Effect of the addition of different concentrations of Solanum glaucophyllum desf. extract on chondrocyte cultures from the growth cartilage of newborn rats.

Solanum glaucophyllum Desf. is a calcinogenic plant responsible for enzootic calcinosis that affects ruminants and causes alterations in bone and cartilaginous tissues, among others. It is believed that changes in cartilage tissue, with reduced bone growth, are due to hypercalcitoninism, caused by excess vitamin D. However, we hypothesized that S. glaucophyllum Desf. can act directly on chondrocytes and therefore, chondrocyte cultures from the epiphysis of the long bones of newborn rats were used as a model to elucidate the direct effects of S. glaucophyllum Desf. on bone growth. Plant samples were collected from Cañuelas, Argentina. An aliquot of the plant extract was used to quantify vitamin D (1,25(OH)2D3). The effects of the three concentrations of the plant extract were tested in cultures of chondrocytes extracted from the epiphyses of the long bones of 32 three-day-old Wistar rats. A control group (without extract), and three groups treated with different concentrations of plant extract were formed: group 1 (100 µL/L); group 2 (1 mL/L), and group 3 (5 mL/L), containing respectively 1 × 10-9 M, 1 × 10-8 M, and 5 × 10-8 M of 1,25(OH)2D3. After 7, 14, and 21 days of culture, MTT assay for cell viability, alkaline phosphatase activity, and quantification of the percentage of areas with glycosaminoglycans (GAG) stained with periodic acid-Schiff (PAS) were performed. On day 7, all chondrocytes in group 3, that is, those with the highest concentration of plant extract, died. On days 14 and 21, groups 1 and 2 showed a significant reduction in chondrocyte viability compared to the control. At 7, 14, and 21 days, groups 1 and 2 showed significantly lower alkaline phosphatase activity than the control. On day 21, group 2 showed a significant reduction in areas with PAS + GAGs. There were no significant differences between the groups in the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan. The S. glaucophyllum Desf. extract directly affected growing rat chondrocytes by reducing viability, alkaline phosphatase activity, and GAG synthesis without altering the expression of gene transcripts for Sox9, Col2, ColX, and aggrecan, which may be one of the mechanisms by which there is a reduction in bone growth in animals intoxicated by the plant.

Proteomics analysis reveals the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets.

1α,25(OH)2VD3 is the most active form of VD3 in animals. It plays an important role in regulating mineral metabolism but also in reproduction. Testes are the main reproductive organs of male mammals. Our research aims to reveal the effect of 1α,25(OH)2VD3-glycosides on development of early testes in piglets. 140 weaned 21-day old piglets were selected. The piglets were randomly divided into four groups and were fed a commercial diet supplemented with 0, 1, 2 and 4 µg/kg of 1α,25(OH)2VD3, provided as 1α,25(OH)2VD3-glycosides. Sixty days after the start of the experiment, at piglet age 82 days, testes were harvested. The morphology and histology of early testicular development were assessed. In addition, the proteomic TMT/iTRAQ labelling technique was used to analyse the protein profile of the testes in each group. Western blotting was applied to verify the target of differentially abundant proteins (DAPs). The analysis of morphology and histology of testes showed that a certain concentration of 1α,25(OH)2VD3-glycosides had a positive and significant effect on testicular development. And the results of proteomics analysis showed that of the identified 132,715 peptides, 122,755 were unique peptides. 7852 proteins, of which 6573 proteins contain quantitative information. Screening for DAPs focused on proteins closely related to the regulation of testicular development such as steroid hormone synthesis, steroid biosynthesis, peroxisome and fatty acid metabolism pathways. These results indicated that 1α,25(OH)2VD3 is involved in the regulation of early testicular development in piglets. At the same time, these findings provide valuable information for the proteins involved in the regulation of testicular development, and help to better understand the mechanisms of 1α,25(OH)2VD3 in regulating the development of piglets' testes.

Enzootic calcinosis in sheep in Uruguay: a brief review and report of two outbreaks / Calcinose enzoótica em ovinos no Uruguai: revisão de literatura e descrição de dois surtos

This study aimed to do a brief review of enzootic calcinosis in sheep and to report two outbreaks of Nierembergia rivularis poisoning in sheep in Uruguay. The outbreaks occurred in farms located on an island (Outbreak A) and on the border (Outbreak B) of the Rincón del Bonete lake. Sheep of all ages were affected, with the exception of suckling lambs. The first clinical signs occurred in early October, and deaths occurred from December to February. Outbreaks A and B had morbidity of 10%, and the mortality was 7.2% and 2.8% in Outbreaks A and B, respectively. The clinical signs included weight loss, retracted abdomen, stiff gait, and kyphosis. An autopsy was performed on one sheep from each outbreak. Pulmonary and arterial calcification, nephrocalcinosis, and osteopetrosis were observed in gross and microscopic examination in both sheep. Thyroid C-cell hyperplasia and carcinoma was observed in sheep A. Sheep B showed thyroid C-cell hyperplasia and parathyroid chief cell atrophy. The parathyroid was not examined in the sheep from Outbreak A. The differential diagnosis of enzootic calcinosis in southern South America should consider four toxic plants in the Solanaceae family: Solanum glaucophyllum, Solanum stuckertii, Nierembergia veitchii, and Nierembergia rivularis.(AU)

Este trabalho faz uma breve revisão da calcinose enzoótica em ovinos e descreve dois surtos de intoxicação por Nierembergia rivularis em ovinos no Uruguai. Os surtos ocorreram em propriedades localizadas em uma ilha (Surto A), e nas margens (Surto B) do lago do Rincón del Bonete. Foram afetados ovinos de todas as idades, exceto cordeiros lactentes. Os primeiros sinais clínicos ocorreram no início de outubro e as mortes de dezembro a fevereiro. Morbidade de 10% foi observada nos Surtos A e B. A mortalidade foi de 7,2% e 2,8% nos Surtos A e B, respectivamente. Os sinais clínicos incluíram perda de peso, abdômen retraído, marcha rígida e cifose. Foram necropsiados um animal de cada rebanho. Observou-se mineralização arterial e pulmonar, nefrocalcinose e osteopetrose no exame macroscópico e histológico dos dois ovinos. Hiperplasia e carcinoma de células C da tireoide foram observados no ovino A. O ovino B apresentou hiperplasia de células C da tireoide e atrofia das células principais da paratireoide. As paratireoides do ovino A não foram examinadas. O diagnóstico diferencial da calcinose enzoótica no Sul da América do Sul deve considerar quatro plantas calcinogênicas da família Solanaceae: Solanum glaucophyllum, Solanum stuckertii, Nierembergia veitchii e Nierembergia rivularis.(AU)

The Solanum glaucophyllum Desf. extract reduces mineralized matrix synthesis in osteogenically differentiated rat mesenchymal stem cells in vitro.

The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 µl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 µl of extract/L containing 0.4 µg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 µg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 µg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.

Targeting 1,25(OH)2D-mediated calcium absorption machinery in proximal colon with calcitriol glycosides and glucuronides.

High intestinal calcium (Ca) absorption efficiency is associated with high peak bone mass in adolescents and reduced bone loss in adulthood. Transepithelial intestinal Ca absorption is mediated by 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) through the vitamin D receptor (VDR). Most research on Ca absorption focuses on the proximal small intestine but evidence shows that large intestine plays a crucial role in whole body Ca homeostasis. We directly assessed and compared Ca absorption capacity at the proximal colon and duodenum using in situ ligated loops (2 mM Ca, 10 min). In C57BL/6 J mice, the proximal colon (26.2 ±â€¯3.7 %) had comparable ability to absorb Ca as the duodenum (30.0 ±â€¯6.7 %). In VDR knockout (KO) mice, Ca absorption efficiency was reduced by 67 % in duodenum and 48 % in proximal colon. These data suggest that large intestine could be targeted to improve Ca absorption and protect bone in at risk-groups (e.g. bariatric patients). Glycoside forms of calcitriol found in Solanum Glaucophyllum (Sg) leaf are biologically inert but can be activated in the colon upon bacterial cleavage of the glycosides. We conducted a study to test whether Sg leaf, as well as a novel, synthetic 1,3-diglucuronide form of calcitriol (1,3-diG) could target the proximal colon and upregulate genes involved in Ca absorption (i.e. Trpv6, S100g). 13-week-old female C57BL6/J mice were fed AIN93 G diet containing increasing levels of one of the two compounds for 2 weeks (delivering 0, 0.25, 0.5, 1, or 2 ng calcitriol equivalent per day). Both compounds induced a dose-dependent upregulation of Cyp24a1 and Trpv6 gene expression in the proximal colon. 1,3-diG also induced S100g gene expression in the proximal colon. Duodenal expression of Trpv6 was upregulated at higher doses of 1,3-diG but not Sg leaf. These data suggest that both glycosylated and glucuronidated calcitriol could be used to target the proximal colon but that dosing must be optimized to limit systemic effects that could cause hypercalcemia. Future studies will test the translational potential of these compounds to determine if they can increase Ca absorption at proximal colon and whether this can help protect bone.

Rat mesenchymal stem cell cultures as a model to elucidate the cellular and molecular pathogenesis of bone metaplasia induced by Solanum glaucophyllum intoxication.

The hypothesis of this experiment is that mesenchymal stem cells (MSCs) are involved in the genesis of the bone metaplasia caused by Solanum glaucophyllum intoxication. We determined using liquid chromatography that 1 mL of plant extract contained 3.8 µl of 1,25(OH)2D3. The ability of 100 µL, 1 mL and 5 mL of extract/L, containing 1 nM (0.4 µg/L), 10 nM (4 µg/L) and 50 nM (20 µg/L) of 1,25(OH)2D3, respectively, in inducing the osteogenic differentiation in bone marrow MSCs from rats was tested. At the concentrations of 1 and 5 mL of extract/L of culture medium without osteogenesis-inducing factors, the plant extract induced the osteogenic differentiation of the MSCs, as was evidenced by the greater synthesis of mineralized matrix. At the higher concentration (5 mL of extract/L), an increase in the relative expression of BMP-2 gene was observed. It was concluded that rat bone marrow MSC culture is a good model for studying the effects of the S. glaucophyllum extract on the osteogenic differentiation of undifferentiated cells. Also, S. glaucophyllum extracts containing 10 nM (4 µg/L) and 50 nM (20 µg/L) of 1,25(OH)2D3 induce the osteogenic differentiation of MSCs, suggesting that this is one of the mechanisms by which S. glaucophyllum causes bone metaplasia.

Enzootic calcinosis in horses grazing Solanum glaucophyllum in Argentina.

Solanum glaucophyllum, a toxic plant known for its calcinogenic effects, causes enzootic calcinosis in ruminant and monogastric animals. We describe an outbreak of enzootic calcinosis that occurred in a herd of 110 horses grazing pastureland heavily contaminated with S. glaucophyllum in Buenos Aires province, Argentina. Ten horses developed clinical signs, and 6 horses died. Clinical signs included abnormal gait (stiff-legged action, short strides), stiffness, thoracolumbar kyphosis, reluctance to move, wide stance, chronic weight loss, weakness, recumbency, and difficulty standing. Autopsy of 2 horses revealed severe mineralization of the aorta, pulmonary arteries, heart, and lungs, consistent with enzootic calcinosis. Although horses usually have very selective grazing behavior, under food restriction conditions, they can ingest the toxic plants and can develop the disease. Enzootic calcinosis should be considered as a differential diagnosis in horses grazing S. glaucophyllum-invaded pasturelands with compatible clinical signs and lesions.

1, 25-dihidroxivitamina-D 3 sobre as características ósseas de frangos de corte fêmeas / 1, 25-dihydroxyvitamin-D3 over bony characteristics of female broilers

Objetivou-se avaliar as características dos tibiotarsos de frangos de corte fêmeas. As variáveis analisadas foram os pesos in natura, secos e desengordurados, o comprimento, os diâmetros, a resistência óssea, o índice de Seedor (IS), os percentuais de proteínas colagenosas (PC), as proteínas não colagenosas (PNC), os minerais (cálcio, fósforo, potássio e sódio) e as cinzas. Foram utilizadas 648 aves, da marca comercial Cobb®, em um delineamento em blocos ao acaso, com seis tratamentos e seis repetições, com 18 aves por unidade experimental. Os tratamentos consistiram na suplementação de 0,00; 0,50; 1,00; 1,50; 2,00 e 2,50 µg de 1,25-dihidroxivitamina-D3 (1,25(OH)2D3)/kg de ração. Aos 21 e 35 dias de idade, as aves foram pesadas e uma ave por unidade experimental (UE) com o peso médio da UE foi eutanasiada para a obtenção dos tibiotarsos e subsequente análise dos parâmetros ósseos. As variáveis métricas, bem como a composição orgânica (PC), a densidade (IS) e a resistência à quebra dos ossos das aves, não foram influenciadas pelos tratamentos. No recebimento de 2,50 µg de 1,25(OH)2D3/kg de ração, observou-se maior retenção mineral (cinzas) nos ossos das aves aos 35 dias de idade.(AU)

This study aimed to evaluate the characteristics of tibiotarsus of female broilers. The variables analyzed were the weights in natura, dry and degreased, length, diameter, bone strength, Seedor index, percentage of collagenous protein (CP), non-collagenous proteins (NCP), minerals (calcium, phosphorus, potassium and sodium), and ash. Six hundred forty-eight, Cobb® birds were used, a design of randomized blocks with six treatments and six replicates of 18 birds each. The treatments consisted of supplementation of 0.00; 0.50; 1.00; 1.50; 2.00 to 2.50 µg of 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3)/kg of ration. At 21 and 35 days of age the birds were weighed and a unit/experimental unit (EU) with the EU average weight was euthanized to obtain the tibiotarsos and subsequent analysis of the bone parameters. The metric variables, as well as the organic composition (CP), density (IS) and resistance to breakage of the bones of the birds were not affected by treatments. Upon reception of 2.50 µg of 1,25(OH)2D3/kg ration, there was a higher mineral retention (ashes) in the bones of female broilers at 35 days of age.(AU)

Tolerance to 1,25 dihydroxyvitamin D3 glycosides from Solanum glaucophyllum by the growing pig.

Solanum glaucophyllum leaves contain high levels of glycosidically bound 1,25 dihydroxyvitamin D3, the most important vitamin D metabolite. The tolerance to this source was evaluated during six weeks with fifty weaned pigs fed increasing levels (0, 2.5, 5, 10 and 20µg 1,25(OH)2D3/kg diet). The diet contained, per kg, 9.7g Ca, 3.5g digestible P and 2000IU cholecalciferol. Ten additional pigs were fed a diet containing 1000IU cholecalciferol/kg, without 1,25(OH)2D3. Weekly plasma and final kidney, bone and urinary mineral contents, bone density and breaking strength served as indicators for possible adverse effects of the supplement. All animals grew well and remained clinically healthy. The measured parameters remained unchanged when 1000 replaced 2000IU cholecalciferol/kg and when 1,25(OH)2D3 was fed up to 10µg/kg. Twenty µg 1,25(OH)2D3 increased plasma Ca and decreased plasma P from the 2nd and the 4th experimental week onwards, respectively. Twenty µg 1,25(OH)2D3 increased final plasma Ca and 1,25(OH)2D3 and reduced final plasma P by respectively 19, 56 and 13%. Twenty µg 1,25(OH)2D3 also increased kidney Ca and urinary Ca by 43 and 69%, respectively, reduced bone breaking strength by 12% and tended to decrease bone ash by 3%. To conclude, 2000IU D3 was not beneficial compared to 1000IU cholecalciferol; up to 10µg 1,25(OH)2D3 per kg diet did not lead to observed adverse effects; 20µg 1,25(OH)2D3 altered the homeostatic regulation of Ca and P thus, may lead to first signs of possible adverse effects, such as soft tissue calcification.

In Pursuit of Vitamin D in Plants.

Vitamin D deficiency is a global concern. Much research has concentrated on the endogenous synthesis of vitamin D in human skin following exposure to ultraviolet-B radiation (UV-B, 280-315 nm). In many regions of the world there is insufficient UV-B radiation during winter months for adequate vitamin D production, and even when there is sufficient UV-B radiation, lifestyles and concerns about the risks of sun exposure may lead to insufficient exposure and to vitamin D deficiency. In these situations, dietary intake of vitamin D from foods or supplements is important for maintaining optimal vitamin D status. Some foods, such as fatty fish and fish liveroils, certain meats, eggs, mushrooms, dairy, and fortified foods, can provide significant amounts of vitamin D when considered cumulatively across the diet. However, little research has focussed on assessing edible plant foods for potential vitamin D content. The biosynthesis of vitamin D in animals, fungi and yeasts is well established; it is less well known that vitamin D is also biosynthesised in plants. Research dates back to the early 1900s, beginning with in vivo experiments showing the anti-rachitic activity of plants consumed by animals with induced rickets, and in vitro experiments using analytical methods with limited sensitivity. The most sensitive, specific and reliable method for measuring vitamin D and its metabolites is by liquid chromatography tandem mass spectrometry (LC-MS/MS). These assays have only recently been customised to allow measurement in foods, including plant materials. This commentary focuses on the current knowledge and research gaps around vitamin D in plants, and the potential of edible plants as an additional source of vitamin D for humans.

Safety profile of 1,25-dihydroxyvitamin D3 of herbal origin in broiler chicken.

INTRODUCTION: The safety of supplementing broiler feed with a standardised herbal extract, Solanum Glaucophyllum Standardised Leaves (SGSL) containing glycosylated 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) and standardised to contain 10 µg/g 1,25(OH)2D3 equivalent, was examined in two studies. In a first study, we examined the potential of SGSL to substitute vitamin D3 (VD3) and the tolerated dose range of SGSL when applied without concomitant VD3 by analyzing performance and blood chemical parameters after 14, 25 and 38 days on diets containing two doses of SGSL (1 and 10 g/kg feed) as source of 1,25(OH)2D3. In the second study, the no adverse effect level of SGSL was determined by analyzing the same parameters after 35 days on diets containing basic VD3 supply and in addition 0.2, 1.0, 2.0 and 4.0 g of SGSL/kg feed. We showed that SGSL was able to substitute VD3 in broilers as far as the performance parameters were concerned. Also, we found that the no adverse effect level is at least 4 g SGSL/kg feed when used with moderate doses of VD3. This is 20 times higher than the upper limit of the commercially recommended dose. We concluded that SGSL is a safe feed additive to use in broiler chicken.

INTRODUCTION: Dans la cadre de deux études, on a examiné la sécurité de l'extrait de plante standardisé Solanum Glaucophyllum Standardised Leaves (SGSL) comme complément alimentaire chez les poulets d'engraissement. Le SGSL contient de façon standardisée 10 µg/g de 1,25(OH)2D3 sous forme glycolysée. Dans la première étude, on a examiné le potentiel d'action en tant que remplaçant de la vitamine D3 (VD3) et le domaine de dose de SGSL toléré, ceci en ne donnant que du SGSL sans addition de VD3 . On a examiné la performance et les paramètres de chimie sanguine après 14, 25 et 38 jours d'affouragement de deux doses différentes (1 et 10 g/kg d'aliment) de SGSL comme source de 1,25(OH)2D3. Dans la seconde étude, on a recherché le No Adverse Effect Level sur la base des mêmes paramètres après 35 jours avec une alimentation contenant, outre une quantité modérée de VD3, 0.2, 1.0, 2.0 et 4.0 g de SGSL/kg. On a pu démontrer que le SGSL peut remplacer la vitamine D3 chez les poulets d'engraissement en ce qui concerne les performances étudiées. Le No Adverse Effect Level se situait aux environs d'au moins 4g de SGSL/kg d'aliment lorsqu'il était associé avec des quantités modérées de Vitamine D3. Cette dose est vingt fois supérieure à la dose maximale recommandée par le fabriquant. Nous en déduisons que le SGSL est un complément alimentaire sûr pour les poulets d'engraissement.

In vitro effects of 1α,25(OH)2D3-glycosides from Solbone A (Solanum glaucophyllum leaves extract; Herbonis AG) compared to synthetic 1α,25(OH)2D3 on myogenesis.

The presence of glycoside derivatives of 1α,25(OH)2D3 endows plants to gradual release of the free bioactive form of 1α,25(OH)2D3 from its glycoconjugates by endogenous animal tissue glycosidases. This results in increased half-life of the hormone in blood when purified plant fractions are administered for therapeutic purposes. In this work, we evaluated the role 1α,25(OH)2D3-glycosides enriched natural product (Solbone A) from Solanum glaucophyllum leaf extract compared with synthetic 1α,25(OH)2D3 on myogenic differentiation in C2C12 myoblasts. For these, differentiation markers and myogenic parameters were studied in C2C12 myoblasts. Results showed that Solbone A, likewise the synthetic hormone, increased creatine kinase activity at day 2 after differentiation induction (60%, p<0.05). Solbone A and synthetic 1α,25(OH)2D3 increased vitamin D3 receptor protein expression at 10nM (50% and 30%, respectively) and the transcription factor myogenin (80%, p<0.05). However, tropomyosin expression was not affected by both compounds. In addition, myosin heavy chain (MHC) protein expression was increased 30% at day 2 of differentiation. Solbone A or synthetic 1α,25(OH)2D3 had no effects on myogenin nor MHC cell localization. Cellular mass increased with myogenesis progression, being Solbone A more effective than synthetic 1α,25(OH)2D3. Finally, Solbone A, as well as synthetic 1α,25(OH)2D3, augmented the index fusion of cultured muscle fibers. In conclusion, these results demonstrated that Solbone A exhibit at least equal or greater effects on early myoblast differentiation as synthetic hormone, suggesting that plant glycosides could be an effective, accessible and cheaper substitute for synthetic 1α,25(OH)2D3 to promote muscle growth.

Feeding Solanum glaucophyllum to preparturient multiparous cows prevents postparturient hypocalcemia.

Solanum glaucophyllum (SG) contains 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) glycosides. We investigated the effect of SG on hypocalcemia in cows. Serum levels of 1,25-(OH)2D3, total calcium and phosphorus dose-relatedly increased after feeding with SG, while serum magnesium and chloride levels fell (P < 0.05). We also performed an ethylenediaminetetraacetic acid (EDTA) infusion to induce artificial hypocalcemia. Cows that had been fed 4.0 mg/kg body weight of SG daily for 2 weeks had a higher serum concentration of total calcium at the end of EDTA infusion than those not fed SG (P < 0.05). In a field trial, multiparous cows were assigned to one of four groups: (1) no SG, (2) 1.3 g or (3) 2.6 g of SG daily from 14 days before the estimated calving day until 3 days after calving, or (4) a single feed of 35.75 g SG at 3 days before the estimated calving day. The concentrations of serum total calcium after the calving in each treatment group were (1) 7.4, (2) 7.9, (3) 8.0 and (4) 8.9 mg/dL and higher for (4) than for (1) (P < 0.05). The data suggests that feeding a high dose of SG before the calving may maintain higher concentrations of serum calcium after the calving.

Targeted delivery of 1,25-dihydroxyvitamin D3 to colon tissue and identification of a major 1,25-dihydroxyvitamin D3 glycoside from Solanumglaucophyllum plant leaves.

Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333µg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000µg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a ß-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and the 1,3-diglycoside is one of the major forms of 1,25D3 found in the leaf. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Human pharmacokinetic profile of 1,25-dihydroxyvitamin D3-glycoside of herbal origin.

A natural form of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, was identified in glycosylated form in Solanum glaucophyllum (SG). Solbone P, an extract of SG with high and homogenous content of glycosylated 1,25(OH)2D3, was chemically characterized and produced under GMP conditions. Three different doses of glycosylated 1,25(OH)2D3 were given as single oral dose to 16 healthy volunteers in a first-in-man trial. The oral pharmacokinetic properties of 1,25(OH)2D3 of SG origin were established and the subjects were monitored until day 28 for safety reasons. This included regular monitoring of vital signs, electrocardiogram (ECG) data, calcium, phosphate and creatinine values. Subjects were exposed to up to the equivalent of a 40-fold level of the recommended human daily dose for synthetic 1,25(OH)2D3 (0.5µg/subject/day) without experiencing any untoward effects. When compared with the historically established pharmacokinetics profile of synthetic 1,25(OH)2D3, glycosylated 1,25(OH)2D3 of herbal origin exhibited delayed absorption characteristics. The phenomenon is species independent, as similar pharmacokinetic patterns were observed in rats and chickens. This modified release pattern may be attributed to the glycosylation of herbal 1,25(OH)2D3 because de-glycosylation by ubiquitous intestinal enzymes prior to intestinal uptake of the unmodified 1,25(OH)2D3 is the rate-limiting step. The major relevance of this finding is that the human pharmacokinetic profile of glycosylated 1,25(OH)2D3 of herbal origin is reminiscent of a delayed release formulation of free 1,25(OH)2D3, resulting in a wider therapeutic window, a potentially longer therapeutic effectiveness, and thus, a better pharmacologic tolerance. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

The efficacy of a standardised product from dried leaves of Solanum glaucophyllum as source of 1,25-dihydroxycholecalciferol for poultry.

1. Chemical characterisation of an extract of Solanum glaucophyllum (SG) leaves affirmed the predominant presence of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) glycosides. The compound 1-(ß-D-glucopyranosyl)-1α,25-dihydroxycholecalciferol was isolated for the first time from a natural source. 2. Vitamin D activity of the extract was confirmed by the calcaemic properties shown in a quail eggshell bioassay. The results suggested a 1,25(OH)2D3 bioavailability of approximately 15%. 3. A broiler feeding experiment replicated in time was carried out with 6 treatments. A basic control diet containing 25 µg cholecalciferol/kg was supplemented with 2.5 and 5 µg free 1,25(OH)2D3/kg, with a product based on dried SG leaves (Panbonis) providing 10 µg of 1,25(OH)2D3-glycosides/kg, with two concentrations of an SG extract providing 8.8 and 37.8 µg of 1,25(OH)2D3-glycosides/kg. 4. Tibia breaking strength and stiffness were numerically greater in all treatment groups with free 1,25(OH)2D3 and with SG products compared to controls, though the overall treatment effects only had probabilities in the range of P = 0.07 to P = 0.1. Values for both characteristics increased progressively, with additions of synthetic 1,25(OH)2D3; values with the dried SG product were similar to those with 5 µg synthetic 1,25(OH)2D3/kg. 5. Plasma calcium was mildly elevated (P < 0.05) in treatment groups. The SG extract treatment containing 37.8 µg 1,25(OH)2D3/kg gave the highest plasma calcium concentration and lowest bodyweight, signs of marginal hypervitaminosis D. Plasma 1,25(OH)2D3 concentrations were in the normal range for all treatments. 6. Tibial dyschondroplasia occurred in only one replicate. The incidences were 31% in controls but considerably lower or zero with all other treatments. 7. Bioavailability of 1,25(OH)2D3 in the SG product seemed to be higher in broiler chickens than in Japanese quails. 8. It is concluded that the inclusion of the dried SG product as a source of vitamin D3 in broiler diets at a dietary concentration of 1 g/kg, providing 10 µg 1,25(OH)2D3/kg, is safe and efficacious.

Quantification of vitamin D3 and its hydroxylated metabolites in waxy leaf nightshade (Solanum glaucophyllum Desf.), tomato (Solanum lycopersicum L.) and bell pepper (Capsicum annuum L.).

Changes in vitamin D(3) and its metabolites were investigated following UVB- and heat-treatment in the leaves of Solanum glaucophyllum Desf., Solanum lycopersicum L. and Capsicum annuum L. The analytical method used was a sensitive and selective liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method including Diels-Alder derivatisation. Vitamin D(3) and 25-hydroxy vitamin D(3) were found in the leaves of all plants after UVB-treatment. S. glaucophyllum had the highest content, 200 ng vitamin D(3)/g dry weight and 31 ng 25-hydroxy vitamin D(3)/g dry weight, and was the only plant that also contained 1,25 dihydroxy vitamin D(3) in both free (32 ng/g dry weight) and glycosylated form (17 ng/g dry weight).

1,25-Dihydroxyvitamin D3-glycoside of herbal origin exhibits delayed release pharmacokinetics when compared to its synthetic counterpart.

Vitamin D requires two metabolic steps to become biologically active. In a first step 25-hydroxyvitamin D3 is formed, which acts as storage form. After a tightly controlled step in kidney the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is formed. Because kidney is the relevant metabolic organ for this conversion, 1,25(OH)2D3 needs to be supplemented in patients with kidney malfunction or kidney failure. Synthetic 1,25(OH)2D3 (calcitriol) has been available as a drug for decades. Due to its high potency and its kinetic profile (fast absorption and rapid elimination) its therapeutic windows has proven to be relatively narrow. A natural form of the active metabolite was identified in a few plants, such as Solanum glaucophyllum (SG) and suggested as alternative for animal and human health. An extract of a SG variety bred for high and uniform level of glycosylated 1,25(OH)2D3 was chemically characterized. Among the typical pharmaceutically inactive plant components (carbohydrates 54.3%, protein 24.9%, minerals 17.1% and water 4.1%) high levels of 1,25(OH)2D3 and a unique flavonoid content was found (1.11mg total quercetin/g extract) consisting exclusively of the quercetin glycosides hyperoside, isoquercetin, rutin and apinosylrutin. The molecular distribution of glycosyl moieties in 1,25(OH)2D3 extracted from SG as determined by gel permeation chromatography was found to be 1-10 hexose units per aglycone. 1,25(OH)2D3-1-ß-glucopyranoside was identified in the SG extract, while a di- and triglycoside have been identified in SG by other groups. The pharmacokinetic properties of synthetic 1,25(OH)2D3 and glycosylated 1,25(OH)2D3 extracted from SG were compared in male rats. When compared to synthetic 1,25(OH)2D3, SG-derived 1,25(OH)2D3 exhibited delayed absorption and elimination characteristics, resulting in delayed Tmax (6-12h vs. 1h) and increased T½ (approximately 30h vs. 23h). This putative modified release pattern may be attributed to the glycosylation of herbal 1,25(OH)2D3 because de-glycosylation by ubiquitous intestinal enzymes prior to intestinal uptake of the aglycone appears to be the rate limiting step. In effect, 1,25(OH)2D3 of herbal origin behaves like a precursor of calcitriol, resulting in a wider therapeutic window and thus better pharmacological tolerance. This article is part of a Special Issue entitled 'Vitamin D Workshop.'.

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits.

Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30 days. A group was treated for 15 days and led to possible recovery for 30 days. Another group was nutritionally restricted for 30 days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.

Intoxicação natural por Solanum glaucophyllum (Solanaceae) em búfalos no Pantanal Matogrossense / Natural poisoning by Solanum glaucophyllum(Solanaceae) in buffaloes in the Brazilian Pantanal

Descreve-se a ocorrência de calcinose enzoótica em búfalos no município de Poconé, Mato Grosso, associado ao consumo de Solanum glaucophyllum (Sg) [=Solanum malacoxylon]. Os casos foram observados entre os anos de 2007 e 2009. Em um rebanho de 40 búfalos, cinco apresentaram emagrecimento progressivo, dorso arqueado, marcha rígida, por vezes com dificuldade para se levantar e locomover, permanecendo apoiando sobre os carpos.Três animais recuperaram-se parcialmente e dois foram eutanasiados in extremis. Os principais achados de necropsia foram calcificação de tecidos moles, principalmente em artérias de grande e médio calibres. A presença de S. glaucophyllum nas pastagens, os sinais clínicos, além dos achados ultrassonográficos e patológicos envolvendo múltiplas calcificações de tendões e outros tecidos, são compatíveis com intoxicação por Solanum glaucophyllum.

This report describes the occurrence of enzootic calcinosis in buffaloes in the municipality of Pocone, Mato Grosso, due to the consumption of Solanum glaucophyllum (Sg) [=Solanum malacoxylon]. The cases were observed in the years 2007 and 2009. In a herd of 40 buffaloes, five showed weight loss, arched back, stiff gait, sometimes difficulty to raise and walk, and leaning on the carpus. Three buffaloes recovered partially and two were euthanized in extremis. The main necropsy findings were calcification of soft tissues, especially of large and medium arteries. The presence of S. glaucophyllum in the pasture, clinical signs, in addition to the sonographic and pathologic calcification involving tendons and other tissues, are consistent with Solanum glaucophyllum poisoning.