Ethnopharmacological and Phytochemical Studies of Tridax Procumbens Linn: A Popular Herb in Ayurveda Medicine

Tridax procumbens (T. procumbens) Linn. is a medicinal plant found in tropical, sub-tropical and mild temperate regions around the world being used in Ayurveda treatment for liver disorders, boils, blisters, cuts, wound healing and as an anticoagulant, antifungal, and insect repellent. The plant is known to contain flavonoids, alkaloids, carotenoids, hydroxycinnamates, lignans, benzoic acid derivatives, phytosterols and tannins. The plant is also associated with endophytes to produce secondary metabolies by endophytes possessing antibacterial and antifungal activities. Different parts of this plant and essential oil are associated with ethnopharmacological properties like wound healing, antibacterial, immunomodulation, antiinflammatory, antidiabetic, vasorelaxant, antihyperlipidemic, anticancer, antiplasmodial, anticoagulation and antihepatic. Most of these studies validate the concept of earlier claims that T. procumbens’s potential as a medicinal plant. Further studies are required to unravel other pharmacological activities as well as the target-based mechanism of actions. The review also highlights the need for exploring lead molecules from these myriad of compounds that are of vital importance in drug discovery strategies.


INTRODUCTION
T. procumbens Linn., though a native of tropical America is also found in India, tropical Africa, Asia, Australia and India as a creeper weed. T. procumbens has been traditionally used in Ayurveda system for centuries and possesses different pharmacological properties including wound healing, anti-oxidant, antibacterial, antifungal, immunomodulatory, antiinflammatory, antidiabetic, vasorelaxant, antihyperlipidemic, analgesic, antiplasmodial, anticoagulation and antihepatic [1]. The diverse biological activities are due to various phytochemicals present in the plant. The aim of this review is to critically evaluate T. procumbens as an important medicinal plant with emphasis on the in-vivo properties of the phytochemicals and their roles in signalling pathways that can be manipulated for specific pharmacological actions 2. METHODS For the review, articles were extracted in Pubmed Central search engine using the key word Tridax Procumbens. In addition, phytochemicals, pharmacology, clinical trials, Flavanoids, Kaempferol, Catechins, alkaloids, antidiabetic, anti-inflammatory, immunomodulatory, antimicrobial, hepatoprotection, antiparasitic and bone homeostasis were also added to the search window to extract all relevant articles pertaining to Tridax Procumbens. The flavonoids detected in T. procumbens are known to mediate pharmacological activities including free radical scavenging, antiinflammatory, antiallergic, antiplatelet aggregation, antimicrobial, antiulcer, antiviral, antitumor and antihepatotoxicity [8]. Two new flavones, 8,3′-dihydroxy-3,7,4′-trimethoxy-6-O-β-D-glucopyranosylflavone and 6,8,3′-trihydroxy-3,7,4′trimethoxyflavone were isolated from the whole plant along with four known compounds puerarin, esculetin, oleanolic acid and betulinic acid (Figure 1) exhibiting antioxidant activity [9]. A new flavonoid procumbenetin, from the aerial parts of T. procumbens, has been characterized as 3,6-dimethoxy-5,7,2',3',4'-pentahydroxyflavone 7-O-β-D-gluco-pyranoside based on spectroscopic techniques and by chemical means [10].

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ISSN: 2278-0181 http://www.ijert.org have shown that EGCG, a derivative of catechin can reduce the effects of food allergen ovalbumin by interacting with its secondary β sheet structure thereby preventing uptake by antigen presenting cells [28].

Alkaloids
A recent study by Ikewuchi et al demonstrated that the total alkaloid content in the leaves of T. procumbens L were 102.421 g/kg and 10.191 g/kg of dry and wet weight respectively (Fig. 4). Akuammidine is known to have biological activities such as antibacterial, antifungal, antimalarial, antiinflammatory, hypotensive, skeletal muscle relaxant, local analgesic and anti-depressant [7]. Other studies also confirmed the presence of different alkaloids in the leaves of T. procumbens L [29,30]. These alkaloids have been isolated using different solvents like chloroform and methanol and using different methods such as infused and decoction in a qualitative manner [29,30].

Wound healing activity
Traditionally, the juice from leaves of T. procumbens has been used for healing dermal wounds. Wound healing process involves three phases namely inflammation, angiogenesis and collagen deposition. In an excision and incision wound model in Wistar rats, both aqueous and ethanolic extracts of T. procumbens increased the tensile strength of the wound compared to control rats. Further, wound healing biomarkers such as hydroxyproline, collagen and hexosamine were significantly increased [38]. The wound healing ability was also confirmed by topical ointment formulation of the leaf extract of T. procumbens in a mouse model, where dose-dependent improvement of cell proliferation and wound remodelling was observed [39]. Lysyl oxidases (LOX) are a group of enzymes catalyzing cross-linking reaction of collagen and elastin to form covalently linked,

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ISSN: 2278-0181 http://www.ijert.org insolubilize extracellular matrix (ECM) proteins thereby facilitating ECM stabilization through ECM formation, development, maturation and remodelling [40]. The increased lysyl oxidase activity on treatment with T. procumbens extract in rat wound healing model suggests their role in wound healing [41]. In C. elegans wound model and cell lines scratch wound healing assay, the wound healing ability of T. procumbens and its phytocompounds Octa decenoic acid (ODA), Pyridine carboxamide oxime, known as Nicotinamide (NA) and Dimethyl Benz[c]acridine (DMB) were studied. Results indicated that the wound healing ability was mainly contributed by NA in the chloromethyl nicotinamide derivative form by interacting with the wound healing biomarker, glycogen synthase kinase 3 (GSK-3) [42].

Antiinflammatory Activities
Since, inflammation is one of the three phases of wound healing, T. procumbens was evaluated for its antiinflammatory potential. An earlier study showed that aqueous extract of T. procumbens exhibited a dose dependant inhibition of paw volume in carrageenan induced rat paw edema and cotton pellet granuloma model as well as significant reduction in cell migration compared to control [43]. In another study, T. procumbens was shown to possess dose dependent analgesic activities in formalin, acetic acid and complete Freund's adjuvant (CFA)-induced pain models. These protective actions against pain may be attributed to flavonoids and sterols present in the extract and have the potential to develop effective analgesics [44]. The antiinflammatory activities of T. procumbens were further corroborated in an in vivo carrageenan-induced rat paw edema model in which standardized EtOAc, MeOH and 70% EtOH extracts of T. procumbens aerial parts displayed anti-inflammatory potential at a median dose of 200 mg/kg with maximum activity by EtOH fraction comparable to the control ibubrufen at 100 mg/kg (% inhibition of paw edema at 5 h: 41.2  2.2 vs. 52.2  1.5) [45]. The EtOAc extract exhibited strong antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and COX1 (Cyclooxygenase 1) and COX2 (Cyclooxygenase 2) inhibitory activities compared to MeOH and 70% EtOH extracts which may be attributed to the flavonoids and other polyphenols present in the extracts [45]. In a similar study, antiinflammatory activities were demonstrated in carrageenan induced mouse model where improved inflammation in histopathological scoring of mice paw, decreased gene expression of TNF-α and COX2 in mouse paw treated with EtOH extract of T. procumbens were observed compared to control [46].

Immunomodulatory Activities
The ability of bioactives in modulating immune response to ameliorate certain diseases reflects its biological or pharmacological effects on humoral or cellular immune response [47]. Intraperitoneal injection of an aqueous extract of T. procumbens Linn. (TPEIF) in Swiss male albino mice exhibited significant increase in immunomodulatory indexes such as phagocytic index, leucocyte count, splenic antibody secreting cells and augmentation of both humoral and cellular immune response as evidenced by elevation of heamagglutination antibody titer and delayed type hypersensitivity reaction respectively [48]. Further, the aqueous extract protected against anaphylactic shock in mice sensitized with 1 mg bovine serum albumin with reduction in number of animals presenting anaphylactic symptoms. The overall immunomodulatory responses may be due to the presence of the sesquiterpene and terpenoids in the aqueous extract. The detection of a specific antibody to tetanus toxoid (TT) in TPEIF treated animals showed significant stimulation of specific antibodies against TT suggesting a role for aqueous extract of T. procumbens Linn. in immuno-compromised patients and as a vaccination adjuvant to reduce the number of nonresponders to vaccines [48]. In another study, oral administration of methanol extract, chloroform fraction (CFTP), ethyl acetate fraction (EFTP), n-Butanol fraction (NFTP) and remnant water soluble fraction (RWSFTP) fractions in Swiss Albino Mice (20-40 mg/kg body weight) exhibited positive response in stimulating immune responses. Notably, EFTP and NFTP fractions rich in flavonoids and triterpenoidal saponin modulated both cell mediated and humoral components of the immune system [49]. Furthermore, ethanolic extract of T. procumbens stimulated humoral response in Swiss albino Rats and phagocytosis and also conferred protection against Pseudomonas aeruginosa infection [50].

Antimicrobial Activity
Bioactives from plants are used in traditional therapies as antimicrobials [51]. Aqueous extracts from different plants including T. procumbens possess antimicrobial activities [52,53]. T. procumbens extract exhibited antibacterial activity against clinically relevant Gram positive and Gram negative bacteria [54][55][56][57][58]. In one study, T. procumbens crude extract of n-hexane was active against pathogens like Mycobacterium smegmatis, Klebsiella species and Salmolella species whereas ethyl acetate extract was active against Mycobacterium smegmatis and S. aureus. These differential antibacterial activities may be due to the presence of mixture of hydrocarbons in n-hexane extracts such as neophytadiene and long chain fatty acids such as hexadecanoic acid. In the case of ethylacetate extract, fatty acids, aromatic compounds, polyaromatic carboxylic acids, polysubstituted phenols and thiols were reported [54]. T. procumbens leaves extracted in different solvents exhibited antibacterial activities. Chloroform extract was effective against B. faecalis and E. coli, the ethanolic extract was moderately active against B. faecalis whereas petroleum ether extract was active against B. faecalis. This study suggested that tannins in chloroform extract and alkaloids in petroleum ether and ethanol extracts were responsible for the antimicrobial activities [55]. Though, the ethanolic extracts of T. procumbens were reported to have antibacterial activity by many researchers [55,56], Saritha et al. have reported no antibacterial activity in whole plant extract which could be due to lower concentration of the actives [57]. Methanolic leaf extract of T. procumbens from one study inhibited S. aureus causing bovine mastitis [58] suggesting that there is wide range of antibacterial activities possessed by this plant. Efforts were also made to prepare nanoparticles from the leaf of T. procumbens. It has been documented that endophytic microorganisms including fungi or bacteria results in the production of antifungals such as pseudomycins, ecomycins [61,62] and antibacterials such as indolosesquiterpenes compounds [63]. Fungal or bacterial endophytes (BEs) are considered as potential sources of novel antibiotics. In a recent study, improvement of experimentally induced dermatophytic lesions by T. procumbens plant extract suggests the presence of antifungal principle to cure dermatophytosis [64]. Further, the plant is also known for its antibacterial properties [65,66]. Analysis of fifty novel endophytes from the leaves and stems of T. procumbens revealed the association of Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus with the plant [67]. Bacterial endophytes associated with roots of T. procumbens can be used for bioremediation of heavy metals [68]. Fungal endophytes in association with T. procumbens have been reported to have antibacterial activity. In an experimental study, out of six endophytic fungi (TP-1 to TP-6) isolated from the leaves of the T. procumbens, TP-1 identified as Alternaria sp. exhibited maximum antibacterial activity against Escherichia coli, Salmonella Typhi, Bacillus sp., Pseudomonas aeruginosa, Staphylococcus aureus and klebsiella pneumonia [69].

Life Style Diseases and T. procumbens
Metabolic syndrome (MetS) is a cluster of metabolic abnormalities having three of five clinical criteria: obesity, dyslipidemia, low serum HDL cholesterol, hypertension and hyperglycemia [70]. Genetic factors, acquired factors, sedentary life style and dysregulated endocrine signalling are primary contributors of MetS [71]. As per American Heart Association (AHA), treatment involves multiple pharmacological interventions to adequately correct the individual risk factors [72]. Natural products are important source of therapeutically effective drugs with more than 80% of world's population depending on plants [73]. FDA (Food and Drug Administration)-approved drugs has revealed that derivatives of natural products represent one-third of the total approved New Molecular Entities (NMEs) of which 50% are from mammals, 25% each from microbes and plants [74].
The antidiabetic activities of T. procumbens were documented in various animal models [75][76][77][78]. Oral administration of whole plant methanolic extract of T. procumbens in alloxan-induced diabetic rats showed a significant decline in fasting blood glucose without much effect on body weight reduction and toxicity [75]. Antidiabetic activity was reported when another aqueous and alcoholic extracts of the leaves of T. procumbens were tested in alloxan-induced diabetic rats [76]. Administration of ethanolic extract of T. procumbens (whole plant) at 250 and 500 mg/kg in streptozotocin-nicotinamide induced Wistar rats have demonstrated significant reduction of blood glucose, serum cholesterol, triglycerides, and LDL-cholesterol compared to the standard anti-diabetic drug glibenclamide at 0.25 mg/kg. Other favourable pharmacological profiles such as increased HDLcholesterol and body weight reduction indicated that standardized extracts of T. procumbens alone or in combination with other herbal extracts may be an excellent choices to treat MetS [77,78]. Polyherbal formulation may bring additive or synergic effects in their pharmacological responses against MetS which were not explained [78]. Ali et al [79] reported that oleanoic acid and its derivatives are inhibitors of α-glucosidase with IC50 as low as 7.97  0.214 µM. Inhibition of α-glucosidase slows the breakdown of carbohydrate, enhances its utilization, lowers insulin levels and maintains glucose homeostasis. In one study, ether, methanol, and chloroform extracts of T. procumbens exhibited a significant reduction of α-glucosidase which may be due to oleanoic acid derivatives [79,80]. In a clinical study, when aqueous whole plant T. procumbens extract was administered for 4 weeks along with prescribed medications to type 2 diabetics, significant lowering of fasting and postprandial blood glucose was observed [81].
Since hypertension (50-75%) and obesity (30-50%) are co-morbid conditions in type 2 diabetics [82], phytotherapeutic intervention is ideal to treat more than one condition of MetS. When 20 mg/kg of N-nitro-L-arginine methyl ester (L-NAME)induced hypertension rats were treated with aerial parts of T. procumbens extracts prepared in different solvents (cyclohexane, micellar, dichloromethane and ethyl acetate) for seven days, ethyl acetate and dichloromethane fractions were most effective in lowering the mean arterial pressure of rats comparable to the standard ACE inhibitor captopril. The hypotensive effects of these fractions may be due to the enriched alkaloids and flavonoids and the probable vasorelaxation effect of flavanoids [83]. The mechanisms of vasodilation of T. procumbens extract include blocking or modulating cGMP and cAMP in rat aorta artery [84], blocking Ca 2+ channels, stimulation of prostacyclin production and opening of small-conductance Ca 2+ activated potassium channels in rat superior mesenteric arteries [85] and by the release of NO from endothelium of rat smooth muscle [86].

Anticancer Activity
T. procumbens flower crude aqueous and acetone extracts when tested against prostate epithelial cancerous cells PC3 in MTT assay, the former demonstrated anti-cancer activity [87]. Sankaranarayanan S. et al. [88] have isolated a compound Lupeol which inhibited cell viability of human lung cancer cell A-549 in MTT assay upto 90%. Further, this compound at 320 µg/ml concentration showed anti-cancer potential in various assays namely colonogenic survival determination, cell cycle control, cell based assay for inhibition of COX-2 activity and DNA fragmentation analysis [88]. significantly prevented B16F-10 cell line-mediated lung metastasis in C57BL/6 mice along with prevention of tumor directed angiogenesis and induction of apoptosis [89]. The crude aqueous extract of T. procumbens when nanoformulated with poly (vinyl pyrrolidone) exhibited superior anticancer, antioxidant and antiinflammatory activity suggesting an effective strategy to enhance therapeutic index [90].

Hepatoprotective Activity
Drug-induced liver injury (DILI) or drug-induced hepatotoxicity is caused by all classes of drugs, herbal and dietary supplements. Specific examples of drug classes include anaesthetics (halothane), non-steroidal anti-inflammatory drugs (diclofenac), antibiotics (rifampicin, beta lactam antibiotics such as penicillin and cephalosporin), antifungals (ketoconazole), highly active antiretroviral therapy (HAART), oral hypoglycemics (PPAR- agonists like troglitazone, rosiglitazone, sulfonylureas such as glimepiride) and lipid lowering drugs (statins and ezetimibe) [91]. The mechanisms of DILI include inhibition of mitochondrial respiratory chain, increased generation of reactive oxygen species (ROS), ATP depletion, triggered apoptosis and immune mediated injury [91]. Though, the global use of herbal therapy for hepatotoxicity is increasing, evidence suggests that these may also cause hepatotoxicity. For instance, herbal toxicity is reported for Herbalife ® products causing differential patterns of liver injury [92]. Thus, phytotherapies to counter hepatotoxicity need a cautious approach.
The oral administration of T. procumbens ethanolic extract at 100, 200, 300 and 400mg/kg body weight for the period of 7 days in paracetamol induced hepatotoxic male albino rats, resulted in the significant reversal of increased alanine aminotransferase, aspartate aminotransferase, serum alkaline phosphatase and enhanced lipid peroxidation. Increase in superoxide dismutase and catalase activities in liver cells suggest antioxidant and hepatoprotective efficacy of T. procumbens L. [93]. When petroleum ether, methanol and chloroform/water extracts were orally administered in D-galactosamine induced hepatotoxic male Wistar Albino rats, significant reduction of liver enzymes alanine transaminase (ALT) and aspartate transaminase (APT) compared to non-treated control (D-galactosamine treated rats) along with dose dependent antioxidant activities in methanolic extract compared to other fractions [94]. Hepatoprotective effects of T. procumbens were also demonstrated against carbon tetrachloride induced liver injury in Wistar rats [95], isoniazid-rifampicin induced toxicity in albino rats [96] and rifampicin induced hepatotoxicity in male Albino rats [97]. Hepatoprotection against experimentally induced hepatitis [98,99] by T. procumbens was also reported. Pretreatment of D-galactosamine/Lipopolysaccharide induced-hepatitis in rats with chloroform insoluble fraction from ethanolic extract of T. procumbens improved liver parameters like aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase, -glutamyl transferase, bilirubin and lipid levels both in serum [98].
In the same animal model, Ravikumar et al. demonstrated the improvement of liver antioxidant defense system such as decreased lipid peroxides, increased activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-Stransferase and the non-enzymatic antioxidants namely reduced glutathione, vitamin C and vitamin D [99].

Bone Homeostasis Modulators
Plant-derived flavonoids are shown to function as bone homeostasis modulators by stimulating osteoblasts function and inhibiting osteoclasts functions either alone or in combination [100]. Primary osteoclastic cells when treated with T. procumbens fraction (TPF), showed significant suppression of Receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced differentiation of osteoclasts and the formation of pits were noticed [101]. The down-regulation of osteoclast differentiation genes like Tartrate resistant acid phosphatase (Trap), Cathepsin K, Matrix metalloprotease-9 (Mmp-9), and Matrix metalloprotease-13 (Mmp-13) and proteins such as Cathepsin K, Mmp-9, and Mmp-13 in primary osteoclast cells treated with TPF suggests that TPF could be a potential anti-bone resorption agent to treat patients with bone loss associated diseases such as osteoporosis [101]. When mice were treated with TPF, bone formation related indices like bone mineral density, bone mineral content, osteoblast number, osteoblast surface, bone volume, mineralizing surface, mineral apposition rate and bone formation rate were significantly increased compared to the control mice [102]. Molecular mechanisms of inhibition of osteoclast differentiation by TPF could be due to the down-regulation of transcription factors such as c-Fos, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and activator protein-1 (AP-1) [103].

Antiparasitic Activity
Parasitic infections caused by protozoa, nematodes, trematodes, and cestodes account for more than 30% of the human population and the plant extracts and their secondary metabolites may be an excellent strategy to target these infections [104]. T. procumbens extracts have been shown to possess antiparasitic activities against Leishmania mexicana [105] and P. falciparum [106]. An active bioactive compound (3S)-16,17-didehydrofalcarinol isolated from T. procumbens exhibited significant in vitro activities against promastigotes and intracellular amastigotes of L. mexicana [105]. Oral administration of L. mexicana-infected mice with T. procumbens extract in combination with Allium sativum extract, known for its immunomodulatory effect, exhibited significant protection against infection as evident by improved anti-inflammatory score in mouse footpad assay and Th1-type immune response [107]. 6. CONCLUSION This is a comprehensive review that highlights the various phytochemicals identified in T. procumbens together with their diverse pharmacological attributes. Since, most of the small molecules approved by FDA are derived from plant sources, it is essential to evaluate the plant in a systematic manner to identify bioactives which could lead to the discovery and development of new drugs against various ailments. Though, T. procumbens has been used in many traditional medicines, scientific data relating their phytochemicals with published pharmacological properties are lacking. Many of the studies have used plant extracts followed by qualitative analysis of its phytochemical constituents. Researchers should try to employ new drug discovery principles like bio-assay guided phytochemical identification, phenotypic screening using relevant cell culture models and if possible demonstrate pharmacokinetic-pharmacodynamic correlation (PK-PD) to show the efficacy of the preparation. Attempts should also be made to identify the active metabolites mediating the in vivo efficacy to ensure standardized extract preparation. In addition, a battery of enzyme targets can be identified for different diseases and the partially purified bioactives checked for inhibitors or activators. Identification of new bioactives may serve as a chemophore or pharmacophore to be developed as a drug using Molecular Modeling, Medicinal Chemistry and Bioinformatics approaches. Since, T. procumbens is reported to have many pharmacological properties, researchers should try to evaluate their properties using modern technologies.