Multi-constituent synergism is responsible for anti-inflammatory effect of Azadirachta indica leaf extract.

CONTEXT: Azadirachta indica A. Juss. (Meliaceaes) leaves have been used traditionally to treat swelling and rheumatism in Indian cultures. OBJECTIVE: To fractionate A. indica leaf extracts using bioactivity guided manner for identification of the active anti-inflammatory principles. MATERIALS AND METHODS: Polarity-gradient sequential extracts (petroleum ether, chloroform, methanol, and water) of A. indica leaves were screened for their anti-inflammatory potential using the carrageenan-induced rat paw edema model (1 g/kg). The chloroform extract was sequentially fractionated to obtain n-hexane (F-1), n-hexane-chloroform (F-2), and chloroform (F-3) fractions and their inhibitory effect on rat paw edema was evaluated (500 mg/kg). Inhibitory effect of F-2 on granuloma formation, plasma interleukin (IL-1), and tumor necrosis factor (TNF-α) was assessed at the doses of 100, 200, and 400 mg/kg using the cotton pellet assay in rats. Three sub-fractions (SF-1, SF-2, and SF-3) were obtained upon chromatography of F-2, and their inhibitory effect on cyclooxygenase was assessed at 200 µg/mL concentration. The sub-fractions were subjected to gas chromatography-mass spectrometry (GC-MS). RESULTS: All the extracts showed significant anti-inflammatory effect; however, chloroform extract was the most effective against paw edema (53.25% inhibition). The three fractions of chloroform extract showed significant effect, while F-2 being the most potent (51.02%). F-2 demonstrated dose-dependent inhibition of granuloma and cytokines. Interestingly, all the sub-fractions of F-2 inhibited COX-1 and COX-2 with almost equal potential. GC-MS revealed that chemically the sub-fractions were totally different from each other. DISCUSSION AND CONCLUSION: Anti-inflammatory effect of A. indica is a result of cumulative and synergistic effects of diversified constituents with varying polarities that collectively exert the effect via suppression of cyclo-oxygenases and cytokines (IL-1 and TNF-α).

Flavonoids eupatorin and sinensetin present in Orthosiphon stamineus leaves inhibit inflammatory gene expression and STAT1 activation.

Cytokines and other inflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO) produced by cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively, activate and drive inflammation and therefore serve as targets for anti-inflammatory drug development. Orthosiphon stamineus is an indigenous medicinal plant of Southeast Asia that has been traditionally used in the treatment of rheumatoid arthritis, gout, and other inflammatory disorders. The present study investigated the anti-inflammatory properties of Orthosiphon stamineus leaf chloroform extract (CE), its flavonoid-containing CE fraction 2 (CF2), and the flavonoids eupatorin, eupatorin-5-methyl ether (TMF), and sinensetin, identified from the CF2. It was found that CE (20 and 50 µg/mL) and CF2 (20 and 50 µg/mL) inhibited iNOS expression and NO production, as well as PGE2 production. Eupatorin and sinensetin inhibited iNOS and COX-2 expression and the production of NO (IC50 5.2 µM and 9.2 µM for eupatorin and sinensetin, respectively) and PGE2 (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively) in a dose-dependent manner. The extracts and the compounds also inhibited tumor necrosis factor α (TNF-α) production (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively). Eupatorin and sinensetin inhibited lipopolysaccharide (LPS)-induced activation of transcription factor signal transducers and activators of transcription 1α (STAT1α). Furthermore, eupatorin (50 mg/kg i. p.) and sinensetin (50 mg/kg i. p.) inhibited carrageenan-induced paw inflammation in mice. The results suggest that CE and CF2, as well as the known constituents of CF2, i.e., eupatorin and sinensetin, have meaningful anti-inflammatory properties which may be utilized in the development of novel anti-inflammatory treatments.

Safety assessment of methanol extract of red dragon fruit (Hylocereus polyrhizus): acute and subchronic toxicity studies.

Recently, the fruits of Hylocereus polyrhizus, known as red dragon fruit, have received much attention from growers worldwide. However, there is little toxicological information regarding the safety of repeated exposure to these fruits. The present study evaluated the potential toxicity of a methanol extract of H. polyrhizus fruit after acute and subchronic administration in rats. In the acute toxicity study, single doses of fruit extract (1250, 2500 and 5000 mg/kg) were administered to rats by oral gavage, and the rats were then monitored for 14 days. In the subchronic toxicity study, the fruit extract was administered orally to rats at doses of 1250, 2500 and 5000 mg/kg/day for 28 days. There was no mortality or signs of acute or subchronic toxicity. There was no significant difference in body weight, relative organ weight or hematological parameters in the subchronic toxicity study. Biochemical analysis showed some significant changes, including creatinine, globulin, total protein and urea levels. No abnormality of internal organs was observed between treatment and control groups. The lethal oral dose of the fruit extract is more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of the extract for both male and female rats is considered to be 5000 mg/kg per day for 28 days.

Potent α-glucosidase and α-amylase inhibitory activities of standardized 50% ethanolic extracts and sinensetin from Orthosiphon stamineus Benth as anti-diabetic mechanism.

BACKGROUND: In the present study, we tested a 50% ethanolic extract of Orthosiphon stamineus plants and its isolated bioactive compound with respect to their α-glucosidase and α-amylase inhibitory activities. METHODS: Bioactive flavonoid sinensetin was isolated from 50% ethanolic extract of Orthosiphon stamineus. The structure of this pure compound was determined on the NMR data and the α-glucosidase and α-amylase inhibitory activities of isolated sinensetin and 50% ethanolic extract of Orthosiphon stamineus were evaluated. RESULTS: In vitro studies of a 50% ethanolic extract of O. stamineus and the isolated sinensetin compound showed inhibitory activity on α-glucosidase (IC50: 4.63 and 0.66 mg/ml, respectively) and α-amylase (IC50: 36.70 mg/ml and 1.13 mg/ml, respectively). Inhibition of these enzymes provides a strong biochemical basis for the management of type 2 diabetes via the control of glucose absorption. CONCLUSION: Alpha-glucosidase and α-amylase inhibition could the mechanisms through which the 50% ethanolic extract of O. stamineus and sinensetin exert their antidiabetic activity, indicating that it could have potential use in the management of non-insulin-dependent diabetes.

Bioactivity-guided isolation of ethyl-p-methoxycinnamate, an anti-inflammatory constituent, from Kaempferia galanga L. extracts.

This study evaluated the anti-inflammatory effect of Kaempferia galanga (KG) using an activity-guided approach. KG rhizomes were serially extracted with petroleum ether, chloroform, methanol and water. These extracts (2 g/kg each) were tested for their ability to inhibit carrageenan-induced rat paw edema. The chloroform extract was found to exert the highest inhibition (42.9%) compared to control (p < 0.001), hence it was further fractionated by washing serially with hexane, hexane-chloroform (1:1) and chloroform. The chloroform fraction (1 g/kg) showed the highest inhibitory effect (51.9%, (p < 0.001), on carrageenan-induced edema. This chloroform fraction was further fractionated with hexane-chloroform (1:3) and chloroform, and of the two fractions, the hexane-chloroform sub-fraction was the most effective in inhibiting edema (53.7%, p < 0.001). GC-MS analysis of the active sub-fraction identified ethyl-p-methoxycinnamate (EPMC) as the major component, which was re-crystallized. EPMC dose-dependently inhibited carrageenan-induced edema with an MIC of 100 mg/kg. Moreover, in an in vitro study, EPMC non-selectively inhibited the activities of cyclooxygenases 1 and 2, with IC50 values of 1.12 µM and 0.83 µM respectively. These results validate the anti-inflammatory activity of KG which may be exerted by the inhibition of cyclooxygenases 1 and 2. EPMC isolated from this plant may be the active anti-inflammatory agent.

A review of the literature and latest advances in research of Piper sarmentosum.

CONTEXT: Piper sarmentosum Roxb. (Piperaceae) is a traditional medicinal as well as a culinary plant in South East Asian countries, whereby aerial parts of the plant are consumed as a vegetable in various forms and the whole plant or parts are used as folk remedies, alone or in combination with other herbs, to treat various ailments. The plant has extensively been investigated in a broad range of studies to provide scientific evidence for folklore claims or to find new therapeutic uses; however, heretofore, a summary of the data are not available. OBJECTIVE: In order to describe nutritional and therapeutic potential of P. sarmentosum and summarize scientific evidence that supports traditional claims, a literature review and latest advances in research of the plant are given herein. MATERIALS AND METHODS: The literature has been retrieved from a number of databases such as Google Scholar, PubMed, Medline, Science Direct and SciFinder. The articles related to synthetic work, ecology and agriculture have been excluded. RESULTS AND DISCUSSION: The review has not only revealed a number of pharmacological activities supporting the traditional claims but indicates new prospects for the plant. Antiangiogenic activity and toxicity studies suggest the usage of the plant in treating diseases involving neo-vascularization. The available efficacy, safety, pharmacokinetic and stability data urge clinical studies on extracts of the plant. CONCLUSION: The present review may be helpful to future researchers intending to investigate the plant and natural pharmaceutical industry for preparing evidence-based formulations.

Rapid separation and determination of betulinic acid from a complex matrix using combination of TLC and RP-HPLC.

Hitherto, only a few studies are reported about using the combination of TLC and RP-HPLC for the separation and determination of analyte(s) from a complex matrix. The present study is aimed to develop a simple and rapid method for the separation and determination of betulinic acid from a complex matrix, extracts of Orthosiphon stamineus, using a combination of the two techniques. The samples having higher contents of the analyte and fewer interfering species were prepared using TLC. The samples were then eluted through C(18) column using isocratic solvent system comprising acetonitrile, methanol and acetic acid acidified water of pH 2.8 in a ratio of 70 : 20 : 10 (v/v/v), respectively, and detection was carried out at 210 nm. The method was validated and applied successfully to quantify betulinic acid in various types of extracts of the plant. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.0005 and 0.0050 µg/ml, respectively. The method exhibited linearity in a concentration range of 0.005-100.00 µg/ml (R(2)= 0.9999). The recovery was found to be 97.10 - 97.60% (RSD < 5%), whereas, intra-day and inter-days accuracy values were 97.13 - 98.67% (RSD < 5%) and 96.45 - 98.00% (RSD < 5%), respectively. The results of the present study indicate that the developed method is simple, rapid, sensitive and accurate, and may be of a value to natural product industry and researchers for the standardization of extracts containing betulinic acid in a lesser time and consuming fewer solvents.

Acute and sub-chronic toxicological evaluation of Averrhoa carambola leaves in Sprague Dawley rats.

Averrhoa carambola is a species of tree native to tropical Southeast Asia. It possesses antioxidant and anti-hyperlipidemia effects and has traditionally been used to treat a variety of ailments. However, the presence of oxalic acid in its fruits might restrict its consumption by individuals suffering from kidney disease, and caramboxin can cause neurotoxicity. In this study, we evaluated the acute and sub-chronic toxicity of the methanolic extract of A. carambola leaves (MEAC) in male and female rats. In the acute study, female rats were given a single oral dose of 5000 mg/kg of MEAC and closely examined for distinct indications of toxic effects during the first 4 h, periodically for 48 h, and daily thereafter for 14 days. Rats of both sexes were employed in the sub-chronic investigation for the 28-day repeated dose oral toxicity study. Results of the acute study revealed the safety of MEAC up to a dose of 5000 mg/kg where the rats did not show changes or signs of toxicity. In the sub-chronic toxicity study, MEAC (250, 500, and 1000 mg/kg) administration did not affect the body weight, food, and water consumption, motor coordination, behavior, or mental alertness in the treated rats. In addition, no variations in hematological or biochemical markers were found in MEAC-treated rats. In conclusion, these findings pinpoint the safety of MEAC at doses up to 5000 mg/kg. The leaves of A. carambola could be safely consumed by people with kidney disease to treat other ailments.

Bioactive Markers Based Pharmacokinetic Evaluation of Extracts of a Traditional Medicinal Plant, Piper sarmentosum.

In vitro assays are economical and easy to perform but to establish relevance of their results to real clinical outcome in animals or human, pharmacokinetics is prerequisite. Despite various in vitro pharmacological activities of extracts of Piper sarmentosum, there is no report of pharmacokinetics. Therefore, the present study aimed to evaluate ethanol extract of fruit of the plant in dose of 500 mg kg(-1) orally for pharmacokinetics. Sprague-Dawley rats were randomly divided into groups 1, 2, and 3 (each n = 6) to study absorption, distribution and excretion, respectively. High performance liquid chromatography (HPLC) with ultraviolet detection was applied to quantify pellitorine, sarmentine and sarmentosine in plasma, tissues, feces and urine to calculate pharmacokinetic parameters. Pellitorine exhibited maximum plasma concentration (C(max)) 34.77 ng mL(-1) ± 1.040, time to achieve C(max) (T(max)) 8 h, mean resident time (MRT) 26.00 ± 0.149 h and half life (t(1/2)) 18.64 ± 1.65 h. Sarmentine showed C(max) 191.50 ± 12.69 ng mL(-1), T(max) 6 h, MRT 11.12 ± 0.44 h and t(1/2) 10.30 ± 1.98 h. Sarmentosine exhibited zero oral bioavailability because it was neither detected in plasma nor in tissues, and in urine. Pellitorine was found to be distributed in intestinal wall, liver, lungs, kidney, and heart, whereas sarmentine was found only in intestinal wall and heart. The cumulative excretion of pellitorine, sarmentine and sarmentosine in feces in 72 h was 0.0773, 0.976, and 0.438 µg, respectively. This study shows that pellitorine and sarmentine have good oral bioavailability while sarmentosine is not absorbed from the gastrointestinal tract.

Antihyperglycemic effect of orthosiphon stamineus benth leaves extract and its bioassay-guided fractions.

Preliminary investigations were carried out to evaluate the antidiabetic effects of the leaves of O. stamineus extracted serially with solvents of increasing polarity (petroleum ether, chloroform, methanol and water); bioassay-guided purification of plant extracts using the subcutaneous glucose tolerance test (SbGTT) was also carried out. Only the chloroform extract, given at 1 g/kg body weight (b.w.), significantly reduced (P < 0.05) the blood glucose level of rats loaded subcutaneously with 150 mg/kg (b.w.) glucose. The active chloroform extract of O. stamineus was separated into five fractions using a dry flash column chromatography method. Out of the five fractions tested, only chloroform fraction 2 (Cƒ2), at the dose of 1 g/kg (b.w.) significantly inhibited (P < 0.05) blood glucose levels in SbGTT. Active Cƒ2 was split into two sub-fractions Cƒ2-A and Cƒ2-B, using a dry flash column chromatography method. The activities Cƒ2-A and Cƒ2-B were investigated using SbGTT, and the active sub-fraction was then further studied for anti-diabetic effects in a streptozotocin-induced diabetic rat model. The results clearly indicate that Cƒ2-B fraction exhibited a blood glucose lowering effect in fasted treated normal rats after glucose-loading of 150 mg/kg (b.w.). In the acute streptozotocin-induced diabetic rat model, Cƒ2-B did not exhibit a hypoglycemic effect on blood glucose levels up to 7 hours after treatment. Thus, it appears that Cƒ2-B functions similarly to metformin, which has no hypoglycemic effect but demonstrates an antihyperglycemic effect only in normogycemic models. The effect of Cƒ2-B may have no direct stimulatory effects on insulin secretion or on blood glucose levels in diabetic animal models. Verification of the active compound(s) within the active fraction (Cƒ2-B) indicated the presence of terpenoids and, flavonoids, including sinensitin.

Standardization and in vivo antioxidant activity of ethanol extracts of fruit and leaf of Piper sarmentosum.

The present study aimed to investigate standardized ethanol extracts of fruit and leaves of Piper sarmentosum for their in vivo antioxidant activity in rats using a CCl (4)-induced oxidative stress model. The standardization was based on the quantification of the markers pellitorine, sarmentine and sarmentosine by high performance liquid chromatography (HPLC), and determination of total primary and secondary metabolites. The rats, divided into 7 groups each (n = 6), were used as follows: group 1 (CCl (4), negative control), group 2 (untreated, control), groups 3 and 4 (fruit extract 250 and 500 mg/kg, respectively), groups 5 and 6 (leaf extract 250 and 500 mg/kg, respectively) and group 7 (vitamin-E 100 mg/kg, positive control). The doses were administered orally for 14 days; 4 h following the last dose, a single dose of CCl (4) (1.5 mg/kg) was given orally to all the groups except group 2, and after 24 h, blood and liver of each animal were obtained. Analysis of plasma and liver homogenate exhibited significant preservation of markers of antioxidant activity, total plasma antioxidant activity (TPAA), total protein (TP), superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid reactive species (TBARS), in the pretreated groups as compared to the CCl (4) group (p < 0.05). Histology of the liver also evidenced the protection of hepatocytes against CCl (4) metabolites in the pretreated groups. The results of this study indicate the IN VIVO antioxidant activity of both extracts of the plant, which may be valuable to combat diseases involving free radicals.

HPLC and anti-inflammatory studies of the flavonoid rich chloroform extract fraction of Orthosiphon stamineus leaves.

The aim of the present study was to verify the anti-inflammatory activity of Orthosiphon stamineus leaf extracts and to identify the active compound(s) contributing to its anti-inflammatory activity using a developed HPLC method. Active chloroform extract of O. stamineus was fractionated into three fractions using a dry flash column chromatography method. These three fractions were investigated for anti-peritoneal capillary permeability, in vitro nitric oxide scavenging activity, anti-inflammatory and nitric oxide (NO) inhibition using carrageenan-induced hind paw edema method. The flavonoid rich chloroform extract fraction (CF2) [containing sinensetin (2.86% w/w), eupatorin (5.05% w/w) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (1.101% w/w)], significantly reduced rat hind paw edema, NO and decreased dye leakage to peritoneal cavity at p < 0.05. IC(50) of in vitro NO scavenging of CF2 was 0.3 mg/mL. These results suggest that the anti-inflammatory properties of these CF2 may possibly be due to the presence of flavonoid compounds capable of affecting the NO pathway.

(E)-N'-(2,4,6-Trihydroxy-benzyl-idene)isonicotinohydrazide sesquihydrate.

In the title compound, C(13)H(11)N(3)O(4)·1.5H(2)O, the pyridine ring forms a dihedral angle of 1.50 (6)° with the benzene ring. An intra-molecular O-H⋯N hydrogen bond forms a six-membered ring with an S(6) ring motif. In the crystal structure, one water mol-ecule is disordered over two positions around an inversion centre with site-occupancy factors of 0.5. Inter-molecular O-H⋯N, O-H⋯O, N-H⋯O and C-H⋯O hydrogen bonds consolidate the structure into a three dimensional network. A π-π stacking inter-action with a centroid-centroid distance of 3.5949 (7) Šis also present.

(E)-N'-(2,3,4-Trimethoxy-benzyl-idene)isonicotinohydrazide.

In the title compound, C(16)H(17)N(3)O(4), the mol-ecule exists in an E configuration with respect to the C=N double bond. The mol-ecule is not planar, the dihedral angle between the pyridine and benzene rings being 71.67 (8)°. In the crystal structure, mol-ecules are linked into chains along the b axis by bifurcated N-H⋯O and C-H⋯O hydrogen bonds. These chains are linked into a three-dimensional network by C-H⋯O and C-H⋯π inter-actions.

(E)-N'-(2,4,5-Trimethoxy-benzyl-idene)isonicotinohydrazide dihydrate.

The asymmetric unit of the title compound, C(16)H(17)N(3)O(4)·2H(2)O, contains one Schiff base mol-ecule and two water mol-ecules. The Schiff base mol-ecule exists in an E configuration with respect to the C=N double bond and is essentially planar, the dihedral angle between the benzene and pyridine rings being 5.48 (8)°. The three meth-oxy groups are also coplanar with the benzene ring [C-O-C-C torsion angles = 3.9 (2), 178.51 (15) and 0.8 (2) Å]. In the crystal structure, the water mol-ecules link the mol-ecules into a three-dimensional network via inter-molecular N-H⋯O, O-H⋯O, O-H⋯N and C-H⋯O hydrogen bonds.

(E)-N'-(2-Benzyl-oxybenzyl-idene)isonicotinohydrazide methanol solvate monohydrate.

The title compound, C(20)H(17)N(3)O(2)·CH(4)O·H(2)O, was synthesized by the condensation reaction of 2-benzyl-oxybenzaldehyde with isoniazid (isonicotinic acid hydrazide). The tricyclic compound displays a trans configuration with respect to the C=N double bond. The central benzene ring makes dihedral angles of 8.83 (7) and 70.39 (8)° with the pyridine ring and the terminal benzene ring, respectively. The dihedral angle between the pyridine ring and the terminal benzene ring is 73.11 (8)°. In the crystal structure, mol-ecules are connected by inter-molecular N-H⋯O, O-H⋯O, O-H⋯(N,N) and C-H⋯O hydrogen bonds, forming a two-dimensional network perpendicular to the a axis.

(E)-N'-[(E)-2-Methyl-pent-2-enyl-idene]isonicotinohydrazide.

The asymmetric unit of the title Schiff base compound, C(12)H(15)N(3)O, contains two crystallographically independent mol-ecules, with both existing in an E configuration with respect to the C=N double bonds. In the crystal structure, inter-molecular N-H⋯N and C-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network.

(E)-N'-(2,4,6-Trimethyl-benzyl-idene)isonicotinohydrazide.

The title isoniazid derivative, C(16)H(17)N(3)O, exists in an E configuration with respect to the Schiff base C=N bond. The pyridine ring is essentially planar [maximum deviation = 0.009 (3) Å]. The mean plane through the hydrazide unit forms dihedral angles of 38.38 (16) and 39.42 (16)°, respectively, with the pyridine and benzene rings. In the crystal structure, symmetry-related mol-ecules are linked via inter-molecular N-H⋯O hydrogen bonds into chains along [100]. The crystal structure is further stabilized by weak inter-molecular C-H⋯π inter-actions.

(E)-N'-[(E)-3-(4-Hydr-oxy-3-methoxy-phen-yl)allyl-idene]isonicotinohydrazide.

In the title compound, C(16)H(15)N(3)O(3), the dihedral angle between the pyridine and benzene rings is 7.66 (5)°. The crystal packing is consolidated by inter-molecular C-H⋯O and O-H⋯N inter-actions, which link the mol-ecules into zigzag chains propagating along [010]. The chains are further linked into a three-dimensional network by N-H⋯O, C-H⋯N, C-H⋯O and C-H⋯π inter-actions.

(E)-N'-(2,4,5-Trifluorobenzyl-idene)isonicotinohydrazide monohydrate.

In the Schiff base mol-ecule of the title compound, C(13)H(8)F(3)N(3)O·H(2)O, the benzene ring and the pyridine ring are nearly coplanar, making a dihedral angle of 6.64 (7)°. The mol-ecule exists in an E configuration with respect to the C=N double bond. In the crystal structure, mol-ecules are linked via the water mol-ecules into two-dimensional planes parallel to the ab plane through inter-molecular N-H⋯O, O-H⋯O O-H⋯N and C-H⋯O hydrogen bonds.