In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

Background

Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.

Methods

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.

Results

Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.

Conclusions

The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.     

The mutagenicity of nitrite-treated aqueous extract of Piper betle L

Betel quid is chewed as a masticatory material by people in certain areas of Asia. The quid chewing has been related to oral cancer by epidemiological study. The mutagenic components in the aqueous extracts of betel quid ingredients were studied. Only nitrite-treated aqueous extract of Piper betle L fruits, leaves or rhizoma were demonstrated to exhibit a mutagenic response, using Salmonella typhimurium strains TA100 and TA1535 in the Ames test. When the aqueous extract of the fruit was nitrosated, the greatest number of mutagenic substances were formed at pH 3. The formation of mutagens was enhanced by increasing the temperature from 5 to 95 degrees C. Maximum production of the mutagens occurred within 15 min when nitrosation was conducted at 35 degrees C. The mutagenic components in nitrite-treated aqueous extract of Piper betle L fruit were found to be N-nitrosopiperidine, N-nitrosopyrrolidine, N-nitrosomorpholine, and other compounds, as determined by gas chromatography-thermal energy analyzer.     

An efficient In Vitro Method for Mass Propagation of Salvadora persica via Apical Meristem

A rapid clonal propagation system has been developed for medicinally potent tree species - Salvadora persica via single step through apical meristem. Shoot tips (1 cm) were inoculated on MS medium incorporated with 17.8 µM benzyladenine and 18.6 µM kinetin along with activated charcoal (0.3%).The shoot tips grew fast (4 cm) within 7 days alongwith well developed roots on the same medium.Thus, the present investigation demonstrates the complete regeneration of Salvadora persicaplants via single step. Such reproducible protocol could be used for the production of virus-free plantlets in quicker time.     

An Improved Method for Large Scale Synthesis of Oligonucleotides Applying the NPE/NPEOC-Strategy

Abstract

Several oligonucleotides were synthesized in scales up to 60 μmol in a standard 10 μmol cartridge on a standard DNA synthesizer. The advantage of a special phosphoramidite approach using only β-eliminating protecting groups over the commonly practised automated oligonucleotide synthesis using ammonia-labile blocking groups could be demonstrated by ¹H-NMR-spectroscopy.     

In Vitro Method for Propagation of Centella asiatica (L) Urban by Shoot Tip Culture

A rapid clonal propagation system has been developed for the medicinally important herb Centella asiatica (L) Urban by shoot tip (2–3 cm long) culture. The shoot tips isolated from mature plants were inoculated on MS medium incorporated with BA alone or in combination with NAA and Kn. The optimum number of shoots (3.38) with optimum number of leaves per shoot (4.25) were attained on MS medium supplemented with 4.0 mg l^?1 BA and 0.1 mg l^?1 NAA. On transferring the microshoots on full strength MS medium supplemented with various concentrations of IBA (1.0-3.0 mg l^?1) and NAA (0.5-2.0 mg l^?1), profuse rooting (46.8 per shoot) was obtained in MS basal medium with 2.0 mg l^?1 IBA with root length of 19.7 cm. Well rooted plantlets were acclimatized successfully by adjusting the temperature and humidity for 3–4 weeks after transfer to pots filled with sterilized vermiculite soil: sand (1:1)mixture. This micropropgation protocol could be useful for raising a stock of genetically homogenous material for field cultivation within a very short period.     

In Vitro Propagation of Potato (Solanum tuberosum L.)

Potato shoots were propagated in vitro by placing nodes fromsprouted tubers on Murashige and Skoog type medium without hormones.The vigour of growth and the rate of node production increasedwith both day-length and temperature over the ranges 8–24h and 15–25 °C respectively. Propagation rates ofup to x 10 per month were obtained. In vitro plantlets spontaneouslyformed roots either in agar or liquid cultures. Plantlets leftin the culture jars for 3–4 months eventually senescedand formed small tubers in 16 and 24 h day-lengths. In a day-lengthof 8 h vegetative growth continued by branching and no tuberswere formed. Solanum tuberosum L., potato, tissue culture, propagation, temperature, day-length     

Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L. leaf

Piper betle L., a dioecious shade-loving perennial climber is one of the important Pan-Asiatic plants. More than hundred landraces having marked variation in leaf chlorophyll (Chl) content are in cultivation in India. In this study, role of chlorophyllase (Chlase) in Chl homeostasis and post-harvest breakdown was investigated in two contrasting P. betle landraces Kapoori Vellaikodi (KV) with light green and Khasi Shillong (KS) with dark green leaves. The two landraces showed negative correlation between Chl content and Chlase activity in fresh as well as stored leaves. Accumulation of chlorophyllide a (Chlid a) was correlated with the level of Chlase activity, which was higher in KV than KS. The overall response of abscisic acid (ABA) and benzylaminopurine (BAP) was similar in KV and KS, however, the time-course was different. ABA-induced Chl loss was accompanied by rise in Chlase activity in KV and KS and the delay in Chl loss by BAP was accompanied by reduction in Chlase activity. While there were significant differences in Chlase activity in KV and KS, only minor differences were observed in the enzyme properties like pH and temperature optima, Km and Vmax. No landrace-related differences were observed on the effect of metal ions and functional group reagents/amino acid effectors on Chlase activity. These results showed that despite significant differences in Chl content and Chlase activity between landraces KV and KS, the properties of Chlase were similar. The findings show that in P. betle Chlase is involved in Chl homeostasis and also in Chl degradation during post-harvest storage and responds to hormonal regulations. These findings might be useful in predicting the stability of Chl during post-harvest storage and also the shelf-life in other P. betle landraces.     

Role of Petiole in Protein Metabolism of Senescing Betel (Piper betle L.) Leaves

Effects of depetiolation on protein metabolism during senescence of detached betel (Piper betle L.) leaves have been studied. In normal petiolated leaves, the level of chlorophyll and proteins and extent of protein synthesis declined, while the protease activity registered manifold increase with the advancement of senescence. All of these changes were delayed by depetiolation/de-midribbing treatments, though without affecting the general pattern of senescence. Thus, the presence of petiole seems to expedite protein degradation, probably due to earlier attainment of optimal concentration of proposed senescence factor(s) (Mishra and Gaur 1970 Science 167: 387).     

HS-SPME-GC-MS分析蒟酱叶挥发性成分

首次采用顶空固相微萃取和气质联用技术(HS-SPME-GCMS),结合保留指数法,用峰面积归一化法测定蒟酱叶中挥发性成分及相对百分含量。结果表明,从蒟酱叶中鉴别出27个化学成分,占峰面积的97.53%,化合物结构类型包括醛、酸、酯、酚、烯烃、芳香烃和(环)烷烃。2-甲氧基-5-甲基苯甲醛(42.89%)、胡椒酚醋酸酯(12.49%)、异丁香酚(13.42%)、4-烯丙基-1,2-二乙酰氧基苯(9.47%)、胡椒酚(2.89%)、γ-毕橙茄烯(2.74%)、丁香酚(2.66%)和乙酸异丁香酚酯(2.06%)是蒟酱叶的主要挥发性成分。     

鱼籽兰(Chloranthus errectus)的离体繁殖

本研究通过几种培养基对鱼籽兰外植体培养,选择出了有效的外植体和适合的培养基,并摸索出了有效的移载和栽培技术,培育出了外植体繁殖的成熟开花植株,为该植物提供了一条新的快速繁殖途径,对同属其它植物的快速繁殖具有参考意义。     

An Improved Cellophane Method for in Vitro Germination of Recalcitrant Pollen

The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper. The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min. This arrangement keeps the cellophane flat as it absorbs medium. The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish. The lower leaves of the booklet keep the cellophane moist for up to 24 hr. Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique. Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.     

Gender-specific responses of Piper betle L. to low temperature stress: changes in chlorophyllase activity

Gender based differences in response to low temperature stress in leaf chlorophyll (Chl), and carotenoids (Car) contents and chlorophyllase (Chlase) activity were monitored in male (Kapoori Vellaikodi and Madras Pan Kapoori) and female (Bangla Mahoba, Desi Bangla and Kaker) betel vine landraces. Although female plants contained nearly two fold more Chl than male counterparts, the low temperature induced Chl loss was comparable, however, male plants showed higher Chl a/b ratio than females. Chlase activity increased due to cold stress in all the landraces. Male plants always showed higher activities of Chlase, which may be one of the reasons for the rather low Chl contents in male plants.     

白檀离体快繁技术

以白檀(Symplocos paniculata)幼嫩茎段为实验材料, 通过对启动培养、增殖、生根培养及移栽的影响因子进行研究, 初步建立了白檀的组织培养体系。结果表明: 白檀外植体最适灭菌方案为0.1%升汞3分钟, 无菌苗获得率达81%; 最适初代启动培养基为1/2MS+30 g∙L^-1蔗糖+8 g∙L^-1琼脂, 出芽率达86.83%; 增殖最适培养基为1/2MS+1.0 mg∙L^-1 6-BA+0.02 mg∙L^-1 IBA+30 g∙L^-1蔗糖+8 g∙L^-1琼脂, 增殖系数达3.57; 最适生根培养基为WPM+0.5 mg∙L^-1 IBA+0.5 mg∙L^-1 NAA+20 g∙L^-1蔗糖+2 g∙L^-1 AC+8 g∙L^-1琼脂, 生根率达93%; 炼苗后, 移入园土:草炭土=1:1 (v/v)的基质中, 成活率达83%。     

柠檬香蜂草离体快速繁殖(简报)

以柠檬香蜂草带腋芽幼嫩茎段为外植体,MS为基本培养基,对其进行离体快繁研究。结果表明,在Ms＋6-BA1.0mgCL＋IBA0．5mg／L培养基上诱导产生不定芽的效果最好;在MS＋6．BA0．5mg/L＋IBAO．1mg／L分化培养基上分化率达3～4倍;在1／2MS＋IBA0．2mg／L生根培养基中正常发根,且根系粗壮。     

莲的离体快速繁殖技术

以莲(Nelumbo nucifera)授粉后18天的莲子胚芽为外植体,通过初代培养、继代培养和炼苗移栽,建立了莲离体快速繁殖体系。结果表明,将胚芽外植体诱导出无菌苗的最适初代培养基为MS固体培养基添加0.5 mg·L–1 6-BA、0.5 mg·L–1NAA、30 g·L–1蔗糖、0.5 g·L–1活性炭和0.8 g...     

Possibility of using a bacterial antagonist against fungal diseases of Piper betle L. and corchorus spp.

Summary A strain ofBacillus subtilis isolated from soil was found effective againstSclerotium rolfsii when grown on 20% potato extract for 48 hours and used along with the medium. Whole boiled potato was also a good medium but the bacteria needed 20 days to grow until insoluble starch was digested.In field trials the antagonist brought down the spread ofSclerotium and anthracnose diseases ofPiper betle. Against seed and air-borne diseases of jute caused byMacrophomina phaseoli,Diplodia corchori andColletotrichum corchori, the antagonist also showed promising results.Concluding part of a scheme supported by Food & Agriculture Council, Pakistan.     

"中芦荟"离体快速繁殖技术研究

探讨用植物组织培养技术进行中芦荟的快速繁殖,为工厂化育苗提供技术依据。用中芦荟幼苗茎尖为外植体进行离体培养,采用单因素试验设计,以MS为基本培养基,进行6-苄基腺嘌呤（6-BA）及吲哚丁酸（IBA）不同浓度用量的配比试验,结果表明,最适宜该品种各阶段快速繁殖的激素配方分别为：（1）丛生芽诱导：MS 6-BA3.0mg/L IBA0.4mg/L;（2）丛生芽继代增殖：MS 6-BA2.0mg/L IBA0.3mg/L;（3）生根诱导：1/2MS IBA0.4mg/L。另移栽试验表明：采用河沙：菌包：腐质土=2：2：1的基质栽植,成活率可达100%。     

In Vitro Propagation Of Four Watsonia Species

The genus Watsonia has a number of species with potential to be developed as new ornamental crop plants, but to date there are no reports on in vitro propagation of any member of this genus. Seeds from four Watsonia species, Watsonia gladioloides, Watsonia lepida, Watsonia laccata, and Watsonia vanderspuyiae were decontaminated and germinated on one-tenth strength MS media without hormones or sucrose. Shoots were induced from seedling hypocotyl segments when both an auxin [α-naphthaleneacetic acid (NAA)] and cytokinin [N⁶−benzylaminopurine (BA)] were present in the medium, while root and leaf explants failed to produce shoots. Multiplication of axillary shoots was greatest when only BA (0.5 mg l⁻¹) was added to the medium. Shoot explants propagated in a ‘liquid-shake’ culture exhibited greater growth rates than those on agar-solidified medium, but shoot production varied between species. Meristemoids were induced in all species, but no significant trend was found between growth index (GI) and meristemoid formation, suggesting that reduction in GI may not necessarily be a prerequisite for producing meristemoids. Corm formation was inconsistent and storage organs could only be induced in one of the four species, W. vanderspuyiae. This occurred best at 25°C with 3% sucrose and an agar level of 15%. Indole-3-acetic acid (IAA) and NAA at 1 mg l⁻¹ significantly increased mean number of roots per shoot explant on all four species, while indole-3-butyric acid (IBA) was more effective when applied at 0.1 mg l⁻¹. Plantlet survival ex vitro was negatively affected when NAA and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to root shoot explants for all species. In W. laccata, all auxin treatments [IAA, IBA, NAA, phenylacetic acid (PAA), and 2,4-D] at a concentration of 1 mg l⁻¹ significantly reduced ex vitro survival of plantlets. Successful micropropagation of Watsonia is an important step in the further development of this genus as a horticultural crop.     

A Novel Method Based on Etiolation for Increasing Efficiency of In Vitro Propagation of Populus tomentosa

This paper describes a novel method based on etiolation treatment for micropropagation of Populus tomentosa. It includes: (1) Antibiotic “cefotaxime” (2.5 ppm in nutrient medium) is used to inhibit bacteria growth and the cultures which have been contaminated after long period subculture are recovered by sterilization with 1/10000 HgCl2(w/v). (2)Low concentration of TDZ (thidiazuron, 0.005 ppm) replaces expensive zeatin for enhancing bud differentiation and multiplication of leaf explants. (3) Shoot elongation is promoted after etiolation treatment, which will increase the number of shoots suitable for rooting, about 50 etiolated shoots are obtained in a 100 ml flask, 3—5 times more than those produced in traditional method. (4) Etiolated shoots or after their greening are used as cuttings for rooting in vivo and over 90% survival rate could be achieved when the medium is sterilized. This method is labour and money saving and high efficient.