Kalanchoe pinnata (Lam.) Pers. Leaf ethanolic extract exerts selective anticancer activity through ROS-induced apoptotic cell death in human cancer cell lines.

BACKGROUND: The leaves of Kalanchoe pinnata (Lam.) Pers. (K. pinnata), a succulent plant native to tropical regions, are used as a medicinal alternative against cancer in several countries worldwide; however, its therapeutic potential to fight cancer has been little addressed. In this study, we analyzed the phytochemical content, antioxidant capacity, and selectivity of K. pinnata leaf ethanolic extract against different human cancer cell lines in vitro. METHODOLOGY: This study subjected the ethanolic extract to enzymatic assays to quantify the phytochemical content (phenolics, flavonoids, and anthraquinones) and its radical scavenging and iron-reducing capacities. Also, the phytoconstituents and major phenolic compounds present in the extract's subfractions were identified by GC-MS, HPLC, and NMR. Human cancer (MCF-7, PC-3, HT-29) and normal colon (CoN) cell lines were treated with different concentrations of K. pinnata leaf ethanolic extract, and the changes in cell proliferation (sulforhodamine B assay), caspases activity (FITC-VAD-FMK reporter), mitochondrial membrane potential (MMP, rhodamine 123 assay), chromatin condensation/fragmentation (Hoechst 33342 stain), and ROS generation (DCFH2 probe assay) were assessed. RESULTS: The results showed that the K. pinnata leaf ethanolic extract is rich in phytoconstituents with therapeutic potential, including phenols (quercetin and kaempferol), flavonoids, fatty acid esters (34.6% of the total composition), 1- triacontanol and sterols (ergosterol and stigmasterol, 15.4% of the total composition); however, it presents a poor content of antioxidant molecules (IC50 = 27.6 mg/mL for H2O2 scavenging activity vs. 2.86 mg/mL in the case of Trolox). Notably, the extract inhibited cell proliferation and reduced MMP in all human cell lines tested but showed selectivity for HT-29 colon cancer cells compared to CoN normal cells (SI = 8.4). Furthermore, ROS generation, caspase activity, and chromatin condensation/fragmentation were augmented significantly in cancer-derived cell lines, indicating a selective cytotoxic effect. CONCLUSION: These findings reveal that the K. pinnata leaf ethanolic extract contains several bioactive molecules with therapeutic potential, capable of displaying selective cytotoxicity in different human cancer cell lines.

Exploring the Pharmacognostical and Phytochemical Profiles of Aqueous Extracts of Kalanchoe.

The use of plants for medicinal purposes has a long history, however it is desirable a continuous evaluation seeking for complementary scientific evidences for their safe application. Species within the Kalanchoe genus are often referred to as "miracle leaf" due to their remarkable healing properties. Traditionally, these plants have been used to treat infections, inflammation, and cancer. Despite their widespread use, the identification of their active components remains incomplete. This study aimed to differentiate K. crenata (KC), K. marmorata (KM), and K. pinnata (KP) by conducting detailed histochemical and phytochemical analyses, and to assess their antioxidant capabilities. The investigation revealed significant differences between the species, highlighting the variability in phenolic (PC) and flavonoid contents (FC) and their distinct antioxidant effects. The KM demonstrated the greatest results (PC: 59.26±1.53 mgEqGA/g; FC: 12.63±0.91 mgEqCQ/g; DPPH⋅ (IC50): 110.66 ug/mL; ABTS⋅+ (IC50): 26.81 ug/mL; ORAC: 9.65±0.75 mmolTE) when compared to KC and KP. These findings underscore a new reference for research within the Kalanchoe genus.

A Nuclear Magnetic Resonance (NMR)- and Mass Spectrometry (MS)-Based Saturation Kinetics Model of a Bryophyllum pinnatum Decoction as a Treatment for Kidney Stones.

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5-10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

A method of genetic transformation and gene editing of succulents without tissue culture.

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

Exposure to Light of the Abaxial versus Adaxial Side of Detached Kalanchoë blossfeldiana Leaves Affects Anthocyanin Content and Composition Differently.

The accumulation and composition of anthocyanins in leaves of Kalanchoë blossfeldiana, detached and kept for five days under natural light conditions, were investigated. The presence of fifteen derivatives of cyanidin, petunidin, and delphinidin was found. Changes in the content of each anthocyanin in the leaves before and after exposure to light on the abaxial (naturally upper) and adaxial (naturally lower) sides of the leaves were compared. When the adaxial side was exposed to light, the anthocyanin contents of the leaves did not change. In contrast, when the abaxial side of detached leaves was exposed to light, there was enhanced accumulation of delphinidin-rhamnoside-glucoside, cyanidin-rhamnoside-glucoside, cyanidin-glucoside-glucoside, and two unknown derivatives of petunidin and delphinidin. Application of methyl jasmonate (JA-Me) on the abaxial side exposed to light inhibited the accumulation of these anthocyanins. This effect could probably be due to the presence of these anthocyanins in the epidermal cells of K. blossfeldiana leaves and was visible in the microscopic view of its cross-section. These anthocyanins were directly exposed to JA-Me, leading to inhibition of their formation and/or accumulation. The lack of significant effects of JA-Me on anthocyanin mono- and tri-glycosides may indicate that they are mainly present in the mesophyll tissue of the leaf.

Spectroscopic investigation of phase transformation of calcium oxalate dehydrates (renal calculi) using acidic Bryophyllum pinnatom powder.

Urolithiasis is one of most common renal disorders, characterized by the formation of kidney stones (renal calculi) through the crystallization process within the urinary system. The frequently observed renal calculi are calcium oxalate renal calculi and treatment is done by shock wave method or lithotripsy which is harmful for other cells of the internal system. The objective of this work was to evaluate in vitro diagnosis of calcium oxalate kidney stones in the aqueous solution of Bryophyllum pinnatum. The B. pinnatum powder was mixed in apple cider vinegar and lemon juice separately to make solution 1 and 2 respectively. Apple cider vinegar and lemon juice were used as solvents due to their acidic and body compatible nature. Two surgically removed stones was dipped in solution 1 and 2. After two weeks, kidney stone of weight 2.7 g is completely dissolved in solution 2 while a considerable weight reduction of other kidney stone has been observed in solution 1. Fourier transform infrared (FTIR) spectroscopy results show the presence of two strong absorption peaks at 610 and 912 (cm-1) in both solutions after dissolution of urinary stones are related to calcium oxalate dehydrate (COD). Raman spectra further confirm the dissolution of COD in solution having Raman shifts at 504 and 910 (cm-1). Cluster formation and aggregation of particles has been observed in scanning electron microscopy images. This in vitro study proves that a mixture of Bryophyllum pinnatum powder and lemon juice is a best remedy to remove kidney stones.

A Review of the Popular Uses, Anatomical, Chemical, and Biological Aspects of Kalanchoe (Crassulaceae): A Genus of Plants Known as "Miracle Leaf".

Species of the genus Kalanchoe have a long history of therapeutic use in ethnomedicine linked to their remarkable healing properties. Several species have chemical and anatomical similarities, often leading to confusion when they are used in folk medicine. This review aims to provide an overview and discussion of the reported traditional uses, botanical aspects, chemical constituents, and pharmacological potential of the Kalanchoe species. Published scientific materials were collected from the PubMed and SciFinder databases without restriction regarding the year of publication through April 2023. Ethnopharmacological knowledge suggests that these species have been used to treat infections, inflammation, injuries, and other disorders. Typically, all parts of the plant are used for medicinal purposes either as crude extract or juice. Botanical evaluation can clarify species differentiation and can enable correct identification and validation of the scientific data. Flavonoids are the most common classes of secondary metabolites identified from Kalanchoe species and can be correlated with some biological studies (antioxidant, anti-inflammatory, and antimicrobial potential). This review summarizes several topics related to the Kalanchoe genus, supporting future studies regarding other unexplored research areas. The need to conduct further studies to confirm the popular uses and biological activities of bioactive compounds is also highlighted.

Metabolomics-based study on the changes of endogenous metabolites during adventitious bud formation from leaf margin of Bryophyllum pinnatum (Lam.) Oken.

Bryophyllum pinnatum (Lam.) Oken is an ornamental and ethno-medicine plant, which can grow a circle of adventitious bud around the leaf margin. The dynamic change of metabolites during the development of B. pinnatum remains poorly understood. Here, leaves from B. pinnatum at four developmental stages were sampled based on morphological characteristics. A non-targeted metabolomics approach was used to evaluate the changes of endogenous metabolites during adventitious bud formation in B. pinnatum. The results showed that differential metabolites were mainly enriched in sphingolipid metabolism, flavone and flavonol biosynthesis, phenylalanine metabolism, and tricarboxylic acid cycle pathway. The metabolites assigned to amino acids, flavonoids, sphingolipids, and the plant hormone jasmonic acid decreased from period Ⅰ to Ⅱ, and then increased from period Ⅲ to Ⅳ with the emergence of adventitious bud (period Ⅲ). While the metabolites related to the tricarboxylic acid cycle showed a trend of first increasing and then decreasing during the four observation periods. Depending on the metabolite changes, leaves may provide conditions similar to in vitro culture for adventitious bud to occur, thus enabling adventitious bud to grow at the leaf edge. Our results provide a basis for illustrating the regulatory mechanisms of adventitious bud in B. pinnatum.

Degradation of Diazo Dye and its Kinetic and Equilibrium Studies Using the Potential of Bryophyllum fedtschenkoiin Aqueous System.

Phytoremediation is emerging as an eco-friendly, innovative, and cost-effective approach for the removal of textile dyes from wastewater from the previous decade. The current research work aims to explore the potential of a terrestrial ornamental plant, Bryophyllum fedtschenkoi (Raym.-Hamet & H. Perrier) Lauz.-March. for remediating the diazo dye as Congo red (CR) in aqueous form. B. fedtschenkoi was grown hydroponically before treatment with 100 mL of a different concentration of CR dye solution. A maximum decolorization potential of 90% was obtained for 10 mg L- 1 after 40 h of equilibrium. The kinetic studies have revealed that the experimental results for the removal of CR dye using the B .fedtschenkoi plant are suitable for Pseudo-first order with R2 ≥ 0.92, while the equilibrium studies agreed with the Freundlich adsorption isotherm with R2 ≥ 0.909. The dye removal by the plant was confirmed with the help of analytical techniques Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). Gas Chromatography-Mass Spectrometry (GC-MS) and High-Performance Liquid Chromatography (HPLC) were also performed on dye-degraded metabolites to explore the mechanism of dye degradation.

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

BACKGROUND AND AIMS: Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis characterized by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as a substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating daytime stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. In this study, we tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been reprogrammed during evolution to enable nocturnal stomatal opening and daytime closure. METHODS: Biochemical and genetic characterization of wild-type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants. KEY RESULTS: Starch deficiency reduced nocturnal net CO2 uptake but had negligible impact on nocturnal stomatal opening. In contrast, daytime stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed daytime stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll. CONCLUSIONS: Nocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring daytime stomatal closure in this CAM species.

Bryophyllum pinnatum leaf extract mediated ZnO nanoparticles with prodigious potential for solar driven photocatalytic degradation of industrial contaminants.

In an era of environment-friendly development plant extract-based biological techniques for synthesizing nanoparticles have gained a lot of attention over traditionally famous chemical and physical synthesis techniques. In the present study we have synthesized biogenic zinc oxide nanoparticles (BPLE-ZnO NPs) using Bryophyllum pinnatum leaf extract, compared its native properties and solar-driven photocatalytic activity with chemically prepared ZnO nanoparticles (Chem-ZnO NPs). In order to characterize and compare the Chem-ZnO and BPLE-ZnO, various techniques were used, including UV-visible spectroscopy, x-ray diffractrometry, photoluminescence spectroscopy, field emission scanning electron microscopy, electron dispersive x-ray spectroscopy, fourier transform infrared spectroscopy, and zeta potential analyzer. The results revealed the formation of hexagonal wurtzite ZnO, with no significant difference between the two methods; however, the use of Bryophyllum pinnatum leaf extract in ZnO NPs synthesis resulted in reduced size, presence of biomolecules on its surface and better monodispersity than purely chemical synthesis. Further, the BPLE-ZnO NPs showed better efficiency in the solar-driven photocatalytic degradation of methylene blue (MB) dye compared to Chem-ZnO NPs. Under solar exposure at a dose of 0.50 mg/mL BPLE-ZnO, resulted in 97.31% photodegradation with a rate constant of 0.06 min-1 of 20 mg/L MB solution within just 60 min which was 9.51% higher compared to the Chem-ZnO NPs. The BPLE-ZnO NPs were also employed to investigate their solar-driven photocatalytic performance for degrading the pharmaceutical (Metronidazole and Amoxycillin) and textile pollutants (Methyl orange dye) under sunlight. The results show that Bryophyllum pinnatum leaf extract-mediated ZnO NPs have an excellent potential in solar-based photocatalytic applications.

Potential of Kalanchoe pinnata as a Cancer Treatment Adjuvant and an Epigenetic Regulator.

Cancer is a global public health problem that is related to different environmental and lifestyle factors. Although the combination of screening, prevention, and treatment of cancer has resulted in increased patient survival, conventional treatments sometimes have therapeutic limitations such as resistance to drugs or severe side effects. Oriental culture includes herbal medicine as a complementary therapy in combination with chemotherapy or radiotherapy. This study aimed to identify the bioactive ingredients in Kalanchoe pinnata, a succulent herb with ethnomedical applications for several diseases, including cancer, and reveal its anticancer mechanisms through a molecular approach. The herb contains gallic acid, caffeic acid, coumaric acid, quercetin, quercitrin, isorhamnetin, kaempferol, bersaldegenin, bryophyllin a, bryophyllin c, bryophynol, bryophyllol and bryophollone, stigmasterol, campesterol, and other elements. Its phytochemicals participate in the regulation of proliferation, apoptosis, cell migration, angiogenesis, metastasis, oxidative stress, and autophagy. They have the potential to act as epigenetic drugs by reverting the acquired epigenetic changes associated with tumor resistance to therapy-such as the promoter methylation of suppressor genes, inhibition of DNMT1 and DNMT3b activity, and HDAC regulation-through methylation, thereby regulating the expression of genes involved in the PI3K/Akt/mTOR, Nrf2/Keap1, MEK/ERK, and Wnt/ß-catenin pathways. All of the data support the use of K. pinnata as an adjuvant in cancer treatment.

Antimicrobial Activity of ß-Sitosterol Isolated from Kalanchoe tomentosa Leaves Against Staphylococcus aureus and Klebsiella pneumonia.

<b>Background and Objective:</b> <i>Kalanchoe tomentosa</i> is identified and their different characteristics regarding the antibacterial and antioxidant properties have a vast effect. Fresh <i>K. tomentosa</i> leaves obtained from Bandung, Indonesia was extracted using n-hexane followed by serial dichloromethane maceration. <b>Materials and Methods:</b> N-hexane and ethyl acetate were used to separate the dichloromethane extract using vacuum liquid chromatography and the isolated compounds were recrystallized with n-hexane. <b>Results:</b> About 37 mg of dichloromethane extract was obtained from the extraction process. Recrystallized compound isolates were identified as stigmast-5-en-3-ol or ß-sitosterol. Both dichloromethane extract and ß-sitosterol isolated compounds showed strong bacteriostatic activity against <i>S. aureus</i> with MIC = 15.63 and 7.81 µg mL¹ and<i> K. pneumonia</i> with MIC = 7.81 and 31.25 µg mL¹, respectively. However, only dichloromethane extract exhibited a bactericidal effect (7.81 µg mL¹). <b>Conclusion:</b> The pure ß-sitosterol compound was isolated from<i> K. tomentosa</i> dichloromethane extract. Both the dichloromethane extract and the isolated ß-sitosterol compound had antibacterial effects against <i>S. aureus</i> and <i>K. pneumonia.</i>.

Kalanchoë blossfeldiana naturally transformed with Rhizobium rhizogenes exhibits superior root phenotype.

Plant transformation with root oncogenic loci (rol) genes and open reading frames (ORFs) from Rhizobium rhizogenes have not yet targeted the underground root phenotype of these transformants. Hence, there is a need to develop plants with more efficient root system architecture (RSA). Here, RSA was assessed in naturally transformed (NT) and single rol/ORF Kalanchoë blossfeldiana 'Molly' lines in an aeroponic growth system combined with gene expression analysis. Three NT lines; 306, 324 and 331; exhibited better-developed RSA with longer roots and increased root biomass. In line 306, longest root was 6.3 ±â€¯0.3 cm while WT had 4.8 ±â€¯0.1 cm. However, root length of all overexpressing lines was ca. 30% shorter than WT. Root fresh weight of NT lines was 4.5-fold higher than WT. The expression of rolB, ∆ORF13a and ORF14 in the leaves of overexpressing lines was many folds higher than in NT lines. Increased expression of ∆ORF13a and ORF14 in leaves and roots may contribute more to a stronger compact phenotype than previously assumed. The moderate compact phenotype of NT lines combined with improved RSA compared to the overexpressing lines and WT strongly indicate that the use of R. rhizogenes has great potential to produce Kalanchoë phenotypes with enhanced RSA.

Bio-fabrication of Bio-inspired Silica Nanomaterials from Bryophyllum pinnatum Leaf for Agricultural Applications.

A green chemistry approach was employed to synthesize silica nanoparticles (SiNPs) using aqueous extract of Bryophyllum pinnatum leaf as capping agents. The novelty of this study was to produce silica nanoparticles using the biological method. An analysis of the physicochemical properties of formed nanoparticles was successfully completed through sophisticated characterization methods, such as UV-Visible absorbance spectroscopy, Fourier transform infra-red spectroscopy, X-ray diffraction, scanning electron microscope, energy dispersive X-ray, zeta potential analysis, and thermo-gravimetric analysis. All the characterization results indicated their spherical morphology and amorphous nature with an average size of 24 nm. FT-IR results highlighted the key bioactive compounds that could be responsible for capping and reducing the formation of SiNPs. Synthesized SiNPs show excellent stability with a negative zeta potential value of - 32 mV. The biomolecules from B. pinnatum were successfully working for the formation of Si NPs with spherical shapes. Moreover, to assess the agricultural application, green-synthesized SiNPs were carried out by seed germination assay on Vigna radiata. The seed germination assay confirms that a low concentration of SiNPs enhances seed germination. Meanwhile, a higher concentration of the SiNPs inhibits seed germination and shoot, and root formation. SiNPs at optimum concentration could be used in the agriculture field as nano growth promoters.

Antiurolithiatic Potential of Three Sri Lankan Medicinal Plants by the Inhibition of Nucleation, Growth, and Aggregation of Calcium Oxalate Crystals In Vitro.

Background: Deposition and formation of stones in any part of the urinary system is called urolithiasis. CaOx is the predominant component of most stones, and the formation of these stones is a multistep process that includes supersaturation, nucleation, aggregation, growth, and retention. In ayurvedic medicine, medicinal plants are used for the management of kidney stones. The objective of this study was to determine the effect of aqueous, ethanol, and hexane extracts of Drymoglossum piloselloides leaves, Kalanchoe laciniata leaves, and Aegle marmelos flowers against CaOx urolithiasis in vitro. Methods: The crystallization of CaOx monohydrate (COM) and dihydrate (COD) was induced in a synthetic urine system. The nucleation, growth, and aggregation of crystals were measured using spectrophotometric methods. The results were compared against the polyherbal drug, Cystone, under identical concentrations. Crystals generated in the urine were also observed under light microscopy. Statistical differences and percentage inhibitions were calculated using standard formulae and compared. A preliminary phytochemical screening was also performed to detect active phytoconstituents present in the three plants used in the study. Results: The results obtained clearly demonstrated that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the capacity to inhibit the nucleation, growth, and aggregation of CaOx crystals. Microscopic examination of crystals revealed the presence of more COM than COD crystals but a dose-dependent reduction in crystals was observed in the presence of plant extracts. Hexane, ethanol, and aqueous extracts of all three plants had different capabilities to inhibit nucleation, growth, and aggregation of CaOx crystals but their activities were different at different concentrations. Preliminary phytochemical screening revealed the presence of reducing sugars, proteins, flavonoids, tannins, and polyphenol compound in Kalanchoe laciniata and Drymoglossum piloselloides and reducing sugars, proteins, anthracene glycosides, and saponins in Aegle marmelos. Conclusions: This study provided evidence that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the potential to be developed as inhibitors of nucleation, growth, and aggregation of CaOx crystals in the treatment of urolithiasis.

An In Vitro Anticancer, Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier.

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC50 values were 1750 ± 140 and 1271.82 ± 53.25 µg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10.

The psychoactive effects of Bryophyllum pinnatum (Lam.) Oken leaves in young zebrafish.

Bryophyllum pinnatum (Lam.) Oken (BP) is a plant that is used worldwide to treat inflammation, infections, anxiety, restlessness, and sleep disorders. While it is known that BP leaves are rich in flavonoids, the extent of the beneficial and toxic effects of its crude extracts remains unclear. Although some neurobehavioral studies using leaf extracts have been conducted, none has examined the effects of water-extracted leaf samples. The zebrafish is a powerful animal model used to gain insights into the efficacy and toxicity profiles of this plant due to its high fecundity, external development, and ease of performing behavioral assays. In this study, we performed behavioral testing after acute exposure to different concentrations of aqueous extract from leaves of B. pinnatum (LABP) on larval zebrafish, investigating light/dark preference, thigmotaxis, and locomotor activity parameters under both normal and stressed conditions. LABP demonstrated dose-and time-dependent biphasic effects on larval behavior. Acute exposure (25 min) to 500 mg/L LABP resulted in decreased locomotor activity. Exposure to 300 mg/L LABP during the sleep cycle decreased dark avoidance and thigmotaxis while increasing swimming velocity. After sleep deprivation, the group treated with 100 mg/L LABP showed decreased dark avoidance and increased velocity. After a heating stressor, the 30 mg/L and 300 mg/L LABP-treated groups showed decreased dark avoidance. These results suggest both anxiolytic and psychoactive effects of LABP in a dose-dependent manner in a larval zebrafish model. These findings provide a better understanding of the mechanisms underlying relevant behavioral effects, consequently supporting the safe and effective use of LABP for the treatment of mood disorders.

Cytotoxic and genotoxic effects of Kalanchoe pinnata (Lam.) Pers. fresh leaf juice in the cultured human blood lymphocytes.

This is the first investigation on the in vitro cytotoxicological and genotoxicological effects of Kalanchoe pinnata (Lam.) Pers. fresh leaf juice, for establishing a safe and effective quantity for use. Peripheral blood of 6 healthy, non-addicted males between 20 and 25 years of age was used for toxicity assessment by 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), mitotic index (MI), sister chromatid exchanges (SCEs) and Cytokinesis Blocked Micronucleus (CBMN) assays. The IC50 of the leaf juice calculated by MTT assay was 155.1 µl. The 50 and70 µl of the juice concentrations were decided after standardization by MI and showed non-significant and significant decrease respectively when compared to control. The SCEs/Cell and SCEs/Chromosome were increased non-significantly (50 µl) and significantly (70 µl),while cell cycle proliferative index, Average generation time and Population doubling time values were non-significant for both doses when compared to the controls. In CBMN assay, the Cytokinesis block proliferation index, cytotoxicity, Micronuclei, Nuclear bud, Nucleoplasmic bridge frequencies and total DNA damage biomarkers showed non-significant changes for both 50 and 70 µl. The changes observed were significant only at 70 µl for MI and SCEs, which were significantly lower than that by positive control indicating a non genotoxic effect. Hence, the fresh leaf juice can be used pharmaceutically as well as traditionally, but for long durations and higher doses should be used with caution, as it can have mutagenic effect at particularly high levels.

Effects of Bryophyllum pinnatum Administration on Wistar Rat Pregnancy: Biochemical and Histological Aspects.

INTRODUCTION: Bryophyllum pinnatum is widely used in folk medicine. It has neuropharmacological, anti-inflammatory, immunomodulatory, antidiabetic, hepatoprotective, and nephroprotective effects, among others. It also acts on uterine contractility. It is prescribed by practitioners of anthroposophic medicine for preterm labor, insomnia, and emotional disorders, and has other potential uses in obstetrics. As all drugs currently used in preterm labor have side effects, new tocolytic agents remain an area of active research. OBJECTIVE: To evaluate the effect of B. pinnatum mother tincture (MT) on albino rats and their offspring throughout pregnancy from a biochemical and histological standpoint. METHODS: Longitudinal, prospective, randomized controlled bioassay. This is the second stage of a trial that investigated 60 animals distributed across six equal groups: controls C1 and C2, which received 1 and 25 times the vehicle dose (30% ethanol), B1 and B2 (1- and 25-fold doses of B. pinnatum MT), and B3 and B4 (which received 50- and 100-fold doses of B. pinnatum concentrate). At this stage, blood chemistry parameters (glucose, alanine aminotransferase [ALT], aspartate aminotransferase [AST], creatinine, and blood urea nitrogen) were measured in dams, as well as histological aspects of dam liver, kidney, placenta, and uterine tissue and fetal liver, kidney, heart, and brain. RESULTS: No differences were found between group B1 (therapeutic dose) and its control C1 in relation to glucose, AST, ALT, and creatinine. Group B2 exhibited lower glucose levels than groups C1, B3, and B4. There was no difference in AST across groups. Groups B3 and B4 exhibited higher ALT levels than groups C1 and B1. Groups B1-B4 exhibited higher urea nitrogen levels than group C1. Creatinine levels were higher in groups B2 and B3 than group C1. On morphological evaluation, fatty infiltration of the liver was observed in the alcoholic vehicle control groups (C1 and C2). CONCLUSIONS: Daily administration of B. pinnatum at therapeutic doses (group B1) to pregnant albino rats appears to be safe, with reduced glucose at dose B2, elevated ALT at doses B3 and B4, and increased urea at doses B1 to B4 and creatinine at B2 and B3, but never exceeding the normal reference range. It was not associated with histological changes in specimens of the maternal or fetal structures of interest.