Beneficial Effects of Natural Alkaloids from Berberis glaucocarpa as Antidiabetic Agents: An In Vitro, In Silico, and In Vivo Approach.

Diabetes, also known as diabetes mellitus (DM), is a metabolic disorder characterized by an abnormal rise in blood sugar (glucose) levels brought on by a complete or partial lack of insulin secretion along with corresponding changes in the metabolism of lipids, proteins, and carbohydrates. It has been reported that medicinal plants play a pivotal role in the treatment of various ailments such as diabetes mellitus, dyslipidemia, and hypertension. The current study involved exploring the acute toxicity and in vivo antidiabetic activity of berberine (WA1), palmatine (WA2), and 8-trichloromethyl dihydroberberine (WA3) previously isolated from Berberis glaucocarpa Stapf using a streptozotocin (STZ)-induced diabetic rat model. Body weight and blood glucose level were assessed on a day interval for 4 weeks. Biochemical parameters, antioxidant enzymes, and oxidative stress markers were also determined. In an acute toxicity profile, the WA1, WA2, and WA3 were determined to be nontoxic up to 500 mg/kg (b.w). After the second and third weeks of treatment (14 and 21 days), the blood glucose levels in the WA1-, WA2-, and WA3-treated groups were significantly lower than those in the diabetic control group (476.81 ± 8.65 mg/dL, n = 8, P < 0.001). On the 21st day, there was a decrease in the blood glucose level and the results obtained were 176.33 ± 4.69, 197.21 ± 4.80, and 161.99 ± 4.75 mg/dL (n = 8, P < 0.001) for WA1, WA2, and WA3 at 12 mg/kg, respectively, as opposed to the diabetic control group (482.87 ± 7.11 mg/dL, n = 8, P < 0.001). Upon comparison with the diabetic group at the end of the study (28 days), a substantial drop in the glucose level of WA3 at 12 mg/kg (110.56 ± 4.11 mg/dL, n = 8, P < 0.001) was observed that was almost near the values of the normal control group. The treated groups (WA1, WA2, and WA3) treated with the samples displayed a significant decline in the levels of HbA1c. Treatment of the samples dramatically lowered the lipid level profile. In groups treated with samples, plasma levels of triglycerides, total cholesterol, and LDL were significantly lowered [F (5, 42) = 100.6, n = 8, P < 0.001]; these levels were also significantly decreased [F (5, 42) = 129.6 and 91.17, n = 8, P < 0.001]. In contrast to the diabetes group, all treated groups had significantly higher HDL levels [F (5, 42) = 15.46, n = 8, P < 0.001]. As a result, hypolipidemic activity was anticipated in the samples. In addition to that, the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was considerably elevated in the groups treated with the sample compared to the diabetic control group (n = 8, P < 0.001).

Corrigendum to "Pharmacognostic evaluation of Artemisia maritima L. a highly medicinal specie of genus Artemisia" [Saudi J. Biol. Sci. 29(10) (2022) 103419].

[This corrects the article DOI: 10.1016/j.sjbs.2022.103419.].

Synthesis and Investigation of the Analgesic Potential of Enantiomerically Pure Schiff Bases: A Mechanistic Approach.

Schiff bases are a class of organic compounds with azomethine moiety, exhibiting a wide range of biological potentials. In this research, six chiral Schiff bases, three 'S' series (H1−H3) and three 'R' series (H4−H6), were synthesized. The reaction was neat, which means without a solvent, and occurred at room temperature with a high product yield. The synthesized compounds were evaluated for analgesic potential in vivo at doses of 12.5 and 25 mg/kg using acetic-acid-induced writhing assay, formalin test, tail immersion and hot plate models, followed by investigating the possible involvement of opioid receptors. The compounds H2 and H3 significantly (*** p < 0.001) reduced the writhing frequency, and H3 and H5 significantly (*** p < 0.001) reduced pain in both phases of the formalin test. The compounds H2 and H5 significantly (*** p < 0.001) increased latency at 90 min in tail immersion, while H2 significantly (*** p < 0.001) increased latency at 90 min in the hot plate test. The 'S' series Schiff bases, H1−H3, were found more potent than the 'R' series compounds, H4−H6. The possible involvement of opioid receptors was also surveyed utilizing naloxone in tail immersion and hot plate models, investigating the involvement of opioid receptors. The synthesized compounds could be used as alternative analgesic agents subjected to further evaluation in other animal models to confirm the observed biological potential.

Effects of Artemisia macrocephala Jacquem on Memory Deficits and Brain Oxidative Stress in Streptozotocin-Induced Diabetic Mice.

Different species of Artemisia have been reported to have therapeutic potential in treating various health disorders, including diabetes and memory dysfunction. The present study was planned to evaluate the effects of Artemisia macrocephala Jacquem crude extract and its subfractions as antiamnesic agents in streptozotocin-induced (STZ) diabetic mice. The in vivo behavioral studies were performed using the Y Maze test and novel object recognition test (NORT) test at doses of 100 and 200 mg/kg of crude extract and 75 and 150 mg/kg of fractions. The in vitro and ex vivo anticholinesterase activities, along with biochemical parameters (superoxide dismutase, catalase, glutathione and lipid peroxidation) in the brain, were evaluated. Blood glucose levels were monitored with a glucometer; crude extract and fractions reduced the glucose level considerably, with some differences in the extent of their efficacies. The crude extract and fractions demonstrated significant inhibitory activity against cholinesterases (AChE and BuChE) in vitro. Crude, chloroform and ethyl acetate extract were found to be more potent than the other fractions, with IC50 of Crd-Am = 116.36 ± 1.48 and 240.52 ± 1.35 µg/mL, Chl-Am = 52.68 ± 1.09 and 57.45 ± 1.39 µg/mL and Et-Am = 75.19 ± 1.02 and 116.58 ± 1.09 µg/mL, respectively. Oxidative stress biomarkers like superoxide dismutase, catalase and glutathione levels were elevated, whereas MDA levels were reduced by crude extract and all fractions with little difference in their respective values. The Y-maze test and novel object recognition test demonstrated declines in memory impairment in groups (n = 6) treated with crude extract and fractions as compared to STZ diabetic (amnesic) group. The most active fraction, Chl-Am, was also subjected to isolation of bioactive compounds; three compounds were obtained in pure state and designated as AB-I, AB-II and AB-III. Overall, the results of the study showed that Artemisia macrocephala Jacquem enhanced the memory impairment associated with diabetes, elevated acetylcholine levels and ameliorated oxidative stress. Further studies are needed to explore the beneficial role of the secondary metabolites isolated in the present study as memory enhancers. Toxicological aspects of the extracts are also important and need to be evaluated in other animal models.

Anatomical Characterization, HPLC Analysis, and Biological Activities of Ilex dipyrena.

Ilex dipyrena Wall (Aquifoliaceae), is a traditional medicinal plant abundantly found in India and Pakistan. In the current research work, initially, the anatomical characteristics were recorded through microscopic examination of selected plant parts, such as leaf, petiole, and midrib. Then, the quantitative phytochemical screening was performed using standard tests reported in literature. The whole-plant powdered sample was then soaked in methanol to obtain crude extract, which was then fractionated into solvents of different polarities to obtain ethyl acetate, chloroform, butanol, hexane, and aqueous extracts. The phytochemical composition of the crude ethyl acetate and chloroform extracts (being the most active fractions) was then confirmed through HPLC analyses, where the possible phytochemical present were predicted through comparison of retention time of a given compound peak with the available standards. The extracts were also evaluated for their in vitro antioxidant and ani-lipoxygenase potentials using standard methods. The microscopic examination revealed the presence of anomocytic type stomata on the abaxial side of the leaf as well as unicellular trichrome and calcium oxalate druses crystals in the midrib and petiole, with a single, centered U-shaped collateral arterial bundle, which was directed toward the adaxial and the phloem toward the abaxial sides of the selected plant parts, respectively. Almost all tested representative groups of phytochemicals and essential minerals were detected in the selected plant, whereas five possible phytochemicals were confirmed in crude and chloroform extract and seven in ethyl acetate fraction. As antioxidant, chloroform fraction was more potent, which exhibited an IC50 value of 64.99, 69.15, and 268.52 µg/mL, determined through DPPH, ABTS, and FRAP assays. Ethyl acetate extract was also equally potent against the tested free radicals. Chloroform and ethyl acetate extracts were also potent against lipoxygenase, with IC50 value of 75.99 and 106.11 µg/mL, respectively. Based on the results of biological studies, Ilex dipyrena was found to good inhibitor of free radicals and lipoxygenase that could be further investigated to isolate compounds of medicinal importance.

GC/MS analysis, anti-leishmanial and relaxant activity of essential oil of Chenopodium ambrosioides (L.) from Malakand region.

Chenopodium ambrosioides is abundantly available in Malakand region. As constituents and concentrations of essential oils vary based on its geographical location, we carried our current study to extract and evaluate its possible relaxant activity in rabbits' jejunum and anti-leishmanial activity against promastigotes of Leishmania tropica. The essential oil was obtained from aerial fresh parts through steam distillation followed by GC/MS analysis. Antispasmodic activity was performed on spontaneous and KCl induced contractions. Curves for calcium concentration response (CCRCs) were prepared with and without different concentrations of essential oils and verapamil - a standard calcium channel blocker as per our reported procedures. GC/MS analysis indicated that the essential oil contains 4-carene (56.59%) and o-cymene (41.46%), the two most abundant compounds previously reported from this species. The LD50 value for acute toxicity is 279.66±2.2mg/kg. The essential oil have significant antileishmanial activity with LC50 of Log10 (1.83±0.0026) ×10-6mg/ml, potent relaxant activity on rabbits' jejunal preparations with respective EC50 = 1.46±0.15mg/ml for spontaneous activity. For KCl (80mM) induced contractions, EC50=0.26±0.02mg/ml. In CCRCs, the oil produced a right shift as exhibited by verapamil. More, its relaxant activity, which is mediated through calcium channel blocking mechanism, proves a rationale for its traditional use in gut spasm.

Evaluation of antinociceptive activity of Ilex dipyrena Wall. in mice.

BACKGROUND: In order to find a new natural resource for pain-relief, the analgesic effects of Ilex dipyrena crude extract, fractions, and subfractions were evaluated in in-vivo mouse models with possible mechanism of action. METHODS: Analgesic effects of crude extract (100 and 200 mg/kg body weight), fractions and subfractions (75 mg/kg body weight) were screened using heat-induced (tail-immersion and hot plate test) and chemical-induced (formalin and acetic acid) nociception models in mice. The samples were also tested for the elucidation of a possible mechanism through opioidergic and GABAergic systems. RESULTS: The administration of crude extract, fractions and subfractions produced analgesic responses in acetic acid, formalin, tail immersion, and hot plate model for pain similar to those obtained with the standard. Naloxone antagonized the antinociceptive effects of the tested samples, whereas bicuculline showed partial inhibition. Considering the analgesic response, crude extract, fractions, and subfractions demonstrated promising inhibitory activity against all test models for pain, which was further supported by the possible involvement of opioidergic and GABAergic systems. CONCLUSION: The results suggest that this plant may be useful in the development of new analgesic drugs. Further research with regard to the isolation of bioactive compounds is required to verify these findings.

A possible mechanistic approach of synthetic flavonoids in the management of pain.

Flavonoids are phenolic compounds that have always attracted pharmaceutical researchers and food manufacturers. Nature has indirectly provided us flavones in our daily diet i.e. tea, fruits, juices and vegetables. Flavones have got special position in research field of natural and synthetic organic chemistry due to their biological capabilities. Three substituted flavone derivatives have been synthesized from substituted O-hydroxy acetophenones and 4-trifluoromethyl benzaldehyde in good yield. The structures have been established by different spectroscopic techniques like 1HNMR 13CNMR, IR spectroscopy. The compounds were then screened for their enzyme inhibition potential and antinociceptive response in mice models with writhings induced by acetic acid, tail immersion and formalin-induced nociception assay procedures and structure activity relationship was established. The effects following pretreatment with naloxone were also studied to reveal the involvement of opioid receptors in the antinociceptive action. The flavone derivatives showed moderate to weak inhibition against LOX. Moreover, significant to moderate decrease in the number of abdominal constrictions, increase in paw-licking response time in both phases and a significant raise in latency time in nociception models. Moreover, the antinociceptive response was significantly attenuated by pretreatment with opioid receptor antagonist suggesting the involvement of opioidergic system in the analgesic action. The flavone derivatives showed analgesic response in all models of nociception suggesting the possible involvement of opioidergic system in the antinociceptive action.

Antidiabetic potential of flavonoids from Artemisia macrocephalla Jaquem in streptozotocin-induced diabetic rats: Pharmacological and biochemical approach.

Plants belongs to Asteraceae family are reported to be rich in major phytochemical including flavonoids and are documented to acquire antidiabetic response. Antidiabetic effects of salvigenin, eupatilin and cirsilineol were screened on in-vitro enzyme inhibition and in-vivo streptozotocin animal models. Administration of salvigenin, eupatilin and cirsilineol (7.5 and 15mg/kg) produced antidiabteic responses in streptozotocin model for diabetes. All natural flavonoids reduces the blood glucose level to a significant level (*P<0.05, **P<0.01, ***P<0.001, n=8) but promising results were observed in eupatilin at dose of 7.5mk/kg (364.12±4.3 to 128.41±4.2mg/dL, n=8) and at dose of 7.5mk/kg 363.65±4.8 to 126.14±5.1mg/dL, n=8). Administration of salvigenin, eupatilin and cirsilineol (7.5 and 15mg/kg) for 28 days showed a substantial fall (*P<0.05, **P<0.01, ***P<0.001, n=8) in total cholesterol, LDL and triglcerides (TGs) in comparison to diabetic model. The isolated flavonoids reduced considerably the serum ALP, SGPT and SGOT in rats intoxicated with streptozotocin. The results indicate that the flavonoids may be useful in the development of new antidiabetic drugs.

Isolation of flavonoides from Artemisia macrocephala anticholinesterase activity: Isolation, characterization and its in vitro anticholinesterse activity supported by molecular docking.

In this study the flavonoids isolated from Artemisia macrocephala were screened out for anticholinesterase activity. The isolated flvanoids were characterized by HNMR, NOESY, COSY, HMBC, HSQC and mass spectroscopy. The compounds (1-4) in appropriate quantities were isolated from chloroform fraction using gravity column chromatography by eluting ethyl acetate/n-hexane solvent system. The flavonoids were characterized and resulted in the form of mono substituted methoxy flavones to tri substituted flavones. Ellman's assay techniques were used to find out enzyme inhibition. Operating environment (MOE) software was used for molecular docking studies. Compounds (1), (2) and (3) showed 88.42±2.76, 84.50±1.60 and 90.16±2.98 percent inhibition of the acetyl cholinesterase (AChE) respectively at 1000µg/mL concentrations with IC50 value 165, 60, 65µg/mL respectively which were comparable to that of standard galanthamine. While for butyryl cholinesterase (BChE), (1), (2) and (3) showed 91.63±4.32, 81.03±3.53 and 87.69±2.84 percent inhibitions respectively at 1mg/mL as compared to the standard galanthamine which caused 96.50±2.41 percent inhibition at the same concentration. Whereas, compound (4) exhibited moderate activity for both the enzymes. Molecular docking studies confirmed the experimental AChE and BChE inhibitory activities of the test samples by their virtue of multiple teractions with target enzymes. The results confirm that the specie has biologically active constituents that are more useful for the management of several neurodegenerative ailments like ataxia, Parkinson's disease, Alzeimer's disease and some other types of dementia.

Spasmogenic and spasmolytic activity of rind of Punica granatum Linn.

BACKGROUND: Rind of Punica granatum is traditionally used in treatment of abdominal cramps and various GIT disorders. So far spasmolytic activity of rind of Punica granatum has been reported using in vitro model. However, its mode of action is not explored yet. Therefore, the current work describes the possible mode of action for spasmolytic activity of methanolic extract of rind of Punica granatum (Pg. Cr). Acute toxicity study is also performed to determine its safe dose range. METHODS: Rind of Punica granatum was subjected to shade drying. Shade dried materials were pulverized using conventional grinder. Grinded materials were macerated in commercial grade methanol. The extract of rind of P. granatum was concentrated using a rotary evaporator. Rabbits' jejunal preparations were mounted in organ bath containing 10 ml Tyrode's solution, constantly aerated with carbogen gas. Pg. Cr was tested on spontaneous rabbits' jejunal preparations in concentrations 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml. Pg. Cr was also tested on KCl (80 mM)-induced contractions in rabbits' jejunal preparations. Since we observed spasmogenic activity for the first time, hence we also determined the effects of Pg. Cr in presence of atropine (0.03 µM). Pg. Cr was also tested in presence of 0.03 µM of loratadine HCl. Pg. Cr was also tested on barium chloride induced contractions. Calcium Concentration Response Curves (CCRCs) were constructed in the absence and presence of test samples of Pg. Cr in decalcified tissues to explore its possible mode of action. Acute toxicity screening was also performed to determine its safe dose range. RESULTS: Phytochemical screening revealed the presence of saponins, tannins, carbohydrates, proteins, flavonoids, saponins and steroids. However, Pg. Cr tested negative for alkaloids and triterpenoids. Pg. Cr was safe up to 100 mg/kg with its LD50 = 1305 mg/kg. Its respective EC50, in the absence and presence of atropine, were 9.7 ± 0.3 and 3.12 ± 0.45 mg/ml. In the presence of 0.02 and 0.08 µM of loratadine HCl, respective EC50 were 5.6 ± 0.4 and 2.8 ± 0.15 mg/ml. EC50 for relaxant effects on KCl-induced contractions was 8.6 ± 1 mg/ml. In the presence of 0.3 mg/ml of Pg. Cr, a right shift was observed with EC50 (log [Ca++]M) = -1.8 ± 0.09 vs. control EC50 -2.6 ± 0.01. Similarly, EC50 for verapamil (0.1 µM) was -2.4 ± 0.011vs. control EC50= -2.4 ± 0.01. The right shift of P. granatum resembled the right shift of verapamil suggesting for inhibition of voltage gated calcium channels. CONCLUSIONS: P. granatum is safe up to 100 mg/kg. In low concentrations, P. granatum produced spasmogenic activity possibly through involvement of cholinergic and histaminergic receptors. The spasmolytic action may follow inhibition of the voltage gated calcium channels.

Sesquiterpene lactone! a promising antioxidant, anticancer and moderate antinociceptive agent from Artemisia macrocephala jacquem.

BACKGROUND: Sesquiterpene lactones (STLs) make a diverse and huge group of bio-active constituents that have been isolated from several plant families. However, the greatest numbers are present in Asteraceae family having more than 3000 different reported structures. Recently several researchers have reported that STLs have significant antioxidant and anticancer potentials. METHODS: To investigate the antioxidant, anticancer and antinociceptive potentials of STLs, gravity column chromatography technique was used for isolation from the biologically rich chloroform fraction of Artemisia macrocephala Jacquem. The antioxidant activity of the isolated STLs was determined by DPPH and ABTS free radical scavenging activity, anticancer activity was determined on 3 T3, HeLa and MCF-7 cells by MTT assay while the antinociceptive activity was determined through acetic acid induced writhings, tail immersion method and formalin induced nociception method. RESULTS: The results showed that the STLs of Artemisia macrocephala possesses promising antioxidant activity and also it decreased the viability of 3 T3, HeLa and MCF-7 cells and mild to moderate antinociceptive activity. CONCLUSION: Sesquiterpenes lactones (STLs) are widely present in numerous genera of the family Asteraceae (compositae). They are described as the active constituents used in traditional medicine for the treatment of various diseases. The present study reveals the significant potentials of STL and may be used as an alternative for the management of cancer. Anyhow, the isolated compound is having no prominent antinociceptive potentials.

Pharmacological profile of the aerial parts of Rubus ulmifolius Schott.

BACKGROUND: As aerial parts of Rubus ulmifolius contains phytochemicals like flavonoids and tannins. And whereas flavonoids and tannins have antioxidant and antipyretic activity, hence, current work is carried out to screen crude methanolic extract of aerial parts of Rubus ulmifolius (Ru.Cr) and crude flavonoids rich extract of Rubus ulmifolius (Ru.F) for possible antioxidant and antipyretic activity. Ru.Cr and Ru.F are also tested for brine shrimps lethality bioassay. Ru.F is tested for the first time for possible antioxidant and antipyretic activity. METHODS: Preliminary phytochemical screening of Ru.Cr and Ru.F was performed as it provides rapid finger printing for targeting a pharmacological activity. Acute toxicity and Brine shrimps' cytotoxicity studies of Ru.Cr and Ru.F were performed to determine its safe dose range. Antioxidant and antipyretic studies were also performed as per reported procedures. RESULTS: Ru.Cr tested positive for presence of tannins, alkaloids, flavonoids and steroids. Ru.Cr is safe up to 6 g/kg following oral doses for acute toxicity study. Ru.Cr is safe up to 75 µg/kg (p.o), LC50 for Ru.Cr and Ru.F are 16.7 ± 1.4 µg/ml 10.6 ± 1.8 µg/ml, respectively (n = 3). Both Ru.Cr and Ru.F demonstrated comparable antioxidant activity using vitamin C as standard (p ≤ 0.05). In test dose of 300 mg of Ru.Cr, rectal temperature was reduced by 74% (p ≤ 0.05) on 4th hour of the administration. More, Ru.F produced 72% reduction in pyrexia (p ≤ 0.05) on 4th hour of administration of paracetamol in Westar rats. CONCLUSIONS: The current work confirms that aerial parts of Rubus ulmifolius contain flavonoids that are safe up to 6 g/kg (p.o). Crude methanolic extract and flavonoids rich fraction of Rubus ulmifolius have significant antioxidant and antipyretic activity. Further work is required to isolate the pharmacologically active substances for relatively safe and effective antipyretics and antioxidants.

Scientific investigation of crude alkaloids from medicinal plants for the management of pain.

BACKGROUND: Tissue damage is associated with pain, which is an alarming sign. Aspirin and morphine have been widely used in recent decades for management of pain. Medicinal herbs have been in use for treatment of different diseases for centuries. Many of these herbs possess analgesic activity with relatively less incidences of adverse effects. The strong positive correlation of alkaloids in medicinal plants for analgesic activity persuades an intention to determine possible analgesic activity of total alkaloids extracted from the selected medicinal plants using animal models to answer its possible mechanisms. METHODS: Crude alkaloids from selected medicinal plants (Woodfordia fruticosa, Adhatoda vasica, Chenopodium ambrosioides, Vitex negundo, Peganum harmala and Broussonetia papyrifera) were extracted as per reported literature. The test crude alkaloids were screened foracute toxicity study. Writhings induced by acetic acid, tail immersion method and formalin-induced nociception assay procedures were used for possible analgesic effects of the crude alkaloids. RESULTS: Crude alkaloids were safe up to dose of 1250 mg/kg body weight in mice. The alkaloids significantly reduced the abdominal constrictions, and increased the time for paw licking response in both phases with a significant raise in latency time in nociception models (P ≤ 0.05). Moreover, the antinociceptive response was significantly attenuated by pretreatment with naloxone suggesting involvement of the opioid receptors for possible antinociceptive action. CONCLUSIONS: Crude alkaloids of Woodfordia fruticosa and Peganum harmala showed prominent analgesic potentials through inhibition of peripheral as well as central nervous system mechanisms. Further work is required for isolation of the pharmacologically active constituents.

Parasiticidal and brine shrimp cytotoxicity potential of crude methanolic extract of rind of Punica granatum Linn against round worms and tape worms.

Rind of Punica granatum is traditionally used for anthelmintic purposes. The current work describes the possible anthelmintic activity of crude methanolic extract of Punica granatum (Pg. Cr) against round worms (Ascaridia galli) and the tape worms (Raillietina spiralis). Brine shrimp cytotoxicity is also performed. Brine shrimp cytotoxic activity was tested using different concentrations (1000 µg/mL, 100 µg/mL and 10 µg/mL) of Pg.Cr. In vitro anthelmintic activity of Pg. Cr was determined against the parasites using albendazole and piperazine citrate as standard anthelmintic drugs in concentration 10 mg/ml. LC50 value for Brine shrimp cytotoxicity was 189.44 ±28 µg/mL. In test concentration of 40mg/ml of the Pg. Cr, Raillietina spiralis was paralyzed in 23 minutes. However, for parasiticidal activity (death of the parasite), it took less time (40 minutes) as compared to standard Albendazole. Time taken for death of the parasite Raillietina spiralis, in concentration 40 mg /ml, is 40 min. While standard drugs took more time to kill the Raillietina spiralis. Pg. Cr took 19 minutes to paralyze the Ascaridia galli at concentration 40 mg/ml whereas; it took 48 minutes for to kill the parasite Ascaridia galli. The current work confirms the traditional use of rind of Punica granatum as anthelmintic against Raillietina spiralis and Ascaridia galli. Results of brine shrimp cytotoxicity assay warrant for the isolation of cytotoxic compounds. List of abbreviation- Pg. Cr = Crude methanolic extract of Punica granatum.

Acute toxicity and antispasmodic activities of Achillea wilhelmsii C. Koch.

Since Achillea wilhelmsii is used as antispasmodic in traditional medicine, we conducted our current work to investigate its rationale on scientific grounds. Acute toxicity studies of crude methanol extract of Achillea wilhelmsii (Aw. CMeOH) is also performed. Effect of Aw. CMeOH and its fractions were tested on isolated sections of rabbits' jejunum at test concentrations 0.01, 0.03, 1.0, 3.0, 5.0 and 10mg/ml. The test extracts, in similar concentrations, were also tested on KCl-induced contractions. Calcium chloride curves were constructed for those fractions which relaxed KCl induced contractions in the absence and presence of the test samples to investigate its possible mode of action through calcium channels. Aw. CMeOH tested positive for flavonoids, saponins, tannins, glycosides, terpenoids, sterols, phenols, carbohydrates and proteins. LD(50) for acute toxicity studies is 2707±12.6 mg/kg. Mean EC(50) values for Aw. CMeOH on spontaneous and KCl-induced contractions are 3.41±0.18 (2.56-3.8, n=6) and 0.68±0.05 (0.6-0.85, n=6) mg/ml, respectively. Respective EC(50) values for n-hexane fraction on spontaneous and KCl-induced contractions are 3.06±0.08 (2.8-3.3, n=6) and 1.68±0.8 (1.4-1.9, n=6) mg/ml, respectively. Corresponding EC(50) (mg/ml) values for chloroformic, ethylacetate and aqueous fractions of Achillea wilhelmsii on spontaneous rabbits' jejunum preparations are 4.8±0.2 (4.41-5.63, n=6), 5.07±0.15 (4.7-5.58, n=6) and 5.2±0.13 (4.91-5.64, n=4), respectively. Constructing calcium chloride curves, in the presence of 0.1 mg/ml of Aw. CMeOH, mean EC(50) value (log molar [Ca(++)]) is-1.98±0.03 (-1.89-2.05, n=6) vs. control EC(50) (log molar [Ca(++)])-2.41±0.02 (-2.32-2.44, n=6). Mean EC(50) value (log molar [Ca(++)]) for 0.3 mg/ml n-hexane fraction is-1.76±0.05 (-1.70 -1.93, n=6) vs. control EC(50) (log molar [Ca(++)]) value-2.18±0.07 (-2.0-2.46, n=6). While in the presence of chloroformic fraction (3 mg/ml), mean EC(50) (log molar [Ca(++)]) value is -2.4±0.1 (-2.78 -2.9, n=6) vs. control EC(50) (log molar [Ca(++)]) value-2.70±0.05 (-2.5-2.8, n=6). Mean EC(50) value (log molar [Ca(++)]) for ethyl acetate fraction (1 mg/ml) is-1.94±0.07 (-1.75-2.05, n=6) vs. control EC(50) (log molar [Ca(++)]) value-2.69±0.04 (-2.57-2.79, n=6). Mean EC(50) (log molar [Ca(++)]) value for residual aqueous fraction (3 mg/ml) is-1.8±0.3 (-1.71-1.84, n=6) vs. control EC(50) (log molar [Ca(++)]) -2.6±0.04 (-2.59-2.76, n=6). Whereas, the verapamil (0.1µM) EC(50) value (log molar [Ca(++)]) is-1.7±0.1 (-1.6-1.8, n=6) vs. control EC(50) value (log molar [Ca(++)])- 2.4±0.09 (-2.3-2.47, n=6). The present research work confirms that the intestinal relaxation effect of Achillea wilhelmsii is supporting its traditional use as antispasmodic. The plant species can be a source for calcium antagonist(s), which can preferably be isolated from n-hexane fraction.

Antioxidant and relaxant activity of fractions of crude methanol extract and essential oil of Artemisia macrocephala Jacquem.

BACKGROUND: The current work is an attempt to know about additional chemical profile of Artemisia macrocephala. Antioxidant activity is performed as the plant is reported to contain flavonoids, which have antioxidant activity in general. Relaxant activity of fractions of crude methanol extract is performed to know in which fraction(s) the relaxant constituents concentrate as we have already reported that its crude methanol has relaxant activity. Antispasmodic activity of essential oil is also performed as the plant is rich with essential oil. METHODS: Phytochemical profile of the plant is performed. Free radical scavenging activity was performed using 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Relaxation activity tests of fractions and essential oil of Artemisia macrocephala were performed on sections of rabbits' jejunum. Calcium chloride curves were constructed to investigate the mode of action of plant extracts and its essential oil. RESULTS: We detected carbohydrates, flavonoids and saponins in A. macrocephala. At concentration 0.005 mg/ml, free radical scavenging activity of ethyl acetate fraction was 121.5 ± 2.02% of ascorbic acid.n- hexane fraction relaxed spontaneous activity with EC50 0.74 ± 0.04 mg/ml. Essential oil relaxed spontaneous activity with EC50 0.8 ± 0.034 mg/ml. Chloroform and ethylacetate fractions relaxed both spontaneous and KCl-induced contractions suggesting its possible mode through calcium channels. Constructing calcium chloride curves, the test fractions showed a right shift in the EC50. Essential oil at concentration 0.1 mg/ml produced right shift with EC50 (log [Ca++]M) -2.08 ± 0.08 vs. control with EC50 -2.47 ± 0.07. The curve resembled the curves of verapamil, which caused a right shift at 0.1 µM, with EC50 -1.7 ±0.07 vs. control EC50 (log [Ca++]M) -2.45 ± 0.06. CONCLUSIONS: Crude methanol and its fractions (ethyl acetate, chloroform and butanol) are rich sources of antioxidant constituents. The relaxing constituents following calcium channel blocking mechanisms are more concentrated in n-hexane, chloroform and ethyl acetate fractions that warrant isolation.

Cytotoxic and anthelmintic potential of crude saponins isolated from Achillea Wilhelmsii C. Koch and Teucrium Stocksianum boiss.

BACKGROUND: Saponins isolated from plant sources have a number of traditional and industrial applications. Saponins have pharmacological effects like anti-inflammatory, molluscicidal, antimicrobial, antispasmodic, antidiabetic, anticancer, anticonvulsant, anthelmintic, antitussive and cytotoxic activities. The current work describes the anthelmintic and cytotoxic activities of crude saponins of Achillea Wilhelmsii and Teucrium Stocksianum as these plants are rich with saponins. METHODS: Brine shrimp cytotoxic activity of crude saponins was determined by Meyer et al. (1982) at test concentrations of 1000 µg/ml, 100 µg/ml, 10 µg/ml, 7.5 µg/ml, 5.0 µg/ml, 2.5 µg/ml and 1.25 µg/ml. Percentage mortality of test concentrations was determined. Similarly, in vitro anthelmintic activity was determined against roundworms, tapeworms and earthworms. Albendazole and piperazine citrate at concentration 10 mg/ml were used as standard anthelmintic drugs. RESULTS: Crude saponins of Achillea wilhelmsii (CSA) and Teucrium stocksianum (CST) had, respectively, cytotoxic activity with LC50 values 2.3±0.16 and 5.23±0. 34 µg/ml. For in vitro anthelmintic activity, time for paralysis and death of parasites (parasiticidal activity) was noted. At concentration 40 mg/ml, crude saponins of Achillea wilhelmsii are 1.96 and 2.12 times more potent than albendazole against Pheretima posthuma and Raillietina spiralis, respectively. Similarly, at concentration 40 mg/ml, crude saponins of Teucrium stocksianum (CST) has 1.89, 1.96 and 1.37 times more parasiticidal activity than albendazole against Pheretima posthuma, Raillietina spiralis and Ascardia galli, respectively. CONCLUSION: Crude saponins of Achillea wilhelmsii and Teucrium stocksianum have cytotoxic and anthelmintic activity. The crude saponins may be excellent sources of cytotoxic and anthelmintic constituents that warrant its isolation and purification for new drug development.

Spasmogenic and spasmolytic activities of Onosma griffithii Vatke.

Methanolic extract of Onosma griffithii and its fractions were evaluated for possible effects on rabbits' jejunum preparations. Rabbits of either sex (weight 1.5-2.0 kg) were used in experiments. Studies were carried out on rabbits' jejunum preparations. Crude methanolic extract of Onosma griffithii (Meth.OG) was tried in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on rabbits' jejunum preparations. Meth.OG was also tried on KCl-induced contractions to explain its possible mode of actions in the presence and absence of atropine (0.03 µM). Fractions of Meth.OG were tried in similar manner. Calcium chloride curves were constructed for Meth.OG treated tissues that were compared with curves constructed for verapamil in same fashion. Preliminary phytochemical screening of the plant was also performed. Meth.OG increased the amplitude of spontaneous activity of rabbits' jejunum preparations at concentrations of 0.1, 0.3 and 1.0 mg/ml. However, spasmolytic effects were observed at higher concentrations 3.0, 5.0 and 10.0 mg/ml. Mean EC(50) values (mg/ml), respectively, in absence and presence of atropine were 7.5 ± 0.25 (6.9-8.4, n=6) and 3.0 ± 0.17 (2.3-3.5, n=6, p<0.05). Mean EC(50) values, respectively, for effects on spontaneous and KCl-induced contractions were 7.5 ± 0.25 (6.9-8.4, n=6) and 7.3 ± 0.35 (6.25-8.2, n=6, p<0.05). n-Hexane, chloroform and ethyl acetate fractions showed their respective EC(50) values (mg/ml) 9.7 ± 0.25 (8.6-10.2, n=6), 4.0 ± 0.2 (3.5-4.6, n=6) and 1.07 ± 0.093 (0.78-1.5, n=6). EC(50) values for calcium chloride curves in presence of 0.3 mg/ml Meth.OG were - 2.27 ± 0.038 (- 2.4 to - 2.10, n=6) vs. control - 2.78 ±0.04 (-2.9 to - 2.6, n=6, p<0.05) Log [Ca(++)]M. Comparing with curves of calcium chloride constructed in presence of 0.1 µM verapamil, the EC(50) (log [Ca(++)] M) values were - 1.82 ± 0.087 (- 2.0 to - 1.65, n=6) vs. control - 2.64 ± 0.089 (- 2.9 to - 2.4, n=6) demonstrated a right shift (p<0.05). Meth.OG tested positive for terpenes, saponins, sterols, flavonoids and carbohydrates. We concluded that the relaxant effect of Meth.OG is exerted through blocking of calcium channels. However, n-butanolic and aqueous fractions produced spasmogenic effects that require further work for isolation of pharmacologically active substances.

Acute toxicity, brine shrimp cytotoxicity and relaxant activity of fruits of callistemon citrinus curtis.

BACKGROUND: Callistemon citrinus Curtis belongs to family Myrtaceae that has a great medicinal importance. In our previous work, fruits of Callistemon citrinus were reported to have relaxant (antispasmodic) activity. The current work describes the screening of fractions of the crude methanol extract for tracing spasmolytic constituents so that it shall help us for isolation of bioactive compounds. Acute toxicity and brine shrimp cytotoxicity of crude methanol extract are also performed to standardize it. METHODS: The crude methanol extract was obtained by maceration with distilled water (500 ml) three times and fractionated successively with n-hexane, chloroform, ethyl acetate and n-butanol (300 ml of each solvent). Phytochemical analysis for crude methanol extract was performed. Acute toxicity studies were performed in mice. Brine shrimp cytotoxicity studies were performed to determine its cytotoxicity and standardize it. In other series of experiments, rabbits' jejunum preparations were used in screening for possible relaxant activities of various fractions. They were applied in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on spontaneous rabbits' jejunum preparations. In similar fashion, fractions were also tested on KCl (80 mM) -induced contractions. Calcium chloride curves were constructed in K-rich Tyrode's solution. The effects of various fractions were tested on calcium chloride curves at concentrations 1.0, 3.0, 5.0 and 10.0 mg/ml. Curves of verapamil used as reference drug at concentration 0.1 µM and 0.3 µM were also constructed. The curves were compared with their respective controls for possible right shift. RESULTS: Methanol extract tested strongly positive for saponins and tannins. However, it tested mild positive for presence of proteins, amino acids, carbohydrates and phenolic compounds. LD(50) value for crude methanol extract is 476.25 ± 10.3 (470-481, n = 4) mg/ml. Similarly, EC(50) value for brine shrimp cytotoxicity is 65.5 ± 7.28 (60.8- 69.4, n = 4) mg/ml. All the fractions relaxed the spontaneous and KCl-induced contractions. EC(50) values (mg/ml) for effects of ethyl acetate fraction on spontaneous and KCl induced contractions are 2.62 ± 0.78 (2.15-3.0, n = 4) and 3.72 ± 0.86 (3.38-4.28, n = 4) respectively. Respective EC(50) values (mg/ml) for n-butanol fraction are 3.59 ± 0.2(3.07-3.9, n = 4) for spontaneous, and 5.57 ± 0.2 (5.07-6.11, n = 4) for KCl- induced contractions. EC(50) value for control calcium chloride curve (without extract) is -2.73 ± 0.19 (-2.6 - -2.81, n = 4) while EC(50) for curves treated with 5.0 mg/ml of chloroform is -2.22 ± 0.02 (-2.16 - -2.3, n = 4). EC(50) value for ethyl acetate treated (1.0 mg/ml) tissues is -1.95 ± 0.10 (-1.88 - -2.0, n = 4) vs. control EC(50) = -2.71 ± 0.08 (-2.66 - -2.76, n = 4). All the fractions, except n-hexane, showed a right shift like that of verapamil (EC(50) = -1.72 ± 0.15 (-1.62 - -1.8, n = 4) vs. Control EC(50) = -2.41 ± 0.06 (-2.38 - - 2.44, n = 4), a standard drug that blocks voltage operated calcium channels. CONCLUSION: Relaxant constituents were more concentrated in ethylacetate fraction followed by chloroform, n -butanol and aqueous fractions that warrant for its isolation. The crude methanol extract is safe at concentration 250 mg/ml or below and results of brine shrimp cytotoxicity assay imply the plant specie may be a source of cytotoxic agents.