Identification of phosphodiesterase type-5 (PDE-5) inhibitors in herbal supplements using a tiered approach and associated consumer risk

Figures & data

Table 1. Prevalence of adulterated herbal products with active pharmacological ingredients.

Country	No. of samples analysed/ No. of samples adulterated (%)	No. of sample adulterated		References
		With approved PDE-5i identified (%)	With analogues of PDE-5i identified (%)
Singapore	175/134 (77)	123 (92)	11 (8)	Li et al. (2009)
India	85/1 (1)	1 (100)	–	Savaliya et al. (2010)
USA	26/15 (58)	3 (20)	12 (80)	Mans et al. (2013)
Netherlands	71/23 (32)	1 (4)	22 (96)	Reeuwijk et al. (2014)
France	150/92 (61)	41 (45)	51 (55)	Gilard et al. (2015)
Malaysia	62/34 (55)	Few^a	Majority^a	Bujang et al. (2017)
Romania	50/11 (23)	8 (73)	3 (27)	Mateescu et al. (2017)
USA	353/166 (47)	166 (100)	–	Tucker et al. (2018)
Korea	404/130 (32)	–	–	Lee et al. (2021)

^aInsufficient information provided to derive the actual number.

Table 2. LC–MS/MS-based levels of individual compound(s) in supplements, their total concentrations, recommended daily doses and EDIs.

Sample ID	Compound concentration (mg g^−1 or mL)			Total compound concentration (mg g^−1 or mL^−1)^b	Average dose (g day^−1)	No. of doses day^−1	EDI (mg sildenafil equivalents day^−1)
	Sildenafil 1^a	Tadalafil 0.1^a	Vardenafil 2^a
S3	13			13	1.01	2	26
S6	123	2		123	0.47	2	116
S10	243	0.2		243	0.46	2	223
S14	14			14	1.16	4	64
S15	322			322	0.36	6	696
S16	18			18	0.65	2	23
S19	135			135	0.43	2	116
S20			1	2	10	3	60
S21	67			67	0.97	4	268
S23	2			2	20	1	40
S37	89	2		89	2.03	4	723
S38	16			16	1.47	4	94

^aRelative potencies of compounds in the bioassay.

^bExample: For S6 (123 × 1) + (2 × 0.1) = 123.2 mg g⁻¹. Meanwhile, the average number of doses per day is 0.47 taken two times per day. Thus, 123.2 × 0.47 × 2 = 115.8 mg g⁻¹. The same calculation procedure was used for the other supplements.

Table 3. Comparison between estimated concentrations in the PDE-Glo assay and the LC–MS/MS analysis. Only supplements classified as resulting in medium or high intake based on the bioassay are shown.

Sample ID	Bioassay concentration (mg g^−1)			LC–MS/MS^b Concentration (mg g^−1)	Evaluation
	100^a	1000^a	10,000^a
S3	>8	36	532	13	Unidentified compounds
S4	1			<1	Comparable
S5	>8	<1		<1	Comparable
S6	>8	31	299	123	Comparable
S10	3	47	377	243	Comparable
S13	5	40	873	<1	Unidentified compounds
S14	4	13		14	Comparable
S15	>8	43	444	322	Comparable
S16	7	26		18	Comparable
S17	>8	<1		<1	Comparable
S19	6	39	326	135	Comparable
S20	6	26		2	Unidentified compounds
S21	5	29		69	Comparable
S22	2			<1	Comparable
S23	1			2	Comparable
S24	2			<1	Comparable
S25	3			<1	Comparable
S27	>8	<1	<7	<1	Comparable
S28	4	<1		<1	Comparable
S30	2			<1	Comparable
S35	4	6		<1	Unidentified compounds
S37	6	32	278	89	Comparable
S38	6	50	127	16	Unidentified compounds
S39	4	<1		<1	Comparable

^aDilution factor applied in the PDE-Glo Bioassay. Estimated levels were corrected for the dilution factor. Empty means that no clear inhibition was observed.

^bLC–MS/MS-based levels are expressed in sildenafil equivalents.

Figure 1. UPLC chromatogram of S13 extract and the various fractions collected.

Figure 2. Inhibition potentials of collected fractions of S13 tested in the PDE-Glo bioassay, fraction c showing the presence of a PDE-5i.

Figure 3. Concentration-dependent reduction in PDE-5 activity. IC₅₀ values of 900 nM and 80 nM were calculated from the fitted dose–response curves for sildenafil and hydroxythiohomosildenafil, respectively, resulting in a REP value for hydroxythiohomosildenafil of about 12.

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