Screening for consistency and contamination within and between bottles of 29 herbal supplements

Maren E Veatch-Blohm; Iris Chicas; Kathryn Margolis; Rachael Vanderminden; Marisa Gochie; Khusmanie Lila

doi:10.1371/journal.pone.0260463

. 2021 Nov 23;16(11):e0260463. doi: 10.1371/journal.pone.0260463

Screening for consistency and contamination within and between bottles of 29 herbal supplements

Maren E Veatch-Blohm ^1,^*, Iris Chicas ¹, Kathryn Margolis ¹, Rachael Vanderminden ¹, Marisa Gochie ¹, Khusmanie Lila ¹

Editor: Hikmet Aydin²

PMCID: PMC8610273 PMID: 34813619

Abstract

In the United States the marketing of dietary supplements, of which the majority are herbal supplements, is currently a multibillion-dollar industry involving use from over half of the adult population. Due to their frequency of use and the lack of regulation of herbal supplements by the Food and Drug Administration (FDA) it is important for the health and safety of consumers to know about consistency of supplements and any possible contamination by harmful products, such as heavy metals or microorganisms. The purpose of the study was to determine consistency and contamination within and between bottles of common herbal supplements. Duplicate bottles of 29 herbal supplements were tested for consistency for antioxidant activity, phenolic concentration and flavonoid concentration under methanolic and water extraction. The supplements were also analyzed for the presence of metals and fungal contaminants. For all of the supplements tested there was high variability around the mean in antioxidant activity, phenolic concentrations and flavonoid concentrations, with coefficients of variation (CV) ranging from 0–120. Zinc was found in almost 90% of the supplements, nickel in about half of the supplements and lead in none of the supplements. Approximately 60% of the supplements contained fungal isolates. Although the majority of the fungi that were found in the supplements are generally not hazardous to human health, many of them could be problematic to sensitive groups, such as immunocompromised individuals. The data, which demonstrates contamination and a lack of consistency, in conjunction with previous studies on supplement contamination, strengthen the case that the FDA should regulate over-the-counter herbal supplements the same way that they regulate food and drugs. Until such time it is crucial that consumers are informed that many of the supplements that they take may lack the standardization that would reduce the chance of contamination and lead to consistency from one pill to the next.

Introduction

Herbal remedies have existed for thousands of years and the demand for herbal supplements worldwide has increased significantly over the past few decades. Every year more consumers use over-the-counter herbal supplements for needs varying from digestive health and improved energy to relief from depression. Herbal medicines are thought to enhance and restore normal physiological functions by facilitating the body’s innate self-healing capabilities [1]. Additionally, many marketed herbal products are mixtures or parts of organic chemicals that come from any raw or processed part of a plant; because these products come from plants consumers assume that they are healthy [2]. The World Health Organization estimates that about 80% of the populations in Asian and African countries take herbal medicine as their only form of medication and treatment [3], while in Europe about 19% of adults use ‘Plant Food Supplements’ [4]. Dietary supplements are used by 50 to 70% of adults in the United States [5, 6] of which herbal supplements are the most popular subset [7]. In 2018, the sales of herbal supplements in the U.S. went up almost 9.4%, which represented the thirteenth straight year of sales increases [8]. Overall net profits from the sale of herbal supplements within the U.S. are over eight billion dollars per year [8]. Over the past ten years herbal supplements that are marketed as adaptogens, to enhance immune function, reduce stress and anxiety have consistently been among the top selling supplements accounting for a high percent of the sales in the supplement market [8, 9].

A 2015 report released by the New York state’s attorney general revealed that store brand supplements did not contain the ingredients listed on the tested product labels up to 80% of the time [10]. Newmaster et al. [11] investigated 44 over-the-counter supplements utilizing DNA barcoding and found that the ingredients listed on the tested product labels were not found in the products. Adulteration using DNA barcoding has been found worldwide with a range of adulteration from 23–79% of tested supplements [12, 13]. In addition, in July 2019, Amazon sent a notice to its customers warning that some nutritional supplements recently sold on their site may have been fake [14]. Unlike pharmaceutical drugs, herbal supplements are not regulated by the Food and Drug Administration (FDA) and can only be recalled when there is substantive proof of harm or when contaminants are present that would change the regulatory status of the supplement [15]. Most Americans consuming over-the-counter herbal supplements often do not realize that the FDA does not regulate herbal supplements the same way that they regulate food and drugs [16].

The lack of regulation of herbal supplements by the FDA only increases the susceptibility of these products to contamination [17] with no assurance of purity, integrity or efficacy. Contamination may be due to growth, processing or storage conditions or may be due to the intentional addition of unlisted ingredients. Geyer et al. [18] studied over 600 nutritional supplements between 2001 and 2002 and found that 15% of the supplements purchased in 13 different countries were contaminated with anabolic steroids. In another study, Chinese herbal medicines (produced and sold in China) also demonstrated contamination with heavy metals and microbes [19], which may come from cultural conditions or production processes. Fungal contamination, particularly fungi that produce mycotoxins, many of which are known carcinogens or neurotoxins, is often associated with storage conditions [20–22]. Contamination may also occur through the accidental or intentional inclusion of other plant species [23–26]. For example, different species of Echinacea are often substituted for each other; even though they do not all have the same efficacy [27]. In 1997, two previously healthy individuals experienced irregular heartbeats after using plantain supplements contaminated with foxglove (Digitalis sp.), which contains cardiac glycosides [24]. In addition, contamination with allergens could lead to severe allergic reactions in sensitive groups. Perhaps the most troubling source of contamination is the addition of pharmaceutical grade drugs to herbal supplements, which could lead to adverse health effects [17, 28–31].

It may also be difficult, in field collected materials, to completely eliminate microbial and mycotoxin contaminants from the area where the plants are grown [32, 33]. Field collections pose other risks as well: plants could naturally take up metal contaminants from the soil and water they are exposed to during growth [32]. Although there have been many studies examining purity and effectiveness of herbal supplements, with hundreds of different supplements from hundreds of suppliers it is critical to evaluate as many supplements as possible. The purpose of the research was to determine the degree of both consistency and contamination within duplicate bottles of 29 herbal supplement products, totaling 58 bottles. The majority of the supplements that were chosen for this analysis were among the most commonly sold on a year-to-year basis in the United States, which typically fall in the categories of general adaptogens, stress and depression relief, and immune response [8, 9].

Materials and methods

ASupplement selection

The supplement manufacturers were Nature’s Way, Spring Valley, NOW, and Sundown Naturals. Two bottles each from a single supplier were tested for the following supplements: Aloe, Biotin, Cranberry, Raspberry, Reishi, Silent Night, Stress Formula, and Yarrow. Two bottles each from two different suppliers were tested for the following supplements: Astragalus, Echinacea, Echinacea Goldenseal, Ginger, Ginseng, and Rhodiola. All Spring Valley supplements were purchased in person at Walmart in Cockeysville, Maryland as were the Sundown Naturals Turmeric and Stress Formula. All other supplements were purchased online through the Amazon Marketplace. Two bottles each from three different suppliers were tested for the following supplements: St. John’s Wort, Turmeric and Valerian Root. All bottles were sealed and stored away from light at room temperature (between 20 and 25°C) until initial analysis. After opening, the bottles were stored with the lids tightly closed under the same temperature and light conditions. The humidity within the lab ranged between 55 and 65%. In addition, each bottle was stored with the included silica pack to reduce moisture accumulation within the bottles. Additional information about each supplement, including lot number and expiration date, is found in Table 1.

Table 1. List of supplements and suppliers for 58 bottles of over the counter herbal supplements.

Supplier^*	Supplement	Bottle	Batch #	EXP Date	Bottles different^*
Nature’s Way	Aloe	1	20043572	Jun-18	Yes
Nature’s Way	Aloe	2	20066063	Oct-19	2/6
Nature’s Way	Astragalus	1	20044498	May-20	Yes
Nature’s Way	Astragalus	2	20066304	Oct-21	4/6
NOW	Astragalus	1	1881061	May-18	Yes
NOW	Astragalus	2	1881061	May-18	4/6
NOW	Biotin	1	1904240–2042	Jan-18	Yes
NOW	Biotin	2	2110421–1022	Jan-19	5/6
Nature’s Way	Cranberry	1	20041000	May-20	Yes
Nature’s Way	Cranberry	2	20067173	Oct-21	4/6
Spring Valley	Echinacea	1	800025	Jul-18	Yes
Spring Valley	Echinacea	2	800025	Jul-18	4/6
Sundown Naturals	Echinacea	1	988248–07	Apr-20	Yes
Sundown Naturals	Echinacea	2	988248–07	Apr-20	5/6
Nature’s Way	Echinacea Goldenseal	1	20066301	Nov-21	Yes
Nature’s Way	Echinacea Goldenseal	2	20066301	Nov-21	3/6
Spring Valley	Echinacea Goldenseal	1	800009	Jun-18	Yes
Spring Valley	Echinacea Goldenseal	2	T800006	Jan-19	4/6
NOW	Ginger	1	2084551–0058	Nov-18	Yes
NOW	Ginger	2	2084551–0121	Nov-18	3/6
Spring Valley	Ginger	1	987324–02	Mar-20	Yes
Spring Valley	Ginger	2	994218–01	Sep-20	2/6
Sundown Naturals	Ginseng Xtra	1	888712–05	Mar-17	Yes
Sundown Naturals	Ginseng Xtra	2	missing	missing	6/6
Spring Valley	Korean Panax Ginseng	1	1039222	May-18	Yes
Spring Valley	Korean Panax Ginseng	2	1054619	Feb-19	6/6
Nature’s Way	Red Raspberry Leaf	1	20065651	Sep-21	Yes
Nature’s Way	Red Raspberry Leaf	2	20065651	Sep-21	4/6
Nature’s Way	Reishi	1	20039110	Feb-18	Yes
Nature’s Way	Reishi	2	20064691	Sep-19	3/6
Nature’s Way	Rhodiola	1	20066944	Nov-18	Yes
Nature’s Way	Rhodiola	2	20066944	Nov-18	4/6
NOW	Rhodiola	1	2025719–0221	Jun-21	Yes
NOW	Rhodiola	2	2105309–2246	Dec-21	6/6
Nature’s Way	Silent Night	1	20056474	Feb-21	Yes
Nature’s Way	Silent Night	2	20064590	May-21	5/6
Nature’s Way	St. John’s Wort	1	20066202	0ct-19	Yes
Nature’s Way	St. John’s Wort	2	20066202	0ct-19	2/6
Spring Valley	St. John’s Wort	1	1041145	Jul-18	Yes
Spring Valley	St. John’s Wort	2	1048622	Aug-18	4/6
Sundown Naturals	St. John’s Wort	1	992660–03	Jun-19	Yes
Sundown Naturals	St. John’s Wort	2	992660–03	Jun-19	6/6
Sundown Naturals	Stress formula	1	983673–01	Feb-19	Yes
Sundown Naturals	Stress formula	2	983245–03	Mar-19	4/6
Nature’s Way	Turmeric	2	20040681	Apr-18	Yes
Nature’s Way	Turmeric	1	20045321	Jul-18	3/6
Spring Valley	Turmeric	1	988358–01	Jun-19	Yes
Spring Valley	Turmeric	2	28008	Oct-19	3/6
Sundown Naturals	Turmeric	1	976191–02	Dec-19	Yes
Sundown Naturals	Turmeric	2	976191–02	Dec-19	2/6
Nature’s Way	Valerian Root	1	20066814	Oct-21	Yes
Nature’s Way	Valerian Root	2	20066819	Oct-21	4/6
Spring Valley	Valerian Root	1	1042635	Aug-18	Yes
Spring Valley	Valerian Root	2	1048489	Nov-18	5/6
Sundown Naturals	Valerian Root	1	987221–01	May-19	Yes
Sundown Naturals	Valerian Root	2	987221–01	May-19	3/6
Nature’s Way	Yarrow	1	20051391	Oct-20	Yes
Nature’s Way	Yarrow	2	20065763	Oct-21	3/6

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*For each bottle of each supplement the batch number and expiration date are included. Also indicated are if the bottles had significant differences on any of the antioxidant, phenolic and flavonoid assays run and on how many of the six assays they had significant differences.

Sample extraction

Ten capsules were removed at random from each bottle and from each capsule sub-samples were weighed out for a variety of extraction methods (methanol, hot water and nitric acid). Four subsamples were taken from each capsule. Two subsamples were taken to analyze the methanolic and water soluble fractions of each supplement and extracted according to the method of Cai et al. [34] with the following modifications such that the assays were conducted on the same day as the extraction. One subsample was extracted with 90% methanol and one with boiling water. After methanol or water addition, the samples were vortexed for 10 seconds and put in a shaker at 150 rpm at 25°C for 60 minutes. The samples were then centrifuged for 5 minutes at 5,000 g followed by removal of the supernatant. Immediately after supernatant removal, three assays were conducted on each fraction. All of the extractions and tests, with the exception of the supplement stability tests were conducted from October 2016 through April 2017. The supplement stability tests were conducted in January and February 2019.

Antioxidant, phenolic and flavonoid assays

Antioxidant capacity, expressed in Trolox equivalents (TE), was tested with the Ferric Reducing Antioxidant Power (FRAP) assay [35]. This assay utilized a standard curve created with Trolox with concentrations ranging from 0–1500 μmol• L^-1 Total phenolic concentration, expressed in gallic acid equivalents (GAE), was tested using the Folin-Ciocalteu method, with a standard curve produced using a range of gallic acid concentration from 0 to 0.4 mg• ml^-1 [36]. Flavonoid concentration, expressed in catechin equivalents (CE), was tested using the AlCl₃ precipitation method, with a standard curve created using a range of (+)-catechin hydrate from 0 to 1000 μg • g^-1 [37]. All assay values were calculated and standardized based on the weight of the sample that was extracted. To assess supplement stability, the three assays were conducted again on methanol and hot water extracts from 5 additional capsules per bottle of the Echinacea, Echinacea-Goldenseal, Turmeric, and Valerian Root two years after the initial testing. These supplements were chosen such that two supplements from two suppliers and two from three suppliers were tested.

Metal extraction and gross physical contamination

Each bottle was also tested for the presence of metals, gross physical contamination and fungi. One subsample from each capsule (0.05 g) was wet digested with nitric acid for flame atomic absorption spectroscopy (FAAS) and prepped according to Pomper and Grusak [38] to measure levels of nickel, zinc, lead, copper, and chromium. To check for gross physical contamination of the samples, the remaining sub-sample from each capsule was screened under a light-dissecting microscope. The samples were scanned quickly for uniformity within a capsule and among capsules within the same bottle and between different bottles. In addition to the above tests, multiple capsules were pooled in order to assess fungal contamination in the supplements.

Fungal isolation

This fungal screen was conducted twice for each bottle based on the method of Singh et al. [39]. For each extraction five grams were taken from each bottle and added to forty-five milliliters of sterile water. Samples were mixed well and allowed to settle for 24 hours. Supernatant was used to inoculate sterile petri dishes, in duplicate, containing Sabouraud Dextrose Agar. In total, four plates were inoculated per bottle. After inoculation, the petri dishes were monitored for fungal growth for five to seven days and contaminants were classified based on visual characteristics. In addition, eight control plates were inoculated with sterile water only under the same plating conditions.

Statistical analysis

For each bottle of each supplement, the means and standard error were calculated for the water, methanol, and nitric acid extractions and are the only calculations done for metal content within the supplements. In addition, the coefficient of variation (CV) was calculated for the water and methanol extractions. Comparisons for the FRAP antioxidant capacity and phenolic and flavonoid contents among suppliers and bottles from the same supplier were done using the general linear model in the FitModel platform of JMP and comparison among bottles and supplements compared using LSD Student’s T-Test (α level of 0.05) [40]. A runs test was also conducted to test for randomness of rankings among bottles, suppliers and supplements using Minitab^® Statistical Software with an α level of 0.05 [41]. A significant runs test means suppliers and/or supplements clump together. To determine if there had been a change in the average values for the FRAP antioxidant, phenolic and flavonoid assays after two years of supplement storage the data was analyzed using a one-tailed t-test with an α level of 0.05 and a test value of < -25% change using Minitab^® Statistical Software [41].

Results

Antioxidant, phenolic and flavonoid assays

There were significant differences not only among suppliers for individual supplements (Table 2), but between bottles from the same supplier (Table 3).

Table 2. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by supplier^*.

		Water Extraction			Methanol Extraction
Supplier	Supplement	A (TE)	P (GAE)	F (CE)	A (TE)	P (GAE)	F (CE)
Nature’s Way	Aloe	76637 ± 4295^{^}	8.94 ± 0.43^{^}	4063 ± 359^{^}	85256 ± 8460^{^}	10.64 ± 0.52^{^}	5646 ± 217^{^}
Nature’s Way	Astragalus	9342 ± 1294^B	1.91 ± 0.08^A	1308 ± 417^A	8794 ± 1827^A	1.09 ± 0.05^B	542 ± 59^B
NOW	Astragalus	22629 ± 1525^A	1.66 ± 0.16^A	675 ± 13^A	10114 ± 520^A	1.89 ± 0.18^A	725 ± 23^A
NOW	Biotin	412 ± 91^{^}	0.23 ± 0.02^{^}	555 ± 241^{^}	4060 ± 915^{^}	0.41 ± 0.04^{^}	1169 ± 348^{^}
Nature’s Way	Cranberry	26359 ± 1781^{^}	3.26 ± 0.26^{^}	2185 ± 240^{^}	25754 ± 1780^{^}	1.55 ± 0.12^{^}	3910 ± 903^{^}
Spring Valley	Echinacea	200190 ± 31988^A	10.59 ± 0.60^A	15962 ± 566^A	11163 ± 170^A	1.04 ± 0.05	1793 ± 101^A
Sundown Naturals	Echinacea	94474 ± 15013^B	6.13 ± 0.61^B	7790 ± 714^B	8311 ± 401^B	1.34 ± 0.51	521 ± 84^B
Nature’s Way	Echinacea Goldenseal	98618 ± 3687^A	3.97 ± 0.41^B	8206 ± 247^b	30641 ± 640^A	4.60 ± 0.72^A	3880 ± 140^A
Spring Valley	Echinacea Goldenseal	57687 ± 9677^B	6.74 ± 0.46^A	9652 ± 664^a	14733 ± 1783^B	1.57 ± 0.19^B	1308 ± 422^B
NOW	Ginger	88335 ± 2353^A	3.43 ± 0.15^A	1243 ± 36^B	373256 ± 46229^A	7.48 ± 0.14^A	6660 ± 1006^A
Spring Valley	Ginger	72285 ± 3836^B	2.98 ± 0.22^A	1444 ± 48^A	140170 ± 46229^B	5.09 ± 0.30^B	3472 ± 117^B
Sundown Naturals	Ginseng Xtra	5950 ± 869^A	0.76 ± 0.10^A	303 ± 68^B	3035 ± 401^B	0.59 ± 0.04^A	455 ± 96^A
Spring Valley	Korean Panax Ginseng	73 ± 23^B	0.18 ± 0.04^B	775 ± 217^A	4770 ± 1102^A	0.32 ± 0.03^B	26 ± 9^B
Nature’s Way	Red Raspberry Leaf	488121 ± 34130^{^}	12.38 ± 0.36^{^}	8272 ± 130^{^}	86122 ± 3019^{^}	4.30 ± 0.18^{^}	4081 ± 89^{^}
Nature’s Way	Reishi	15575 ± 1729^{^}	2.29 ± 0.18^{^}	1469 ± 194^{^}	5536 ± 1153^{^}	0.56 ± 0.04^{^}	1768 ± 505^{^}
Nature’s Way	Rhodiola	853458 ± 28330^A	15.59 ± 0.52^A	15566 ± 315^A	757413 ± 109746^A	17.07 ± 0.88^A	18741 ± 1404^A
NOW	Rhodiola	602570 ± 46729^B	7.59 ± 1.88^B	7764 ± 739^B	222758 ± 42227^B	13.66 ± 0.96^B	10941 ± 852^B
Nature’s Way	Silent Night	70378 ± 4516^{^}	3.38 ± 0.36^{^}	5324 ± 336^{^}	12044± 899^{^}	3.58 ± 0.22^{^}	1515 ± 203^{^}
Nature’s Way	St. John’s Wort	206984 ± 12697^A	8.00 ± 0.47^B	8755 ± 255^B	53124 ± 3352^C	4.14 ± 0.16^C	4278 ± 98^C
Spring Valley	St. John’s Wort	180944 ± 21774^A	17.00 ± 0.68^A	16036 ± 714^A	93851 ± 15547^B	16.64 ± 0.45^A	10028 ± 760^B
Sundown Naturals	St. John’s Wort	196151 ± 23313^A	17.42 ± 0.71^A	16816 ± 1013^A	154977 ± 12703^A	13.77 ± 0.49^B	11177 ± 301^A
Sundown Naturals	Stress formula	93095 ± 15299^{^}	6.53 ± 0.34^{^}	10698 ± 366^{^}	18168 ± 1524^{^}	1.23 ± 0.25^{^}	1176 ± 164^{^}
Nature’s Way	Turmeric	1762 ± 84^C	0.68 ± 0.07^C	0 ± 0^B	183344 ± 10703^C	14.03 ± 0.71^B	339869 ± 21356^A
Spring Valley	Turmeric	5948 ± 693^B	1.03 ± 0.06^B	2984 ± 794^A	125930 ± 17877^B	8.56 ± 1.69^C	49705 ± 7829^C
Sundown Naturals	Turmeric	17353 ± 543^A	1.33 ± 0.16^A	1786 ± 208^A	266502 ± 18262^A	17.90 ± 1.11^A	176657 ± 7828^B
Nature’s Way	Valerian Root	29662 ± 1460^B	2.77 ± 0.08^B	3106 ± 101^B	11684 ± 767^B	1.47 ± 0.06^B	1808 ± 76^A
Spring Valley	Valerian Root	10390 ± 3289^C	1.23 ± 0.20^C	2633 ± 416^C	6401 ± 381^C	0.79 ± 0.18^C	355 ± 59^B
Sundown Naturals	Valerian Root	43176 ± 2577^A	3.88 ± 0.24^A	3424 ± 99^A	15973 ± 772^A	1.81 ± 0.09^A	1854 ± 84^A
Nature’s Way	Yarrow	141389 ± 10630^{^}	7.67 ± 0.66^{^}	11126 ± 665^{^}	37482 ± 913^{^}	2.72 ± 0.13^{^}	3100 ± 300^{^}

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* The assays include the Ferrric Reducing Antioxidant Power (FRAP) assay for antioxidant capacity expressed as Trolox equivalents per gram, the Folin-Ciocalteu method for phenolics expressed as gallic acid equivalents per gram (GAE), and the AlCl3 precipitation assay for flavonoids expressed as +-catechin equivalents per gram. The values are expressed as the mean ± SE (n = 20) for each extraction from each supplier of each supplement. Means within the same supplement with different capital letters are significantly different based on the LSD Student’s T-test (± of 0.05). For specific p-values for each supplement supplier see S1 Table.

^—Means followed by this symbol indicate a supplement where there were no duplicate suppliers.

Table 3. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by bottle and supplier^*.

			Water Extraction			Methanol Extraction
Supplier	Supplement	Bottle	A (TE)	P (GAE)	F (CE)	A (TE)	P (GAE)	F (CE)
Nature’s Way	Aloe	1	66460 ± 7203^a	8.54 ± 0.86^a	4193 ± 735^a	496322 ± 1549^a	11.20 ± 0.80^a	5276 ± 315^a
Nature’s Way	Aloe	2	86814 ± 1728^b	9.34 ± 0.13^a	3933 ± 33^a	120880 ±4217^b	10.08 ± 0.67^a	6016 ± 264^a
Nature’s Way	Astragalus	1	13014 ± 2001^a	1.81 ± 0.14^a	2030 ± 786^a	16712 ± 364^a	1.21± 0.08^a	308 ± 43^b
Nature’s Way	Astragalus	2	5670 ± 269^b	2.02 ± 0.09^a	587 ± 22^b	875 ± 185^b	0.97 ± 0.02^b	776 ± 28^a
NOW	Astragalus	1	28942 ± 857^a	2.05 ± 0.25^a	667 ± 15^a	11878 ± 477^a	1.16 ± 0.06^b	757 ± 22^a
NOW	Astragalus	2	16316 ± 482^b	1.26 ± 0.11^b	685 ± 21^a	8351 ± 475^b	2.62 ± 0.12^a	692 ± 40^a
NOW	Biotin	1	32 ± 9^b	0.22 ± 0.03^a	1111 ± 421^a	8044 ± 105^a	0.29 ± 0.03^b	2338 ± 456^a
NOW	Biotin	2	792 ± 56^a	0.24 ± 0.01^a	0 ± 0^b	76 ± 39^b	0.52 ± 0.04^a	0 ± 0^b
Nature’s Way	Cranberry	1	24622 ± 3459^a	4.21 ± 0.29^a	2255 ± 487^a	19145 ± 1250^b	1.34 ± 0.17^b	589 ± 64^b
Nature’s Way	Cranberry	2	28095 ± 869^a	2.31 ± 0.06^b	2114 ± 71^a	32362 ± 1455^a	1.77 ± 0.15^a	7230 ± 994^a
Spring Valley	Echinacea	1	330437 ± 19358^a	9.49 ± 1.00^a	14186 ± 663^b	11249 ± 231	1.01 ± 0.05^a	1421 ± 95^b
Spring Valley	Echinacea	2	69943 ± 13259^b	11.70 ± 0.52^a	17738 ± 460^a	11077 ± 258	1.07 ± 0.09^a	2164 ± 61^a
Sundown Naturals	Echinacea	1	29672 ± 1656^b	4.16 ± 0.33^b	5358 ± 405^b	9212 ± 565^a	0.94 ± 0.09^a	700 ± 136^a
Sundown Naturals	Echinacea	2	159277 ± 3976^a	8.10 ± 0.77^a	10222 ± 821^a	7409 ± 423^b	1.73 ± 1.03^a	341 ± 62^b
Nature’s Way	Echinacea Goldenseal	1	98028 ± 7015^a	5.53 ± 0.39^a	7797 ± 395^a	32553 ± 929^a	2.36 ± 0.10^b	3956 ± 70^a
Nature’s Way	Echinacea Goldenseal	2	99208 ± 2847^a	2.42 ± 0.07^b	8615 ± 252^a	28728 ± 230^b	6.84 ± 1.04^a	3804 ± 277^a
Spring Valley	Echinacea Goldenseal	1	85953 ± 14036^a	6.55 ± 0.73^a	10709 ± 797^a	21867 ± 1432^a	2.33 ± 0.13^a	2093 ± 722^a
Spring Valley	Echinacea Goldenseal	2	29421 ± 1134^b	6.94 ± 0.59^a	8477 ± 986^a	7599 ± 265^b	0.80 ± 0.05^b	436 ± 114^b
NOW	Ginger	1	81890 ± 2301^b	3.25 ± 0.13^a	1181 ± 35^a	559238 ± 35560^a	7.38 ± 0.17^a	3803 ± 207^b
NOW	Ginger	2	94782 ± 2976^a	3.60 ± 0.26^a	1306 ± 59^a	187273 ±8513^b	7.58 ± 0.22^a	9516 ± 1554^a
Spring Valley	Ginger	1	70175 ± 2565^a	3.24 ± 0.41^a	1367 ± 67^a	144331 ± 4122	5.87 ± 0.21^a	3203 ± 192^b
Spring Valley	Ginger	2	74395 ± 7388^a	2.72 ± 0.13^a	1521 ± 62^a	136009 ± 1325	4.31 ± 0.46^b	3741 ± 72^a
Sundown Naturals	Ginseng Xtra	1	9324 ± 423^a	1.09 ± 0.09^a	577 ± 16^a	4547 ± 399^a	0.73 ± 0.04^a	739 ± 31^a
Sundown Naturals	Ginseng Xtra	2	2202 ± 156^b	0.39 ± 0.05^b	0 ± 0^b	1524 ± 109^b	0.46 ± 0.04^b	138 ± 138^b
Spring Valley	Korean Panax Ginseng	1	146 ± 32^a	0.32 ± 0.04^a	1550 ± 255^a	9540 ± 261^a	0.40 ± 0.02^a	52 ± 14^a
Spring Valley	Korean Panax Ginseng	2	0 ± 0^b	0.05 ± 0.03^b	0 ± 0^b	0 ± 0^b	0.23 ± 0.03^b	0 ± 0^b
Nature’s Way	Red Raspberry Leaf	1	614243 ± 29370^a	13.76 ± 0.30^a	8506 ± 112^a	73693 ± 1121^b	4.46 ± 0.33^a	3752 ± 53^b
Nature’s Way	Red Raspberry Leaf	2	362000 ± 22822^b	11.00 ± 0.18^b	8038 ± 216^a	98551 ± 1702^a	4.14 ± 0.15^a	4410 ± 83^a
Nature’s Way	Reishi	1	16681 ± 3489^a	2.87 ± 0.24^a	1784 ± 370?	10549 ± 137^a	0.59 ± 0.04^a	3088 ± 830^a
Nature’s Way	Reishi	2	14469 ± 415^a	1.72 ± 0.09^b	1154 ± 12	521 ± 82^b	0.53 ± 0.06^a	447 ± 23^b
Nature’s Way	Rhodiola	1	859558 ± 40568^a	14.32 ± 0.67^a	16018 ± 439^a	451224 ± 109504^b	14.73± 0.67^b	15684 ± 1917^a
Nature’s Way	Rhodiola	2	847358 ± 41650^a	16.86 ± 0.59^a	15114 ± 424^a	1063602 ± 134270^a	19.41 ± 1.27^a	21798 ± 1602^b
NOW	Rhodiola	1	739303 ± 57664^a	0.22 ± 0.04^b	5292 ± 512^b	45552 ± 4538^b	11.72 ± 1.43^b	8223 ± 386^b
NOW	Rhodiola	2	465838 ± 41714^b	14.97 ± 1.70^a	10235 ± 827^a	399962 ±23023^a	15.60 ± 1.01^a	13659 ± 1129^a
Nature’s Way	Silent Night	1	87025 ± 2987^a	1.47 ± 0.03^b	6372 ± 256^a	10799 ± 1694	4.91 ± 0.11^a	2354 ± 70^a
Nature’s Way	Silent Night	2	53731 ± 3951^b	5.70 ± 0.42^a	4277 ± 407^b	13289 ± 443	1.86 ± 0.10^b	675 ± 114^b
Nature’s Way	St. John’s Wort	1	166933 ± 3218^b	8.22 ± 0.37^a	9002 ± 360^a	66065 ± 1080^a	4.04 ± 0.11^a	4368 ± 161^a
Nature’s Way	St. John’s Wort	2	247036 ± 17717^a	7.79 ± 0.89^a	8509 ± 364^a	40182 ± 3011^b	4.24 ± 0.30^a	4188 ± 113^a
Spring Valley	St. John’s Wort	1	90260 ± 8356^b	17.62 ± 1.25^a	16212 ± 1355^a	29367 ± 3146^b	17.58 ± 0.57^a	6862 ± 318^b
Spring Valley	St. John’s Wort	2	271628 ± 10226^a	16.38 ± 0.56^a	15860 ± 559^a	158334 ± 9309^a	15.70 ± 0.59^b	13195 ± 335^a
Sundown Naturals	St. John’s Wort	1	104370 ± 8184^b	15.56 ± 1.01^b	13471 ± 857^b	103772 ± 4655^b	12.37 ± 0.61^b	1115 ± 437^b
Sundown Naturals	St. John’s Wort	2	287932 ± 18868^a	19.29 ± 0.56^a	20160 ± 1057^a	206181 ± 8777^a	15.18 ± 0.45^a	10539 ± 320^a
Sundown Naturals	Stress formula	1	39304 ± 1662^b	7.63 ± 0.34^a	10223 ± 335^a	24750 ± 220^a	0.76 ± 0.05^b	472 ± 30^b
Sundown Naturals	Stress formula	2	160332 ± 10585^a	5.43 ± 0.31^b	11174 ± 634^a	11586 ± 363^b	1.70 ± 0.45^a	1880 ± 52^a
Nature’s Way	Turmeric	1	1555 ± 76^b	0.69 ± 0.04^a	0 ± 0^a	205518 ± 18668^a	14.51 ± 1.33^a	389853 ± 36119^a
Nature’s Way	Turmeric	2	1969 ± 119^a	0.67 ± 0.07^a	0 ± 0^a	161170 ± 5090^b	13.55 ± 0.59^a	289884 ± 8117^a
Spring Valley	Turmeric	1	4816 ± 1234^a	0.98 ± 0.10^a	5384 ± 1275^a	51320 ± 3681^b	1.69 ± 0.14^b	9548 ± 1036^b
Spring Valley	Turmeric	2	7081 ± 466^a	1.08 ± 0.07^a	824 ± 26^b	200538 ± 9945^a	15.44 ±1.25^a	89862 ± 7177^a
Sundown Naturals	Turmeric	1	16762 ± 520^a	0.86 ± 0.08^b	1236 ± 73^b	337434 ± 15251^a	18.26 ± 2.16^a	168328 ± 13461^a
Sundown Naturals	Turmeric	2	17944 ± 946^a	1.81 ± 0.23^a	2336 ± 333^a	195569 ± 7574^b	17.55 ± 0.73^a	184986 ± 7883^a
Nature’s Way	Valerian Root	1	35271 ± 384^a	3.06 ± 0.07^a	3319 ± 154^a	13286 ± 770^a	1.41 ± 0.09^a	1929 ± 79^a
Nature’s Way	Valerian Root	2	24052 ± 1366^b	2.48 ± 0.08^b	2893 ± 95^b	10081 ± 1148^b	1.53 ± 0.08^a	1687 ± 121^a
Spring Valley	Valerian Root	1	8208 ± 6651^a	0.52 ± 0.03^b	4091 ± 505^a	7993 ± 132^a	0.33 ± 0.01^b	105 ± 18^b
Spring Valley	Valerian Root	2	12573 ± 625^a	1.94 ± 0.25^a	1176 ± 65^b	4808 ± 180^b	1.25 ± 0.30^a	606 ± 24^a
Sundown Naturals	Valerian Root	1	53318 ± 1286^a	3.46 ± 0.11^a	3625 ± 147^a	17338 ± 997	1.59 ± 0.12^b	1796 ± 139^a
Sundown Naturals	Valerian Root	2	33033 ± 1876^b	4.30 ± 0.43^a	3224 ± 104^b	14606 ± 1054	2.04 ± 0.11^a	1913 ± 32^a
Nature’s Way	Yarrow	1	114887 ± 12562^b	7.45 ± 0.91^a	11819 ± 1050^a	35094 ± 1356^b	2.69 ± 0.20^a	1840 ± 39^b
Nature’s Way	Yarrow	2	167892 ± 12775^a	7.89 ± 0.98^a	10433 ± 809^a	39870 ± 641^a	2.74 ± 0.19^a	4360 ± 108^a

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* The assays include the Ferrric Reducing Antioxidant Power (FRAP) assay for antioxidant capacity expressed as Trolox equivalents per gram, the Folin-Ciocalteu method for phenolics expressed as gallic acid equivalents per gram (GAE), and the AlCl3 precipitation assay for flavonoids expressed as +-catechin equivalents per gram. The values are expressed as the mean ± SE (n = 10) for each extraction from each bottle. Means within the same supplier for a supplement with different lowercase letters are significantly different based on LSD Student’s T-Test (α of 0.05). For specific p-values for each supplement see S2 Table.

There were significant differences between bottles for at least two assays for each supplement (Tables 2 and 3). When there were 2 or more suppliers per supplement there were always differences among suppliers for at least two of the assays. Ten of the supplements that were tested had duplicate bottles that were from the same batch number (Table 1). The least variation between bottles was Ginger from Spring Valley and St. John’s Wort from Nature’s Way (only a significant difference in a third of the tests), while for Ginseng Xtra, Korean Panax Ginseng, St. John’s Wort from Sundown Naturals the bottles were significantly different from each other in 100% of the tests. Three of the supplements (St. John’s Wort, Turmeric and Valerian Root) came from the three main suppliers. Data from the methanolic extraction of these three supplements is found in (Fig 1a–1c). Similar differences between bottles and suppliers were obtained for the water extraction; therefore, only the data from the methanolic extractions is shown. Firstly, the figures illustrate the differences among supplements. Turmeric had the highest antioxidant capacity and flavonoid concentration, while valerian root was the lowest for all three assays. The data also illustrate differences among suppliers for the same supplement. For example, for both St. John’s Wort and Turmeric there was almost always a supplier that was significantly lower for the tested compounds than the other suppliers; however, the supplier with the lowest values was not consistent across supplements. Finally, the data illustrate the differences that could occur between bottles from the same supplier. This was particularly evident in the Turmeric samples, where bottle 1 from Spring Valley had significantly lower antioxidant capacity (Fig 1a), phenolic concentration (Fig 1b) and flavonoid concentration (Fig 1c) than not only other suppliers, but also the other bottle from the same supplier. This bottle was also found to be much lighter in color than all other Turmeric bottles that were sampled.

Fig 1 — The mean ± se from each bottle was calculated from 10 pills per bottle. An * indicates that bottle 1 is significantly different from bottle 2 according to ANOVA (p < 0.05).

The coefficient of variation (CV) was used to show the relative standard deviation among suppliers, bottles and supplements (Table 4), which is particularly valuable when the values involved can be different by orders of magnitude and demonstrate how much variability there were within each supplement. There is a wide range of CVs (S3 Table) for each supplement with the greatest CV found in Valerian Root, where the most consistent bottle had a CV for antioxidant activity that was 74 times lower than the bottle with the highest CV. This inconsistent pattern was observed throughout most of the bottles within the same supplier as well as different supplements within different suppliers. In addition, the rank runs test showed that for the bottles from the same supplier the bottles did not generally rank together (NS runs tests, Table 4).

Table 4. Coefficient of variation (CV) for the FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) for 58 bottles of over the counter herbal supplements extracted with hot water and methanol.

			Water Extraction			Methanol Extraction
Supplier	Supplement	Bottle	A (TE)^*	P (GAE)	F (CE)	A (TE)	P (GAE)	F (CE)
Nature’s Way	Aloe	1	34.27	31.93	55.40	9.87	22.60	18.89
Nature’s Way	Aloe	2	6.29	4.23	2.67	11.03	21.01	13.85
Nature’s Way	Astragalus	1	48.62	23.81	122.55	6.88	20.67	44.39
Nature’s Way	Astragalus	2	15.00	13.39	11.93	66.72	7.85	11.25
NOW	Astragalus	1	9.37	38.60	7.23	12.69	15.35	9.24
NOW	Astragalus	2	9.34	27.84	9.94	18.00	14.53	18.24
NOW	Biotin	1	89.13	38.71	120.00	4.13	30.10	61.70
NOW	Biotin	2	22.23	18.81	0	161.93	25.24	0
Nature’s Way	Cranberry	1	44.42	22.15	68.28	20.64	39.01	34.59
Nature’s Way	Cranberry	2	9.79	8.39	10.65	14.22	26.90	43.47
Spring Valley	Echinacea	1	18.53	33.17	14.77	6.49	15.99	21.20
Spring Valley	Echinacea	2	59.95	14.16	8.21	7.38	27.26	8.90
Sundown Naturals	Echinacea	1	17.65	24.75	23.89	19.41	30.35	61.54
Sundown Naturals	Echinacea	2	7.89	29.93	25.39	18.06	187.57	57.86
Nature’s Way	Echinacea Goldenseal	1	22.63	22.36	16.03	9.03	12.89	5.59
Nature’s Way	Echinacea Goldenseal	2	9.07	9.59	9.27	2.53	47.92	23.05
Spring Valley	Echinacea Goldenseal	1	48.99	33.33	23.54	20.71	17.53	109.15
Spring Valley	Echinacea Goldenseal	2	11.57	25.67	34.89	11.02	18.91	78.70
NOW	Ginger	1	8.89	13.08	9.40	20.11	7.44	17.21
NOW	Ginger	2	9.93	22.92	14.38	14.37	9.24	51.64
Spring Valley	Ginger	1	11.56	40.34	15.49	9.03	11.37	18.94
Spring Valley	Ginger	2	31.40	15.15	12.96	3.08	33.48	6.07
Sundown Naturals	Ginseng Xtra	1	14.36	26.78	9.05	27.77	19.56	13.50
Sundown Naturals	Ginseng Xtra	2	21.23	39.37	0.00	22.53	24.74	300.00
Spring Valley	Korean Panax Ginseng	1	68.47	35.99	52.03	8.64	16.34	84.05
Spring Valley	Korean Panax Ginseng	2	0	169.19	0	0	40.26	0
Nature’s Way	Red Raspberry Leaf	1	15.12	6.80	4.18	4.81	23.30	4.43
Nature’s Way	Red Raspberry Leaf	2	19.94	5.26	8.49	5.46	11.82	5.95
Nature’s Way	Reishi	1	66.15	26.32	65.56	4.10	21.29	85.03
Nature’s Way	Reishi	2	9.08	15.65	3.33	49.90	37.46	16.58
Nature’s Way	Rhodiola	1	14.92	14.74	8.67	76.74	14.49	38.66
Nature’s Way	Rhodiola	2	15.54	11.11	8.88	39.92	20.68	23.25
NOW	Rhodiola	1	24.67	61.57	30.61	31.51	38.45	14.84
NOW	Rhodiola	2	28.32	35.93	25.55	18.20	20.38	26.13
Nature’s Way	Silent Night	1	10.86	6.76	12.69	49.61	6.99	9.37
Nature’s Way	Silent Night	2	23.26	23.57	30.14	10.54	17.10	53.23
Nature’s Way	St. John’s Wort	1	6.10	14.19	12.64	5.17	9.00	11.68
Nature’s Way	St. John’s Wort	2	22.68	36.18	13.53	23.70	22.51	8.52
Spring Valley	St. John’s Wort	1	29.28	22.40	26.44	33.89	10.29	14.67
Spring Valley	St. John’s Wort	2	11.91	10.84	11.15	18.59	11.85	8.03
Sundown Naturals	St. John’s Wort	1	24.80	20.51	20.13	14.19	15.51	11.69
Sundown Naturals	St. John’s Wort	2	20.72	9.14	16.58	13.46	9.42	9.60
Sundown Naturals	Stress formula	1	13.37	14.29	10.36	2.81	21.63	19.89
Sundown Naturals	Stress formula	2	18.67	17.96	17.95	9.91	84.20	8.70
Nature’s Way	Turmeric	2	15.44	18.38	0.00	28.72	28.90	29.30
Nature’s Way	Turmeric	1	19.19	31.13	0.00	9.99	13.82	8.85
Spring Valley	Turmeric	1	81.06	31.66	71.04	22.68	25.95	34.31
Spring Valley	Turmeric	2	20.81	21.45	10.13	15.68	25.50	25.26
Sundown Naturals	Turmeric	1	9.81	30.67	18.66	14.29	37.41	25.29
Sundown Naturals	Turmeric	2	16.68	40.59	45.14	12.25	13.10	13.48
Nature’s Way	Valerian Root	1	3.45	7.56	14.70	18.34	19.56	12.90
Nature’s Way	Valerian Root	2	17.96	9.84	10.37	36.01	17.17	22.74
Spring Valley	Valerian Root	1	256.21	15.81	39.06	5.23	12.28	54.16
Spring Valley	Valerian Root	2	15.71	41.21	17.44	11.86	75.11	12.73
Sundown Naturals	Valerian Root	1	7.63	10.20	12.86	18.19	23.35	24.48
Sundown Naturals	Valerian Root	2	17.96	31.90	10.20	22.82	16.68	16.32
Nature’s Way	Yarrow	1	34.58	38.81	28.12	12.22	23.18	21.63
Nature’s Way	Yarrow	2	24.06	39.45	24.53	5.09	21.86	7.82
Rank Runs Test (p-values)			0.114	0.114	0.993	0.993	0.008	0.002

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ASupplement stability

To get an indication of supplement stability, the antioxidant, phenolic and flavonoid assays on nine of the supplements (a total of 18 bottles) were conducted two years after the initial assays were completed (Table 5). A total decrease of greater than 25% in antioxidant capacity, phenolic content and flavonoid content was considered a significant change according to a 1-tailed t-test (S4 Table). Phenolic content was the most stable as the phenolic assay indicated a decreased in only 50% of the methanolic extracts, and only about 10% of the water extracts. Flavonoid concentration decreased in only 28% of the methanolic extracts, while it decreased in 100% of all of the water extracts. Antioxidant capacity demonstrated the least stability with a decrease in the majority of both the water and methanol extracts. The most stable supplement appeared to be supplements containing Echinacea.

Table 5. Change in the mean FRAP antioxidant capacity, phenolic and flavonoid concentration of two bottles each of nine herbal supplements^*.

			Water Extractions			Methanol Extractions
Supplier	Supplement	Bottle	A(TE)	P (GAE)	F (CE)	A (TE)	P (GAE)	F (CE)
Spring Valley^{^}	Echinacea	1		-
Spring Valley^{^}	Echinacea	2	-		-			-
Sundown Naturals	Echinacea	1			-	-
Sundown Naturals	Echinacea	2	-		-	-
Nature’s Way	Echinacea Goldenseal	1	-		-	-	-
Nature’s Way	Echinacea Goldenseal	2	-		-	-
Spring Valley^{^}	Echinacea Goldenseal	1			-	-	-
Spring Valley^{^}	Echinacea Goldenseal	2			-	-
Spring Valley	Turmeric	1			-	-		-
Spring Valley	Turmeric	2	-		-	-		-
Sundown Naturals	Turmeric	1	-		-	-	-	-
Sundown Naturals	Turmeric	2	-	-	-	-		-
Nature’s Way	Valerian	1	-		-	-
Nature’s Way	Valerian	2	-		-	-
Spring Valley^{^}	Valerian	1			-	-
Spring Valley^{^}	Valerian	2	-		-	-
Sundown Naturals	Valerian	1	-		-	-
Sundown Naturals	Valerian	2	-		-	-

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* Assays were conducted on two bottles each 5 capsules per bottle two years after the initial supplement testing conducted January to February 2019. Data was analyzed with a one-tailed t-test with an α level of 0.05 and a test value of < -25% change.

^Supplements that had expired at the time of the retest.

Metals and gross physical contamination

Zinc was the most abundant metal (Table 6), which was found in 88% of the bottles. Nickel was the next most common, found in about 40% of the samples. By contrast, lead (Pb) was found in none of the samples. Chromium was present in 5 bottles, but always at very low concentrations (<0.05 μg • g^-1). Copper was found in the highest overall concentration of all the metals, but only in St. John’s Wort from Spring Valley. The vast majority of bottles were uniform in texture and color, as observed under the dissecting microscope. There were a few notable exceptions. In one of the biotin bottles a thin black line of unknown origin was observed under the dissecting microscope, which was particularly notable because it is a bright white powder. In all the Turmeric bottles there was evidence of leaf particles in a few of the capsules; although, the capsules were listed as all being derived from the rhizome. Of the six bottles of Turmeric that were examined, one of the bottles was a dull yellow color rather than a bright orange and had very low values for all the assays (Fig 1), being significantly different from the other bottle from the same supplier. One other notable item is that batch number and the expiration date are completely absent from one of the bottles (Table 1) of Sundown Naturals Ginseng Xtra. The bottle was purchased at Walmart in Maryland at the same time as the purchase of the replicate bottle.

Table 6. The mean ± SE concentrations of Ni, Cr, Cu, Zn and Pb in (μg • g^-1) isolated from 58 bottles of over the counter herbal supplements.

Supplier	Supplement	Bottle	Ni	Cr	Cu	Zn	Pb
Nature’s Way	Aloe	1	0 ± 0^*	0 ± 0	0 ± 0	2.65 ± 0.08	0
Nature’s Way	Aloe	2	0.06 ± 0.05	0 ± 0	0 ± 0	0 ± 0	0
Nature’s Way	Astragalus	1	0 ± 0	0 ± 0	0 ± 0	4.10 ± 2.98	0
Nature’s Way	Astragalus	2	0 ± 0	0 ± 0	0 ± 0	0.71 ± 0.05	0
NOW	Astragalus	1	0 ± 0	0 ± 0	0 ± 0	0.68 ± 0.18	0
NOW	Astragalus	2	0 ± 0	0 ± 0	0 ± 0	0.38 ± 0.10	0
NOW	Biotin	1	0.11 ± .08	0 ± 0	0 ± 0	0.16 ± 0.08	0
NOW	Biotin	2	0 ± 0	0 ± 0	0 ± 0	0.69 ± 0.04	0
Nature’s Way	Cranberry	1	0.45 ± 0.21	0 ± 0	0 ± 0	0.60 ± 0.19	0
Nature’s Way	Cranberry	2	0.05 ± 0.05	0.04 ± 0.02	0 ± 0	1.06 ± 0.05	0
Spring Valley	Echinacea	1	0 ± 0	0 ± 0	0 ± 0	0.10 ± 0.07	0
Spring Valley	Echinacea	2	0.61 ± 0.34	0.03 ± 0.02	0 ± 0	0.34 ± 0.03	0
Sundown Naturals	Echinacea	1	0 ± 0	0 ± 0	0 ± 0	0.77 ± 0.07	0
Sundown Naturals	Echinacea	2	0 ± 0	0 ± 0	0 ± 0	0.75 ± 0.02	0
Nature’s Way	Echinacea Goldenseal	1	0 ± 0	0 ± 0	0 ± 0	1.56 ± 0.08	0
Nature’s Way	Echinacea Goldenseal	2	0.68 ± 0.31	0 ± 0	0 ± 0	1.19 ± 0.18	0
Spring Valley	Echinacea Goldenseal	1	0.02 ± 0.02	0 ± 0	0 ± 0	0.63 ± 0.07	0
Spring Valley	Echinacea Goldenseal	2	0 ± 0	0 ± 0	0 ± 0	0.32 ± 0.09	0
NOW	Ginger	1	0 ± 0	0 ± 0	0 ± 0	0.08 ± 0.03	0
NOW	Ginger	2	0 ± 0	0 ± 0	0 ± 0	0.08 ± 0.05	0
Spring Valley	Ginger	1	0 ± 0	0 ± 0	0 ± 0	1.01 ± 0.04	0
Spring Valley	Ginger	2	0 ± 0	0 ± 0	0 ± 0	1.20 ± 0.10	0
Sundown Naturals	Ginseng Xtra	1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0
Sundown Naturals	Ginseng Xtra	2	0 ± 0	0 ± 0	0 ± 0	0.07 ± 0.04	0
Spring Valley	Korean Panax Ginseng	1	0 ± 0	0 ± 0	0 ± 0	0.33 ± 0.23	0
Spring Valley	Korean Panax Ginseng	2	0 ± 0	0 ± 0	0 ± 0	0.08 ± 0.08	0
Nature’s Way	Red Raspberry Leaf	1	0 ± 0	0 ± 0	0 ± 0	1.90 ± 0.04	0
Nature’s Way	Red Raspberry Leaf	2	0 ± 0	0.42 ± 0.09	0 ± 0	0 ± 0	0
Nature’s Way	Reishi	1	0 ± 0	0 ± 0	0 ± 0	0.23 ± 0.11	0
Nature’s Way	Reishi	2	0.31± 0.20	0.17 ± 0.17	0 ± 0	5.15 ± 3.56	0
Nature’s Way	Rhodiola	1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0
Nature’s Way	Rhodiola	2	0.20 ± 0.12	0 ± 0	0 ± 0	0 ± 0	0
NOW	Rhodiola	1	0.13 ± 0.10	0 ± 0	0 ± 0	0.93 ± 0.80	0
NOW	Rhodiola	2	0 ± 0	0.08 ± 0.08	0 ± 0	2.29 ± 0.04	0
Nature’s Way	Silent Night	1	0.23 ± 0.23	0 ± 0	0 ± 0	1.79 ± 0.40	0
Nature’s Way	Silent Night	2	0 ± 0	0 ± 0	0 ± 0	1.51 ± 0.27
Nature’s Way	St. John’s Wort	1	0 ± 0	0 ± 0	0 ± 0	1.00 ± 0.06	0
Nature’s Way	St. John’s Wort	2	0.26 ± 0.19	0 ± 0	0 ± 0	0.91 ± 0.06	0
Spring Valley	St. John’s Wort	1	0 ± 0	0 ± 0	19.97 ± 0.85	1.93 ± 0.17	0
Spring Valley	St. John’s Wort	2	0.01 ± 0.01	0 ± 0	15.00 ± 0.53	0.79 ± 0.05	0
Sundown Naturals	St. John’s Wort	1	0.08 ± 0.08	0 ± 0	0 ± 0	0.78 ± 0.14	0
Sundown Naturals	St. John’s Wort	2	0.18 ± 0.18	0 ± 0	0 ± 0	0.72 ± 0.08	0
Sundown Naturals	Stress formula	1	0.02 ± 0.02	0 ± 0	0 ± 0	1.02 ± 0.04	0
Sundown Naturals	Stress formula	2	0 ± 0	0 ± 0	0 ± 0	0.67 ± 0.16	0
Nature’s Way	Turmeric	2	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0
Nature’s Way	Turmeric	1	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0
Spring Valley	Turmeric	1	0.09 ± 0.06	0 ± 0	0 ± 0	1.69 ± 0.31	0
Spring Valley	Turmeric	2	0 ± 0	0 ± 0	0 ± 0	1.33 ± 0.23	0
Sundown Naturals	Turmeric	1	0 ± 0	0 ± 0	0 ± 0	0.08 ± 0.04	0
Sundown Naturals	Turmeric	2	0.01 ± 0.01	0 ± 0	0 ± 0	0.13 ± 0.05	0
Nature’s Way	Valerian Root	1	0 ± 0	0 ± 0	0 ± 0	1.68 ± 0.11	0
Nature’s Way	Valerian Root	2	0.16 ± 0.16	0 ± 0	0 ± 0	1.41 ± 0.08	0
Spring Valley	Valerian Root	1	0.12 ± 0.12	0 ± 0	0 ± 0	1.02 ± 0.12	0
Spring Valley	Valerian Root	2	0 ± 0	0 ± 0	0 ± 0	0.31 ± 0.11	0
Sundown Naturals	Valerian Root	1	0.01 ± 0.01	0 ± 0	0 ± 0	1.89 ± 0.05	0
Sundown Naturals	Valerian Root	2	0.08 ± 0.08	0 ± 0	0 ± 0	1.81 ± 0.09	0
Nature’s Way	Yarrow	1	0.04 ± 0.04	0 ± 0	0 ± 0	3.44 ± 0.95	0
Nature’s Way	Yarrow	2	0.09 ± 0.09	0 ± 0	0 ± 0	0.96 ± 0.19	0
Bottles where element present			24/58	5/58	2/58	51/58	0/58

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*The values are expressed as the mean ± SE (n = 5) for each extraction from each bottle.

Fungal isolation

All control plates were clear of fungal growth. Four of the tested supplements were completely clear of fungal growth but were not isolated to any one supplier (Table 7). Of the bottles tested, 37 of the 58 had fungal contamination, with 21 of the supplements containing multiple microbial isolates, ranging from two to six species. The types of fungi that were isolated from the samples varied, but the most common fungi were in the genus Aspergillus. Other commonly isolated fungi include species from the genus Candida, Microsporum, and Nocardia. In addition, there was some bacterial growth on the SDA plates, such as Bacillus and Streptomyces.

Table 7. Microbial species identified from four samples from each of two bottles from 29 herbal supplements.

Isolates are separated by supplier and supplement type.

Nature’s Way	Bottles	Identified Contaminant
Aloe	2/2	Aspergillus sp., Sporobolomyces salmonicolor, Trichophyton sp.
Astragalus	1/2	Bacillus subtilis, Candida tropicalis
Cranberry	1/2	Aspergillus fumig\atus, Microsporum sp., Trichophyton terrestre
Echinacea	2/2	Candida albicans, Sporobolomyces, Microsporum sp.
Echinacea Goldenseal	2/2	Bacillus subtilis, Rhodotorula
Red Raspberry Leaf	2/2	Aspergillus fumigatus, Microsporum sp., Nocardia brasiliensis
Reishi	1/2	Bacillus subtilis
Rhodiola	2/2	Sporobolomyces salmonicolor
Silent Night	1/2	Candida sp., Candida tropicalis, Nocardia brasiliensis, Streptomyces
St. John’s Wort	0/2	none
Turmeric	1/2	Chlorociboria aeruginascens
Valerian Root	2/2	Aspergillus sp., Bacillus subtilis, Crytococcus neoformans, Candida krusei, Nocardia brasiliensis
Yarrow	0/2	none
NOW
Astragalus	2/2	Aspergillus fumigatus, Aspergillus sp., Bacillus subtilis, Sporobolomyces sp., Sporotrichum sp., Trichophyton rubrum
Biotin	1/2	Lycogala epidendrum, Rhodotorula sp.
Ginger	2/2	Acremonium sp., Bacillus subtilis, Chromelosporium fulva, Cryptococcus sp., Nocardia brasiliensis, Sporobolomyces salmonicolor
Rhodiola	0/2	none
Spring Valley
Echinacea	1/2	Rhodotorula sp., Streptomyces sp.
Echinacea Goldenseal	2/2	Aspergillus sp., Beauveria sp., Trichophyton sp.
Ginger Root	1/2	Bacillus subtilis, Microsporum sp., Nocardia brasiliensis, Sporobolomyces salmonicolor
Korean Panax Ginseng	1/2	Cryptococcus neoformans
St. John’s Wort	1/2	Aspergillus niger, Aspergillus sp., Cryptococcus sp.
Turmeric	1/2	Aspergillus sp., Candida tropicalis, Penicillium sp., Nocardia brasiliensis
Valerian Root	2/2	Aspergillus fumigatus, Aspergillus niger, Candida sp., Cryptococcus sp., Pseudomonas sp.
Sundown Naturals
Echinacea	1/2	Bacillus subtilis, Nocardia brasiliensis
Ginseng Xtra	1/2	Aspergillus flavus, Bacillus subtilis, Nocardia brasiliensis, Rhizopus sp.
St. John’s Wort	1/2	Aspergillus fumigatus
Stress Formula	2/2	Bacillus subtilis, Cryptococcus sp., Nocardia brasiliensis, Trichophyton sp.
Turmeric	0/2	none
Valerian Root	1/2	Aspergillus fumigatus, Bacillus subtilis, Candida sp., Nocardia brasiliensis, Trichophyton terrestre

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Discussion

Assays and supplement stability

Recognizing that there are multiple methods of measuring antioxidant capacity, phenolics and flavonoids it was decided that one type of assay for each should be conducted to more quickly determine if there is justification for further investigation into the purity and consistency of the supplements. An important next step is testing the supplements for the presence of and concentration of the active ingredient if present. The data included in this analysis, in conjunction with previous studies on supplement contamination, strengthen the case that the FDA should regulate over-the-counter herbal supplements the same way that they regulate food and drugs and that further testing involving different assays for antioxidant capacity and specific phenolics and flavonoids is justified for these supplements. The inconsistency between different bottles of the same supplement from the same supplier (as demonstrated by the large standard errors) demonstrates that there seems to be little consistency in the production of herbal supplements among all the suppliers that were tested. Bottles from the same batch theoretically should have low differences, as they should have been produced side by side with the same manufacturing process [2]. Of the 18 bottles that were retested after two years, 6 were past their expiration date, while 12 were well within their use by date, so it was anticipated that there would be less change for these supplements, but this was not observed. There was no consistent pattern displayed by bottles within their use by date versus those that had expired. When there was a significant difference between the supplements that had expired and those that had not in all but one case the expired supplement had more stability than the non-expired supplement (data not shown). Valerian root that had expired had a more significant decrease in flavonoid content in the water soluble fraction; however, all bottles saw a decrease of more than 25% (data not shown). The least stable values between the two testing periods were antioxidant activity in both the methanolic and water extracts and the flavonoid content in the water extract (Table 4). Phenolic content in both extracts and flavonoid content of the methanol extract were the most stable after two years (Table 4). The stability of antioxidant capacity can be susceptible to temperature, light and humidity, depending on the types of antioxidants in the supplements [42]. If this was the case in these data, which mimicked storage conditions in a home, consumers should be made aware that stability and efficacy of the products could be impacted by storage conditions.

Metal analysis

In addition to issues with consistency within and between bottles, was the data associated with the presence of metals and microbial isolates in the supplements. The recommended daily allowance (RDA) for adults for copper is 700 μg per day with an upper limit of 10 mg per day [43]. The RDA for adults for zinc is 15 mg per day with an upper limit of 40 mg per day [43]. The RDA for adults for chromium is 24 μg per day with no established upper limit due to lack of research [39]. The upper limit for nickel for adults is 1 mg per day [43]. These RDA values are usually met by taking a single dose of a daily multivitamin and most often will be met by eating a well-balanced diet. Surpassing the upper limit of minerals is not typically through diet, but by ingesting contaminated water supplies or from their presence within supplements [43, 44]. The main risk factor leading to metal overdose is that people who take supplements often take multiple supplements at the same time [45–47]. Overconsumption of these supplements when taken together, particularly over a prolonged period, can lead to excess zinc, copper, chromium and nickel build up, which could lead to mineral toxicity.

The metal screens indicated a low concentration of metals in general; apart from copper from St. John’s Wort produced by Spring Valley (Table 6), with values that ranged from 13–22 μg • g^-1. High levels of copper intake can lead to copper toxicity, which has been implicated in a few psychiatric disorders such as Alzheimer’s and Parkinson’s [48]. In addition, high copper intake in conjunction with a high fat diet has been linked to reductions in cognition in the general population [46]. This is particularly problematic with the high copper levels found in St. John’s Wort from Spring Valley. Although the amount of copper within the supplement is most likely not problematic for the majority of the population, it could unintentionally lead to dangerous copper levels in individuals who already have higher than normal copper uptake or susceptible populations. The St. John’s Wort bottles from Spring Valley, furthermore, were from different batch numbers, which indicates that this may be a chronic issue with the supplement supply or with the processing of the supplement. High amounts of dietary zinc, which could be exacerbated by taking multiple supplements, alters intestinal bacteria and can increase susceptibility to Clostridium difficile infection [49]. Caution is indicated with consumption of supplements given the above findings because other minerals that were not tested may also be present in supplements, which may have as of yet unknown and unintended negative health consequences.

Fungal isolation

About 80% of the microbial screens were contaminated with fungus, with a few bacteria showing up in the fungal screens. Morphological identification of the species indicates that there were potential species of concern, which indicate targets for further research to determine if the species tentatively identified are indeed problematic. Of the microbial isolates that were classified only one, Aspergillus flavus, is known to produce mycotoxins [50]. Isolates from three genera, Candida, Trichophyton and Microsporum are known to cause infections and can be particularly problematic for immunocompromised individuals [51–53]. Most of the isolates that were identified, such as Nocardia, Rhodotorula, and Sporobolomyces, are generally safe, but may act as opportunistic pathogens in immunocompromised individuals [54–56]. Other isolates, such as Chlorociboria aeruginascens, Lycogala epidendrum, Chromelosporium fulva, Beauveri and B. subtilis generally have no impact on human health as noted by a search of the literature.

Conclusion

Due to the high demand for herbal supplements and other natural products, suppliers should follow strict manufacturing practices that ensure consistency, purity, and safety of their products. The data presented here strengthens the case that lack of regulation leads to products that are not standardized and have the potential to either be ineffective or to cause harm. Currently in the United States manufacturers are to ensure safety of their products, but the FDA can only legally act to remove supplements when there is proof of harm, false statements or ineffective labelling [57]. Until the regulation of herbal supplements falls under the jurisdiction of the FDA, the general public should be informed that if they are consuming herbal supplements they may not be getting what they are paying for.

Supporting information

S1 Table. P-values of the analysis of herbal supplements from two or more suppliers.

P-values are based on ANOVA with an α level of 0.05.

(PDF)

Click here for additional data file.^{(75.2KB, pdf)}

S2 Table. P-values of the analysis of bottles within a supplier for supplement.

P-values are based on ANOVA with an α level of 0.05.

(PDF)

Click here for additional data file.^{(86.6KB, pdf)}

S3 Table. Range of coefficient of variations (CV) from 58 bottles of over the counter herbal supplements.

Antioxidant capacity, phenolic concentrations, and flavonoid concentrations were measured from both hot water and methanolic extractions of 10 pills from each of two bottles per supplement per supplier. Results of the Rank Runs test are also included with p-value of < 0.05. indicating non-randomness of distribution among the results.

(PDF)

Click here for additional data file.^{(84.9KB, pdf)}

S4 Table. P-value from a 1 tailed t-test.

T-tests indicated a change in antioxidant capacity, phenolic and flavonoid concentration of -25% from the original test.

(PDF)

Click here for additional data file.^{(76.5KB, pdf)}

Data Availability

All data files are available from the Harvard Dataverse database (https://doi.org/10.7910/DVN/TRLTI3).

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0260463.r001

Decision Letter 0

Catherine Nkirote Kunyanga

14 Sep 2020

PONE-D-20-06630

Screening for consistency and contamination within and between bottles of 29 herbal supplements.

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It is a very good initiation to work on ensuring the consistency, purity, and safety of herbs relating to heavy metals and microorganisms, which have a role in adaptogens, stress and depression relief, and immune response. However, I have doubtful to consider this manuscript as a full research paper. If there is a room, PLOS can also handle as a short communication or letter.

In addition, I do have the following comment.

Line 21-23, Please state the quantitative value/data (mean±sd) for antioxidant activity, phenolic concentrations, and flavonoid concentrations.

Line 15. Please put in bracket (FDA) after the Food and Drug Administration (FDA), as this is the 1st appearance.

Line 33 Key Words, please make Keywords, please also check for the number of the keywords included, from PLOS authors guide.

Line 99. Please make subsections for Materials and Methods.

Example

Sample collection

Laboratory analysis

Sample extraction

Antioxidant

Stability test

Metals test

Physical contamination

Fungi test

Please also make the subsections for your Discussion; based on the content of your Discussion.

The authors repeatedly use personal pronouns (we) in a different part of the manuscript that needs to be changed/modified, Line 101, 102, 104 122, 141, 152, 158, 169, 180, 201, 204, 219, 224, 241, 245, 282, 285.

Line 107, the title of Table 1 is lengthy, please revise, and if need be, put all the other descriptions at the bottom of the table in the bracket or asterisk.

Line 113 please indicate the source/reference for extraction, which consists of the detailed protocol.

Line 118 state the source, at the end of the sentence.

Line 119. FRAP please write Ferric reducing antioxidant power and FRAP in the bracket.

Line 119. For antioxidant in vitro test, at least two assays needed; in addition, better also to look the Effective concentration (EC50). These make the antioxidant activity test/data to the standard and trustful.

Line 130, please state a source for the physical contamination test.

Line 139 and 140 please state a source.

Line 148,149, please state the alpha level for the degree of significance.

Line 170. Table 2 needs a general modification.

This table should be with the numerical value (mean±se for each supplement and parameters). The mean values should be also differentiated using superscript letters if significant differences found.

Line 170. Please state the title of Table 2, and put the others, as the footnote at the bottom of the Table.

Line 188 please rewrite the title of Table 3.

Table 3. Better to describe as mean±sd, please.

Table 3. needs to have superscript letters to show the degree of significance.

Table 3. An antioxidant is a broad term that does not described as a single parameter. It needs to be specific. In your case, FRAP. Please know that a single in vitro parameter is not to the standard to support the antioxidant activity of your samples. Please refer to the comment give in Line 119.

Line 209 Table 4, please modify the title of the Table, state mean±sd with significant superscript letters. The range value can be indicated in your discussion.

Line 237, Our data, in conjunction with previous studies on supplement contamination, …..

Please substitute Our data, ……

The findings of this study/data

Please apply these changes, wherever needed.

Line 281 please apply the above comment (Line 237).

Page 24 I am not clear for the question mark found in Figure 1.

Thank you

Abera Belay

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Nov 23;16(11):e0260463. doi: 10.1371/journal.pone.0260463.r002

Author response to Decision Letter 0

29 Sep 2020

Reviewer #1: No – Actually the manuscript is written in standard English; although, grammatical issues related to use of first person rather than passive voice have been addressed. However, some of the reviewer’s comments were not written using correct English grammar and syntax.

5. Review Comments to the Author

1. Reviewer #1: It is a very good initiation to work on ensuring the consistency, purity, and safety of herbs relating to heavy metals and microorganisms, which have a role in adaptogens, stress and depression relief, and immune response. However, I have doubtful to consider this manuscript as a full research paper. If there is a room, PLOS can also handle as a short communication or letter. – Hopefully the addition of the complete statistical analysis will allow the paper to be considered as a full research paper.

2. Line 21-23, Please state the quantitative value/data (mean±sd) for antioxidant activity, phenolic concentrations, and flavonoid concentrations. – Included one the range of CVs as an example, but it would be impractical to include the quantitative data for all of the supplements in a meaningful way within the abstract of the manuscript. In this case the CV is ideal as it gives an idea of variability that is standardized across values that are different by orders of magnitude.

3. Line 15. Please put in bracket (FDA) after the Food and Drug Administration (FDA), as this is the 1st appearance. - Done

4. Line 33 Key Words, please make Keywords, please also check for the number of the keywords included, from PLOS authors guide. – There are already keywords included in the paper. I could not find information about key words in the authors guide

5. Line 99. Please make subsections for Materials and Methods.

Example

Sample collection

Laboratory analysis

Sample extraction

Antioxidant

Stability test

Metals test

Physical contamination

Fungi test

Please also make the subsections for your Discussion; based on the content of your Discussion. – Done, the number of subsections included is not as numerous as suggested in the above example as this would have impeded the flow of the paper. However, there are now subsections for the Materials and Methods, Results and Discussion

6. The authors repeatedly use personal pronouns (we) in a different part of the manuscript that needs to be changed/modified, Line 101, 102, 104 122, 141, 152, 158, 169, 180, 201, 204, 219, 224, 241, 245, 282, 285. – Done

7. Line 107, the title of Table 1 is lengthy, please revise, and if need be, put all the other descriptions at the bottom of the table in the bracket or asterisk. -Done

Line 113 please indicate the source/reference for extraction, which consists of the detailed protocol. – Done - Cai, Y., Luo, Q., Sun, M., Corke, H., 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 74, 2157-2184.

8. Line 118 state the source, at the end of the sentence. – In the original manuscript only line 118 refers to an extraction protocol, while line 113 is the title for table 1. So I’ve included the source for the extraction protocol, but there was no source for what supplements were tested. These were chosen by the lab group and the subsampling was done to ensure that each pill had all 4 extractions conducted.

9. Line 119. FRAP please write Ferric reducing antioxidant power and FRAP in the bracket. - Done

10. Line 119. For antioxidant in vitro test, at least two assays needed; in addition, better also to look the Effective concentration (EC50). These make the antioxidant activity test/data to the standard and trustful. – The reason for choosing only one type of assay for each type is included at the beginning of the discussion. “Recognizing that there are multiple methods of measuring antioxidants, phenolics and flavonoids it was decided that one type of assay for each should be conducted to more quickly determine if there is justification for further investigation into the purity and consistency of the supplements.”

11. Line 130, please state a source for the physical contamination test. – There is no source for this. We just examined the samples under the dissecting microscope to see if anything looked unusual or out of place.

12. Line 139 and 140 please state a source. – The source was added. Singh P, Srivastava B, Kumar A, Dubey NK. Contamination of raw materials of some herbal drugs and recommendation of cinnamomum camphora oil as herbal fungitoxicant. Micro Ecol. 2008; 56: 555-560.

13. Line 148,149, please state the alpha level for the degree of significance. - Done

14. Line 170. Table 2 needs a general modification.

This table should be with the numerical value (mean±se for each supplement and parameters). The mean values should be also differentiated using superscript letters if significant differences found. – This data is now split between 2 tables. One based on the means and SE for each bottle of each supplement and supplier. The other is based on the overall means for each supplier of each supplement (now tables 2 and 3)

15. Line 170. Please state the title of Table 2, and put the others, as the footnote at the bottom of the Table. - Done

16. Line 188 please rewrite the title of Table 3. - Done

17. Table 3. Better to describe as mean±sd, please. – The table has been changed to show the CV for all values, but we believe that the CV is valuable in evaluating supplements with a wide range of values. Therefore we are including an expanded CV table in addition to the means and standard errors with accompanying superscripts in two new tables. See 15 above.

18. Table 3. needs to have superscript letters to show the degree of significance. - Done

19. Table 3. An antioxidant is a broad term that does not described as a single parameter. It needs to be specific. In your case, FRAP. Please know that a single in vitro parameter is not to the standard to support the antioxidant activity of your samples. Please refer to the comment give in Line 119. – See response to comment 11 above.

20. Line 209 Table 4, please modify the title of the Table, state mean±sd with significant superscript letters. The range value can be indicated in your discussion. – The title has been modified

21. Line 237, Our data, in conjunction with previous studies on supplement contamination, …..

Please substitute Our data, ……

The findings of this study/data

Please apply these changes, wherever needed. - Done

22. Line 281 please apply the above comment (Line 237). – Done

23. Page 24 I am not clear for the question mark found in Figure 1. – I have carefully looked over the manuscript figure file and found no question mark in Figure 1.

Attachment

Submitted filename: PLOS One Herbals - Response to reviewer comments.docx

Click here for additional data file.^{(21.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0260463.r003

Decision Letter 1

Catherine Nkirote Kunyanga

24 Nov 2020

PONE-D-20-06630R1

Screening for consistency and contamination within and between bottles of 29 herbal supplements.

PLOS ONE

Dear Dr. Veatch-Blohm,

In addition to responding the points raised by the reviewer, please address the following items:

We note that line 68 is not supported by citation to a reference, but this could be because it may be intended to set up the reference in line 76. Please confirm or revise as appropriate. As currently phrased, it could reads like manufacturers intentionally contaminated supplements. Is this what the authors intended to say? If not, perhaps revise like this: “Contamination may be due to growth, processing or storage conditions or may be intentional addition of unlisted ingredients.”
Lns 83-86: Please provide links to FDA sources describing the three 2015 recalls reported.
In the Materials and Methods section, please specify exactly when the testing was done.
Lns 184-85: Please clarify whether any differences reported were statistically significant.
Lns 237-38 & 297-306: Is the suggestion that ingesting copper levels detected in Spring Valley’s St. John’s Wort over time result in copper toxicity bad enough to cause Alzheimer’s, Parkinson’s or cognitive decline? The current phrasing seems to imply this is the case, even when taken a “correct dosage” (at least with respect to cognitive decline). This should be substantiated.
Lns 244-48: Please indicate where the bottle described in these lines was purchased to avoid any argument that the bottle tested was not a genuine bottle but a counterfeit.

Please submit your revised manuscript by Jan 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Jamie Males, Ph.D.

Senior Editor

PLOS ONE

on behalf of

Catherine Nkirote Kunyanga, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

**********

6. Review Comments to the Author

Reviewer #1: This is my 2nd review for the authors, please.

Hopefully, the authors will consider all the comments to maintain the quality of the manuscript for the readership of the PLOS community.

As a reviewer, I am still sticking to my previous recommendation. Additional data is necessary to consider this paper as a full research manuscript, and need to improve and thoroughly discuss the tables and figures.

2. Line 21-23, Please state the quantitative value/data (mean±sd) for antioxidant

activity, phenolic concentrations, and flavonoid concentrations.

– Included one the range of CVs as an example, but it would be impractical to include the quantitative data for all of the supplements in a meaningful way within the abstract of the manuscript. In this case the CV is ideal as it gives an idea of variability that is standardized across values that are different by orders of magnitude.

My request is to include the mean±sd of antioxidant activity, phenolic concentrations, and flavonoid concentrations in the abstract section. The authors do not address this.

– The reason for choosing only one type of assay for each type is included at the beginning of the discussion. “Recognizing that there are multiple methods of measuring antioxidants, phenolics and flavonoids it was decided that one type of assay for each should be conducted to more quickly determine if there

is justification for further investigation into the purity and consistency of the

supplements.”

13. Line 148,149, please state the alpha level for the degree of significance.

- Done

The authors said done for this comment; however, the alpha level for the degree of significance is not in place.

14. Line 170. Table 2 needs a general modification. This table should be with the numerical value (mean±se for each supplement and parameters). The mean values should be also differentiated using superscript letters if significant differences are found.

– This data is now split between 2 tables. One based on the means and SE for each bottle of each supplement and supplier. The other is based on the overall means for each supplier of each supplement (now tables 2 and 3).

The superscript stated in Table 2 and 3 are not correct, and is not statistical accepted. Some of the values do not have also the superscripts.

17.Table 3. Better to describe as mean±sd, please.

– The table has been changed to show the CV for all values, but we believe that the CV is valuable in evaluating supplements with a wide range of values. Therefore we are including an expanded CV table in addition to the means and standard errors with accompanying superscripts in two new tables. See 15 above.

18. Table 3. needs to have superscript letters to show the degree of significance. –Done.

This is not properly done.

19. Table 3. An antioxidant is a broad term that does not described as a single

parameter. It needs to be specific. In your case, FRAP. Please know that a single in vitro parameter is not to the standard to support the antioxidant activity of your samples. Please refer to the comment give in Line 119. – See response to comment 11 above.

This is not addressed.

20.Line 209 Table 4, please modify the title of the Table, state mean±sd with significant superscript letters. The range value can be indicated in your discussion.

– The title has been modified.

The title is modified; however, it is necessary to address the other requests.

Thank you

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. 2021 Nov 23;16(11):e0260463. doi: 10.1371/journal.pone.0260463.r004

Author response to Decision Letter 1

2 Jan 2021

Response to Reviewer comments

• We note that line 68 is not supported by citation to a reference, but this could be because it may be intended to set up the reference in line 76. Please confirm or revise as appropriate. As currently phrased, it could reads like manufacturers intentionally contaminated supplements. Is this what the authors intended to say? If not, perhaps revise like this: “Contamination may be due to growth, processing or storage conditions or may be intentional addition of unlisted ingredients.” – The wording has been changed as per your suggestion. The sentence was to provide a set up for the rest of the paragraph.

• Lns 83-86: Please provide links to FDA sources describing the three 2015 recalls reported. – The archived links are now included in the reference section under reference 28.

• In the Materials and Methods section, please specify exactly when the testing was done. – The following statement was added at the end of the first paragraph of the materials and methods. – “ All of the extractions and tests, with the exception of the supplement stability tests were conducted from October 2016 through April 2017. The supplement stability tests were conducted in January and February 2019.”

• Lns 184-85: Please clarify whether any differences reported were statistically significant. – The wording has been changed to show that differences reported among bottles indicate a significant difference.

• Lns 237-38 & 297-306: Is the suggestion that ingesting copper levels detected in Spring Valley’s St. John’s Wort over time result in copper toxicity bad enough to cause Alzheimer’s, Parkinson’s or cognitive decline? The current phrasing seems to imply this is the case, even when taken a “correct dosage” (at least with respect to cognitive decline). This should be substantiated. – The wording here has been changed to the following, which hopefully removes a causalconnection – “Although the amount of copper within the supplement is most likely not problematic for the majority of the population, it could unintentionally lead to dangerous copper levels in individuals who already have higher than normal copper uptake or susceptible populations”

• Lns 244-48: Please indicate where the bottle described in these lines was purchased to avoid any argument that the bottle tested was not a genuine bottle but a counterfeit. – The following was added “The bottle was purchased at Walmart in Maryland at the same time as the purchase of the replicate bottle.”

Hopefully, the authors will consider all the comments to maintain the quality of the manuscript for the readership of the PLOS community.

2. Line 21-23, Please state the quantitative value/data (mean±sd) for antioxidant

activity, phenolic concentrations, and flavonoid concentrations.

My request is to include the mean±sd of antioxidant activity, phenolic concentrations, and flavonoid concentrations in the abstract section. The authors do not address this. – The coefficient of variation is calculated as a ratio of the mean to the standard deviation. It is specifically used in cases where the means are so different in a scale of magnitude that comparing one mean to the other would not be feasible. To include any kind of single overall mean ± std for antioxidants, phenolics and flavonoids would be useless. The values between supplements are so different that any sort of composite mean would not be representative of any of the values presented within the tables in the manuscript. Because the coefficient of variation is a measure of the relative standard deviation it is a much more efficient way to encapsulate what the reviewer wants in a very concise format that is also meaningful.

The description of statistical variation needs to be based on mean±sd, and the superscript should be stated for each treatment, which is based on the treatment variation. (This is also seen in the new document, line number 163 page 10). – I’m not sure how the reviewer expects for this to be included within the abstract since the values for each of these are so vastly different and that any composite mean would be meaningless. See comment above.

is justification for further investigation into the purity and consistency of the

supplements.”

Phenol and flavonoids are phytochemicals, which are considered as antioxidant content. My request is for antioxidant activity, like FRAP. In line with this calculating EC50 increases the readability of the manuscript, and also makes the data trustful. – The samples and supplements that we tested have long since passed the time where an additional antioxidant test, such as the EC50 could be performed on these bottles. However, in addition to the statement included in the first revision this statement has also been added to the discussion to indicate that further testing is justified and needed – “and that further testing involving different assays for antioxidant capacity and specific phenolics and flavonoids is justified for these supplements”

13. Line 148,149, please state the alpha level for the degree of significance.

- Done

The authors said done for this comment; however, the alpha level for the degree of significance is not in place. – The alpha level for each test is listed in the section under statistical analysis which is copied here. The alpha level was also included in the footnotes of the tables where appropriate. “Statistical Analysis. For each bottle of each supplement, the means and standard error were calculated for the water, methanol, and nitric acid extractions. In addition, the coefficient of variation (CV) was calculated for the water and methanol extractions. Comparisons among suppliers and bottles from the same supplier were done using the general linear model in the FitModel platform of JMP with an � level of 0.05 [38]. A runs test was also conducted to test for randomness of rankings among bottles, suppliers and supplements using Minitab� Statistical Software with an � level of 0.05 [39]. A significant runs test means suppliers and/or supplements clump together. To determine if there had been a change in the average values for the antioxidant, phenolic and flavonoid assays after two years of supplement storage the data was analyzed using a one-tailed t-test with an � level of 0.05 and a test value of < -25% change using Minitab� Statistical Software [39].

The superscript stated in Table 2 and 3 are not correct, and is not statistical accepted. Some of the values do not have also the superscripts. – I’m not really sure why the reviewer states that the superscripts are not correct. I have made the following modifications to hopefully address the reviewers concerns. In table 2 a ^ was placed next to values from supplements where there is only one supplier so that comparisons among suppliers is not possible. In addition, I have placed a superscript letter next to all values, even those that are not significantly different. In addition our data set with statistical analysis has been reorganized and updated in the open source data link to make it easier to see all of the analysis. https://doi.org/10.7910/DVN/TRLTI3

17.Table 3. Better to describe as mean±sd, please.

To my knowledge, CV does not indicate a statistical variation between treatments. For a reputable journal like PLOS ONE, a description of treatments using a statistical variation is important for the readers, and maintain the quality of the journal. – The purpose of the coefficient of variation is to allow for a snapshot of how much variation there is around a mean without reference directly to the mean and it’s attendant units. It is also useful in examining variation in tests that do not have similar means as it is a measure of the relative standard deviation (RSD)

•

18. Table 3. needs to have superscript letters to show the degree of significance. –Done.

This is not properly done. – The table has been modified to include superscript letters for all values, even those without significant differences between bottles. Initially we had not included superscripts for the insignificant values to reduce the amount of clutter within the table.

19. Table 3. An antioxidant is a broad term that does not described as a single

This is not addressed. – To address this FRAP is included in all table titles in association with antioxidant capacity, but within the body of the tables we use only ‘A’ to symbolize this category in keeping with the single letter symbols ‘P’ and ‘F’ for phenolics and flavonoids.

20.Line 209 Table 4, please modify the title of the Table, state mean±sd with significant superscript letters. The range value can be indicated in your discussion.

– The title has been modified.

The title is modified; however, it is necessary to address the other requests. – Table 4 in the old manuscript is now table 6 in revision 1. For the metals we were simply testing for the presence versus absence of the metals within the supplements and did not conduct an ANOVA on the data.

Attachment

Submitted filename: Response to Reviewer comments_Plos R2.docx

Click here for additional data file.^{(20KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0260463.r005

Decision Letter 2

Catherine Nkirote Kunyanga

22 Jan 2021

PONE-D-20-06630R2

Screening for consistency and contamination within and between bottles of 29 herbal supplements.

PLOS ONE

Dear Dr. Veatch-Blohm,

1. Supplement stability tests were in some cases performed after the expiration date listed, but in others before. Please include some text discussing this as a limitation, and consider whether you could include subgroup analysis separating out the supplements that had expired from those that had not in the stability tests.

2. Please clarify whether the supplements were stored according to the package instructions, and were they unopened before testing? Storage conditions are currently described as having “mimicked storage conditions in a home”, but there is no formal description of these conditions (in terms of temperature, humidity etc.)

3. Please describe where all of the tested supplements were purchased e.g. online or a local store (please provide names).

4. Regarding the recalls (lines 83-88), in reading the archived FDA alerts, it seems that the companies themselves voluntarily recalled the products, not the FDA. We would recommend removing this sentence and the next in their entirety – they are not necessary to support the claims in this paper.

Please submit your revised manuscript by Mar 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Jamie Males

Senior Editor

PLOS ONE

on behalf of

Catherine Nkirote Kunyanga, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Attachment

Submitted filename: Submission Report PONE D PONE-D-20-06630R2.pdf

Click here for additional data file.^{(295.2KB, pdf)}

PLoS One. 2021 Nov 23;16(11):e0260463. doi: 10.1371/journal.pone.0260463.r006

Author response to Decision Letter 2

14 Feb 2021

Changes have been made to the manuscript according to reviewer comments. A point by point response to the comments is included in the uploaded files

Attachment

Submitted filename: Response to Reviewer Comments_Plos R3.docx

Click here for additional data file.^{(13.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0260463.r007

Decision Letter 3

Hikmet Aydin

4 Oct 2021

PONE-D-20-06630R3Screening for consistency and contamination within and between bottles of 29 herbal supplements.PLOS ONE

Dear Dr. Veatch-Blohm,

Please submit your revised manuscript by Nov 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Hikmet Aydin, MD, FAACC

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Reviewer #2: The authors have addressed the comments appropriately and I recommend the manuscript be accepted for publication.

Reviewer #3: 1) line 75: cancel comma before the references

2)Introduction: the cited references are a little bit old. Could you find newer literature?

3)Tables 3 and 6: what do you mean "0" is the same as not determined?

4)in Tables with results SD is very high in some cases.

5) Please, rewrite the conclusion.

6) Lack of novelty.

7) Lack of the validation of the methods.

Reviewer #4: Did the extraction protocol performed according to the total weight of the supplement, or according to weight of extract per gram in a supplement? No information is provided in the methods section in terms of the extract amount per weight.

Eventhough, morphological identification of fungal species is important and routinely used, for fungal systematics, taxonomic classification may not always perform well for species classifications. This would be a limitation which should be further addressed.

Similarly, the gross physical contamination of the samples were screened under a light-dissecting microscope; while no information regarding the standards for the methodology (such as AOAC International, American Spice Trade Association, pharmacopeia’s, FDA’s Macroanalytical Procedures Manual etc) is provided. Also comprehensive investigation are also based on chemical techniques along with Fourier transform-infrared (FTIR) analysis; which is also a limitation and is expected to be addressed.

Legal aspects in terms of chemical and microbiological quality of herbal supplements should be discussed within the respective directives.

Reviewer #5: The required revisions are made on the manuscript. It is really very important to search for the quality of herbal supplements and share the gathered results with public.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Attachment

Submitted filename: Academic editor report PONE D 20 06630.pdf

Click here for additional data file.^{(151.7KB, pdf)}

PLoS One. 2021 Nov 23;16(11):e0260463. doi: 10.1371/journal.pone.0260463.r008

Author response to Decision Letter 3

3 Nov 2021

6. Review Comments to the Author

Reviewer #2: The authors have addressed the comments appropriately and I recommend the manuscript be accepted for publication.

Reviewer #3: 1) line 75: cancel comma before the references – Done

2)Introduction: the cited references are a little bit old. Could you find newer literature? – Four additional references from 2019 through 2021 were added to the introduction and one reference from 2020 was added to the discussion. The rest of the references we wanted to keep as they contribute to the justification for the importance of the research conducted. When the paper was first submitted almost 2 years ago at least some of the references were more recent.

3)Tables 3 and 6: what do you mean "0" is the same as not determined? – It is unclear to me what the reviewer is asking here. It is never stated in the paper that 0 is the same as not determined. When zero is listed as the mean of the value for the supplement it indicates that none of the pills tested of that supplement had any detectable amount of either the antixodant activity, phenolic or flavonoids or the metals in question.

4)in Tables with results SD is very high in some cases. – This is addressed briefly in the discussion with the following statement -- (as demonstrated by the large standard errors)

5) Please, rewrite the conclusion. - done

6) Lack of novelty.

7) Lack of the validation of the methods.

Reviewer #4:

1. Did the extraction protocol performed according to the total weight of the supplement, or according to weight of extract per gram in a supplement? No information is provided in the methods section in terms of the extract amount per weight.

- Added to the method sections- ‘All assays values were calculated and standardized based on the weight of the sample that was extracted.’

2. Even though, morphological identification of fungal species is important and routinely used, for fungal systematics, taxonomic classification may not always perform well for species classifications. This would be a limitation which should be further addressed. – The following statement was added to the discussion – “Morphological identification of the species indicates that there were potential species of concern, which indicate targets for further research to determine if the species tentatively identified are indeed problematic.”

3. Similarly, the gross physical contamination of the samples were screened under a light-dissecting microscope; while no information regarding the standards for the methodology (such as AOAC International, American Spice Trade Association, pharmacopeia’s, FDA’s Macroanalytical Procedures Manual etc) is provided.

- The following line was added to the methods. “The samples were scanned quickly for uniformity within a capsule and among capsules within the same bottle and between different bottles.”

- Within the results the following statement was changed “lacked gross physical contamination” and replaced with “were uniform in texture and color”

4. Also comprehensive investigation are also based on chemical techniques along with Fourier transform-infrared (FTIR) analysis; which is also a limitation and is expected to be addressed. – Included the following statement in the discussion - “An important next step is testing the supplements for the presence of and concentration of the active ingredient if present.”

5. Legal aspects in terms of chemical and microbiological quality of herbal supplements should be discussed within the respective directives.

- It wasn’t exactly clear to me what the reviewer meant by this comment. I had it checked by the university’s research counsel and they also were not certain. In order to try to address it I added the following to the conclusion –‘The data presented here strengthens the case that lack of regulation leads to products that are not standardized and have the potential to either be ineffective or to cause harm. Currently in the United States manufacturers are to ensure safety of their products, but the FDA can only legally act to remove supplements when there is proof of harm, false statements or ineffective labelling [57].’

Reviewer #5: The required revisions are made on the manuscript. It is really very important to search for the quality of herbal supplements and share the gathered results with public.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Attachment

Submitted filename: Response to reviewers Plos One_R4.docx

Click here for additional data file.^{(36.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0260463.r009

Decision Letter 4

Hikmet Aydin

11 Nov 2021

Screening for consistency and contamination within and between bottles of 29 herbal supplements.

PONE-D-20-06630R4

Dear Dr. Veatch-Blohm,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hikmet Aydin, MD, FAACC

Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0260463.r010

Acceptance letter

Hikmet Aydin

15 Nov 2021

PONE-D-20-06630R4

Screening for consistency and contamination within and between bottles of 29 herbal supplements.

Dear Dr. Veatch-Blohm:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Hikmet Aydin

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. P-values of the analysis of herbal supplements from two or more suppliers.

P-values are based on ANOVA with an α level of 0.05.

(PDF)

Click here for additional data file.^{(75.2KB, pdf)}

S2 Table. P-values of the analysis of bottles within a supplier for supplement.

P-values are based on ANOVA with an α level of 0.05.

(PDF)

Click here for additional data file.^{(86.6KB, pdf)}

S3 Table. Range of coefficient of variations (CV) from 58 bottles of over the counter herbal supplements.

(PDF)

Click here for additional data file.^{(84.9KB, pdf)}

S4 Table. P-value from a 1 tailed t-test.

T-tests indicated a change in antioxidant capacity, phenolic and flavonoid concentration of -25% from the original test.

(PDF)

Click here for additional data file.^{(76.5KB, pdf)}

Attachment

Submitted filename: PLOS One Herbals - Response to reviewer comments.docx

Click here for additional data file.^{(21.9KB, docx)}

Attachment

Submitted filename: Response to Reviewer comments_Plos R2.docx

Click here for additional data file.^{(20KB, docx)}

Attachment

Submitted filename: Submission Report PONE D PONE-D-20-06630R2.pdf

Click here for additional data file.^{(295.2KB, pdf)}

Attachment

Submitted filename: Response to Reviewer Comments_Plos R3.docx

Click here for additional data file.^{(13.7KB, docx)}

Attachment

Submitted filename: Academic editor report PONE D 20 06630.pdf

Click here for additional data file.^{(151.7KB, pdf)}

Attachment

Submitted filename: Response to reviewers Plos One_R4.docx

Click here for additional data file.^{(36.4KB, docx)}

Data Availability Statement

All data files are available from the Harvard Dataverse database (https://doi.org/10.7910/DVN/TRLTI3).

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PERMALINK

Screening for consistency and contamination within and between bottles of 29 herbal supplements

Maren E Veatch-Blohm

Iris Chicas

Kathryn Margolis

Rachael Vanderminden

Marisa Gochie

Khusmanie Lila

Roles

Abstract

Introduction

Materials and methods

ASupplement selection

Table 1. List of supplements and suppliers for 58 bottles of over the counter herbal supplements.

Sample extraction

Antioxidant, phenolic and flavonoid assays

Metal extraction and gross physical contamination

Fungal isolation

Statistical analysis

Results

Antioxidant, phenolic and flavonoid assays

Table 2. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by supplier*.

Table 3. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by bottle and supplier*.

Fig 1. The mean ± SE FRAP antioxidant capacity (a), phenolic concentration (b), and flavonoid concentration (c) of the methanolic fraction of two bottles each of St. John’s Wort (SJW), Turmeric (T), and Valerian Root (VR) from three different suppliers.

Table 4. Coefficient of variation (CV) for the FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) for 58 bottles of over the counter herbal supplements extracted with hot water and methanol.

ASupplement stability

Table 5. Change in the mean FRAP antioxidant capacity, phenolic and flavonoid concentration of two bottles each of nine herbal supplements*.

Metals and gross physical contamination

Table 6. The mean ± SE concentrations of Ni, Cr, Cu, Zn and Pb in (μg • g-1) isolated from 58 bottles of over the counter herbal supplements.

Fungal isolation

Table 7. Microbial species identified from four samples from each of two bottles from 29 herbal supplements.

Discussion

Assays and supplement stability

Metal analysis

Fungal isolation

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Catherine Nkirote Kunyanga

Roles

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Catherine Nkirote Kunyanga

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Hikmet Aydin

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Table 2. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by supplier^*.

Table 3. The mean ± SE FRAP antioxidant capacity (A), phenolic content (P) and flavonoid content (F) from 58 bottles of over the counter herbal supplements extracted with hot water and methanol by bottle and supplier^*.

Table 5. Change in the mean FRAP antioxidant capacity, phenolic and flavonoid concentration of two bottles each of nine herbal supplements^*.

Table 6. The mean ± SE concentrations of Ni, Cr, Cu, Zn and Pb in (μg • g^-1) isolated from 58 bottles of over the counter herbal supplements.