Periplocymarin is a potential natural compound for drug development: highly permeable with absence of P-glycoprotein efflux and cytochrome P450 inhibitions.

Periplocymarin, a cardiac glycoside isolated from Periploca sepium (P. sepium) and Periploca graeca (P. graeca), is a potential anti-cancer compound. The aim of the study was to investigate the potential for periplocymarin to interact with P-glycoprotein (P-gp) and to inhibit cytochrome P450s known to be expressed in the human small intestine. The in vitro and in situ permeability of periplocymarin were studied using Madin-Darby canine kidney (MDCK-II-WT) cells transfected with or without the human multidrug resistance (MDR1) gene and the single-pass perfused rat intestinal model. The cell system exhibited high functional activity and a net efflux ratio (NER) of 4.32 after transport of Rhodamine 123 (R123) (the P-gp substrate). Periplocymarin is highly permeable (Papp > 10 × 10(-6) cm/s; Peff(rat) > 5.09 × 10(-5) cm/s) and independent of P-gp influences. The NER at 100 µm periplocymarin (0.8) was unchanged in the presence of cyclosporine A (a non-specific P-gp inhibitor) (0.82). In the single-pass intestinal model, the Peff (rat) of 5 µg/ml periplocymarin (5.490 × 10(-5) cm/s) did not change in the presence of cyclosporine A (5.394 × 10(-5) cm/s). In the R123-inhibition assay, periplocymarin did not competitively inhibit P-gp. The inhibitory potential of periplocymarin on cytochrome P450 (CYP450s) was also studied. Periplocymarin (5, 50 µm) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4. Thus, periplocymarin is unlikely to encounter drug-drug interactions with P-gp and CYP450s. Periplocymarin could be taken forward for further studies in drug development to test bioavailability, Phase II enzyme interactions and additional transporter interactions.

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Background: Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. Materials and Methods: After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Results: Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. Conclusion: LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Wound Healing Properties and Antimicrobial Effects of Parkia clappertoniana Keay Fruit Husk Extract in a Rat Excisional Wound Model.

Background: Parkia clappertoniana Keay (Family: Fabaceae) (P. clappertoniana) fruit husk is commonly used in northern Ghana for wound treatment. However, this folk claim remains to be confirmed scientifically. Objective: This study investigated wound healing and antimicrobial effects of P. clappertoniana fruit husk extract (PCFHE) by using excision wound model in rats. Materials and Methods: After preparation and phytochemical analysis of PCFHE, it was reconstituted in purified water and emulsifying ointment yielding a wound healing formula (0.3, 1, and 3%). Excision wounds were established in healthy male Sprague-Dawley rats (aged 8-10 weeks; weighing 150-200 g). Rats were randomly assigned into six groups (model, 1% silver sulfadiazine [SSD], vehicle, and PCFHE [0.3, 1, and 3%, respectively]) and topically treated daily until complete wound healing. The endpoints (period of epithelialization, wound contraction, collagen content, erythema index, oedema index, inflammatory cell infiltration, and antimicrobial activity) were assessed for all groups. Minimum fungicidal concentration (MFC), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill were assessed. Results: Quercetin and catechin were detected in PCFHE. Compared to model and vehicle groups, PCFHE-treatment groups improved wound healing and antimicrobial (MBC, MFC, and MIC) endpoints. PCFHE demonstrated bacteriostatic and fungicidal effects against identified wound contaminants (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Candida albicans). Conclusion: P. clappertoniana fruit husk possesses wound healing and antimicrobial effects in excisional wounds in rats that confirms its folk use, and the reported pharmacological properties of PCFHE are attributable to its quercetin and catechin phyto-constituents.

Flavonoid-Rich Extract of Dissotis rotundifolia Whole Plant Protects against Ethanol-Induced Gastric Mucosal Damage.

Dissotis rotundifolia is a plant in the family Melastomataceae. The methanolic extract of the whole plant is reported to be rich in C-glycosylflavones such as vitexin and orientin. Though there are several reports on the ethnomedicinal use of this plant extract in stomach ulcers, experimental-based data is unavailable. The drive for carrying out this research was to obtain data on the possible ameliorative effect of the whole plant extract of Dissotis rotundifolia (DRE) in gastric ulcerations induced by ethanol in Sprague Dawley (SD) rats. SD rats were pretreated with 100, 300, and 500 mg/kg of DRE for 14 days after which an ulcerogen-ethanol was administered. Gross examinations of the stomach lining and histological analysis of gastric lesions were carried out coupled with an assessment of the antioxidant activity of gastric mucosa using MDA, GSH, CAT, and SOD as indicators. The data suggested a significant attenuation in gastric mucosal damage in DRE-pretreated ethanol-induced gastric ulcer reflected in the antioxidant status. There was also a reduction or absence of hemorrhage, edema, and leucocytes infiltration in DRE-treated groups compared to the negative control group. DRE conserved glutathione (GSH) levels, reduced malondialdehyde (MDA) levels, and enhanced catalase (CAT) and superoxide dismutase (SOD) enzyme levels. The present study shows that DRE possess protective effects against ethanol-induced ulcer damage in the stomach of rats, which could be attributed to its antioxidant activity.

A multivariate statistical analysis of the effects of styrene maleic acid encapsulated RL71 in a xenograft model of triple negative breast cancer.

We have previously shown that the curcumin derivative 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71), when encapsulated in styrene maleic acid micelles (SMA-RL71), significantly suppressed the growth of MDA-MB-231 xenografts by 67%. Univariate statistical analysis showed that pEGFR/EGFR, pAkt/Akt, pmTOR/mTOR and p4EBP1/4EPBP1 were all significantly decreased in tumors from treated mice compared to SMA controls. In this study, multivariate statistical analyses (MVAs) were performed to identify the molecular networks that worked together to drive tumor suppression, with the aim to determine if this analysis could also be used to predict treatment outcome. Linear discriminant analysis correctly predicted, to 100% certainty, mice that received SMA-RL71 treatment. Additionally, results from multiple linear regression showed that the expression of Ki67, PKC-α, PP2AA-α, PP2AA-ß and CaD1 networked together to drive tumor growth suppression. Overall, the MVAs provided evidence for a molecular network of signaling proteins that drives tumor suppression in response to SMA-RL71 treatment, which should be explored further in animal studies of cancer.

Does the mouse tail vein injection method provide a good model of lung cancer?

Lung cancer drug development requires screening in animal models. We aimed to develop orthotopic models of human non-small lung cancer using A549 and H3122 cells delivered by tail vein injection. This procedure has been used previously for a mouse lung cancer (Lewis lung carcinoma) and as a model of human breast cancer metastasis to lung. We report that the procedure led to poor animal condition 7-8 weeks after injection, and produced lesions in the lungs visible at necropsy but we were unable identify individual cancer cells using immunohistochemistry. We conclude that if this method is to produce a model that can be used in drug experiments, improvements are required for cancer cell detection post mortem, such as by using of a fluorescently tagged human lung cancer cell line.

Styrene maleic acid-encapsulated RL71 micelles suppress tumor growth in a murine xenograft model of triple negative breast cancer.

Patients with triple negative breast cancer have a poor prognosis due in part to the lack of targeted therapies. In the search for novel drugs, our laboratory has developed a second-generation curcumin derivative, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71), that exhibits potent in vitro cytotoxicity. To improve the clinical potential of this drug, we have encapsulated it in styrene maleic acid (SMA) micelles. SMA-RL71 showed improved biodistribution, and drug accumulation in the tumor increased 16-fold compared to control. SMA-RL71 (10 mg/kg, intravenously, two times a week for 2 weeks) also significantly suppressed tumor growth compared to control in a xenograft model of triple negative breast cancer. Free RL71 was unable to alter tumor growth. Tumors from SMA-RL71-treated mice showed a decrease in angiogenesis and an increase in apoptosis. The drug treatment also modulated various cell signaling proteins including the epidermal growth factor receptor, with the mechanisms for tumor suppression consistent with previous work with RL71 in vitro. The nanoformulation was also nontoxic as shown by normal levels of plasma markers for liver and kidney injury following weekly administration of SMA-RL71 (10 mg/kg) for 90 days. Thus, we report clinical potential following encapsulation of a novel curcumin derivative, RL71, in SMA micelles.

Assessment of an aqueous seed extract of Parkia clappertoniana on reproductive performance and toxicity in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Parkia clappertoniana Keay (Family: Fabaceae) are extensively used in food in the form of a local condiment called 'Dawadawa' in Ghana and consumed by all class of people including sensitive groups such as pregnant women and children. Also, crudely pounded preparations of P. clappertoniana seeds are used as labor inducing agent in farm animals by local farmers across northern Ghana where nomadism is the livelihood of most indigenes. Ecologically, P. clappertoniana is extensively distributed across the savannah ecological zone of many African countries where just like Ghana it enjoys ethnobotanical usage. Although, many studies have investigated some aspects of the pharmacological activity of P. clappertoniana, none of these studies focused on the reproductive system, particularly its effects on reproductive performance and toxicity. To contribute, this study assessed the effect of aqueous seed extract of P. clappertoniana (PCE) on reproductive performance and toxicity in Sprague-Dawley rats and ICR mice. METHODS: After preparation of PCE, it was then tested on rodents at different gestational and developmental windows (1-7, 8-14, and 15-term gestational days) to assess the following: mating behavior, implantation rate, maternal and developmental toxicities. Generally, animals were randomly grouped into five and treated as follows: normal saline group (5ml/kg po), cytotec (misoprostol) group (200mg/kg po), folic acid group (5mg/kg po), and PCE groups (100, 200, and 500mg/kg po), however, these groupings were varied to suit the specific requirements of some parameters. For acute toxicity, animals were orally administered PCE (3 and 5g/kg for mice and rats respectively). RESULTS: PCE-treated rats showed improved mating behavior compared to control rats. PCE improved implantation rate compared to misoprostol-treated rats. On the average, PCE-treated rats delivered termed live pubs at 21 days compared to that of folic acid-treated rats at 23 days. Also, PCE-treated rats showed no observable maternal and developmental toxicities compared to folic acid and control rats. PCE (3-5g/kg po) was orally tolerated in rodents. CONCLUSION: Oral administration of Parkia clappertoniana seed extract improves reproductive performance in rodents with no observable maternal and developmental toxicity.

P-glycoprotein- and organic anion-transporting polypeptide-mediated transport of periplocin may lead to drug-herb/drug-drug interactions.

Periplocin, an active and toxic component of the traditional Chinese herbal medicine Periploca sepium Bge, is a cardiac glycoside compound that has been implicated in various clinical accidents. This study investigated the role of transporters in the intestinal absorption and biliary excretion of periplocin, as well as the possible metabolic mechanism of periplocin in liver S9. In a bidirectional transport assay using Madin-Darby canine kidney (MDCK) and MDCK multidrug-resistance protein (MRP)-1 cell monolayers, both in situ intestinal and liver-perfusion models were used to evaluate the role of efflux and uptake transporters on the absorption and biliary excretion of periplocin. In addition, in vitro metabolism of periplocin was investigated by incubating with human/rat liver S9 homogenate fractions to evaluate its metabolic mechanisms in liver metabolic enzymes. The results showed that P-glycoprotein (P-gp) was involved in the intestinal absorption of periplocin, whereas MRP2 and breast cancer-resistance protein were not. The efflux function of P-gp may be partly responsible for the low permeability and bioavailability of periplocin. Moreover, both inhibitors of P-gp and organic anion-transporting polypeptides (OATPs) increased periplocin biliary excretion. No obvious indications of metabolism were observed in the in vitro incubation system, which suggests that periplocin did not interact with the hepatic drug metabolic enzymes. The results of this study showed that the efflux and uptake transporters P-gp and OATPs were involved in the absorption and biliary excretion of periplocin, which may partially account for its low permeability and bioavailability. As a toxic compound, potential drug-herb/herb-herb interactions based on OATPs and P-gp should be taken into account when using P. sepium Bge in the clinic.

Absence of organ specific toxicity in rats treated with Tonica, an aqueous herbal haematinic preparation.

The sub-chronic toxicity of Tonica, an aqueous herbal haematinic prepared from the stem barks of Khaya senegalensis, Mitragyna stipulosa and Kigelia africana, was investigated in male Sprague-Dawley rats at 28, 280 and 560 mg kg(-1) day(-1), representing the normal human dose, 10x and 20x that dose, respectively for 6 weeks. The growth rate of animals over the period of treatment and certain serum biochemical and haematological indices as well as urinalysis and weight of selected organs at termination, were determined. Results show that the extract did not affect the weight gain of the animals with time or the mean wet weights of selected organs. Although there were slight but insignificant (p>0.05) elevations in WBC (16-27%) and PLT (8-11%) counts in Tonica-treated animals compared to controls at 10x and 20x the normal dose, most serum biochemical, haematological and urinalysis data indicated no significant differences (p>0.05) between tests and control rats. There were also no changes in the morphology of liver, kidney, lung and heart tissues as a result of Tonica treatment. These findings suggest that Tonica is safe at the dosage regimens administered to the animals in this study, and there appears to be no overt organ specific toxicity associated with it.