Role of Vacha (Acorus calamus Linn.) in Neurological and Metabolic Disorders: Evidence from Ethnopharmacology, Phytochemistry, Pharmacology and Clinical Study.

Vacha (Acorus calamus Linn. (Acoraceae)) is a traditional Indian medicinal herb, which is practiced to treat a wide range of health ailments, including neurological, gastrointestinal, respiratory, metabolic, kidney, and liver disorders. The purpose of this paper is to provide a comprehensive up-to-date report on its ethnomedicinal use, phytochemistry, and pharmacotherapeutic potential, while identifying potential areas for further research. To date, 145 constituents have been isolated from this herb and identified, including phenylpropanoids, sesquiterpenoids, and monoterpenes. Compelling evidence is suggestive of the biopotential of its various extracts and active constituents in several metabolic and neurological disorders, such as anticonvulsant, antidepressant, antihypertensive, anti-inflammatory, immunomodulatory, neuroprotective, cardioprotective, and anti-obesity effects. The present extensive literature survey is expected to provide insights into the involvement of several signaling pathways and oxidative mechanisms that can mitigate oxidative stress, and other indirect mechanisms modulated by active biomolecules of A. calamus to improve neurological and metabolic disorders.


Introduction
Globally, an estimated 450 million people are suffering from mental disorders and about 425 million are known diabetics [1,2]. In 2016, 650 million adults were obese and about 23.6 million people were estimated to die of cardiovascular diseases (CVDs) by the year 2030 [3]. Metabolic disorders are characterized by hypertension, hyperglycemia, abdominal obesity, and hyperlipidemia, which may worsen the neurological disease risk. Improper diet (high calorie intake), lifestyle (e.g., smoking, chronic alcohol consumption, sedentary habits), and/or low level of nitrosamines (through processed food, tobacco smoke, and nitrate-containing fertilizers) affect the liver and can further lead to fatty liver disease [4,5]. In this condition, fatty changes may be due to increased production or decreased use of fatty Acorus calamus Linn. (Acoraceae), also known as Vacha in Sanskrit, is a mid-term, perennial, fragrant herb which is practiced in the Ayurvedic (Indian traditional) and the Chinese system of medicine. The plant's rhizomes are brown in color, twisted, cylindrical, curved, and shortly nodded. The leaves are radiant green, with a sword-like structure, which is thicker in the middle and has curvy margins ( Figure 2) [8]. Several reports ascertained a wide range of biological activities involving its myriad of active phytoconstituents. In this sense, the intent of this review is to assemble and summarize the geographical distribution, ethnopharmacology, phytochemistry, mechanism of action of A. calamus along with preclinical and clinical claims that are relevant to manage neurological and metabolic disorders. To the best of our knowledge, so far, none of the published reviews has described all the characteristics of this medicinal plant [9][10][11]. The present report is expected to produce a better understanding of the characteristics, bioactivities, and mechanistic aspects of this plant and to provide new leads for future research.

Methodology
The literature available in the Ayurvedic classical texts, technical reports, online scientific records such as SciFinder, Google Scholar, MEDLINE, EMBASE, Scopus directory were explored for ethnomedicinal uses, geographical distribution, phytochemistry, pharmacology, and biomedicine by applying the following keywords: "Acorus calamus", "Vacha", "Medhya", "neuroprotective", "phytochemistry", "obesity", "oxidative stress", "anticonvulsant", "antidepressant", "antihypertensive", "anti-inflammatory", "immunomodulator", "antioxidant", "diabetes", "mechanism of action" with their corresponding medical subject headings (MeSH) terms using conjunctions OR/AND. The search was focused on identifying Ayurvedic claims in the available ethnomedicinal, phytochemical, preclinical, clinical, and toxicity reports to understand the role of A. calamus in neurological and metabolic disorders. This search was undertaken between January 2018 and January 2020. Searches were restricted to the English language. The search methodology as per the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) is stipulated in the flowchart in Figure 3.

Methodology
The literature available in the Ayurvedic classical texts, technical reports, online scientific records such as SciFinder, Google Scholar, MEDLINE, EMBASE, Scopus directory were explored for ethnomedicinal uses, geographical distribution, phytochemistry, pharmacology, and biomedicine by applying the following keywords: "Acorus calamus", "Vacha", "Medhya", "neuroprotective", "phytochemistry", "obesity", "oxidative stress", "anticonvulsant", "antidepressant", "antihypertensive", "anti-inflammatory", "immunomodulator", "antioxidant", "diabetes", "mechanism of action" with their corresponding medical subject headings (MeSH) terms using conjunctions OR/AND. The search was focused on identifying Ayurvedic claims in the available ethnomedicinal, phytochemical, preclinical, clinical, and toxicity reports to understand the role of A. calamus in neurological and metabolic disorders. This search was undertaken between January 2018 and January 2020. Searches were restricted to the English language. The search methodology as per the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) is stipulated in the flowchart in Figure 3.

Methodology
The literature available in the Ayurvedic classical texts, technical reports, online scientific records such as SciFinder, Google Scholar, MEDLINE, EMBASE, Scopus directory were explored for ethnomedicinal uses, geographical distribution, phytochemistry, pharmacology, and biomedicine by applying the following keywords: "Acorus calamus", "Vacha", "Medhya", "neuroprotective", "phytochemistry", "obesity", "oxidative stress", "anticonvulsant", "antidepressant", "antihypertensive", "anti-inflammatory", "immunomodulator", "antioxidant", "diabetes", "mechanism of action" with their corresponding medical subject headings (MeSH) terms using conjunctions OR/AND. The search was focused on identifying Ayurvedic claims in the available ethnomedicinal, phytochemical, preclinical, clinical, and toxicity reports to understand the role of A. calamus in neurological and metabolic disorders. This search was undertaken between January 2018 and January 2020. Searches were restricted to the English language. The search methodology as per the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) is stipulated in the flowchart in Figure 3.

Geographical Distribution
A. calamus grows in high (1800 m) and low (900 m) altitudes and it is found to be geographically available in 42 countries [8]. Furthermore, as per the Global Biodiversity Information Facility records [12], the distribution of this plant in several parts of the world, as well as in India, is highlighted in Figure 4.

Ethnomedicinal Use
This plant is being practiced traditionally in the Indian Ayurvedic tradition, as well as in the Chinese system of medicine for analgesic, antipyretic, tonic, anti-obesity, and healing purposes; it is highly effective for skin diseases, along with neurological, gastrointestinal, respiratory, and several other health disorders. Rhizomes and leaves are found to be profusely practiced in the form of infusion, powder, paste, or decoction . The ethnomedicinal uses of the A. calamus are detailed in Table 1.
A. calamus rhizomes and leaves are also used as an active pharmaceutical ingredient in various Ayurvedic formulations (Table 2). Cold, cough, and fever Rhizomes paste of A. calamus is given to children with mother's milk, Myristica fragrance, and Calunarejan spinosa fruits [15] Two teaspoonfuls of herbal powder containing A. calamus rhizomes, Boerhaavia diffusa roots, Calonyction muricutum flower pedicles, Ipomoea muricate seeds, Senna leaves, Cassia fistula fruits pulp, Curcuma longa rhizomes, Helicteres isora fruits, and Mentha arvensis leaves, black pepper is taken with lukewarm water [16] Gastric disorders A. calamus rhizomes paste is given with cow milk [17] Carminative, flavoring, tonic, and head lice infestation Infusion of a dried rhizomes (collected and stored in the autumn season) [17][18][19] Epilepsy, dysentery, mental illnesses, diarrhea, kidney and liver disorders A. calamus rhizomes paste is given with honey [20] Wounds, fever, body pain Rhizomes [21,22] Dysentery Fresh ground rhizomes is mixed with hot water and given for 3 days [23] Stimulant Dry powder of A. calamus is given with honey [24] Injuries External application of the A. calamus rhizomes paste Dermal [25] Stomachache Ash of the A. calamus rhizomes paste [26] Otitis externa A. calamus roots paste is given with coconut husk juice [27] Lotion Fresh leaves of A. calamus [28] Cough, cancer, and fever A. calamus roots juice is given with honey and MyristicaDactyloides [29]

Phytochemistry
The phytochemical investigation of this plant has been ongoing since the year 1957 [73,74]. To date, about 145 compounds were isolated from A. calamus rhizomes and leaves, viz. phenylpropanoids, sterols, triterpene glycosides, triterpenoid saponins, sesquiterpenoids, monoterpenes, and alkaloids (Table 3). Amongst those, phenylpropanoids (chiefly, asarone and eugenol) and sesquiterpenoids have been considered the principal effective compounds of A. calamus. Chemical structures of isolated compounds from A. calamus are illustrated in Figure 5.

Pharmacological Properties
Diverse bioactivities of A. calamus extracts are evident from preclinical (in vitro and in vivo) and clinical reports, such as antidiabetic, anti-obesity, antihypertensive, antioxidant, anti-inflammatory, immunomodulatory, anticonvulsant, and neuroprotective . The summarized information on A. calamus botanical parts, extract type, and their bioactivities in neurological and metabolic disorders is stipulated in Table 4.

Antidiabetic Effect
The antidiabetic effect of A. calamus ethyl acetate fraction was evaluated in streptozotocin (STZ)induced and diabetic (db/db) mice. Glucagon-like peptide-1 (GLP-1) levels, plasma insulin, "and related gene expression were evaluated. The fraction (100 mg/kg, intragastric (i.g.)) indicated a significant reduction in blood glucose levels. For in vitro, at the concentration of 12.5 μg/mL, a significant increment in GLP-1 levels was found in the insulin-secreting L-cell culture medium [108]. The ethyl acetate radix fraction exhibited a significant effect on the HIT-T15 cell line and αglucosidase enzyme. The ethyl acetate fraction also enhanced insulin secretion in HIT-T15 cells and blocked the α-glucosidase in vitro activity with 0.41 μg/mL of inhibitory concentration (IC50) [109]."

Anti-Obesity Effect
The β-asarone compound isolated from the rhizome was investigated against high-fat diet (HFD)-induced obesity in animals. β-Asarone-treated adipose rats showed weight loss, but also inhibited metabolic transformations, as well as glucose intolerance, elevated cholesterol, and adipokine variance [143]. The in vitro investigation on the A. calamus aqueous extract showed lipidlowering activity through inhibition of the pancreatic lipase percentage (28.73%) [144].

Antihypertensive Effect
The antihypertensive effects of A. calamus were studied on their own, in isolation, and in combination with Gymnema sylvestre in the HFD-induced hypertension in rats. The HFD was given for 4 weeks, which significantly increased the average systolic blood pressure (SBP). At a 200 mg/kg

Phenylpropanoids
Phenylpropanoids have an aromatic ring with a structurally diverse group of phenylalaninederived secondary plant metabolites (C 6 -C 3 ), like α-asarone, β-asarone, eugenol, isoeugenol, etc. [75]. A number of phenylpropanoids have been identified from A. calamus rhizome and leaves (1-45). α and β-asarone isolated from the rhizome are the predominant compounds present in this plant. A series of aromatic oils from the rhizome with diverse structures are also reported .

Triterpenoid Saponins
Triterpenoid saponins are made up of a pentacyclic C-30 terpene skeleton as a pillar. Limited reports studying triterpenoid saponins in A. calamus are available, and only two triterpenoid saponins (124,125) have been isolated from A. calamus rhizomes (Table 3) [85].

Pharmacological Properties
Diverse bioactivities of A. calamus extracts are evident from preclinical (in vitro and in vivo) and clinical reports, such as antidiabetic, anti-obesity, antihypertensive, antioxidant, anti-inflammatory, immunomodulatory, anticonvulsant, and neuroprotective . The summarized information on A. calamus botanical parts, extract type, and their bioactivities in neurological and metabolic disorders is stipulated in Table 4.

Antidiabetic Effect
The antidiabetic effect of A. calamus ethyl acetate fraction was evaluated in streptozotocin (STZ)-induced and diabetic (db/db) mice. Glucagon-like peptide-1 (GLP-1) levels, plasma insulin, "and related gene expression were evaluated. The fraction (100 mg/kg, intragastric (i.g.)) indicated a significant reduction in blood glucose levels. For in vitro, at the concentration of 12.5 µg/mL, a significant increment in GLP-1 levels was found in the insulin-secreting L-cell culture medium [108]. The ethyl acetate radix fraction exhibited a significant effect on the HIT-T15 cell line and α-glucosidase enzyme. The ethyl acetate fraction also enhanced insulin secretion in HIT-T15 cells and blocked the α-glucosidase in vitro activity with 0.41 µg/mL of inhibitory concentration (IC 50 ) [109]."

Anti-Obesity Effect
The β-asarone compound isolated from the rhizome was investigated against high-fat diet (HFD)-induced obesity in animals. β-Asarone-treated adipose rats showed weight loss, but also inhibited metabolic transformations, as well as glucose intolerance, elevated cholesterol, and adipokine variance [143]. The in vitro investigation on the A. calamus aqueous extract showed lipid-lowering activity through inhibition of the pancreatic lipase percentage (28.73%) [144].

Antihypertensive Effect
The antihypertensive effects of A. calamus were studied on their own, in isolation, and in combination with Gymnema sylvestre in the HFD-induced hypertension in rats. The HFD was given for 4 weeks, which significantly increased the average systolic blood pressure (SBP). At a 200 mg/kg dose, A. calamus in combination with G. sylvestre reduced the SBP and heart rate significantly. A. calamus with G. sylvestre exhibited synergistic effect as compared with individual herbs [145].

Anti-Inflammatory and Immunomodulatory Effect
The methanolic A. calamus rhizome extract (12.5 µg/mL) prevented the VCAP-1 and intercellular expression on the surface of mouse myeloid leukemia cells and murine endothelial cells, respectively [146].
In an in vitro anti-inflammatory study (Red blood cell membrane stabilization method), the A. calamus aqueous rhizome extract at the highest concentration of 10 mg/mL showed insignificant activity against hemolysis inhibition and the RBC membrane stabilization percentage [144]. Aqueous A. calamus leave extract was studied on HaCaT cells and restricted the characteristics of interleukin (IL)-8, IL-6 RNA protein levels alongside interferon regulatory factor 3 (IRF3) and nuclear factor kB (NF-κB) activation [147]. N-hexane, butanolic, and aqueous fractions of A. calamus were evaluated against cyclooxygenase (COX) and lipoxygenase (LOX)-mediated eicosanoid production by arachidonic acid. The butanolic fraction inhibited the COX-mediated production of thromboxane B2 (TXB2) and lipoxygenase product 1 (LP1). Investigation of the underlying signaling pathways revealed that the butanolic fraction inhibited phospholipase C (PLC) pathway in platelets, presumably acting on protein kinase C (PKC) [148]. The essential oil isolated from A. calamus was evaluated by protein denaturation assay, where at the concentration level of 300 µg/mL, 69.56% of the inhibition level was observed [149].

Antioxidant Effect
The in vitro antioxidant activity of acetone, acetonitrile, alcoholic, and aqueous extracts of A. calamus rhizomes exhibited free radical scavenging activity on the [2,2 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid)] free radical scavenging activity assay (ABTS), the (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity assay (DPPH), and the ferric ion reducing antioxidant power assay (FRAP). Strong antioxidant effect was noticed in the acetone extract, followed by acetonitrile and methanol, while in the aqueous extract, poor antioxidant activity was found [150]. The aqueous extract exhibited superior antioxidant effects in metal ion chelation, lipid peroxidation (LPO), and DPPH assays [144,151]. The in vitro antioxidant activity of ethanol, hydro-ethanol, and aqueous whole plant extracts of A. calamus was investigated using FRAP, DPPH, nitric oxide, hydroxyl radical, reductive ability, and superoxide radical scavenging activity. The existence of phenolics and flavonoids in A. calamus are believed to contribute to the promising antioxidant effect. IC 50 values of the ethanol extract were found to be 54.82, 109.85, 38.3, 118.802 µg/mL for the scavenging activities of DPPH, hydroxyl radical, superoxide radical, and nitric oxide, respectively. The irreversible potential of the above results and the FRAP values of the extracts were found to augment in a concentration-dependent manner [152]. "Ethanol and hydro-alcoholic extracts of A. calamus roots and rhizomes were studied for antioxidant potential against DPPH compared with butylated hydroxyanisole (BHA) and silymarin. Ethanol and hydro-alcoholic extracts showed free radical scavenging activity of 59.13 ± 18.95 and 56.71 ± 19.54, respectively [153][154][155]. The essential oil isolated from A. calamus showed strong antioxidant efficacy against the β-carotene/linoleic acid bleaching test and DPPH free radicals [156]. The methanol extract of the A. calamus rhizome was evaluated against the free radical scavenging activity, and the reported IC 50 value was 704 µg/mL [157]. The IC 50 of the essential oil was 1.68 µg/mL, which showed virtuous free radical scavenging activity in the DPPH test [149]."

Anticonvulsant Effect
The methanol extract shows anticonvulsant effects feasibly through potentiating the action of gamma-aminobutyric acid (GABA) pathway in the central nervous system [124]. When it comes to the purification of A. calamus rhizome in cow urine, it is advocated in the Ayurvedic pharmacopoeia of India (API) before its therapeutic use. The purified rhizome was investigated in a maximal electroshock (MES) seizure model, and phenytoin was used as the standard drug. The raw and processed rhizome (11 mg/kg, p.o.) exhibited notable anticonvulsant activity by minimizing the span of the tonic extensor period in rats, whereas the processed rhizome showed better therapeutic activity than when it was raw [158]. The calamus oil isolated from the A. calamus rhizome was evaluated at varying dose levels of 30, 100, and 300 mg/kg, p.o., body weight (b.w.), against MES, pentylenetetrazol (PTZ), and minimal clonic seizure (MCS) models. The calamus oil was found to be neurotoxic at 300 mg/kg, though it was effective in the MCS test at 6 Hz. The protective index value of calamus oil was found to be 4.65 [125].

Antidepressant Effect
Interaction of the methanolic A. calamus rhizome extract with the adrenergic, dopaminergic, serotonergic, and GABAergic system was found responsible for the expression of antidepressant activity [128]. In another study, the methanolic A. calamus leave extract showed significant activity through a reduction in the immobility period in the TST and FST [129]. Through interaction with the adrenergic and dopaminergic system, the hydro-alcoholic extract was normalized to the over-activity of the hypothalamic pituitary adrenal (HPA) axis [131]. Sobers capsules (a herbo-mineral formulation containing A. calamus) were evaluated by tail suspension and forced swimming tests in mice. At the oral dose of 50 mg/kg for 14 days, capsules exhibited insignificant impact on locomotor activity, and caused antidepressant effects in experimental animals [159]. Tensarin (the traditional medicine of Nepal containing A. calamus) was evaluated for the anxiolytic effect in mice using the open field test (OFT), activity monitoring along with the passive avoidance test. At all three dose levels (50, 100, 200 mg/kg), Tensarin produced an anxiolytic effect in a dose-dependent way by an improvement in rearing, number of passages, and duration of the period employed by mice [160].

Neuroprotective Effect
The ethanolic extract was studied (25, 50, and 100 mg/kg doses, oral and intraperitoneal routes) for learning and memory-enhancing activity. The subjects used consisted of male rates, through Y maze and shuttle box tests models. The findings showed an increase in acquisition-recalling and spatial recognition data [161]. The ethanolic A. calamus rhizome extract (0.5 mL/kg, i.p.) potentiated pentobarbitone-created sleep periods, which caused significant inhibition of conditioned avoidance response in rats and marked (40-60%) protection against PTZ-induced convulsions, although it did not show any spontaneous motor activity and impact the aggressive or fighting behavior response in male rat pairs [162].

Cardioprotective Effect
The alcoholic A. calamus rhizome extract (100 and 200 mg/kg) considerably attenuated isoproterenol-led cardiomyopathy in rats and showed a significant reduction in the heart/body weight ratio, level of serum calcineurin, serum nitric oxide, serum lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances (TBARS) level. However, the level of the antioxidant enzyme was found increased at the 100 mg/kg extract dose level [163]. The crude extract and its fractions (0.01-10 mg/mL) were investigated in an isolated rabbit heart, which showed mild reduction in the force of forced vital capacity (FVC), hazard ratio (HR), and cystic fibrosis (CF), while the ethyl acetate extract exhibited complete suppression, and the n-hexane fraction showed the same effect on FVC and HR, but enhanced CF. The extract and its fractions exhibited controlled coronary vasodilator effect, interceded maybe by an endothelial-derived hyperpolarizing factor [164]. The cardioprotective potential of the whole plant's ethanolic extract (100 and 200 mg/kg) reduced serum enzyme levels and shielded the myocardium from the lethal effect of DOX [141].

Cytochrome Inhibitory Activities
Cytochromes P450 (CYPs) are the prime enzymes that catalyze the oxidative metabolism of a wide variety of xenobiotics. It is known that 2,4,5-trimethoxycinnamic acid is the main metabolite of αor βasarone [165]. The metabolism rate of αand β-asarone was shown to be directly proportional to the CYPs concentration in rat hepatocytes and liver microsomes [166,167]. CYP3A4 (CYP isoforms) has been reported for bioactivation of α-asarone [168]. The hydro-alcoholic A. calamus extract and α-asarone were evaluated by the CYPs-carbon monoxide complex method. The extract exhibited moderate potential interaction in CYP3A4 (IC 50 = 46.84 µg/mL) and CYP2D6 (IC 50 = 36.81 µg/mL), while α-asarone showed higher interaction in CYP3A4 (IC 50 = 65.16 µg/mL) and CYP2D6 (IC 50 = 55.17 µg/mL) [169]. These outcomes indicated that both extracts and α-asarone interacted quite well in drug metabolism and also had an inhibitory effect on CYP3A4 and CYP2D6. The drug-drug interaction effect of the A. calamus extract and its main chemical constituent (α and β-asarone) needs to be studied in more CYPs isomers, like CYP2C9 and CYP2E1.

Toxicity and Safety Concerns
In acute and sub-acute toxicity of the hydro-alcoholic extract of A. calamus in rats, at the highest dose level of 10 gm/kg, no severe changes were observed, and the lethal dose (LD 50 ) was found to be 5 g/kg [170]. The petroleum ether extracts (obtained by cold rolling, water distillation, and Soxhlet extraction methods) of the A. calamus rhizome showed mild toxicity in two-day-old oriental fruit flies [171]. The ethanolic extract of the A. calamus rhizome at oral dosage of 175, 550, 1750, and 5000 mg/kg b.w. was given for 14 days within an acute toxicity study, while at the dose level of 0, 200, 400, and 600 mg/kg, p.o., the extract was given for 90 days within a chronic toxicity study. At the doses of 1750 and 5000 mg/kg, piloerection, tremors, and abdominal breathing were found for 30 min [172]. In that study, A. calamus was purified for 3 h in cow urine, decoction of Sphaeranthus indicus, and decoction of leaves of Mangifera indica, Eugenia jambolana, Feronia limonia, Citrus medica, and Aegle marmelos, followed by fomentation with Gandhodaka (decoction of six aromatic herbs) for 1 h. The acute oral toxicity test of raw and purified A. calamus was performed in albino rats at 2000 mg/kg for 2 weeks. At the 2000 mg/kg dose, A. calamus did not produce any toxic symptoms within 14 days [173].
The β-asarone compound isolated from A. calamus was found to be carcinogenic and toxic [174]. The LD 50 value of β-asarone by oral and intraperitoneal route was found to be 1010 and 184 mg/kg, respectively, in mice and rats [175]. The LD 50 of calamus oil was found to be 8.88 gm/kg b.w. [176], while in the calamus oil obtained from Jammu, India, the LD 50 was 777 mg/kg b.w. [177]. Overall, several investigations have been carried out on A. calamus regarding its toxicity; however, no noticeable data on toxicity have been found so far.

Clinical Reports
A. calamus has also been clinically investigated as a monotherapy as well as in combination with other medicinal herbs in healthy subjects and sufferers of various metabolic and neurological ailments. Most clinical research has looked at the A. calamus effect on obesity, depression, neuroprotection, and cardiovascular disease [178][179][180][181][182][183][184][185][186][187][188][189][190][191]. The data obtained so far can be found in Table 5. Furthermore, a systematic review reveals that A. calamus (alone or in combination therapy) exhibits anti-obesity, antidepressant, and cardioprotective effects, as well as helps physical and mental performance.   [191] ADAS-cog, alzheimer's disease assessment scale-cognitive subscale; BMI, body mass index; BPRS, brief psychiatric rating scale; CDR-SOB, clinical dementia rating scale sum of boxes; DBP, diastolic blood pressure; ECG, electrocardiogram; Ib, evidence from at least one randomized study with control; HDL, high-density lipoprotein; Ig, immunoglobulin; III, evidence from well-performed nonexperimental descriptive studies, as well as from comparative studies, correlation studies, and case studies; LDL, low-density lipoprotein; MAP, mean arterial pressure; SBP, systolic blood pressure.

Mechanistic Role
The proposed mechanism of action of A. calamus in neurological and metabolic disorders includes a synergic integration of antioxidant defense, GABAergic transmission, brain stress hormones modulation, pro-inflammatory cytokines, leptin and resistin levels, adipocytes inhibition, calcium channel blocker effect, protein synthesis, oxidative stress, acetylcholinesterase (AChE) inhibition, and anti-dopaminergic properties. A compendium of mechanisms of action of A. calamus in neurological and metabolic protection is illustrated in Figure 6 and Table 6. A. calamus significantly affects fasting blood sugar, insulin resistance, HbA1c, and the adipogenic transcription expression factor through various mechanisms, viz. antioxidant, anti-inflammatory, β-cells regeneration, improving insulin sensitivity, gluconeogenesis, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and glucose transporter type 4 (GLUT-4)-mediated transport inhibition.
PPAR gene and C/EBP are involved in the differentiation process. PPAR-δ and PPAR-γ promote adipogenesis. In the same way, amino acids and glucose react with C/EBP-δ and C/EBP-β. If low levels of glucose induce gadd153, the inactive dimer is formed, with C/EBP-β inhibiting the progress of adipocyte development. C/EBP delta activates C/EBP-α. This is mainly involved in the formation of mature adipocytes and lipid accumulation in adipose tissue. In 3T3-L1 preadipocytes, α-asarone and β-asarone inhibited adipocyte differentiation and reduced the intracellular lipid accumulation, and also decreased the expression levels of adipogenic transcription factors (PPARγ and C/EBPα). These phytochemicals significantly promoted adenosine monophosphate-activated protein kinase (AMPK), which is known to suppress adipogenesis. It was also found that pretreatment with α-asarone and β-asarone, a typical inhibitor of AMPK, attenuated the inhibitory effect of asarone on AMPK phosphorylation. The asarone-induced AMPK activation leads to a decrease in adipogenic transcription factor expression, and suppresses adipogenesis.

Ethnomedicinal Use
This plant is being practiced traditionally in the Indian Ayurvedic tradition, as well as in the Chinese system of medicine for analgesic, antipyretic, tonic, anti-obesity, and healing purposes; it is highly effective for skin diseases, along with neurological, gastrointestinal, respiratory, and several other health disorders. Rhizomes and leaves are found to be profusely practiced in the form of infusion, powder, paste, or decoction . The ethnomedicinal uses of the A. calamus are detailed in Table 1.
A. calamus rhizomes and leaves are also used as an active pharmaceutical ingredient in various Ayurvedic formulations (Table 2).

Perspectives and Future Directions
The present review provides a plethora of information apropos ethnomedicinal uses, marketed formulations, geographical distribution, chemical constituents, pharmacological activities of crude, n-hexane, ethyl acetate, methanolic, ethanolic, hydro-alcoholic, aqueous extracts along with pure compounds, and clinical trials related to A. calamus.
Investigations on extracts and compounds of A. calamus suggested antidiabetic, anti-obesity, antihypertensive, anti-inflammatory, antioxidant, anticonvulsant, antidepressant, neuroprotective, and cardioprotective potentials with distinct underlying signaling pathways. The biological potential and mechanisms of action of some of the chemical constituents (α-asarone, β-asarone, eugenol) are known. However, other compounds need to be scientifically explored for their bioactivities and molecular modes of action, which could provide a lead for further development into therapeutics. More systematic, well-designed, and multi-center clinical studies are warranted to evaluate standardized extracts of A. calamus therapeutically and to identify the pharmacokinetic-dynamic roles of pharmacologically active biomolecules. There is scarce data from experimental and clinical reports on hypertension, diabetes, and atherosclerosis, and less supporting evidence is available on the use of A. calamus to treat hypertension and diabetes. Based on the available data, it is suggested that this plant could be used as an adjuvant to the established targeted drugs for neurological and metabolic disorders.
In 1974, United States food & drug administration (USFDA) banned A. calamus due to its carcinogenic effects following animal studies. They reported β-asarone as a carcinogenic agent, but the study was conducted on the calamus oil which consists of β-asarone in about 80%, while its different genotype in Europe and India contains β-asarone in lower concentrations. A. calamus cultivated in various geographical regions may have different chemical compositions along with therapeutic properties challenging quality control, toxicity, and safety concerns of A. calamus. In addition, the heavy metal, mycotoxin, and pesticide concentrations are required to be addressed in all toxicity studies.

Conclusions
Compelling in vitro, in vivo and clinical evidence suggests that the potential role of A. calamus rhizomes for modulating metabolic and neurological disorders could be due to their richness in several classes of active phytoconstituents. The predominant compounds present in rhizomes and leaves responsible for expression of potent bioactivities include α-asarone, β-asarone, eugenol, and calamine. The present report is expected to fill the gaps in the existing knowledge and could provide a lead for researchers working in the areas of phytomedicine, ethnopharmacology, and clinical research. Funding: This paper was supported by the UHK Excellence project.