Pharkphoom Panichayupakaranant

Antimicrobial activity and stability of rhinacanthins-rich Rhinacanthus nasutus extract.

Rhinacanthins-rich Rhinacanthus nasutus (RRn) extract was prepared and standardized to contain total rhinacantins not less than 70% w/w. In this study, antimicrobial activities of the RRn extract as well as rhinacanthin-C against Streptococcus mutans, Propionibacterium acnes, Helicobacter pylori, Staphylococcus aureus, S. epidermidis and Candida albicans were evaluated by microdilution assay. It was found that the RRn extract exhibited potent bactericidal activity against S. mutans with MIC and MBC of 4 microg/ml, and potent bacteriostatic activity against S. epidermidis, P. acnes and S. aureus with the MICs of 8-16 microg/ml. However, the RRn extract was not active against C. albicans at concentration up to 2000 microg/ml. The antimicrobial activities of the RRn extract was almost equal to those of rhinacanthin-C. Stability evaluations of the RRn extract through a period of 4 months found that the RRn extracts were stable when kept in a well-closed container protected from light and stored either under 4+/-2 degrees C or 30+/-2 degrees C. The RRn extracts that were exposed to light were not stable after 1 week of storage. Under accelerated conditions at 45 degrees C with 75% relative humidity, the RRn extracts were not stable after 8 weeks of storage. Study on the effect of pH on the stability of the aqueous methanolic solution of the RRn extract found that the solutions were not stable at pH 5.5, pH 7.0, and pH 8.0. However, the rhinacanthin solution at pH 5.5 was more stable than those at pH 7.0 and pH 8.0.

Plumbagin production by root cultures of Plumbago rosea

Root cultures of Plumbago rosea Linn. were established from young leaf explants on solid Gamborgs B5 (B5) medium supplemented with the combination of α-naphthalene acetic acid (NAA) and kinetin in the concentration ranges of 0.5-2.0 mg/l and 0.1-0.5 mg/l, respectively. The production of plumbagin, determined by TLC-densitometry was higher [0.016 ± 0.0030% dry weight (DW)] in cultured roots obtained from B5 medium supplemented with 1.0 mg/l NAA and 0.1 mg/l kinetin. Plant selection increased the plumbagin production to 0.129 ± 0.0139% DW, while variation of sucrose and nitrogen (as (NH 4 ) 2 SO 4 ) concentration in B5 media slightly increase the plumbagin synthesis to 0.023 ± 0.0017 and 0.020 ± 0.0015% DW, respectively.

Anti-allergic principles of Rhinacanthus nasutus leaves

Three naphthoquinone derivatives, rhinacanthin-C (1), -D (2) and -N (3) were isolated from the extract of Rhinacanthus nasutus Kurz leaves and were tested for anti-allergic effect. The result indicated that all three compounds possessed very potent anti-allergic activity against antigen-induced beta-hexosaminidase release as a marker of degranulation in RBL-2H3 cells with IC(50) values of 6.9, 8.9 and 6.4 microM, respectively. In addition, the effects of rhinacanthin-C, -D and -N on antigen-induced release of TNF-alpha and IL-4 were also examined. It was found that rhinacanthin-C showed the most potent on antigen-induced TNF-alpha release with an IC(50) value of 0.7 microM, followed by rhinacanthin-D (IC(50)=3.8 microM) and rhinacanthin-N (IC(50)=10.3 microM), whereas those for IL-4 were rhinacanthin-D (IC(50)=5.4 microM), rhinacanthin-C (IC(50)=7.0 microM) and rhinacanthin-N (IC(50)=12.0 microM), respectively. The mechanisms in the late phase reaction of rhinacanthin-C, -D and -N were found to inhibit TNF-alpha and IL-4 gene expression in antigen-induced TNF-alpha and IL-4 releases on from RBL-2H3 cells as dose-dependent manners. The structure-activity trends of rhinacanthin-C,-D and-N on the inhibition of TNF-alpha release are as follow; substitution with octadienoic acid (rhinacanthin-C) conferred much higher activity than that of benzodioxo carboxylic acid ester (rhinacanthin-D) as well as naphthalene carboxylic acid ester (rhinacanthin-N). For the inhibition of IL-4 release, the substitution with octadienoic acid (rhinacanthin-C) and benzodioxo carboxylic acid ester (rhinacanthin-D) possessed the effect two-fold higher than that of naphthalene carboxylic acid ester (rhinacanthin-N). As regards active constituents for anti-allergic activity of R. nasutus, rhinacanthin-C, -D and -N are responsible for anti-allergic effect of this plant on both the early phase and late phase reactions. The finding may support the traditional use of R. nasutus leaves for treatment of allergy and allergy-related diseases.

Topical anti-inflammatory and analgesic activities of standardized pomegranate rind extract in comparison with its marker compound ellagic acid in vivo

ETHNOPHARMACOLOGICAL RELEVANCE In Chinese traditional medicine, the peels of Punica granatum L. have been used to treat traumatic hemorrhage, burn, and ulcers. AIMS OF THE STUDY This study aimed to assess the topical anti-inflammatory and analgesic activities of a standardized pomegranate rind extract (SPRE) of which ellagic acid (EA) was the major antioxidant constituent and the marker compound. MATERIAL AND METHODS The topical anti-inflammatory effects of SPRE were evaluated against acute models (croton oil-induced mouse ear edema and carrageenan-induced rat paw edema) and chronic model (complete Freunds adjuvant (CFA)-induced polyarthritis). The topical analgesic activities of SPRE were investigated in the rat punctuate mechanical hyperalgesia test and in the mouse formalin test. All studies of SPRE were carried out in parallel with its marker compound EA. RESULTS SPRE (5%, 2.5%, and 1%, w/w) and the equivalent EA (0.65%, 0.325%, and 0.13%, w/w) dose-dependently reduced the croton oil-induced mouse ear edema with a maximal inhibition of 86.30% and 80.82%, respectively. SPRE dose-dependently attenuated the inflammatory responses in the carrageenan-induced rat paw edema and in the CFA-induced polyarthritis but the equivalent EA were effective only at the doses of 0.65% and 0.325%. Both SPRE (5%) and EA (0.65%) showed significant topical analgesic activities in the rat punctuate mechanical hyperalgesia test and in the mouse formalin test. CONCLUSIONS SPRE was more active as an anti-inflammatory agent than EA. The anti-inflammatory and analgesic effects of SPRE were achieved through inhibiting the leukocyte infiltration and modulating the pro-inflammatory cytokines IL-β and TNF-α. These results clearly demonstrated that SPRE is a promising phytomedicine that could find use in the treatment of inflammatory diseases.

Comparative antiulcer effect of Bisdemethoxycurcumin and Curcumin in a gastric ulcer model system.

The antiulcer effect of bisdemethoxycurcumin, a yellow pigment found mainly in rhizomes of Curcuma longa, was compared with curcumin in gastric ulcer model systems to validate its clinical application as a remedy for peptic ulcer. Western blot analysis of mouse macrophage cell line RAW 264.7 activated with lipopolysaccharide showed that bisdemethoxycurcumin inhibited inducible nitric oxide synthase (iNOS) production significantly but had no effect on tumor necrosis factor-alpha (TNF-alpha) production, whereas curcumin showed stronger suppression of iNOS protein production and inhibited TNF-alpha protein production significantly. However, bisdemethoxycurcumin and curcumin possessed similar potency in scavenging nitric oxide generated from mouse macrophage cell line RAW 264.7. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that both curcuminoids inhibited the induction of iNOS dose-dependently at the transcriptional level and curcumin also appeared to inhibit the induction of TNF-alpha at post-transcriptional level. In an animal model, intraduodenal administration of bisdemethoxycurcumin (5-80 mg/kg body wt.) showed a strong inhibitory effect on gastric acid secretion in pylorus-ligated rats whereas curcumin (5-20 mg/kg body wt.) showed a less inhibitory effect, with maximum potency at a dose of 20mg/kg body wt. Moreover, oral administration of bisdemethoxycurcumin at doses of 20-80 mg/kg body wt. twice daily for 10 days showed a significant curative efficacy in accelerating the healing of acetic acid-induced chronic gastric ulcer and promotion of mucosal regeneration in the ulcerated portion in a dose-related manner with potency equal to curcumin. In contrast, the curative potency of curcumin tended to decrease at doses over 160 mg/kg body wt./day. Western blot analysis in ulcerated gastric mucosa showed that bisdemethoxycurcumin dose-dependently reduced the increased protein expression level of iNOS but not TNF-alpha. These results indicated that bisdemethoxycurcumin directly accelerates gastric ulcer healing with potency equal to curcumin. Its antiulcer effect might be due to its properties of decreasing gastric acid secretion and enhancing the mucosal defensive mechanism through suppression of iNOS-mediated inflammation.

Comparison of antimicrobial activities of naphthoquinones from Impatiens balsamina

Lawsone (1), lawsone methyl ether (2), and methylene-3,3′-bilawsone (3) are the main naphthoquinones in the leaf extracts of Impatiens balsamina L. (Balsaminaceae). Antimicrobial activities of these three naphthoquinones against dermatophyte fungi, yeast, aerobic bacteria and facultative anaerobic and anaerobic bacteria were evaluated by determination of minimal inhibitory concentrations (MICs) and minimal bactericidal or fungicidal concentrations (MBCs or MFCs) using a modified agar dilution method. Compound 2 showed the highest antimicrobial activity. It showed antifungal activity against dermatophyte fungi and Candida albicans with the MICs and MFCs in the ranges of 3.9–23.4 and 7.8–23.4 µg mL–1, respectively, and also had some antibacterial activity against aerobic, facultative anaerobic and anaerobic bacteria with MICs in the range of 23.4–93.8, 31.2–62.5 and 125 µg mL–1, respectively. Compound 1 showed only moderate antimicrobial activity against dermatophytes (MICs and MFCs in the ranges of 62.5–250 and 125–250 µg mL–1, respectively), but had low potency against aerobic bacteria, and was not active against C. albicans and facultative anaerobic bacteria. In contrast, 3 showed significant antimicrobial activity only against Staphylococus epidermidis and Bacillus subtilis (MIC and MBC of 46.9 and 93.8 µg mL–1, respectively).

Preparation method and stability of ellagic acid-rich pomegranate fruit peel extract.

A simple one-step purification using liquid–liquid extraction for preparing pomegranate peel extract rich in ellagic acid has been demonstrated. The method involved partitioning of the 10% v/v water in methanol extract of pomegranate peel between ethyl acetate and 2% aqueous acetic acid. This method was capable of increasing the ellagic acid content of the extract from 7.06% to 13.63% w/w. Moreover, the antioxidant activity of the extract evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was also increased (ED50 from 38.21 to 14.91 μg/mL). Stability evaluations of the ellagic acid-rich pomegranate peel extract in several conditions through a period of four months found that the extracts were stable either kept under light or protected from light. The extracts were also stable under 4° ± 2°C, 30° ± 2°C and accelerated conditions at 45°C with 75% relative humidity. However, study on the effect of pH on stability of the extract in the form of solution revealed that the extract was not stable in all tested pH (5.5, 7 and 8). These results indicated that the ellagic acid-rich pomegranate peel extract was stable when it was kept as dried powder, but it was not stable in any aqueous solution.

Effects of rhinacanthins from Rhinacanthus nasutus on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha releases using RAW264.7 macrophage cells

Three naphthoquinone derivatives, rhinacanthin-C (1), -D (2) and -N (3) were isolated from the leaves of Rhinacanthus nasutus extract and were tested for anti-inflammatory activity. The result indicated that all three compounds possessed very potent anti-inflammatory activity against lipopolysaccharide (LPS)-induced nitric oxide release with IC(50) values of 1.8, 6.2 and 3.0 microM, respectively. In addition, the effects of rhinacanthin-C, -D and -N on LPS induced release of prostaglandin E(2) (PGE(2)) and tumor necrosis factor (TNF-alpha) were also examined. It was found that rhinacanthin-C exhibited the most potent on PGE(2) release with an IC(50) value of 10.4 microM, followed by rhinacanthin-D (IC(50)=14.4 microM) and rhinacanthin-N (IC(50)=52.1 microM), whereas those for TNF-alpha were inactive (IC(50)>100 microM). The mechanisms in transcriptional level of rhinacanthin-C were found to inhibit iNOS and COX-2 gene expressions in LPS-induced NO and PGE(2) releases from RAW264.7 cells in concentration-dependent manners. Regarding active constituents for anti-inflammatory activity of R. nasutus, rhinacanthins are responsible for this effect through the inhibition of NO and PGE(2) releases. The finding may support the traditional use of R. nasutus leaves for treatment of the inflammatory-related diseases.

Factors affecting the content of pentacyclic triterpenes in Centella asiatica raw materials

Context: Pentacyclic triterpenes, mainly, asiatic acid, madecassic acid, asiaticoside, and madecassoside are the active constituents of Centella asitica (L.) Urban. (Apiaceae). These compounds possess various pharmacological activities that have been shown to assist with wound healing and brain enrichment. Objective: Determination of these active pentacyclic triterpenes in extracts from the various parts of C. asiatica plants harvested at different times of the year and grown in different environments. Materials and methods: The separate plant parts selected were leaves, stolons, petioles, flowers, fruits, and nodes with roots. Dried powder from each part was extracted with ethanol by microwave-assisted extraction and subjected to determination of their content of the four pentacyclic triterpenes using a HPLC method. The effects of the places of cultivation as well as harvesting periods on the content of the four pentacyclic triterpenes in the extracts were also determined. Results and discussion: Among the various parts of C. asiatica, the leaves contained the highest amount of pentacyclic triterpenes with a total content of pentacyclic triterpenes of 19.5 mg/g dry powder. However, the contents of the pentacyclic triterpenes in C. asiatica varied according to the place of cultivation and the harvesting period. C. asiatica collected from Trang, Thailand gave the highest content of total pentacyclic triterpenes (37.2 mg/g dry powder) when harvested in March, while those collected from Songkhla, Thailand gave the highest value (37.4 mg/g dry powder) when collected in December. C. asiatica collected from Nakornsrithammarat and Ratchaburi, Thailand gave the lowest content of total pentacyclic triterpenes in all experimental harvesting periods.

Studies on complex formation between curcumin and Hg(II) ion by spectrophotometric method: a new approach to overcome peak overlap.

Complex formation between curcumin and Hg(II) ion MeOH/H2O (1: 1 v/v) was investigated and monitored by the spectrophotometric method. The absorption peak of unreacted curcumin which was close and overlapped with that of the complex, was removed by calculation using Microsoft Excel, thereby, allowing determination of the stoichiometry of the complex by the mole-ratio and the Job’s continuous variation methods. Both methods indicated that a 1:1 complex of curcumin and Hg(II) was formed in solution. The formation constant of the 1:1 Hg(II) complex was obtained from two methods, the equilibrium concentration calculation and the linear plot of Benesi-Hildebrand equation, as log K = 4.44 ± 0.16 and 4.83 ± 0.02, respectively. The structure is proposed as a tetrahedral complex of Hg(II) with one curcumin and two chloride ions as ligands.