Optimisation of asarone removal from Piper sarmentosum Roxburgh leaves using supercritical carbon dioxide extraction: the Box-Behnken design

Abstract Piper sarmentosum is a herbaceous shrub with numerous pharmacological benefits. However, the presence of two toxic phenylpropanoids (α- and β-asarone) limits the medicinal usage of the plant. In this study, the extraction of three asarone isomers, namely α-, β-, and -asarone was optimised using supercritical carbon dioxide extraction (SC-CO2) combined with Box-Behnken experimental design. Comparison of asarone contents in different conventional solvent extracts of P. sarmentosum leaves prior to and after SC-CO2 extraction was performed. The SC-CO2 method successfully maximised the extraction of α-, β-, and ɣ-asarone at P = 81.16 bar, T = 50.11°C, and t = 80.90 min, yielding 13.91% α-asarone, 3.43% β-asarone, and 14.95% ɣ-asarone. The SC-CO2 residue of the leaves re-extracted with conventional solvents showed a significant decrease of asarone ranging from 45% to 100% (p<0.001) compared to their counterparts without SC-CO2 treatment. α-, β-, and ɣ-asarone were completely removed in the ethanol extract of the residue. These findings suggested that the optimised SC-CO2 extraction parameters may serve as a quick treatment step for the selective removal of asarone from P. sarmentosum to develop safer extracts for the food and nutraceutical industries applications.

Anti-acanthamoebic activity of methanolic extract of Piper sarmentosum leaves

Aims@#Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii.@*Methodology and results@#Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba.@*Conclusion, significance and impact of study@#This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.

Factors Regulating Nitric Oxide Production in Spontaneously Hypertensive Rats Treated with Piper Sarmentosum Aqueous Extract

@#Introduction: Hypertension is a major risk factor for cardiovascular diseases which is one of the leading causes of death worldwide. Piper sarmentosum (PS) has been widely used in traditional medicine with proven antihypertensive and antioxidant effects. This study aims to evaluate the antihypertensive potential of PS aqueous extract (PSAE) and to investigate the factors modulating nitric oxide (NO) production through its anti-oxidant activities. Methods: PS leaves were extracted with distilled water, freeze-dried and examined to quantify their antioxidant activities through 2,2-diphenyl-1-picrylhydrazyl and ferric reducing ability plasma test. The antihypertensive effect of PSAE in spontaneous hypertensive rats (SHR) was evaluated using four different groups (n=6); C (negative control), K (PSAE 500mg/kg), P (3 mg/kg perindopril) and M (PSAE 500 mg/kg + 1.5 mg/kg perindopril). PSAE and other treatments were given via oral gavage for 28 days. The blood pressure (BP) was determined using the non-invasive BP monitoring tail cuff technique and recorded weekly. SHR’s blood was collected to determine the serum NO level using Griess assay. Asymmetric dimethylarginine (ADMA) and arginine levels were determined using high performance liquid chromatography. Results: The extract showed good in-vitro antioxidant activities and a significant reduction in both systolic and diastolic BP compared to control group. They were also a decrease in plasma ADMA and an increase in serum NO level. Meanwhile, arginine level does not change significantly. Conclusion: High in-vitro antioxidant activities in PSAE enhances the clearance of ADMA that leads to an increase in serum NO production hence ameliorating the blood pressure of SHR.

Qualitative Phytochemical Analysis and Antimicrobial Activity of Piper sarmentosum Leaves Extract against Selected Pathogens / Jurnal Sains Kesihatan Malaysia

@#The controversial usage of antiseptic in treating wound infections had become a huge issue over the years due to its minimal effectiveness and high toxicity level that are harmful to humans. Hence, numerous studies had been carried out to determine other possible approaches including herbal remedies. The alarming situation had led us to study on Piper sarmentosum and its antimicrobial activity against selected pathogens. In present study, methanol extract of Piper sarmentosum leaves were prepared to investigate the presence of phytochemical compounds. The in vitro antimicrobial activity of crude methanolic extract was evaluated using disc diffusion and microdilution broth methods. The study demonstrated the antimicrobial activity of leaves extract against Staphylococcus aureus (7 mm) and Escherichia coli (6.5 mm). However, no zone of inhibition was observed against Pseudomonas aeruginosa. Meanwhile, the MIC values for Staphylococcus aureus was 6.25 mg/mL whilst Escherichia coli was 12.5 mg/mL. In addition, the phytochemical screening results revealed that the extract contained glycosides, flavonoids, terpenoids, alkaloids, and phenolics. In conclusion, methanolic extract of Piper sarmentosum leaves has the potential as a new, effective alternative towards current drugs that are available for skin-associated infection. The findings from this study are crucial in providing latest information of the plant’s additional values that can be incorporated as a baseline for current and future studies as well as in investigating other possible plants that are beneficial for health purposes, particularly for combating skin-associated infection.

A medium supplemented with vegetable extract used for cellulose production of Rhodococcus SP. MI 2 / Meio suplementado com extrato vegetal utilizado na produção de celulose de Rhodococcus SP. MI 2

In this study, the most suitable vegetable extract was screened to use as non-conventional nutrient sources for cellulose production of Rhodococcus sp. MI 2. SH medium or a synthetic medium was used as a conventional or control medium. Cha-poo (Piper sarmentosum Roxb.) and sweet potato (Ipomoea batatas Lam.) were 2 out of 14 vegetable extracts chosen as medium supplements. Rhodococcus sp. MI 2 gave the highest cellulose yield in a medium supplemented with Cha-poo extract. The optimum culture conditions in the medium supplemented with Cha-poo extract at room temperature (25o C) under static condition were 5% (v v-1) inoculum size, a 6 -day -incubation period, pH 3, 3% sucrose, and 0.5% (NH4)2SO4. The cellulose yield in the medium supplemented with Cha-poo extract was increased about 3 times (6.83 g L-1 during 6 days) higher than that obtained before optimizing (2.39 g L-1 during 6 days). The medium supplemented with Cha-poo extract cost one quarter (0.5 USD L-1 of medium) of the SH medium (1.9 USD L-1 of medium). The structure of the microfibrils of cellulose produced by Rhodococcus sp. MI 2 in a medium supplemented with Cha-poo extract observed by SEM had larger, less crowded fibrils than those produced in the medium supplemented with sweet potato extract. In addition, the microfibrils of the former had many beehive shaped knots whereas those of the latter had mantle-like surrounding the fibrils.

Neste estudo, o extrato vegetal mais adequado foi triado para uso como fontes não convencionais de nutrientes para produção de celulose de Rhodococcus sp. MI 2. Utilizou-se um meio SH ou meio sintético como meio convencional ou de controle. Cha-poo (Piper sarmentosum Roxb.) e batata-doce (Ipomoea batatas Lam.) foram 2 dos 14 extratos vegetais escolhidos como suplementos do meio. Rhodococcus sp. MI 2 deu o maior rendimento de celulose em um meio suplementado com extrato de Cha-poo. As condições ótimas de cultivo no meio suplementado com extrato de Cha-poo em temperatura ambiente (25 ºC) em condição estática foram 5% (v v-1) do tamanho do inóculo, um período de 6 dias de incubação, pH 3, 3% de sacarose, e 0,5% (NH4) 2SO4. O rendimento de celulose no meio suplementado com extrato de Cha-poo foi aumentado cerca de 3 vezes (6,83 g L-1 durante 6 dias), maior do que o obtido antes da otimização (2,39 g L-1 durante 6 dias). O meio suplementado com extrato de Cha-poo custou um quarto (0,5 USD L-1 de meio) do meio SH (1,9 USD L-1 de meio). A estrutura das microfibrilas de celulose produzidas por Rhodococcus sp. MI 2 em meio suplementado com extrato de Cha-poo, observado por MEV, apresentou fibrilas maiores e menos congestionadas do que aquelas produzidas no meio suplementado com extrato de batata-doce. Além disso, as microfibrilas do primeiro possuíam muitos nós em forma de colmeia, enquanto os do último tinham um aspecto tipo manto ao redor das fibras.

Piper sarmentosum Improves Bone Structure and Biomechanical Strength of Rats Given Excess Glucocorticoid.

Aims: To determine the effect of Piper sarmentosum (Ps) leaf extract on biomechanical strength and trabecular structure of the bones of glucocorticoid-induced osteoporotic rats. Study Design: Administration of crude extract to rats with excessive glucocorticoids. Place and Duration of Study: Department of Anatomy and Pharmacology, National University of Malaysia, between September 2010 and December 2011. Methodology: Three-month old male Sprague-Dawley rats were adrenalectomized to remove the main source of circulating glucocorticoids. The animals were replaced with dexamethasone 120 μg/kg body weight/day. Treatment with P. sarmentosum 125 mg/kg body weight and glycirrhizic acid (GCA) 120 mg/kg body weight were given simultaneously for 2 months. After been sacrificed, a three-point bending configuration test for assessing the biomechanical properties of the right femoral bones was done using an Instron Universal testing machine equipped with Instron Bluehill software. The left undecalcified femoral bones were embedded in resin, sectioned and stained with Von Kossa for structural histomorphometric measurements. Results: P. sarmentosum extract had significantly increased the intrinsic parameter (flexure modulus) and extrinsic parameter (energy at break) of the biomechanical properties of the bone. It had also significantly improved the trabecular structure by increasing the BV/TV, Tb.Th, Tb.N and by reducing the Tb.Sp based on histomorphometric analysis. Conclusion: P. sarmentosum extract was able to protect bone biomechanical strength in glucocorticoid-induced osteoporotic bone, as confirmed by the structural histomorphometric finding. Therefore, Ps extract has the potential to be used as an agent to protect the bone strength and structure against osteoporosis due to chronic glucocorticoid treatment. These results however, need further study for better justification.

Piper sarmentosum enhances fracture healing in ovariectomized osteoporotic rats: a radiological study

INTRODUCTION: Osteoporotic fractures are common during osteoporotic states. Piper sarmentosum extract is known to possess antioxidant and anti-inflammatory properties. OBJECTIVES: To observe the radiological changes in fracture calluses following administration of a Piper sarmentosum extract during an estrogen-deficient state. METHODS: A total of 24 female Sprague-Dawley rats (200-250 g) were randomly divided into 4 groups: (i) the sham-operated group; (ii) the ovariectomized-control group; (iii) the ovariectomized + estrogen-replacement therapy (ovariectomized-control + estrogen replacement therapy) group, which was supplemented with estrogen (100 μg/kg/day); and (iv) the ovariectomized + Piper sarmentosum (ovariectomized + Piper sarmentosum) group, which was supplemented with a water-based Piper sarmentosum extract (125 mg/kg). Six weeks after an ovariectomy, the right femora were fractured at the mid-diaphysis, and a K-wire was inserted. Each group of rats received their respective treatment for 6 weeks. Following sacrifice, the right femora were subjected to radiological assessment. RESULTS: The mean axial callus volume was significantly higher in the ovariectomized-control group (68.2 + 11.74 mm³) than in the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups (20.4 + 4.05, 22.4 + 4.14 and 17.5 + 3.68 mm³, respectively). The median callus scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups had median (range, minimum - maximum value) as 1.0 (0 - 2), 1.0 (1 - 2) and 1.0 (1 - 2), respectively, which were significantly lower than the ovariectomized-control group score of 2.0 (2 - 3). The median fracture scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups were 3.0 (3 - 4), 3.0 (2 - 3) and 3.0 (2 - 3), respectively, which were significantly higher than the ovariectomized-control group score of 2.0 (1 - 2) (p<0.05). CONCLUSION: The Piper sarmentosum extract improved fracture healing, as assessed by the reduced callus volumes and reduced callus scores. This extract is beneficial for fractures in osteoporotic states.

Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells

OBJECTIVE: Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were divided into four groups: control, treatment with 180 ìM hydrogen peroxide (H2O2), treatment with 150 ìg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H2O2 for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. RESULTS: Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. CONCLUSION: Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.