Bioassay-guided isolation of anti-inflammatory, antinociceptive and wound healer glycosides from the flowers of Verbascum mucronatum Lam.

Ethnopharmacological relevance

The leaves, flowers and whole aerial parts of Verbascum L. species have been used to treat respiratory problems, haemorrhoids and other types of inflammatory conditions in traditional Turkish medicine.

Aim of the study

In order to evaluate this traditional information, the anti-inflammatory, antinociceptive and wound healing activities of Verbascum mucronatum Lam. which is used as haemostatic in Turkish folk medicine were investigated.

Materials and methods

In vivo inhibitory effect of the extracts on the carrageenan-induced hind paw edema model in mice was studied for the assessment of anti-inflammatory activity. Moreover, the wound healing potential of the plant were evaluated by using in vivo wound healing experimental models, i.e. incision and excision models on mice and rats, were comparatively assessed with a reference ointment Madecassol^®. Skin samples were also evaluated histopathologically.

Results

The results of these experimental studies exhibited that Verbascum mucronatum displays anti-inflammatory, antinociceptive and wound healing activities. Through bioassay-guided fractionation and isolation procedures four iridoid glucosides, ajugol (1), aucubin (2), lasianthoside I (3), catalpol (4), two saponins, ilwensisaponin A (5) and C (6) and a phenylethanoid glycoside, verbascoside (7) were isolated and their structures were elucidated by spectral techniques. Verbascoside (7) was found to possess significant wound healing activity as well as antinociceptive and anti-inflammatory potentials, per os without inducing any apparent acute toxicity or gastric damage.

Conclusion

The experimental study revealed that Verbascum mucronatum displays remarkable antinociceptive, anti-inflammatory and wound healing activities.     

Bioactive styryllactones and alkaloid from flowers of Goniothalamus laoticus

Ethnopharmacological relevance

Goniothalamus laoticus (Annonaceae) is being used traditionally as a tonic and a febrifuge by the local people in the northeastern part of Thailand.

Aim of study

To investigate the Thai medicinal plant, Goniothalamus laoticus, for antiplasmodial, antimycobacterial and cytotoxicity activities.

Materials and methods

The flowers extracts of Goniothalamus laoticus were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The antiplasmodial, antimycobacterial and cytotoxicity evaluation of styryllactone derivatives and alkaloid were also performed.

Results

Ten compounds, cinnamic acid (1); dihydrochrysine (2); β-sitosterol (3); six styryllactones, (+)-3-acetylaltholactone (4), goniotriol (5), (+)-altholactone (6), (+)-goniofufurone (7), 9-deoxygoniopypyrone (8), howiinin A (9); and an aporphine alkaloid; (−)-nordicentrine (10) were isolated from flowers of Goniothalamus laoticus. Among these, compounds 1, 3–5, 8–10 are first isolated from the Goniothalamus laoticus. Besides, compound 10 is the first report from the Goniothalamus genus. The isolated compounds were evaluated in antiplasmodial, antimycobacterial and anticancer cell lines tests. Compounds 4–6 and 10 exhibited antiplasmodial activity against Plasmodium falciparum (IC₅₀ 2.6, 7.9, 2.6 and 0.3 μg/mL, respectively), while 5, 6, 9 and 10 showed antimycobacterial activity against Mycobacterium tuberculosis (MIC 100, 6.25, 6.25 and 12.5 μg/mL, respectively). In addition, compounds 4–10 showed cytotoxicity against cancer cells, KB, BC1, NCI-H187, and MCF-7 with IC₅₀ ranging from 0.4 to 22.7 μg/mL.

Conclusion

This finding showed that the styryllactone derivatives and alkaloid isolated from the flowers of Goniothalamus laoticus exhibited antiplasmodial activity against Plasmodium falciparum, antimycobacterail against Mycobacterium tuberculosis and cytotoxicity against four cancer cell lines.     

In vitro activity of Tridax procumbens against promastigotes of Leishmania mexicana

Ethnopharmacological relevance

Tridax procumbens is an active herb against leishmaniasis.

Aim of the study

Leishmaniasis is a group of diseases caused by Leishmania protozoa. We investigated the antileishmanial activity of Tridax procumbens extracts and a pure compound against promastigotes of Leishmania mexicana, the causative agent of cutaneous leishmaniasis in the New World.

Materials and methods

Extracts and (3S)-16,17-didehydrofalcarinol (1) were obtained by chromatographic methods from Tridax procumbens, and the latter identified by spectroscopic analysis. The effect of these extracts and 1 on the growth inhibition of promastigotes of Leishmania mexicana was evaluated. In order to test the safety of extracts and 1, mammalian cells were treated with them, and cell viability was assessed using trypan blue and MTT.

Results

We demonstrated that extracts of Tridax procumbens and 1 showed a pronounced activity against Leishmania mexicana. The methanol extract inhibited promastigotes growth of Leishmania mexicana with a 50% inhibitory concentration (IC₅₀) of 3 μg/ml, while oxylipin 1 exhibited the highest inhibition at IC₅₀ = 0.478 μg/ml.

Conclusions

In this study we report the biological activity of extracts and (3S)-16,17-didehydrofalcarinol (1), obtained from Tridax procumbens, on the promastigote form of Leishmania mexicana, with no effect upon mammalian cells.     

Isolation and identification of antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Ethnopharmacological relevance

Muntingia calabura (Elaeocarpaceae) is one of the most common roadside trees in Malaysia. Its leaves, barks, flowers and roots have been used as a folk remedy for the treatment of fever, incipient cold, liver disease, as well as an antiseptic agent in Southeast Asia. The aim of this study is to isolate and identify the antibacterial and cytotoxic compounds from the leaves of Muntingia calabura L.

Materials and methods

Antibacterial and cytotoxic activities were determined by micro-broth dilution and MTT assays, respectively. Seven fractions (F1–F7), three flavones and a chalcone were isolated from the active EtOAc extract using bioassay-guided screening. The structures of four compounds were elucidated by spectroscopic methods and compared with published data. The compounds were further tested for their antibacterial and cytotoxic activities.

Results

Three flavones and a chalcone [5,7-dihydroxy-3,8-dimethoxyflavone (1), 2′,4′-dihydroxychalcone (2), 5-hydroxy-3,7-dimethoxyflavone (3) and 3,5,7-trihydroxy-8-methoxyflavone (4)] were isolated from the active fraction F5 of EtOAc extract. Compounds 1 and 3 were isolated for the first time from Muntingia calabura L. Antibacterial activity indicates that compound 2 exhibited the most significant activity with MIC value of 50 μg/mL and 100 μg/mL against MSSA and MRSA, respectively. Cytotoxic activity indicates that compounds 2 and 3 exhibited very strong activity against HL60 with IC₅₀ values of 3.43 μg/mL and 3.34 μg/mL, respectively.

Conclusion

The antibacterial activity of the leaves of Muntingia calabura L. is ascribable to the active compound 2 while the cytotoxic activity is ascribable to the active compounds 2 and 3.     

Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents

Ethnopharmacological relevance

Arctium lappa and Tussilago farfara (Asteraceae) are two plant species used traditionally as antitubercular remedies. The aim of this study was (i) to screen Arctium lappa and Tussilago farfara extracts for activity against Mycobacterium tuberculosis and (ii) to isolate and identify the compound(s) responsible for this reputed anti-TB effect.

Materials and methods

The activity of extracts and isolated compounds was determined against Mycobacterium tuberculosis H₃₇R_v using a high throughput spot culture growth inhibition (HT-SPOTi) assay.

Results

The n-hexane extracts of both plants, the ethyl acetate extract of Tussilago farfara and the dichloromethane phase derived from the methanol extract of Arctium lappa displayed antitubercular activity (MIC 62.5 μg/mL). Further chemical investigation of Arctium lappa led to the isolation of n-nonacosane (1), taraxasterol acetate (2), taraxasterol (3), a (1:1) mixture of β sitosterol/stigmasterol (4), isololiolide (5), melitensin (6), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10). Compounds isolated from Tussilago farfara were identified as a (1:1) mixture of β sitosterol/stigmasterol (4), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10), loliolide (11), a (4:1) mixture of p-coumaric acid/4-hydroxybenzoic acid (12), p-coumaric acid (13). All compounds were identified following analyses of their physicochemical and spectroscopic data (MS, ¹H and ¹³C-NMR) and by comparison with published data. This is the first report of the isolation of n-nonacosane (1), isololiolide (5), melitensin (6) and kaempferol-3-O-glucoside (10) from Arctium lappa, and of loliolide (11) from Tussilago farfara. Amongst the isolated compounds, the best activity was observed for p-coumaric acid (13) (MIC 31.3 μg/mL or 190.9 μM) alone and in mixture with 4-hydroxybenzoic acid (12) (MIC 62.5 μg/mL).

Conclusions

The above results provide for the first time some scientific evidence to support, to some extent, the ethno-medicinal use of Arctium lappa and Tussilago farfara as traditional antitubercular remedies.     

Topical analgesic,anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: Phytochemical characterization of active fractions

Ethnopharmacological relevance

Oxybaphus nyctagineus (Michx.) Sweet has traditionally been used by several Native American tribes predominantly as a topical anti-inflammatory and analgesic agent.

Aim of the study

To evaluate the antioxidant, analgesic and anti-inflammatory activity of the extracts prepared from the aerial parts of Oxybaphus nyctagineus and to characterize the major chemical constituents of the bioactive extracts.

Materials and methods

Crude polar and apolar extracts (PCE and ACE) of the herb of Oxybaphus nyctagineus were prepared and tested in the models of the CFA-induced hyperalgesia in rat knee and carrageenan-induced paw edema in rat. To identify the active compounds, subfractions were prepared by column chromatography and subjected in vitro assays, such as antioxidant assays (DPPH, peroxynitrite (ONOO⁻) scavenging), and the LPS-induced IL-1β release test in human monocytes. Preparative HPLC was employed for the isolation of active substances, while phytochemical analysis was performed by mean of LC–MS/MS and NMR.

Results

The topically administered PCE and ACE of Oxybaphus nyctagineus demonstrated a significant analgesic and anti-inflammatory effect in the inflammation animal models. The subfraction A4 of ACE and the subfraction P5 of PCE considerably inhibited the LPS-induced IL-1β release in human monocytes, while the strongest activity was localized in the subfraction P5 in the antioxidant assays. The HPLC–MS/MS and NMR analysis revealed that 6-methoxyflavonol diglycosides, namely patuletin-3-O-robinobioside (1), 6-methoxykaempferol-3-O-robinobioside (2), spinacetin-3-O-robinobioside (3), and hydroxy-polyenoic fatty acids, namely corchorifatty acid B (4), 9-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9-HOT acid) (5), and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HOD acid) (6) were present in PCE, and in ACE as major compounds.

Conclusion

The results of this study established a pharmacological evidence for the traditional use of Oxybaphus nyctagineus as an anti-inflammatory agent used topically, and provided data on its phytochemical composition for the first time.     

Antiplasmodial activity of sesquiterpene lactones and a sucrose ester from Vernonia guineensis Benth. (Asteraceae)

Ethnopharmacological relevance

Aqueous preparations of Vernonia guineensis Benth. (Asteraceae) are used in Cameroonian folk medicine as a general stimulant and to treat various illnesses and conditions including malaria, bacterial infections and helminthic infestations.

Materials and methods

Ten gram samples of the leaf and tuber powders of Vernonia guineensis were extracted separately using dichloromethane, methanol and distilled water. The extracts were dried in vacuo and used in bioassays. These extracts and three compounds previously isolated from Vernonia guineensis [vernopicrin (1), vernomelitensin (2) and pentaisovalerylsucrose (3)] were screened for antiplasmodial activity against chloroquine (CQ)-sensitive (Hb3) and CQ-resistant (Dd2) Plasmodium falciparum lines.

Results

Crude extracts and pure compounds from Vernonia guineensis showed antiplasmodial activity against both Hb3 and Dd2. The IC₅₀ values of extracts ranged from 1.64 to 27.2 µg/ml for Hb3 and 1.82–30.0 µg/ml for Dd2; those for compounds 1, 2 and 3 ranged from 0.47 to 1.62 µg/ml (1364–1774 nM) for Hb3 and 0.57–1.50 µg/ml (1644–2332 nM) for Dd2. None of the crude extracts or pure compounds was observed to exert toxic effects on the erythrocytes used to cultivate the Plasmodium falciparum lines.

Conclusion

In Cameroonian folk medicine, Vernonia guineensis may be used to treat malaria in part due to the antiplasmodial activity of sesquiterpene lactones (1, 2), a sucrose ester (3) and perhaps other compounds present in crude plant extracts. Exploring the safety and antiplasmodial efficacy of these compounds in vivo requires further study.     

Vitexin and isovitexin from the Leaves of Ficus deltoidea with in-vivo α-glucosidase inhibition

Ethnopharmacological relevance

The leaves of Ficus deltoidea are used as a traditional medicine by diabetes patients in Malaysia.

Aim of the study

The objective of the study is to identify and evaluate bioactive constituents with in vivo α-glucosidase inhibition.

Materials and Methods

The partitioned extracts, subfractions and pure bioactive constituents were subjected to α-glucosidase inhibition assay. The identified bioactive constituents were administered orally to sucrose loaded normoglycemic mice and induced diabetic rats. The postprandial blood glucose levels were monitored at 30 min interval. Acute toxicity was evaluated in both normoglycemic mice and induced diabetic rats.

Results

Bioactivity guided fractionation led to the isolation of both vitexin (1) and isovitexin (2). Oral administration of 1 mg/kg of either vitexin (1) or isovitexin (2) significantly (p<0.05) reduced the postprandial blood glucose level in sucrose loaded normoglycemic mice at 30 min. The percentage of postprandial blood glucose reduction was highest in sucrose loaded induced diabetic rats administered orally with 200 mg/kg of vitexin (1) or 100 mg/kg of isovitexin (2). Both vitexin (1) and isovitexin (2) did not exert any signs of toxicity at the highest dose of 2 g/kg administered orally to normoglycemic mice and induced diabetic rats.

Conclusion

Both the C-glycosyl bioflavonoids, namely, vitexin (1) and isovitexin (2) exhibited in vivo α-glucosidase inhibition.     

Antimicrobial activity of the crude extract,fractions and compounds from stem bark of Ficus ovata (Moraceae)

Aim of the study

This study was designed to investigate the antimicrobial activities of the methanol extracts from the stem bark of Ficus ovata (FOB), fractions (FOB1–6) and compounds isolated following bio-guided fractionation [3-friedelanone (1), taraxeryl acetate (2), betulinic acid (3), oleanoïc acid (4), 2-hydroxyisoprunetin (5), 6,7-(2-isopropenyl furo)-5,2,4-trihydroxyisoflavone (6), Cajanin (7) and protocatechuic acid (8)].

Materials and Methods

The micro-dilution method was used for the determination of the minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC) against fungi (two species), Gram-positive (three species) and Gram-negative bacteria (five species).

Results

The results of the MIC determinations indicated that the crude extract (FOB), fractions FOB2 and FOB4 as well as compound 5 were active on the entire studied organisms. Other samples showed selective activity, fractions FOB1, FOB3 and FOB5 being active against 50% of the tested microbial species while FOB6 was active on 40%. Compounds 8, 6, 2 and 7 prevented the growth of 80%, 70%, 50% and 20% of the organisms respectively. The lowest MIC value (156g/ml) observed with the crude extract was recorded on Streptococcus faecalis, Candida albicans and Microsporum audouinii. The corresponding value for fractions (39 μg/ml) was noted with FOB4 against Staphylococcus aureus, while that of the tested compounds (10 μg/ml) was observed with compound 8 on Microsporum audouinii. The results of the MMC determination suggested that the cidal effect of most of the tested samples on the studied microorganisms could be expected.

Conclusions

The overall results provided evidence that the studied plant extract, as well as some of the isolated compounds might be potential sources of new antimicrobial drug.     

Antinociceptive and anti-inflammatory kaempferol glycosides from Sedum dendroideum

Aim of the study

To identify the compounds responsible for the antinociceptive and anti-inflammatory effects previously described for Sedum dendroideum, through bioassay-guided fractionation procedures.

Materials and methods

Antinociceptive activity was evaluated through mouse acetic acid-induced writhing model. The anti-inflammatory activity was assessed through croton oil-induced mouse ear oedema and carrageenan-induced peritonitis.

Results

The Sedum dendroideum juice afforded seven known flavonoids identified with basis on NMR data. The oral administration of the major kaempferol glycosides kaempferitrin [1] (17.29 μmol/kg), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside [2] (16.82 μmol/kg), kaempferol 3-O-neohesperidoside-7-O-α-rhamnopyranoside [3] (13.50 μmol/kg) or α-rhamnoisorobin [5] (23.13 μmol/kg) inhibited by 47.3%, 25.7%, 60.2% and 58.0%, respectively, the acetic acid-induced nociception (indomethacin: 27.95 μmol/kg, p.o.; 68.9%). Flavonoids 1, 2, 3 or 5, at the same doses, reduced by 39.5%, 46.5%, 35.6% and 33.3%, respectively, the croton oil-induced oedema (dexamethasone: 5.09 μmol/kg, s.c.; 83.7%) and impaired leukocyte migration by 42.9%, 46.3%, 50.4% and 49.6%, respectively (dexamethasone: 5.09 μmol/kg, s.c.; 66.1%).

Conclusions

Our findings show that the major kaempferol glycosides may account for the renowned medicinal use of Sedum dendroideum against pain and inflammatory troubles.     

4-Methylthio-butanyl derivatives from the seeds of Raphanus sativus and their biological evaluation on anti-inflammatory and antitumor activities

Ethnopharmacological relevance

Raphanus sativus seeds (Brassicaceae) known as Raphani Semen have long been used as anti-cancer and/or anti-inflammatory agents in Korean traditional medicine. This study was designed to isolate the bioactive constituents from the seed extracts of Raphanus sativus and evaluate their anti-inflammatory and antitumor activities.

Material and methods

Bioassay-guided fractionation and chemical investigation of a methanolic extract of the seeds of Raphanus sativus led to the isolation and identification of seven 4-methylthio-butanyl derivatives. Structural elucidation of the isolated compounds was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques (¹H, ¹³C, COSY, HMQC and HMBC experiments) and mass spectrometry.

Results

The isolated compounds were characterized as in the following: three new 4-methylthio-butanyl derivatives, sinapoyl desulfoglucoraphenin (1), (E)-5-(methylsulfinyl)pent-4-enoxylimidic acid methyl ester (2), and (S)-5-((methylsulfinyl)methyl)pyrrolidine-2-thione (3), together with four known compounds, 5-(methylsulfinyl)-4-pentenenitrile (4), 5-(methylsulfinyl)-pentanenitrile (5), sulforaphene (6), and sulforaphane (7). Full NMR data assignments of the three known compounds 4–6 were also reported for the first time. We evaluated the anti-neuroinflammatory effect of 1–7 in lipopolysaccharide-stimulated murine microglia BV2 cells. Compound 1 significantly inhibited nitrite oxide production with IC₅₀ values of 45.36 μM. Moreover, it also reduced the protein expression of inducible nitric oxide synthase. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and all of them showed antiproliferative activity against the HCT-15 cell, with IC₅₀ values of 8.49–23.97 μM.

Conclusions

4-Methylthio-butanyl derivatives were one of the main compositions of Raphanus sativus seeds, and activities demonstrated by the isolated compounds support the ethnopharmacological use of Raphanus sativus seeds (Brassicaceae) as anti-cancer and/or anti-inflammatory agents.     

Bioactivity-guided isolation of polyacetylenes with inhibitory activity against NO production in LPS-activated RAW264.7 macrophages from the rhizomes of Atractylodes macrocephala

Ethnopharmacological relevance

The rhizome of Atractylodes macrocephala (Compositae) is one of the most well-known traditional Chinese medicine in China, Japan and Korea, which has a long history of use for the treatment of splenic asthenia, edema, anorexia, and excessive perspiration, etc. As active compounds of anti-inflammatory activity of this medicinal plant have not been fully elucidated, the aim of this study was to isolate and identify the active constituents inhibiting nitric oxide (NO) production from the rhizomes of A. macrocephala.

Materials and methods

Inhibitory activity against NO production in lipopolysaccharide-activated RAW264.7 macrophages was evaluated by Griess reaction. Fifteen polyacetylenes were isolated from the active ethyl acetate extract using activity-guided screening. The structures of all compounds were elucidated by spectroscopic methods and comparison with published data. The compounds were further tested for their inhibitory activity against NO production.

Results

Seven new polyacetylenes, named atractylodemaynes A–G (1–7), along with eight known ones (8–15) were isolated. Compound 14 was isolated for the first time from the rhizomes of A. macrocephala. The study showed that the tested compounds exhibited inhibitory activity against NO production in a dose-dependent manner. Among them, compounds 10, 11 and 12 had relatively stronger inhibitory effect with IC₅₀ values of 28, 23 and 19 μM, respectively.

Conclusion

The results demonstrated that the polyacetylenes might greatly contribute to the anti-inflammatory activity of the rhizomes of A. macrocephala.     

HPLC-based activity profiling for antiplasmodial compounds in the traditional Indonesian medicinal plant Carica papaya L

Ethnopharmacological relevance

Leaf decoctions of Carica papaya have been traditionally used in some parts of Indonesia to treat and prevent malaria. Leaf extracts and fraction have been previously shown to possess antiplasmodial activity in vitro and in vivo.

Materials and methods

Antiplasmodial activity of extracts was confirmed and the active fractions in the extract were identified by HPLC-based activity profiling, a gradient HPLC fractionation of a single injection of the extract, followed by offline bioassay of the obtained microfractions. For preparative isolation of compounds, an alkaloidal fraction was obtained via adsorption on cationic ion exchange resin. Active compounds were purified by HPLC–MS and MPLC–ELSD. Structures were established by HR-ESI-MS and NMR spectroscopy. For compounds 5 and 7 absolute configuration was confirmed by comparison of experimental and calculated electronic circular dichroism (ECD) spectroscopy data, and by X-ray crystallography. Compounds were tested for bioactivity in vitro against four parasites (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum), and in the Plasmodium berghei mouse model.

Results

Profiling indicated flavonoids and alkaloids in the active time windows. A total of nine compounds were isolated. Four were known flavonols – manghaslin, clitorin, rutin, and nicotiflorin. Five compounds isolated from the alkaloidal fraction were piperidine alkaloids. Compounds 5 and 6 were inactive carpamic acid and methyl carpamate, while three alkaloids 7–9 showed high antiplasmodial activity and low cytotoxicity. When tested in the Plasmodium berghei mouse model, carpaine (7) did not increase the survival time of animals.

Conclusions

The antiplasmodial activity of papaya leaves could be linked to alkaloids. Among these, carpaine was highly active and selective in vitro. The high in vitro activity could not be substantiated with the in vivo murine model. Further investigations are needed to clarify the divergence between our negative in vivo results for carpaine, and previous reports of in vivo activity with papaya leaf extracts.     

Phenological and geographical influence in the concentration of selected bioactive 4-phenylcoumarins and chlorogenic acid in Hintonia latiflora leaves

Ethnopharmacological relevance

Hintonia latiflora is a Mexican medicinal plant with well-documented ethnomedical record comprising more than 400 years; in modern Mexico is used for treating several maladies such as diabetes and gastric ulcers. Although the pharmacological actions of the stem-bark and leaves have been demonstrated, the phenological and geographical effect on the concentration of active principles remains unexplored.

Aim of the study

The main goals of this study were to analyze the amount of selected 4-phenylcoumarins and chlorogenic acid in the leaves in order to assess the best harvesting period, and consequently their pharmacological efficacy. In addition, the preclinical antidiabetic efficacy of the infusion of the leaves was corroborated using standard pharmacological tests.

Materials and methods

The aqueous extracts from the leaves of Hintonia latiflora were prepared by infusion. For phenological and geographical comparison, leaves of Hintonia latiflora were collected in two different regions in Chihuahua and Michoacán. The material was analyzed by UPLC applying an analytical method that developed and validated for this purpose following the ICH guidelines. Investigation of the antidiabetic action was accomplished using an acute hypoglycemic test and oral glucose and sucrose tolerance tests.

Results

The validated analytical method was successfully applied for quantifying chlorogenic acid (1) and 4-phenylcoumarins (2–5) in the leaves of 12 different batches (1–12) during one-year period, and seven different batches for each geographical region; the concentration of the metabolites at the phenological cycle was significantly different, their concentration increased during the pre-senescence phase whereas in the leaf renovation stage the highest concentration of 2–5 was reached. The overall analysis of the active compounds concentration between the two populations investigated seems to be less important than the phenological variations. The aqueous extract of the leaves of Hintonia latiflora exerted its antidiabetic effect by different mechanisms showing comparable effect to the organic extract.

Conclusions

The findings of the present investigation reveal that the best harvest season for the leaves of Hintonia latiflora is between the leaves renovation and senescence stages avoiding the flowering period. In addition, no significant differences were found among the two different geographical populations analyzed. The infusions of the leaves, rich in 4-phenylcoumarins and chlorogenic acid, showed comparable antidiabetic action than the organic extract.     

Estrogenic and serotonergic butenolides from the leaves of Piper hispidum Swingle (Piperaceae)

Ethnopharmacological relevance

Our previous work has demonstrated that several plants in the Piperaceae family are commonly used by the Q’eqchi Maya of Livingston, Guatemala to treat amenorrhea, dysmenorrhea, and pain. Extracts of Piper hispidum Swingle (Piperaceae), bound to the estrogen (ER) and serotonin (5-HT7) receptors.

Aim of the study

To investigate the estrogenic and serotonergic activities of Piper hispidum extracts in functionalized assays, identify the active chemical constituents in the leaf extract, and test these compounds as agonists or antagonists of ER and 5-HT7.

Materials and methods

The effects of the Piper hispidum leaf extracts were investigated in estrogen reporter gene and endogenous gene assays in MCF-7 cells to determine if the extracts acted as an estrogen agonist or antagonist. In addition, the active compounds were isolated using ER- and 5-HT7 receptor bioassay-guided fractionation. The structures of the purified compounds were identified using high-resolution LC–MS and NMR spectroscopic methods. The ER- and 5-HT7-agonist effects of the purified chemical constituents were tested in a 2ERE-reporter gene assay in MCF-7 cells and in serotonin binding and functionalized assays.

Results

Three butenolides including one new compound (1) were isolated from the leaves of Piper hispidum, and their structures were determined. Compound 1 bound to the serotonin receptor 5-HT₇ with IC₅₀ values of 16.1 and 8.3 μM, respectively, and using GTP shift assays, Compound 1 was found to be a partial agonist of the 5-HT₇ receptor. The Piper hispidum leaf extracts, as well as Compounds 2 and 3 enhanced the expression of estrogen responsive reporter and endogenous genes in MCF-7 cells, demonstrating estrogen agonist effects.

Conclusions

Extracts of Piper hispidum act as agonists of the ER and 5-HT₇ receptors. Compound 1, a new natural product, identified as 9,10-methylenedioxy-5,6-Z-fadyenolide, was isolated as the 5-HT₇ agonist. Compounds 2 and 3 are reported for the first time in Piper hispidum, and identified as the estrogen agonists. No inhibition of CYP450 was observed for any of these compounds in concentrations up to 1 μM. These activities are consistent with the Q’eqchi traditional use of the plant for the treatment of disorders associated with the female reproductive cycle.     

The constituents of Cibotium barometz and their permeability in the human Caco-2 monolayer cell model

Ethnopharmacological relevance

Cibotium barometz (L.) J. Sm. (Dicksoniaceae) has been traditionally used as anti-inflammatory and anodyne.

Aim of the study

To investigate the constituents in the rhizomes of Cibotium barometz, and evaluate their permeability in the human Caco-2 model.

Materials and methods

The rhizomes extracts of Cibotium barometz were isolated by chromatographic techniques. Structures of isolated compounds were identified by spectroscopic methods. The permeability of the main constituents was evaluated using human Caco-2 cell monolayer as a model system.

Results

Three unusual sesquiterpenes having 1-indanone nucleus (1, 3 and 4) and an unusual orthoester spiropyranosyl derivative of protocatechuic acid (2) were isolated from the rhizomes of Cibotium barometz. Among these, the bilateral permeation of 1, 3 and 4 in Caco-2 model was examined. The apparent permeability coefficients (P_app) of 1 was identical with those of propranolol, which is often used as reference standard of high permeability. The P_app values of 3 and 4 were in agreement with those of atenolol, which is often used as reference standard of poor permeability. The permeation rates of 1, 3 and 4 increased linearly as a function of time up to 180 min and with the concentration within the test range of 25–200 μM.

Conclusions

This is the first report on the presence of compounds 2 and 3 in this plant and 4 was a new compound. Compound 1 is assigned for a well-absorbed, and 2 and 3 are assigned for the poorly absorbed compounds in human intestine. A passive diffusion mechanism for transport of 1, 3 and 4 in Caco-2 model was proposed. The results provided some useful information for predicting the oral bioavailability of 1, 3 and 4.     

Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals

Ethnopharmacological relevance

The rhizomes of Kaempferia parviflora Wall. ex Baker have been used in Thailand for treatment of gout, apthous ulcer, peptic ulcer and abscesses.

Aim of the study

In our previous study, the crude ethanol extract of Kaempferia parviflora and its compound (5, 5-hydroxy-3,7,3′,4′-tetramethoxyflavone), was reported to show nitric oxide (NO) inhibition in RAW 264.7 cells. The present study is thus investigated the anti-inflammatory mechanism of Kaempferia parviflora extract and compound 5 against inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expressions.

Materials and methods

The extract of Kaempferia parviflora and its compound were tested against NO and prostaglandin E₂ (PGE₂) releases using RAW264.7 cells as well as studied on anti-inflammatory activity in carrageenan-induced rat paw edema and acute toxicity in mice.

Results

The results revealed that the ethanol extract of Kaempferia parviflora markedly inhibited PGE₂ release with an IC₅₀ value of 9.2 μg/ml. This plant extract and compound 5 also suppressed mRNA expression of iNOS in dose-dependent manners, whereas COX-2 mRNA expression was partly affected. According to the in vivo study, chloroform and hexane fractions greater decreased rat paw edema than ethanol, ethyl acetate and water fractions.

Conclusion

The mechanisms for anti-inflammatory activity of Kaempferia parviflora and compound 5 are mainly due to the inhibition of iNOS mRNA expression but partly through that of COX-2 mRNA.     

Constituents isolated from Cordyceps militaris suppress enhanced inflammatory mediator's production and human cancer cell proliferation

Aim of the study

The purpose of this study is to isolate the pure compounds from the extracts of Cordyceps militaris obtained through solid-state cultivation process, and evaluate their anti-inflammatory and anticancer properties.

Materials and methods

Silica gel column chromatographic purification of Cordyceps militaris extracts resulted in the isolation of 10 pure compounds (1-10). The compounds 1-10 were examined for their growth inhibitory properties against nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-12 enhanced production from LPS/IFN-γ-stimulated macrophages. Additionally, the anti-proliferation effects of 1-10 on human cancer cell lines, colon (colon 205), prostate (PC-3), and hepatoma (HepG2) cells were also analyzed.

Results

Compound 8 displayed potent growth inhibition on NO, TNF-α and IL-12 production with an IC₅₀ value of 7.5, 6.3, and 7.6 μg/ml, respectively. A similar inhibitory trend on these inflammatory mediators was observed for 3, 7, 9 and 10 with an IC₅₀ values ranging from 10.8 to 17.2 μg/ml. On the other hand, compounds 3 and 8 were potent anti-proliferative agents with an IC₅₀ value of 35.6 and 32.6 μg/ml toward PC-3 and colon 205 cell lines, respectively. The compounds 1 and 2 showed potent anti-proliferation in PC-3 and colon 205 cells, while only 3 displayed such effect in HepG2 cells.

Conclusion

The present study provides scientific supporting information for the ethnopharmacological use of Cordyceps militaris as an anti-inflammatory and anticancer agent.