Unlocking the Medicinal Potential of Plant-Derived Extracellular Vesicles: current Progress and Future Perspectives.

Botanical preparations for herbal medicine have received more and more attention from drug researchers, and the extraction of active ingredients and their successful clinical application have become an important direction of drug research in major pharmaceutical companies, but the complexity of extracts, multiple side effects, and significant individual differences have brought many difficulties to the clinical application of herbal preparations. It is noteworthy that extracellular vesicles as active biomolecules extracted from medicinal plants are believed to be useful for the treatment of a variety of diseases, including cancer, inflammation, regenerative-restorative and degenerative diseases, which may provide a new direction for the clinical utilization of herbal preparations. In this review, we sort out recent advances in medicinal plant extracellular vesicles and discuss their potential as disease therapeutics. Finally, future challenges and research directions for the clinical translation of medicinal plant extracellular vesicles are also discussed, and we expect that continued development based on medicinal plant extracellular vesicles will facilitate the clinical application of herbal preparations.

Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application.

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4-5 years). This research shows that H+-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell's resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K+ and Ca2+ trans-membrane flows using a patch-clamp method, together with OA modulation of H+-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH4Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La3+ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca2+, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Biotechnological Exploration of Transformed Root Culture for Value-Added Products.

Medicinal plants produce valuable secondary metabolites with anticancer, analgesic, anticholinergic or other activities, but low metabolite levels and limited available tissue restrict metabolite yields. Transformed root cultures, also called hairy roots, provide a feasible approach for producing valuable secondary metabolites. Various strategies have been used to enhance secondary metabolite production in hairy roots, including increasing substrate availability, regulating key biosynthetic genes, multigene engineering, combining genetic engineering and elicitation, using transcription factors (TFs), and introducing new genes. In this review, we focus on recent developments in hairy roots from medicinal plants, techniques to boost production of desired secondary metabolites, and the development of new technologies to study these metabolites. We also discuss recent trends, emerging applications, and future perspectives.

A new anti-inflammatory ß-carboline alkaloid from the hairy-root cultures of Eurycoma longifolia.

One new ß-carboline alkaloid 7-methoxy-(9H-ß-carbolin-1-il)-(E)-1-propenoic acid (1) together with 9-methoxycanthin-6-one (2) and 9-hydroxycanthin-6-one (3) were isolated from the hairy-root cultures of Eurycoma longifolia. The effects of these compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells were investigated. Compound 1 strongly inhibited the production of NO while 2 and 3 having weak or inactive effect. Consistently, compound 1 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase.

Phenolic compound production in relation to differentiation in cell and tissue cultures of Larrea divaricata (Cav.).

The lignan nordihydroguaiaretic acid (NDGA) and its derivatives existing in Larrea divaricata species show a wide range of pharmacological activities which makes this genus an interesting target to consider the plant in vitro cultivation systems as a feasible alternative source for their production. These compounds are potentially useful in treating diseases related to heart condition, asthma, arteriosclerosis, viral and bacterial infections, inflammation and cancer. In the present study, calli, cell suspension cultures, and in vitro and wild plants of L. divaricata were investigated for their potential to synthesize phenolic compounds. Calli, both with and without organogenesis, produced NDGA and quercetin, as did plantlet and wild plants. NDGA was also produced by the cell suspension cultures, together with p-coumaric acid, ferulic acid and sinapyl alcohol. The capacity of undifferentiated tissues to form phenolic compounds is very limited, but when the calli underwent organogenesis, developing mainly adventitious shoots, the phenolic compound production increased significantly. Plantlets regenerated from adventitious shoots of L. divaricata calli did not show the same phenolic pattern as wild plants, with levels of NDGA and quercetin being 3.6- and 5.9-fold lower, respectively.

Neolignans and sesquiterpenes from cell cultures of Stellera chamaejasme.

Two new neolignans (1, 2) and six sesquiterpenes (3- 8) were isolated from the cell cultures of Stellera chamaejasme. Their structures and absolute configurations were elucidated by extensive spectroscopic and computational methods. Compound 4 exhibited significant protective effects against CCl (4)-induced hepatotoxicity in HepG2 cells, reducing aspartate aminotransferase release by 29.49 % at 10 µM. These compounds have not been isolated from plant material, which implies that in vitro plant cell cultures may offer alternative and effective sources of bioactive natural compounds.

Nain-e Havandi Andrographis paniculata present yesterday, absent today: a plenary review on underutilized herb of Iran's pharmaceutical plants.

Nain-e Havandi (Andrographis paniculata Nees.) (AP) is an annual herbaceous plant belonging to the family Acanthacea. Only a few species of Andrographis genus out of 28 are medicinally concerned of which AP is the most important. Knowledge about the arrival of AP to Iran is extremely lacking but most probably it has been imported from India. However, evidence implies the familiarity of Iran's folkloric medicine with this plant, but it has been disappeared from contemporary medicine for unknown reasons. Presence of active ingredients from diterpenoids group such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide has given incredible unique medicinal properties to the plant. Traditionally, Nain-e Havandi has been used in the role of a non-farm plant as a remedy for skin problems, flu, respiratory disease, and snakebite in East and Southeast Asia for centuries. Recently, it has been utilized as a treatment for HIV, hepatitis, diabetes, cancer and kidney disorders. Intensive cultivation of the herb started only in the past decade in countries such as China, India, Thailand, Indonesia, West Indies, Mauritius and to some extent, in Malaysia. Availability of different ecological zones in Iran complies with reestablishment of AP in tropical and temperate regions of the country. This is killing two birds with one stone, supporting the conservational and economic aspects.

Effects of Indian herbal formulation Body Revival on human platelet aggregation and myocardial ischemia in rats.

OBJECTIVE: To study the effect of Body Revival (BR), a compound traditional Indian herbal medicine, on human platelet aggregation and isoproterenol (IS)-induced myocardial ischemia (MI) damage in male Wistar rats. METHODS: BR suspension 10, 20 and 30 µg was mixed with platelet-rich plasma and incubated at 37 degrees centigrade for 30 min, respectively. Then, adenosine diphosphate (ADP, 20 mmol/L) or collagen (2 µg) was added in the mixture and the aggregation was observed against platelet-poor plasma mixed with equal volume of suspension of the same test samples. Wistar rats divided into 4 groups were used to investigate BR's effects on IS-induced MI. Levels of serum creatinine kinase (CK), aspartate transaminase (AST) and alanine transaminase (ALT) were estimated by standard commercial biological kits. Serum nitric oxide (NOx) was also measured. The lipid peroxides (LPO) and protein concentrations in heart tissues were measured. RESULTS: BR could inhibit ADP- or collagen-induced human platelet aggregation dose-dependently. Moreover, it could protect MI caused by IS in rats. BR reduced the levels of serum CK, AST, ALT and NOx dose-dependently and also lowered LPO in heart tissues in comparison with the MI control (P<0.01). CONCLUSION: BR can inhibit human platelet aggregation and protect MI caused by IS in rats.

Biotransformation of valdecoxib by plant cell cultures.

Valdecoxib is a new anti-inflammatory drug that is highly selective for inhibition of the inducible form of cyclooxygenase (COX-2). In the present study, biotransformation of valdecoxib was investigated in cell cultures of five medicinal plants, viz., Catharanthus roseus, Azadirachta indica, Capsicum annuum, Ervatamia heyneana, and Nicotiana tabacum. Identification of the biotransformed products was carried out by using high-performance liquid chromatography coupled with diode array detection and liquid chromatography--tandem mass spectrometry analysis. All the cultures transformed valdecoxib into more polar compounds, and C. roseus also produced one unknown compound that is less polar than the substrate. The reactions performed by these plant cell cultures include hydroxylation, methylation, and demethylation. Optimization studies were performed to investigate the effect of the day of extraction and substrate concentration on biotransformation.

[Pharmacognostic identification of Rodersia aesculifolia].

OBJECTIVE: To establish pharmacognostical methods of Rodersia aesculifolia. METHODS: Macroscopical, microscopic and physicochemical identification were used to authenticate this crude drug, and the identification characteristics were studied. RESULTS: There were some palea on the rhizoma superior extremity and some white flare dot on transverse section. Vascular bundle formed into interval circularity in rhizoma transverse section, and some small atypia bundle appeared in lateral of core. Xylem of root was tetrarch. A lot of starch grain and acicular cyrstal were observed in parenchyma cell. Brown cell was present as single or several linked. CONCLUSION: These characteristics can be used as identification basis for Rodersia aesulifolia Batal.

Actividad antimicrobiana de plantas medicinales argentinas sobre bacterias antibiótico-resistentes / Antimicrobial activity of Argentine medicinal plants on antibiotic-resistant bacteria

El propósito del presente trabajo fue determinar la potencia antimicrobiana de extractos alcohólicos de plantas utilizadas popularmente en Argentina como antisépticos y antiinflamatorios: Dasyphyllum diaconthoides, Erythrina cristagalli, Larrea cuneifolia, Larrea divaricata, Phytolacca dioica, Pithecoctenium cynanchoides, Prosopanche americana, Schinus molle, Schkuhria pinnata, Senna aphylla y Solidago chilensis. La inhibición del crecimiento bacteriano se determinó a través de ensayos de difusión en agar, macrodilución en medio sólido y microdilución en medio líquido frente a 47 aislamientos clínicos multirresistentes a antibióticos, obtenidos de pacientes de un hospital de Tucumán, Argentina: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. De acuerdo con los valores de concentración inhibitoria mínima (CIM), tres de las once especies ensayadas fueron las más activas: L. divaricata, L. cuneifolia, y S. aphylla (CIM de 25 a 200 µg/mL). P. mirabilis, A. baumanni y S. maltophilia fueron las cepas más susceptibles con valores de CIM entre 25 y 50 µg/mL seguido por P. aeruginosa con valores de CIM de 50 a 100 µg/mL. Los valores de concentración bactericida mínima (CBM) fueron semejantes o dos veces superiores a los valores de CIM. Mediante ensayos bioautográficos se comprobó que los extractos más activos presentaban al menos dos principios antimicrobianos. Análisis fitoquímicos indican que estos compuestos son de naturaleza fenólica. Los resultados obtenidos justificarían el uso de estos extractos para el tratamiento de infecciones bacterianas, especialmente aquellas de origen dérmico.

The present study was conducted to investigate antimicrobial activity of alcoholic extracts of Argentine medicinal plant species (Dasyphyllum diacanthoides, Erythrina cristagalli, Larrea cuneifolia, Larrea divaricata, Phytolacca dioica, Pithecoctenium cynanchoides, Prosopanche americana, Schinus molle, Schkuhria pinnata, Senna aphylla and Solidago chilensis) against multidrug resistant human pathogen gram negative bacteria isolated from a Hospital in Tucumán, Argentina. Inhibition of bacterial growth was investigated using disc diffusion, agar macrodilution and broth microdilution methods against multiresistant clinical isolates of nine different specie of gram negative bacteria: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. A significant antimicrobial activity was found in three of the eleven plant species studied. Based on minimal inhibitory concentration (MIC) values, three plant species, L. divaricata, L. cuneifolia, and S. aphylla were the most potent ones with MIC values between 25-200 µg/mL. Overall, P. mirabilis, M. morganii and P. aeruginosa isolates were the most susceptible to these extracts with MIC values of 25 to 50 µg/mL. All extracts showed significant inhibitory activities on bacteria growth in a dose phenolic compound-dependent fashion. The minimal bactericidal concentration (MBC) values were identical to the MIC values or twofold higher than the corresponding MIC. Contact bioautography indicated that crude extracts possess several major antibacterial components. Phytochemical screening showed that the bioactive compounds correspond to polyphenols. Investigations are in progress to purify the bioactive principles.

Isolation and characterization of a novel glutathione S-transferase-activating peptide from the oriental medicinal plant Phellodendron amurense.

The aim of this study was to elucidate the characteristics of glutathione S-transferase (GST)-activating compounds from medicinal plants. Among 265 kinds of medicinal plants, Phellodendron amurense showed the highest GST activity at 174.8%. The GST-activating compound of P. amurense was maximally extracted when treated with distilled water at 30 degrees C for 12 h. The compound was purified by ultrafiltration, Sephadex G-10 gel filtration chromatography, and reverse-phase HPLC. The purified GST-activating compound from P. amurense was a novel tetrapeptide with an amino acid sequence of Ala-Pro-Trp-Cys and its molecular weight was estimated to be 476 Da. It also displayed a clear detoxicative effect in 1-chloro-2,4-dinitrobenzene treated mice at a dosage of mg/kg body weight.

Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin.

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.

Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model.

We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.

[Comparison on ingredients between cultured cell and cultivated Saffron pistils].

OBJECTIVE: To compare the differences of active ingredients between tissue cultured cells and cultivated saffron pistils. METHOD: The experiment was carried out by the Fourier transform infrared spectroscopy (FTIR) spectra and high performance liquid chromatography (HPLC). RESULT: The data indicated that the species and contents active ingredients in saffron pistils from different places were different. The species of active ingredients in tissue cultured cells are less than those in cultivated saffron pistils. However, the quantity of crocin A, which showed good anticancer effect, is 2-3 times more than that in cultivated saffron pistils. CONCLUSION: The active ingredients of the tissue cultured cells are similar to those of saffron pistils, but their contents are different. Therefore, the tissue cultured cells can only be the part-substitutes of cultivated saffron pistils.

Ultrasound-induced stress responses of Panax ginseng cells: enzymatic browning and phenolics production.

The stress metabolic activities of Panax ginseng (P. ginseng) cells induced by low-energy ultrasound (US) were examined. P. ginseng cells in suspension cultures were exposed to 38.5 kHz US at two power levels (power density 13.7 and 61 mW/cm(3)) for 2 min. The US treatment caused rapid increase in the intracellular levels of polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) and the production of polyphenols (PP) and phenolic compounds. The US-induced enzyme activities and phenolics production are part of plant stress responses to a mechanical stimulus. The much higher PPO activity and rate of PP production in the sonicated cultures are correlated to enzymatic browning, suggestive of physical damage and membrane permeabilization of the cells by US. The cells after sonication also showed decreased water content and cell volume, which may also be attributed to US-induced cell membrane permeabilization and water release. High-pressure shock and fluid shear stress arising from acoustic cavitation were regarded as the major causes of the responses. Nevertheless, the US exposure caused only temporary cell growth depression but no net loss of biomass yield of the culture.

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus.

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.

[Morphological and histological study on Pteris multifida].

The thesis studies on the pharmacognostical characteristics of Pteris multifida were carried out in this paper. The blade has special vein-like cell. The stomas in dorsal epidermis are regular. The petiol has V-shaped amphicribral bundles. The rhizome has 6-8 meristeles. The triangle-form spore has tumor-shaped or pellet-shaped decorations on its surface.