammation through a reduction in the actions of IL-6 and TNF-α have been demonstrated and antibacterial actions on Gram-positive and Gram-negative bacteria. These actions suggest that a beneficial effect could be demonstrated on wound healing cell models (Long et al., 2019).

Regarding metabolites levels of interest in the bark of the Amur cork tree, berberine and palmatine contents differ significantly depending on the botanical origin (P. chinense or P. amurense), with a lot of berberine and no palmatine for P. chinense, lower concentrations of berberine and the presence of palmatine for P. amurense (Li et al., 2006). Again, these variabilities suggest that one of the levers for rationalizing Kampo’s use of ointments is a better understanding of the influencing factors on herbal crude drugs’ chemical composition. Here, the botanical species used will be decisive for the composition of therapeutic metabolites in the Kampo ointments.

Dahurian angelica root comes from Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav. (Apiaceae) (The society of Japanese pharmacopeia, 2016; WFO, 2022). Dahurica refers to the mountainous region beyond Lake Baikal (Transbaikalia), to the east of it, in Russia. The crude drug is named byakushi (ビャクシ; 白芷) in Japanese. Its flavor is characteristic and slightly bitter (The society of Japanese pharmacopeia, 2016). The root was used by Kampo medical doctor Hanaoka Seishū (who created the two remedies Shiunkō and Chuōkō) for the preparation of anesthetic medication (Tsūsensan or Mafutsu-tō) used for surgery (Izuo, 2004). Hanaoka Seishū was the first doctor to perform a breast tumor resection under this kind of anesthesia. The use of Dahurian angelica root in this preparation suggests analgesic properties from the crude drug, which is of great interest from an ethnopharmacological perspective.

The root contains more than 300 compounds including 150 coumarins. Among them, there are furanocoumarins such as imperatorin (C₁₆H₁₄O₄; 270.0892), byakangelicin (C₁₇H₁₈O₇; 334.1052), and oxypeucedanin (C₁₆H₁₄O₅; 286.0841). These furanocoumarins are lipophilic compounds and would thus be highly represented in the Kampo oily extracts (Deng et al., 2015). A. dahurica root also contains volatile oils (terpenoids, aromatic compounds, and aliphatic compounds) with, for example, α-pinene (C₁₀H₁₆; 136.1252), myrcene (C₁₀H₁₆; 136.1252), (+)-terpinen-4-ol (C₁₀H₁₈O; 154.1358), and 1-dodecanol (C₁₂H₂₆O; 186.1984). In the same way as in the roots of A. acutiloba, there is lipophilic ferulic acid (C₁₀H₁₀O₄; 194.0579), which is an important phenolic compound. Typical psoralens such as byakangelicol (C₁₇H₁₆O₆; 316.0947) compose the root. This kind of metabolites is photosensitizing, and its presence as an ingredient should be monitored (Keio University Kampo Department; Zhao et al., 2022).

Studies carried out on A. dahurica root extract for topical uses (also combined to the Chinese rhubarb root extract, another ingredient from Shinsen taitsukō) demonstrated significantly wound closure with more inflammatory cell infiltration, collagen fibers, and myofibroblasts a few days after the treatment on rats. It manifested an accelerated wound healing process during the inflammation and proliferation phases (Yang et al., 2017). Other studies made in vitro demonstrated an accelerated proliferation of fibroblasts and an upregulated expression of collagen I and III, inducing a better wound healing status (Zhi et al., 2012). Angelica dahurica root ethanolic extracts led to an increased proliferation of keratinocytes. Real-time fluorescent quantitative PCR revealed that the cyclin D1 and caspase-3 mRNA levels were downregulated, indicating that apoptosis was inhibited (Bai et al., 2012).

Associated treatment with Chinese rhubarb and A. dahurica roots has been studied in more specific chronic wound models with diabetic rats. The mix of herbal extracts improved wound healing in diabetic conditions while increasing vascular endothelial growth factor (VEGF) activities (Chao et al., 2021).

By the way, oral intake of the ethanolic extract from A. dahurica roots accelerated diabetic wound healing as well through induced angiogenesis and granulation tissue formation in streptozotocin-induced diabetic rats (Zhang et al., 2017). Angiogenesis is a pivotal process in wound repair, and this therapeutic property of A. dahurica root makes its use relevant for wound healing. The oral intake of A. dahurica also regulates the polarization of M1 and M2 subtypes of macrophages and thus inflammation (Hu et al., 2021). On the diabetic mice model, oral intake of the A. dahurica root extract activated the phosphoinositide 3-kinase/protein kinase B signaling pathway (PI3K/AKT). In vitro, the promotion of the hypoxia-inducible factor-1α/platelet derived growth factor ß pathway (HIF-1α/PDGF-β) efficiently enhanced vascularization in regenerated tissue. This increased neovascularization and PDGF-β expression thus facilitated wound healing (Guo et al., 2020).

The benefits brought by A. dahurica root oral intake suggest that the topical use may be beneficial, especially its effects on vessel injury-related wounds through the modulation of PDGF-β and VEGF.

Cinnamon bark comes from the trunk of Cinnamomum cassia Blume (Lauraceae) with or without the periderm removed (The society of Japanese pharmacopeia, 2016). This species is a synonym of Cinnamomum cassia (L.) J. Presl (WFO, 2022). The tree is endemic from Southwest China and North Vietnam, and the bark started to be imported into Japan in the 8th century. Named keihi (ケイヒ; 桂皮) in Japanese, it presents a characteristic aromatic, sweet, and pungent taste which is then mucilaginous and slightly astringent (Keio University Kampo Department). Traditionally, the bark is used for its stomachic, hypoglycemic, hypotensive, sedative, and antipyretic activities (Keio University Kampo Department). The bark contains essential oil with aldehydes and esters such as cinnamaldehyde (C₉H₈O; 132.0575), 2-methoxy cinnamaldehyde (C₁₀H₁₀O₂; 162.0681), and cinnamyl acetate (C₁₁H₁₂O₂; 176.0837) as main compounds (Keio University Kampo Department). There are also diterpenoids such as cinncassiol A (C₂₀H₃₀O₇; 382.1991), cinncassiol E (C₂₀H₃₀O₈; 398.1941), and cinnzeylanol (C₂₀H₃₂O₇; 384.2148). The bark contains several sesquiterpenoids such as cinnamosides and sugars. Tannins are found in the bark such as (-)-epicatechin (C₁₅H₁₄O₆; 290.0790) or proanthocyanidins such as procyanidin B2 and procyanidin B4 (C₃₀H₂₆O₁₂; 578.1424) (Keio University Kampo Department; Daemi et al., 2019).

The most well-known compound from cinnamon is probably cinnamaldehyde, which has been largely studied in vitro and in vivo for its wound healing properties. Studies carried out on human umbilical vein endothelial cells (HUVECs) showed that cinnamaldehyde stimulated cell migration and proliferation. It induced a wound healing effect by promoting angiogenesis through activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Cinnamaldehyde induced in vivo, an angiogenic effect on the zebrafish model pre-treated with PTK787 which is a selective inhibitor for VEGFR (Yuan et al., 2018). Cinnamaldehyde as well presented a favorable activity with regards to wound healing in Pseudomonas aeruginosa-infected mice. Daily topical application on these induced wounds promoted wound healing and reduced bacteria load. Looking at biochemical parameters, lower IL-17, VEGF, and NO levels have been observed in the cinnamaldehyde-treated wounds (Ferro et al., 2019). In a same way, wound healing activity from the Cinnamomum genus has been studied in C. verum. Topical application of a hydroethanolic extract enhanced re-epithelialization and keratin biosynthesis in streptozotocin-induced diabetic mice (Daemi et al., 2019).

Epicatechin, a well-known flavonoid from tea leaves, has been tested on radiation-induced cellular damage in vitro on fibroblasts and in vivo in a zebrafish model. The metabolite increased, in vitro, the survival rate and restored the migration ability of the fibroblasts after irradiation. The mechanism is an inhibition of ROS generation, mitochondrial dysfunction, and cell death. Epicatechin reduced the expression of protein kinase p-JNK, p-38 MAPKs, and cleaved caspase-3. In addition, it lowered the cellular damage, improved wound healing after stress such as radiation exposure, and reduced the reprotoxicity in the zebrafish model (Shin et al., 2014). The epicatechin content in cinnamon bark, therefore, suggests that it has important effects on the wound healing process.

Peony root from Shinsen taitsukō is the root of Paeonia lactiflora Pall. (Paeoniaceae) (The society of Japanese pharmacopeia, 2016; WFO, 2022). It contains not less than 2.0% of paeoniflorin (C₂₃H₂₈O₁₁; 480.1632) (The society of Japanese pharmacopeia, 2016). The plant is originally distributed from Northeast China to the eastern part of Siberia, and medicinal hybrids are cultivated in Japan, in Nara and Hokkaido prefectures. P. lactiflora root, named shakuyaku (シャクヤク; 芍薬), should not be mistaken for the tree peony, P. suffruticosa Andrews, whose root cortex is used as a different crude drug. P. lactiflora root is traditionally harvested after 5 years of cultivation and then used for its sedative, immunomodulatory, and anti-inflammatory activities (Keio University Kampo Department; He and Dai, 2011).

More distinctive metabolites from the root are monoterpene glycosides such as the isomers, paeoniflorin and albiflorin (C₂₃H₂₈O₁₁; 480.1632), oxypaeoniflorin (C₂₃H₂₈O₁₂; 496.1581), or benzoylpaeoniflorin (C₃₀H₃₂O₁₂; 584.1894). Furthermore, root periderm treatments made on the raw material lead to a loss of bioactive constituents such as paeoniflorin and albiflorin. This has been demonstrated using MALDI MSI studies (Li et al., 2021). Some other important compounds are phenylpropanoids such as paeonol (C₉H₁₀O₃; 166.06299), hydrolyzable tannins including casuariin (C₃₄H₂₄O₂₂; 784.0759), glycosides such as paeonoside (C₁₅H₂₀O₈; 328.1158), and sucrose (C₁₂H₂₂O₁₁; 342.1162) (Keio University Kampo Department; Parker et al., 2016). Some other compounds such as ß-glucogallin (C₁₃H₁₆O₁₀; 332.0743), a gallate ester, and benzoic acid (C₇H₆O₂; 122.0368) characterize the P. lactiflora root (Sawada et al., 2018). Majority of them are water-soluble sugars, but they will not be included in Kampo oily extracts. Therefore, these compounds mostly represent the singularity of this crude drug. Hence, one of the challenges will be to better understand how oil extraction will influence the composition of the final ointments, especially for sugar-rich crude drugs like this one.

Paeoniflorin has shown effective anti-inflammatory and immunosuppressive activities in a large number of studies. The active mechanisms are associated with regulation of lymphocytes and dendritic cells with an enhancement of several pathways. Thus, protein kinase B (Akt), peroxisome proliferator-activated receptor (PPARγ), protein kinase (PKA), IL-4, IL-10, and TGF-β through an inhibition of JNK, ERK, iNOS, cyclooxygenase 2 (COX-2), IL-1β, IL-6, IL-17, and interferon γ (IFN-γ) are intensified by paeoniflorin. Some other signaling pathways such as the G protein-coupled receptor (GPCR), NF-κB, MAPK, and PI3K/Akt are also involved (Zhou et al., 2020). Regarding specific therapeutic actions on chronic wounds, paeoniflorin has been tested on streptozotocin-induced diabetic rat models and high-glucose-treated HaCaT cells. In this study, paeoniflorin improved wound healing in diabetic rat models (diabetic foot ulcer, DFU) and activated the expression of nuclear factor-E2-related factor 2 (Nrf2). In vitro experiments showed that paeoniflorin accelerated wound healing through this Nrf2 pathway and an increased expression of VEGF and TGF-β1. It reduced oxidative stress, increased cell proliferation and migration, and decreased apoptosis levels (Sun et al., 2020). A similar study on streptozotocin rat models using skin biopsy punches and high-glucose-treated HaCaT showed a downregulation of IL-β, IL-18, and TNF-α in paeoniflorin-treated DFU rats. Paeoniflorin decreased the expression levels of the chemokine receptor C-X-C motif chemokine receptor 2 (CXCR2), NF-κB, and p-IκB (Ser36) and increased the IkappaB kinase (IκB) level (Sun et al., 2021).

Paeoniflorin has also been tested with topical applications coupled with hyaluronic acid gel. The metabolite promoted a modulation of macrophages which are well involved in diabetic wounds. The topical application of the paeoniflorin-enriched gel brought better inflammation management, improved angiogenesis, re-epithelialization, and collagen deposition (Yang et al., 2021). Other studies using a paeoniflorin-sodium alginate-gelatin skin scaffold for treating diabetic wounds in a rat model gave positive results on macrophage modulation as well (Yu et al., 2022).

Oxypaeoniflorin, a metabolite found in P. lactiflora root, has been studied for its antioxidative and anti-inflammatory activities when it has been associated with paeoniflorin. Observed effects modulated glycation end products and induced inflammatory and oxidative stress responses (Zhanghua et al., 2013). On another note, ß-glucogallin reduces the expression of lipopolysaccharide-induced inflammatory markers by inhibiting aldose reductase. Moreover, because ß-glucogallin reduces LPS-induced activation of JNK and p38, we could imagine that this kind of plant metabolite may have a positive action on dermatological inflammation (Chang et al., 2013).

All these interesting therapeutic properties for wound healing deserve further research studies, especially a better understanding of what metabolites of interest would pass into the oil fraction during extraction.

Rehmannia root is one of the 50 fundamental herbs in TCM, and it is cultivated in the Yamato Valley in Japan and in Northern China (Ergil et al., 2002). According to the Japanese Pharmacopeia, accepted species are Rehmannia glutinosa Liboschitz var. purpurea Makino and R. glutinosa Liboschitz. However, the purpurea variety might be considered as a synonym, and nowadays, only the R. glutinosa (Gaertn.) DC. (Orobanchaceae) denomination is accepted (The society of Japanese pharmacopeia, 2016; WFO, 2022). The species name comes from the word glutinous because of the sticky aspect of the root (Zhang et al., 2008). Two processes could be used to prepare the crude drug, with the application of steaming (processed: juku-jiō) or without it (non-processed: kan-jiō) (The society of Japanese pharmacopeia, 2016). The plant is also known as Chinese fox glove and in Japanese jiō (ジオウ; 地黄). An interesting fact in relation to R. glutinosa is that the plant is sensitive to phytoviruses. Because its reproduction is realized through vegetative propagation, it may facilitate the spread of viral infections (Wu et al., 2002). For Kampo manufacturers, producing virus-free root stocks of Rehmannia is therefore important. Regarding pharmacognosy, the virological status of Rehmannia plants may be considered an influential factor of metabolites diversity (Matsumoto et al., 1989). The root is traditionally used for its effects on cardiovascular, digestive, and immune systems (Keio University Kampo Department; Zhang et al., 2008).

It contains monoterpenoids, phenethylalcohol glycosides, and triterpenes. Some metabolites are characteristic of the Rehmannia botanical genus such as catalpol (C₁₅H₂₂O₁₀; 362.1213) and acteoside (named verbascoside, C₂₉H₃₆O₁₅; 624.2054) (Zhou et al., 2018). These secondary metabolites are sensitive to growing conditions, processing treatments carried out after cultivation, and also the type of virus contamination (Matsumoto et al., 1989; Chang et al., 2006). Among the iridoids, monotermenes and glycosides which distinguish R. glutinosa root are the catalpol, dihydrocatalpol (C₁₅H₂₄O₁₀; 364.1369), ajugol (named leonuride, C₁₅H₂₄O₉; 348.1420), aucubin (C₁₅H₂₂O₉; 346.1264), melittoside (C₂₁H₃₂O₁₅; 524.1741), rehmaglutin A (C₉H₁₄O₅; 202.0841), rehmaglutin B (C₉H₁₃ClO₅; 236.0451), rehmaglutin C (C₉H₁₂O₅; 200.0685), rehmaglutin D (C₉H₁₃ClO₄; 220.0502), rehmannioside A (C₂₁H₃₂O₁₅; 524.1741), rehmannioside B (C₂₁H₃₂O₁₅; 524.1741), rehmannioside C (C₂₁H₃₄O₁₄; 510.1949), and rehmannioside D (C₂₇H₄₂O₂₀; 686.2269) (Zhang et al., 2008). Moreover, the root is rich in saccharides with three types of monosaccharides extracted (glucose, galactose, and fructose) and five kinds of oligosaccharides. Amino acids like arginine (C₆H₁₄N₄O₂; 174.1117) and organic acids like benzoic, linoleic, and stearic acids are also a part of the root (Keio University Kampo Department). In a way, the compounds present in Rehmannia root are either hydrophilic with the wide variety of compounds such as glycosides or more lipophilic with the organic acids.

Iridoids are a metabolites group characteristic of the Rehmannia botanical genus and the Rubiaceae and Scrophulariaceae botanical families. This group presents anti-inflammatory activity that may be beneficial in the treatment of inflammation (Viljoen et al., 2012). Moreover, the wound-healing activity of acylated iridoid glycosides was shown in vitro to stimulate the growth of human dermal fibroblasts (Nishimura et al., 1989; Stevenson et al., 2002). Amino acids in the root, even at low concentration, could therefore have a positive action on wound strength and collagen deposition. These therapeutic effects brought about by oral supplementation have been tested in artificial incisional wounds in animal models (Stechmiller et al., 2005).

In the same way, as discussed earlier for the compounds present in sesame oil, R. glutinosa root is rich in omega 6. It has favorable properties such as modulation of cell migration and proliferation, phagocytic capacity, and the production of inflammatory mediators (Silva et al., 2018). The underground parts of Rehmannia therefore include both lipophilic and hydrophilic compounds (with the iridoids in particular). Further research may reveal the metabolites present in Kampo oil extracts and thus provide a better understanding of the therapeutic actions of Kampo ointments.

Chinese rhubarb root usually comes from species Rheum palmatum L., R. tanguticum Maxim. ex Balf., R. officinale Baill., or R. coreanum Nakai (Polygonaceae) (The society of Japanese pharmacopeia, 2016; WFO, 2022). The plant also spreads to Eastern Europe, Northern America, and the cold regions of Asia. Since ancient times, it has been used as a remedy in these areas. The plant is named daiō (ダイオウ; 学名) in Japanese. Roots are usually used after a few years of cultivation. The interspecific hybrids made from Rheum coreanum Nakai x R. palmatum, named shinsyu-daiō, are now largely cultivated in Japan.

Chinese rhubarb root is one of the most commonly used Kampo ingredients, traditionally chosen for its laxative and tonic properties (Keio University Kampo Department). The crude drug presents antibacterial, digestive, anti-inflammatory, anti-fibrosis, and antitumor activities (Xiang et al., 2020).

At least 30 compounds could be identified in the Chinese rhubarb root. From anthraquinones and associated glucosides, dianthrones, phenylbutanones, stilbenes, flavan-3-ols, procyanidins, glucogallin, acyl-glucoses, gallic acid, or polymeric procyanidins were identified. Major differences regarding compounds concentrations exist depending on the botanical and geographical origins of the plant (Komatsu et al., 2006). According to the Japanese Pharmacopeia, the Chinese rhubarb root contains not less than 0.25% of sennoside A (C₄₂H₃₈O₂₀; 862.1956) (The society of Japanese pharmacopeia, 2016). This sennoside is from the anthraquinones group, but some other representatives are part of the root such as emodin (C₁₅H₁₀O₅; 270.0528), rhein (C₁₅H₈O₆; 284.0321), aloe-emodin (C₁₅H₁₀O₅; 270.0528), chrysophanol (C₁₅H₁₀O₄; 254.0579), or physcion (C₁₆H₁₂O₅; 284.0685). The root contains diantrone compounds such as sennoside F (C₄₄H₃₈O₂₃; 934.1804) or sennidin A (C₃₀H₁₈O₁₀; 538.0900) (Koyama et al., 2007). Other metabolites, such as tannins or other glycosides, are also typical, but they will not be easily extracted with sesame oil. The tannin group from Chinese rhubarb includes (+)-catechin and (−)-epicatechin (C₁₅H₁₄O₆; 290.0790). Both of these enantiomeric compounds are antioxidant flavonoids. Furthermore, some other glycosides such as lindleyin (C₂₃H₂₆O₁₁; 478.1475), trans-stilbene (C₁₄H₁₂; 180.0939), naphthalene (C₁₀H₈; 128.0626), and phenylbutanone are representatives of Chinese rhubarb root (Keio University Kampo Department). An important compound from the root is gallic acid, a trihydroxybenzoic acid (C₇H₆O₅; 170.0215).

Anthraquinones have broad biological activities that do not concern therapeutic applications for the digestive sphere. Indeed, emodin, a trihydroxyanthraquinone, has shown in vitro action on the NF-κB and phosphoinositide 3-kinase/Akt pathways, which are well implicated in the cell cycle. In vitro and in vivo studies have shown an anti-fibrosis action at the skin level via treatments of hypertrophic scars involving fibroblast deregulation (Mehta et al., 2016). This was confirmed in another study conducted on rats with topically applied emodin that enhanced the repair of excisional wounds. In this study, tissue regeneration stimulation implicated the SMAD-mediated TGF-β signaling pathway (Tang et al., 2007). According to recent in vitro studies, some other anthraquinone metabolites such as rhein stimulate HaCaT cell proliferation through the activation of the estrogen signaling pathway. This induces the expression of proto-oncogene c-myc in collaboration with the protein FosB and proto-oncogene JunD. Together, these activations accelerate re-epithelialization, an important step in the wound healing process (Xu et al., 2022).

In addition to this, gallic acid showed a beneficial role in wound healing. Thanks to the regulation and activation of the wound healing mechanisms surrounding the fibroblasts which are observed in vivo after per os treatment in experimentally induced hyperglycemic animals. The favorable factors observed included better wound edge cohesion, a smaller wound area, and a shorter time to reach re-epithelialization after wound induction (Liu et al., 2022).

The fact that Chinese rhubarb contains such compounds favorable for wound healing deserves metabolomics analyses of these compounds which are lipophilic (rhein or gallic acid) or amphiphilic (emodin) to understand their distribution in Kampo oily extracts.

The genus Scrophularia gathers more than 100 species of herbaceous flowering plants commonly known as figwort (Scrophulariaceae). There are two main species used in TCM and Kampo. First one is the Scrophularia ningpoensis Hemsl, with a species’ name which is refering to Ningbo, a city from Zhejiang province in China, also named Ningpo in the past. The second is S. buergeriana Miq. (WFO, 2022). Both species are named genjin (ゲンジン; 玄参) in Japanese. The root part is essential in traditional medicine from East Asia and has been used there for 2,000 years (Zhang et al., 2021). According to the Chinese Pharmacopoeia, Scrophularia roots are used to treat inflammatory and infectious pathologies. The crude drug is however not referenced by the Japanese Pharmacopoeia (The society of Japanese pharmacopeia, 2016). The name of the genus Scrophularia is also related to its topical use on scrofula lesions (tuberculous cervical lymphadenitis) (Lu et al., 2019). This suggests its properties as a relevant ingredient for skin wound healing treatments and as herbal antiseptic. Moreover, both S. ningpoensis and S. buergeriana have shown anti-inflammatory effects in in vitro and in vivo studies (Lee et al., 2021).

The Scrophularia botanical genus is a rich source of iridoids, terpenes, phenolic glycosides, alkaloids, and flavonoids (Pasdaran and Hamedi, 2017; Zhang et al., 2021). These compounds play a role as antioxidants, as proven in vivo through metabolomic approaches (Lu et al., 2019). Therapeutic applications of S. ningpoensis root, in addition to those for infectious lesions, have shown an effect on skin inflammations caused by allergic reactions in mice (Zhang et al., 2021). The anti-inflammatory properties studied in aqueous extracts affected the MAPK pathway and inhibited the NF-κB pathway (Shen et al., 2012). However, these are not easily extrapolated to Kampo extracts made with sesame oil.

Another characteristic compound is 6’-O-cinnamoylharpagide (C₂₄H₃₀O₁₁; 494.1788), which has been isolated from the ethanolic extracts made using the roots of S. ningpoensis. It is accompanied by other metabolites such as harpagide (C₁₅H₂₄O₁₀; 364.1369), harpagoside (C₂₄H₃₀O₁₁; 494.1788), 8-O-feruloylharpagide (C₂₅H₃₂O₁₃; 540.1843), 8-p-coumaroylharpagide (C₂₄H₃₀O₁₂; 510.1738), 6-O-methylcatalpol (C₁₆H₂₄O₁₀; 376.1369), aucubin (C₁₅H₂₂O₉; 346.1264), buergerinin B (C₉H₁₄O₅; 202.0841), teuhircoside (C₁₅H₂₀O₉; 344.1107), and 6-O-cinnamoyl-d-glucopyranose (C₂₉H₂₆O₁₅; 310.1052) (Niu et al., 2009). The properties of acylated iridoid glycosides from S. nodosa, a close species, were studied in vitro and showed a beneficial, dose-dependent effect on human fibroblasts (Stevenson et al., 2002).

The history of herbal medicines’ uses from the genus Scrophularia and its specific metabolites suggest that the root plays an important role in wound healing and the management of dermatological superinfections.