Secondary metabolites with diversified structures from an endophytic fungus Colletotrichum gloeosporioides associated with a toxic medicinal plant Tylophora ovata.

Six new secondary metabolites, including two new nor-triterpenes (1 and 2), one new sesquiterpene (4), two new α-pyrone derivatives (6 and 7), and one new natural product (5) along with two known compounds (3 and 8) were isolated from an endophytic fungus Colletotrichum gloeosporioides obtained from a toxic medicinal plant Tylophora ovata. Their structures were elucidated by spectroscopic data analyses, while their absolute configurations were determined by CD and X-ray diffraction analyses. The in vitro anti-inflammatory activities of these compounds were evaluated.

Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure-activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.

Design, Synthesis and in Vitro Evaluation of Tylophorine Derivatives as Possible Antitumor Agents.

Structural simplification and modification of natural products are always very important resources to antitumor drugs. By introducing various aminomethyl groups and amide groups into the phenanthrene ring of tylophorine, a novel series of tylophorine derivatives have been designed and synthesized, and their antiproliferative activities against MCF-7, A549 and HepG-2 cells have been evaluated, too. The results indicated that most of the prepared compounds exhibited good antitumor activities. Especially, one compound with an {ethyl[2-(morpholin-4-yl)ethyl]amino}methyl group at the side chain exhibited the most significant cytotoxic effects.

[Investigation on secondary metabolites of endophytic fungus Talaromyces purpurogenus hosted in Tylophora ovate].

Eight compounds,(R)-2-[5-(methoxycarbonyl)-4-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]acetic acid(1),(3S,4R)-3,4-dihydro-3,4-epoxy-5-hydroxynaphthalen-1(2H)-one(2),(-)-mitorubrinol(3),(-)-mitorubrin(4),(±)-asperlone A(5), terreusinone(6), verrucisidinol(7) and cerebroside C(8) were isolated from the endophytic fungus Talaromyces purpurogenus by using various column chromatographic techniques. Their structures were identified by NMR, MS, CD and optical rotation. Compounds 1 and 2 were new compounds. Their anti-diabetic activities in vitro were evaluated, and compound 1 showed moderate inhibitory activity toward XOD at 10 µmol·L~(-1) with the inhibition rate of 69.9%.

Nonadride and Spirocyclic Anhydride Derivatives from the Plant Endophytic Fungus Talaromyces purpurogenus.

Six new nonadride derivatives (1-6) and three new spirocyclic anhydride derivatives (7-9) were isolated from the endophytic fungus Talaromyces purpurogenus obtained from fresh leaves of the toxic medicinal plant Tylophora ovata. The structures of these compounds were determined by spectroscopic analyses including 1D and 2D NMR, HRESIMS, and ECD techniques. Maleic anhydride derivatives 1-9 were evaluated for their in vitro anti-inflammatory activities. Compound 1 showed significant inhibitory activity against NO production in LPS-induced RAW264.7 cells with an IC50 value of 1.9 µM. Compounds 2 and 6 showed moderate inhibitory activities toward XOD and PTP1b, respectively, at 10 µM with inhibition rates of 67% and 76%.

[Investigations on secondary metabolites of endophyte Diaporthe sp. hosted in Tylophora ovata].

One new compound (2S, 3S)-2,3-dihydroxybutyl 2-hydroxy-3,5,6-trimethylbenzoate (1) and six known compounds xylariphthalide A (2), convolvulol (3), cis-4-hydroxy-6-deoxytalone (4), phomoxydienes B (5), 5,6-dihydroxy-2,3,6-trimethylcyclohex-2-enone (6), trans-cyclo-(D-tryptophanyl-L-tyrosyl) (7) were isolated from Diaporthe sp., an endophytic fungus hosted in the leaves of the toxic Chinese folk medicine Tylophora ouata, using the combination methods of silica gel column chromatography, medium-pressure ODS column chromatography and RP-preparative HPLC. The structure of compound 1 was elucidated by NMR and MS data analyses. The absolute configurations were established according to the ¹H-NMR data and exciton chirality method. Compound 1 inhibited the activation of human lung fibroblasts MRC-5 cells by 64.0% at 10 µmol·L⁻¹. The MTT assay showed that compounds 2 and 4 displayed cytotoxic activity against human tumor cell lines BGC-823 cells with IC50 values of 1.5 and 8.6 µmol·L⁻¹, respectively.

A General Cp*CoIII -Catalyzed Intramolecular C-H Activation Approach for the Efficient Total Syntheses of Aromathecin, Protoberberine, and Tylophora Alkaloids.

Herein, we report a Cp*CoIII -catalyzed C-H activation approach as the key step to create highly valuable isoquinolones and pyridones as building blocks that can readily be applied in the total syntheses of a variety of aromathecin, protoberberine, and tylophora alkaloids. This particular C-H activation/annulation reaction was achieved with several terminal as well as internal alkyne coupling partners delivering a broad scope with excellent functional group tolerance. The synthetic applicability of this protocol reported herein was demonstrated in the total syntheses of two Topo-I-Inhibitors and two 8-oxyprotoberberine cores that can be further elaborated into the tetrahydroprotoberberine and the protoberberine alkaloid core. Moreover these building blocks were also transformed to six different tylophora alkaloids in expedient fashion.

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

KEY MESSAGE: This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Cytotoxic potential and metabolomic profiling of alkaloid rich fraction of Tylophora indica leaves.

Tylophora indica (Burm f.) Merrill, belong to family Asclepiadaceae, is considered to be a natural remedy with high medicinal benefits. The objective of this work is to assess the metabolomic profile of T. indica leaves enriched in alkaloids, as well as to evaluate the in vitro cytotoxicity of these leaves using the MTT assay on human breast MCF-7 and liver HepG2 cancer cell lines. Dried leaves of T. indica were extracted by sonication, using methanol containing 2 % (v/v) of acetic acid and obtained fraction was characterized by HPTLC and UPLC-MS. The UPLC-MS study yielded a preliminary identification of 32 metabolites, with tylophorine, tylophorine B, tylophorinine, and tylophorinidine being the predominant metabolites. The cytotoxicity of the extract of T. indica was evaluated on HepG2 and MCF-7 cell lines, yielding inhibitory concentration (IC50) values of 75.71 µg/mL and 69.60 µg/mL, respectively. Data suggested that the phytochemical screening clearly showed presence of numerous secondary metabolites with moderate cytotoxic efficacy. In conclusion, the future prospects of T. indica appear promising for the advancement of phytopharmaceutical-based anticancer medications, as well as for the design of contemporary pharmaceuticals in the field of cancer chemotherapy.

c-Jun-mediated anticancer mechanisms of tylophorine.

Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb Tylophora indica. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein 1 (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun N-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation activating transcription factor (ATF) site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: (i) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus c-Jun protein translation. To the best of our knowledge, this report is the first to demonstrate the involvement of c-Jun in the anticancer activity of tylophorine and the release of c-Jun translation from a global translational blockade via the PI3K_PDK1_eEF2 signaling cascade.

Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts antiangiogenic and antitumor activity by targeting vascular endothelial growth factor receptor 2-mediated angiogenesis.

BACKGROUND: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy. Tylophorine is known to possess anti-inflammatory and antitumor activity, but its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is still unknown. Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo. METHODS: We used tylophorine and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro and Ehrlich ascites carcinoma (EAC) tumor in vivo. RESULTS: Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and its downstream signaling pathways including Akt, Erk and ROS in endothelial cells. Using HUVECs we demonstrated that tylophorine inhibited VEGF-stimulated inflammatory responses including IL-6, IL-8, TNF-α, IFN-γ, MMP-2 and NO secretion. Tylophorine significantly inhibited neovascularization in sponge implant angiogenesis assay and also inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit. CONCLUSION: Tylophorine exerts anti-angiogenesis effects via VEGFR2 signaling pathway thus, may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Left, right, or both? On the configuration of the phenanthroindolizidine alkaloid tylophorine from Tylophora indica.

The alkaloid (-)-tylophorine was isolated from a sample of Tylophora indica, and the crude extract was analyzed by HPLC/MS(n) and chiral HPLC/MS. While the literature states that the naturally occurring form of this alkaloid is the R-enantiomer and that its S-antipode is usually not found in nature, we confirmed the hypothesis of Govindachari and Nagarajan that natural levorotatory tylophorine is indeed a nearly racemic mixture with a slight excess of the R-enantiomer.

Brine shrimp cytotoxicity of crude methanol extract and antispasmodic activity of α-amyrin acetate from Tylophora hirsuta wall.

BACKGROUND: We have previously reported that aerial parts of Tylophora hirsuta have antispasmodic profile. The current work is an attempt for isolation of pharmacologically active compound(s) that contribute for its antispasmodic activity. METHODS: Preliminary phytochemical screening for crude methanol extract of Tylophora hirsuta (Th.Cr) is performed. Brine shrimp cytotoxicity of crude methanol extract is performed. Column chromatography was used for isolation of compounds. Mass spectroscopy, H(1) NMR and C(13) NMR were used for structural determination of compounds. α-amyrin acetate was tried for possible spasmolytic activity in rabbit's jejunal preparations and KCl-induced contractions. RESULTS: Th.Cr tested positive for saponins, alkaloids, flavonoids and terpenoids. Compound 1 was isolated as α-amyrin acetate. Compound 2 was heptaeicosanol. Crude methanol extract tested positive for brine shrimp cytotoxicity with LC(50) 492.33± 8.08 mg/ml. Compound 1 tested positive for antispasmodic activity on spontaneous rabbits' jejunum preparations with EC(50) (60 ± 2) × 10(-5)M. The compound also tested positive on KCl induced contractions with EC(50) (72 ± 3) × 10(-5)M. CONCLUSIONS: The present work confirms that α-amyrin acetate is has antispasmodic profile and the relaxant effect may be attributed to α-amyrin acetate which is a major compound.

Elicitor mediated enhancement of shoot biomass and lupeol production in Hemidesmus indicus (L.) R. Br. ex. Schult. and Tylophora indica (Burm. F.) Merrill using yeast extract and salicylic acid.

Hemidesmus indicus (L.) R. Br. ex Schult. and Tylophora indica (Burm. F.) Merrill shoot cultures were treated with different concentrations of yeast extract (YE; 25-200 mg/L) and salicylic acid (SA; 50-200 µM), and their effect on lupeol production was assessed. The maximum dry weight (DW) biomass was recorded when H. indicus shoots were treated with SA (50 µM) and T. indica shoots with YE (200 mg/L). Highest lupeol yield (335.40 ± 0.04 µg/g DW) was obtained in H. indicus shoots after treatment with 50 µM of SA for 3 weeks. Whereas in T. indica, maximum lupeol content (584.26 ± 8.14 µg/g DW) was recorded by giving treatment with 25 µM of SA for 6 weeks.

Tylophora indica (Burm. f.) Merr alleviates tracheal smooth muscle hyperresponsiveness in ovalbumin-induced allergic-asthma model in guinea-pigs: Evidences from ex vivo, in silico and in vivo studies.

BACKGROUND: Tylophora indica (Burm. f.) Merr is a climbing perennial plant reported in Indian traditional system of medicine for its use in allergy and asthma. However, only few scientific studies have been performed in the past to validate its antiasthmatic potential. OBJECTIVES: The present study deals with investigation of airway smooth muscle relaxant and antiasthmatic potential of extract and subsequent fractions prepared from T. indica. METHODS: The most active fraction of T. indica leaves selected through bio-guided activity was subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for chemical profiling. The binding affinity of identified compounds in fraction towards M3 and H1 receptors was determined by molecular docking study. F-2 (chloroform fraction prepared from methanolic extract of T. indica leaves) was examined for its smooth muscle relaxant properties using isolated trachea of guinea-pig. Further, F-2 was evaluated through in vivo studies employing ovalbumin-induced asthma model in guinea-pigs. RESULTS: F-2 was found most effective in bioassay-guided fractionation. Characterization by LC-MS analysis revealed presence of five major bioactive compounds in F-2 that showed good docking interactions with M3 and H1 receptors. The ex vivo study demonstrated that F-2 could significantly relax tracheal rings via targeting multiple signalling pathways videlicet, namely, noncompetitive antagonism of the histamine and muscarinic receptors, ß2-adrenergic stimulation and activation of soluble guanylyl cyclase. In in vivo studies, F-2 ameliorated airway hyperresponsiveness and decreased broncho alveolar lavage fluid (BALF) levels of inflammatory cytokines and immunoglobulin E (IgE). CONCLUSION: These results confirm the traditional use of T. indica as an antiasthmatic agent which are evidenced through ex vivo, in silico and in vivo studies.

Effects of Tylophora yunnanensis Schltr on regulating the gut microbiota and its metabolites in non-alcoholic steatohepatitis rats by inhibiting the activation of NOD-like receptor protein 3.

ETHNOPHARMACOLOGICAL RELEVANCE: Tylophora yunnanensis Schltr (TYS) is widely distributed in Yunnan, Guizhou, and other places in China. It is commonly used by folks to treat hepatitis and other liver-related diseases; however, its mechanism of action is still unclear. AIM OF THE STUDY: This study aimed to determine the effects of TYS on regulating gut microbiota and its metabolites in non-alcoholic steatohepatitis (NASH) rats by inhibiting the activation of NOD-like receptor protein3 (NLRP3). MATERIAL AND METHODS: An HFD-induced rat model was established to investigate if the intragastric administration of TYS could mediate gut microbiota and their metabolites to ultimately improve the symptoms of NASH. The improving effects of TYS on NASH rats were assessed by measuring their body weight, lipid levels, histopathology, and inflammatory factor levels in the rat models. The regulatory effects of TYS on NLRP3 in the NASH rats were analyzed using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), which determined the levels of NLRP3-related factors. The changes in the composition of the gut microbiota of NASH rats were analyzed using 16S rRNA gene sequencing technology. Meanwhile, the Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for the non-targeted analysis of metabolites in the cecum contents. RESULTS: The results showed that TYS could improve NASH by decreasing the body weight and levels of lipid, AST, ALT, LPS, FFA, VLDL, IL-1ß, IL-6, TNF-α, TGF-ß, NLRP3, ASC, and Caspase-1 in the NASH rats. The analysis of gut microbiota showed that TYS could improve the diversity and abundance of gut microbiota and alter their composition by decreasing the Firmicutes/Bacteroidetes (F/B) ratio and relative abundances of Lachnospiraceae, Christensenellaceae, Blautia, etc. while increasing those of Muribaculaceae, Rumiaococcus, Ruminococcaceae, etc. The analysis of metabolites in the cecum contents suggested that the arachidonic acid metabolism, bile secretion, serotonergic synapse, Fc epsilon RI signaling pathway, etc. were regulated by TYS. The metabolites enriched in these pathways mainly included chenodeoxycholic acid, prostaglandin D2, TXB2, 9-OxoODE, and 13(S)-HOTrE. CONCLUSIONS: These findings suggested that TYS could alleviate the NASH symptoms by decreasing the body weight, regulating the lipid levels, reducing the inflammatory response, and inhibiting the expression levels of NLRP3, ASC, and Caspase-1 in the NASH rats. The changes in the composition of gut microbiota and their metabolic disorder were closely related to the activation of NLRP3. TYS could significantly inhibit the activation of NLRP3 and regulate the composition of gut microbiota and the disorder of metabolites during NASH modeling.

Interaction studies of an anticancer alkaloid, (+)-(13aS)-deoxytylophorinine, with calf thymus DNA and four repeated double-helical DNAs.

BACKGROUND: Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity. The specific biomolecular targets of these alkaloids have not yet been clearly identified. (+)-(13aS)-deoxytylophorinine is a new phenanthroindolizidine alkaloid originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata in our institute. (+)-(13aS)-deoxytylophorinine exerts both in vitro and in vivoanticancer activities. METHODS: The in vivo anticancer effects and toxicity of this compound were investigated in mice, and interactions between this compound and double-helical DNA sequences were studied in detail with circular dichroic spectroscopy and fluorescence spectroscopy. Viscosity measurements were applied to check the interactive mode between this compound and DNA. RESULTS: Potent anticancer effects were observed in vivo. Also, concentration-dependent interactions were observed and this compound seemed to interact in a sequence-specific manner with AT-repeated sequences of double-helical DNA. Such interactions were proved to be intercalating by viscosity measurements. CONCLUSIONS: Anticancer alkaloid (+)-(13aS)-deoxytylophorinine can have sequence-specific interactions with DNA in an intercalating manner.

Isolation and biological activities of phenanthroindolizidine and septicine alkaloids from the Formosan Tylophora ovata.

An investigation of alkaloids present in the leaves and stems of Tylophora ovata led to the isolation of two new septicine alkaloids and one new phenanthroindolizidine alkaloid, tylophovatines A, B, C (1, 2, and 5), respectively, together with two known septicine and six known phenanthroindolizidine alkaloids. The structures of the new alkaloids 1, 2, and 5 were established by means of spectroscopic analyses. These eleven alkaloids show in vitro anti-inflammatory activities with IC50 values ranging from 84 nM to 20.6 µM through their suppression of nitric oxide production in RAW264.7 cells stimulated by lipopolysaccharide and interferon-γ. Moreover, these substances display growth inhibition in HONE-1, NUGC-3, HepG2, SF-268, MCF-7, and NCI-H460 cancer cell lines, with GI50 values ranging from 4 nM to 24.2 µM. In addition, tylophovatine C (5) and 13a(S)-(+)-tylophorine (7) were found to exhibit potent in vivo anti-inflammation activities in a rat paw edema model. Finally, structure­activity relationships were probed by using the isolated phenanthroindolizidines and septicines. Phenanthroindolizidines are suggested to be divided into cytotoxic agents (e.g., 10 and 11) and anti-inflammation based anticancer agents (e.g., 5­9).

Anticancer effect and neurotoxicity of S-(+)-deoxytylophorinidine, a new phenanthroindolizidine alkaloid that interacts with nucleic acids.

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity as well as other effects like antiamebicidal, antiviral, and anti-inflammatory activities. The specific biomolecular targets of these compounds have not yet been clearly identified. S-(+)-Deoxytylophorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata. Potent anticancer activity was observed in vitro and in vivo. Neurotoxicity of CAT was also studied and it was far less serious than that of vinblastine. Interactions between this compound and DNA had been studied in detail in our laboratory previously, and we further studied its interactions with RNA.

Tylophora hirsuta (Wall.) Extracts Ameliorate Diabetes Associated with Inflammation in Alloxan-induced Diabetic Rats.

BACKGROUND: Tylophora hirsuta (Wall) has long been used as traditional medicine for the treatment of diabetes. The current study is designed to evaluate the anti-diabetic and anti-inflammatory activity of different extracts of aerial parts of Tylophora hirsuta. METHODS: Sequential maceration was conducted to obtain extracts. Total phenolic contents were determined by the Folin-Ciocalteau method. The anti-oxidant activity was assessed by DPPH free radical scavenging assay. The extracts were tested for its inhibitory activity against α-amylase in-vitro. In-vivo anti-diabetic assay was conducted using alloxan-induced diabetic model and OGTT was conducted on normal rats. ELISA was used to determine the pro-inflammatory cytokines (TNF-α and IL-6). The polyphenolic composition of the extract was analyzed using an HPLC system. RESULTS: Aqueous extract exhibited highest total phenolic contents (985.24± 3.82 mg GAE/100 g DW), antioxidant activity (IC50 = 786.70 ± 5.23 µg/mL), and alpha-amylase inhibition (IC50 =352.8 µg/mL). The aqueous extract of Tylophora hirsuta showed remarkable in-vivo anti-diabetic activity. Results were compared with standard drug glibenclamide. Alloxan induced diabetic mediated alterations in liver function enzymes, renal function determinants, and lipid parameters were significantly restored in aqueous extract treated diabetic rats. A significant reduction in pro-inflammatory cytokines (p<0.001) was observed when compared to the control group. HPLC analysis confirms the presence of quercetin, gallic acid, cinnamic acid, and p-coumaric acid. CONCLUSION: These results showed that Tylophora hirsuta possesses strong anti-diabetic and anti-inflammatory potentials and justify its folklore use for the management of diabetes.