Planta Med
DOI: 10.1055/a-2591-3765
Original Papers

Passionflower (Passiflora incarnata): Quality of Food Supplements Versus Registered Herbal Medicinal Products

Alexa Brouns
Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Münster, Germany
,
Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Münster, Germany
,
Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Münster, Germany
› Author Affiliations

Financial support from the Apothekerstiftung Westfalen-Lippe (www.akwl.de/apothekerstiftung/) to AH is acknowledged.
 

Abstract

The above-ground plant material from Passiflora incarnata is used for relief of symptoms of mental stress and to aid sleep. In Europe, passionflower products are marketed either as registered herbal medicinal products or as food supplements. Passionflower products for sleep disorders are increasingly recommended to patients by physicians or by social advertisement, but the potential consumers are in most cases not able to differentiate between food supplements or licensed herbal medicinal products. Analytical investigations by validated protocols on passionflower food supplements and registered medicinal products from different sources were performed to obtain an insight into the actual quality situation. TLC fingerprinting revealed the non-identity of five food supplements, while six products met the specification (five registered herbal medicinal products and one food supplement). A validated UHPLC method confirmed this result. LC-MS identified one food supplement containing only hyperoside and lacking other passionflower-related compounds. Quantitative determination of flavones by photometric protocol, as well as by a calibrated UHPLC, indicated that five out of six food supplements did not meet the specified content and identity, suggesting instances of food fraud. All registered herbal medicinal products conformed to the specification. As this analytical investigation is in line with other reports on the low quality of food supplements, transparent and intensified quality control is recommended. In addition, routine analyses of every batch using validated procedures by manufacturers on a batch-by-batch basis should provide a secure basis for improved product quality and for the safety of the consumer.


#

Abbreviations

HMPC: Committee on Herbal Medicinal Products of the European Medicines Agency EMA
Ph. Eur.: European Pharmacopoeia
TLC: thin-layer chromatography
WHO: World Health Organisation
 

Introduction

The above-ground parts from Passiflora incarnata L. (Passifloraceae) are traditionally used for the relief of mild symptoms of mental stress and to aid sleep [1]. In addition, the World Health Organisation (WHO) recommends the herbal material for nervous restlessness, insomnia, and anxiety based on well-established documents [2]. Passionflower can be used either as herbal tea for oral use (daily dose up to 8 g per day) or by use of concentrated extract preparations. Passionflower products are in many cases used as mono-preparations, but also, combinations with other medicinal plants for treatment of sleep disorders are found. From the phytochemical point of view, the above-ground parts of passionflower are characterized by the C-glycosides of the flavones apigenin and luteolin (0.2 to 2%). Both, C6- and C8-linked glycosides have been identified, with isovitexin, isoorientin, and their 2″-β-D-glucosides (= sophorosides) as main compounds, in addition to schaftoside, isoschaftoside, vicenin-2, and swertisin ([Fig. 1]) [3].

Zoom Image
Fig. 1 Structural features of flavones characterizing passionflower herbal material from Passiflora incarnata. Glc: β-D-glucose, Ara: α-L-arabinose, Soph: 2-O-β-D-glucopyranosyl-β-D-glucopyranose.

Other constituents include cyanogenic glycosides [4], maltol (3-hydroxy-2-methyl-γ-pyrone gamma-pyrone) [5], [6], and significant amounts of mono-, oligo-, and polysaccharides [7], [8]. β-Carbolin- and 3,4-dihydro-β-carbolin alkaloids have been claimed to be part of passionflower, but intensive research could not confirm the presence of these harman-like alkaloids [9]. P. incarnata is known to exist in two chemotypes. The isovitexin-type with isovitexin/schaftoside/isoschaftoside as the main compounds represents the most prominent type, while the swertisin-type with swertisin as the main compound and minor amounts of schaftoside and isoschaftoside is found less frequently [10]. The herbal material of both chemotypes originates mostly from controlled farming in India, the U. S.A, and South America. For analytical quality control, passionflower dry extract is monographed by the European Pharmacopoeia [11]. Both the swertisin and isovitexin chemotypes, as well as mixtures of both, are accepted, as long as the content of total flavonoids, calculated as isovitexin, exceeds 1.0%. Although a direct correlation between the listed flavonoids and the pharmacological activity of the herbal material has not been proven, the flavonoids are still used as marker compounds for authentication and for quality assessment [10]. Also, WHO uses the flavone-C-glycosides as analytical marker compounds and specifies ≥ 1.5% for the chemical assay, expressed as vitexin and analyzed by spectrophotometry [2]. In addition to the passionflower herbal material, the Ph. Eur. lists a monograph for passionflower dry extract, which should contain ≥ 1.5% of total flavonoids, calculated as vitexin [11]. As the recommended daily intake of passionflower is quite high (up to 8 g of herbal material), most products on the market are using concentrated extracts for oral use in capsules or tablets. Interestingly, in Europe, passionflower products are marketed either as registered herbal medicinal products or as food supplements (please note: according to European legislation, these products are referred to as food supplements, whereas in the United States, they are commonly known as dietary supplements). It has to be kept in mind that food supplements are regulated by the respective food laws, while herbal medicinal products are controlled by the national medicinal laws. Both pieces of legislation are based on different quality control systems, which can lead to a high quality of medicinal products but also to lower quality of food supplements [12], [13]. In general, a consensus has been made that botanicals should be regulated in a more harmonized way in the EU, e.g., in terms of labeling, nutrivigilance, or quality control, to enhance product transparency and patient safety [14]. It should always be clear to the consumer whether the product is an herbal medicinal product or a food supplement [14]. Additionally, the recommended dosage should be based on evidence-based data, such as those, for example, displayed by the HMPC monographs. Recent investigations on the quality of food supplements containing botanicals indicated relevant quality problems. This includes instances of food fraud, the wrong identity of the claimed active ingredient, discrepancies between the actual content and the labeling, and products that are significantly under-dosed. [15], [16]. However, the use of passionflower products for sleep disorders is often recommended to patients by physicians or social advertisement, but the potential consumers are in most cases not able to differentiate between food supplements or licensed herbal medicinal products [13]. Both product categories can in general have similar appearance and presentation. Based on this situation, and realizing a growing market for passionflower products, the following study aimed to perform an analytical investigation by validated protocols on passionflower food supplements and registered herbal medicinal products obtained from community pharmacies in Germany, internet shopping, or health food stores. The outcome of this study explicitly confirmed the quality for all registered herbal medicinal products, whereas only one (!) of the investigated food products met the specifications of the European Pharmacopoeia. Consequences have to be considered to overcome this non-acceptable situation.


#

Results and Discussion

Analytical investigations of a series of 11 passionflower-containing samples, representing the passionflower market in the German-speaking regions, was performed: six food supplements (P01 to P06) and five registered herbal medicinal products (P07 to P11) were obtained from internet trading, as well as from a community pharmacy. [Table 1] displays an overview on the different products. Freshly harvested, authenticated, and air-dried plant material from P. incarnata served as reference.

Table 1 Passionflower-containing test samples: six food supplements (P01 to P06) and five registered herbal medicinal products (P07 to P11) were obtained from internet trading, as well as from a community pharmacy. Data indicate supplier, specifications, and composition of the respective products.

No.

Supplier/Distributor

Brand name

Category: Food supplement (FS), registered medicinal product (D)

Shelf life1
Lot/batch number2

Declaration, content, serving size per day

Further labeling

a Passionflower extract per capsule [mg]; b total flavon/flavonoide content [mg] per capsule; c DER Medicinal Herbal material – extract ratio; d serving size

P01

FairVital b. v., Netherlands

Passionsblume Extrakt 750 mg

FS

03/20261
8 882 5192

Passionflower extract: 750 mga
15 mg Flavoneb
3 capsulesd

Contains additional L-leucin and excipients

P02

Sunday Natural Products GmbH
Germany

Passionsblumen Extrakt

FS

20. 04. 20251
04 3042

Passionsblumen-Extrakt: 375 mg
15 mg Flavonoidsb
10 : 1c
1 capsuled

Contains additional excipients

P03

Warnke GmbH & Co. KG
Germany

Passionsblumen-Extrakt

FS

02/20251
2 051 5192

Passionflower extract: 750 mga
15 mg Flavoneb
3 capsulesd

Contains additional excipients

P04

Narayana Verlag GmbH
Germany

Passionsblume Kapseln

FS

11/20241
LO1 – 29 112 0222

Passionflower extract: 375 mga
Flavone: 7.5 mg (2%)b
12 : 1c
2 capsulesd

Contains additional excipients

P05

Nature Love
Tauron Ventures GmbH
Germany

Passionsblume

FS

06/20241
NL22 230 050 0212

Passionflower extract: 375 mga
DER 5 : 1c
2 capsulesd

Contains additional excipients

P06

Natureʼs Answer
U. S. A.

Passionflower – natural Calming Botanical

FS

08/20251
222 7552

Passionflower Aerial Parts Extract: 250 mga
standardized for 3.5% flavonoids as Vitexinb
2 capsulesd

Contains additional excipients

P07

Walther Schoenenberger Pflanzensaftwerk GmbH & Co. KG
Germany

Naturreiner Heilpflanzensaft Passionsblumenkraut

D

05/20241
AB-05072

100 mL pressed juice of Passionflower
1 : 0,7 – 0,9c
3 × 10 mL per dayd

P08

Doppelherz Pharma
Queisser Pharma GmbH & Co. KG
Germany

Passiflora

D

01/20251
223 0022

Passionflower extract: 425 mga
DER: 5 – 7 : 1c
1 – 2 × 1 tablet per day

Contains additional excipients

P09

Salus Haus GmbH & Co. KG
Germany

Passionsblumenkraut

D

03/20251
U0455 A2

Passionflower herba
Drink 2 – 4 times a day a cup of tea (pour boiling water over 1 teaspoon (150 mL) and strain after 10 – 15 min)d

P10

Ceres Heilmittel GmbH
Germany

Passiflora incarnata Urtinktur

D

08/20261
1 938 540 M2

1 – 3 × daily 3 dropsd

Passiflora incarnata Urtinktur, 65% (vol) ethanol

P11

Cesra Arzneimittel GmbH & Co. KG Germany

Lioran classic

D

03/20251

Passionflower extract: 260 mga
5 – 7 : 1c
2 × 2 capsules per dayd

Contains additional excipients

As the presentation of the food supplements is similar to that of medicinal products, a test on the uniformity of mass for single-dose preparations according to the European Pharmacopoeia [17] was performed for the solid oral dosage forms (P01 – P06, P08 – P09, and P11). All tested samples were found to be in compliance with their respective declarations.

Thin layer chromatography (TLC) fingerprinting according to the methodology of the Ph. Eur. was employed for the identity testing of the samples, using isovitexin, orientin, and homoorientin as reference compounds and detection with diphenylboryloxyethylamine ([Fig. 2]).

Zoom Image
Fig. 2 TLC analysis of passionflower test samples P01 to P11 after detection with diphenylboryloxyethylamine (λ = 366 nm). Reference extract PFf, reference compounds: isovitexin, homoorientin (HO), orientin (O).

Systematic investigation of the passionflower test products indicated that five out of eleven samples (P01 to P05) did not correspond to the requirements of positive identity for P. incarnata ([Fig. 2]). While P01 and P03 did not show any fluorescent zones that can be related to the relevant marker flavonoids, P04 and P05 showed only weak zones, which do not correspond to the reference spectrum. P02 showed a conspicuous orange zone, which could not be assigned to any of the spots in the reference chromatogram. In contrast, test samples P06 to P11 referred exactly to the reference chromatograms of P. incarnata and the requirements of the pharmacopoeia ([Fig. 2]). Interestingly, P01 to P06 are all classified as food supplements, while P07 to P11 are registered herbal medicinal products.

Subsequently, UHPLC was used for the qualitative and quantitative determination of the relevant flavone glycosides. Based on the protocol of Ph. Eur. 11.3 [11], the HPLC method was optimized toward a UHPLC protocol for better resolution. Comparisons of the different parameters between the pharmacopoeial method and the optimized protocol are displayed in the Supplementary File, Table 1S. The optimized UHPLC method was validated and qualified for system precision (± 0.79%), method precision (< ± 1%%), recovery rate (108%), and linearity of detection signals (concentration range from 5 to 100 µg/mL, R2 = 0.9997) (Supplementary File, Table 2S, Figure 1S.

Typical chromatograms are displayed in Fig. [3] Samples P01, P03, and P04 had identical fingerprints, which are not related to the typical passionflower flavonoids. P02 showed only a single peak, which was subsequently identified as hyperoside (quercetin-3-O-galactoside) based on its UV-spectra (λ max = 254 and 354 nm) and MS characteristics (m/z = 465.1 (M+H)). A hyperoside reference standard was also used to confirm this falsification (Supplementary Data, Figure 2S). Peaks in P05 could not be correlated to any natural product, but the existence of typical passionflower compounds can definitely be excluded for this sample. Based on these data and the TLC investigations, products P01 to P05 are to be assessed as not compliant with their respective specifications and must therefore be considered counterfeit.

Zoom Image
Fig. 3 Chromatograms (UHPLC) of flavone glycosides in passionflower test solutions from food supplements (P01 to P06) and registered herbal medicinal products (P07 to P11). Reference standards: homoorientin, orientin, isovitexin, reference extract from P. incarnata extract (PFf). Detection at λ = 338 nm.

Samples P06 to P11 met the specifications and confirmed the identity of passionflower, which is also consistent with the results obtained from the TLC analysis ([Fig. 3]).

For quantitation of flavone glycosides in the test samples, two different methods were applied. The photometric method determines the total flavone content by measuring their borinic acid complexes, with the results expressed as vitexin [18].

The HPLC protocol quantifies flavone glycosides within a defined retention time range using isovitexin as a reference compound [11]. The present work developed and improved this HPLC method into a modern UHPLC method, where the flavone glycosides elute baseline separated between 4 and 10 minutes. The calculation is performed using a five-point isovitexin calibration curve (Supplementary Data, Fig. 1S).

The results of both quantitations are presented in [Table 2]. For P01 to P04, the photometric flavone quantitation did not meet the flavone content specified for the respective products. Content values obtained are much lower than specified by the respective manufacturers. The UHPLC investigations indicated–as expected from TLC data–the absence of any relevant flavone content. P06 was the only food supplement found to comply with the declaration, achieving a specified flavone content within the range of 90 to 100%. The liquid preparation P07 was only investigated by UHPLC, as no exact weight-in quantity could be achieved due to the presence of excipients. The flavone contents of P08, P09, and P11, all registered herbal medicinal products, were in accordance with the specified values. The sample P10, a homeopathic mother tincture, still has relevant flavone content.

Table 2 Summary of quality investigation of passionflower-based preparations with regard to identity and flavone content.

No.

Identity
[UPLC/TLC]

Uniformity of mass

Declaration

Calculated content

Conforms to dosing recommendation of HMPC*

Overall Assessment

Flavones [mg] of manufacturerʼs specifications per unita; passionflower extract per unitb

Recommended dose per day (capsules, flavone content [mg])

Total flavones [mg] and [% w/w declared passionflower extract], per unit

Total flavones [mg], as determined (UHPLC), per day

photometric

UHPLC

*daily dose 1 to 8 g passionflower herbal material per day; **declaration conforms to HMPC recommendation, but for this product identity of passion flower negative

P01

+

15a
750b

3 capsules

45

3.1 mg ± 0.8; 0.4%

No passionflower flavones

9.4

** Flavone content per caps. equiv. to 1 g herbal material

Does not conform

P02

+

15a
375b

1 capsule

15

4.8 mg ± 0.4; 1.3%

No passionflower flavones

4.8

+**

Does not conform

P03

+

15a
750b

3 capsules

45

1.2 mg ± 0.4; 0.15%

No passionflower flavones

3.5

** Flavone content per caps. equiv. to 1 g herbal material

Does not conform

P04

+

7.5a
375b

2 capsules

15

1.3 mg ± 0.2 0.35%

No passionflower flavones

2.5

+**

Does not conform

P05

+

375b

2 capsules

1.9 mg ± 0.4; 0.5%

3.7

+**

Does not conform

P06

+

+

3.5%, equiv. to 8.75 mga
250b

2 capsules

17.5

9.2 ± 0.3 mg; 3.7%

8.1 ± 0.34 mg; 3.2%

16.2

+

Conforms

P07

+

+

No declaration

30 mL

Not performed

1.7 ± 0.1 mg/mL

52.2

No evaluation possible

Conforms

P08

+

+

425b

2 tablets

27.2 ± 0,9 mg; 6.4%

25.6 ± 0.5 mg;
6.0%

51.1

+

Conforms

P09

+

+

No declaration

2 – 4 × 2 g

61 ± 1 mg; 3.1%

61.5 ± 1.5 mg;
3.1%

123 – 246

+

Conforms

P10

+

+

No declaration

1 – 3 × 3 drops (99 µL)

Not performed

3.4 ± 0.3 mg/mL; 0.3%

0.3 – 1

Critical

P11

+

+

260b

4 capsules

14.2 ± 0.2 mg; 5.5%

15.6 ± 0.1 mg; 6.0%

62.5

+

Conforms

However, by comparing the investigated food supplements with the registered medicinal products, notable differences regarding the daily intake are obvious ([Table 2]). HMPC recommends a daily intake of 1 to 8 g of herbal material, which corresponds to a daily intake of 10 to 80 mg flavones by using the Ph. Eur. specification for passionflower of ≥ 1% total flavonoids, calculated as isovitexin. The daily flavone intake from medicinal herbal products P07 to P11 is in the range of between 50 and > 123 mg, with the exception of P10, which is a homeopathic mother tincture that has to be evaluated in a different way and not according to HMPC. In contrast, the only food supplement with positive identity of passionflower is P06, but, using the recommended intake of 2 capsules per day, only about 16 mg flavones are administered, indicating relevant lower dosage.

From these data, it can be concluded that a significant proportion of food supplements labeled as “passionflower” do not contain the claimed herbal material and can be considered food fraud. On the other hand, the registered herbal products fully conform to the specifications and the specific requirements for medicinal products. Summarizing these data, it is obvious that a much more stringent quality control of manufacturers of food supplements has to be initiated by the respective national governmental institutions. It is possible that the food industry is using the pharmacological and clinical particularities for herbal medicinal products, as stated in the respective HMPC monographs, for intensified marketing. However, at the same time, the fundamental basics of quality control–such as identity, purity, content, and correct dosages–are completely ignored. It raises questions as to why the relevant authorities, responsible for food control and inspection, do not appear to fully utilize their capacities to increase pressure on manufacturers engaging in food fraud, in order to ensure the safety of consumers and patients. As stated recently, the scientific community pleads for a clear distinction of food supplements from medicinal products [13]. The consumption of food supplements is intended only for healthy humans and for people interested in maintaining their health. Therefore, such products should be clearly identifiable as food in their external form, including the respective primary packaging, and should not have the appearance of medicinal products. In relation to the European Health Claims Regulation (EC) No. 1924/2006, food supplements may only claim health benefits when this claim has been positively assessed by the European Food Safety Agency (EFSA), based on substantial scientific data [19], [20].

Although the European Federation of Association of Health Products Manufacturers developed the first pan-European quality guideline as early as 2007 (updated in the second edition in 2014 (European Federation of Association of Health Products Manufacturers)), the very many quality deficiencies range from incorrect content values or contamination to ingredients that are not detectable despite being declared on the label [21].

The scientific community therefore calls for more transparent and significantly intensified quality testing of food supplements on the market. In addition, routine analyses of every batch using validated procedures should ensure that quality controls are carried out by manufacturers on a batch-by-batch basis, thus providing a secure data basis for product quality. Food supplements that, after inspection, show the above-mentioned deficiencies may no longer be placed in the market. More and intensified work has to be invested into consumer and patient safety. And a clear recommendation for users of passionflower products can be made: use high-quality registered herbal medicines instead of inadequate food supplements.


#

Materials and Methods

Materials

If not stated otherwise, solvents, reagents, and consumables were obtained from VWR International. All solvents and reagents were of analytical quality. Water was produced by a Millipore Simplicity 185 system (Millipore). Orientin (lot 69 101) and homoorientin (lot 69 102) were obtained in reference standard quality from Phytolab; isovitexin CRS (batch 1) was obtained from the European Directorate for the Quality of Medicines and Healthcare (EDQM). Dried herbal material Passiflora herba (batch 19 000 739 005) (Ph. Eur. 11) was purchased from Caesar & Loretz. Authenticated plant material from P. incarnata was obtained from the medicinal plant garden of University of Münster, Institute of Pharmaceutical Biology and Phytochemistry. Authentication was done by AH and AB. Voucher samples are stored at the archives of the institute (IPBP No. 911 – 915; 920 – 929).


#

Uniformity of mass for single-dose preparations [17]

Twenty capsules were randomly selected and weighed. The respective content was removed from every individual capsule using a gentle stream of air. Empty capsule shells were weighed to determine the net filling mass. For capsules with a net mass > 300 mg, the test meets the requirements if no more than two capsules deviate ± 7.5% and none deviate more than ± 15% from the mean value. For average filling mass < 300 mg, the respective limits are ± 10% and ± 20%.


#

Thin-layer chromatography

Test solution: 0.1 g test sample was mixed with 5.0 mL methanol and treated for 15 min in an ultrasonic bath. The suspension was centrifuged (5 min, 3600 × g). The clear supernatant was used for further analysis. For reference isovitexin (100 µg/mL in methanol), orientin and homoorientin (150 µg/mL in methanol) were used. A P. incarnata reference extract (PFf) from authenticated plant materials was prepared as follows: 0.5 g of the plant material was mixed with 5.0 mL methanol and treated for 15 min in an ultrasonic bath. After centrifugation (5 min, 3600 × g), the supernatant was used for analysis.

Stationary phase: TLC silica gel F254 (2 – 10 µm); mobile phase: anhydrous formic acid, water, ethyl methyl ketone, ethyl acetate (10 : 10 : 30 : 50 v/v/v/v); spotting volume (8 mm): isovitexin and test samples P01 – P11, PFf, 4 µL each; homoorientin and orientin 20 µL; development: 70 mm; drying: 5 min in an air stream at RT; detection: 100 to 105 °C, 5 min, followed by spraying with a solution of diphenylboryloxyethylamine (10 g/L) in methanol, and further followed by fixation with macrogol 400 (50 g/L) in methanol. Evaluation at λ = 366 nm.


#

Photometric determination of total flavones

In a 100 mL round-bottomed flask, 0.200 g powdered herbal material was mixed with 40 mL ethanol 60% (v/v) and heated to 60 °C for 30 min with frequent shaking and reflux cooling. After cooling, the suspension was filtered through a cotton ball into a 100 mL flask. The cotton ball was added to the residue in the round-bottomed flask and heated again with 40 mL EtOH 60% to 60 °C for 10 min. The first filtrate and the second mixture were filtered through a paper filter in vacuo, transferred to a volumetric flask, and made up to 100 mL with ethanol 60%. Two flasks each containing 5.0 mL of this stock solution were evaporated using a rotary evaporator. The first residue was taken up with 10 mL of a mixture of 10 parts by volume methanol and 100 parts by volume acetic acid 99%. After adding 10 mL of a solution of boric acid (25 g/L) and oxalic acid (20 g/L) in anhydrous formic acid, it was diluted with acetic acid to 25 mL (test solution). The second residue was treated with 10 parts of a mixture of 10 parts by volume methanol and 100 parts by volume acetic acid 99%. After adding 10 mL of anhydrous formic acid, the flask was filled up to 25 mL with anhydrous acetic acid (compensation liquid). The absorbance of the test solution was measured after 30 min at λ = 401 nm against the compensation liquid. The flavonoid content (calculated as vitexin) was calculated using the following formula: [%] = A × 0,8/m where A is the absorbance and m is the weight of the sample in [g].


#

UHPLC

Acquity UHPLC H-Class (Waters) equipped with PDA detector (210 – 400 nm), QDa detector (ESI, positive mode), stationary phase: Waters Acquity HSS T3, 1.8 µm, 2.1 × 100 mm. Mobile phase: (A) water, formic acid (99.9/0.1 v/v); (B) acetonitrile, formic acid (99.9/0.1 v/v) (C) MeOH with the following gradient: Initial: 97.5 – 0 – 2.5 (A, B, C); 0 – 0.5 min: 97.5 → 84.0 (A), 0 → 13.5 (B), 2.5 (C); 0.5 – 10 min: 84 → 82.5 (A) 13.5 → 15.0 (B); 2.5 (C); 10 – 12 min: 82.5 → 0 (A); 15 → 100 (B); 2.5 → 0 (C); 12 – 13 min: 0 – 100 – 0 (A, B, C); 13.1: back to initial condition and equilibration for 2 min. Flow rate: 0.4 mL/min; injection volume: 2 µL; oven temperature: 40 °C; detection: λ = 338 nm. Evaluation by Empower 3 Software.

Test solution: 0.45 g of the sample material was mixed with 25.0 mL water: methanol (20 : 80 v/v). The suspension was sonicated for 40 min. Every 10 minutes, the samples were shaken to avoid caking. After sonication, the samples were centrifuged 5 min at 3200 × g. The supernatant was used for analysis. Liquid samples were either directly injected or diluted 1 : 1 with methanol.

Reference solution: 1.0 mg isovitexin was mixed to 10.0 mL with water/methanol (20 : 80 v/v). The solution was then diluted to a concentration of 5 to 75 µg/mL for calibration. Additionally, authenticated plant material of P. incarnata served as reference material (PFf); 0.8 g of pulverized plant material was mixed with 25.0 mL of water: methanol (20 : 80 v/v). The suspension was sonicated for 40 min and shaken every 10 minutes. It was then centrifuged for 5 minutes at 3200 × g. The clear supernatant was used for analysis.


#

Supplementing information

Table 1S: Comparison of the HPLC protocol of the European Pharmacopoeia 11.3 for the Passiflora incarnata herbal material (Passiflorae herba) and P. incarnata dry extract (Passiflorae herbae extractum siccum) method with the adapted and improved UHPLC method. Table 2S: Validation data of adapted UHPLC protocol. Figure 1S: Five-point calibration line of isovitexin. Figure 2S: UV spectrum and MS data from test sample P02 and identification of the main peak in P02 as hyperoside (quercetin-3-O-galactoside) by comparison of retention time, UV spectra, and MS characteristics with hyperoside reference standard.


#
#

Contributorsʼ Statement

Conception and design of the work: AB, ML, AH; data collection AB, ML, analysis and interpretation of data AB, ML, AH; drafting the manuscript AH, AB, critical revision of the manuscript ML, AH.


#
#

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The technical assistance of Mr. L. Krüger for providing plant material is acknowledged.

Supporting Information


Correspondence

Prof. Dr. Andreas Hensel
Institute of Pharmaceutical Biology and Phytochemistry
University of Münster
Corrensstraße 48
48149 Münster
Germany   
Phone: + 49 251 8333381   

Publication History

Received: 04 March 2025

Accepted after revision: 17 April 2025

Accepted Manuscript online:
17 April 2025

Article published online:
07 May 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany


Zoom Image
Fig. 1 Structural features of flavones characterizing passionflower herbal material from Passiflora incarnata. Glc: β-D-glucose, Ara: α-L-arabinose, Soph: 2-O-β-D-glucopyranosyl-β-D-glucopyranose.
Zoom Image
Fig. 2 TLC analysis of passionflower test samples P01 to P11 after detection with diphenylboryloxyethylamine (λ = 366 nm). Reference extract PFf, reference compounds: isovitexin, homoorientin (HO), orientin (O).
Zoom Image
Fig. 3 Chromatograms (UHPLC) of flavone glycosides in passionflower test solutions from food supplements (P01 to P06) and registered herbal medicinal products (P07 to P11). Reference standards: homoorientin, orientin, isovitexin, reference extract from P. incarnata extract (PFf). Detection at λ = 338 nm.