Extraction of Essential Oil from a Leaf of Piper betle L. (Paan) by Hydro-distillation Method

Piper betle leaf commonly known to be a Paan in India is eaten raw and sometimes with a mixture of areca nut. The leaf is consumed usually after a lunch or dinner. Traditionally the leaf is well known for a health benefits and also chewed as a mouth freshener. A wide range of researches on a Piper betle leaf suggest excellent nutritional benefits on the health. The edible leaf is also known for its specialized essential contents. The current research attempt is carried out to extract the essential oil from the leaf of Piper betle L. by a Hydro distillation method (Clevenger method). The research successfully concludes the extraction of oil with its efficacy towards the percentage of oil achieved during an extraction process.

Antibacterial activity of Sireh (Piper betle L.) leaf extracts for controlling bacterial leaf blight diseases in rice plant

Aims@#This study aimed to determine the antibacterial activity of Piper betle L. leaf extract against Xanthomonas oryzae pv. oryzae that causes bacterial leaf blight in rice plant.@*Methodology and results@#The antibacterial activity of the P. betle leaf extract (100, 50, 25 and 12.25 mg/mL) with four different solvents (methanol, ethyl acetate, hexane and acetone) was evaluated using a disc diffusion assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. The inhibition zone of methanolic extract appeared to have the maximum diameter compared to those of other extracts, which is 32.67 mm at a concentration of 100 mg/mL, followed by 30.33 mm, 22.00 mm and 20.30 mm for the concentrations of 50 mg/mL, 25 mg/mL and 12.5 mg/mL, respectively. The MIC and MBC values of the methanolic extract were 0.625 mg/mL suggesting that the extract has a bactericidal effect on X. oryzae pv. oryzae (Xoo). The time-kill curve studies revealed that the 1× MIC (0.625 mg/mL) concentration of methanolic extract had a time and concentration-dependent killing effect on Xoo. Gas chromatography-mass spectrometry (GC-MS) analysis of methanol extract revealed the presence of eugenol acetate (29.53%), 4-allyl-1,2-diacetoxybenzene (29.51%) and 2,3-dimethyl benzoic acid (22.82%) as major compounds.@*Conclusion, significance and impact of study@#The methanolic leaf extract of P. betle was proven to have an effective inhibitory effect on Xoo and may have the potential to be used as an alternative management strategy for controlling rice diseases. In the future study, the methanolic leaf extract of P. betle is one of the recommendations to be applied in glasshouse and field trials.

Antimicrobial efficacy and activity of ethanolic extract of Piper betle L. on Staphylococcus aureus-infected wound in mice and clinical isolates of multiple drug-resistant bacterial pathogens

@#This study aimed to determine the in vivo effectiveness of the ethanolic extract of Piper betle L. leaves against Staphylococcus aureus-infected wounds in mice and its antimicrobial properties on clinical isolates of multiple drug-resistant bacterial pathogens. Twenty mice were divided into four groups. Wounds were created in all mice under anesthesia by excision from the dorsal skin down to the subcutaneous fat and inoculating with S. aureus. After 24 h, the wound of each mouse was treated once daily by application of the respective cream. Group I was treated with mupirocin antibacterial cream; Group II received a cream base containing no active ingredient; Groups III and IV were treated with 2.5% and 5.0% concentrations of P. betle cream, respectively. Further, an in vitro study was performed by adding undiluted, 1:50 and 1:100 dilutions of the four studied creams in normal saline containing 1.5 × 108 CFU/mL of the following bacteria: antimicrobial-susceptible S. aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, extended-spectrum β-lactamase-producing Escherichia coli, vancomycin-resistant Enterococcus, metallo-βlactamase-producing P. aeruginosa and carbapenem-resistant Klebsiella pneumoniae. The mice in Groups III and IV had significantly faster wound contraction and significantly shorter reepithelialization time than Group II (p < 0.05), which were not significantly different from Group I (p > 0.05). P. betle creams inhibited all studied bacterial strains at full concentration and at a dilution of 1:50. The inhibitory effect was more significant than Groups I and II (p < 0.05), except on S. aureus. Specifically, S. aureus inhibition was not significantly different for Groups III and IV (p > 0.05) when compared with Group I. Cream formulations derived from P. betle ethanolic extract have great potential as antimicrobial agents for the treatment of wound infection. Further clinical tests are recommended to determine the safety and efficacy of these formulations in other mammalian species.

Hydroxychavicol, a polyphenol from / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol (HC). Its effects on cell cycle, apoptosis, and the expression of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) in HT-29 colon cancer cells were investigated. HC was isolated from

Anti-migratory effects of Piper betle leaf aqueous extract on cancer cells and its microtubule targeting properties / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Piper betle (PB), also known as "betel" in Malay language, is a tropical Asian vine. PB leaves are commonly chewed by Asians along with betel quid. It contains phenols such as eugenol and hydroxychavicol along with chlorophyll, β-carotene, and vitamin C (Salehi et al., 2019). Extracts from PB leaves have various medicinal properties including anticancer, antioxidant, anti-inflammatory, and antibacterial effects (Salehi et al., 2019). Previous research has shown that PB induces cell cycle arrest at late S or G2/M phase and causes apoptosis at higher doses (Wu et al., 2014; Guha Majumdar and Subramanian, 2019). A combination of PB leaf extract has also been shown to enhance the cytotoxicity of the anticancer drug, 5-fluorouracil (5-FU), in cancer cells (Ng et al., 2014).

Bioanalytical HPLC method of Piper betle L. for quantifying phenolic compound, water-soluble vitamin, and essential oil in five different solvent extracts

A reversed-phase high-performance liquid chromatography with diode-array detection (HPLC-DAD) was developedand validated to estimate the phenolic acids (gallic acid, caffeic acid, syringic acid, p-coumaric acid, sinapic acid, andferrulic acid), flavonoids (catechin rutin, myricetin, quercetin, apigenin, and kaempferol), ascorbic acid, and eugenol.The chromatogram condition was set in suitable wavelength 272 nm and run flow rate 0.7 µl/minutes using HPLCAgilent Technologies 1260 Infinity, a reversed-phase Zorbax SB-C18 column (3.5 µm particle size, i.d. 4.6 mm × 250mm) with the mobile phase solution (1:9, HPLC-grade acetonitrile:1% acetic acid). The linearity, precision, limit ofdetection, limit of detection, and accuracy were R2 > 0.9907, relative standard deviation < 1%, 0.005 µg/ml, 0.015 µg/ml, and 96%–102%, respectively. As a result, all the selective compounds were successfully separated, identified, andquantified. The enormous contents were found in quercetin and eugenol, expressing crude content (mean, 5.989 mg/g)and residue content (mean, 1.934 mg/g) for quercetin, while crude content (mean, 3.209 mg/g) and residue content(mean, 0.184 mg/g) for eugenol. Consequently, this method could be applied, repeated, and developed for the laterobservation, especially in commercially inclination of Piper betle analysis

Preclinical evaluation of antidepressant activity of aqueous extract of Piper betle leaves in Swiss albino mice

Background: There is a growing demand for alternative medicines derived from indigenous plants having natural antioxidants and neuroprotective actions for the treatment of many behavioural disorders such as anxiety and depression. This study was designed to screen antidepressant activity of aqueous extract of Piper betle L. leaf (betel leaf) in Swiss albino mice.Methods: Swiss albino mice of both sexes weighing 25-30grams were used in the present study. Piper betle leaves aqueous extract (PBAE) was administered to the animals at a dose of 100, 200mg/kg body weight orally for 14 days. On the 14th day, after 1 hour of PBAE administration, experiments on force swim test (FST) and tail suspension (TST) were carried out for studying the level of depression. In FST and TST, time of immobility was noted for a period of 5 minutes.Results: Data was analyzed by one-way ANOVA followed by Tukey Kramer’s multiple comparison test at P = 0.05. The results were represented as Mean±SE. PBAE at a dose of 100mg/kg has shown significant antidepressant activity, as evidenced by decrease in the immobility time in both the screening tests of depression.Conclusions: Present results demonstrated that PBAE possess potent antidepressant property. The exact mechanism(s) related to the active compound(s) in Piper betle leaf extract have to be elucidated in future studies.

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

Background: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. Aim: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. Methods: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by fl ow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2’-7’-dichlorodihydrofl uorescein diacetate. Results: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fl uorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 μg/ml signifi cantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fl uorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. Limitations: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. Conclusion: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was signifi cantly reduced following ex vivo treatment with Piper betle.

Biotechnological intervention in betelvine (Piper betle L.): A review on recent advances and future prospects

Betelvine (Piper betle L.) is cultivated for its deep green heart shaped leaf for (15–20) million Indian and 2 billion foreign consumers annually. The crop provides Rs (6 000–7 000) million of national income per year and at the same time leaves worth Rs (30–40) million is exported to other countries. The leaves are not only used directly for chewing purposes but also possesses antioxidant, anti-inflammatory, anti-apoptotic, anti-cancer and anti-microbial properties. Besides, the leaves also contain eugenol rich essential oil (1%–3%) which is the source for medicine, stimulant, antiseptic, tonic and other ayurvedic formulations. The essential oil also contains chavibetol, caryophyllene and methyl eugenol which are the potent source for preparation in ayurvedic medicine and herbal products. Cost of betelvine essential oil is 10$ per 5 mL. In spite of its great economical and medicinal importance betelvine is still neglected by the researchers for proper characterization and authentication for selection of elite landraces. Lack of awareness among people, use of same planting material for many generations, existing of many synonyms for a single landraces, no proper characterization of available landraces are some of the significant constraints for its commercialization. Our review endeavours a complete advance in the research on betelvine, existing lacunae for its proper characterization and commercial cultivation. It also attempts to provide a comprehensive account on biotechnological interventions made in betelvine aimed at complementing conventional programmes for improvement of this nutraceutically important cash crop.

Antimicrobial, total phenolic and total flavonoid properties of leaves and seed of Jatropha curcas, Piper nigrum L. and Piper betle methanolic crude extracts

Aims: Herbal and medicinal plants bioactive compounds of Jatropha curcas, Piper nigrum and P. betle have been shown to possess therapeutic properties. This study investigates the presence and characterization of phytochemical compounds as well as to evaluate the antimicrobial activities of the methanolic crude extract of the leaves and seeds of J. curcas, P. nigrum L. and P. betle. Methodology and results: The study on antibacterial and antifungal efficacy of the crude extracts of leaves and seeds were carried out using standard disc diffusion method. The crude extracts were found to exhibit an average response of antimicrobial activity with the inhibition zones ranged from 3% to 28% for antibacterial and from 21% to 79% for antifungal activity. Among all extracts, the leaf extract of P. betle showed a good antibacterial activity against Staphylococcus aureus and excellent antifungal properties against Aspergillus niger and A. flavus. The phytochemical screening analysis revealed the presence of saponin, tannins, glycosides, terpenoids, reducing sugar, flavonoid and anthraquinones. However, phlobatannins was not present. Total phenolic content (TPC) and total flavonoid content (TFC) were highly detected in the crude extract of P. betle and recorded as 13.33 mg of gallic acid equivalents, GAE (mg/ 100 mg sample) and 0.88 mg of RE (mg/100 mg sample), respectively. GC-MS analysis of the bioactive compounds reveals the presence of diethyl phthalate, 2-hexadecen-1-ol (Phytol), hexadecanoic acid, piperine, phenol and other minor compounds. Conclusion, significance and impact of study: The study suggested that P. betle has a potential as a source for antimicrobial agent from plants extracts. Nevertheless, further studies are needed to elucidate their precise mechanism of action.

Biotechnological intervention in betelvine (Piper betle L.): A review on recent advances and future prospects

Betelvine (Piper betle L.) is cultivated for its deep green heart shaped leaf for (15-20) million Indian and 2 billion foreign consumers annually. The crop provides Rs (6000-7000) million of national income per year and at the same time leaves worth Rs (30-40) million is exported to other countries. The leaves are not only used directly for chewing purposes but also possesses antioxidant, anti-inflammatory, anti-apoptotic, anti-cancer and anti-microbial properties. Besides, the leaves also contain eugenol rich essential oil (1%-3%) which is the source for medicine, stimulant, antiseptic, tonic and other ayurvedic formulations. The essential oil also contains chavibetol, caryophyllene and methyl eugenol which are the potent source for preparation in ayurvedic medicine and herbal products. Cost of betelvine essential oil is 10$ per 5 mL. In spite of its great economical and medicinal importance betelvine is still neglected by the researchers for proper characterization and authentication for selection of elite landraces. Lack of awareness among people, use of same planting material for many generations, existing of many synonyms for a single landraces, no proper characterization of available landraces are some of the significant constraints for its commercialization. Our review endeavours a complete advance in the research on betelvine, existing lacunae for its proper characterization and commercial cultivation. It also attempts to provide a comprehensive account on biotechnological interventions made in betelvine aimed at complementing conventional programmes for improvement of this nutraceutically important cash crop.

Antibacterial activities of ethanol extracts of Philippine medicinal plants against multidrug-resistant bacteria

Objective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillin- resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16-33 mm inhibition diameter), minimum inhibitory concentration (19-156 μg/mL) and minimum bactericidal concentration (312 μg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-c-lactamase-producing) in the disk diffusion (17-21 mm inhibition diameter), minimum inhibitory concentration (312-625 μg/mL) and minimum bactericidal concentration (312-625 μg/mL) assays. Conclusions: P. betle had the greatest potential value against both Gram-negative and Gram-positive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.

Gastroprotective effect of Piper betle Linn. leaves grown in Sri Lanka.

Background: Piper betle Linn. (Piperaceae) is used as a remedy for gastric ulcers in traditional medicinal systems in Sri Lanka. However, the gastroprotective activity has never been proven scientifi cally using betel leaves grown in Sri Lanka. Objective: To evaluate the gastroprotective activity of hot aqueous extract (HAE) and cold ethanolic extract (CEE) of P. betle in rats as the experimental model. Materials and Methods: Three doses (200, 300, and 500 mg/kg/bw) of both extracts were evaluated for the gastroprotective activity against ethanol induced gastric ulcers in rats. The parameters evaluated were (a) effects of HAE on mucus content adhering to the wall of the gastric mucosa, (b) acidity (total and free), (c) volume and (d) pH of the gastric juice. Results: Oral administration of HAE and CEE provided marked dose dependent (HAE: r2 = 0.97; CEE: r2 = 0.96) and signifi cant (P ≤ 0.05) protection against gastric damage caused by absolute ethanol. The gastroprotective effect of CEE was comparable with that of HAE. Further, gastroprotective activity of the highest dose of both extracts were signifi cantly greater (P ≤ 0.05) than that of misoprostol, the reference drug. The HAE signifi cantly (P ≤ 0.05) increased the mucus content adhering to the wall of the gastric mucosa and inhibited the volume of gastric acid. However, acidity (total and free) and pH of the gastric juice remained unaltered. Conclusion: It is concluded that both HAE and CEE of P. betle leaves have a strong gastroprotective activity.

Inhibitory effect of Piper betle Linn. leaf extract on protein glycation - Quantification and characterization of the antiglycation components.

Piper betle Linn. is a Pan-Asiatic plant having several beneficial properties. Protein glycation and advanced glycation end products (AGEs) formation are associated with different pathophysiological conditions, including diabetes mellitus. Our study aims to find the effect of methanolic extract of P. betle leaves on in vitro protein glycation in bovine serum albumin (BSA)-glucose model. The extract inhibits glucose-induced glycation, thiol group modification and carbonyl formation in BSA in dose-dependent manner. It inhibits different stages of protein glycation, as demonstrated by using glycation models: hemoglobin-d-gluconolactone (for early stage, Amadori product formation), BSA-methylglyoxal (for middle stage, formation of oxidative cleavage products) and BSA-glucose (for last stage, formation of AGEs) systems. Several phenolic compounds are isolated from the extract. Considering their relative amounts present in the extract, rutin appears to be the most active antiglycating agent. The extract of P. betle leaf may thus have beneficial effect in preventing protein glycation and associated complications in pathological conditions.

Characteristic differences in metabolite profile in male and female plants of dioecious Piper betle L.

Piper betle is a dioecious pan-Asiatic plant having cultural and medicinal uses. It belongs to the family Piperaceae and is a native of the tropics although it is also cultivated in subtropical areas. Flowering in P. betle occurs only in tropical regions. Due to lack of inductive floral cycles the plant remains in its vegetative state in the subtropics. Therefore, due to lack of flowering, gender distinction cannot be made the in the subtropics. Gender distinction in P. betle in vegetative state can be made using Direct Analysis in Real Time Mass Spectroscopy (DARTMS), a robust highthroughput method. DARTMS analysis of leaf samples of two male and six female plants showed characteristic differences in the spectra between male and female plants. Semi-quantitative differences in some of the identified peaks in male and female landraces showed gender-based differences in metabolites. Cluster analysis using the peaks at m/z 151, 193, 235 and 252 showed two distinct clusters of male and female landraces. It appears that male and female plants besides having flowers of different sexes also have characteristic differences in the metabolites representing two metabolic types.

Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice

OBJECTIVE: The aim of this study was to determine the erythrocyte antioxidant enzyme activity and the superoxide dismutase, catalase, glutathione peroxidase, and plasma malondialdehyde levels in aging mice and to evaluate how these measures are modulated by potential antioxidants, including the tocotrienol-rich fraction, Piper betle, and Chlorella vulgaris. METHOD: One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level. RESULTS: Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments. CONCLUSION: We found equivocal age-related changes in erythrocyte antioxidant enzyme activity when mice were treated with Piper betle, the tocotrienol-rich fraction, and Chlorella vulgaris. However, Piper betle treatment showed increased antioxidant enzymes activity during aging.

Antioxidant effect of ethanolic extract of Piper betle Linn. (Paan) on erythrocytes from patients with HbE-beta thalassemia.

HbE-beta thalassemia is caused by an interaction between HbE and defective b globin gene of thalassemia. Repeated blood transfusions cause an iron overload, triggering an enhanced generation of free radicals. In the present study, the anti-oxidant property of ethanolic extract of the leaves of Piper betle Linn. (PB) was evaluated in the erythrocytes from patients with HbE-beta thalassemia. In patients with HbE-beta thalassemia (n = 30) and age- and sex-matched healthy individuals (n = 30), the baseline level of reactive oxygen species (ROS) and free radical scavenging activity in the erythrocytes was measured by flow cytometry using dihydrodichlorofluorescein diacetate (H2DCFDA), in terms of the geometric mean fluorescence channel (GMFC). The baseline generation of ROS was significantly higher in the erythrocytes from patients with HbE-beta thalassemia, as compared to healthy volunteers, the GMFC being 67.20 ± 4.64 vs. 23.03 ± 1.88 (p<0.001), which was effectively decreased by PB. Similarly, H2O2 (0.5-1.0 mM) induced a higher increase in the GMFC in the erythrocytes from patients with HbE-beta thalassemia, as compared to controls which was effectively reduced by PB. Taken together, PB showed promising anti-oxidant activity against the erythrocytes from patients with HbE-beta thalassemia.

In Vitro Effect of Fungicides, Plant Extracts and Smoke on Conidial Germination of Fusarium oxysporum Root Rot Pathogen of Piper betle

Five fungicides such as rovral, bavistin, cupravit, dithane M-45 and thiovit were tested against conidial germination of Fusarium oxysporum. Dithane M-45 was the most effective against the fungus. Rests of the fungicides were more or less effective in the inhibition of conidial germination after 5~30 minutes immersion in 500~2500 ppm concentration. Five plant extracts(leaf extracts of Ocimum sanctum, Lantana camera, Calotropis procera, Azadirachta indica and Vinca rosea) found to be more or less effective against the fungus. 'Dhup' somke have good inhibitory effect on conidial germination of F. oxysporum.

Chemical components of essential oil of Piper nigrum L. and essential oil of Piper betle L. in Nghe An province

Chemical components of essential oil of Piper nigrum L. and essential oil of Piper betle L. were identified by GC/MS method. The main constituents in essential oil of Piper nigrum L. are D-limonene, alpha-pinene, beta-pinene and D3-carene. The main components of essential oil of Piper betle L. are eugenyl acetate or isoeugenyl acetate, alylpyrocatechol diacetate, eugenol or isoeugenol, caryophylen