Protective effect of ethyl acetate fraction of Rhododendron arboreum flowers against carbon tetrachloride-induced hepatotoxicity in experimental models

Objective:

To evaluate the hepatoprotective potential of ethyl acetate fraction of Rhododendron arboreum (Family: Ericaceae) in Wistar rats against carbon tetrachloride (CCl₄)-induced liver damage in preventive and curative models.

Materials and Methods:

Fraction at a dose of 100, 200, and 400 mg/kg was administered orally once daily for 14 days in CCl₄-treated groups (II, III, IV, V and VI). The serum levels of glutamic oxaloacetic transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase (SALP), γ-glutamyltransferase (γ -GT), and bilirubin were estimated along with activities of glutathione S-transferase (GST), glutathione reductase, hepatic malondialdehyde formation, and glutathione content.

Result and Discussion:

The substantially elevated serum enzymatic activities of SGOT, SGPT, SALP, γ-GT, and bilirubin due to CCl₄ treatment were restored toward normal in a dose-dependent manner. Meanwhile, the decreased activities of GST and glutathione reductase were also restored toward normal. In addition, ethyl acetate fraction also significantly prevented the elevation of hepatic malondialdehyde formation and depletion of reduced glutathione content in the liver of CCl₄-intoxicated rats in a dose-dependent manner. Silymarin used as standard reference also exhibited significant hepatoprotective activity on post-treatment against CCl₄-induced hepatotoxicity in rats. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicate that ethyl acetate fraction has a potent hepatoprotective action against CCl₄-induced hepatic damage in rats.     

Antidiarrheal and antiulcer activity of Amaranthus spinosus in experimental animals

The ethanol extract (50%) of the whole plant of Amaranthus spinosus Linn. (Amaranthaceae) (ASE) significantly inhibited travel of a charcoal meal at three different doses of ASE, but when 400?mg/kg of ASE was repeated in the presence of yohimbine, intestinal propulsive inhibition decreased, while morphine reversed the activity. The percentages related to controls for the onset of diarrhea were 16.58, 83.42, and 116.18% at doses of 100, 200, and 400?mg/kg of ASE, while with morphine this value was 123.93% compared to controls. The percentage purging frequency related to controls was 41.09, 64.38, 71.23, and 86.30% at three different doses of ASE and with morphine, respectively. The inhibitions in intestinal accumulation were 8.9, 48.16, and 68.06% at doses of 100, 200, and 400?mg/kg of ASE, respectively, compared to control, while inhibition with yohimbine was 50.78%. Antidiarraheal indices of ASE were 23.55, 49.16, and 76.53 at the three different doses of ASE, while morphine had a maximum index of 88.45. Protection in ethanol- induced ulcer was 51.07, 55.91, 77.95, and 60.75%, but in aspirin-induced ulcer it was 41.33, 61.77, 80.88, and 74.66% at doses of 100, 200, and 400?mg/kg of ASE and with cimetidine, respectively. Lipid peroxidation was also associated with a concomitant decrease in ulcer index, while protection was 56.96, 63.29, 81.01, and 52.32% at three different doses of ASE and with cimetidine in cold restraint-induced ulcer.     

Evaluation of the cytotoxic potential of a new pentacyclic triterpene from Rhododendron arboreum stem bark

Context: Traditionally, Rhododendron arboreum Sm. (Ericaceae) is a very important medicinal plant having oxytocic, estrogenic, anti-inflammatory, analgesic and hepatoprotective activities; it also inhibits the prostaglandin synthetase.

Objectives: This study determines the cytotoxic potential of 15-oxoursolic acid isolated from R. arboreum against selected human cancer cell lines.

Materials and methods: Extraction from stem bark (5?kg) of R. arboreum was performed with methanol, which was successively partitioned into hexane, dichloromethane and ethyl acetate fractions, respectively. The new antitumor agent [15-oxoursolic acid (1)] was isolated from ethyl acetate fraction through column chromatography. Structure elucidation of new compound was performed through extensive spectroscopy i.e., IR, MS and 1D and 2D NMR. Cytotoxicity of isolated compound was determined at doses 5–100?μM for a period of 72?h on specified human cancer cell lines [renal cell carcinoma (A498), non-small cell lung (NCI-H226), squamous cell carcinoma (H157) and human ovarian carcinoma (MDR-2780AD)].

Results: Structure of isolated compound was characterized as 15-oxoursolic acid on the basis of various extensive spectroscopic techniques. 15-Oxoursolic acid revealed considerable anticancer activity with IC₅₀ values of 2.3?±?0.1?μM, 4.9?±?0.2?μM, 9.2?±?0.2?μM and 10.3?±?0.1?μM against MDR 2780AD, Hep G2, H157 and NCI-H226, respectively, while in the case of A498, the activity was good (IC₅₀ 32.8?±?1.2?μM).

Conclusions: This study highlighted the potential of 15-oxoursolic acid to be further explored as a new lead compound for cancer chemotherapy.     

Antinociceptive grayanane-derived diterpenoids from flowers of Rhododendron molle

Twelve new grayanoids (1−12) along with five known compounds were isolated from flowers of Rhododendron molle. Their structures were fully characterized using a combination of spectroscopic analyses, computational calculations, and single crystal X-ray diffraction. Rhomollone A (1) possesses an unprecedented 5/6/6/5 tetra-cyclic ring system (B-nor grayanane) incorporating a cyclopentene-1,3-dione scaffold. Rhodomollein XLIII (2) is a dimeric grayanoid, containing a novel 14-membered heterocyclic ring with a C₂ symmetry axis. The antinociceptive activities of compounds 3, 4, 6, 7, and 12−17 were evaluated by an acetic acid-induced writhing test. Among them, compounds 3, 7, 12, 15 and 16 displayed significant antinociceptive activities at a dose of 20 mg/kg with inhibition rates ranging from 41.9% to 91.6%. Compounds 6 and 13 inhibited 46.0% and 39.4% of the acetic acid-induced writhes at a dose of 2 mg/kg, while compound 17 inhibited 34.3% of the writhes at a dose of 0.4 mg/kg.     

Evaluation of the anti-pyretic potential of Orthosiphon stamineus Benth standardized extract

The anti-pyretic activity of a standardized methanol/water (50/50) extract of Orthosiphon stamineus Benth. (SEOS) was investigated for its effect on normal body temperature and yeast-induced pyrexia in Sprague Dawley (SD) rats. The SEOS showed no effect on normal body temperature. Doses of 500 and 1000 mg/kg body weight of SEOS significantly reduced the yeast-induced elevation in body temperature. This effect persisted up to 4 h following the administration of the extract. The anti-pyretic effect of SEOS was comparable with that of paracetamol (acetaminophen in U.S) (150 mg/kg p.o.), a standard anti-pyretic agent. HPLC study revealed that rosmarinic acid, sinensetin, eupatorin and tetramethoxyflavone were present in SEOS in the amounts of 7.58 %, 0.2 %, 0.34 % and 0.24 % respectively. The LD₅₀ of the extract in rats was higher than 5000 mg/kg body weight. Therefore, the present study ascertained that SEOS possesses a significant anti-pyretic activity. Received 10 July 2008; accepted 10 October 2008     

Immunomodulatory activity of methanolic fruit extract of Aegle marmelos in experimental animals

Aim

The aim of the present study was to investigate the immunomodulatory action of methanolic extract of Aegle marmelos fruit (FEAM) in experimental model of immunity.

Methods

Cellular immunity was carried out by neutrophil adhesion test and carbon clearance assay, whereas, humoral immunity was analyzed by mice lethality test and indirect haemagglutination assay. FEAM dose was selected by Stair case method (up and down) and administered at 100 and 500 mg/kg orally. The Ocimum sanctum (OSE, 100 mg/kg, p.o) was used as standard.

Results

FEAM at 100 and 500 mg/kg produced significant increases in adhesion of neutrophils and an increase in phagocytic index in carbon clearance assay. Both high and low doses of FEAM significantly prevented the mortality induced by bovine Pasteurella multocida in mice. Treatment of animals with FEAM and OSE significantly increased the circulating antibody titre in indirect haemagglunation test. Among the different doses, low one was more effective in cellular immunity models than the high. However, all the doses exhibited similar protection in humoral immunity procedures.

Conclusion

From the above findings, it is concluded that FEAM possesses potential for augmenting immune activity by cellular and humoral mediated mechanisms more at low dose (100 mg/kg) than high dose (500 mg/kg).     

Gastroprotective effect of standardized extract of Amukkara choornam on experimental gastric ulcer in rats

Amukkara choornam ethanolic extract (ACE) was investigated for phytochemical screening, content of total phenolics and flavonoids, in vitro radical scavenging activity (RSA), quantification of various antiulcer marker compounds (i.e., eugenol, piperine, trans-caryophyllene, and withaferine A) by a validated HPTLC method, and evaluated for its in vivo gastroprotective ability against ethanol (EtOH)-induced and pylorus ligation (PL)-induced ulcer models in rats. Phytochemical screening revealed the presence of flavonoids, saponins, phenols, bitter principles, and steroids. Total phenolic and flavonoid content was found to be 61.12 ± 0.72 mg GAE/g of ACE and 24.06 ± 1.07 mg RE/g of ACE, respectively; this was found to be very high in plant extracts showing very good antioxidant and antiulcerogenic effect. RSA of ACE appeared significantly (p < 0.05) lower than that of ascorbic acid (AA), but higher than that of ranitidine (RAN). In vivo the pretreatment of rats with RAN (100 mg/kg) and 50, 100, and 200 mg/kg doses of ACE significantly reduced the ulcer index in a dose-dependant manner in both the models by blocking lipid peroxidation and by significant increases in superoxide dismutase and catalase activity. But rats treated with AA (200 mg/kg) did not have any effect on the ulcer induced by EtOH or PL as it has very good in vitro and in vivo antioxidant activity. HPTLC analysis showed the presence of 0.198 ± 0.01 μg/g, 0.754 ± 0.06 mg/g, 3.50 ± 0.04, and 0.854 ± 0.04 μg/g of eugenol, piperine, trans-caryophyllene, and withaferine A per gram of Amukkara choornam (AC). So the antiulcerogenic activity of ACE might be due to a possible synergistic antioxidant, supported by the holistic approach of polyherbal formulations, i.e., systematism, multi-target and multi-channel owing to their complex chemical constituents and antihistaminic-like effects.     

Analgesic activity of the ethanolic extract of Shorea robusta resin in experimental animals

Aim:

Shorea robusta (Sal), an important traditional Indian medicinal plant used in various ailments and rituals and the indigenous use of the resin of this plant as a medicament for treatment of various inflammatory conditions is well documented in literature. In the present study, ethanolic extract of S. robusta resin (SRE) was evaluated for its analgesic activity by making use of different central and peripheral pain models.

Materials and Methods:

The analgesic activity of SRE was assessed by employing different pain models such as, i) hot plate and tail flick tests for central analgesia, ii) acetic acid- induced writhing (peripheral analgesic model), iii) formalin-induced hind paw licking (both central and peripheral model), iv) carrageenan-induced hyperalgesia (peripheral analgesic model) and v) post-surgical pain (peripheral analgesic model).

Results:

The extract produced significant central and peripheral analgesic effects, as is evident from increase in reaction time in hot plate and tail flick tests, inhibition in writhing counts in acetic acid-induced writhing test, inhibition of licking time in formalin-induced hind paw licking, increased pain threshold in paw withdrawal latency in carrageenan-induced hyperalgesia and increased paw withdrawal threshold in post-surgical pain.

Conclusion:

The results of the present study demonstrate marked antinociceptive effects of SRE.KEY WORDS: Carrageenan, hot plate, post-surgical pain, resin, Shorea robusta, tail flick     

Antinociceptive and anti-inflammatory activities of Dicranopteris linearis leaves chloroform extract in experimental animals

The present study was carried out to establish the antinociceptive and anti-inflammatory properties of Dicranopteris linearis leaves chloroform extract in experimental animals. The antinociceptive activity was measured using the abdominal constriction, formalin and hot plate tests, while the anti-inflammatory activity was measured using the carrageenan-induced paw edema. The extract, obtained after 72 h soaking of the air-dried leaves in chloroform followed by evaporation under vacuo (40 degrees C) to dryness, was dissolved in dimethyl sulfoxide to the doses of 20, 100 and 200 mg/kg and administered subcutaneously 30 min prior to subjection to the above mentioned assays. The extract, at all doses used, was found to exhibit significant (p<0.05) antinociceptive activity in a dose-dependent manner. However, the significant (p<0.05) anti-inflammatory activity observed occur in a dose-independent manner. As a conclusion, the chloroform extract of D. linearis possesses antinociceptive and anti-inflammatory activity and thus justify its traditional uses by the Malays to treat various ailments.     

Anti-inflammatory and anti-nociceptive activities of Fumaria indica whole plant extract in experimental animals

The 50% ethanolic extract of Fumaria indica was investigated for its anti-inflammatory and antinociceptive potential in animal models. Oral administration of F. indica dry extract (100, 200 and 400 mg kg-1) exhibited dose dependent and significant anti-inflammatory activity in acute (carrageenean and histamine induced hind paw oedema, p < 0.05) and chronic cotton pellet granuloma models of inflammation, p < 0.01). The extract (400 mg kg-1) exhibited maximum anti-inflammatory effects of 42.2 and 42.1% after 3 h with carrageenean and histamine, respectively. The same dose of extract showed 38.9% reduction in granuloma mass in a chronic condition. A significant anti-nociceptive activity was evidenced in mice; 6.6-67.7% (p < 0.01) protection in mechanical, 33.9-125.1% (p < 0.05) protection in thermal induced pain and 22.2-73.9% (p < 0.05) protection in acetic acid-induced writhing.     

Two new grayanane diterpenoids from the flowers of Rhododendron molle

A phytochemical investigation of the flowers of Rhododendron molle has yielded two new grayanane diterpenoids, rhodomolleins F and G (1, 2). Compounds 1 and 2 possessed an oxo-bridge between C-5 and C-9, and their structures were elucidated on the basis of interpretation of spectroscopic data, including 1D and 2D NMR techniques.     

Angiotensin-converting enzyme (ACE) inhibitory potential of standardized Mucuna pruriens seed extract

Abstract

Context: Mucuna pruriens Linn. (Fabaceae) is a tropical legume, traditionally used for controlling blood pressure. Inhibition of angiotensin-converting enzyme (ACE) is one of the successful strategies for controlling hypertension.

Objective: The present study evaluated the ACE inhibition potential of the standardized extract of M. pruriens seeds.

Materials and methods: Standardization of the extract and its fractions were carried out by RP-HPLC method [methanol and 1% v/v acetic acid in water (5:95 v/v)] using levodopa as a marker. The ACE inhibition activity of the extract and fractions was evaluated at different concentrations (20, 40, 60, 80, and 100?µg/mL) using the HPLC-DAD and the UV spectrophotometric method. The liberation of hippuric acid (HA) from hippuryl-l-histidyl-l-leucine (HHL) was estimated in the spectrophotometric method and RP-HPLC assay at 228?nm.

Results: Methanol extract and aqueous fraction showed a maximum activity with IC₅₀ values of 38.44?±?0.90 and 57.07?±?2.90?µg/mL (RP-HPLC), and 52.68?±?2.02 and 67.65?±?2.40?µg/mL (spectrophotometry), respectively.

Discussion: The study revealed that the aqueous extract contains the highest amount of levodopa. Eventually the methanol extract showed highest ACE inhibition activity except levodopa alone. It was further observed that the inhibition was altered with respect to the change in the content of levodopa in the extract. Thus, it can be assumed that levodopa may be responsible for the ACE inhibition activity of M. pruriens seeds.

Conclusion: It can be concluded that M. pruriens seed is a potential ACE inhibitor can be explored further as an effective antihypertensive agent.     

藏药髯花杜鹃花中化学成分的研究

目的 研究藏药髯花杜鹃Rhododendron anthopogon D.Don花中的化学成分.方法 采用95%乙醇提取制备总浸膏,经萃取及柱层析分离,核磁共振鉴定结构.结果 分离得14个已知化合物,分别为三油酸甘油酯、3-羟基-3-苯基丙酰胺、齐墩果酸甲酯、熊果酸、2-羟基-4-甲氧基苯酚、5-甲氧基糠醛、7-羟基香豆素、23-羟基乌苏酸、β-谷甾醇、槲皮素、杨梅素、桃叶珊瑚苷、杨梅素-3'-O-β-D-吡喃木糖苷、金丝桃苷.结论 所有化合物均为首次从该植物花中分离得到.     

Antidiarrheal evaluation of the ethanol extract of Nauclea latifolia root bark

Nauclea latifolia (Rubiaceae) is a medicinal plant used in Nigeria folk medicine for the treatment of gastrointestinal disorders. The root bark of the plant was extracted with 70% ethanol and the extract screened for antidiarrheal activity by investigating castor oil-induced diarrhea and small intestinal motility in mice. The effects of the extract on isolated rat ileum were also investigated. The extract (125, 250 and 500 mg/kg) caused a significant decrease in the frequency of diarrhea and conferred protection following castor oil administration (P < 0.05). It also significantly (P < 0.0001) inhibited small intestinal motility in mice at the same doses. Its activity was dose-dependent and when compared to atropine, its antidiarrheal effects at 500 mg/kg were 179% and 165% respectively, in castor oil-induced diarrhea and small intestinal motility experiments. Extract effects on rat ileum revealed a significant (P < 0.0001) inhibition of acetylcholine-induced contractions at 0.2 and 2 mg/mL final bath concentrations used. These findings confirm the antidiarrheal activity of the root bark extract of N. latifolia.     

Gastroprotective effect of standardized leaf extract from Careya arborea on experimental gastric ulcers in rats

Context: The leaf of Careya arborea Roxb. (Lecthidaceae) has been advocated in Ayurveda for the treatment of various disorders, including ulcers, healing of wounds and several skin diseases.

Objective: The 70% ethanol (EtOH) extract of C. arborea leaves (CALE) was investigated for its gastroprotective effect in different gastric ulcer models.

Materials and methods: CALE (100, 200, and 400?mg/kg body weight) was administered orally, twice daily for 5?d, for preventing aspirin (ASP)-, EtOH-, pylorus ligation (PL)-, and cold restraint stress (CRS)-induced ulcer in rats. The status of the antioxidant enzymes in CRS-induced ulcers, H⁺K⁺ATPase activity, gastric wall mucous in EtOH-induced ulcer, and gastric secretion parameters were estimated in the PL-induced ulcer model.

Results: CALE exhibited significant (p?<?0.01) dose-dependent inhibition of ulcer index in ASP 12.90–51.61%, EtOH 11.97–40.35%, PL 28.63–63.92%, and CRS 38.30–66.37%, respectively. A significant (p?<?0.001) decrease occurred in the level of H⁺K⁺ATPase, volume of gastric juice, and acid output. Simultaneously, the level of gastric wall mucus was increased significantly (p?<?0.05). The antioxidant enzyme levels of LPO and SOD were decreased with concomitant increase in catalase activity in CRS-induced ulcers. High-performance thin-layer chromatography (HPTLC) showed the presence of quercetin, ellagic acid, and gallic acid (0.31%, 0.24%, and 0.71% w/w, respectively) in CALE.

Conclusions: Our results show that C. arborea possesses significant gastro-protective activity, probably due to its free radical scavenging activity, and validate the folklore claim.     

Matrix metalloproteinase,hyaluronidase and elastase inhibitory potential of standardized extract of Centella asiatica

Abstract

Context: Centella asiatica (L.) Urban (Apiaceae), a valuable herb described in Ayurveda, is used in the indigenous system of medicine as a tonic to treat skin diseases.

Objective: Centella asiatica methanol extract and its ethyl acetate, n-butanol and aqueous fraction, were subjected for the evaluation of skin care potential through the in vitro hyaluronidase, elastase and matrix metalloproteinase-1 (MMP-1) inhibitory assay.

Materials and methods: The C. asiatica plant was extracted with methanol and fractionated with ethyl acetate, n-butanol and water. The enzymatic activities were evaluated using ursolic acid and oleanolic acid as standards. Isolate molecule asiaticoside was quantified in the crude extract and fractions through high-performance liquid chromatography (HPLC) and structural was characterized by liquid chromatography–mass spectroscopy (LC–MS) and ¹H nuclear magnetic resonance (NMR). Isolated compound was also evaluated for in vitro enzyme assays.

Results: Extract exhibited anti-hyaluronidase and anti-elastase activity with IC₅₀ of 19.27?±?0.37 and 14.54?±?0.39?µg/mL, respectively, as compared to ursolic acid. Centella asiatica n-butanol fraction (CAnB) and isolated compound showed significant hyaluronidase (IC₅₀?=?27.00?±?0.43 and 18.63?±?0.33?µg/mL) and elastase (IC₅₀?=?29.15?±?0.31 and 19.45?±?0.25?µg/mL) inhibitory activities, respectively, and also showed significant MMP-1 inhibition (p?<?0.05 and p?<?0.01).

Discussion and conclusion: n-Butanol fraction was found to be most effective among the all fractions from which asiaticoside was isolated and further quantified by HPLC. This work concludes that the asiaticoside from C. asiatica may be a prospective agent for skin care.     

Toxicological studies of gambogic acid and its potential targets in experimental animals

Acute and chronic toxicity of gambogic acid, a promising novel anticancer agent, was determined using albino mice and Beagle dogs as model animals. Histopathological examination and viscera parameter investigation were also carried out after autopsy. The LD50 of gambogic acid was found to be 45 approximately 96 mg/kg and the 95% confidence limit was determined to be 43.18 approximately 48.45 mg/kg. The results from the chronic toxicity studies demonstrated that the toxicity targets in the experimental animals were liver and kidney. The innocuous dose was established to be 4 mg/kg after administration to dogs for a total of 13 weeks at a frequency of one injection every other day. This dose (4 mg/kg) was approximately 9.6 (body weight) or 5.1 (body surface area) times the dosage (25 mg/60 kg, every other day) recommended for human trials. Our results provide the theoretical foundation for clinical applications of this promising natural anticancer agent and will likely bring about considerable economic and social benefits.     

羊踯躅的化学成分研究

对羊踯躅植物花蕾的化学成分进行研究,应用多种色谱技术进行分离纯化、波谱技术鉴定其结构。从羊踯躅花蕾中分离并鉴定了15个化合物,分别为2E,4Z-脱落酸（1）,2α-羟基齐墩果酸（2）,齐墩果酸（3）,积雪草酸（4）,苯甲基糖苷（5）,邻苯二甲酸二丁酯（6）,β-谷甾醇（7）,牡荆素（8）,槲皮素（9）,硬脂酸（10）,rhodomollein Ⅰ（11）,rhodojaponin Ⅵ（12）,rhodomollein Ⅺ（13）,rhodojaponin Ⅱ（14）,kalmanol（15）。其中,化合物1 10为首次从羊踯躅植物中得到。     

Anticonvulsive activity of Butea monosperma flowers in laboratory animals

The bioassay-guided fractionation of dried flowers of Butea monosperma (BM) was carried out to isolate the active principle responsible for its anticonvulsant activity. The petroleum ether extract was fractionated by column chromatography using solvents of varying polarity such as n-hexane, n-hexane:ethyl acetate, ethyl acetate, and methanol. The anticonvulsive principle of B. monosperma was found to be a triterpene (TBM) present in the n-hexane:ethyl acetate (1:1) fraction of the petroleum ether extract. TBM exhibited anticonvulsant activity against seizures induced by maximum electroshock (MES) and its PD(50) was found to be 34.2+/-18.1 mg/kg. TBM also inhibited seizures induced by pentylenetetrazol (PTZ), electrical kindling, and the combination of lithium sulfate and pilocarpine nitrate (Li-Pilo). However, TBM was not effective against seizures induced by strychnine and picrotoxin. TBM exhibited depressant effect on the central nervous system. After repeated use for 7 days, the PD(50) (MES) of TBM increased to 51.5+/-12.1 mg/kg. Similarly, after repeated use of TBM, the duration of sleep induced by pentobarbital was not reduced significantly. Further studies are required to investigate its usefulness in the treatment of epilepsy.