Phytochemistry and Therapeutic Potential of Medicinal Plant: Dioscorea bulbifera

Medicinal plants are rich source of diverse phytochemicals with multifaceted therapeutic potential [1]. Dioscorea bulbifera is one of the unique medicinal plants among 600 species in the family Dioscoreaceae which has found its importance in traditional medicine throughout the world. Aglycons of its steroid saponins like diosgenin have gained attention as precursors in the synthesis of sex hormones, cardiatonic glucosides, fertility control compounds, corticosteroids and anabolic agents [2]. In Bangladesh it is used for treatment of leprosy and tumours while in Chinese medicine as a remedy for sore throat and for struma [3,4]. Similarly, it is applied on cuts and sores as infusion in Zimbabwe, while for treatment of abscesses, boils and wound infections in Cameroon and Madagascar [5]. It has got its wide applications in piles, ulcers, pain and inflammation in Indian traditional medicine [6,7]. Ancient and modern literature on its medicinal use have indicated that its tubers have therapeutic benefits as purgative, anthelmintic, diuretic, deflatulent, rejuvenating tonic, aphrodisiac and can also be used for treatment in scrofula, haematological disorders, diabetic disorders, worm infestations, haemorrhoids, skin disorders, general debility as well as polyurea (Figure 1). Crushed tubers and decoction are emulsified into oil to treat infected ulcers and sinus [8,9]. It is profusely used in both Indian and Chinese traditional medicine to cure gastric cancer and carcinoma of rectum and goitre [10]. Similarly, its extracts are reported to be antihyperlipidemic, antitumor, antioxidant, anorexiant, analgesic, antiinflammatory, plasmid curing, antidiabetic and antihyperglycemic [11-17]. Dried yam is known to dissolve toxins, cures carbuncles, scrofula and purulent infections. It is considered as remedy against dysentery and diarrhoea in addition to syphilis in Java and Brazil. Interestingly, it is used against dog bites, snake bites and food poisoning in China, in addition to hepatic fibrosis by protecting the liver [18] It is also reported to have remedial potential against conjunctivitis, leucoderma, dyspepsia, urinary discharges, jaundice, diabetes, asthma, bronchitis, strangay and vata biliousness [19]. More recently, we have shown that its unique phytochemistry enables it to synthesize both gold (AuNPs) and silver nanoparticles (AgNPs) [20,21]. In the recent past, D. bulbifera has emerged as a promising complementary and alternative source of traditional medicine attracting prime attention towards spectacular scientific advancement in exploration of new biomolecules.


Introduction
Medicinal plants are rich source of diverse phytochemicals with multifaceted therapeutic potential [1]. Dioscorea bulbifera is one of the unique medicinal plants among 600 species in the family Dioscoreaceae which has found its importance in traditional medicine throughout the world. Aglycons of its steroid saponins like diosgenin have gained attention as precursors in the synthesis of sex hormones, cardiatonic glucosides, fertility control compounds, corticosteroids and anabolic agents [2]. In Bangladesh it is used for treatment of leprosy and tumours while in Chinese medicine as a remedy for sore throat and for struma 1 9.

Medicinal Use
Goitre Tubers of D. bulbifera are steeped in white wine for a week which is traditionally recommended as infused wine and taken daily to get benefits in treatment of goitre. Formulations have profound applications as clinical medicine to treat thyroid glands [9,18,25].

Respiratory disorders
Fresh tuber pieces are pounded with black pepper followed by mixing with curd is taken against cough and cold in India. Similarly, roasted, peeled and dried powder is known to cure respiratory problems when taken with honey. Bitter bulbils are useful against severe cough while edible normal tubers are used against asthmatic conditions. In China, it is considered as a styptic against lung bleeding and epistaxis [18].

Eye disorder
D. bulbifera serves as a well-known ophthalmic remedy. Purulent ophthalmia is treated with sap from stem as eye drops in Congo. Wakefulness is promoted in Ivory Coast by stillation of leaf sap into the eye. A potion made out of steamed leaf is used in east Africa for treating a type of conjunctivitis called "pink eye" [18].

Parasitic infections
D. bulbifera from Southwest Nigeria is reported to show anthelmintic activity as one of its significant ethnobotanical application when eaten after roasting. Scientific evidence to the fact was provided by a recent study confirming in vitro anthelmintic activity of methanol extracts of the flesh and peel of D. bulbifera bulbils. The extracts rich in phenolics (tannins, flavonoids), saponins as well as other secondary metabolites could efficiently show anthelmintic activity against Pheritima posthuma and Fasciola gigantic. Earthworms were paralysed in 5.6 min and death was observed in 10 min when treated with peel extract, while treatment with flesh extract showed paralysis in 8.4 min and death in 13.8 min at 100 mg/ml. Likewise, liverflukes were paralyzed after 10.2 min and died after 15.81 min at a concentration of 100 mg/ml of peel extract. The probable mechanism was explained to be the binding of phenolic and tannin compounds to glycoprotein present on the cuticle together with saponin mediated alteration of permeability and pore formation in the membrane of the parasite leading to paralysis and death [10].

Bacterial and fungal infections
D. bulbifera is known to be used for treatment of sexually transmitted diseases like gonorrhoea and syphilis in addition to sore throat in Chinese medicine. In Congo it is used against parasitic and fungal infections [18]. Its aqueous extract showed superior activity against Escherichia coli while ethanol extract was found to be potent against Staphylococcus aureus and Candida albicans [37]. Bacterial pathogens have posed a threat to the human health by developing multidrug resistance leading to re-emergence of diseases once controlled. Genetic determinants conferring resistance to one or more antibiotics are mostly located on plasmids which being extra chromosomal DNA, can be effectively transferred to other bacteria, co-existing in the same environment. This underlying mechanism for spread of antimicrobial resistance is an acute difficulty for treating infectious diseases which has led to the search of new drugs called plasmid-curing agents, although many of which such as acridine orange, ethidium bromide and sodium dodecyl sulphate, are toxic, mutagenic and carcinogenic. Hence, plasmid curing agents isolated from medicinal plants have gained more importance and are being investigated owing to their biocompatibility and minimum toxicity. Recently, norditerpene compound, 8-epidiosbulbin E acetate from D. bulbifera bulbs has exhibited significant broad spectrum potential to cure antibiotic resistance plasmids (R-plasmids) from clinical isolates although it showed low antimicrobial activity (MIC > 400 µg/mL) for all tested pathogens except Escherichia coli (pUC18) which showed an MIC = 200 µg/mL. However, R-plasmids in clinical strains of Enterococcus faecalis, E. coli, Shigella sonnei, Pseudomonas aeruginosa and Bacillus subtilis were effectively cured. Plasmid curing by 8-epidiosbulbin E acetate resulted in effective reversal of bacterial resistance to multiple antibiotics in an E. coli strain that was resistant to gentamicin, kanamycin, neomycin, streptomycin, tetracycline, novobiocin, ciprofloxacin, cefoperazone, oxacillin, ceftazidin, co-trimazine, imipenem, cefalexin, cefotaxime, oxytetracycline, cloxacillin, doxycycline, levofloxacin, ofloxacin, gatifloxacin, moxifloxacin, norfloxacin, cefpirome and cotrimoxazole. Further, S. sonnei was sensitised to ampicillin, gentamicin, tetracycline, novobiocin, ciprofloxacin, cefoperazone, ceftazidime, oxacillin, ceftazidin, co -trimazine, imipenem, cefazolin, cefalexin, cefotaxime, levofloxacin, ofloxacin, norfloxacin, and cefpirome as a result of 8-epidiosbulbin E acetate mediated plasmid curing. Vancomycinresistant Enterococcus faecalis, resistant to roxithromycin, cloxacillin, cefalexin and clindamycin became sensitive to these antibiotics after plasmid curing by 8-epidiosbulbin E acetate. Similarly, reference R-plasmids such as RP4, RMS163, RIP64 and pUB110 were also cured by 8-epidiosbulbin E acetate at curing efficiencies of 44%, 30%, 64% and 48%, respectively. Reduction of minimal inhibitory concentration (MIC) of antibiotics against MDR bacteria on curing of R-plasmid by 8-epidiosbulbin E acetate provided strong evidence for the novel mechanism behind the effective antibacterial treatment by D. bulbifera [16]. Additionally, vanillic acid and isovanillic acid have shown antibacterial activity [9]. In the western highlands of Cameroon, tubers of D. bulbifera are used against typhoid fever caused by Salmonella typhi and paratyphoid fever caused by Salmonella paratyphi A and Salmonella paratyphi B. Two clerodane diterpenoids, bafoudiosbulbins A and B were reported to have anti-bacterial activity when tested against by crude extracts can be considered as significant. Bafoudiosbulbin C was found to be active against M. smegmatis and M. tuberculosis ATCC and MTCS2 strains at a value as low as 8 μg/mL. Inhibitory effect of the compounds isolated from D. bulbifera against MDR bacteria such as E. aerogenes EA289, CM64, K. pneumoniae KP63 and P. aeruginosa PA124 was better than that of standard antibiotic chloramphenicol in absence as well as in presence of efflux pump inhibitor, phenylalanine arginine β-naphthylamide [28].

Inflammation and pain
D. bulbifera is used to treat inflammation associated dispersal of "lumps", hernia, sprain, injury, testicular inflammations, in China. Rheumatic pain and breast problems are relieved in Congo and Gabon, respectively by an ointment prepared by incorporation of bulbils into palm oil [18] It is also used as analgesic and antispasmodic. Vanillic acid and isovanillic acid present in D. bulbifera are reported to exhibit anti-inflammatory activity [9]. Recent in vivo studies of aqueous and methanol extracts from bulbils of D. bulbifera var sativa confirmed potent antinociceptive effect. A dose dependent reduction in the acetic acid induced abdominal constriction was observed in white adult mice Mus musculus (weighing 25-30 g), on oral administration of aqueous and methanol extracts. Similarly, in case of formalin-induced paw licking test, intraplantar injection of formalin (2.5%) into the right hind paw of adult Wistar rats (weighing 180-200 g), generated a classical biphasic nociceptive response that was significantly inhibited by aqueous and methanol extracts as compared to indomethacin. These extracts also presented important anti-inflammatory effects on pressure induced pain sensitivity, acute oedema induced by carrageenan, histamine, serotonin, formalin and chronic oedema induced by formalin [5]. Pronounced hypernociception induced by intraplantar injection of complete Freud's adjuvant (CFA) in Swiss mice, could be countered significantly (61 % inhibition) owing to the antinociceptive effect of methanol extract of D. bulbifera (MEDB) administered orally. Hypersensitivity in mice due to neuropathic pain induced by partial ligation sciatic nerve (PLSN) was significantly reduced (38 %) followed to oral administration of MEDB. Similarly, the extract even reduced LPS and PGE2 induced mechanical hypernociception and further study helped in partial prediction that the antinociceptive activities of D. bulbifera both in inflammatory and neuropathic models of pain were attributed to its ability to activate the NO-cGMP-ATP-sensitive potassium channels pathway [15].

Diabetes and digestive problems
Traditional use of D. bulbifera is known to lower glycemic index in Diabetes mellitus. Diosgenin has shown to ameliorate diabetic neuropathy [18]. A study carried out to evaluate aqueous extract of D. bulbifera tubers (DBEA003) for antihyperglycemic activity on streptozotocin (STZ) treated Wistar rats, provided scientific rationale behind the traditional use. A higher dose (1000 mg/kg p.o.) resulted in highly significant antihyperglycemic effect in glucose tolerance test bringing down the blood glucose level from 129±8.81 mg/dL (30 min) to 97±5.83 mg/dL (90 min). Remarkably, a long lasting glycemic control in diabetic condition was observed over administration of DBEA003, which might be attributed due to its action at the tissue or receptor level [17]. However, the actual mechanism of action by which D. bulbifera exerts the antidiabetic effect was furnished in our previous report where we confirmed the inhibition of two key enzymes, α-amylase and α-glucosidase. Petroleum ether, ethyl acetate and methanol extracts of D. bulbifera bulbs were obtained by sequential hot Soxhlet extraction among which ethyl acetate extract showed most superior activity of 73.39 % against porcine pancreatic α-amylase. Strongest inhibition against α-glucosidase was found for ethyl acetate extract (99.6 %) of D. bulbifera bulb [38]. Similarly, even among cold extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. The bioactive principle was identified as diosgenin which in its isolated form showed superior α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. The mechanism was established to be uncompetitive mode of binding to α-amylase as confirmed by decrease in both Km and Vm values. Further, hydrogen bonding between carboxyl group of Asp300 and hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase was indicated by molecular docking. Similarly, two catalytic residues (Asp352 and Glu411) from α-glucosidase were found to be the target of interaction with diosgenin [35]. Inhibition of digestive enzymes like α-amylase (132 ± 2%) and trypsin (4.3 ± 0.2 %) were also reported by crude extract of D. bulbifera tubers collected from the central region (Narayani Zone) of Nepal which were found to be rich in oxalate (67 ± 9 mg/100g), phytate (184 ± 14 mg/100g) and cyanogens (3.3 ± 0.9 mg HCN/Kg FW) [13].

Oxidative stress and degenerative diseases
Free radicals are key mediators for emergence of disease, progression and its associated pathology including diabetes, cancer and even AIDS [39]. Antioxidants, namely, epicatechin, isovanillic acid, vanillic acid, myricetin are important bioactive principles in D. bulbifera that are responsible for protection against cardiovascular diseases and styptic activities [9]. Tubers of D. bulbifera collected from Nepal showing superior DPPH radical scavenging, ferrous ion chelating, reducing power, and total antioxidant activity contained high oxalic acid (67 ± 9 mg/100g), citric acid (282 ± 24 mg/100g), malic acid(266 ± 20 mg/100g), succinic acid(2510 ± 108 mg/100g) and olyphenols (166 ± 10 mg/100g). Thus the chemical constituents might be playing the key role behind its pronounced antioxidant activity [13]. D. bulbifera from Guangzhou of China showed the highest phenolic content (59.43 mg GAE/g), that further exhibited most superior antioxidant property in terms of ABTS •+ radical scavenging activity (708.73 μmol Trolox/g) as well as ferric reducing antioxidant power (FRAP) assay (856.92 μmol Fe 2+ /g) [40]. Ethyl acetate fraction of hydro alcoholic extract of D. bulbifera from China, yielded a bibezyl compound, 2,5,2',5'-tetrahydroxy-3'-methoxybibenzyl and diobulbinone A amongst which Trolox-equivalent antioxidant capacity of the new bibenzyl 7 was found to be 0.52 ± 0.01 by FRAP at a concentration of 1 mM. Similarly it showed an EC50 value of 2.57 ± 0.06 mM for DPPH radical scavenging. However, the new diarylheptanone, diobulbinone A did not show significant antioxidant activity at the same concentration by either of the methods [33]. Dried bulbs of D. bulbifera from India were reduced to fine powder and extracted with 70% (v/v) ethanol in distilled water which was further sequentially extracted with petroleum ether, ethyl acetate and methanol. Among the extracts, methanolic extract with maximum phenolic content (145.44 ± 3.29 µg/mL) showed most superior antioxidant activity proved to be a function of phenolic content. Percentage scavenging activity of methanolic extract against DPPH, hydroxyl, superoxide anion radical and nitric oxide was found to be 84.94 ± 0.62 %, 76.11 ± 1.26 %, 59.75 ± 0.98 % and 57.59 ± 0.64 %, respectively. Simultaneously, it scavenged pulse radiolysis generated ABTS •+ radical with a second order rate constant of 1.72×10 6 , respectively. Similarly, ethyl acetate extract also showed higher percent scavenging activity of free radicals owing to its high phenolic content (98 ± 1.17 µg/mL) and 94.05 % diosgenin [41]. D. bulbifera (DB) tubers obtained from Mumbai, India, were extracted using a Soxhlet-extractor by 70% ethanol. Sprague-Dawley male rats were administered with 1 ml of DB extract dissolved in water (150 mg kg -1 of body weight) for 30 days, which resulted in significantly improved performance towards aortic flow (AF), left ventricular developed pressure (LVDP) and the first derivative of developed pressure (LVmax dp/dt) in DB treated hearts during post ischemic reperfusion. Additionally, marked reduction in myocardial infarct size (20 ± 2.64%) was observed in the treated group. Significant reduction of apoptotic cardiomyocytes (16.89 ± 1.7%) confirmed its anti-apoptotic activity. Increased Bcl2 expression and decreased Bax expression leading to reduction in Bax/Bcl2 ratio was evidenced. Further, upregulation of procaspase 3 and downregulation of cleaved caspase 3 coupled with prevention of loss of phase II enzyme HO-1 with proven cardioprotective ability considerably, provided a strong scientific rationale that D. bulbifera has the potential to ameliorate myocardial ischemia and reperfusion injury by improving ventricular function and inhibition of necrosis and apoptosis in cardiomyocytes [42].

Tumor and cancer
Aborigines from Tully district in North Queensland use a decoction of D. bulbifera against skin cancer [18]. Similarly it is also used in traditional Chinese medicine against cancer [25].

Synthesis of Nanoparticles
In spite of having a treasure of diverse groups of phytochemicals, D. bulbifera was not explored in the field of nanobiotechnology, until recently. We had reported for the first time the potential of its tuber extract to synthesize AuNPs of exotic shapes, namely gold nanotriangles, nanoprisms, nanotrapezoid and spheres in a range from 50 to 300 nm. Maximum synthesis of AuNPs was achieved at 50ºC by complete reduction of 1 mM chloroauric acid at 90 mins which was found to be faster as compared to maximum synthesis of AuNPs after 5 h by Plumbago zeylanica root extract (PZRE) [20,43]. Rapid reduction by D. bulbifera tuber extract (DBTE) might be due to its high total reducing sugar content (3.41± 0.15 mg/mL) and high flavonoid content (4 ± 0.12 mg/mL) while PZRE contained 1.25 ±0.04 mg/mL and 0.95 ± 0.05 mg/mL of total reducing sugar and total flavonoids, respectively. Further, the aqueous tuber extract synthesized anisotropic AgNPs in the form of rare nanotriangles, nanorods, spheres and hexagons in the size range of 8-20 nm at 50°C with 0.7 mM AgNO 3 solution in 5 h. Bioreduced AgNPs possessed potent antibacterial activity against both Gram-negative bacteria, namely Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Haemophilus influenza, Klebsiella pneumonia Neisseria mucosa, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Serratia odorifera, Vibrio parahaemolyticus and Gram-positive bacteria, namely, Bacillus subtilis, Paenibacillus koreensis and Staphylococcus aureus. A selective synergistic activity was observed where β-lactam, piperacillin exhibited 3.6 fold increase while macrolide, erythromycin showed 3 fold increase in potency against multidrug-resistant A.baumannii when combined with AgNPs. Likewise, a combination of AgNPs with chloramphenicol and vancomycin against P. aeruginosa resulted in 4.9-fold and 4.2fold increase in zone diameter, respectively. 11.8-fold increase in zone diameter of streptomycin in combination with AgNPs against E. coli was found to be most significant. The unique phytochemistry of D. bulbifera with both reducing agents like ascorbic acid, citric acid and phenolics along with starch as capping agent and saponins helped in reduction, stabilization and shape evolution of nanoparticles [21].

Toxicity and Detoxification
Roots of D. bulbifera from China are reported as toxic, thereby might have achieved limited focus on its use in traditional medicine. Dioscin and diosbulbin B, derived from D. bulbifera roots (Huang-yao-zi) are responsible for liver toxicities, nausea, abdominal pain, coma and even death. Oral administration of decoction (400 g/kg) in male Sprague-Dawley rats for 72 h, exhibited elevated levels of taurine, creatine, betaine, dimethylglycine (DMG), acetate and glycine. On the contrary, a reduction in the levels of succinate, 2-oxoglutarate, citrate, hippurate and urea was observed. Marked increase in the organ coefficients of liver and kidney of rats was noticed on exposure to Huang-yao-zi. Histopathological alterations of liver included diffuse hepatocyte degeneration, apoptosis, necrosis in addition to ballooning degeneration of hepatocytes and vascular congestion of hepatic sinusoidal and portal areas. Impairment of mitochondrial energy metabolism indicated by reduction in citrate, 2-oxoglutarate and succinate levels coupled with inhibition of ornithine cycle and urea production confirmed oxidative injury of hepatic mitochondria leading to inhibition of ATP formation resulting in further inhibition of hippurate synthesis. Similarly, elevation in taurine levels due to glutathione depletion also indicated oxidative hepatic damage [44]. Another study, revealed that ethyl acetate fraction (EF) of hydroalcoholic extract of D. bulbifera rhizome from China exhibited dose dependent hepatotoxicity in ICR male and female mice when administered consecutively for fourteen days. Maximum elevation in levels of biomarkers of liver injury, namely, alanine transaminase (ALT) and aspartate transaminase (AST) were observed at a highest dose of 480 mg/kg. Reduction in the levels of glutathione-related enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR) and glutamate-cysteine ligase (GCL) of hepatic tissues of EF treated mice indicated oxidative stress mediated liver injury in mice [45]. Hepatotoxicity against normal human liver cell line L-02 was reported by diosbulbin D isolated from the rhizome of D. bulbifera from China. A time and dose dependent manner of reduction in cell viability was shown by the benzene fraction, highest being 4.32% ± 1.87 at a dose of 800 µg/ml. AnnexinV and propidium iodide assay, Hoechst 33258 staining and the occurrence of a sub-G1 peak indicated diosbulbin D mediated apoptosis induction which was further confirmed to be caspase 3 dependent [46].
Traditional food processing techniques were scientifically validated as most efficient for removal of bitter and toxic compounds from D. bulbifera tubers collected by aboriginal people from two locations in northeast Arnhem Land, NT, Australia. Bitterness due to presence of saponins and sapogenins in Central American, South African and Indian species, tannins and polyphenols in Indo-Chinese varieties and furanoid norditerpenes (diosbulbins) mostly in China, can be effectively removed by the traditional techniques like leaching, thereby, rendering palatability to the processed sliced tubers, otherwise described as "cheeky", meaning bitter or poisonous in aboriginal English. Treatment practices varying from baking, followed by overnight leaching of the sliced tubers for 12 h in running water, resulted in reduction of major bitter and toxic compound, diosbulbin D (0.07 mg/g) decreasing it to a very low level under the taste threshold rendering the final food palatable [47].

Conclusions
Since ancient times, D. bulbifera is being used in different parts of the world as traditional medicine against several diseases. Numerous studies on its therapeutic potential are accomplished, as it is considered to be a natural treasure of bioactive principles which can be used for production of new medicines with higher efficacy and biocompatibility with least side effects. Recent researches are continuously adding up new attributes to its pre-existing wide spectrum therapeutic window. As detailed in this review, the comprehensive classical compilation of promising medicinal applications, in-vitro and in-vivo studies on D. bulbifera will enable to search for the untapped pool of novel lead compounds. Further intensive study on isolation and identification of novel compounds with antiviral and antibiofilm properties can broaden its horizon against infectious diseases. Similarly, this review will encourage the screening of more compounds from different groups for plasmid curing efficacy and even for synthesis of novel multifunctional nanomedicine by conjugating with nanocarriers towards rational drug designing and targeted drug delivery. Simultaneously, more scientific experimentation can be initiated with different parts of D. bulbifera as well as its isolated phytochemicals to generate evidences supporting its traditional applications, deciphering the underlying mechanism and searching for its utility as nutraceuticals.