Phytochemical, Toxicological and Pharmacological Studies of Asiasari Radix et Rhizoma : A Review

Herba Asari and Asiasari Radix (AR) have a centuries-old history as a folk medicine in China, Korea and Japan. AR has long been used in combination with other herbs to treat cough, toothache, headache, neuralgia, aphthous stomatitis, gingivitis, chronic bronchitis, asthma and allergies. AR from three species of the genus Asarum: A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., A. sieboldii Miq. var. seoulense Nakai and A. sieboldii Miq are recognized as original “Asiasari Radix et Rhizoma”. These species do not contain aristolochic acids (nephrotoxins present in the Asarum species) and therefore the roots of AR are recommended for use. Asiasari Radix is rich in volatile oils, phenylpropanoids, terpenoids, flavonoids, glycosides and lignins. The reported toxicological and pharmacological activities of AR, including antifungal, antimicrobial, antilisterial, antiallergic, acarcidal, larvicidal, anti-caries, anti-inflammatory, hepatoprotective, neuroprotective


INTRODUCTION
Traditional, indigenous systems of medicines are as old as human civilizations that have provided us with novel concepts and modalities for the treatment of various ailments [1].The use of natural agents derived from fruits, vegetables, spices, legumes and cereals in traditional oriental medicine are attractive sources for developing novel therapeutics or prophylactics because of their safety, affordability, long-term use and ability to target multiple pathways [2].In the past decades, extensive research and development have generated over 25 % of the approved clinical drugs that are derived or tailored from natural products [3].
The species of genus Asarum (Aristolochiaceae) are distributed throughout Asia (65 species), and in eastern and western North America (15 species), and a single species in Europe [4].Nowadays, the plant sources of Herba Asari are recorded as comprised of three species of the genus Asarum, namely, A. heterotropoides Fr.Schmidt var.mandshuricum (Maxim.)Kitag., A. sieboldii Miq.var.seoulense Nakai, and A. sieboldii Miq.The medicinal part of these plants in use is the root "Asiasari Radix et Rhizoma" (AR).AR is a nearly cylindrical rhizome with numerous thin and long roots, externally light brown to dark brown.The AR is called seshin in Korean, saishin in Japanese, Xixin in Chinese or Chinese wild ginger root in English [2].AR has long been used in traditional Chinese and Korean medicine to treat cough, toothache, headache, neuralgia, aphthous stomatitis, gingivitis, chronic bronchitis, asthma and allergies, as well as for its anti-bacterial and analgesic effects [5,6].

QUALITY AND SAFETY
The rapid popularity and demand of commercial herbal medicine has led to the indiscriminate and unscientific collection without much consideration on the quality and safety [7].Specifically, aristolochic acid (aristolic acid; AA) containing herbs such as Aristolochia and Asarum species from the Aristolochiaceae family [8] have been prohibited for therapeutic use in Australia.However, these were not included in the toxic herb schedules in Asian countries [9].The toxicity of Asarum is much lower than that of the Aristolochia species [9].Asarum species contains aristolochic acids, mainly consisting of aristolochic acid I (AA-I) and aristolochic acid II (AA-II) [10].However, non-official species such as A. himalaicum Hook.f. et Thoms.Ex Klotzsch and A. forbesii Maxim., presented higher AAs concentrations than official species of Asiasari Radix [8,11].Studies revealed that the toxic components in Herba Asari is greatly reduced during the preparation of the decoction [12] and the decoction of the root part of Asarum is recommended for traditional usage [9].

PHYTOCHEMISTRY
Plants produce a huge array of chemicals, numbering tens of thousands, primarily for defense against herbivores and pathogens as well as for the production of floral fragrance to attract pollinators [13]

Compound
References Flavonoids and their glycosides

Acute and subacute toxicity
Increasing concentrations of methanol extract of AR orally administered to mice for 7 days to measure LD 50 was calculated to be 3400 mg/kg.There were no significant behavioral changes in the mice for 240 min following oral administration of 100 mg/kg of AR methanol extract [5].Acute oral toxicity study of AR extract showed no mortality of mice at the doses of 1000, 3000, and 5000 mg/kg body weight [28].In 28 days of subacute toxicity study, rats received repeated doses of AR methanol extract (50, 250 and 500 mg/kg body weight/day) and showed no mortality [29].Besides, no signs of toxicity, such as convulsion, vomiting, diarrhea, paralysis, breathing difficulties, bleeding, restless, irritation, and abnormal posture, were observed in AR extract-treated mice and rats in acute and subacute toxicity studies respectively [28,29].

Food consumption, weight changes and histopathology
As proper intake of nutrients are essential to the physiological status of the animals, AR extract treatment did not show significant differences in food consumption, body weights and organs weights in male and female mice and rats for 14 days in acute toxicity and 28 days in subacute toxicity studies at all doses tested [28,29].Organs weights revealed that the AR methanol extract did not produce organ swelling, atrophy, or hypertrophy.Moreover, gross examination of internal organs of all animals revealed no detectable abnormalities.Histopathologic evaluation did not reveal any observable damages in all the vital organs.Thus, it can be suggested that AR extract is virtually nontoxic in mice and rats [28,29].

Blood chemistry
Blood is an important index and the parameters usually measured are haemoglobin (HB), red blood cell (RBC) count, white blood cell (WBC) count, and differential leukocyte count.These blood indices were all measured after 28 days of oral administration without any significant alterations from the control values, again corroborating the wide safety margin of the AR methanol extract.Biochemical parameters are an important marker to evaluate the organs and cellular functions.The results obtained from the biochemical evaluation, showed no significant difference between all doses administered and between sexes [29].Among the evaluated parameters, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), total and conjugated bilirubin, total protein, albumin, globulin, and A/G ratio are considered as liver function markers because several reports of liver toxicity are related to the use of phytotherapeutic products.Any marked necrosis of the liver cells can lead to a significant change of these parameters in the blood serum.No changes were observed in these parameters showed that the AR methanol extract has no adverse effect on the hepatocytes [29].
Kidney toxicity has also been reported after use of phytotherapeutic products.In this case, urea, creatinine, and electrolyte determinations are vital, as these substances are markers of kidney function.No significant differences in the parameters were detected.AR methanol extract did not cause any effect on kidney function [29].Moreover, glucose was estimated as a marker for pancreatic damage.Total cholesterol, triglycerides, and HDL-cholesterol were determined to evaluate whether the AR methanol extract has hypo-or hyperlipidemic properties.Inorganic phosphorus and calcium were measured for bone-demineralization properties.There were no significant differences observed in the above biochemical parameters.Collectively, these data demonstrated that the methanol extract of AR has a high margin of drug safety [29].

PHARMACOLOGICAL PROPERTIES Antifungal, antimicrobial and anti-listerial activities
Colletotrichum orbiculare (Berk & Mont) Arx is the causal agent of anthracnose which is probably the most destructive disease of cucurbits in warm seasons with frequent rain.The infection of cucumber leaves by C. orbiculare and anthracnose development was reduced as the concentrations of kakuol and chlorothalonil gradually increased.Kakuol did not show any phytotoxic symptoms on cucumber leaves suggesting that the kakuol could be developed as an agricultural fungicide for the control of plant diseases [30].The methanolic extract of AR showed strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) KCCM 11812, 40510 and S. aureus ATCC 25923 [31].

Acaricidal and larvicidal activities
House dust mite, Dermatophagoides farinae (Hughes), causes major allergic diseases, including asthma, allergic rhinitis, and atopic dermatitis.Petroleum ether extract from AR was toxic to D. farinae adults [34].Methyleugenol, one congener of eugenol which is known to be toxic to D. farinae [35] was weaker than AR essential oil [34].Mosquitoes are prevalent worldwide and are common and serious disease vectoring insect pests.The toxicity of several compounds isolated from AR steam distillate to third-instar larvae of Culex pipiens pallens Coquillett, Aedes aegypti (L.), and Ochlerotatus togoi Theobald has been reported [36].Methyleugenol, α-asarone, pentadecane, (-)asarinin and pellitorine isolated from AR were effective against C. p. pallens larvae resistant to various insecticides [37].The methanol extract of AR showed the strongest larvicidal activity against larvae of the house fly, Musca domestica (L.) [38].Eucarvone and safrole extracted from AR were reported for their significant insecticidal activities against the rice weevil Sitophilus oryzae (L.), a most widespread and destructive primary insect pest of stored cereals [39].

Anti-caries activity
Dental caries is the most common chronic oral disease.Streptococcus mutans is the most important bacterium in the formation of dental plaque and dental caries.Ethanol and aqueous extracts of A. sieboldii reduced the acid production and inhibited the growth and adhesion of S. mutans.The synthesis of water-insoluble glucan by crude GTFase was also suppressed during A. sieboldii treatment [40].
Both histamine-and bradykinin-induced guinea pig ileum contractions were considerably inhibited, suggesting that the AR extract could interfere with the binding of histamine and bradykinin to its corresponding receptors, thereby inhibiting pain and inflammation reactions [41].Higenamine and methyleugenol mainly play important roles in the antitussive action of AR.It has been clarified that higenamine is an adrenergic β-stimulant and that methyleugenol relaxes directly the tracheal muscle [14].
A hot-plate test to clarify the supra-spinal action of ethanol extract of AR has been reported and these results suggest that AR may stimulate GABA A receptors at postsynaptic sites rather than affect the supra-spinal nervous system.AR ethanol extract suppresses tonic pain induced by the intraplantar injection of formalin and Nmethyl-D-aspartate (NMDA)-induced pain-related responses through activation of ɣ-aminobutyric acid (GABA) receptors

Hair growth promoting activity
It is important to develop novel therapies that prevent hair loss and enhance hair growth.Ethanol extract of AR has outstanding hair growth-promoting effects in rodent models.In addition, the AR extract regulates the expression of growth factors in dermal papilla (DP) cells that are cultured in vitro.Various cytokines and growth factors play important roles in hair growth control.Insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) have stimulatory effects on hair follicle growth, whereas epidermal growth factor (EGF) and tranforming growth factor-β (TGF-β) has inhibitory effects on hair follicle growth.It is noteworthy that the AR extract has the potential to increase the expression of VEGF in DP cells.But, the AR extract did not show an inhibitory effect on the expression of 5α-reductase [47].

Anti-melanogenesis activity
Transcriptional regulation of melanogenic proteins is primarily dependent on the expression of microphthalmia-associated transcription factor (MITF), which is a master transcriptional regulator of key melanogenic enzymes.The suppressive activity of AR on melanogenesis is linked to down-regulation of MITF-tyrosinase expression signaling pathways and not due to the direct down-regulation of tyrosinase activity.Activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling reduced melanin synthesis through MITF degradation and inhibition, respectively.AR inhibits pigmentation by indirectly regulating tyrosinase via ERK activation and subsequent MITF down-regulation [6].AR also strongly inhibited tyrosinase activity, tyrosinase-related protein (TRP)-1, dopachrome tautomerase (Dct) and MITF and leads to decreased melanin synthesis in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells [48].

Vascular inflammatory conditions
Nuclear DNA-binding protein high mobility group box 1 (HMGB1) protein acts as a late mediator of severe vascular inflammatory conditions, such as sepsis and septic shock [49].Extracellular HMGB1, released from necrotic cells and/or from inflammation-stimulated immune cells, functions as a pro-inflammatory cytokine and elicits proinflammatory responses of macrophages and endothelial cells [50].The epi-sesamin isolated from AR treatment resulted in marked inhibition of cecal ligation and puncture (CLP)-induced release of HMGB1 in septic mice and LPSinduced HMGB1 in HUVECs.
Moreover, epi-sesamin resulted in significant inhibition of toll-like receptor 4 (TLR4) expression without affecting cell viability in HUVECs.Activation of nuclear factor-κB (NF-κB) and ERK1/2 increased by HMGB1 were significantly reduced by epi-sesamin.Mice pretreated with epi-sesamin survived longer after CLP than untreated mice suggests that epi-sesamin suppressed HMGB1 and of HMGB1-mediated inflammatory responses to be a therapeutic strategy for management of sepsis and septic shock [49].

Hepatoprotective and anti-HPV activities
The aqueous extract of AR decreased markedly the increase of serum and liver ALT, AST, lactate dehydrogenase (LDH) and ALP activities caused by benzo[a]pyrene (BP) in rats.The increase of serum total cholesterol and phospholipid contents in BP-injection were also decreased by treatment of the aqueous extract of AR.From these results, the aqueous extracts of AR have significant liver-protective effects against BPinduced hepatotoxicity [51].The aqueous extracts from AR are also effective on anti-Human papillomavirus [52].

Neuroprotective activities
An increase in acetylcholine levels induced by decreased activity of acetylcholinesterase following AR extract treatment may cause activation of acetylcholine receptors, thereby stimulating extracellular signal-regulated kinase (ERK).The prolongation of survival time by AR extracts following sodium nitrite (NaNO 2 ) administration could be regarded as an increase of learning and memory possibly by increasing acetylcholine release or synthesis [5].AR enhances the transcripts neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophin factor (BDNF) and neurotrophin-3 (NT-3) in C2C12 cells-cultured medium and stimulate neurite outgrowth and stabilized the neurite formation in PC12D cells [53].
The AR methanol extract and its fractions showed decreasing working and reference errors and time spent collecting all baits, suggesting these fractions of AR significantly increase memory formation.In the passive avoidance test with mouse and rats, administration of AR caused a considerable increase in memory activity indicating that species differences are minimal in terms of memory formation by AR extracts.AR extracts have the ability to stimulate the tyrosine kinase activity of hippocampal insulin receptor in mouse as well as in rat, subsequently cause the activation of ERK, which eventually leads to the activation of subsequent signal transduction pathways in the hippocampus, thereby inducing memory enhancement later [5].
AR extracts has significantly inhibited the cell death induced by α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) in PC12 and glial cells.Moreover, with a grease-gap recording techniques, the methanol extract and methanol fraction obtained from the chloroform-methanol insoluble fraction of AR contains active components responsible for the inhibition of AMPA receptors [54].

Cytotoxic and anticancer activities
AR induced apoptosis preceded by a tight cell cycle arrest in the G2/M phase suggests that AR prevents the growth of HCT-116 cells.AR activated the caspases involved in the intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways and further elevates the expression of p53.Thus, the active p53 upregulates the expression of pro-apoptotic proteins, such as Bax and p53 upregulated modulator of apoptosis (PUMA), thereby inhibiting the anti-apoptotic survival protein Bcl-2 [2].The ethyl acetate soluble fraction of AR has been found to exhibit cytotoxic activity against human cancer cell lines such as A549 (human lung carcinoma), SK-OV-3 (human ovary adenocarcinoma), and SK-MEL-2 (human malignant melanoma) [55].The ethanol extract of AR significantly enhanced the sensitivity to paclitaxel, which is a MDR1 substrate, of HeLa cells [47].Moreover, It did not affect the sensitivity to 5-fluorouracil, which is not a MDR1 substrate [56].
[45].Mice administered with water extracts of AR delayed the latency times in the hot plate test than ethanol extracts of AR [42].Methyleugenol inhibited Ca 2+ influx by directly inhibiting nerve depolarization via the stimulation of postsynaptic GABAA receptors in the dorsal horn, preventing hyperalgesia [45].Methyleugenol (1-allyl-3,4-dimethoxybenzene), also has antinociceptive effect on the second phase of formalin-induced nociception without affecting the first phase [46].Acetic acid-induced mouse writhing assay is used for detecting peripheral analgesia whereas tail-flick is more sensitive in centrally acting analgesics.Results indicate that some active principles of AR methanol extract could bind to the µ, κ and δ opioid receptors.The methanol extract of AR contains morphine-like component and other peripherally acting principles.AR extracts are more potent in the anti-nociceptive actions compared to aspirin [41].

Table 2 :
Other compounds isolated from Asiasari Radix

Table 3 :
Toxicological activities of the extracts from Asiasari Radix