Clinical Potential of Himalayan Herb Bergenia ligulata: An Evidence-Based Study

Herbal products have been used in traditional systems of medicine and by ethnic healers for ages to treat various diseases. Currently, it is estimated that about 80% of people worldwide use herbal traditional medicines against various ailments, partly due to easy accessibility and low cost, and the lower side effects they pose. Bergenia ligulata, a herb ranging from the Himalayas to the foothills, including the north-eastern states of India, has traditionally been used as a remedy against various diseases, most prominently kidney stones. The medicinal properties of B. ligulata have been attributed to bergenin, its most potent bioactive component. Apart from bergenin, the other compounds available in B. ligulata are arbutin, gallic acid, protocatechuic acid, chlorogenic acid, syringic acid, catechin, ferulic acid, afzelechin, paashaanolactone, caryophyllene, 1,8-cineole, β-eudesmol, stigmasterol, β-sitosterol, parasorbic acid, 3-methyl-2-buten-1-ol, phytol, terpinen-4-ol, tannic acid, isovalaric acid, avicularin, quercetin, reynoutrin, and sitoinoside I. This review summarizes various medicinal properties of the herb, along with providing deep insight into its bioactive molecules and their potential roles in the amelioration of human ailments. Additionally, the possible mechanism(s) of action of the herb’s anti-urolithiatic, antioxidative, antipyretic, anti-diabetic, anti-inflammatory and hepatoprotective properties are discussed. This comprehensive documentation will help researchers to better understand the medicinal uses of the herb. Further studies on B. ligulata can lead to the discovery of new drug(s) and therapeutics for various ailments.


Introduction
Traditional herbal medicines are plant-derived natural products that have been used by rural communities for ages for the management of various diseases [1]. Particularly in the tropics and the sub-tropics, an abundance of medicinal plants offers access to effective prevention and management of diseases through self-medication using plant-based medicines. It is estimated that 80% of people worldwide depend on such plant-based articles, and abstracts were screened, and unrelated articles, as well as publications on languages other than English, were excluded from the study as those publications do not relate to the specific aim of the review article. Thus selected articles ( Figure 1) were critically analyzed, and results were organized into several sections in the manuscript. Finally, a possible mechanism of action of B. ligulata was also speculated as an outcome of the study. At the end of the article future perspective is also considered.

Bergenia ligulata as Traditional Herbal Medicine
A huge recovery in the interest and use of medicinal plants has been witnessed in the previous decade. For a considerably long time, the plants belonging to the genus Bergenia have received notable attention for their restorative properties against ailments and have been broadly utilized in traditional medicines in various regions, particularly in the Asian continent, including India, Pakistan and Nepal [20]. Rhizomes of the species belonging to the genus Bergenia have been used in folk medicine for their antiscorbutic, astringent, diuretic, antipyretic, and ophthalmic properties [21]. The rhizomes are also used in dissolving kidney and gall bladder stones apart from healing cuts, burns, wounds, inflammation, cold, and cough [21]. The first use of the plant, "Paashanbheda", for dissolving calculi and treatment of painful urination was narrated in ancient Charaka Samhita as early as 600 BC [5]. Ayurveda mentions the use of sap prepared from the leaves of B. ligulata for the treatment of urinary diseases, stomach problems, epilepsy, and cold [9]. Similarly, Ayurveda and Unani medicine systems also mention the utilization of B. ligulata roots in the management of vesicular calculi, urinary discharge, exorbitant uterine hemorrhage, ailments of the bladder, diarrhea, menorrhagia, excessive splenic growth, and cardiovascular diseases [7,22]. Sushruta Samhita mentions its use in the management of kidney and bladder stones and blood sugar. Some other indigenous Indian literature, including Bhavaprakash, Rajnighantu, and Chakradatta, also prescribed the use of B. ligulata in the management of urinary diseases and stones and the purification of the urinary bladder. Various local communities in the central Himalayan region consume different parts of the plant, such as the root rhizome, leaf, or whole plant in the form of sap or liquor or powder against dizziness, headache, vertigo, and kidney stones [9]. For example, the Bhotia tribes of central Himalayas consume dried rhizomes powder to treat kidney stones [23]. The tribal communities of Dharchula, Uttar Pradesh, India use roots of B. ligulata in healing cuts and wounds,

Bergenia ligulata as Traditional Herbal Medicine
A huge recovery in the interest and use of medicinal plants has been witnessed in the previous decade. For a considerably long time, the plants belonging to the genus Bergenia have received notable attention for their restorative properties against ailments and have been broadly utilized in traditional medicines in various regions, particularly in the Asian continent, including India, Pakistan and Nepal [20]. Rhizomes of the species belonging to the genus Bergenia have been used in folk medicine for their antiscorbutic, astringent, diuretic, antipyretic, and ophthalmic properties [21]. The rhizomes are also used in dissolving kidney and gall bladder stones apart from healing cuts, burns, wounds, inflammation, cold, and cough [21]. The first use of the plant, "Paashanbheda", for dissolving calculi and treatment of painful urination was narrated in ancient Charaka Samhita as early as 600 BC [5]. Ayurveda mentions the use of sap prepared from the leaves of B. ligulata for the treatment of urinary diseases, stomach problems, epilepsy, and cold [9]. Similarly, Ayurveda and Unani medicine systems also mention the utilization of B. ligulata roots in the management of vesicular calculi, urinary discharge, exorbitant uterine hemorrhage, ailments of the bladder, diarrhea, menorrhagia, excessive splenic growth, and cardiovascular diseases [7,22]. Sushruta Samhita mentions its use in the management of kidney and bladder stones and blood sugar. Some other indigenous Indian literature, including Bhavaprakash, Rajnighantu, and Chakradatta, also prescribed the use of B. ligulata in the management of urinary diseases and stones and the purification of the urinary bladder. Various local communities in the central Himalayan region consume different parts of the plant, such as the root rhizome, leaf, or whole plant in the form of sap or liquor or powder against dizziness, headache, vertigo, and kidney stones [9]. For example, the Bhotia tribes of central Himalayas consume dried rhizomes powder to treat kidney stones [23]. The tribal communities of Dharchula, Uttar Pradesh, India use roots of B. ligulata in healing cuts and wounds, ophthalmic problems, and dissolution of kidney stones and in urinary diseases [9]. The tribal communities in the Chamba district of Himachal Pradesh, India, use leaves of B. ligulata in the preparation of tea and consume it to treat common cold [24]. In the eastern Himalayas, leaves of this plant are used to treat cuts, wounds, and boils by the Monpa tribes of Arunachal Pradesh, India, whereas the Naga tribes of India use the roots in the management of liver diseases and tuberculosis [9]. Similarly, Mizo tribes in India use the decoction prepared from the roots of B. ligulata in the management of diarrhea and infection of pulmonary system. Juice prepared from the leaf is used against boils and is also taken orally to dissolve kidney stones [25]. B. ligulata is available as an over-the-counter herbal product, particularly as powder [26], as well as stems, roots, and seeds [27].

Bioactive Compounds in Bergenia ligulata
Ultra-high-performance liquid chromatography coupled with hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) has been able to quantify eight major bioactive compounds from the rhizome of the plant: bergenin, arbutin, gallic acid, protocatechuic acid, chlorogenic acid, catechin, syringic acid, and ferulic acid [10]. In comparison with other medicinally important Bergenia species such as B. ciliata, B. purpurascens, and B. stracheyi, the highest total contents of these eight compounds have been noted in B. ligulata [10]. Other important phenolic compounds of B. ligulata include (+)-afzelechin [28,29], paashaanolactone [30], caryophyllene, 1,8-cineole, β-eudesmol, β-sitosterol, (+)-(6S)-parasorbic acid, 3-methyl-2-buten-1-ol, phytol, and tannic acid [31], isovalaric acid [32], stigmasterol [14], avicularin [7,33] terpinen-4-ol [34], quercetin [35], reynoutrin [36,37], and sitoinoside I [38]. These bioactive compounds, as presented in Table 1, are believed to be responsible for the medicinal properties of the plant [9]. ophthalmic problems, and dissolution of kidney stones and in urinary diseases [9]. The tribal communities in the Chamba district of Himachal Pradesh, India, use leaves of B. ligulata in the preparation of tea and consume it to treat common cold [24]. In the eastern Himalayas, leaves of this plant are used to treat cuts, wounds, and boils by the Monpa tribes of Arunachal Pradesh, India, whereas the Naga tribes of India use the roots in the management of liver diseases and tuberculosis [9]. Similarly, Mizo tribes in India use the decoction prepared from the roots of B. ligulata in the management of diarrhea and infection of pulmonary system. Juice prepared from the leaf is used against boils and is also taken orally to dissolve kidney stones [25]. B. ligulata is available as an over-the-counter herbal product, particularly as powder [26], as well as stems, roots, and seeds [27].

Bioactive Compounds in Bergenia ligulata
Ultra-high-performance liquid chromatography coupled with hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) has been able to quantify eight major bioactive compounds from the rhizome of the plant: bergenin, arbutin, gallic acid, protocatechuic acid, chlorogenic acid, catechin, syringic acid, and ferulic acid [10]. In comparison with other medicinally important Bergenia species such as B. ciliata, B. purpurascens, and B. stracheyi, the highest total contents of these eight compounds have been noted in B. ligulata [10]. Other important phenolic compounds of B. ligulata include (+)afzelechin [28,29], paashaanolactone [30], caryophyllene, 1,8-cineole, β-eudesmol, β-sitosterol, (+)-(6S)-parasorbic acid, 3-methyl-2-buten-1-ol, phytol, and tannic acid [31], isovalaric acid [32], stigmasterol [14], avicularin [7,33] terpinen-4-ol [34], quercetin [35], reynoutrin [36,37], and sitoinoside I [38]. These bioactive compounds, as presented in Table  1, are believed to be responsible for the medicinal properties of the plant [9]. Arbutin [10] Bergenin [10] ophthalmic problems, and dissolution of kidney stones and in urinary diseases [9]. The tribal communities in the Chamba district of Himachal Pradesh, India, use leaves of B. ligulata in the preparation of tea and consume it to treat common cold [24]. In the eastern Himalayas, leaves of this plant are used to treat cuts, wounds, and boils by the Monpa tribes of Arunachal Pradesh, India, whereas the Naga tribes of India use the roots in the management of liver diseases and tuberculosis [9]. Similarly, Mizo tribes in India use the decoction prepared from the roots of B. ligulata in the management of diarrhea and infection of pulmonary system. Juice prepared from the leaf is used against boils and is also taken orally to dissolve kidney stones [25]. B. ligulata is available as an over-the-counter herbal product, particularly as powder [26], as well as stems, roots, and seeds [27].

Bergenin
Bergenin is a C-glucoside of 4-O-methyl gallic acid, a colorless crystalline polyphenol that comprises an aromatic ring, an annellated δ-lactone ring, and a glucopyranose ring [39]. It shows strong effectiveness in averting stress-induced gastric ulcers in rat models and is widely used as an ingredient of folk medicine for gastritis [40]. Cell line studies revealed that bergenin can induce apoptosis in HeLa (cervical cancer) cells by arresting the cell cycle at G0/G1 phase and can also promote anti-cancer activity by inhibiting the expression of signal transducer and activator of transcription 3 (STAT3) protein and metastasis of cancer cell [41]. Bergenin introduced the highest antioxidant and lipophilic properties which played a vital role in averting neuronal diseases and death [42]. It also accelerates the osteogenesis of bone mesenchymal stem cells through the upregulation of the sirtuin 1 (SIRT1) expression [43]. Strong antimicrobial activity of bergenin has been recorded against Aspergillus flavus, Aspergillus niger, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans [44,45].

Catechins
Catechins are composed of two aromatic rings with hydroxyl group. This colorless crystalline polyphenol [46] has a molecular weight of 290 g/mol [47]. Depending on the distribution of hydroxyl group in aromatic ring, catechins are categorized into two types: i) free catechins and ii) esterified catechins [48]. Several studies show strong antiviral properties of catechins against adenovirus [49], enterovirus [50], human immunodeficiency viruses (HIV) [51], influenza virus [52], and tobacco mosaic virus (TMV) [53]. Catechins and their derivatives are effective scavengers of reactive oxygen species (ROS) [54]. Catechins moiety can function as free radical scavengers by requisitioning metal ions, and the B-ring site serves as a major site where the scavenging reaction is carried out [55][56][57].

Bergenin
Bergenin is a C-glucoside of 4-O-methyl gallic acid, a colorless crystalline polyphenol that comprises an aromatic ring, an annellated δ-lactone ring, and a glucopyranose ring [39]. It shows strong effectiveness in averting stress-induced gastric ulcers in rat models and is widely used as an ingredient of folk medicine for gastritis [40]. Cell line studies revealed that bergenin can induce apoptosis in HeLa (cervical cancer) cells by arresting the cell cycle at G0/G1 phase and can also promote anti-cancer activity by inhibiting the expression of signal transducer and activator of transcription 3 (STAT3) protein and metastasis of cancer cell [41]. Bergenin introduced the highest antioxidant and lipophilic properties which played a vital role in averting neuronal diseases and death [42]. It also accelerates the osteogenesis of bone mesenchymal stem cells through the upregulation of the sirtuin 1 (SIRT1) expression [43]. Strong antimicrobial activity of bergenin has been recorded against Aspergillus flavus, Aspergillus niger, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans [44,45].

Catechins
Catechins are composed of two aromatic rings with hydroxyl group. This colorless crystalline polyphenol [46] has a molecular weight of 290 g/mol [47]. Depending on the distribution of hydroxyl group in aromatic ring, catechins are categorized into two types: (i) free catechins and (ii) esterified catechins [48]. Several studies show strong antiviral properties of catechins against adenovirus [49], enterovirus [50], human immunodeficiency viruses (HIV) [51], influenza virus [52], and tobacco mosaic virus (TMV) [53]. Catechins and their derivatives are effective scavengers of reactive oxygen species (ROS) [54]. Catechins moiety can function as free radical scavengers by requisitioning metal ions, and the B-ring site serves as a major site where the scavenging reaction is carried out [55][56][57]. Manikandan et al. (2012) reported that catechins possess significant anti-cancer properties and may reduce the proliferation of HCT 116, HCT 15 (human colon adenocarcinoma) and Hep G-2 (human larynx carcinoma) cell lines and are able to induce apoptosis [58].

Arbutin
Additionally known as p-hydroxyphenyl-β-d-glucopyranoside, arbutin is a bioactive hydrophilic polyphenol that has two isomers such as α-arbutin and β-arbutin [59]. Arbutin and its derivatives can directly control the overproduction of melanin by converting tyrosinase into L-DOPA (levodopa) and obstructing the tyrosinase activity without altering the mRNA expression [60]. Arbutin can introduce anti-inflammatory activity by decreasing the production of nitric oxide (NO) and expression of iNOS and cyclooxygenase-2 (COX-2) in lipopolysaccharide-stimulated BV2 cells (murine microglial cells) which also simultaneously suppressed the production of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) [61]. Li et al. (2011) reported that arbutin has antitumor activity by inducing TCC-SUP (human bladder cancer) cell proliferation by inhibiting extracellular signal-regulated kinase (ERK) and by accelerating p21 protein expression [62].

Gallic Acid
Gallic acid is a secondary metabolite present in most plants and is also known as 3,4,5 trihydroxybenzoic acid [63]. Gallic acid alters the integrity of bacterial cell membranes by penetrating the bacterial cell wall and disrupting the cellular respiration and electron transport chain. This compound also negatively alters DNA cleavage by affecting dihydrofolate reductase activity in bacteria [64]. It also shows antiviral activity with respect to hepatitis C virus (HCV) [65] and herpes simplex virus (HSV) [66]. Gallic acid showed anti-cancer activity by arresting the cell cycle and promoted apoptosis by activating the caspases pathway and can also reduce metastasis [64]. Gallic acids also possess gastroprotective activity [67], anti-hypertriglyceridemia activity, and diet-induced anti-hyperglycemic activity [68].

Protocatechuic Acid
Protocatechuic acid is a secondary metabolite that consists of an aromatic ring and one or more hydroxyl groups and is also known as 3,4-dihydroxybenzoic acid [69]. Protocatechuic acid possesses free radical scavenging property and shows antioxidant activity through reduction of ROS generation in different parts of the body such as the brain, heart, kidney, and liver, and prevents different degenerative diseases [70]. Protocatechuic acid reportedly shows preventive activity against neurodegenerative diseases such as Alzheimer's and Parkinson's diseases by interrupting the aggregation of β-amyloid plaques in brain tissues and preventing hyperphosphorylation of tau protein in neurons [71]. It has the ability to inhibit the growth of Gram-positive and Gram-negative bacteria and also shows antifungal activity [70]. In addition to antioxidant and anti-inflammatory activities, protocatechuic acid produced a significant positive effect in hyperglycemic conditions by increasing plasma insulin level [72]. Lende et al. (2011) suggested that protocatechuic acid can introduce promising anti-inflammatory properties by reducing carrageenan-induced paw edema and Freund's adjuvant arthritis [73].

Chlorogenic acid
Chlorogenic acid is a phenolic secondary metabolite and is also known as 5-Ocaffeoylquinic acid (5-CQA). Chlorogenic acid plays a role in glucose metabolism through the activation of AMP-activated protein kinase (AMPK), which leads to glucose transport from intracellular membrane to plasma membrane by glucose transporter-4 (GLUT4) and increases cardiac glycolysis by activating phosphofructokinase 2 [74]. It can inhibit glucose-6-phosphatase activity and reduce glucose level in circulation, which causes less deposition of fatty acids in the adipose tissue and simultaneously utilizes the stored fat in the body, resulting in a reduction in body weight. It also shows anti-diabetic properties [75]. In addition to antioxidant activity, chlorogenic acid also possesses antibacterial activity against Escherichia coli, Klebsiella pneumonia, Staphylococcus epidermidis and antifungal activity against Candida albicans and antiviral activities against HIV, HSV-1, and HSV-2 and adenovirus [75].

Syringic Acid and Ferulic Acid
Syringic acid and ferulic acid are phenolic compounds. Syringic acid contains methoxy groups on the aromatic ring at positions of 3 and 5 [76]. Ferulic acid is also known as 4hydroxy-3-methoxycinnamic acid [77,78]. Ferulic acid is a potent photoprotective and brightening agent for skin care; it nourishes the skin by balancing collagen and elastin activity [79]. Ferulic acid is also a potent scavenger of free radicals, and it can decrease lipid peroxidation rate in the rat brain by donating electrons from hydroxy and phenoxy groups to neutralize ROS [80]. Ferulic acid has also demonstrated antimicrobial activities against Enterococcus faecalis, Staphylococcus aureus [81], and Cronobacter sakazakii [82]. Treating Cronobacter sakazakii infection with syringic acid deteriorated bacterial cell membrane structure and halted bacterial growth, and this compound can be used as a natural preservative, too [82]. In addition to antioxidant activity, syringic acid appeared effective against acute pancreatitis [83], renal ischemia-reperfusion injury [84], and demyelination and inflammation in sciatic nerves [85]. Syringic acid enhances the working capacity of β-cells in the pancreas and increases the plasma insulin level, which induces more deposition of glycogen in peripheral tissue [85,86].

Potential Clinical Use
Bergenia species have been well known since ancient times for their potential curative effects on different human ailments [37]. B. ligulata is one of the important members of this genus and, also shows an extensive range of pharmacological activities. It is widely used in Indian traditional medicine as well as other traditional medicine systems of the world. The bioactive compounds found in the plant are diverse and may be responsible for the pharmacological activity of the plant. In traditional medicine systems, the plant is used mainly for its antilithiatic activity; additionally, the whole plant or parts of the plant (root and rhizome) have been used in the management of fever, inflammation, diabetes, microbial infections, wounds, burns [21], amelioration of liver disease, urinary crux, and abdominal and heart diseases [7].

Antilithiatic Activity
In Indian traditional medicine, the rhizomes of B. ligulata have been considered a potential drug in the management of renal stones [87]. Dichloromethane, a bioactive fraction of B. ligulata extract showed high efficiency against kidney stone aggregation when administered orally for 21 days at a dose of 7 mg/kg body weight [88]. Spectroscopic analysis revealed bergenin to be the potent antilithiatic bioactive molecule isolated from rhizomes of B. ligulata [89]. Methanolic extract of B. ligulata and bergenin exhibited marked dissolution of urinary calculi both in kidney and urine constituents [90]. Treatment of rats with aqueous-methanolic extract of B. ligulata rhizomes at a dose range of 5-10 mg/kg body weight for 21 days was able to prevent ethylene glycol-induced urolithiasis by inhibiting CaC 2 O 4 crystal deposition in the renal tubules and simultaneously improved renal function [15]. An in vitro study confirmed that supplementation of dried leaves aqueous extract of B. ligulata has strong inhibitory potential against calcium oxalate monohydrate (COM) and hydrogen phosphate dehydrate crystal formation [91,92]. In ethylene glycol-induced hyperoxaluric rats, the dysfunction of mitochondria during stone crystal formation was manifested by reducing activities of electron transport chain complexes I, II and IV and, also by increasing mitochondrial oxidative stress. Oral administration of bergenin at a dose of 10 mg/kg for 28 days in ethylene glycol-induced hyperoxaluric rats showed amelioration of the damages to mitochondrial complexes as well as the alleviation of oxidative stress showing its potential effectiveness against urolithiasis [93]. A recent study on renal epithelial NRK-52E cells showed that ethanolic extract of B. ligulata significantly inhibits the nucleation and aggregation process of calcium oxalate crystals, further modulating the crystal structure by converting COM to the less pernicious form of calcium oxalate dihydrate (COD) [16].

Antipyretic Activity
Pyrexia or fever may occur due to acute infection or inflammation, or even due to any injury to tissue leading to the release of cytokines that initiate the synthesis of prostaglandin E2 (PgE2) in the hypothalamic area [94]. B. ligulata is considered an antipyretic herbal drug, and particularly the dried rhizome is used to prevent such fever [9]. Another study showed that ethanolic extract of roots and rhizomes of B. ligulata exhibit antipyretic activity at a dose of 500 mg/kg body weight in albino Wistar rats against yeast-induced fever. The rectal temperatures were recorded at time intervals of 1, 2, 3, 4 and 5 h after administration of B. ligulata extract [95].

Anti-Diabetic Activity
Plants belonging to the genus Bergenia play a crucial role in reducing the hyperglycemic condition. Rigorous studies on animal models revealed that B. ligulata possesses strong anti-diabetic activity [37]. Another possible mechanism of anti-diabetic action of B. ligulata may be attributed to its bioactive compound (+)-afzelechin, which acts as an inhibitor of α-glucosidase enzyme, as ascertained by enzyme inhibition assay [9,96]. The inhibition of α-glucosidase enzyme has been found to be effective in the treatment of hyperglycemia by delaying the absorption of carbohydrates in rat small intestines [96].

Anti-Inflammatory Activity
Bergenia species have strong anti-inflammatory activity as both the aqueous as well as ethanolic extracts of rhizomes showed effective anti-inflammatory potential in rat models [37]. B. ligulata is a well-known herb among folklore medical practitioners due to its anti-inflammatory potential. A study regarding the anti-inflammatory activity ensured its bioactive effect, where the oral application of aqueous, as well 50% ethanolic extracts of B. ligulata at a dose of 1 g/kg body weight of male Wistar rats, was able to attenuate the inflammatory response by reducing the level of succinate dehydrogenase (SDH), a key enzyme the level of which has been reported to rise during inflammation [6]. A possible explanation behind the therapeutic effect may be attributed to the bioactive molecule bergenin [97]. A new study on NRK-52 E cells revealed the reduction in inflammatory mediators such as MAPK, OPN, and nuclear factor kappa B (NF-kB) in presence of B. ligulata extract [16].

Hepatoprotective Activity
Bergenia species also have a hepato-protective effect. The administration of ethanolic root extract of B. ligulata to albino Wistar rats at a dose range of 25-35 g/kg body weight for 10 days exerted hepatoprotective activity, which was assessed by measuring the levels of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), serum alkaline phosphatase (ALP), and total bilirubin. All these parameters were significantly lower in the B. ligulata-treated group as compared to standard drugs [95].

Cardioprotective Activity
The cardioprotective potential of B. ligulata is also attributed to its phytochemical constituents. Several studies have highlighted its effects on different mammalian models. The administration of B. ligulata extract at a dose of 50 mg/kg body weight in dogs through an intravenous route showed effective hypotensive activity [7,98]. Alcoholic extract of rhizome also exhibited anti-bradykinin action without altering the action of 5-hydroxytryptamine (5-HT) receptor and acetylcholine as reported from isolated guinea pig ileum [7,9]. The scientific experiments that are carried out in mammalian models shows various pharmacological activities of the B. ligulata (Table 2). However, further studies are required for validation of these activities of the bioactive molecules present in the herb.

Possible Mechanism(s) of Action
Bioactive molecules of B. ligulata include polyphenols, flavonoids, and quinones that contribute to the pharmacological properties of the plant. The major ingredients of polyphenols include bergenin, arbutin, and catechin, which mostly add value to the medicinal value of the plant [37,100].

Anti-Urolithiatic Mechanism
The composition of kidney stones mainly depends on the physiologically and chemically altered urine. Depending on the chemical deposition, kidney stones can be classified as calcium stones-i.e., stones formed due to aggregation of calcium oxalate (CaOx) and calcium phosphate (Ca 3 (PO 4 ) 2 ) in renal calculi [101]. Other forms of stone include magnesium ammonium phosphate stones; uric acid stones; cystine stones; and drug-induced stones [101]. The general process of stone formation is common for all types of kidney stones. The most common, i.e., the calcium stone formation process, is mainly attributed to the crystallization of calcium oxalate, and the process involves nucleation, crystal development, aggregation, and the retention of crystal in the renal duct [93,101]. The wide range of action of bergenin can be demonstrated by its inhibitory effect on formation of kidney stones. Bergenin is a C-glycoside of 4-O-methyl gallic acid with an ionizing ability in buffer medium under neutral pH, whereas 4-O-methylglycoside (4-OMG) [102], the hydrolysis product of bergenin has negatively charged free carboxylate group, which enables it to bind with the calcium site of CaOx crystals and inhibit the growth and precipitation of CaOx crystals [89]. Similarly, other phenolic groups of B. ligulata such as arbutin and catechin also has the ability to interact with negatively charged oxalate ions by hydrogen bonds, which ultimately modulates the growth of CaOx crystals. Treatment with B. ligulata extract directly reduces the activity of lactate dehydrogenase (Figure 2), which is required for the formation of oxalate crystals in kidney [89].

Anti-Urolithiatic Mechanism
The composition of kidney stones mainly depends on the physiologically and chemically altered urine. Depending on the chemical deposition, kidney stones can be classified as calcium stones-i.e., stones formed due to aggregation of calcium oxalate (CaOx) and calcium phosphate (Ca3(PO4)2) in renal calculi [101]. Other forms of stone include magnesium ammonium phosphate stones; uric acid stones; cystine stones; and drug-induced stones [101]. The general process of stone formation is common for all types of kidney stones. The most common, i.e., the calcium stone formation process, is mainly attributed to the crystallization of calcium oxalate, and the process involves nucleation, crystal development, aggregation, and the retention of crystal in the renal duct [93,101]. The wide range of action of bergenin can be demonstrated by its inhibitory effect on formation of kidney stones. Bergenin is a C-glycoside of 4-O-methyl gallic acid with an ionizing ability in buffer medium under neutral pH, whereas 4-O-methylglycoside (4-OMG) [102], the hydrolysis product of bergenin has negatively charged free carboxylate group, which enables it to bind with the calcium site of CaOx crystals and inhibit the growth and precipitation of CaOx crystals [89]. Similarly, other phenolic groups of B. ligulata such as arbutin and catechin also has the ability to interact with negatively charged oxalate ions by hydrogen bonds, which ultimately modulates the growth of CaOx crystals. Treatment with B. ligulata extract directly reduces the activity of lactate dehydrogenase (Figure 2), which is required for the formation of oxalate crystals in kidney [89]. , has negatively charged free carboxylate group, which enables it to bind with the calcium site of calcium oxalate (CaOx) crystals and inhibit the precipitation of these crystals, thereby impeding renal stone formation.

Antioxidative Mechanism
Biological reactions in living organisms produce unpaired-electron-containing molecules called free radicals, referred to as ROS and reactive nitrogen species (RNS). , has negatively charged free carboxylate group, which enables it to bind with the calcium site of calcium oxalate (CaOx) crystals and inhibit the precipitation of these crystals, thereby impeding renal stone formation.

Antioxidative Mechanism
Biological reactions in living organisms produce unpaired-electron-containing molecules called free radicals, referred to as ROS and reactive nitrogen species (RNS). Antioxidants are the main defensive mechanism that neutralizes the action of free radicals. A balance between ROS and antioxidants is crucial for maintaining normal biological activity in living organisms. A minimal amount of free radicals are essential for immune responses, phagocytosis, activation of cellular receptors, processing of cellular signaling, and other important biological activities [103], whereas an excessive amount of free radical generation makes cells susceptible to oxidative stress, which ultimately promotes lipid peroxidation, DNA damage, oxidative modification of amino acid, and oxidative-stress-mediated peptide cleavage [103,104]. Bergenin and catechin are two major compounds found in B. ligulata that may mostly contribute to the antioxidant property of the plant [14]. Bergenin introduces free-radical-scavenging capacity against hydroxyl radicals by forming aromatic conjugated dienes, and it can also form a complex with Fe (II) that ultimately blocks the generation of hydroxyl radicals in the Fenton reaction [89]. The superoxide anion radical scavenging property of bergenin was assessed through the NADH radical scavenging assay, and the reducing property of the bioactive molecule was attributed to its electron-shifting ability [105]. The 11-O-galloylbergenin of B. ligulata has a benzoyl moiety with three hydroxyl groups, i.e., two meta and one para group, which provides 11-O-galloylbergenin a special structural orientation to easily interact with free radicals and scavenge on them [11] ( Figure 3).
Molecules 2022, 27, x FOR PEER REVIEW 1 Antioxidants are the main defensive mechanism that neutralizes the action of free cals. A balance between ROS and antioxidants is crucial for maintaining normal biol activity in living organisms. A minimal amount of free radicals are essential for im responses, phagocytosis, activation of cellular receptors, processing of cellular sign and other important biological activities [103], whereas an excessive amount of free cal generation makes cells susceptible to oxidative stress, which ultimately promote peroxidation, DNA damage, oxidative modification of amino acid, and oxidative-s mediated peptide cleavage [103,104]. Bergenin and catechin are two major compo found in B. ligulata that may mostly contribute to the antioxidant property of the [14]. Bergenin introduces free-radical-scavenging capacity against hydroxyl radic forming aromatic conjugated dienes, and it can also form a complex with Fe (II) tha mately blocks the generation of hydroxyl radicals in the Fenton reaction [89]. The s oxide anion radical scavenging property of bergenin was assessed through the N radical scavenging assay, and the reducing property of the bioactive molecule w tributed to its electron-shifting ability [105]. The 11-O-galloylbergenin of B. ligulata benzoyl moiety with three hydroxyl groups, i.e., two meta and one para group, provides 11-O-galloylbergenin a special structural orientation to easily interact wit radicals and scavenge on them [11] (Figure 3). Figure 3. Antioxidative mechanism of B. ligulata. Bergenin has been attributed to the free scavenging capacity against hydroxyl radicals through the formation of aromatic conjugated and Fe (II) complex, which ultimately block the generation of hydroxyl radicals. One of th pounds of B. ligulata, 11-O-galloylbergenin, has a special structural orientation due to the pr of two meta and one para group, which induces free radical scavenging by the herb. ROS-re oxygen species.

Antipyretic Mechanism
Pyrexia is defined as the abnormal elevation of body temperature, where ex temperature is more than 38 °C or internal temperature is above 38.4 °C [106]. In th of pyrogenic pyrexia, the generation of fever involves several mechanisms, most monly exogenous pyrogens such as microbes or endogenous pyrogens such as interl 1 (IL-1) or interleukin 6 (IL-6). TNF-α interacts with organum vasculosum of the l terminalis (OVLT), leading to the development of pyrexia. On the contrary, most a flammatory conditions are genetic and also related to malfunctioning of pro-infla tory cytokines such as interlukin-1 or interferon signaling or constitutive NF-kB (n Figure 3. Antioxidative mechanism of B. ligulata. Bergenin has been attributed to the free radical scavenging capacity against hydroxyl radicals through the formation of aromatic conjugated dienes and Fe (II) complex, which ultimately block the generation of hydroxyl radicals. One of the compounds of B. ligulata, 11-O-galloylbergenin, has a special structural orientation due to the presence of two meta and one para group, which induces free radical scavenging by the herb. ROS-reactive oxygen species.

Antipyretic Mechanism
Pyrexia is defined as the abnormal elevation of body temperature, where external temperature is more than 38 • C or internal temperature is above 38.4 • C [106]. In the case of pyrogenic pyrexia, the generation of fever involves several mechanisms, most commonly exogenous pyrogens such as microbes or endogenous pyrogens such as interleukin 1 (IL-1) or interleukin 6 (IL-6). TNF-α interacts with organum vasculosum of the lamina terminalis (OVLT), leading to the development of pyrexia. On the contrary, most autoinflammatory conditions are genetic and also related to malfunctioning of pro-inflammatory cytokines such as interlukin-1 or interferon signaling or constitutive NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, which are the most potent targets for the management of pyrexia [107]. In pyrexic conditions, there is an increase in prostaglandins (PGE2), which control the thermoregulatory center in the hypothalamus. Some antipyretic drugs introduce their antipyretic activity by blocking prostaglandin biosynthesis [108]. B. ligulata has antipyretic properties, particularly ethanolic root and rhizome extracts of the plant contain steroids, alkaloids, flavonoids, and terpenoids, which may block the activity of pyrogens on temperature-sensitive neurons in the preoptic part of the hypothalamus [17]. However, more detailed study is needed to reveal the bioactive molecules that are responsible for the antipyretic property ( Figure 4). factor kappa-light-chain-enhancer of activated B cells) activation, which are the most potent targets for the management of pyrexia [107]. In pyrexic conditions, there is an increase in prostaglandins (PGE2), which control the thermoregulatory center in the hypothalamus. Some antipyretic drugs introduce their antipyretic activity by blocking prostaglandin biosynthesis [108]. B. ligulata has antipyretic properties, particularly ethanolic root and rhizome extracts of the plant contain steroids, alkaloids, flavonoids, and terpenoids, which may block the activity of pyrogens on temperature-sensitive neurons in the preoptic part of the hypothalamus [17]. However, more detailed study is needed to reveal the bioactive molecules that are responsible for the antipyretic property ( Figure 4).

Anti-Diabetic Mechanism
Diabetes mellitus has several etiologies and is characterized by high blood glucose level resulting from the destruction of pancreatic beta cells, defects in insulin secretion, and abnormalities in insulin receptors [109]. Diabetes can also trigger other diseases such as cardiovascular diseases, neuropathy, retinopathy, and nephropathy [96]. B. ligulata extract may stimulate the pancreatic islet cells and increase insulin secretion to maintain normal blood glucose levels [7]. Afzelechin from B. ligulata extract exhibited anti-diabetic activity by inhibiting the enzymatic action of α-glucosidase, thus delaying the absorption of dietary carbohydrates in the small intestine and reducing postprandial hyperglycemia and hyper-insulinemia [96,110]. Similarly, (-)-3-O-galloylepicatechin and (-)-3-O-galloylcatechin isolated from B. ligulata have demonstrated inhibitory effects against porcine pancreatic α-amylase ( Figure 5), which also delays the absorption of glucose in the intestine [9,111].

Anti-Diabetic Mechanism
Diabetes mellitus has several etiologies and is characterized by high blood glucose level resulting from the destruction of pancreatic beta cells, defects in insulin secretion, and abnormalities in insulin receptors [109]. Diabetes can also trigger other diseases such as cardiovascular diseases, neuropathy, retinopathy, and nephropathy [96]. B. ligulata extract may stimulate the pancreatic islet cells and increase insulin secretion to maintain normal blood glucose levels [7]. Afzelechin from B. ligulata extract exhibited anti-diabetic activity by inhibiting the enzymatic action of α-glucosidase, thus delaying the absorption of dietary carbohydrates in the small intestine and reducing postprandial hyperglycemia and hyper-insulinemia [96,110]. Similarly, (-)-3-O-galloylepicatechin and (-)-3-O-galloylcatechin isolated from B. ligulata have demonstrated inhibitory effects against porcine pancreatic α-amylase ( Figure 5), which also delays the absorption of glucose in the intestine [9,111].

Anti-Inflammatory Mechanism
Inflammation is a response of the immune system to foreign substances and injuries, which is essential for tissue homeostasis under different noxious conditions [112]. Betasitosterol is an important constituent isolated from B. ligulata extract that possesses antiinflammatory properties. Beta-sitosterol directly induces the proliferation rate of T cells and releases interferon and increases natural killer cell activity as well. Anti-inflammatory properties of the B. ligulata plant may also be attributed to the synergistic effect of proinflammatory enzyme inhibitors that are responsible for the reduction in inflammatory response [6]. Bergenin also inhibits the formation of proinflammatory cytokines such as IL-2, interferon gamma (INF-ɣ), and TNF-α [45]. It also shows anti-microbial properties by inhibiting the growth of microbes in both in vivo and in vitro ( Figure 6); however, the exact mechanism of action is yet unknown [37].

Anti-Inflammatory Mechanism
Inflammation is a response of the immune system to foreign substances and injuries, which is essential for tissue homeostasis under different noxious conditions [112]. Betasitosterol is an important constituent isolated from B. ligulata extract that possesses antiinflammatory properties. Beta-sitosterol directly induces the proliferation rate of T cells and releases interferon and increases natural killer cell activity as well. Anti-inflammatory properties of the B. ligulata plant may also be attributed to the synergistic effect of proinflammatory enzyme inhibitors that are responsible for the reduction in inflammatory response [6]. Bergenin also inhibits the formation of proinflammatory cytokines such as IL-2, interferon gamma (INF-

Hepatoprotective Mechanism
Several studies have demonstrated the hepatoprotective activity of B. ligulata, although the mechanism of action is poorly understood. The antioxidant properties and cellular restoration capacity of the plant may play significant roles in the recovery of damaged liver tissues. Free radicals such as hydroxyl radicals, hydrogen peroxide, superoxide radicals, and lipid peroxide are predominant in liver diseases. These free radicals are normally generated during the biochemical process of the body or due to exposure to different environmental toxicants or pathological states [113]. Excess amounts of free radicals generate oxidative stress that alters the membrane structure and damage other important components of the cell including lipids, proteins, and nucleic acids [114]. The bioactive compounds of B. ligulata may exert hepatoprotective activity (Figure 7) through antioxidant and free-radical scavenging properties. Similarly, it can normalize increased Kuffer cells number and lymphocytic infiltration in infected mice [19]. In Wistar albino rats, B. ligulata root extract ameliorated carbon tetrachloride (CCl4)-induced liver damage along with a reduction in the level of alkaline phosphatase (ALP), total bilirubin level, SGPT, and SGOT [95]. The major bioactive component of B. ligulata, i.e., bergenin, might be responsible for the hepatoprotective properties of the plant. Bergenin at doses of 50, 100, and 200 mg/kg body weight showed a strong hepatoprotective effect when administrated orally for 7 successive days in CCl4-induced liver damage in rats. The administration of bergenin subsequently normalizes the increasing serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase, and γ-glutamyltransferase in CCl4-), and TNF-α [45]. It also shows anti-microbial properties by inhibiting the growth of microbes in both in vivo and in vitro ( Figure 6); however, the exact mechanism of action is yet unknown [37].

Hepatoprotective Mechanism
Several studies have demonstrated the hepatoprotective activity of B. ligulata, although the mechanism of action is poorly understood. The antioxidant properties and cellular restoration capacity of the plant may play significant roles in the recovery of damaged liver tissues. Free radicals such as hydroxyl radicals, hydrogen peroxide, superoxide radicals, and lipid peroxide are predominant in liver diseases. These free radicals are normally generated during the biochemical process of the body or due to exposure to different environmental toxicants or pathological states [113]. Excess amounts of free radicals generate oxidative stress that alters the membrane structure and damage other important components of the cell including lipids, proteins, and nucleic acids [114]. The bioactive compounds of B. ligulata may exert hepatoprotective activity (Figure 7) through antioxidant and free-radical scavenging properties. Similarly, it can normalize increased Kuffer cells number and lymphocytic infiltration in infected mice [19]. In Wistar albino rats, B. ligulata root extract ameliorated carbon tetrachloride (CCl 4 )-induced liver damage along with a reduction in the level of alkaline phosphatase (ALP), total bilirubin level, SGPT, and SGOT [95]. The major bioactive component of B. ligulata, i.e., bergenin, might be responsible for the hepatoprotective properties of the plant. Bergenin at doses of 50, 100, and 200 mg/kg body weight showed a strong hepatoprotective effect when administrated orally for 7 successive days in CCl 4 -induced liver damage in rats. The administration of bergenin subsequently normalizes the increasing serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase, and γ-glutamyltransferase in CCl 4 -treated rats in a dose-dependent manner. In contrast, the recuperation of the activities of glutathione S-transferase and glutathione reductase was also reported. Additionally, bergenin can prevent the elevation of hepatic malondialdehyde formation and the depletion of reduced glutathione content in the liver of CCl 4 -intoxicated rats [115,116].

Hepatoprotective Mechanism
Several studies have demonstrated the hepatoprotective activity of B. ligulata, although the mechanism of action is poorly understood. The antioxidant properties and cellular restoration capacity of the plant may play significant roles in the recovery of damaged liver tissues. Free radicals such as hydroxyl radicals, hydrogen peroxide, superoxide radicals, and lipid peroxide are predominant in liver diseases. These free radicals are normally generated during the biochemical process of the body or due to exposure to different environmental toxicants or pathological states [113]. Excess amounts of free radicals generate oxidative stress that alters the membrane structure and damage other important components of the cell including lipids, proteins, and nucleic acids [114]. The bioactive compounds of B. ligulata may exert hepatoprotective activity (Figure 7) through antioxidant and free-radical scavenging properties. Similarly, it can normalize increased Kuffer cells number and lymphocytic infiltration in infected mice [19]. In Wistar albino rats, B. ligulata root extract ameliorated carbon tetrachloride (CCl4)-induced liver damage along with a reduction in the level of alkaline phosphatase (ALP), total bilirubin level, SGPT, and SGOT [95]. The major bioactive component of B. ligulata, i.e., bergenin, might be responsible for the hepatoprotective properties of the plant. Bergenin at doses of 50, 100, and 200 mg/kg body weight showed a strong hepatoprotective effect when administrated orally for 7 successive days in CCl4-induced liver damage in rats. The administration of bergenin subsequently normalizes the increasing serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase, and γ-glutamyltransferase in CCl4treated rats in a dose-dependent manner. In contrast, the recuperation of the activities of glutathione S-transferase and glutathione reductase was also reported. Additionally, bergenin can prevent the elevation of hepatic malondialdehyde formation and the depletion of reduced glutathione content in the liver of CCl4-intoxicated rats [115,116].

Hepatoprotective Mechanism
Several studies have demonstrated the hepatoprotective activity of B. ligulata, although the mechanism of action is poorly understood. The antioxidant properties and cellular restoration capacity of the plant may play significant roles in the recovery of damaged liver tissues. Free radicals such as hydroxyl radicals, hydrogen peroxide, superoxide radicals, and lipid peroxide are predominant in liver diseases. These free radicals are normally generated during the biochemical process of the body or due to exposure to different environmental toxicants or pathological states [113]. Excess amounts of free radicals generate oxidative stress that alters the membrane structure and damage other important components of the cell including lipids, proteins, and nucleic acids [114]. The bioactive compounds of B. ligulata may exert hepatoprotective activity (Figure 7) through antioxidant and free-radical scavenging properties. Similarly, it can normalize increased Kuffer cells number and lymphocytic infiltration in infected mice [19]. In Wistar albino rats, B. ligulata root extract ameliorated carbon tetrachloride (CCl4)-induced liver damage along with a reduction in the level of alkaline phosphatase (ALP), total bilirubin level, SGPT, and SGOT [95]. The major bioactive component of B. ligulata, i.e., bergenin, might be responsible for the hepatoprotective properties of the plant. Bergenin at doses of 50, 100, and 200 mg/kg body weight showed a strong hepatoprotective effect when administrated orally for 7 successive days in CCl4-induced liver damage in rats. The administration of bergenin subsequently normalizes the increasing serum enzymatic activities of alanine/aspartate aminotransferase, sorbitol dehydrogenase, and γ-glutamyltransferase in CCl4treated rats in a dose-dependent manner. In contrast, the recuperation of the activities of glutathione S-transferase and glutathione reductase was also reported. Additionally, bergenin can prevent the elevation of hepatic malondialdehyde formation and the depletion of reduced glutathione content in the liver of CCl4-intoxicated rats [115,116].

Future Perspectives
Plant-based natural remedies have been used worldwide for the management of various human ailments for ages. In developing countries, approximately 8 out of 10 people opt for herbal treatment for their primary health care, due to easy accessibility, low toxicity, and inherited traditional knowledge of using plant and their derivatives in the form of either the whole plant or part of the plant, such as root, rhizome, leaf, fruit, or flower. In this modern era, researchers are focusing on herbal medicines by taking reference from ligulata. The plant extract shows hepatoprotective properties through antioxidant activity and free radical scavenging activity. Bioactive compounds reduce the number of Kuffer cells in the liver and inhibit the infiltration of lymphocytes. It also reduces alkaline phosphate, total bilirubin, serum glutamic-pyruvic transaminase (SGPT) and serum glumatic-oxaloacetic transaminase (SGOT), alanine aminotransferase (ALT), sorbitol dehydrogenase, and γ-glutamyl transferase. Further, it helped resist the depletion of reduced glutathione content and inhibit malondialdehyde formation in the liver of CCl 4 -treated rats.

Future Perspectives
Plant-based natural remedies have been used worldwide for the management of various human ailments for ages. In developing countries, approximately 8 out of 10 people opt for herbal treatment for their primary health care, due to easy accessibility, low toxicity, and inherited traditional knowledge of using plant and their derivatives in the form of either the whole plant or part of the plant, such as root, rhizome, leaf, fruit, or flower. In this modern era, researchers are focusing on herbal medicines by taking reference from indigenous ancient traditional literature such as Ayurveda, Unani, Siddha, traditional Chinese medicine, and others, as a reverse pharmacology approach. Cutting-edge analytical tools and techniques are paving the way for the identification and validation of bioactive molecules from herbs and their use on par with standard modern synthetic drugs. The bioactive molecules of various traditionally used plants have been clinically proven as effective against a range of acute and chronic diseases. Further studies on the traditional herbs may lead to development of several novel drugs against various long-standing human ailments. In this review, we attempted to provide the mechanism(s) of action of B. ligulata and/or its phytoconstituents to better understand how the plant executes its anti-urolithiatic, antioxidative, antipyretic, anti-diabetic, anti-inflammatory, and hepatoprotective properties, as well as discuss the important bioactive compounds of clinical importance. The studies conducted on rats and other mammalian models provide a deep insight into the therapeutic potential of B. ligulata in the management of various ailments. Despite limited data, B. ligulata has been shown to exert antioxidant properties, reduce cellular oxidative stress, and modulate enzyme action (α-glucosidase) under in vivo and in vitro conditions, which renders the herb interesting for further clinical studies.