Advances for anti-inflammatory ingredients and activities of Fuzi (lateral root of Aconitum carmichaelii Debx.)

ABSTRACT

The rising prevalence and detrimental impact of inflammatory diseases have spurred the exploration of novel therapeutic approaches to tackle these conditions. Fuzi, lateral root of Aconitum carmichaelii Debx. has been used as a traditional Chinese medicine (TCM) for the treatment of inflammatory diseases for a long time. This article provides a comprehensive review of the medicinal applications, anti-inflammatory constituents, analytical techniques for detecting anti-inflammatory components, molecular mechanisms, and anti-inflammatory effects of Fuzi and its formulas. The information on Fuzi was collected from a variety of resources including classical works on Chinese herbal medicine, scientific databases (PubMed, Google Scholar, ACS, Web of Science, Science Direct, CNKI), TCMSP, NCBI, PubChem databases and others. About 122 compounds were isolated and identified from Fuzi, with alkaloids constituting the primary constituents that demonstrate potent anti-inflammatory activity. Specifically, among the identified compounds, there was one C-18 type, nineteen C-19 type, four C-20 type diterpenoid alkaloids, one other alkaloid, and 6 non-alkaloid constituents, all of which exhibited noteworthy anti-inflammatory and pain-alleviating properties in both in vivo and in vitro models of inflammation. These effects were mainly attributed to the reduction of pro-inflammatory cytokines, including IL-1, TNF-α, NO, IL-Iβ, and IL-6, as well as the inhibition of MAPK, NF-κB or PI3K/Akt signaling pathways, which effectively suppressed the inflammatory response and alleviated pain. Additionally, the anti-inflammatory effects were also achieved through the regulation of TLR4 expression and its downstream factors, or by modulating the homeostasis of the gut microbiota.

KEYWORDS:

• Fuzi
• botany
• medicinal and food use
• anti-inflammatory
• molecular mechanism

Introduction

As is widely known, inflammatory reactions play a crucial role under both normal and pathological physiological conditions, as one of the most important defense reactions of body against tissue damage or invasive microbes^[1]. However, persistent inflammation can lead to tissue and organ dysfunction, which contributes to the development of numerous acute and chronic diseases.^[2] These diseases include cardiovascular diseases,^[3] obesity, diabetes,^[4] dyslipidemia,^[5] and degenerative diseases.^[6] Currently, pharmaceutical treatments for inflammation in clinical practice include steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and corticosteroid injection. These treatments are generally effective in alleviating symptoms but carry the risk of additional side effects.^[7] These side effects may include gastrointestinal, cardiovascular, and renal complications, hepatotoxicity, and even the development of malignant tumors. Therefore, the development and utilization of natural and low side effect anti-inflammatory drugs are urgently needed to address clinical needs.

Traditional Chinese medicine (TCM) has a long history of extensive use spanning thousands of years.^[8] Fuzi, lateral root of Aconitum carmichaelii Debx., is extensively used in TCM due to its significant medical and edible value, as well as its stable efficacy. It has a rich historical tradition of medicinal application in China and is intriguingly utilized as a condiment in Southwest China. Fuzi was documented in the earliest existing pharmaceutical work “Shennong’s classic of materia medica” for its good therapeutic effect against energy losses, cold limbs, sweating, rapid breathing, feeble and impalpable pulse.^[9] Two thousand years ago, Fuzi was advocated to combine with other herbs, such as Paeoniae Radix Alba (Baishao), Glycyrrhizae Radix et Rhizoma (Gancao), and Zingiberis rhizome recens (Shengjiang). This combination aimed to enhance efficacy and reduce toxicity.^[10] In clinical practice, the processed Fuzi and Fuzi-based formulas have been prescribed to improve inflammation-associated diseases. These conditions may include heat, pain, redness, and swelling, such as diarrhea-predominant irritable bowel syndrome,^[11] osteoarthritis,^[12] and as rheumatoid arthritis.^[13] Therefore, conducting in-depth research on Fuzi is valuable for uncovering and developing novel natural anti-inflammatory compounds derived from medicinal plants.

The aim of this review was to summarize the anti-inflammatory properties, anti-inflammatory ingredients and mechanisms of Fuzi and its formulas. Additionally, it aimed to provide documented evidence for the clinical use of Fuzi and its preparations against inflammation.

Botany of Fuzi and its processing products

According to a study by Wang,^[14] there are probably 167 Aconitum species growing in China, of which are widely used in folk medicine. Among these species, only two Aconitum species, Aconitum carmichaeli Debx. and Aconitum kusnezoffii Reichb., are extensively cultivated in the eastern of Yunnan province, Sichuan province, Hubei province, and are widely applied as essential drugs in the 2020 edition of the Chinese Pharmacopoeia. The dried mother root of former is called “Chuanwu,” while the processed product of the daughter root is called “Fuzi.” The raw Fuzi, known as “ShengFuzi,” is usually excavated during the period from late June to early August. After being removed from the mother root, fibrous root, and sediment, the preliminary processed product “NiFuzi” can be further processed into “YanFuzi,” “Heishunpian” or “Baifupian,” according to different applying purposes (Table 1). The processing of Fuzi can effectively reduce its toxicity and optimize its efficacy.

Table 1. Fuzi recorded in Chinese Pharmacopoeia 2020.

Species	Chinese name	Medicinal part	Processing	Processed products
Aconitum carmichaeli. Debx.	Chuanwu	Mother root	Unprocessed
	Fuzi	Daughter root	Unprocessed	ShengFuzi
			Removed from the mother root, fibrous root and sediment	NiFuzi
			Wash, soak in salt, and dry in the sun	YanFuzi
			Soak, steam, water bleach, cut into 0.5 cm slices, soak, dye, steam, and dry in the sun	Heishunpian
			Soak, boil, peel off, cut into 0.3 cm slices, soak, steam, dry in the sun	Baifupian

Traditional application in Chinese history

In traditional folk medicine, phytotherapy is highly valued and widely utilized. As one of controversial Chinese herbs, Fuzi has been used in China for over 2000 years, since the Qin Dynasty. It was firstly recorded in the Shennong’s Classic of Materia Medica, which described that Fuzi could warm the body and prevent patients from bloody mass, cold, dampness, and cold cough. Another monograph, Ben cao zheng yi, elucidated that Fuzi was warm in nature, pungent, and sweet in flavor, and attributive to the heart, kidney and spleen meridians. The effects recorded in Shennong’s Classic of Materia Medica were consistent with those described in Ben cao zheng yi. Not long afterward, there was another Chinese medical classic, the “Compendium of Materia Medica (Ben cao gang mu),” which emphasized that Fuzi could be used to treat serious diseases or death due to its toxicity. In fact, the application of Fuzi-based formulas was advocated in Shang Han Lun by Zhong-jing Zhang in the Eastern Han Dynasty, because the combination of Fuzi with other herbs could enhance efficacy and reduce toxicity based on the TCM theory. Zhang introduced many Fuzi-based formulas, including Fuzi decoction, Shaogan Fuzi decoction, Mahuang Xixin Fuzi decoction, Guizhi Fuzi decoction, and Mahuang Fuzi Shenzhuo decoction, all exert good anti-inflammatory functions clinically. Additionally, other Fuzi-based formulas with anti-inflammatory effects were also recorded in classics and circulated come down, including Fuzi Lizhong pill, Fujiang decoction, Shenfu injection/decoction, Da buxin Tang/buxin wan, and Yiyi Fuzi Baijiang formula. With the development of TCM modernization, their anti-inflammatory activity has been further explored and investigated (Table 2).

Table 2. The traditional and clinical usage of Fuzi in China.

Formulas name	Compositions		Efficacy and application	Ancient books	Ref.
Fuzi Lizhong pill	Fuzi, Zingiberis rhizoma (Ganjiang), Glycyrrhizae radix et rhizoma. (Gancao), Codonopsis radix (Dangshen), Atractylodis macrocephalae rhizoma (Baizhu)		Warm stomach and spleen, for abdominal pain, vomiting, diarrhea, cold limbs with deficient cold of spleen and stomach, and now for enteritis, diarrhea, and gastritis	Taiping Huimin Heji Ju Fang	^[Citation11]
Fuzi decoction	Fuzi, Poria (Fuling), Codonopsis radix (Dangshen), Atractylodis macrocephalae rhizoma (Baizhu), Paeoniae radix alba or Paeoniae radix rubra (Shaoyao)		warming yang, dispersing cold, dispelling dampness and relieving pain	Shanghan Lun	^[Citation12]
Fujiang decoction	Fuzi, Zingiberis rhizoma (Ganjiang)		Restore yang for spontaneous sweating, cold extremities or dysphoria, and now for ulcerative colitis, abdominal pain, bloody stools, and vomiting	Fa Lv	^[Citation15]
Shenfu injection/decoction	Fuzi, Panax ginseng C. A. Mey. (Hongshen)		Tonify qi and yang, and now for arthritis, rheumatoid arthritis, rheumatoid arthritis, shoulder periarthritis, etc.	Sheng Ji Zong Lu	^[Citation16,Citation17]
Mahuang Xixin Fuzi decoction	Ephedrae herba (Mahuang), Asari radix et rhizoma (Xixin), Fuzi		Treat kidney-yang deficiency syndrome, and now for kidney-yang deficiency syndrome infected with influenza A virus	Treatise on Febrile Diseases (Shang Han Lun)	^[Citation18,Citation19]
Shaogan Fuzi decoction	Paeoniae Radix Alba (Baishao), Glycyrrhizae Radix et Rhizoma (Gancao), Fuzi	reinforcing deficiency and reducing excess, warming and activating meridians, nourishing blood, extending sinew, relieving spasm and pain, and now for rheumatoid arthritis		Treatise on Febrile Diseases (Shang Han Lun)	^[Citation20]
Guizhi Fuzi decoction	Cinnamomi ramulus (Guizhi, 12 g), Fuzi (15 g), Zingiberis rhizome recens (Shengjiang, 9 g), Glycyrrhizae radix et rhizoma (Gancao, 6 g), Jujubae fructus (Dazao, 12 g)	warm channel and expelling cold, nourish blood, and now for rheumatoid arthritis		Treatise on Febrile Diseases (Shang Han Lun)	^[Citation21]
Mahuang Fuzi Shenzhuo Decoction	Ephedrae herba (Mahuang), Fuzi, Zingiberis rhizoma (Ganjiang), Poria (Fuling), Atractylodis macrocephalae rhizoma (Baizhu), Glycyrrhizae Radix et Rhizoma (Gancao)	warm yang and resolve exterior methods, and now for passive Heymann nephritis		Treatise on Febrile Diseases	^[Citation22]
Da buxin Tang/buxin wan	Fuzi, Pinelliae rhizome (Banxia), Scutellariae radix (Huangqin), Glycyrrhizae radix et rhizoma (Gancao), Poria (Fuling), Cinnamomi Cortex (Rougui), Gypsum fibrosum (Shigao), Polygalae radix (Yuanzhi), Zingiberis rhizome recens (Shengjiang), Jujubae fructus (Dazao), maltose (Yitang), Qiandihuang, Asini corii colla (Ejiao), Ophiopogonis radix (Maidong)	warming yang to resolve phlegm, and now for lowering inflammation in heart failure with preserved ejection fraction (HFpEF)		Beiji Qianjin Yaofang	^[Citation23]
Yiyi Fuzi Baijiang formula	Coicis semen (Yiyiren), Fuzi, Patrinia villosa (Baijiangcao)	treat intestinal carbuncles, and now for enteritis, chronic appendicitis, bronchiectasis, chronic prostatitis and chronic pelvic inflammatory disease		Jingui Yaolue	^[Citation17]

Medicinal uses of Fuzi in the treatment of inflammation

Fuzi Lizhong pill, a classic TCM prescription for treating spleen – yang deficiency recorded in Taiping Huimin Heji Ju Fang in the Song Dynasty, which consists of five herbs, including Aconiti lateralis radix praeparata (Fuzi), Zingiberis rhizoma (Ganjiang), Glycyrrhizae radix et rhizoma. (Gancao), Codonopsis radix (Dangshen), Atractylodis macrocephalae rhizoma (Baizhu). Currently, Fuzi Lizhong pill is clinically applied for the treatment of enteritis, diarrhea, and gastritis.^[11] When used for warming yang and dispelling cold, dispelling dampness, and relieving pain, the PI3K-Akt signaling pathway is regulated by Fuzi decoction. This decoction is always comprised of Fuzi, Poria (Fuling), Codonopsis radix (Dangshen), Atractylodis macrocephalae rhizoma (Baizhu), Paeoniae radix alba and Paeoniae radix rubra (Shaoyao).^[12] In clinical practice, Fuzi and Zingiberis rhizome (Ganjiang) are usually prescribed in pairs to exert a synergistic anti-inflammatory effect on ulcerative colitis, abdominal pain, bloody stools, and vomiting. The involved mechanism may be related to the down-regulation of inflammatory mediators and the inhibition of the activation of corresponding NF-κB, MAPK, and STAT3 signaling pathways.^[15] As a modern preparation of Shenfu decoction, Shenfu injection, mainly prepared from Fuzi, Panax ginseng C. A. Mey. (Hongshen), by using countercurrent extraction and macroporous resin adsorption chromatography, can improve lung and liver damage through inhibiting the activity of NF-кB, and reducing the expressions of TNF-α and IL-6 in rats with systemic inflammatory response syndrome.^[16] It can also treat LPS-induced cardiac dysfunction, and attenuate the accompanying inflammation and apoptosis via downregulating the MEK and ERK signaling pathways.^[17]

Mahuang Xixin Fuzi decoction, composed of Ephedrae herba (Mahuang) and Asari radix et rhizoma (Xixin), has been shown to exert obviously protective effects on kidney-yang deficiency syndrome mice infected with influenza A virus, as it alleviates inflammatory reaction and inhibits Toll-like receptors (TLRs) response.^[18,19] During the treatment of collagen-induced arthritis, Shaogan Fuzi decoction and its modification have displayed good efficacy and safety. The mechanism may be related to the regulation of TLR4/MAPKs/NF-κB signaling.^[20]

Dahuang Fuzi decoction has a relatively good therapeutic effect in treating severe acute pancreatitis. Its mechanism of action may involve enhancing intestinal peristalsis, protecting the gastrointestinal barrier function, inhibiting bacterial and endotoxin translocation, and releasing inflammatory mediators, which ultimately protects the body’s immune function.^[24]

Guizhi Fuzi decoction, consisting of Cinnamomi ramulus (Guizhi, 12 g), Fuzi (15 g), Zingiberis rhizome recens (Shengjiang, 9 g), Glycyrrhizae radix et rhizoma (Gancao, 6 g), Jujubae fructus (Dazao, 12 g), has a significant therapeutic effect on rheumatoid arthritis by inhibiting the activities of interleukin-6 and tumor necrosis factor-α in the serum of adjuvant-induced arthritis rats. Additionally, it inhibits the formation of synovitis and pannus.^[21]

Mahuang Fuzi Shenzhuo decoction, a classic formula in Treatise on Febrile Diseases, can regulate autophagy and Wnt/βcatenin pathway in the treatment of passive Heymann nephritis (PHN) with inflammation, thereby providing therapeutic benefits.^[22] Although Fuzi and Pinelliae rhizome (Banxia) combination therapy elicit toxic effects based on the 18 antagonisms theory, the concurrent protective effect appears to predominate and is associated with suppression of PKA/β2-AR-Gs signaling pathway. Fuzi is often used in combination with Banxia in classic formulas such as Da buxin Tang and Buxin Wan to treat heart failure with preserved ejection fraction due to their encouraging anti-inflammatory effect.^[23]

Modern pharmacological studies have shown that Yiyi Fuzi Baijiang formula and its modifications have anti-inflammatory effect on ulcerative colitis by regulating the balance of Th17/Treg cells in rats. This is achieved by increasing the content of SOD, reducing TNF-α and IL-1β levels of two proinflammatory factors significantly with dose dependence.^[25]

Above all, Fuzi is a widely used herb in TCM, which is often combined with other herbs to enhance its efficacy and reduce its toxicity. Its traditional efficacy is focused on warming yang, dispersing cold, dispelling dampness and relieving pain. A considerable amount of evidence from ancient works, modern literature and clinical prescriptions investigation supports the synergistic interaction of Fuzi and other herbs in combination therapy, which enhances efficacy and reduces toxicity in diseases associated-inflammation.

Anti-inflammatory active compounds of Fuzi

Fuzi alkaloids

Fuzi contains more than 100 chemical components, with the majority being diterpenoid alkaloids and the rest being flavonoids, saponins, benzoic acid, polysaccharide, fatty acids, and ceramides glycosides. The alkaloids in Fuzi are divided into three categories based on the number of carbon atoms on the mother nucleus: C18, C19, and C20 diterpenoid alkaloids.^[26] Modern pharmacological research has shown that Fuzi alkaloids possess significant anti-inflammatory activity through the MAPK/NF-κB/STAT3 signaling pathway.^[27] The compounds are listed in Table 3.

Table 3. The anti-inflammatory properties of Fuzi compounds in vivo or in vitro.

Compounds	Inflammation model	Cell/animals	Treatment	Effect	Ref
C18 Diterpenoid Alkaloids
Total alkaloids	2% (DSS)-induced ulcerative colitis	C57BL/6 mice	gavage with 3, 6, and 12 g/kg crude drug dose	↓MPO, TNF-α, IL-1β, IL-6, IFN-c, and IL-17A, ↓pp38, ERK, JNK, NF-κB, IκB-α, STAT3	^[Citation15]
Lappaconitine	CFA-induced inflammatory pain model	Wistar rats	i.p. with 4 and 8 mg/kg	↑alpha-linolenic acid metabolism, ↓ retinol metabolism	^[Citation28]
	LPS-induced inflammatory model	RAW264.7 cells	30 μM	↓ iNOS, COX-2, p-NF-κB, NF-κB, and p-IκBa/IκBa	^[Citation29]
	LPS-induced acute lung injury	male mice	gavage with 5, 10, and 15 mg/kg	improved ici, lung edema, alveolar hemorrhage, interalveolar septal thickening	^[Citation29]
C19 Diterpenoid Alkaloids
14-veratroylpseudaconin	TNBS-induced colitis model	C57BL/6 mice	i.p. with 10, 20 and 50 μg	↓proinflammatory cytokines	^[Citation30]
14-O-acetylneoline	TNBS-induced colitis model	C57BL/6 mice	i.p. with 10, 20 and 50 μg	↓IFN-γ and proinflammatory cytokines	^[Citation30]
8-O-Cinnamoylneoline	-	ddY mice	i.h. with 0.86 mg/kg	significant analgesic effect	^[Citation31]
Meaconitine	-	ddY mice	i.h. with 0.86 mg/kg	significant analgesic effect	^[Citation31]
Benzoylmesaconitine	-	ddY mice	i.h. with 0.86 mg/kg	significant analgesic effect	^[Citation31]
Mesaconitine	hind-paw edema model	mice/Rats	i.c. with 40 or 80 ng	↓histamine, serotonin and prostaglandin E1	^[Citation32]
Hypaconitine	carrageenan/histamine	CD1 mice/CBA mice	administer to mice for 5 days	↑antiexudative effect; ↓edema	^[Citation33]
Deoxyaconitine	xylene- induced ear edema	Kunming mice	administer to mice	↓ear swelling	^[Citation34]
Guiwuline	mouse hot-plate test	Kunming mice	intravenous injection 15 mg/kg	significant analgesic effect	^[Citation35]
Mesaconine	TNF-α-stimulated	EAhy926 cells	0.1-1ug/ml	↓NF–κB activity	^[Citation36]
Fuziline	TNF-α-stimulated	EAhy926 cells	0.1-1ug/ml	↓NF–κB activity	^[Citation36]
Neoline	TNF-α-stimulated	EAhy926 cells	0.1-1ug/ml	↓NF–κB activity	^[Citation36]
Talatizamine	TNF-α-stimulated	EAhy926 cells	0.1-1ug/ml	↓NF–κB activity	^[Citation36]
Isotalatizidine	constrictive injury	ICR/C57BL/6 mice	Intrathecal injection 100 μl	↑ERK1/2,CREB; ↑dynorphin A release	^[Citation37]
Aconitine	adjuvant arthritis model	Wistar rats	fuzi extract orally at a daily dose 10 ml/kg for 3 d	↓nitric oxide and TNFα	^[Citation38]
	nerve-ligated neuropathic rats	Rats	i,t, with 35 pmol or 39 pmol	↑mechanical allodynia; ↑ heat hyperalgesia	^[Citation22]
Benzoylmesaconine	LPS-induced RAW264.7 macrophages	RAW264.7 cells	treated with Benzoylmesaconine (40, 80, or 160 μM) for 1 h	↓IL-1β, IL-6, TNF-α, PGE2, NO, ROS COX-2, and iNOS	^[Citation39]
Benzoylaconine	LPS- induced RAW264.7 macrophages	RAW264.7 cells	treated with Benzoylaconine (1, 100 μM) for 1 h	↓IL-1β, IL-6, TNF-α, PGE2, NO, ROS COX-2, and iNOS.	^[Citation40]
Benzoylhypaconine	LPS- induced RAW264.7 macrophages	RAW264.7 cells	treated with Benzoylhypaconine	↓NO; ↓ iNOS, ↓IL-1β; IL-6, and TNF-α.	^[Citation41]
Aconine	RANKL-induced os- teoclast differentiation	RAW264.7 cells	treated with aconine (0.125 or 0.25 Μm)	↓NF-κB and NFATc1 activation; ↓DC-STAMP	^[Citation42]
C20-Diterpenoid Alkaloids
Ignavine	Writhing induced by acetic acid	mice/Wistar	gavage with 50 or 100 mg/kg	↓writhing syndrome; paw edema	^[Citation43]
songorine	carrageenan/histamine	CD1 mice/CBA mice	administer to mice for 5 days	↑antiexudative effect; ↓edema	^[Citation33]
napelline	carrageenan/histamine	CD1 mice/CBA mice	administer to mice for 5 days	↑antiexudative effect; ↓edema	^[Citation33]
Aconicarchamine A	LPS-induced inflammatory model	RAW264.7 cells	treat with 6.25 μM	↓IL-6, IL-1β, COX-2, and iNOS	^[Citation44]
Other alkaloids and non-alkaloid constituents
higenamine	(IL)-1β-induced inflammation cell model	NPCs cell	IL-1β (10 ng/ml) for 24 h	↓iNOS，↓NO, ↓PGE2, ↓COX-2, TNF-α, and IL-6	^[Citation45]
	LPS-induced inflammatory cell model	BV2 cell	treat with higenamine (0. 5 μM)	↓TNF-α, NO, PGE2, COX-2, IL-6 and NF-κB, ↑Nrf2/HO-1	^[Citation46]
	A type II collagen ‑induced arthritis model	DBA/1J mice	injected with higenamine (10 mg/kg) for 2 weeks	↓TNF-α, IL-1β, Caspase-3, and Caspase-9; ↑tMDA and GSH; PI3K/AKT/Nrf2	^[Citation47]
Daucosterol	DSS-induced colitis in mice	C57BL/6J mice	gavage with 3 times every week at 10 mg/kg	↓infiltration of macrophages, ↓TNF-α, IL-6, and IL-1β	^[Citation48]
6”-O-Acetylliquiritin	ear edema experiments	mice	gavage for 7 days	↓ear edema	^[Citation49]
isoliquiritigenin	LPS-induced inflammatory model	RAW264.7 cells	3.125–50 g/mL	↓NO release	^[Citation50]
Liquiritigenin	LPS-induced inflammatory model	RAW264.7 cells	3.125–50 g/mL	↓NO release	^[Citation50]
Liquiritin	LPS-induced inflammatory model	BV2 cells	treat with 50 μM and 100 μM for 22 h	↓iNOS, COX-2, TNF-α, IL-1β and IL-6	^[Citation51]
Gracillin	LPS-induced inflammatory model	C57BL/6 mice	i.p. at 10 mg/kg for 7 days	block NF-κB signaling pathway	^[Citation52]

Complete Freund’s adjuvant (CFA); intraperitoneal injection (i.p.); inflammatory cell infiltration (ici), subcutaneous injection (i.h); intracutaneous injection (i.c.); trinitro-benzene-sulfonic acid (TNBS), Intrathecal administration (i,t); human nucleus pulposus (NPCs); inducible nitric oxide synthase (iNOS), nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), dextran sulfate sodium (DSS).

C18 diterpenoid alkaloids

Lappaconitine (LA) is a representative component of the C18 diterpenoid alkaloids isolated from Fuzi and possess a wide range of pharmacological activities. LA has antipyretic, analgesic, anti-inflammatory, and anti-swelling effects.^[53,54] Most of the pharmacological activity studies have focused on anti-inflammatory and analgesic effects of LA (Figure 1) and its derivatives. The analgesic effect of LA is equivalent to that of pethidine,^[53] and it could exert anticancer and analgesic effects by inhibiting voltage-gated sodium channels, stimulating the release of norepinephrine, and suppressing the release of substance P in the synaptic cleft. LA, acting as a sodium channel antagonist, can enter the pore from the intracellular side while the voltage-gated human heart (hH1) sodium channel is open. It can then bind to the site 2 receptor and irreversibly block the pore open, which slows the onset of pain.^[55] Additionally, LA has been shown to significantly inhibit the writhing response induced by acetic acid, the pain response induced by formaldehyde and hot plate methods. LA also suppress the egg albumen-induced paw edema in the rat and the xylene induced-ear edema in mice after intraperitoneal injection or gavage.^[56] Furthermore, LA can ameliorate the excessive inflammatory situation of severely burned rats by inhibiting the expression of P2X(4) receptor in microglia in the dorsal horn of the spinal cord and reducing the release of inflammatory factors TNF-α and IL-1β.^[57] Metabolomics technology has revealed that LA can effectively alleviate inflammatory pain by up-regulating alpha-linolenic acid metabolism and down-regulating retinol metabolism in the rat dorsal root ganglion.^[28]

Figure 1. The structure of C18-diterpenoid alkaloid lappaconitine in Fuzi.

C19-diterpenoid alkaloids

To date, researchers have extracted nearly seventy-six C19-diterpenoid alkaloids from Fuzi. Among these, the most well-studied pharmaceutical ingredients are aconitine (AC), mesaconitine (MA), hypaconitine (HA), and their decomposition products (monoester-diterpenoid alkaloids), for instance, benzoylaconine (BAC), benzoylmesaconine (BMA) and benzoylhypaconine (BHA). These compounds all share a common C19-norditerpenoid skeleton with different substituents. Specifically, C-1 is typically attached to -OH or -OCH₃; C-3 and C-13 to -OH; C-6, C-16 and C-18 to -OCH₃; C-15 to -OH, and ester bond frequently attaches to C-8 and C-14. C19-diterpenoid alkaloids are the major alkaloids responsible for its wide range of pharmacological properties and toxicity.^[58–61] Of these seventy-six C19-diterpenoid alkaloids, 19 have been reported to possess anti-inflammatory and pain-alleviating effects in vivo and in vitro models of inflammation (Table 3). The structures of these active ingredients are shown in Figure 2.

Figure 2. The structure of C19-diterpenoid alkaloids and specific groups of anti-inflammatory active constituents in Fuzi.

C20-diterpenoid alkaloids

So far, nearly thirty C20-diterpenoid alkaloids have been isolated from Fuzi, with their structures primarily based on nine types of core structures: actaline,^[62] atisine,^[63] arcutine,^[64] denudatine,^[62] hetisine,^[65] napelline,^[66] rearranged napelline,^[67] and vakognavine types.^[68] Among them, ignavine,^[43] songorine,^[33] napelline, and aconicarchamine A^[44] have been reported to possess anti-inflammatory properties (Table 3). The chemical structures of these bioactive ingredients are illustrated in Figure 3.

Figure 3. C20-diterpenoid alkaloids’ skeleton of anti-inflammatory active constituents in Fuzi.

Other alkaloids

Fuzi contains eight non-diterpenoid alkaloids, namely aconicaramide, 5-hydroxymethylpyrrole-2-carbaldehyde, oleacein E, yokonoside, higenamine, coryneine chloride, salsolinol, and fuzitine. Among them, only the anti-inflammatory effect of higenamine has been fully explored and elucidated. Higenamine has been found to inhibit iNOS expression and reduce NO production in LPS activated-RAW 264.7 cells,^[69] indicating its obvious anti-inflammatory activity (Figure 4). It is worth mentioning that further studies are needed to assess the anti-inflammatory activity of other non-diterpenoid alkaloids, despite their limited content and types. Figure 4 depicts the structure of higenamine.

Figure 4. The chemical structures of other alkaloid or non-alkaloid of anti-inflammatory active constituents of Fuzi.

Non-alkaloid constituents

Besides alkaloid, other non-alkaloid components such as daucosterol, 6”-O-acetylliquiritin, isoliquiritigenin, liquiritigenin, liquiritin, gracillin, uracil, fuzinoside, adenosine, and Fuzi polysaccharides have garnered attention from scholars and become a popular topic of study, particularly Fuzi polysaccharides for their potential immunomodulators. FPS-1, a water-soluble polysaccharides, has been reported to exert potent stimulating effects on the immune system, including murine lymphocyte proliferation induced by concanavalin A or lipopolysaccharide and splenocyte antibody production.^[70] Fuzi neutral polysaccharide (FNPS) is a mixture of rhamnose, arabinose, galactose, glucose, and mannose with a molecular weight of 94 kDa. In vitro experiments demonstrated that FNPS could promote phagocytosis, proliferation, and migration of macrophages. While in vivo experiments showed that it could stimulate the serum levels of Th1 immune-related IL-1β and IL-6, and suppress Th2 immune-related IL-10 and TNF-α.^[71] Among the non-alkaloid components, six have been found to exert significant anti-inflammatory activity, namely daucosterol, 6”-O-acetylliquiritin, isoliquiritigenin, liquiritigenin, liquiritin, and gracillin (Table 3). The structures of these active ingredients are depicted in Figure 4.

Analysis methods

The significant variation of alkaloids in Fuzi necessitates the establishment of quality analysis methods. Various methods have been reported to identify and quantitatively analyze diester-diterpenoid alkaloids, including gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis (CE), mass spectrometry (MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), HPLC combined with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS-MS). These methods ensure the stability of diester-diterpenoid alkaloids by selecting optimized solvents and pH values of the buffer.^[72–75] Additionally, HPLC coupled with photodiode array detector (DAD) has been used to simultaneously determine six aconitum alkaloids in Fuzi and establish a quality control standard.^[14,76] Aconitine-type alkaloids were also determined as markers in Fuzi by LC-ESI-MS, successfully used in phase I clinical trial for the pharmacokinetic evaluation of “SHEN-FU” injectable powder after intravenous drop administration,^[77,78] because processed and unprocessed Fuzi samples could be differentiated by controlling the ratio of aconitine and mesaconitine to hypaconitine.^[79] Recently, the reaction products of aconitine（8-ethyoxyl-14-benzoylaconitine, benzoylaconine, pyraconitine, 8-acetyl-14-ethyoxylaconitine）in dilute ethanol have been analyzed respectively, by using electrospray ionization-triple quad time-of-flight mass spectrometry (ESI-Q-TOF-MS). 8-acetyl-14-ethyoxylaconitine is attributed as indicator in quality control of Fuzi to ensure its quality and safety.^[80] However, the quantitative and qualitative analysis of a specific ingredient in blood or urine using these analytical methods poses a challenge due to the requirement of large volumes of pretreated test samples. To address this issue, a reliable enzyme immunoassay (EIA) method has been established for rapid quantitative determination of small amount of aconitine in sera after intravenous and oral administration, utilizing a highly selective antigen-antibody reaction with high sensitivity and specificity.^[81]

Main anti-inflammatory mechanisms of Fuzi and its formulas

Figure 5 illustrates the anti-inflammatory mechanism of Fuzi. It exerts pronounced regulatory effects on the gut microbiota, MAPK signaling pathway, PI3K/Akt signaling pathway, NF-kB signaling pathway, and TLR4.

Figure 5. Molecular mechanism of anti-inflammatory effect of Fuzi.

Regulate gut microbiota

The gut microbiota is an integral component of the human body that can modulate the disease and health status of the host by regulating various signaling pathways.^[82] The underlying mechanism involves the disruption of gut microbiota, which can lead to sustained inflammatory and immune responses in the intestinal mucosa.^[83] Fuzi-Lizhong pill (FZLZ) was found to reduce the abundance of gut microbiota, including Bacteroidetes, Blautia, Turicibacter, and Ruminococcus_torques_group, thereby inhibiting the release of inflammatory factors such as TNF, IL-Iβ, IFN-γ and IL-6. This, in turn, regulates the activity of NF-κB, Toll-like receptor, and other related pathways, thereby alleviating inflammatory response.^[84,85] Moreover, FZLZ inhibits the apoptosis of intestinal mucosal epithelial cells by promoting the proliferation of Lactobacillus and Lachnospiraceae_NK4A136_group, thereby restoring intestinal dysfunction.^[86] These Lactobacillus species can suppress the release of pro-inflammatory TNF,^[87] reduce the expression of pro-inflammatory surface proteins,^[88] promote the expression of IgA antibody,^[89] activate the AMPK/PGC1α signaling pathway,^[90] and strengthen intestinal immune barrier function. FZLZ has been demonstrate to inhibit persistent systemic inflammation and renovate the intestinal immune barrier in rats with diarrhea-predominant irritable bowel syndrome by modulating the bacterial diversity and community structures of Blautia, Turicibacter, and Ruminococcus_torques_group, as well as Lactobacillus in the host.^[11]

Regulate the MAPK signaling pathway

Mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK), play essential roles in the initiation and progression of ulcerative colitis and other inflammatory diseases.^[91,92] Upon phosphorylated, p38 MAPK promotes the release of intracellular cytokines, such as TNF-α and IL-1β, which subsequently activate p38 MAPK through paracrine action.^[93] The JNK and ERK signaling pathways regulate the expression and secretion of inflammatory cytokines, such as IL-6 and IFN-γ. Fuzi, a traditional Chinese medicine, exert significant anti-inflammatory effects against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice by reducing the expressions of total p38 MAPK, STAT3, and the phosphorylation of ERK and JNK.^[15] Moreover, the total alkaloids of Fuzi not only inhibited the levels of inflammatory cytokines but also suppressed the phosphorylation of p38 MAPK, ERK, and JNK, thereby alleviating the symptoms of ulcerative colitis.^[27] These findings suggest that Fuzi and its active ingredients possess potent anti-inflammatory properties through repressing the activation of MAPK signaling.

Regulate the NF-κB signaling pathway

NF-κB is widely acknowledged as a critical signaling pathway involved in various inflammatory responses.^[94] Upon activation, NF-κB promotes the expression of pro-inflammatory factors (TNF-α, IL-6, and IL-1β), as well as chemokines, adhesion molecules (ICAM-1, VCAM-1, and ELAM-1), and enzymes associated with inflammatory cascade amplification (iNOS and COX-2), ultimately leading to chemotactic neutrophil infiltration and aggregation at the site of inflammation, resulting in an inflammatory response.^[95,96] Recent studies have shown that LA can attenuate the expression of iNOS and COX-2, and downregulate the relative protein expression of p-NF-κB, P65/NF-κB P65, and p-IκBa/IκBa in LPS-induced macrophage and models in vitro models, as well as in an acute lung injury mouse model in vivo.^[29] Furthermore, higenamine, the active ingredient of Fuzi, suppressed interleukin (IL)-1β-induced inflammation in human nucleus pulposus cells (NPCs) by regulating the NF-κB signaling pathway.^[45] Additionally, the active ingredients benzoylaconitine in Fuzi can also reduce IL-6-induced inflammatory response by inhibiting the NF-κB signaling.^[97]

Regulate the PI3K/Akt signaling pathway

The PI3K family plays a prominent role in intracellular signal transduction and the pathogenesis of inflammatory diseases.^[98] In the context of osteoarthritis, the phosphorylated activation of PI3K and Akt has been significantly associated with cartilage injury, resulting in the upregulation of their downstream proteins such as Col2, Col10, MMP9 and MMP13 in chondrocytes stimulated with IL-1β.^[12,99] Notably, the abnormal expression of these downstream proteins was effectively attenuated by Fuzi decoction (benzoylaconitine, benzoylhypaconitine) and the PI3K inhibitor, implying that the anti-osteoarthritis mechanism of Fuzi decoction is mediated, at least in part, by the PI3K/Akt signaling pathway. Moreover, higenamine has been shown to exert a protective effect against hypoxic damage and inhibit high mobility group box-1 (HMGB1) through PI3K/Akt/erythroid 2-related factor 2 (Nrf-2) signaling pathway, thereby demonstrating its potential as a therapeutic agent for brain ischemia, such as stroke.^[100] Finally, the analgesic effect of aconitine has been linked to its ability to activate intracellular G protein/PI3K/phosphatidylinositol 4,5-bisphosphate (PIP2) signaling pathways and increase intracellular PIP2 levels.^[101]

Regulate the expression of Toll-like receptor 4 (TLR4)

TLR4 is a crucial pattern recognition receptor that governs the inflammatory response in vivo. Upon activation, TLR4 triggers downstream transcription factors NF-κB and AP-1 via MyD88, which subsequently translocate into the nucleus to initiate the expression of inflammatory cytokines.^[102,103] The activation of the TLR4 signaling pathway results in the upregulation of inflammatory factors, including IL-6, TNF-α, and IL-12, which subsequently contribute to the mediation of inflammation.^[104] Songorine, benzoylaconine, and aconitine can inhibit the release of inflammatory cytokine and downregulate TLR4, thereby alleviating inflammatory reactions in LPS-induced proliferation of HFLS-RA fibroblast-like synoviocytes.^[105] Yan et al. reported that Fuzi and its formulas exert an anti-inflammatory effect by repressing the TLR4/NF-κB pathway in transverse aortic constriction mice, thereby protecting the heart.^[106] In the treatment of collagen-induced arthritis (CIA), Shaogan Fuzi decoction and its modification exhibit favorable efficacy in ameliorating symptoms and reducing the release of inflammatory factor (TNF-α, IL-1β, IL-6, IFN-γ and IL-17) by suppressing the TLR4/MAPKs/NF-κB signaling pathway.^[20] Furthermore, Fuzi Lizhong pill can regulate gut microbiota, promote the generation of short chain fatty acid, and thereby inhibit TRL4 to alleviate inflammatory responses.^[90]

Conclusion

Fuzi is a traditional Chinese medicine that has been used clinically for thousands of years. It has been discovered to have a broad spectrum of pharmacological effects, particularly in the areas of anti-inflammatory and analgesic activity. In this review, we have investigated the anti-inflammatory chemical constituents of Fuzi, the methods used for their analysis and detection, the molecular mechanisms underlying the anti-inflammatory effects, and the anti-inflammatory effects of Fuzi-containing prescription. The primary constituents of Fuzi are alkaloids, which exhibit potent anti-inflammatory activity. Among these, C-18 type diterpenoid alkaloid (LA), C-19 type diterpenoid alkaloids (14-veratroylpseudaconin, 14-O-acetylneoline, 8-O-cinnamoylneoline, meaconitine, mesaconitine, mesaconine, hypaconitine, deoxyaconitine, guiwuline, fuziline, neoline, talatizamine, isotalatizidine, aconitine, aconine, benzoylmesaconitine, benzoylmesaconine, benzoylaconine, benzoylhypaconine), and C-20 type diterpenoid alkaloids (ignavine, songorine, napelline and aconicarchamine A) exhibit anti-inflammatory and pain-alleviating. In general, Fuzi or its formulations exhibit a reduction in the release of proinflammatory cytokines, including but not limited to IL-1, TNF-α, NO, IL-Iβ, and IL-6 through the inhibition of the MAPK, NF-κB or PI3K/Akt signaling pathways. As a result, Fuzi suppresses the inflammatory response and provides pain relief. Additionally, Fuzi’s anti-inflammatory effects can be attributed to the downregulation of TLR4 expression and its downstream factors, as well as the regulation of gut microbiota homeostasis. Although there have been reports of cardiotoxicity, neurotoxicity, and other adverse effects associated with Fuzi (as mentioned in the supplementary materials), the Fuzi used in clinical settings undergoes processing and strict dose control to ensure safety. As the pharmacological properties, safety and molecular mechanisms of Fuzi continue to be elucidated, its therapeutic applications in clinical settings are expected to expand.

Abbreviations

TCM	=	Traditional Chinese medicine
LA	=	Lappaconitine
TNF-α	=	Tumor necrosis factor alpha.
DSS	=	Dextran sulfate sodium
FNPS	=	Fuzi neutral polysaccharide
MAPK	=	Mitogen-activated protein kinases
MS	=	Mass spectrometry
BAC	=	Benzoylaconine
TLR4	=	Toll-like receptor 4

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10942912.2023.2293461