Tannic acid-rich porcupine bezoars induce apoptosis and cell cycle arrest in human colon cancer cells Peng-Nian Yew, Yau-Yan Lim, Wai-Leng Lee Pharmacognosy Magazine 2019 15(65):523-531 Background: Porcupine bezoar, a phytobezoar used as traditional medicine, was recently claimed to effectively treat cancer. However, there is a lack of scientific evidence to prove the claim. Objectives: This study aimed to scientifically examine porcupine bezoars as a potential anticancer agent and to investigate their principal bioactive constituents. Materials and Methods: The porcupine bezoars were extracted using methanol and further Sephadex LH-20 column chromatography was used to enrich the tannins content. The inhibitory effects of the crude extracts on a panel of cancer cell lines were first determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then, the anticancer activities of the enriched fractions in selected cell lines were analyzed, while the chemical composition of the active fraction was identified using liquid chromatography––electrospray ionization-tandem mass spectrometry. Results: Crude extracts of black date and powdery date effectively inhibited colon cancer cell lines HT-29 and HT-116, but not the normal colon cells, and their tannin-enriched fractions demonstrated higher inhibitory effects when compared to the extracts. Further, the fractions arrested cell cycle at S phase and induced apoptosis in treated colon cancer cells with a similar effect to that of commercial tannic acid. Lipoxygenase activity which plays a role in tumorigenesis of colon cancer was also inhibited by these fractions. Chemical analysis found that both the enriched fractions and commercial tannic acid share similar chemical constituents, including gallic acid and its derivatives (polygalloyl glucose). Conclusion: Together, the results suggest that tannic acid in porcupine bezoars may inhibit colon cancer cells by interfering cell proliferation and triggering program cell death in the cells.

In vitro UDP-Glucuronosyltransferase and Cytochrome P450 Enzymes Activities of Clinacanthus nutans Leaf Juice and Aqueous Extract Gabriel Akyirem Akowuah, Jin Han Chin, Siew Wei Yeong, Suk Yen Quah, Mariam Ahmad Pharmacognosy Magazine 2019 15(65):532-537 Aim: The objective of the present study was to evaluate the in vitro effect of aqueous extract of Clinacanthus nutans leaves and the juice on the activity of UDP-glucuronosyltransferase (UGT), cytochrome P (CYP) 3A4, and CYP2E1 in human liver microsomes (HLMs). Materials and Methods: The herb-drug interactions of the leaf extracts and juice were determined by a specific enzyme activity of CYP isoforms with specific probe substrate using spectrophotometry. CYP3A4 activity was measured for aminopyrine-specific metabolite (formaldehyde) at 415 nm. CYP2E1 activity was determined using p-nitrophenol-specific metabolite (p-nitrocatechol) at 535 nm. UGT activity was quantified through the consumption of p-nitrophenol by UGT at 405 nm. Results: Results obtained showed that the juice and aqueous extract of C. nutans leaves exhibited significant inhibition (P < 0.05) in CYP3A4 and CYP2E1 activity in HLMs. The aqueous extract of C. nutans showed statistically significant (P < 0.05) activation on UGT activity at the concentration of 1000 ng/mL as compared to the negative control. Conclusion: There is a possibility that herb-drug interaction could occur with C. nutans through inhibitory effects on CYP3A4 and CYP2E1. The leaf preparation also activated UGT catalyzed metabolism which may result in a reduction of the potency of the drug metabolized by UGT pathway.

Effect of Piper nigrum ethanolic extract on human breast cancer cell growth and cell migration Benjaporn Buranrat, Supavadee Boontha Pharmacognosy Magazine 2019 15(65):538-546 Background: Piper nigrum (PN) is widely used as a traditional medicine, which has anti-cancer activity among others. Objective: Our study purposes to illuminate the inhibition of PN effects on breast cancer cells growth and migration along with its mechanisms. Materials and Methods: The piperine in the young fruit of PN extract was determined by the high-performance liquid chromatography method. Growth inhibition with a mechanism of PN extract was studied in the MCF-7 cells using sulforhodamine B assay, cell cycle analysis, colony formation, caspase-3 activity, and reactive oxygen species formation. Furthermore, the anti-migratory effects of PN were investigated using wound healing, matrigel migration, and gelatin zymography assay. Finally, PN mechanism was determined by reverse transcription polymerase chain reaction and Western blotting for the gene and protein levels. Results: Piperine level was showed at a high concentration in the PN extract. Further, the PN extract suppressed Rac1 mRNA expression in the mevalonate (MVA) pathway as well as repressed Rac1 and RhoA protein expression. Interestingly, PN stimulated growth inhibition in dose- and time-dependent as well as being accompanied by increasing the G1 phase arrest and inhibiting cyclin D1 and NF-κB as well as inducing caspase-3 expression. The PN extract inhibited MCF-7 cell migration by reducing matrix metalloproteinases (MMP) 9 protein expression as well as MMP 2, MMP 9, VEGFA, and ICAMP1 gene expression. Conclusion: PN could be herbal medicine for anti-cancer and anti-migratory activities with a correlation to the MVA pathway; therefore, PN maybe deserving further to study as a new candidate for treating breast cancer.

Optimization of ultrasonic-assisted extraction of flavonoids and anti-oxidant capacity from the whole plant of Andrographis echioides (L.) nees by response surface methodology and chemical composition analysis Jayalakshmi Ramasamy, Ruckmani Kandasamy, Selvamani Palanisamy, Subramanian Nadesan Pharmacognosy Magazine 2019 15(65):547-556 Background: Andrographis echioides (L.) Nees is an annual herb mainly distributed in India and Sri Lanka. In traditional medicine system, the plant is used for treating various ailments such as fevers, skin diseases, stomach ache, toothache, snake bite, and eczema. The whole plant of A. echioides was reported as the rich source of flavonoids. Ultrasound-assisted extraction (UAE) is an effective extraction method used for secondary metabolite extraction from various plant materials over conventional methods. Today, the response surface methodology (RSM) is a successful statistical tool used to optimize the various extraction conditions of the secondary metabolite from various sources. Objective: The objective of this study is to optimize the UAE conditions such as ethanol concentration (50%–100%), solvent-to-solid ratio (10–50 mL/g), and sonication time (20–60 min) for the extraction of flavonoids and anti-oxidant capacity (AOC) from A. echioides (L.) Nees whole plant (AEWP) using the RSM strategy with Box–Behnken design (BBD). Materials and Methods: UAE conditions, i.e. ethanol concentration, solvent-to-solid ratio, and sonication time, were optimized with the corresponding responses of flavonoid yield and %DPPHAOCand %ABTSAOCby RSM. The effect of ultrasound on plant material was analyzed using Scanning electron microscope (SEM). The efficiency of the optimized extract was analyzed using Fourier-transformed infrared spectroscopy (FTIR) and liquid chromatography-mass spectra (LC-MS). Results: The BBD provided adequate mathematical models that accurately describe the behavior of the technique and help to predict the flavonoid yield, %DPPHAOCand %ABTSAOCfrom AEWP. The optimized UAE conditions were 77% of ethanol concentration, 35 mL/g of solvent-to-solid ratio, and 41 min of sonication time. Under these extraction conditions, UAE would obtain a maximum of 10.91 ± 0.04 mg CE/g for flavonoid yield, 87.36 ± 0.06% for %DPPHAOC, and 85.14 ± 0.03% for %ABTSAOC.The obtained experimental results of all the responses are in good agreement with the predicted values. SEM analysis explores the effect of UAE compared with the conventional extraction. The FTIR and LC-MS analysis revealed that the optimized extract of AEWP is rich in flavonoids; apart from the known flavonoids, five new flavonoids were identified from this optimization study. Conclusion: The study confirmed that UAE was the effective extraction method for the extraction of flavonoids from AEWP with ethanol as a solvent of choice with a low solvent usage in a reasonable time.

Study on pharmacokinetics and tissues distribution of neomangiferin, mangiferin, timosaponin BII, Timosaponin BIII, and timosaponin AIII after oral administration of Anemarrhenae rhizoma extract in rats De Ji, Jin-Chun Qiu, Xiao-Nan Su, Yu-Wen Qin, Min Hao, Lin Li, Tu-Lin Lu, Xiao-Kun Li, Cheng-Xi Jiang Pharmacognosy Magazine 2019 15(65):557-567 Background: Anemarrhenae rhizoma (AR) is widely used for the treatment of febrile diseases, cough, and diabetes in traditional Chinese medicines. AR mainly contains flavonoids and steroidal saponins, such as neomangiferin, mangiferin, timosaponin BII, timosaponin BIII, and timosaponin AIII, which showed various biological activities. Objective: The main objective of the study is to establish an ultra-high-performance liquid chromatography-tandem mass spectrometry (MS/MS) method to determine the concentrations of five bioactive constituents in rats' plasma and various tissues. Materials and Methods: The analytes were separated on a C18reversed-phase column. A triple-quadrupole MS/MS equipped with an electrospray ionization source was used as a detector. The main pharmacokinetic parameters were estimated with Drug and Statistics 2.0 Software Package. Results: Neomangiferin and mangiferin exhibit poor oral absorption and slow clearance from the body. Timosaponin BII and timosaponin BIII could be quickly absorbed into the blood circulation and showed double plasma concentration peaks. Timosaponin AIII exhibited a single peak in the plasma concentration-time plot and pharmacokinetic parameters of timosaponin AIII indicated slower absorption, longer body residence time, and slower elimination than timosaponin BII and timosaponin BIII. The five analytes were widely distributed to most of the tissues. Neomangiferin and mangiferin exhibited the maximum concentration in the lung at 6 h after oral administration, the highest levels of timosaponin BII and timosaponin BIII were also observed in the lung at 1 h after oral administration, and the maximum concentration of timosaponin AIII was observed in the liver. Conclusion: The findings of the present study might be helpful to better understand the pharmacokinetics and distribution of AR bioactive constituents in vivo, which would facilitate the clinical application of AR.

In vitro antitoxoplasmal activity of some medicinal plants Ibrahim S Al Nasr, Waleed S Koko, Tariq A Khan, Gamal E Elghazali Pharmacognosy Magazine 2019 15(65):568-572 Background: Toxoplasmosis is a serious zoonotic protozoal disease that is distributed worldwide and can infect almost all warm-blooded animals, including humans. In most cases, it is asymptomatic, but in immunocompromised individuals, it is associated with severe neurological and gastrointestinal disorders. A previous serological prevalence investigation in Saudi Arabia indicated that the disease prevalence ranged between 25% and 51% in various areas. Recommended commercial drugs cannot achieve 100% clearance due to side effects. Hence, the development of new safe and affordable drugs is an important goal. Aim: In the present study, extracts from the leaves and fruit of Azadirachta indica A. Juss. collected from different areas, along with two other medicinal plants (Argemone mexicana L. and Xanthium brasilicum Vell.) with established antiprotozoal activity, will be evaluated for antitoxoplasmal activity using an in vitro technique. Materials and Methods: All plants were extracted with 100% methanol and examined for activity against the Toxoplasma gondii RH strain via an intracellularly invaded Vero cell line with calculated inhibition percentages. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay against the Vero cell line was used for cytotoxic evaluation, followed by selectivity index (SI) calculation. Results: X. brasilicum exhibited the best antitoxoplasmal activity with an IC50of 7.19 μg/ml, followed by the A. indica fruits collected from Qassim, Kingdom of Saudi Arabia, and leaves collected from Central Sudan with an IC50of 17.26 and 18.43 μg/ml, respectively. The best SI was obtained from the leaves of A. indica (6.28) collected from Sudan. Conclusion: Although X. brasilicum proved to have very potent antitoxoplasmal activity, the cytotoxicity was also very high, so the isolation of active compounds is highly recommended.

Juglone Induces Michigan Cancer Foundation-7 Human Breast Cancer Cells Apoptosis through Bcl-2-Associated X protein/B-cell lymphoma/leukemia-2 Signal Way Qing Luo, Kang Hu, Fang Chen, Feng-Jiao Gan, Yuan-Xiu Leng, Xu-Mei Chen, Su-Hong Sun Pharmacognosy Magazine 2019 15(65):573-578 Background: Juglone is a natural pigment, which has a cytotoxic effect against tumor cells. However, its cytotoxicity to human breast cancer cells (Michigan Cancer Foundation-7 [MCF7]) has not been demonstrated. Objective: The objective was to observe the effect of Juglone on the protein expression of caspase-3, 9 in the apoptosis of Human Breast Cancer Cells (MCF7) and apoptosis-related protein, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2) and investigate in the inhibitory effect of MCF7. Materials and Methods: Methyl thiazolyl tetrazolium experiment was performed for the detection of the cell growth inhibition effect of MCF7. The apoptosis rate was detected by flow cytometry. Western Blot was used to detect the protein expression of Bax, Bcl-2, and caspase. The messenger RNA (mRNA) was tested using quantitative real-time polymerase chain reaction. Results: Juglone inhibited the growth of MCF7 cells in a concentration- and time-dependent manner and promotes the apoptosis of MCF7 cells in a concentration-dependent manner. Compared with control group, the Juglone group raised the expression of Bax protein (*P < 0.05), and the protein expression of Bcl-2 was decreased (*P < 0.05). The expression of caspase-3 protein was not changed, and the caspase-9 was significantly elevated in the high concentration of Juglone. Cleaved caspase-3, -9 protein was significantly raised (*P < 0.05). The mRNA levels of Bax, caspase-3, and caspase-9 in MCF7 cells of Juglone group were significantly increased compared with control, while the expression of Bcl-2 was suppressed obviously. Conclusion: Juglone inhibited the growth of MCF7 cells and promotes the apoptosis of MCF7 cells. Its mechanism in promoting MCF7 cell apoptosis may be related to the decrease of the expression of the mitochondrial pathway-associated apoptosis factor Bcl-2, increase of the protein expression of Bax, and mitochondrial pathway downstream caspase-3, 9.

Effects of Atractylodes macrocephala rhizoma on isoproterenol-induced myocardial hypertrophy in mice Ke-Zhao Wei, Xiao-Hua Cui, Jia-Hua Feng, Ping-An Yao, Jian-Ping Gao Pharmacognosy Magazine 2019 15(65):579-585 Background: The studies about the protective effect on the heart of single Atractylodes macrocephala rhizoma (AMR) herb and mechanisms have not been reported. Objective: The purpose of this study was to assess the effects of AMR on attenuating myocardial hypertrophy induced by isoproterenol (ISO) in mice. Materials and Methods: Mice were randomly divided into normal control group, ISO control group, ISO plus metoprolol (60 mg/kg) group, ISO plus AMR (2, 4, and 8 g/kg) groups, and AMR (4 g/kg) control group. The mice with myocardial hypertrophy were established by subcutaneous (s.c.) injection with ISO (2 mg/kg/d) and administered intragastrically with the corresponding drugs in the volume of 0.2 mL/10 g/d for 7 days. In the normal and AMR control groups, mice were injected (s.c.) with physiological saline (the solvent for ISO) and administered intragastrically with drinking water for 7 days. Results: Compared with the ISO-induced group, AMR significantly decreased heart weight index, left ventricular weight index, and average transverse area of cardiomyocytes, significantly increased the activity of total superoxide dismutase in serum and the level of the angiotensin II receptor type (AT) gene expression in myocardium and significantly decreased the contents of malondialdehyde, cyclic adenosine monophosphate , and aldosterone in serum and angiotensin II (Ang II) in myocardium. Conclusion: The ability of AMR to mitigate myocardial hypertrophy is partly associated with its anti-oxidative effect, restraining excessive secretion or activation of neuroendocrine factors, and the stronger upward effect on AT2gene expression than AT1.

Anti-eczematic and molecular modeling of anthraquinones isolated from the seeds of Asphodelus microcarpus salzm. viv. growing in Egypt Abd El-Salam I.Mohammed, Arafa Musa, Marwa S Abu-Bakr, Hatem S Abbass Pharmacognosy Magazine 2019 15(65):586-591 Background: Eczema or atopic dermatitis is a widely spread skin disorder; the topical application of corticosteroids is the first choice for treatment. Natural products have a great contribution in the treatment of this disease; Asphodelus microcarpus seeds are rich in anthraquinones and known to possess both anti-inflammatory and antidermatitis effects. Objective: The objective of the study is to investigate the anti-eczematic activity, acute toxicity, and molecular modeling of A. microcarpus seeds. Materials and Methods: Nuclear magnetic resonance, ultraviolet, and mass spectroscopy were applied for characterization of isolated metabolites; induction of eczema was conducted by 2% and 0.2% w/v dinitrochlorobenzene in acetone; eczema was treated with topical application of the different seed extracts in the form of ointments (1% w/w); Swiss albino mice (25–30 g) were used for the determination of LD50and anti-eczematic effect. Docking studies were performed by Molecular Operating Environment software. Results: A. microcarpus seed extract exhibited promising ant-eczematic activity, six anthraquinones were isolated from chloroform portion and characterized as 10,7'-bichrysophanol (1), asphodelin (2), chrysophanol-8-O-methyl ether (3), chrysophanol (4), physcion (5), and emodin (6). Compounds 1, 3, and 5 exerted significant anti-eczematic effect. Conclusion: Six known anthraquinone derivatives were isolated and characterized for the first time from the seeds of A. microcarpus. Chloroform fraction (1% w/w) showed significant anti-eczematic effect compared to standard mometasone furoate (0.1 w/w). The docking study proved the anti-eczematic activity of anthraquinone content by their affinity to the target human histamine H1receptor.

Antioxidant flavonoids from Alhagi maurorum with hepatoprotective effect Muneera S M. Al-Saleem, Lamya H Al-Wahaib, Wael M Abdel-Mageed, Yaser G Gouda, Hanaa M Sayed Pharmacognosy Magazine 2019 15(65):592-599 Background: Alhagi maurorum, commonly used in folk medicine, has been reported to have several biological activities. Objective: We have studied the antioxidant chemical components from A. maurorum to determine their in vitro antiproliferative and hepatoprotective activities. Materials and Methods: The alcoholic extract of A. maurorum root was subjected to a successive solvent fractionation and various chromatographic techniques guided by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay to isolate their antioxidant active compounds. The structures of the isolated compounds were identified through the extensive use of nuclear magnetic resonance and mass spectroscopy coupled with correlation to known compounds. The antioxidant and cytotoxic activities of the isolated compounds were quantified using DPPH and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays, respectively. The hepatoprotective activity of each extract and the total flavonoid fraction were assessed quantitatively on carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Results: Fourteen flavonoids, including four aglycones (1–4) and ten glycosides (5–14), were isolated. The flavonoid glycosides (6–14) are being reported for the first time to our knowledge. The free aglycones, those of the flavonol type, exhibited strong antioxidant and antiproliferative activities. The flavonoid glycosides exhibited weak cytotoxic activity against the hepatocellular carcinoma cell line. The total flavonoid fraction showed the strongest hepatoprotective activity against CCl4-induced hepatotoxicity. Conclusion: A total of 14 flavonoids were identified from A. maurorum; nine of them were isolated for the first time. Flavonoids were the main chemical group identified from the A. maurorum root extracts, and they are responsible for the hepatoprotective activity. The findings set up a scientific explanation for the folkloric administration of A. maurorum in the treatment of hepatic disorders.