Release Of Alkaloids Research Articles

The Effect of Gendarussin a Isolates of Justicia gendarussa Burm.f. Leaf in Reverse Transcriptase Inhibition of HIV Type I In Vitro

Screening has been done to a few extracts from the leaves Justicia gendarussa Burm.f to see the growth rate of the virus from the blood plasma of HIV patients at Dr Soetomo Hospital. It is known that J. gendarussa leaf extract inhibits HIV type 1 reverse transcriptase. In addition, its main content is gendarussin A, besides gendarussin B, JGF1, JGF2 and JGF3, which have just identified. At the beginning, extraction and fractionation were performed with 3 models that highlight the absolute methanol, 70% methanol and 70% ethanol with the release of alkaloids. Furthermore, samples of each fraction were incubated in plasma of HIV patients with a titer of 3.6 10^6 copies for 1 h in concentrations of 1.64 ppm, 4.1 ppm, 8.2 ppm, 16.4 ppm and 41.0 ppm. After incubation, examination was performed by using Nucli sens a machine, which is a combination of PCR and Elisa, thus avoiding direct contact with the highly pathogenic virus. The result showed that the activity sequence from the most potential to the weak, among others, was 1.64 ppm >4.1 ppm > 8.2 ppm > 16.4 ppm > 41.0 ppm, each with barriers value of 0.62 10^6, 1.4 10^6, 1.6 10^6, 2.4 10 cells/ml. In conclusion, highest anti-HIV activity comes from the concentration of gendarussin A isolate at 1.64 ppm. Furthermore, after linear regression of y = -3.063 x + 81.37 was done, the IC50 of 10.24 ppm was obtained.

Design, Development and Rationalization of Sarpagandha Ghanvati.

Sarpagandha ghanvati is a classical Ayurvedic formulation widely prescribed for anxiety and insomnia. It contains Sarpagandha (roots of Rauwolfia serpentina L. (Benth.) Ex Kurz; Family: Apocyanaceae), Khurasani ajowan (Hyocyamus niger L.; Family: Solanaceae) seeds, Jatamansi (Nardostachys jatamansi DC. Family: Valerianaceae) roots and Pipplamul (root of Piper longum L.; Family: Piperaceae). The objective of this study was to make a comparative evaluation of Ghanvatis and tablets of this formulation. Two tablet formulations were prepared; one incorporating only powders of all ingredients; the other with ethanol extracts of the first three ingredients and powder of Piper longum root. Similarly, two types of Sarpagandha ghanvati pills were prepared; one as per Ayurvedic Formulary of India; the other with ethanol extracts of the first three ingredients and powder of Piper longum root. Alcohol extracted 0.22% w/w of total alkaloids as against 0.061% w/w extracted by water. Tablets prepared with powders of all the ingredients had friability more than 3.0% where as those prepared with ethanol extract had very low friability. Ghanvatis, prepared as per the Ayurvedic formulary, did not show reserpine although other alkaloids were present. They showed less content uniformity and lower drug release. Ethanol extracted reserpine along with other alkaloids. Ghanvatis made with the alcoholic extracts exhibited better content uniformity and drug release than the traditional formulation. Tablets prepared with powders or extracts of the ingredients exhibited good content uniformity but the release of alkaloids from the tablets of powders was only 80%. Tablets of the extracts had good content uniformity with 90% release of the total alkaloids. Tablets prepared with alcoholic extracts using 1% polyvinylpyrrolidone as binder and 5% dried starch powder as disintegrating agent confirmed to all the requirements. Thus, the study shows tablets made with the extracts are superior to Ghanvatis and powder tablets.

Herbal delivery system for treatment of obesity administration of encapsulated khat-extracts on body weight of rats

Obesity is one of the most important problems worldwide. Khat (Catha edulis), an evergreen shrub, is thought to reduce body-weight. Its effect is more prominent when khat leaves are chewed. Thus, anti-obesity effects of khat and its associated side effects may depend on the release rate of its active constituents. The present study aimed to investigate the effect of a selected low dose of dried-khat, extracted, formulated as controlled release delivery systems on the body weight (BW), food intake (FI), cholesterol (CS) and triglyceride (TG) levels in rats. Khat extract (KE) was microencapsulated (KE235) and formulated into a parenteral implant (InjKE235). The effects of KE, KE235 and InjKE235 on BW, FI, CS and TG in rats were investigated. The results showed that microcapsules sustained the khat alkaloid release with T50% 1.58 h for KE235 and 14.41 days for InjKE235. KE and KE235 caused maximum reduction in BW, FI, CS and TG during the first to third weeks but rebound gradually thereafter. On the contrary, InjKE235 exhibited a sustained reduction in BW, FI, CS and TG levels for 2 months. The T50% of KE, KE235 and InjKE235 correlated with the reduction in BW, CS and TG but not with FI. In conclusion, the subcutaneous injection and sustained release rate of khat extract play an important role in enhancing the anti-obesity effect in SD rats.:

Extraction and Microencapsulation of Khat: Effects on Sexual Motivation and Estradiol Level in Female Rats

Khat (Catha edulis) is an evergreen tree/shrub that is thought to affect sexual motivation or libido. Its positive effect on sexual desire is more frequently observed in females than in males and occurs when khat is chewed. Thus, khat's effects on sexual behavior may depend on the release mode of its active constituent. This study aimed to investigate the effect of dried khat alkaloids on the sexual motivation and estradiol levels of female rats, with special emphasis on the importance of the sustained release effect. Dried khat leaves were extracted and isolated. The alkaloids in khat extract were identified and calculated using thin layer chromatography and high-performance liquid chromatography. The isolated khat extract was microencapsulated using a phase separation coacervation method. The morphology, particle size, yield, drug loading, and entrapment efficiency were evaluated. The in vitro release and stability of alkaloids in khat extract and in khat extract microcapsules were determined. The effect of khat extract microcapsules and varying doses of khat extract on sexual motivation in female rats were investigated. Additionally, estradiol levels, vaginal secretions and vaginal pH were determined. The differences in the effect of khat extract and khat extract microcapsules on sexual motivation, vaginal secretion and estradiol levels in female rats were compared. Results. Cathine and norephedrine were identified in the isolated khat extract at composition of 81.3% and 17.2%, respectively. Among the formulations studied, khat extract microcapsules of formulation 2:3:5 (containing a ratio of khat extract to ovalbumin to gelatin of 2:3:5) were found to exhibit higher yield, loading, and entrapment efficiency. Khat extract microcapsules showed sustained in vitro release and were more stable than khat extract. In addition, khat extract microcapsules enhanced sexual motivation, increased vaginal secretions, and upregulated estradiol level in female rats. The sustained release of alkaloids from dried khat has significantly enhanced the sexual motivation and increased the estradiol level of female rats. Thus the release of dried khat alkaloids from microcapsules might be an effective means of enhancing the libido in females.

Responses of transformed root culture of Atropa belladonna to salicylic acid stress

The effect of salicylic acid (SA) on tropane alkaloid production and the responses to SA stress of transformed root cultures of Atropa belladonna (belladonna) were investigated. Treatment of A. belladonna transformed roots with 0.2 mM SA did not have any effect on tropane alkaloid production, but two compounds were produced in the medium. These were identified as the SA derivatives methylsalicylate and methyl- o-methoxybenzoate by high-resolution mass spectrometry and UV spectrometry. In contrast, treatment with 2 mM salicylic acid stimulated tropane alkaloid release from the transformed roots into the medium by up to 35% of the total alkaloids after 24 h, and the SA derivatives were not observed in the medium. These results revealed that transformed root of A. belladonna exhibits distinct by different responses to SA stress depending on the SA concentration.

Responses of Transformed Root Culture of Atropa belladonna to Salicylic Acid Stress.

The effect of salicylic acid (SA) on tropane alkaloid production and the responses to SA stress of transformed root cultures of Atropa belladonna (belladonna) were investigated. Treatment of A. belladonna transformed roots with 0.2 mM SA did not have any effect on tropane alkaloid production, but two compounds were produced in the medium. These were identified as the SA derivatives methylsalicylate and methyl-o-methoxybenzoate by high-resolution mass spectrometry and UV spectrometry. In contrast, treatment with 2 mM salicylic acid stimulated tropane alkaloid release from the transformed roots into the medium by up to 35% of the total alkaloids after 24 h, and the SA derivatives were not observed in the medium. These results revealed that transformed root of A. belladonna exhibits distinct by different responses to SA stress depending on the SA concentration.

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l -1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l -1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine.

Effects of chemicals on alkaloid production by transformed roots of belladonna

Abstract The growth and tropane content in transformed root cultures of Atropa belladonna (strain M8) grown in 300 ml flasks were studied. After one month in culture, the biomass of the transformed roots increased 15 times and reached 5 g dry wt l −1 ; the alkaloid contents were 0.7% for (−)-hyoscyamine, 0.1% for 6 β -hydroxyhyoscyamine, 0.02% for scopolamine and 0.02% for littorine. During the culture period, no alkaloid could be detected in the medium. Seven chemicals were individually added to 18-day-old cultures. Glutathione (5 mM), chitin (0.1%), chitosan (0.1%) or yeast extract (0.1%) had no effect on the release of alkaloids into the medium. However, treatment with 5 mM H 2 O 2 induced a transient release of tropane alkaloids from transformed roots and the maximum amount of alkaloids in the medium was ca 0.35 mg per flask (10% of alkaloid contained in tissues); tissue viability was still preserved. Cu 2 and Cd 2 (5 mM) also improved the excretion of tropane alkaloids into the medium. However, their addition led to lysis of transformed roots.

Study on Oscillatory Behavior and Dynamic Release of Alkaloids of Cultured Cαthαrαnthus roseus Cells

Study on Oscillatory Behavior and Dynamic Release of Alkaloids of Cultured Cαthαrαnthus roseus Cells

Novel biotechnological approaches to obtain scopolamine and hyoscyamine: the influence of biotic elicitors and stress agents on cultures of transformed roots of Brugmansia candida

Scopolamine and hyoscyamine are tropane alkaloids employed in medicine as antispasmodics, preoperative medication and for the treatment of motion sickness. Since their chemical synthesis is difficult and expensive, these compounds are still extracted from plants that belong to several species of the Solanaceae. In this research, a biotechnological system for the production of both alkaloids was employed. Hairy roots of Brugmansia candida (syn. Datura candida), obtained through transformation with the soil pathogen Agrobacterium rhizogenes, were cultured in vitro. In an attempt to increase the productivity of the cultures, they were exposed to biotic elicitors (hemicellulase and homogenized B. candida roots) and stress agents (CuSO4). It was observed that hemicellulase had a positive effect on scopolamine (2-fold increase) and, especially, hyoscyamine accumulation (4-fold increase) in the roots. The homogenized roots also had a positive effect on alkaloid accumulation in the roots throughout the experiment (approximately 2.5-fold). The main effects of CuSO4 were seen in the release of alkaloids to the medium, particularly at 24 and 48 (6 to 10-fold increase). © 1998 John Wiley & Sons, Ltd.

Inducer effect of Tween 20 permeabilization treatment used for release of stored tropane alkaloids inDatura innoxia Mill. hairy root cultures

The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.

An integrated “Root tube” bioreactor/separator for transformed root cultures

The special morphology and biochemical characteristics of transformed hairy roots demand novel approaches in bioreactor concept to provide a favorable environment for root growth and product extraction. This paper describes our evaluation of the performance of an integrated bioreactor (1 l) and separation system. Growth rates of transformed Nicotiana glauca roots were comparable to those achieved in Erlenmeyer flasks, but the release of alkaloids was enhanced using production medium and heat treatment. Incorporation of an XAD-4 resin packed column separator also facillitated alkaloid release and improved recovery. In addition transformed roots of N. glauca, Nicotiana rustica and Datura stramonium were cultured in a 8 l bioreactor, achieving high growth rates and productivities comparable to reported values.

Permeabilization ofDatura innoxiaHairy Roots for Release of Stored Tropane Alkaloids

The effects of tween 20 as permeabilizing agent on tropane alkaloids from Datura innoxia Mill, hairy roots have been studied. For various tween 20 concentrations both hyoscyamine and scopolamine accumulated in the culture medium. Plant material viability could be preserved after a 24 hours-2% tween 20 concentration treatment. The time-course study of alkaloid release showed that the maximum of excretion occurred after a 20 hour contact with tween 20. At that time, a concentration of hyoscyamine superior to 25 mg/l was detected in the medium.

Effect of elicitation on the accumulation of solasodine by immobilized cells of Solanum eleagnifolium Cav.

The effect of a fungal elicitor obtained from Alternaria sp. on growth and solasodine production by free and alginate-entrapped cells of Solanum eleagnifolium Cav. was studied. Fourteen-day-old cultures were elicited with 1% FW/V autoclaved homogenates. The solasodine production increased from 0.9 to 1.5 mg g -1 DW (65%) in suspension cultures and from 0.75 to 1.4 mg g -1 DW (about 95%) in entrapped cells. The maximum accumulation was obtained after 72 h of elicitation. In order to induce alkaloid release from cells (suspension and entrapped cells), permeabilization with 10% dimethylsulfoxide (DMSO) for 30 min was used. In both cases (free and entrapped cells), about 50–60% of intracellular solasodine was released into the medium. The reuse of elicited and permeabilized entrapped cells was also carried out for three production cycles.

Biologically active secondary metabolites of barley. II. Phytotoxicity of barley allelochemicals

The release of alkaloids by barley was quantified by HPLC. Hordenine was released from the roots of barley in a hydroponic system for up to 60 days. The amount reached a maximum, 2μg/plant/day, at 36 days, then declined. Effects on white mustard by hordenine and gramine included reduction of radicle length and apparent reduction in health and vigor of radicle tips. Transmission electron microscopic examination of white mustard radicle tips exposed to hordenine and gramine showed damage to cell walls, increase in both size and number of vacuoles, autophagy, and disorganization of organelles. The evidence of the morphological and primary effects of barley allelochemicals at the levels released by living plants indicates that the biologically active secondary metabolites of barley may lead to a significant role in selfdefense by the crop.

Effect of the medium pH on the release of secondary metabolites from roots ofDatura stramonium, Catharanthus roseus, andTagetes patula cultured in vitro

The release of alkaloids from root culturesDatura stramonium andCatharanthus roseus and thiophenes from root cultures ofTagetes patula was found to increase when the pH of the culture media (ranging from 4.8 to 7.0) was reduced to 3.5. The extent of the effect was different in each type of culture. Increases ranged from 4- to 20-fold, which in some cases accounted for 75% of the total secondary metabolite pool produced per flask. When the release of individual metabolites was measured, even larger increases, were observed (nearly 400-fold for ajmalicine). Increased release of alkaloids fromC. roseus roots were also observed in cultures growing in a 14-L fermentor, when the medium pH was reduced. Reduction of the pH of the media did not affect growth of the root cultures in subsequent subcultures. The importance of this treatment as a stategy to improve the recovery of secondary metabolites from producing cultures is discussed.

Characteristics of Scopolamine-releasing Hairy Root Clones ofDuboisia leichhardtii

A good scopolamine-releasing hairy root clone, DL47-1, was developed from Duboisia leichhardtii. Hyoscyamine 6β-hydroxylase activity of the clone DL47-1 in the medium for alkaloid release was 2-fold higher than that in the medium for hairy root growth. Other high scopolamine-producing clones also tended to have a high enzyme activity.

Effect of polymeric adsorbents on the production of sanguinarine by Papaver somniferum cell cultures

The suitability of adsorbent polymeric resins, Amberlite XAD-4 and XAD-7 (Rohm and Hass, Inc.), was investigated for the accumulation of sanguinarine from Papaver somniferum cell cultures. The adsorption and desorption of sanguinarine from aqueous solution was most effective with XAD-7. In addition to sanguinarine, the resins were found to absorb growth regulators and vitamins from the culture medium. Growth inhibition was overcome by delaying for approximately 4 days resin addition after cell inoculation in fresh medium. Resin addition (5% wt/vol) to actively growing uneclicited cultures led to increases in sanguinarine production and release of 30% to 40% and 60%, respectively. The addition of resins to elicited cultures led to increases in alkaloid production of up to 50% to 85% with similar increases in alkaloid release as observed for nonelicited cells. Overall yield of sanguinarine increased from 21 mg . g biomass dry weight(-1) (dw) for elicited cultures to more than 39 mg . gdw(-1) when elicitation was combined with resin addition. Higher quantities of resin (10% to 20% wt/vol) increased marginally the release of sanguinarine into the medium, and on the resin, up to 85% of total production. The use of resin appears promising for the development of a bioprocess for sanguinarine production by cultured plant cells.

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 μg l −1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (< 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increases in total alkaloid (from ∼ 5–10 mg l −1 to 15 mg l −1), ajmalicine (from 0 to ∼ 0.29 mg l −1) and serpentine (from 0 to ∼ 0.20 mg l −1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.