Correction: Authentication of Herbal Supplements Using Next-Generation Sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0156426.].

Authentication of Herbal Supplements Using Next-Generation Sequencing.

BACKGROUND: DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. METHODS: We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. RESULTS: All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. CONCLUSION: Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.

Fish oil, atherogenesis, and thrombogenesis.

Marine fish consumption is known to reduce mortality from ischemic heart disease. The use of fish oil as a dietary supplement, however, is not universally recommended. In large doses, fish oil reduces plasma cholesterol and triacylglycerol but increases low density lipoprotein (LDL) levels and the potential for free radical generation and bleeding. Moderate marine fish consumption is known to reduce mortality without altering commonly measured variables, i.e., plasma cholesterol levels, in vitro platelet aggregation, and bleeding times. In swine, we observed that monocyte adhesions and platelet clumps over the lesion surface of proximal left anterior descending (LAD) coronary arteries are markedly reduced when an atherogenic diet was supplemented with cod-liver oil, even when the cholesterol levels were equalized with the untreated group. These findings suggest that fish oil is hypothrombogenic. We developed an in vitro assay to delineate the mechanism whereby fish oil reduced monocyte-endothelial cell interactions in vivo. The effects of supplementing the culture medium with different fatty acids on adhesions between lipopolysaccharide (LPS) stimulated swine aortic endothelial cells (SAEC) and the human monocyte-like cell line, U937, was investigated in a 10 minute adhesion assay at 37 degrees C. Exposure of SAEC for 6 hours to media containing 50-200 microMs eicosapentaenoic (EPA), stearic, oleic, linoleic, and arachidonic acid, respectively, revealed that only EPA reduced U937-SAEC adhesion. Exposure of U937 to EPA also reduced adhesions. EPA was not effective when added to the SAEC more than 2 hours after they were stimulated with LPS. Exposure of human umbilical vein endothelial cells (HUVEC) to EPA reduced the expression of VCAM-1, ELAM-1, and ICAM-1 after 5 hours of stimulation with LPS. These results suggest that EPA may functionally impair the induction/expression of adhesion molecules.

Dietary fish oil reduces microthrombi over atherosclerotic lesions in hyperlipidemic swine even in the absence of plasma cholesterol reduction.

We have investigated in swine the effect of fish oil additives to a butter-cholesterol hyperlipidemic diet (BT) on atherogenesis and thrombogenesis when average plasma cholesterol levels were kept similar in fish oil-treated and untreated BT groups. The studies included evaluation of lesion sizes and cell numbers, counts of adherent monocytes over lesions, and counts of platelet clumps (microthrombi) over lesions either attached directly to endothelium or to adherent monocytes. Anatomic sites studied for lesion development were the left anterior descending coronary artery (LAD), the distal 1/5 of the abdominal aorta, and a proximal portion of the thoracic aorta. Counts of attached monocytes and platelet clumps were made by scanning electron microscopy only for the LAD and expressed per mm2 of surface. The most striking new result was in regard to the platelet clumps. These were reduced by the fish oil from 996 +/- 295/mm2 in the untreated BT group to 313 +/- 59 and 364 +/- 105 in BT+cod liver oil and BT+menhaden oil groups, respectively. Most of the platelet clumps were adherent to attached monocytes in all groups and the number of attached monocytes were greatly reduced by the fish oil additive. Thus there were close relationships among platelet clumps, monocytes, and lesion endothelium. Numbers of attachments over nonlesion endothelium were much less than those over lesions in all dietary groups. The most surprising result was the lack of retardation of lesion growth by the fish oil additives in spite of the reduction in attached monocytes and platelet clumps. In previous studies where the high plasma cholesterol levels in the BT swine had been modestly reduced (about 25%) there had been a marked retardation of lesion growth. The current result suggests that plasma cholesterol is the major factor controlling lesion growth in this model through under milder conditions and longer observation periods other factors might become apparent.

Reductions in serum thromboxane, prostacyclin, and leukotriene B4 levels in swine fed a fish oil supplement to an atherogenic diet.

We have reported previously that fish oil rich in omega-3 fatty acids added to a butter-cholesterol atherogenic diet for swine resulted in marked retardation of the atherosclerotic process which many regard as largely an inflammatory response to injury by excessive lipids in the intima. In this report on the same swine we present serum levels of several eicosanoids derived from arachidonic acid via the cyclooxygenase and lipoxygenase pathways. The study involves six swine fed a high fat, high cholesterol diet (BT group) for 4 months, six swine fed the same diet but with 30 ml/day fish oil added (BT + FO), and five swine fed a low fat, low cholesterol mash diet (MA). The serum eicosanoids were measured by radioimmunoassay. Thromboxane B2 levels (ng/dl: means +/- SEM) were 543 +/- 49 for MA, 231 +/- 12 for BT, and 105 +/- 20 for BT + FO, and all differences were statistically highly significant, 6-Keto PGF1 alpha (a relatively stable prostacyclin metabolite) levels were 249 +/- 31 for MA, 184 +/- 12 for BT, and 101 +/- 10 for BT + FO, and all differences were significant. Leukotriene B4 levels at 4 months were 151 +/- 25 for MA, 112 +/- 11 for BT, and 84 +/- 11 for BT + FO. BT + FO was significantly different from both MA and BT, but BT was not significantly different from MA. Leukotriene C4 levels were not significantly different among the three groups. Of special interest was the effect of the BT diet without the FO additive in reducing several eicosanoid levels compared to MA values. The affected eicosanoid levels were reduced still further by the fish oil additive, indicating its ability to inhibit both the cyclooxygenase and the lipoxygenase pathways. The relation of the fish oil-induced inhibition to the observed retardation of atherogenesis is not as yet clear but there are several theoretical possibilities, including reduction in recruitment of monocytes and in proliferation of smooth muscle cells.

Comparison of effects of fish oil and corn oil supplements on hyperlipidemic diet induced atherogenesis in swine.

The addition of a fish oil supplement rich in n - 3 unsaturated fatty acids to a high cholesterol, high saturated fat (BT) diet for swine has been shown previously to result in modest lowering of plasma cholesterol levels and in marked retardation of atherogenesis. It has been suggested that the effect was due to the change in polyunsaturated (PUFA) to saturated fatty acid ratios (P/S) and that a supplement of PUFA of the n - 6 series might have the same effect as the fish oil. We have tested this hypothesis in swine fed an atherogenic diet by comparing the effect of a fish oil supplement producing a P/S ratio of 0.28 to that of corn oil in the same amount producing a ratio of 0.46. The P/S ratio of the atherogenic diet without supplements was 0.16. Thirteen young male Yorkshire swine were fed either BT alone (n = 4), BT + cod liver oil (n = 4) or BT + corn oil (n = 5) for 6 months and then killed for quantitative studies of atherosclerosis in the aortas and coronary arteries including lesion areas, number of lesion cells, and number of monocytes attached to endothelium. Plasma cholesterol levels were determined periodically and lipoproteins were separated terminally by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. The fish oil supplement resulted in a 30% reduction in time-weighted average plasma cholesterol levels, and a marked shift in terminal lipoprotein patterns from predominantly apo B and E containing ones to predominantly apo B only ones. Atherogenesis was reduced by the fish oil supplement as judged by several morphometric criteria including size of lesions, number of lesion cells, and number of monocytes attached to lesion endothelium. The corn oil supplement produced no significant reductions in any of these variables from those in swine fed the atherogenic BT diet without the supplement. We conclude that the n - 3 fatty acid rich fish oil supplemented diet retarded atherogenesis, but that this effect was not shared by the corn oil supplemented diet which had an even higher P/S ratio.

Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine.

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

Association of plasma intermediate density lipoproteins with atherogenic intimal proliferative activity in abdominal aortas of hyperlipidemic swine.

In an earlier study of swine fed hyperlipidemic (HL) diets containing either butter or corn oil we found wide differences in atherogenic intimal proliferative responses among the swine, especially in the corn oil group, that could not be entirely accounted for by differences in serum cholesterol levels. We hypothesized that there might be differences in some lipoprotein constituents other than total cholesterol that might better account for the differences in intimal proliferative responses. In the current experiment, 11 swine were fed HL diets containing butter (n = 5) or corn oil (n = 6). At 90 days on HL diet, plasma was obtained for lipoprotein fractionation. At 120 days the swine were killed and the lesion areas and number of nuclear profiles in the intima or lesions in multiple cross-sections of the distal abdominal aorta were determined under light microscopy. Tritiated thymidine labeling indices of the intima or lesion cells were also determined. On comparing the butter and corn oil groups, only IDL-cholesterol and tritiated thymidine labeling indices of the cells in the lesions were significantly different. Based on the number of nuclear profiles per cross-section (Np/Cx) 7 were classified as high intimal proliferative responders (5 butter, 2 corn oil) and 4 as low responders (4 corn oil). The high-proliferative responders had plasma IDL levels 6-fold greater than those of the low responders with no overlaps. LDL, VLDL and HDL were not significantly different between the high- and low-proliferative responders. Among correlation coefficients between Np/Cx and plasma lipoprotein variables for all swine (n = 11), IDL cholesterol level was best correlated with Np/Cx. However, in the high responder group LDL values showed a highly significant correlation with the amount of intimal proliferation.

Hypo-atherogenic effect of dietary corn oil exceeds hypo-cholesterolemic effect in swine.

Three groups of young swine were fed semi-purified diets enriched in fat and cholesterol for 90 days. In one group 40% of calories were from butter; in another 40% were from corn oil; in a third group 20% were from corn oil. Mean serum cholesterol values were respectively 530, 458 and 400 mg/dl. Amounts of atherosclerosis in the abdominal aortas expressed as mean number of nuclear profiles per cross-section were respectively 1611, 734 and 585; expressed as mean lesion area per cross section 46.7, 21.4 and 19.4 mm2 X 10(2). All differences between the butter group and either of the two corn oil groups were statistically significant. We found that intimal response to the butter containing diet was significantly greater than to the corn oil containing diet for equivalent hypercholesterolemic response. We present detailed statistical validation of the disproportionately larger intimal response to butter containing diet. Furthermore, we suggest that estimates of comparative atherogenic risk of food fats based on response of serum cholesterol may mask important atherogenic differences.