In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Impacts of supplementing Cannabis sativa byproducts during the transition period on behaviour, feed consumption, constipation levels, colostrum production and piglet performance in hyperprolific sows.

In the modern swine industry, inflammation and pain in sows after farrowing represent a crucial animal welfare concern. Cannabis sativa, a medicinal plant, has analgesic, anti-inflammatory and antipyretic properties and is rich in fibre. The objective of this study is to examine the impacts of supplementing sows with Cannabis sativa byproducts during transition periods (7 days before and after farrowing) on various aspects including postpartum behaviour, feed intake, constipation, farrowing duration, colostrum yield and piglet performance. The experiment involved a total of 100 Landrace × Yorkshire sows. The sows were distributed according to parity numbers into two groups, i.e., control (n = 54) and treatment (n = 46). The control group was provided with a lactation diet at 3.0-3.5 kg per day for a period of 7 days before and after farrowing. The treatment groups received the same quantity of the diet but with an additional supplementation of 150 g/d of Cannabis sativa byproduct. The byproduct was analysed and contained 0.24 % (w/w) cannabidiol (CBD), resulting in a daily intake of 360 mg of CBD per sow. The conventional lactational diet had a dietary fibre content of 4.3 %, whereas the diet supplemented with Cannabis sativa byproduct had a higher dietary fibre content of 16.9 %. Video cameras were used to observe and document the behaviour of sows within the initial 24 h after farrowing. The duration in which sows engaged in activities such as sleeping, sitting, standing, feeding and nursing their piglets was quantified. Additionally, the rectal temperature of the sows was measured, and a temperature equal to or exceeding 39.5 °C was considered indicative of fever. The faecal score of the sows was assessed, and a faecal score of ≤2 was classified as constipation. On the third day postpartum, the proportion of sows with fever in the treatment group was lower than that in the control group (20.0 % and 38.9 % respectively, P = 0.051). Sows receiving supplementation with Cannabis sativa byproducts exhibited increased durations of standing and feeding compared to those in the control group (P < 0.05). Notably, overall, sows without constipation issues spent more time consuming feed than those experiencing constipation (P = 0.006). The prevalence of constipation was significantly lower in the treatment group compared to the control group (17.4 % and 81.5 %, respectively, P < 0.001). Furthermore, the postpartum sows demonstrated increased feed intake following supplementation with Cannabis sativa byproducts (P < 0.05). Sow colostrum yield, piglet colostrum intake, piglet mortality and other piglet traits did not differ between the control and treatment groups (P > 0.05). In conclusion, supplementing Cannabis sativa byproducts during the transition periods in peri-parturient sows under tropical conditions resulted in a reduction in constipation issues and improved sow activities, such as increased time spent standing and consuming feed within the first 24 h postpartum.

Chemical compositions, antioxidant, antimicrobial, and mosquito larvicidal activity of Ocimum americanum L. and Ocimum basilicum L. leaf essential oils.

BACKGROUND: Ocimum americanum L. (O. americanum) and Ocimum basilicum L. (O. basilicum) are highly valued aromatic medicinal plants. Their leaves are widely used as spices in traditional cuisine. Their essential oils (EOs) are extensively used in food, cosmetic, and pharmaceutical industries. This study aimed to investigate the main chemical profiles of O. americanum and O. basilicum leaf EOs and assess their effects on antibacterial, antioxidant, and larvicidal properties. METHODS: EOs were extracted from the leaves of O. basilicum and O. americanum using steam distillation in a Clevenger-type apparatus. The chemical constituents of the EOs were analyzed using gas chromatography-mass spectrometry. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and metal-chelating techniques were used to assess the free-radical scavenging capability of the oils. The extracted oils were also tested for their antibacterial activities via a disk-diffusion test and the broth microdilution method. Furthermore, the mosquito larvicidal (Aedes aegypti) activity was tested using standard protocols. RESULTS: Camphor (33.869%), limonene (7.215%), longifolene (6.727%), caryophyllene (5.500%), and isoledene (5.472%) were the major compounds in O. americanum leaf EO. The EO yield was 0.4%, and citral (19.557%), estragole (18.582%) camphor (9.224%) and caryophyllene (3.009%) were the major compounds found among the 37 chemical constituents identified in O. basilicum oil. O. basilicum exhibited a more potent antioxidant activity in DPPH, FRAP, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid tests than O. americanum. The zones of inhibition and minimum inhibitory concentration of the oils in the microdilution and disk diffusion methods were 8.00 ± 0.19 mm to 26.43 ± 2.19 mm and 3.12-100 µg/mL, respectively. At 400 ppm, O. basilicum and O. americanum EOs demonstrated larvicidal activity, with mortality ratios of 73.60% ± 0.89% and 78.00% ± 1.00%, respectively. Furthermore, after 30 min of exposure to O. americanum and O. basilicum EOs, the larval death rates were 73.60% ± 0.89% and 78.00% ± 1.00%, respectively. CONCLUSIONS: The findings revealed that the EOs extracted from the leaves of O. basilicum and O. americanum exhibited reasonable antioxidant, antibacterial, and mosquito larvicidal potentials, and can be used as alternative medicine for the treatment of human health and larvicidal mosquito control.

Elicitation enhances the production of friedelin and epifriedelanol in hairy root cultures of Cannabis sativa L.

Cannabis sativa L. (hemp) has a global distribution and social impact, and it is widely used as a medicinal plant, food ingredient, and textile fiber. Its roots have received less attention than other parts, especially the inflorescence, leaves, and shoots. Triterpenoids, including friedelin and epifriedelanol, have been found in hemp roots, and their anti-inflammatory effects have been reported. In this study, the potential enhancement of triterpenoid accumulation in the roots of C. sativa by elicitation was examined. Hairy roots were successfully established, and they contained 2.02-fold higher triterpenoid levels than natural roots. Furthermore, hairy roots treated with 75 µM salicylic acid had 1.95-fold higher friedelin levels (0.963 mg/g DW) and 1.4-fold higher epifriedelanol levels (0.685 mg/g DW) than untreated hairy roots. These results suggested that the elucidation of hairy root cultures using an optimized elicitor could represent an alternative strategy to produce the valuable triterpenoids friedelin and epifriedelanol.

Antioxidant, Anti-Skin-Aging, Anti-Inflammatory, and Anti-Acetylcholinesterase Activities of Rourea oligophlebia Extracts.

The objective of this study was to evaluate the antioxidant, anti-skin-aging, anti-inflammatory, and anti-acetylcholinesterase activities of the hexane (n-hex), AcOEt, BuOH, MeOH, and aqueous extracts from R. oligophlebia roots. The total phenolic and flavonoid contents (TPC and TFC) were determined using Folin-Ciocalteu and AlCl3 colorimetric assays. The antioxidant capacity was examined by reducing power (RP), ferric reducing antioxidant power (FRAP), ABTS⋅+ , and DPPH⋅+ radical cation assays. All extracts potentially exhibited antioxidant activity with IC50 values ranging from 2.93 to 5.73 µg/mL for ABTS⋅+ and from 5.69 to 7.65 µg/mL for DPPH⋅+ except the n-hex extract. The BuOH, MeOH, and aqueous extract possess promising anti-skin-aging activities, as observed by an attenuation of UV-A toxicity on human keratinocytes. We proposed that these anti-skin-aging properties are possibly due to direct scavenging activity against reactive oxygen species and upregulate cellular antioxidant machinery. Moreover, we found that the antioxidant capacity was well correlated with anti-inflammatory capacity against nitric oxide (NO) production in terms of the n-hex, AcOEt, and BuOH extracts with IC50 values from 23.21 to 47.1 µg/mL. In contrast, these activities were found to be poorly correlated with AchE activity. To the best of our knowledge, this is the first report of the antioxidant, anti-skin-aging, anti-inflammatory, and anti-acetylcholinesterase activities of the extracts of R. oligophlebia roots. These findings indicated that this species could be a potential source of natural antioxidant, anti-aging, and anti-inflammatory agents. Consequently, it may be suggested as a medicinal plant that prevents diseases related to oxidative stress and inflammatory responses.

Enhanced Physicochemical Stability of the L-DOPA Extract of Mucuna pruriens Seeds by Adding Phyllanthus emblica.

Levodopa (L-DOPA) is an essential drug for the treatment of Parkinson's disease. Currently, L-DOPA can be produced by chemical synthesis and can also be found naturally in many herbs, especially Mucuna Pruriens (MP). According to clinical research, the MP extract containing L-DOPA for the treatment of Parkinson's disease could reduce side effects more than the synthetic one. Unfortunately, MP extracts can be easily degraded. Changes in physical and chemical properties such as the appearance (color, melt, solid lump) and the reduction of L-DOPA content in the extract were commonly observed. Therefore, it is necessary to develop an extraction procedure to stabilize the extract of L-DOPA. This study attempted to enhance the extraction process by modifying the traditional acidification approach using hydrochloric acid, citric acid, or ascorbic acid. According to the stability test results, using Phyllanthus emblica water (PEW) as a solvent improved the preservative properties more than other solvents. The color of the PEW-MP powder changed slightly after 12 months of accelerated storage, but the amount of L-DOPA remained the highest (73.55%). Moreover, L-DOPA was only detected in MP and PEW-MP, but not PEW alone (the HPTLC chromatogram at Rf 0.48 and the HPLC chromatogram at Rt 6.0 min). The chemical profiles of PEW and L-DOPA observed in the chromatograms indicated that they are independently separated. As a result, they can be applied to a quality control process. Therefore, PEW was proven to be a powerful solvent for L-DOPA herbal extract that could be readily used as a raw material for herbal products.

Harnessing the advances of genetic engineering in microalgae for the production of cannabinoids.

Cannabis is widely recognized as a medicinal plant owing to bioactive cannabinoids. However, it is still considered a narcotic plant, making it hard to be accessed. Since the biosynthetic pathway of cannabinoids is disclosed, biotechnological methods can be employed to produce cannabinoids in heterologous systems. This would pave the way toward biosynthesizing any cannabinoid compound of interest, especially minor substances that are less produced by a plant but have a high medicinal value. In this context, microalgae have attracted increasing scientific interest given their unique potential for biopharmaceutical production. In the present review, the current knowledge on cannabinoid production in different hosts is summarized and the biotechnological potential of microalgae as an emerging platform for synthetic production is put in perspective. A critical survey of genetic requirements and various transformation approaches are also discussed.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products.

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

Some Antioxidant Properties of Components from the Flower of Ochna integerrima and Their Beneficial Effects on HaCaT Keratinocytes and in Silico Analysis on Tyrosinase.

Four compounds, luteolin (1), 6-γ,γ-dimethylallylquercetin 7-O-ß-D-glucopyranoside (2), 6-γ,γ-dimethylallylkaempferol 7-O-ß-D-glucopyranoside (3), and 6-γ,γ-dimethylallyldihydrokaempferol 7-O-ß-D-glucoside (4), were isolated for the first time from AcOEt extract of the O. integerrima flower. We then evaluated the antioxidant effects of AcOEt, butanol, and MeOH extracts and their effects on H2 O2 against oxidative stress in HaCaT keratinocyte cell lines. Furthermore, 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH⋅) radical scavenging activities of 1-4 were determined and their mechanisms of action on tyrosinase were predicted by in silico studies. The results revealed that the AcOEt extract and 1-3 exhibited good DPPH⋅ radical scavenging activity. Furthermore, this extract also had a significant protective effect against H2 O2 -induced oxidative stress in HaCaT cells. In silico studies indicated that the activity of 1-3 may be due to tyrosinase inhibition with MM-GBSA free binding energies of -78.9, -70.1, and -71.1 kcal mol-1 , respectively, compared to 4 with an energy -56.9 kcal mol-1 .

Insights into antioxidant activities and anti-skin-aging potential of callus extract from Centella asiatica (L.).

Formation of oxidative stress in dermal fibroblasts plays crucial roles in aging processes of skin. The use of phytochemicals that can promote capacity of fibroblasts to combat oxidative stress is an attractive strategy to prevent skin aging and promote skin beauty. Centella asiatica has been used to treat multitude of diseases for centuries. Previous investigations demonstrated that extracts from C. asiatica have a broad range of beneficial activities through their antioxidant activity. Hence, the extract from this medicinal plant could be a great candidate for anti-skin-aging agent. Callus culture offers a powerful platform for sustainable, rapid and large-scale production of phytochemicals to serve extensive demands of pharmaceutical and cosmeceutical industries. Here, we demonstrated the application of callus culture of Centella asiatica to produce bioactive metabolites. The 50% ethanolic extract of callus culture has distinctive features of chemical compositions and biological profiles. Information from HPTLC-DPPH and HPLC analysis suggested that the callus extract comprises distinctive antioxidant compounds, compared with those isolated from authentic plant. Moreover, results from cell culture experiment demonstrated that callus extract possesses promising antioxidant and anti-skin-aging activities. Pre-treatment with callus extract attenuated H2O2-induced-cytotoxicity on human dermal fibroblasts. The results from RT-qPCR clearly suggested that the upregulation of cellular antioxidant enzymes appeared to be major contributor for the protective effects of callus extract against oxidative stress. Moreover, supplementation with callus extract inhibited induction of matrix metalloprotease-9 following H2O2 exposure, suggesting its potential anti-skin-aging activity. Our results demonstrate the potential utility of C. asiatica callus extract as anti-skin-aging agent in cosmeceutical preparations.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Correction to: Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

The article Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites, written by Seiichi Sakamoto, Waraporn Putalun, Sornkanok Vimolmangkang, Waranyoo Phoolcharoen, Yukihiro Shoyama, Hiroyuki Tanaka and Satoshi Morimoto, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 21 November 2017 without open access.

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

Analysis of Isoquinoline Alkaloid Composition and Wound-Induced Variation in Nelumbo Using HPLC-MS/MS.

Alkaloids are the most relevant bioactive components in lotus, a traditional herb in Asia, but little is known about their qualitative and quantitative distributions. Here, we report on the alkaloid composition in various lotus organs. Lotus laminae and embryos are rich in isoquinoline alkaloids, whereas petioles and rhizomes contain trace amounts of alkaloids. Wide variation of alkaloid accumulation in lamina and embryo was observed among screened genotypes. In laminae, alkaloid accumulation increases during early developmental stages, reaches the highest level at full size stage, and then decreases slightly during senescence. Vegetative and embryogenic tissues accumulate mainly aporphine-type and bisbenzylisoquinoline-type alkaloids, respectively. Bisbenzylisoquinoline-type alkaloids may be synthesized mainly in lamina and then transported into embryo via latex through phloem translocation. In addition, mechanical wounding was shown to induce significant accumulation of specific alkaloids in lotus leaves.

Productivity and quality of volatile oil extracted from Mentha spicata and M. arvensis var. piperascens grown by a hydroponic system using the deep flow technique.

The purpose of this study was to determine the differences between spearmint (Mentha spicata L.) and Japanese mint (M. arvensis L. var. piperascens Malinv.) cultivated in either soil or nutrient solution using the deep flow technique (DFT). The differences were measured in terms of harvest period (full bloom period) and quantity and chemical components of volatile oils. The spearmint and Japanese mint were cultivated in four different nutrient formulas: plant standard nutrient, plant standard nutrient with an amino acid mixture, plant standard nutrient with a sulphur compound, and a combination of plant standard nutrient with an amino acid mixture and a sulphur compound. We observed that cultivation of spearmint and Japanese mint in nutrient solution using DFT is an effective method to provide high production of volatile oil, since it results in an earlier harvest period and higher quantity of volatile oil. We determined that for spearmint an amino acid mixture is an appropriate nutrient supplement to enhance production of volatile oil with optimum carvone content. Finally, we observed high menthol content in Japanese mint grown in all four nutrient formulas; however, supplementation with a combination of sulphur fertilisation and amino acid mixture yields the highest quantity of volatile oil.