Sambucus nigra: A traditional medicine effective in reducing inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. AIM OF THE STUDY: The aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. MATERIAL AND METHODS: The anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1ß, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 µg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. RESULTS: The oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1ß and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2-, TNF, IL-1ß and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2-, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2-, TNF, IL-1ß, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. CONCLUSIONS: S. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Improvement in phloroglucinol compound extracted from different parts of Eugenia umbelliflora monitored by LC-UV and antimicrobial activity.

The phloroglucinol eugenial C, eugenial D and eugenial E are the main active compounds in Eugenia umbelliflora fruits. This study aims to evaluate the extraction conditions of E. umbelliflora, using ethanol as solvent, focusing on the phloroglucinol and antimicrobial activity. In order to optimize the extraction conditions, ethanol 50, 70 and 90 oGL was used as a solvent in the proportions of 1:20 (w/v) of drug:solvent ratio (D:S), stirring (330 rpm) at room temperature during 4 h, monitored by LC-UV and antimicrobial assay. The LC-UV method developed was linear over a concentration range of 3.4-68.0, 5.3-106.0 and 5.0-100.8 µg.mL-1 of eugenial C, eugenial D and eugenial E, having LOQ of 1.68, 1.33 and 0.8 µg.mL-1, respectively. The fruits showed the best herbal raw material and showed the highest phloroglucinol concentration and activity against S. aureus, when extracted with ethanol 90oGL, during 4 h, at 1:20 of D:S.

Corticoid-like anti-inflammatory effect of Vochysia bifalcata Warm.: Preclinical evidence of efficacy and safety.

ETHNOPHARMACOLOGICAL RELEVANCE: Vochysia bifalcata is a Brazilian native tree commonly used for economic purpose in the reforestation and in the manufacture of products. However, the potential usage of other parts of the plant is usually wasted. Besides, other species of Vochysia are well known for its anti-inflammatory action. AIM OF THE STUDY: In this study we evaluate the possible anti-inflammatory activity of the hydroethanolic extract from the leaves of V. bifalcata in models of mice skin inflammation. MATERIALS AND METHODS: Effects of V. bifalcata were evaluated in croton oil-induced acute and chronic skin inflammation. The role of glucocorticoid receptors in the extract effect was assessed by using a glucocorticoid receptor antagonist and by a specific binding assay. Possible adverse effects were evaluated after multiple treatments with the extract in a skin atrophy model. RESULTS: Topical application of V. bifalcata reduced ear edema formation, cell infiltration and interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels. In the chronic model, besides edema formation and cell infiltration, the extract inhibited epidermal hyperproliferation and Proliferating Cell Nuclear Antigen expression. V. bifalcata seems to act by biding to corticoid receptors, however it did not induce corticoid related undesirable effects. CONCLUSION: Hydroethanolic extract from leaves of V. bifalcata could be an interesting tool in the search for new anti-inflammatory and antiproliferative agents for the treatment of skin disorders.

Effects of Eugenia umbelliflora O. Berg (Myrtaceae)-leaf extract on inflammation and hypersensitivity.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Eugenia species are widely used in popular medicine to treat several diseases, such as arthritis, rheumatism and diabetes. Eugenia umbelliflora O. Berg is popularly known in Brazil as "baguaçu", name also conferred to Eugenia jambolana probably due to their apparent similarity. Although the popular use scientifically proved of E. jambolana as anti-diabetes and also as anti-inflammatory, there are only two scientific studies demonstrating anti-ulcer and bactericide activities of E. umbelliflora leaves extract, without reference to its possible anti-inflammatory activity. AIM OF THE STUDY: The aim of this study was to show the anti-oxidant and anti-inflammatory activity of the methanol extract obtained from E. umbelliflora leaves (EuL) using in vitro and in vivo protocols. MATERIALS AND METHODS: The total phenolic content was evaluated using the folin-Ciocalteu colorimetric method and phloroglucinols content by HPLC. The anti-oxidant activity was evaluated by ORAC, ABTS•+, DPPH, and metal chelation methods. The anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally pre-treated with the EuL (0.3, 1 or 3 mg/kg). The leukocyte influx (optical microscopy) and secretion of chemical mediators (TNF, IL-6, IL-1ß and CXCL1, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. Histological analysis of the pouches was also performed. The anti-hypersensitive activity was investigated using carrageenan-induced mechanical hypersensitivity and mice were then evaluated using the von Frey filaments. The Open Field test was used to evaluate possible interference of adverse effect of EuL on locomotor activity that could lead to misinterpretation of the hypersensitivity evaluation. RESULTS: The EuL demonstrated important and moderate reducing capacity on ABTS•+ and DPPH assays, respectively, but with slight activity in ORAC test. It reflects low protection against cell damage. The EuL also presented 30% of phenolic compounds. The phloroglucinols content of EuL was 25.9 mg/g, 18.4 mg/g and 16.6 mg/g of eugenial C, eugenial D and eugenial E, respectively. The in vivo analysis of the inflammatory exudate of EuL-treated mice demonstrated reduction in the polymorphonuclear cells (PMN) migration to the inflamed tissue, as well as the reduction of the pro-inflammatory cytokine IL-1ß. Histologically, it was observed evident decrease in the oedema, formed essentially by non-haemorrhagic fibrin exudate, as well as PMN infiltrate, when compared with control mice injected with carrageenan. Furthermore, the extract also presented effective reduction of the mechanical hypersensitivity induced by carrageenan without any interference in animal's locomotor and exploratory activity. CONCLUSIONS: Together, the results herein obtained show that EuL presented anti-inflammatory activity by decreasing the influx of PMN to the inflamed tissue, as well as the cytokine IL-1ß level. This anti-inflammatory activity was also accompanied by significant anti-hypersensitive effect. The effects presented by EuL seem not to be correlated with an antioxidant activity. However other extract chemical compounds could be responsible for its important anti-inflammatory effects.

Aleurites moluccanus and its main active constituent, the flavonoid 2″-O-rhamnosylswertisin, in experimental model of rheumatoid arthritis.

ETHNOPHARMMACOLOGICAL RELEVANCE: Aleurites moluccana is used in folk medicine to treat pain, fever, asthma, hepatitis, gastric ulcer and inflammatory process in general, and the nut oil had been topically applied to treat arthritis and other joint pain, however the seeds are classified as toxic for oral use. AIM: Faced with the need for new alternative to treat the symptoms and modify rheumatoid arthritis (RA) the aim of this work was to evaluate the effects of A. moluccanus' leaves dried extract in rats and mice submitted to complete Freund adjuvant (CFA)-induced RA. MATERIAL AND METHODS: Wistar Rats and Swiss mice were submitted to CFA-induced RA in the right hindpaw. They received A. moluccanus extract (orally; p.o.), dexamethasone (subcutaneously), 2″-O-rhamnosylswertisin (p.o.) or vehicle (p.o.), from the 14th day after the CFA injection for up to 8 days. The mechanical hypersensitivity was evaluated using the von Frey filaments and the paw-oedema was measured using a plethysmometer. The rats' injected hindpaw was used to perform the histological analysis. RESULTS: A. moluccanus was able to significantly reduce the mechanical hypersensitivity in both ipsi- and contralateral hindpaws of mice injected with CFA, in a dose dependent manner. Furthermore, the paw-oedema was progressively reduced by A. moluccanus. Similar results were obtained for the positive-control drug dexamethasone and the isolated compound 2″-O-rhamnosylswertisin. Besides the effects mentioned above, the extract was also effective to repair the joint damage in CFA-induced RA rats, including reduction of fibrosis, cartilage degradation and bone erosion scores. CONCLUSION: These results together with the literature data reinforce the anti-hypersensitivity and anti-inflammatory activity of A. moluccanus extract. Part of the observed effects is due to the presence of the compound 2″-O-rhamnosylswertisin. The fact that the extract acted as a disease modifier point this herbal product as a promisor and safe tool to treat RA and other associated chronic diseases.

Hepatoprotective effect of Maytenus robusta Reiss extract on CCl4-induced hepatotoxicity in mice and HepG2 cells.

We investigated the hepatoprotective effect of methanolic extract from Maytenus robusta leaves in mice and HepG2 cells. The administration of CCl4 in mice promoted a deep destruction of the histological lobular structure and increased the alanine aminotransferase (ALT) serum levels by 46.25% compared with the control group (p < 0.05). The M. robusta extract reduced the hepatic histological changes and normalization the ALT levels. The antioxidant effect of M. robusta in liver tissue promoted the reduction in 31.5% on lipoperoxides levels (p < 0.05), increased by 101.5% the reduced glutathione content (p < 0.05) and increased the activity of superoxide dismutase, catalase, and glutathione-S-transferase by 21.3% (p < 0.05), 49.3% (p < 0.05), and 27.6% (p < 0.05), respectively, compared with the vehicle group. Moreover, the extract reduced hepatic inflammation by diminishing myeloperoxidase activity, TNF and interleukin-6 levels by 29.4% (p < 0.05), 46.1% (p < 0.01), and 59.5% (p < 0.0001), respectively, compared with the vehicle group. The viability of HepG2 cells after incubation with CCl4 was 29.56± 3.07%, whereas the extract (300 µg/mL) restored the viability to 65.27± 8.75% and aspartate aminotransferase levels to 41.82 ± 4.41 U/L. The extract scavenged DPPH (IC50 = 14.44 µg/mL) and ABTS (IC50 = 3.00 µg/mL) radicals and did not produce acute toxicity in mice at 2000 mg/kg. In conclusion, was confirmed the hepatoprotective potential of M. robusta by its antioxidant effects.

Biomonitored UHPLC-ESI-QTOF-MS2 and HPLC-UV thermostability study of the aerial parts of Sphagneticola trilobata (L.) Pruski, Asteraceae.

As the temperature of extraction and processing could impact the biological effects of herbal extracts, which have wide chemical diversity, the aim of this work were to investigate the thermostability of herbal derivatives of the aerial parts of Sphagneticola trilobata, a reputed medicinal plant; to biomonitor its oral anti-hyperalgesic effect; and to elucidate the degradation pathways of the major components by UHPLC-ESI-QTOF-MS. The lipophilic markers (kaurenoic acid-KA) and hydrophilic markers [chlorogenic acids; measured as total phenolic compounds (PC), expressed in caffeic acid] were also monitored through a validated HPLC-UV methodology, suitable for quality control and stability studies. The soft extract (drug:solvent ratio of 1:10, ethanol 60% v/v) was exposed to high temperatures (50-180°C). PC showed high thermolability (27.4% of degradation at 150°C), compared to KA (16.5%, at 180°C). These results suggest that the loss of oral anti-hyperalgesic activity observed in the spray-dried extracts (dried at 170°C), compared with the soft and the lyophilized extract may be related to degradation of the active components, especially the polar compounds, i.e. chlorogenic acid derivatives and the four oleanane type triterpenoid saponins. These latter degraded at temperatures above 150°C and vanished at 180°C, as demonstrated in the UHPLC-ESI-QTOF-MS analyses. These results provide a relevant guide for the extraction process of S. trilobata, aimed at preserving the antinociceptive effect.

Photochemoprotective effects against UVA and UVB irradiation and photosafety assessment of Litchi chinensis leaves extract.

Actually, there has been an increase in the use of natural products as skin photoprotective agents. In this way, the aim of present study was to investigate the L. chinensis leaves extract photochemoprotection potential and photosafety using in vitro methods. The extract cytotoxicity, cytoprotection and photochemoprotection against UVA and UVB radiation were assayed in L929 cells. The DNA damage was evaluated by comet assay. DCFH-DA, SOD, CAT and GSH assays were employed to verify the oxidative stress. We also determined the spectrophotometric Solar Protection Factor (SPF) of the extract. The photosafety was evaluated in red blood cells (RBC). In addition, the HET-CAM and agarose overlay tests were employed to evaluate the irritant potential. The results obtained show that extract is not cytotoxic and present cytoprotective activity against H2O2 and is able to protect the cells against DNA damage induced by UVA and UVB radiation. The extract was able to reduce the ROS production. The SPF obtained was 18.9 at 1mg/mL. We demonstrate that L. chinensis extract is photosafe and protect RBCs against oxidative damage, and did not induce irritation. Data herein obtained pointed out the potential of L. chinensis extract for photochemoprotection against UVA/UVB radiation and its damaging effects on human skin.

Hydroalcoholic extract of Sapium glandulatum (Vell.) Pax displays potent anti-inflammatory activities through a glucocorticoid receptor-dependent pathway.

BACKGROUND: Ethnobotanical studies of the Sapium genus reveal that many species are widely used in several countries as therapeutic drugs and they are widely used in folk medicine for treatment of different diseases, including skin inflammation. This raises interest in the study of the pharmacological properties and phytochemical composition of these plants. The biological properties of Sapium glandulatum, a native species of southern Brazil, has not been reported in the literature. PURPOSE: The aim of the present study was to investigate the anti-inflammatory action of the hydroalcoholic extract of Sapium glandulatum (EHSG) leaves in mouse models of acute or chronic skin inflammation. STUDY DESIGN/METHODS: Topical effects of EHSG were evaluated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema in the ear. Systemic effects of the extract were studied in a TPA-induced ear edema model, as well as in a carrageenan-induced paw edema model. To gain insight into the mechanism by which EHSG blocked inflammation, we evaluated the role of glucocorticoid receptors (GR) using the TPA-induced ear edema model and also measured specific binding in a glucocorticoid assay. Possible adverse effects of EHSG were evaluated after multiple treatments with the extract in the skin atrophy model on the ear and with the alkaline comet assay. RESULTS: EHSG presented potent anti-inflammatory activity when applied topically in acute and chronic models, inhibiting edema formation and leukocyte migration as well as expression pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in the tissue. Similar anti-inflammatory effects were found following oral treatment in both ear and paw edema models. Strikingly, the EHSG-induced blockade of leukocyte migration was reversed by mifepristone, a GR antagonist. Additionally, a specific binding assay revealed that ESGH interacts with GR. Multiple treatments with EHSG failed to induce adverse effects when evaluated in the skin atrophy model and bone marrow genotoxicity test. CONCLUSION: Taken together, our data suggest that EHSG is a potential source of anti-inflammatory tool compounds for the treatment of pro-inflammatory-derived skin diseases, and its mechanism of action may be, at least in part, via the GR pathway.

Topical anti-inflammatory phytomedicine based on Sphagneticola trilobata dried extracts.

CONTEXT: The aerial parts of Sphagneticola trilobata (L.) Pruski (Asteraceae) are popularly used to treat topical inflammation, but have not been fully investigated. OBJECTIVE: To identify polar compounds in S. trilobata extracts and develop a new topical phytomedicine based on the kaurenoic acid (KA) content while monitoring and demonstrating its topical anti-inflammatory activity. MATERIALS AND METHODS: Ethanol spray-dried extract of S. trilobata was analysed by LC-MS while the KA content from semisolid was analysed by LC-UV. The extent of ear edema induced by applying 20 µL of croton oil (2.5%), arachidonic acid (AA; 2 mg/ear) and decanoylphorbol-13-acetate (TPA; 2.5 mg/ear) in mice was used to evaluate the biological activity of the semisolids, which were applied 30 min before the phlogistic agents. RESULTS: Eight phenylpropanoids and four oleanane-type triterpenoid saponins were identified, majority of them reported for the first time in this species, in addition to KA. The semisolid containing 1.0% of dried extract reduced the ear edema induced by croton oil [77.2 ± 4.5%; ID50 = 0.49 (0.28-0.87%)], TPA (81.5 ± 2.4%) and AA (39.1 ± 6.9%), with decreasing effect at higher KA concentrations. This was accompanied by neutrophil migration inhibition as investigated by biochemical and histological assays. DISCUSSION AND CONCLUSION: The anti-inflammatory effects were (at least in part) due to the interference in protein kinase C (PKC) activation, AA-cascade products and neutrophil migration inhibition, demonstrating the efficacy of the folk topical usage of this plant. The results support the development of a novel topical anti-inflammatory phytomedicine properly standardized to treat inflammatory dermatological diseases.

Development of an oral suspension containing dry extract of Aleurites moluccanus with anti-inflammatory activity

Abstract Aleurites moluccanus L. (Willd.), Euphorbiaceae, is a tree that is native to Indonesia and India. Various parts of this tree are commonly used in traditional medicine to treat pain, fever, inflammation, hepatitis, gastric ulcer and other ailments. An oral suspension containing dried extract of A. moluccanus was developed and in vivo anti-inflammatory activity was evaluated. Extract 100 and 50 mg/ml loaded oral suspensions were prepared using different suspending agents. The formulations were analysed by their appearance, pH, density, redispersion time, rate of settling, rheological behaviour, distribution of particle size and zeta potential. The dose uniformity was determined by measuring the content of total phenolic compounds expressed in swertisin by a validated HPLC method, as well as the dissolution profile. The stability of oral suspensions was analysed in accelerated studies (40 °C for 6 months). The anti-inflammatory activity was analysed using an in vivo paw oedema model. The taste and odour of the suspensions were shown to be characteristic of the extract. Carmellose sodium (CS; 0.5%) and microcrystalline cellulose and carmellose sodium mixture (MCCS; 1%) showed better physical behaviour. The content of total phenolic compounds was 1.6 mg/ml and approximately 100% of the total phenolic compounds dissolved within 10 min. During the stability study, the formulations were approved by their physical–chemical properties and were shown to lose 12–14% of total phenolic compounds at 40 °C after 6 months. Suspensions containing 50 mg/ml of standardised dried extract inhibited around 35 ± 7.6% of paw oedema. Formulations containing CS showed more anti-inflammatory activity. Suspensions containing dry extract of A. moluccanus were successfully obtained and showed physical and physical–chemistry properties that were appropriate and characteristic of this dosage form, suitable for administration in paediatric and elderly populations, making this an alternative to tablets.

Reproductive toxicology and clastogenic evaluation in mice of a phytotherapeutic formulation obtained from Cinchona calisaya Weddel (Rubiaceae) used in Brazilian folk medicine as female fertility stimulant.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, a phytotherapeutic preparation produced from a standardized tincture of Cinchona calisaya Weddel such that each mL of product contains 400µg of quinine, known in Portuguese as Água Inglesa(®) (English water), is indicated by the manufacturer as a tonic, appetite stimulant, and digestive. However, this preparation has long been used in folk medicine as a female fertility stimulant. Despite its widespread use in folk medicine to stimulate female fertility, no study has been undertaken to assess the potential teratogenic and genotoxic effects of this phytotherapeutic preparation. The aim of the present study was to investigate possible toxic reproductive effects in mice caused by exposure to Água Inglesa(®), either before mating or during the pre- and post-embryo implantation periods. The genotoxic potential was evaluated using the micronucleus assay. MATERIAL, METHODS, AND RESULTS: Virgin female mice, with at least one estrous cycle evidenced by vaginal cytology, were divided into five groups of 15 individuals each (Group I - control, Group II - treated with ethanol solution at 16%, Groups III, IV and V treated with phytotherapeutic preparation at 1.5mL/kg/day, 3.0mL/kg/day and 4.5mL/kg/day, respectively). After the first 28 days of treatment, females were caged individually with adult fertile males. Pregnant females continued to receive treatment for seven days (preimplantation period). Body weight was recorded weekly during treatment. Signs of toxicity (weight loss, food intake, piloerection, apathy, prostration, diarrhea, seizures, behavioral changes, and locomotion) were also observed. The females were sacrificed on the 15th day of pregnancy, uterine horns were evaluated for implantation, and the placental index was recorded. In the micronucleus test, 2000 polychromatic erythrocytes (PCE) per animal, obtained from bone marrow, were scored. Results The results showed that exposure of the females during the pre- and post-implantation periods did not significantly alter the reproductive capacity (p<0.05); however, in higher dose (three times human dose)reduction of fetal weight was observed . There was no difference between the control and phytotherapeutic preparation (p>0.05) in terms of the average number of micronucleated polychromatic erythrocytes. CONCLUSIONS: Although folk medicine suggests that the Água Inglesa(®) preparation is useful as a female fertility stimulant, no such effect was confirmed in mice.

Role of gastric mucus secretion, oxinitrergic system and sulfhydryl groups on the gastroprotection elicited by Polygala cyparissias (Polygalaceae) in mice.

OBJECTIVES: This study has aimed to assess the mechanisms of action for the gastroprotective effect of the acetone extract (PCAE) and methanol fraction (PCMF) of Polygala cyparissias, as well as to evaluate the activity of 1,3,6,8-tetrahydroxy-2,7-dimethoxyxanthone (1), 1,7-dihydroxy-2,3-dimethoxyxanthone (2) and astragalin (3). METHODS: Gastric secretion and mucus content were determined by pylorus ligation in mice. Nitric oxide (NO) and sulfhydryl group participation were observed by the pretreatment of mice with L-NAME or NEM. Acute ulcer was induced by ethanol/HCl and chronic ulcer by acetic acid. Anti-Helicobacter pylori activity was evaluated by the agar solid dilution assay. KEY FINDINGS: Neither PCAE nor PCMF had the ability to reduce H(+) concentration. However, both of them enhanced mucus secretion. PCAE demonstrated its gastroprotection in a NO-dependent manner, while PCMF exerted the activity depending on the sulfhydryl group. In chronic ulcer, the curative ratios for the PCAE and PCMF were 67.5 and 58.4%, respectively. No effect over H. pylori was detected. Compounds 1, 2 and 3 were able to reduce lesions in the order of 79.6, 73.8 and 67.6%, respectively. CONCLUSIONS: The data suggested that PCAE and PCMF displayed antiulcer activity due to different mechanisms and with the participation of phenolic compounds obtained from the plant.

Development and validation of a stability indicative HPLC-PDA method for kaurenoic acid in spray dried extracts of Sphagneticola trilobata (L.) Pruski, Asteraceae.

A gradient stability indicative HPLC-UV method was developed and validated for assay of the marker kaurenoic acid (KA) in spray dried extract of Sphagneticola trilobata (L.) Pruski. The marker, and another unidentified polar component, were separated on a Luna Phenomenex C(18) column (250×4.6 mm, 5 µm) with mobile phase composed of acetonitrile:acidified water pH 3.0 with phosphoric acid, in a gradient run of 40 min; at a flow rate of 1.0 mL min(-1), 35 °C, using wavelengths of 210 and 338 nm. The method was linear over a KA concentration range of 4.5-30.0 µg mL(-1), without interference of the herbal matrix on the linearity of the method. The RSD% values for the intra- and inter-day precision studies were <2.0 and <8.0% for inter-laboratorial study. The method showed excellent KA recovery (99.0%). The LOQ value was found to be 1.13 µg mL(-1) and the method proved to be robust for small, deliberate changes in temperature and pH of the mobile phase with RSD%<2.5% for the KA assay. A forced degradation study of S. trilobata dried extract was conducted under conditions of visible light (1.200.000 l×h(-1)) and UV (200 Whm(-2)) irradiation, acid (0.5 mol L(-1) HCl, 30 min), basic (1 mol L(-1) NaOH, 2 h) and oxidative (30% H(2)O(2), 4h) hydrolysis, in order to develop a gradient stability-indicating LC-UV method for KA quantification, the selected marker, and also to detect the major polar components of the extract, under investigation. The KA contents remaining after these stress conditions were 72.3, 70.0, 97.6, 65.8 and 87.0%, respectively. The alkaline conditions resulted in higher degradation for the unknown polar components of the extract, without interference of supplementary peaks at the retention time of the KA. This method can be used for the KA assay and qualitative analysis of polar components in stability study of spray dried extracts of S. trilobata, for subsequent use in the quality control of dosage forms.