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HPTLC HYPHENATIONS FOR PHYTOCHEMICAL QUALITY ANALYSIS: MS AND BIOAUTPGRAPHY ENLARGE THE ANALYTICAL INFORMATION
© Evelyn Wolfram, Ivana Kroslakova, Samuel Peter, Sarah Bräm
Zürich University of Applied Science (ZHAW), Phytopharmacy and Natural Products Research Group, Wädenswil, Switzerland
HPTLC is widely used in phytochemical analysis, especially for the identification of herbal drug raw material [1] or stability studies. Methods are rapid and simple, but for quality control, a high degree of standardization is required concerning the chromatographic layers and automated state of the art TLC equipment to standardize chromatographic development and detection. The analytical information that is gained by HPTLC analysis of multicompound natural mixtures is a fingerprint picture, which shows the result of a separation (Rf values) and subsequent derivatization reaction. In contrast to other chromatographic techniques, the analytes are preserved on the thin layer plate and a coupling, also called hyphenation, with further analysis techniques or even bioassays is possible. This opens opportunities for gaining additional molecular and bioactivity information to the visual results. The presentation demonstrates examples of own studies of coupling of HPTLC with MALDI-TOF-MS [2], with an enzymatic (XOD optimized on basis of [3]) and a reporter gene bioassay (planar Yeast Estrogen Screen [4]).
HPTLC standardized fi ngerprint analysis was performed with automated equipment from (CAM AG, Muttenz) and silica HPTLC plates (Merck, Darmstadt). TLC-MALDI-TOF-MS coupling (Bruker, Daltonics). As positive controls, Allopurinol (Sigma-Aldrich) for XOD, Estradiol and the Isoflavones Daidzein and Genistein (Extrasynthese) for YES assay have been used.
Xanthin Oxidase inhibiting zones can be detected in plant extracts for screening and can be correlated with the concentration of the positive control Allopurinol.
However, the technique could deliver false positive results due to feigned inhibition spots. Control tests have to be introduced.
The coupling of the planar YES can help to detect phytoestrogen^ compounds and might serve as quality control method of standardized extracts of soy and red clover extracts.
Direct coupling of HPTLC and MALDI-TOF MS for the analysis of flavonol aglycones and glycosides demonstrates the suitability of the method for screening of HPTLC chromatograms from start to front zone. This gives more insight in flavonoid identity fingerprints and QC and might be applied for investigations of degradation in the extract mixture during stability studies of herbal drug and preparations. Also, active zones from bioautographic assays can be targeted to gain mass information.
It can be concluded that the presented hyphenation methods offer rapid and simple tools for enlarged quality assessment and bioactivity standardization of herbal products. However, the method design has to exclude artefacts and active spots need to be examined critically.
References:
1. EDQM Ph. Eur. 9th Edition.
2. Kroslakova I, Pedrussio S, Wolfram E. 2016. Phyto-chem. Anal. 27:222-228.
3. Bräm S, Wolfram E. 2017. Phytochem. Anal. 28:74-86.
4. Schönborn A, Grimmer A. 2013. J.Plan.Chrom. 26(5):402-408.
ANTIDIABETIC ACTIVITY OF PROTEIN EXTRACTED FROM CLITORIA TERNATEA BLUE PETAL FLOWER
© Yanti12, Marstella Minelko2, Agustin Wydia Gunawan3, Antonius Suwanto2
1 Food Technology, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia;
2 Master of Science in Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia;
3 Biology Program, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia
Clitoria ternatea is a well-known plant in the traditional Ayurvedic medicine that posseses a wide range pharmacological activities. This study was conducted to determine the antidiabetic activity of protein extracted from C. ternatea blue petal flower (PCT) to inhibit the development of diabetes in vitro by a-amylase inhibition assay, and in vivo by measurement of blood glucose level, and the diabetes-related genes expression, such as peroxisome proliferator-activated receptors gamma
(PPARy), glucose transporter 2 (Glut2), transcription factor 7-like 2 (Tcf7l2), calpain-10 (Capn10), and monocyte chemoattractant protein-1 (MCP-1) in alloxan-induced diabetic mice. PCT was extracted by using isoelectric precipitation and confirmed by by SDS-PAGE. The result showed that there were 2 protein fractions with molecular size of 60 dan 80 kDa. For in vitro study, we employed PCT at 0.2-1.0 mg/ml. For in vivo study, we used oral PCT with doses of 100 and
Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy] vol. 15/2017/suppLement 1
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500 mg/kg bw. The inhibition of a-amylase activity by PCT and acarbose standard was dose-dependent from 0.2 to 1.0 mg/ml. The highest a-amylase inhibition was observed at 1.0 mg/ml of PCT, which was 20.63%, where acarbose standard with the similar concentration only showed 14.30% inhibition of a-amylase. The in vivo study showed that PCT at 500 mg/kg bw decreased the blood glucose level in alloxan-induced diabetic mice, and its efficacy was comparable to metformin
at 100 mg/kg bw. PCT (100 and 500 mg/kg bw) also significantly modulated the expression of diabetes-related genes (P < 0.05). In adipose and skeletal muscle tissues, PCT upregulated the expression of PPARy and Tcf7l2 genes, meanwhile MCP-1 gene expression was downregulated. In the pancreas, PCT upregulated the expression of Glut2, Capn10, and Tcf7l2 genes. These results indicate that PCT has antidiabetic potential for management of type 2 diabetes.
IN VITRO ANTIOBESITY AND ANTIDIABETIC ACTIVITIES OF ACTIVE FRACTIONS FROM ZANTHOXYLUM ACANTHOPODIUM FRUITS
© Yanti12, Riyardi Wijaya Limas2, Yenny Novita2
1 Food Technology, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia;
2 Biology Program, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia
Therapeutic strategy using spice-derived compounds which are naturally occurring phytochemicals and biomolecules may elicit anti-obesity and anti-diabetic properties. In this study, Zanthoxylum acanthopodium, known as lemon pepper, was isolated for its active fractions (protein, polysaccharide, polyphenol, and essential oil) and investigated for its actions for treatment of obesity and diabetes via regulating the secretion of pro-inflammatory mediators and lipogenic proteins in cell culture system of 3T3-L1 pre-adipocytes and Chang hepatocytes in vitro. In obesity system, ELISA profiles showed that protein fraction from lemon pepper at 1-100 ig/ml significanlty increased up to 140% of adiponectin level in 3T3-L1 adipocytes compared to the untreated cells. Protein fraction at various concentrations (1-100 ig/ml) also attenuated the expression of leptin and interleukin (IL)-6 proteins up to 30% in cells, indicating that it may be potential as antiobesity candidate by reducing and regulating caloric intake,
body weight, and lipid metabolism. In diabetic system, ELISA profiles demonstrated that all lemon pepper fractions, including protein and polysaccharide fractions at 1-100 ig/ml, polyphenol and essential oil fractions at 1-25 ig/ml, effectively reduced the expression of C-reactive protein (CRP) level up to 25% in the dose-dependent manner in Chang cells. CRP is known as one of the inflammatory factors produced by liver in an acute phase of inflammation and its concentration can increase in plasma. Meanwhile, polysaccharide (1-100 ig/ml) and polyphenol fractions (1 -25 ig/ml) were found to be active on inhibiting tumor necrosis factor (TNF)-a and monocyte chemoattractant protein (MCP)-1 levels at range of 20-45% compared to untreated Chang cells. Both TNF-a and MCP-1 are recognized as pro-inflammatory cytokines in obesity-related diseases. Therefore, selected fractions from Z. acanthopodium may possess antiobesity and antidiabetic potentials on hepatocytes and adipocytes in vitro.
ANTI-INFLAMMATORY EFFECT OF ANTHOCYANIN EXTRACTED FROM CLITORIA TERNATEA BLUE POWER PETAL IN CARRAGENAN-INDUCED RAT PAW EDEMA
© Yanti123, Yoseph Toni Wijaya2, Adi Yulandi23, Agustin Wydia Gunawan3, Devi Anggraini Ngandiri3, Dina Sabella3
1 Food Technology, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia;
2 Master of Science in Biotechnology, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia;
3 Biology Program, Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta, Indonesia
Inflammation therapeutics could be targeted by non- of these drugs have been increasing. This study was steroidal anti-inflammatory drugs (NSAIDs), however, aimed to evaluate the anti-inflammatory activity of several gastrointestinal side effects caused by the use anthocyanin fraction isolated from Clitoria ternatea blue
Obzory po kliniceskoj farmacologii i lekarstvennoj terapii [Reviews of clinical pharmacology and drug therapy]
vol. 15/2017/suppLeMEnT 1
