Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Corrigendum: A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

[This corrects the article DOI: 10.3389/fmicb.2023.1018242.].

A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

Introduction: Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic epithelium and has a blurred etiology. A western diet and microbial dysbiosis in the colon were reported to play a role in UC development. In this study, we investigated the effect of a westernized diet, i.e., increasing fat and protein content by including ground beef, on the colonic bacterial composition in a dextran sulfate sodium (DexSS) challenged pig study. Methods: The experiment was carried out in three complete blocks following a 2×2 factorial design including 24 six-week old pigs, fed either a standard diet (CT) or the standard diet substituted with 15% ground beef to simulate a typical westernized diet (WD). Colitis was induced in half of the pigs on each dietary treatment by oral administration of DexSS (DSS and WD+DSS, respectively). Samples from proximal and distal colon and feces were collected. Results and discussion: Bacterial alpha diversity was unaffected by experimental block, and sample type. In proximal colon, WD group had similar alpha diversity to CT group and the WD+DSS group showed the lowest alpha diversity compared to the other treatment groups. There was a significant interaction between western diet and DexSS for beta diversity, based on Bray-Curtis dissimilarly. The westernized diet and DexSS resulted in three and seven differentially abundant phyla, 21 and 65 species, respectively, mainly associated with the Firmicutes and Bacteroidota phyla followed by Spirochaetota, Desulfobacterota, and Proteobacteria. The concentration of short-chain fatty acids (SCFA) was lowest in the distal colon. Treatment had a slight effect on the estimates for microbial metabolites that might have valuable biological relevance for future studies. The concentration of putrescine in the colon and feces and that of total biogenic amines was highest in the WD+DSS group. We conclude that a westernized diet could be a potential risk factor and an exacerbating agent for UC by reducing the abundance of SCFA-producing bacteria, increasing the abundance of pathogens such as Helicobacter trogontum, and by increasing the concentration of microbial proteolytic-derived metabolites in the colon.

Role of Short Chain Fatty Acids to Counteract Inflammatory Stress and Mucus Production in Human Intestinal HT29-MTX-E12 Cells.

Short chain fatty acids (SCFAs), especially butyrate (BUT), are known to promote intestinal health, but their role in the protection of intestinal barrier integrity is poorly characterized. The aim of the study was to set up an in vitro model of human colon epithelium using HT29-MTX-E12 cells to delineate the potential role of SCFAs under stress conditions. Accordingly, the HT29-MTX-E12 cells were differentiated for 42 days and subsequently exposed to dextran sulphate sodium (DSS). Further, the effects of BUT or its mixture with acetate and propionate (SCFAs-MIX) were tested to study proliferation, epithelial integrity and mucus production. The results showed that the concentration of 10% DSS for 24 h decreased the TEER about 50% compared to the control in HT29-MTX-E12 cells. The pre-treatment on HT29-MTX-E12 cells with BUT or SCFAs-MIX at specific concentrations significantly (p < 0.05) reduced the DSS-induced damage on epithelial cell integrity and permeability. Further, the treatment with specific concentrations of BUT and SCFAs-MIX for 24 h significantly promoted ZO-1, MUC2 and MUC5AC mRNA expression (p < 0.005). The present study demonstrated the suitability of HT29-MTX-E12 cells treated with DSS as an in vitro stress model of inflammatory bowel disease, which enabled us to understand the effect of bioactive SCFAs on the intestinal barrier.

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus):A Preliminary Protocol.

This study describes a successful protocol for establishing cell lines from the threatened Triturus cristatus in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a combination of ITS (insulin/transferrin/selenite) and 2-mercaptoethanol had a positive effect on growth. Real-time measurements on the cell lines were used, for the first time in amphibian cells, to investigate the effect of different treatments such as media change with or without washing. Media change had a positive impact on the cells, whereas the effect was negative when combined with washing. It is concluded that establishment of cell lines is possible from the great crested newt, especially when using fresh tissue, but much more challenging if the tissue has been cryopreserved. Real-time measurement during cell culture is a useful tool to visualize the sensitivity of amphibian cells during different culture treatments.

Dietary Red Meat Adversely Affects Disease Severity in a Pig Model of DSS-Induced Colitis Despite Reduction in Colonic Pro-Inflammatory Gene Expression.

Diet plays a substantial role in the pathogenesis and management of ulcerative colitis (UC), and epidemiologic studies indicate an association between red meat intake and increased risk of UC development. Therefore, we evaluated the effect of a red meat diet on dextran sulfate sodium (DSS)-induced colitis in pigs. Weaned pigs (42 days old) were fed either a control diet or a diet substituted with 15% minced, cooked and dried beef from experimental day 0 to 14. From day 14 to 18, half of the pigs on each diet received a daily oral dose of DSS. Dietary red meat aggravated the severity of colitis based on clinical signs of disease (negative performance score) and histopathological parameters in the colon such as erosion/ulceration and the overall inflammation score but no negative effects were observed on systemic health or small intestinal permeability. Importantly, dietary meat also caused a potential beneficial reduction in the colonic expression of the pro-inflammatory cytokines IL-17A and IL-6, the pro-inflammatory enzyme PTGS2 and in the chemokine IL-8. The present study emphasizes the potential of diet to modulate mucosal inflammation and that a red meat diet might be a risk factor for the development of inflammatory bowel disease.

Comparative Nutrient Profiling of Retail Goat and Cow Milk.

Goat milk is globally consumed but nutritional profiling at retail level is scarce. This study compared the nutrient composition of retail cow and goat milk (basic solids, fatty acids, minerals, and phytoestrogens) throughout the year and quantified the potential implications on the consumers' nutrient intakes. When compared to cow milk, goat milk demonstrated nutritionally desirable traits, such as lower concentrations of C12:0, C14:0, C16:0 and Na: K ratio, and the higher concentrations of cis polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), isoflavones, B, Cu, Mg, Mn, P and I, although the latter may be less desirable in cases of high milk intakes. However, in contrast with nutritional targets, it had lower concentrations of omega-3 PUFA, vaccenic acid, lignans, Ca, S and Zn. The extent of these differences was strongly influenced by season and may demonstrate a combination of differences on intrinsic species metabolism, and farm breeding/husbandry practices.

Concentrations of phytoestrogens in conventional, organic and free-range retail milk in England.

The effect of dairy management system (conventional, CNV; organic, ORG; free-range, FRG) and month on retail milk phytoestrogen composition was assessed for 12 consecutive months. ORG milk contained more secoisolariciresinol, matairesinol, lariciresinol, sum of plant lignans, daidzein, genistein, formononetin, naringenin, equol, sum of isoflavones and coumestrol, than CNV and FRG milk. This may be explained by the higher supply of pasture, and grazed or ensiled clover, in ORG dairy diets. Seasonal variation in milk phytoestrogen concentrations was higher for ORG than CNV and FRG systems. Phytoestrogen composition did not vary between FRG and CNV milk. Consuming organic milk can increase intake of potentially beneficial lignans and isoflavonoids, and in particular equol; but, any effects on human health from such milk compositional differences cannot be implied.

Effect of Casein Hydrolysates on Intestinal Cell Migration and Their Peptide Profiles by LC-ESI/MS/MS.

Potential beneficial effects of bioactive peptides derived from casein on epithelial cellular wound healing in the gastrointestinal tract were studied. Bovine casein was digested by a combination of pepsin and pancreatic proteases at different time intervals to represent ranges of duration of gastrointestinal digestion. Intestinal epithelial cells were used as an in vitro model of the small intestine. The effect of casein hydrolysates on cell migration was studied by scratch assay as a model of wound healing. Casein digested by pepsin and pancreatin for 10 to 30 min were found to have a significant stimulatory effect of >40% on cell migration relative to the control. A potential effect of casein gastrointestinal digests on gastro-intestinal wound healing has not previously been reported. The peptide profiles of active as well as inactive casein hydrolysates were characterised by liquid chromatography coupled to ion trap tandem mass spectrometry. By comparison of identified peptides in active and inactive casein hydrolysates, a pool of 11 peptides derived from casein were identified as potential candidates for effects on cell migration. Searching the milk bioactive peptide database (MBPDB) showed that 15 of the identified peptides had known biological functions such as antimicrobial, antioxidant, and immunomodulatory activity.

Mammary metabolism and colostrogenesis in sows during late gestation and the colostral period.

The aims of this study were to investigate 1) the effect of high dietary fiber (DF; 19.3% to 21.7%) supplemented to late gestating sows on mammary uptake and metabolism of energy substrates as well as colostrum production and 2) the ontogeny of colostral fat and lactose synthesis using mammary carbon balance, and colostral protein using IgG as a biomarker. Sows were fed either a control diet (CON) consisting of a standard gestation diet (14.6% DF) until day 108 of gestation and a transition diet (16.8% DF) from day 109 of gestation until farrowing or a high DF treatment where part of the daily ration was replaced with a high DF supplement (FIB). The FIB sows received 19.3% and 21.7% DF in the last 2 wk prior to farrowing. Sows were surgically implanted with permanent indwelling catheters at day 75 ± 2 of gestation and blood samples were collected at 6 different time points in late gestation and at 11 different time points within 24 h after the onset of farrowing. Colostrum samples were collected at 0, 12, and 24 h after the onset of farrowing. Arterial concentration of acetate (P = 0.05) and colostral fat content (P = 0.009) were greater in FIB sows compared with CON sows. Plasma IgG dropped from day -10 relative to farrowing (P < 0.001), suggesting an uptake by the mammary glands. Mammary plasma flow (P = 0.007) and net mammary uptake of glucose (P = 0.04) increased during farrowing while dietary treatment had no effect on net mammary uptake of other energy substrates during late gestation and farrowing. The net mammary uptake of carbon from glucogenic precursors did not equate to the sum of carbons secreted in colostral lactose and released as CO2, indicating that carbons from ketogenic precursors were likely used for colostral fat and for oxidation. Mammary nonprotein carbon uptake matched the mammary output, indicating that the majority of colostral fat and lactose were produced after the onset of farrowing. In conclusion, high DF included in the diet for late gestating sows increased colostral fat content by 49% but this substantial dietary response could not be explained by the increased carbon uptake from short chain fatty acids during the colostral period. The nonprotein carbon balance of mammary glands during farrowing suggests that the majority of colostral fat and lactose were produced after the onset of farrowing, whereas the drop in plasma IgG in late gestation suggests that the mammary glands take up this colostral component prior to farrowing.

Obesity Development in a Miniature Yucatan Pig Model: A Multi-compartmental Metabolomics Study on Cloned and Normal Pigs Fed Restricted or Ad Libitum High-Energy Diets.

Miniature-pig models for human metabolic disorders such as obesity and metabolic syndrome are gaining popularity. However, in-depth knowledge on the phenotypic and metabolic effects of metabolic dysregulation is lacking, and ad libitum feeding is not well-characterized in these pig breeds. Therefore, an investigation was performed into the metabolome of Yucatan minipigs fed ad libitum or restricted diets. Furthermore, we used cloned and conventional minipigs to assess if cloning reflects a presumably lowered variation between subjects. For 5 months, 17 female Yucatan minipigs were fed either ad libitum or restricted Western-style diets. Serum, urine, and liver tissues were collected and analyzed by non-targeted liquid chromatography-mass spectrometry metabolomics and by biochemical analyses. Several metabolic pathways were deregulated as a result of obesity and increased energy-dense feed intake, particularly the hepatic glutathione pathway and the pantothenic acid and tryptophan metabolic pathways in serum and urine. Although cloned minipigs were phenotypically similar to wild-type minipigs, the metabolomics analysis of serum and liver tissues showed several altered pathways, such as amino acid and purine metabolism. These changes, as an effect of cloning, could limit the use of cloned models in dietary intervention studies and provides no evidence of decreased variability between subjects.

Impact of Diet-Modulated Butyrate Production on Intestinal Barrier Function and Inflammation.

A major challenge in affluent societies is the increase in disorders related to gut and metabolic health. Chronic over nutrition by unhealthy foods high in energy, fat, and sugar, and low in dietary fibre is a key environmental factor responsible for this development, which may cause local and systemic inflammation. A low intake of dietary fibre is a limiting factor for maintaining a viable and diverse microbiota and production of short-chain fatty acids in the gut. A suppressed production of butyrate is crucial, as this short-chain fatty acid (SCFA) can play a key role not only in colonic health and function but also at the systemic level. At both sites, the mode of action is through mediation of signalling pathways involving nuclear NF-κB and inhibition of histone deacetylase. The intake and composition of dietary fibre modulate production of butyrate in the large intestine. While butyrate production is easily adjustable it is more variable how it influences gut barrier function and inflammatory markers in the gut and periphery. The effect of butyrate seems generally to be more consistent and positive on inflammatory markers related to the gut than on inflammatory markers in the peripheral tissue. This discrepancy may be explained by differences in butyrate concentrations in the gut compared with the much lower concentration at more remote sites.

Bioactive Whey Protein Concentrate and Lactose Stimulate Gut Function in Formula-fed Preterm Pigs.

OBJECTIVE: Formula feeding is associated with compromised intestinal health in preterm neonates compared with maternal milk, but the mechanisms behind this are unclear. We hypothesized that the use of maltodextrin and whey protein concentrates (WPCs) with reduced bioactivity owing to thermal processing are important factors. METHOD: Ninety-two cesarean-delivered preterm pigs were fed increasing doses of formulas for 5 days (24-120 mL ·â€Škg ·â€Šday). In experiment 1, 4 groups of pigs (n = 15-16) were fed lactose- or maltodextrin-dominant formulas (lactose/maltodextrin ratios 3:1 or 1:3, respectively), containing WPC with either high or low levels of IgG (WPC1 or WPC2, respectively). In experiment 2, 2 groups of pigs (n = 15-16) were fed lactose-dominant formulas with either a bioactive WPC (BioWPC, produced by reduced thermal-processing) or a conventional WPC (ConWPC). RESULTS: In experiment 1, pigs fed formula with WPC1 had higher villi, hexose absorption, and lactase activity in small intestine, relative to WPC2, but predominantly with the lactose-dominant formula (all P < 0.05). In experiment 2, the BioWPC product had higher bioactivity, as indicated by higher IgG, lactoferrin, and TGF-ß2 levels, and better enterocyte proliferation in vitro. Pigs fed the BioWPC formula showed better feeding tolerance and higher intestinal villi and lactase activity (all P < 0.05). The BioWPC formula-fed pigs also had greater physical activity (P < 0.05 on day 4) and tended to show improved hexose absorption and decreased gut permeability (both P ≤ 0.09). CONCLUSIONS: Infant formulas containing lactose as the main carbohydrate, and WPC with reduced thermal processing, may support gut maturation and health in sensitive, preterm neonates.

Administration of Protein Kinase D1 Induces a Protective Effect on Lipopolysaccharide-Induced Intestinal Inflammation in a Co-Culture Model of Intestinal Epithelial Caco-2 Cells and RAW264.7 Macrophage Cells.

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases involving all or part of the gastrointestinal tract. The stress-activated serine-threonine protein kinase D1 (PKD1) protein has previously been implicated in intestinal immune regulation. The objective of this study was to evaluate the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF-α), interleukin- (IL-) 1ß, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10-100 ng/ml) following induction of inflammation induced downregulation of TNF-α expression in RAW264.7 cells. In addition, PKD1 administered for 3 h prior to LPS stimulation reduced the subsequent inflammatory response through downregulation of TNF-α, IL-1ß, and IL-6 in RAW264.7 cells. These results demonstrate a potential role of PKD1 in the intercellular communication between intestinal epithelial and immune cells, proposing a protective effect of PKD1 on the induction of an inflammatory response in macrophages, an important aspect during the pathogenesis of IBD.

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and ß1.

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and ß1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology.

Effects of vitamin D and its metabolites on cell viability and Staphylococcus aureus invasion into bovine mammary epithelial cells.

Vitamin D has been found have various biological effects that may be potent in preventing bovine mastitis. Two forms of vitamin D, vitamin D2 (D2) and vitamin D3 (D3), can be hydroxylated to functional metabolites in cattle. The objectives of the present study were to investigate the potential of vitamin D compounds for controlling bovine mastitis using in vitro cell models, and to compare the differences between D2 and D3 compounds. Results showed that D2 compounds have comparable effects to their D3 analogues on inhibiting MAC-T cell viability in vitro. S. aureus growth was inhibited by high concentrations of D2, D3, 25(OH)D2 and 25(OH)D3. 25(OH)D2 and 25(OH)D3 induced CYP24A1 expression but reduced VDR mRNA expression, whereas the expression of CYP27B1, occludin, and E-cadherin did not change. Additionally, the induction of CYP24A1 expression by 25(OH)D3 was higher than that of 25(OH)D2, which may contribute to their differences in inhibiting cell viability. S. aureus invaded into MAC-T cells and universally inhibited gene expressions. Pre-treat MAC-T cells with 25(OH)D2 reduced S. aureus adhesion while pre-treatment with 25(OH)D3 inhibited S. aureus invasion, but neither of the compounds attenuated the S. aureus-induced gene expression reduction. In conclusion, the present study shows that D2 compounds have comparable effects on inhibiting cell viability and S. aureus invasion to their D3 analogues in vitro, suggesting that D2 and its metabolites have potential in controlling bovine mastitis.

A search for synbiotics: effects of enzymatically modified arabinoxylan and Butyrivibrio fibrisolvens on short-chain fatty acids in the cecum content and plasma of rats.

Identification of dietary strategies to increase large intestinal production and absorption of short-chain fatty acids (SCFAs), especially butyrate, is of great interest due to the possible health promoting effects. We explored the effect of an enzymatically modified arabinoxylan-rich diet (EAXD) versus a Western-style control diet (WSD) low in dietary fiber with or without orally administrated Butyrivibrio fibrisolvens, a butyrate producer, on the SCFA pool in the cecal content and feces and the SCFA concentration in the blood of rats. The pool of acetate, butyrate and total SCFA was more than double in the cecal content from EAXD-fed rats compared with WSD-fed rats, and this was also reflected as an increase in portal plasma SCFA concentrations. Acetate, propionate and total SCFA concentrations were higher in mixed venous plasma following the EAXD. The number of B. fibrisolvens did not increase significantly in cecal content following administration of the bacteria. Furthermore, there was no interaction between the EAXD and B. fibrisolvens on the measured parameters.

Spray Dried, Pasteurised Bovine Colostrum Protects Against Gut Dysfunction and Inflammation in Preterm Pigs.

OBJECTIVE: Feeding bovine colostrum (BC) improves gut maturation and function and protects against necrotizing enterocolitis, relative to formula in newborn preterm pigs. Before BC can be used for preterm infants, it is important to test if the milk processing, required to reduce bacterial load and increase shelf life, may affect bioactivity and efficacy of a BC product. METHODS: We investigated if spray dried, pasteurised BC had protective effects on gut function in preterm pigs, relative to formula. After a 2-day total parenteral nutrition period, preterm pigs were fed formula for a few hours (to induce a proinflammatory state) followed by 2 days of formula (FORM, n = 14), BC (colostrum [COLOS], n = 14), spray-dried BC (POW, n = 8), or pasteurised, spray-dried BC (POWPAS, n = 9). RESULTS: Spray drying and pasteurisation of BC decreased the concentration of transforming growth factor-ß1, -ß2 and increased protein aggregation. All of the 3 BC groups had reduced necrotizing enterocolitis severity, small intestinal levels of IL-1ß, -8, and colonic lactic acid levels, and increased intestinal villus height, hexose absorption, and digestive enzyme activities, relative to the FORM group (all P < 0.05). All of the 3 BC diets stimulated epithelial cell migration in a wound-healing model with IEC-6 cells. CONCLUSIONS: Spray drying and pasteurisation affect BC proteins, but do not reduce the trophic and anti-inflammatory effects of BC on the immature intestine. It remains to be studied if BC products will benefit preterm infants just after birth when human milk is often not available.

Effects of Resistant Starch and Arabinoxylan on Parameters Related to Large Intestinal and Metabolic Health in Pigs Fed Fat-Rich Diets.

This study compared the effects of a resistant starch (RS)-rich, arabinoxylan (AX)-rich, or low-DF Western-style control diet (all high-fat) on large intestinal gene expression, adiposity, and glycemic response parameters in pigs. Animals were slaughtered after 3 weeks of treatment. Plasma butyrate concentration was higher following the high-DF diets, whereas plasma glucose, insulin, and insulin resistance increased after 3 weeks irrespective of diet. The mRNA abundance in the large intestine of genes involved in nutrient transport, immune response, and intestinal permeability was affected by segment (cecum, proximal, mid or distal colon) and some genes also by diet. In contrast, there was no diet-induced effect on adipose mRNA abundance or adipocyte size. Overall, a high level of RS or AX did not demonstrate strong beneficial effects on large intestinal gene expression as indicators of colonic health or glycemic response parameters when included in a high-fat diet for pigs as a model of healthy humans.

Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity.

A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1-4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 µg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 µg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3-10) and their anti-inflammatory activity against RAW 264.7 cells (1-10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.