Phytochemistry 215 (2023) 113854 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Towards the development of analytical monograph specifications for the quality assessment of the medicinal plant Phyllanthus urinaria Emmanuel Orman a,b,c, Samuel Oppong Bekoe c, Samuel Asare-Nkansah c, Ina Kralisch d, Jonathan Jato a,e, Verena Spiegler a, Christian Agyare f, Emelia Oppong Bekoe g,**, Andreas Hensel a,* a Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Corrensstraße 48, Münster, Germany b Department of Pharmaceutical Chemistry, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana c Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana d Chemical and Veterinary Inspection Office, Münsterland-Emscher-Lippe (CVUA-MEL) – AöR, Joseph-König-Str. 40, Münster, Germany e Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana f Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana g Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Ghana, Legon, Ghana A R T I C L E I N F O A B S T R A C T Keywords: Many people in developing countries rely on herbal remedies for their primary healthcare needs. The challenge Phyllanthus urinaria, phyllanthaceae however is that several of these products lack proper documentation of quality and safety. To ensure consistent Quality control quality, validated methods are needed to establish and control quality attributes associated with identity, purity, Specifications and levels of bioactive constituents of the respective herbal materials. The present study focused on Phyllanthus Analytical method validation Contaminants urinaria (PU), a widely used medicinal plant in Ghana and West Africa that lacks the necessary quality control Metabolomics standards. The study aimed to develop an HPTLC identification method, which together with UPLC-ESI-Q-TOF- Design of experiment MS/MS analysis established the identity of PU samples and differentiated PU from other closely related Phyl- lanthus species. Quantitative UPLC and HPTLC methods were developed to assess the contents of selected active markers in the PU samples, which invariably led to the proposal of acceptance criteria for the active markers. Prior to the content analyses, the sample extraction procedure was optimized through the use of Design of Experiment method. The effects of harvest time and geographic origin on the content of active compounds were demonstrated in the investigations. PU samples were also found to be contaminated with higher levels of pes- ticides like chlorpyrifos and folpet. Essentially, this study provides analytical protocols, insights into the quality status of PU samples in Ghana, and analytical specifications contained in a drafted monograph for future consideration in regional and subregional African pharmacopoeias. 1. Introduction healthcare needs (Yeboah et al., 2020), the absence and/or limitations in the establishment of the quality and safety statuses of most of these Over the years, concerns and calls for the need to monitor the quality medicines have served as barriers to the general adoption by all and full status of herbal medicines (HM) have increasingly been made by integration of herbal medicine use and practice into the formal health- different categories of stakeholders in the healthcare industry and value care systems in several countries (Zhang et al., 2012). chain (Ghosh, 2018; Govindaraghavan and Sucher, 2015). Although the It must be emphasised however, that the role of herbal medicines in available data show an encouraging proportion of the public, especially achieving Universal Health Coverage (UHC) is critical. As the focus of in the developing world, patronising herbal medicines for their basic the World Health Organisation (WHO) shifts towards primary * Corresponding author.Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Corrensstrasse 48, D-48149, Münster, Germany. ** Corresponding author.Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Ghana, P. O. Box LG 43, Legon, Accra, Ghana. E-mail addresses: eorman@uhas.edu.gh (E. Orman), sobek03@gmail.com (S.O. Bekoe), asn12002@yahoo.com (S. Asare-Nkansah), Ina.Kralisch@cvua-mel.de (I. Kralisch), jjato@uhas.edu.gh (J. Jato), verena.spiegler@uni-muenster.de (V. Spiegler), chrisagyare@yahoo.com (C. Agyare), eoppongbekoe@ug.edu.gh (E.O. Bekoe), ahensel@uni-muenster.de (A. Hensel). https://doi.org/10.1016/j.phytochem.2023.113854 Received 31 May 2023; Received in revised form 7 September 2023; Accepted 7 September 2023 Available online 15 September 2023 0031-9422/© 2023 Elsevier Ltd. All rights reserved. E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 healthcare in the quest to realise UHC by the year 2025 (World Health develop analytical specifications based on the results of a representative Organization WHO, 2020a), the wide availability, easy accessibility as batch analysis of many PU samples. Additionally, an analytical mono- well as the relative affordability of herbal medicines as compared to graph capturing specifications from the present study is proposed for orthodox medicines (Yeboah et al., 2020) become the more important possible consideration into relevant regional pharmacopoeia such as the deciding factors in healthcare delivery in resource-challenged settings. It Ghana, West Africa, and African herbal pharmacopoeias. The outcomes is for these and many more reasons (World Health Organization WHO, of our analytical investigations on PU are hereby presented and 2013) that the quality and safety of HM has become a great concern in discussed. the area of pharmaceutical and medical development. The incidences of plant misidentification, poor quality and the presence of harmful con- 2. Results and discussion taminants such as heavy metals and pesticides therefore remain as threats in achieving UHC with HM. 2.1. Phytochemical characterization of P. urinaria by LC-MS/MS In the current study, we consider an important medicinal plant, Phyllanthus urinaria L. (Phyllanthaceae) (PU) (Fig. S1), which is widely The phytochemical characterization of the acetone: water (7:3) used in Ghana and other parts of Africa, Asia and America, as a model extract of PU by UPLC/+ESI-QqTOF-MS (Fig. 1) resulted in the tentative herbal remedy, to show how analytical specifications and state-of-the art identification of hydrolysable tannins, flavonoids, lignans, phenolics, analytical protocols can be developed by a systematic work-flow to and terpenoids. In summary, 56 compounds were tentatively identified perform quality assessment of HM (Xu et al., 2007). PU is a tropical or by comparing their spectral data with literature and the online database, perennial herb used traditionally in the management of worm in- Reaxys (https://www.reaxys.com/). Most of these compounds identi- festations (Agyare et al., 2014), as well as in the treatment of enteritis, fied had previously been reported in the plant species and other Phyl- hepatitis, jaundice, renal diseases, malaria, diarrhoea, hypertension, lanthus species (Geethangili and Ding, 2018; Mao et al., 2016), showing among others (Geethangili and Ding, 2018). Ethnopharmacological in- a high degree of similarity in the phytochemistry of the species in the vestigations have demonstrated the following biological effects: genus. Table 1 describes the compounds identified in PU extract. anti-inflammatory, antioxidant, antibacterial, antiviral, antihelminthic, anticancer, and antidiabetic effects (Geethangili and Ding, 2018). PU is 2.2. Phytochemical differences between P. urinaria and closely related reported to contain several classes of secondary metabolites including Phyllanthus species hydrolysable tannins, flavonoids, lignans, coumarins, triterpenoids and phenolic natural products – structural features of typical marker com- Building on the knowledge from the phytochemical characterization pounds see Supplementary Data, Fig. S2 (Chang et al., 2003; Hu et al., of the PU extract, further investigations into the similarities and differ- 2014; Jikai et al., 2002; Spiegler et al., 2015; Xu et al., 2007). Some of ences in the phytochemistry of eight different Phyllanthus species these secondary products are responsible for the observed pharmaco- including PU was carried out through metabolomic analysis. Other logical effects of the plant. For example, the hepatoprotective effects of species investigated were also from the region of West Africa, namely the plant have been attributed to the presence of phyllanthin (Chird- P. amarus, P. fraternus, P. niruri, P. muellerianus, and three unknown chupunseree and Pramyothin, 2010), hypophyllanthin (Chirdchu- species were labelled as Unknown A, B, and C. They were collectively punseree and Pramyothin, 2010), corilagin (Liu et al., 2017), and labelled as non-PU samples for the purpose of the analysis. In addition to geraniin (Londhe et al., 2012). Similarly, phyllanthin from the plant has examining the similarities and differences between PU and non-PU been shown to possess antiamnestic, antiaging, antiapoptotic, and samples, feature selection was also carried out to identify molecular antibacterial effects (Geethangili and Ding, 2018). Norsecurinine has features that could be used to distinguish one group from the other been shown to possess antifungal effects (Sahni et al., 2005). A number (Fig. 2). of the pharmacological effects of the natural products present in PU have The volcano plot obtained from the t-test statistics showed some been detailed in the works of Mao et al. (2016), Geethangili & Ding significant differences in terms of the phytochemical composition of PU (Geethangili and Ding, 2018), Seyed (2019) among others. samples when compared to that of non-PU samples (Fig. 2A). The key Due to the enumerated therapeutic effects of the plant, P. urinaria information in the plot is defined as molecular features with high fold together with related species in the Phyllanthus genus have become changes [log2(FC)] and significant p-values, and these are summarized mainstay components in several commercial herbal formulations in in the plot in red (for features in PU) and blue (for features in non-PU). Ghana (Komlaga et al., 2015; Osei-Djarbeng et al., 2015). Locally, PU is For instance, PU samples were characterized by the presence of signif- known as ‘Bɔwomaguwakyi’, which literally means ‘carry your progeny icant intensities of the molecular features m/z: 703.3431 (cleistantho- behind you’ (Agyare et al., 2014). It is a name that is loosely used also side A, [M+H]+), m/z: 407.1992 (urinatetralin, [M+Na]+), m/z: for related species like P. fraternus G.L.Webster, P. niruri L., P. amarus 203.2416 (norsecurinine, [M+H]+), and m/z: 302.1972 (ellagic acid, Schumach. & Thonn., and at times for P. muellerianus (Kuntze) Exell. [M+H]+). On the other hand, non-PU samples were seen to possess Compounded by the similarities in their morphological features, these molecular features like m/z: 685.5153 (phainanoid G, [M+H]+), m/z: plants are likely to be mistaken for the other. Thus, in the formulation of 729.5425 (phyllaemblicin H5, [M+H]+), m/z: 303.1350 (quercetin, commercial herbal products that contain any of the above-mentioned [M+H]+), m/z: 328.3184 (phyllangin, [M + NH4]+), m/z: 941.0945 species, there stands a risk of collecting the wrong species when the (mallotusinin, [M+Na]+) among others, which were present in signifi- harvesting of the plant is based only on the knowledge of the local name. cantly higher amounts when compared to the PU samples. Additionally, Therefore, the need to develop analytical specifications to control the a PLS-DA classification model with an accuracy of 99% (R2 = 0.9639 and identity of the plant species is obvious. Additionally, some studies have Q2 = 0.7942) was able to discriminate PU samples from non-PU samples demonstrated concerns with the quality of medicinal plant materials based on their LC-MS spectral data (Fig. 2B). The model also showed the originating from different geographical locations and collections at molecular features with very high variable importance in projection different times and seasons of the year (Orman et al., 2023). These (VIP) scores summarized in Fig. 2C. Some of the molecular features variations tend to affect the phytochemical content of the plants, espe- predicted from the VIP scores plot were also identified from the volcano cially that of the bioactive compounds and may invariably affect their plot as characteristic of either PU or non-PU samples, for example, m/z: therapeutic potential. As a result, there is also the need to establish 703.3431 (cleistanthoside A, [M+H]+), m/z: 407.1992 (urinatetralin, validated analytical standards to ensure batch-to-batch consistency of [M+Na]+) and m/z: 203.2416 (norsecurinine, [M+H]+) in PU, and m/z: levels of phytochemical constituents, among others. 685.5153 (phainanoid G, [M+H]+) in non-PU samples. Other charac- The present study was therefore carried out to develop new analyt- teristic features also predicted to contribute to the distinct classification ical protocols for the quality assessment of PU plant material, and also to of the two set of samples were mostly lignans and included the likes of 2 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Fig. 1. UPLC-ESI-QTOF-MS analysis of acetone: water extract (7:3) from the aerial parts of Phyllanthus urinaria. Interpretation of compounds 1 to 55 see Table 1. m/z: 383.1853 ((iso)lariciresinol, [M+Na]+) and its glycoside m/z: NP-PEG and detection at λ = 366 nm and at λ = 254 nm for plates from 515.3261 (isolariciresinol-9′-O-β-D-xylopyranoside, [M+Na]+), m/z: the first and second sets respectively, provided a good characteristic 401.1642 (urinaligran, [M+H]+), m/z: 355.1561 (1-O-galloyl-β-gluco- profile of the plant showing part of its phenolic composition as well as pyranose, [M+Na]+), m/z: 417.2248 (phyltetralin, [M+H]+). providing a basis for its representative quality assessment. Fig. 3 depicts Based on these observations, it is proposed that the different Phyl- the HPTLC profiles developed along with some highlighted analytical lanthus species (as considered in the study) possessed similar hydro- investigations carried out during the validation of the method. The lysable tannins and flavonoid compositions. The lignans present HPTLC method was subsequently validated in accordance with ICH Q2 however tend to differ to a certain extent and contribute to the (R1) guidelines (ICH, 2005) and recommendations from literature distinction of mostly PU from non-PU samples. For this reason, it could (Reich et al., 2008; Renger et al., 2011). Table 2 summarizes the results also be argued that biological activities of the plant which are a result of from the validation carried out. the hydrolysable tannins and flavonoids may be similar across the The method was found to be specific for both qualitative and quan- different species, but those activities as accounted for by the presence of titative purposes. The presence of the marker compounds in the plant lignans could vary across the different species. That notwithstanding, extract was confirmed from simultaneous development of the profiles further studies could be performed to confirm this. with the reference markers (Table 2). Additionally, for the qualitative use of the method, profiles of different Phyllanthus species, including P. urinaria, P. amarus, P. fraternus, P. muellerianus, P. niruri and three un- 2.3. Validated HPTLC method known species, were simultaneously developed and also evaluated. Both visual inspection and chemometric-assisted analysis of the grayscale The HPTLC method was developed with mobile phase systems video-densitometric like profile showed the presence of some differ- optimized from the knowledge of Snyder’s classification of solvents ences in the fingerprints for the different species (Fig. 3E). Within the (Snyder, 1974) and their corresponding polarity indices. The two HPTLC PCA (Fig. 3F), most of the profile for PU were clustered together, and the protocols involved one system developed from a mobile phase con- cluster was quite far off the samples of other Phyllanthus species. Thus, taining ethylacetate, water and formic acid (75:15:10, v/v/v). This the profiles as developed and analysed chemometrically establish intra- system was adopted for both qualitative (fingerprint analysis) and species similarities and inter-species differences which confirms the quantitative purposes where the assay of marker compounds (including specificity and selectivity of the method for chemical authentication rutin, isoquercitrin, and gallic acid) were carried out (structural for- purposes. For the assay of marker compounds, the relationship between mulas of marker compounds are shown in Fig. S2, Supplementary Data concentration and peak areas from densitometric scans were found to be File). The second system involved the use of toluene: ethylacetate: for- linear for gallic acid (r2 = 0.9992) and non-linear for rutin (adj. r2 = mic acid (69:30:1, v/v/v) as mobile phase for the assay of phyllanthin. 0.9940), isoquercitrin (adj. r2 = 0.9976), and phyllanthin (adj. r2 = The HPTLC profiles as developed after derivatization of the plates with 3 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Table 1 Compounds identified in the aerial parts of Phyllanthus urinaria from UPLC-ESI-QTOF-MS/MS analysis. No tR Observed m/z Monoisotopic MS/MS Fragments Accuracy Molecular Compound name Ref. [min] [Adduct(s)] mass (mDa) Formular 1 1.508 236.0983 [M+H] 235.0913 100.0775; 4 C13H17NO3 4-Methoxydihydronorsecurinine Hassarajani and 122.0559; 197.1255 Mulchandani (1990) 2 3.774 485.0945 [M+H] 484.0875 315.0735; 153.0264 5 C20H20O14 Di-O-galloyl-β-glucopyranose Huang et al. (1998) isomer 3 4.029 293.0311 [M+H] 292.0241 191.0380; 107.0455 2 C13H8O8 Brevifolin carboxylic acid Huang et al. (2009) 4 4.122 668.1137 [M + 650.0799 481.0628; 209.0829 13 C27H22O19 Furosin (Agyare et al., 2011; NH4] Xu et al., 2007) 5 4.182 975.0810 [M+Na] 952.0918 783.0729; 10 C41H28O27 Phyllanthusiin A Yoshida et al. (1992) 303.0162; 277.0385; 337.0189 6 4.336 465.0689 [M+H] 464.0619 277.0304; 5 C21H20O12 Myricitrin Tram et al. (2017) 303.0204; 259.0242 7 4.336 652.1187 [M + 634.0849 277.0358; 3 C27H22O18 Corilagin (Agyare et al., 2011; NH4] 303.0182; 127.0349 Xu et al., 2007) 8 4.369 971.1248 [M+H] 970.1203 783.0747; 5 C41H30O28 Repandusinic acid A Xu et al. (2007) 463.0456; 303.0149 9 4.419 411.1676 [M+Na] 388.1784 4 C19H32O8 Dendranthemoside B Van Thanh et al. (2014) 10 4.419 970.1206 [M + 952.0868 783.0749; 10 C41H28O27 Geraniin (Agyare et al., 2011; NH4] 463.0453; 303.0135 Xu et al., 2007) 11 4.470 388.2541 [M + 370.2203 355.0702 4 C16H18O10 4-Acetyl-bergenin Wu et al. (2018) NH4] 12 4.470 422.2379 [M + 404.1986 393.2541 9 C16H20O12 Mucic acid 1-ethyl 6-methyl ester 2- Zhang et al. (2017) NH4] O-gallate 13 4.470 975.0802 [M+Na] 952.0910 783.0710 19 C41H28O27 Geraniinic acid B (Foo, 1995; Yoshida et al., 1992) 14 4.520 387.2046 [M+H] 386.1976 95.0851; 149.0996 4 C22H26O6 Cubebin dimethyl ether Conrado et al. (2020) 15 4.575 944.1405 [M + 926.1067 757.0946; 5 C40H30O26 Phyllanthusiin C Huang et al. (2009) NH4] 437.0786; 277.0351 16 4.728 581.1550 [M+H] 580.148 303.0519 3 C30H28O12 Naringenin-7-O-(6″-O-trans-p- Zhang et al. (2002) coumaroyl)-glucoside 17 4.780 972.1337 [M + 954.0999 785.0854; 10 C41H30O27 Chebulagic acid Luo et al. (2012) NH4] 277.0374; 429.0633; 153.0211 18 4.810 249.1115 [M+Na] 226.1223 246.8356; 105.0761 4 C13H22O3 Boscialin Busch et al. (1998) 19 4.989 611.1656 [M+H] 610.1586 303.0515 6 C27H30O16 Rutin Van Thanh et al. (2014) 20 5.022 303.0188 [M+H] 302.0118 4 C14H6O8 Ellagic acid Huang et al. (2009) 21 5.130 465.1061 [M+H] 464.0991 85.0301; 303.0516; 5 C21H20O12 Isoquercetin Yao and Zuo (1993) 153.0197 22 5.214 197.1177 [M+H] 196.1107 100.1177 4 C12H8O6 Brevifolin Wu et al. (2012) 23 5.285 119.0853 [M+H] 118.0783 4 C4H6O4 Succinic acid Wei et al. (2005) 24 5.285 524.2501 [M + 506.2163 303.0505; 10 C23H22O13 Tri-O-methyl ellagic acid 4-O- Tuchinda et al. NH4] 204.0992; 153.0135 β-glucopyranoside isomer (2008) 25 5.318 435.0959 [M+H] 434.0889 303.0517 4 C21H22O10 Naringenin-7-O-glucoside Zhang et al. (2002) 26 5.434 449.1109 [M+H] 448.1039 303.0512 4 C21H20O11 Kaempferol-3-glucoside Van Thanh et al. (2014) 27 5.434 540.2493 [M + 522.2155 345.1695; 331.1584 10 C26H34O11 Isolarisiresinol 9′-O- Cai et al. (2009) NH4] β-glucopyranoside 28 5.581 393.1930 [M+Na] 370.2038 355.1742 4 C21H22O6 Dextrobursehernin Chang et al. (2003) 29 5.581 800.3019 [M + 782.2681 371.1193, 8 C32H30O23 Emblicannin A Usharani et al. NH4] 251.0582; (2013) 311.0850; 517.1747; 637.3690 30 5.625 461.1436 [M+Na] 438.1544 464.2577; 4 C30H62O Triacontanol Satyan et al. (1995) 253.1086; 331.1042 31 5.731 261.1591 [M+Na] 238.1699 159.0948; 118.0688 4 C15H26O2 Cloven-2β,9α-diol Hu et al. (2014) 32 6.851 926.3330 [M + 908.2992 111.0445; 5 C40H28O25 Acalyphidin M1 Matou (2019) NH4] 171.0651; 231.0861; 371.1129 33 7.138 313.2070 [M+Na] 290.2430 8 C15H14O6 (Epi)catechin (Ishimaru et al., 1992; Zhang et al., 2001) 34 7.138 365.0327 [M+H] 364.0257 271.0609; 4 C16H12O10 Phyllangin Wei et al. (2004) 240.0432; 133.0658 35 8.379 401.1687 [M+H] 400.1617 151.0729 4 C23H28O6 (Iso)lintetralin (Chang et al., 2003; Huang et al., 1992) 36 8.577 333.2046 [M+H] 332.1976 8 C13H16O10 β-glucogallin Subeki et al. (2005) 37 8.577 329.1928 [M+H] 328.1858 247.1323 8 C14H16O9 Bergenin Tanaka and Matsunaga (1988) 38 8.637 455.2032 [M+Na] 432.2140 332.1731; 231.1030 4 C24H32O7 Niranthin (Chang et al., 2003; Van Thanh et al., 2014) (continued on next page) 4 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Table 1 (continued ) No tR Observed m/z Monoisotopic MS/MS Fragments Accuracy Molecular Compound name Ref. [min] [Adduct(s)] mass (mDa) Formular 39 8.863 355.1168 [M+H] 354.1098 135.0441; 231.0875 2 C20H18O6 Hinokinin Huang et al. (1992) 40 8.863 415.2115 [M+H] 414.2045 4 C29H50O Amarosterol B Ahmad and Alam (2003) 41 9.062 437.1590 [M+H] 414.1698 4 C29H50O β-sitosterol Hu et al. (2014) 42 9.197 439.2022 [M+Na] 416.2130 355.1415; 4 C24H32O6 Phyltetralin Fang et al. (2008) 242.8953; 130.0669 43 9.429 441.2251 [M+Na] 418.2359 151.0728; 2 C24H34O6 Phyllanthin Wei et al. (2004) 56 anf 57 have been 411.1771; 355.1912 eliminated 44 9.572 529.2114 [M+Na] 506.2222 238.1266; 3 C26H34O10 Phyllanembloid B Lv et al. (2015) 117.0690; 379.2043 45 9.679 453.1893 [M+Na] 430.2001 261.1125; 115.0529 2 C24H30O7 Hypophyllanthin Chang et al. (2003) 46 9.748 201.0908 [M+H] 200.0838 2 C12H24O2 Lauric acid Islam et al. (2022) 47 9.810 423.1803 [M+Na] 400.1911 295.1796; 100.1105 4 C22H24O7 Urinaligran Chang et al. (2003) 48 9.849 293.2107 [M+H] 292.2037 277.2166 8 C13H8O8 Phyllanthusiin E Wu et al. (2012) 49 9.931 425.1952 [M+Na] 402.2060 135.0412 2 C23H30O6 5-Demethoxyniranthin Chang et al. (2003) 50 10.306 407.1474 [M+Na] 384.1582 231.1016 4 C22H24O6 Urinatetralin Chang et al. (2003) 51 10.743 297.2480 [M+H] 296.2362 183.1379; 100.1120 4 C20H24O2 Phyllane B Duong et al. (2017) 52 11.554 323.1969 [M+Na] 300.2077 279.2323 4 C20H28O2 Spruceanol Hu et al. (2014) 53 12.228 353.2673 [M+Na] 330.2781 195.1227 4 C15H22O8 3,4-dimethoxy benzyl alcohol-7-O- Yu et al. (2016) β-glucopyranoside 54 12.228 452.3978 [M + 434.3640 313.2724 9 C22H26O9 Phyllaemblic acid methyl ester Liu et al. (2009) NH4] 55 12.228 496.4214 [M + 478.3876 10 C26H38O8 19-Hydroxyspruceanol-19-O-β-D- Lan et al. (2010) NH4] glucopyranoside Fig. 2. Results from metabolomic analysis (LC-MS) from eight different Phyllanthus species. [A] Volcano plot of molecular features present in PU as compared to non- PU. Buckets with a high fold change >2 and p-value ≤0.05 in PU and non-PU samples are shown in red and blue colours respectively; [B] PLS-DA score plot showing the discrimination of PU samples from the non-PU samples. Classification model achieved with 3 components and had an accuracy of 99% with R2 = 0.9639 and Q2 = 0.7942. [C] VIP scores obtained from PLS-DA showing the most important features that contribute to the classification of PU samples different from the non- PU samples. 0.9980). The respective quantitative models described for the relation- 2.4. Validated UPLC method ship between the concentration and response and their consequent use in content assays are shown in Table 2. With recoveries ranging between Existing HPLC methods for the quality assessment of PU and other 98.5 and 101.7%, the use of these quantitative models was also shown to Phyllanthus species mostly target the estimation of selected lignans be accurate from the recovery studies. Additionally, the RFs and the peak (Murugaiyah and Chan, 2007; Shanker et al., 2011). However, due to area estimates were demonstrated to be precise (RSD <2%) from both the presence of other classes of bioactive compounds in the herbal ma- repeatability and intermediate precision tests. The method was then terial, developing a more specific UPLC method focused on the selection shown to be robust when deliberate changes to the ambient temperature of representative analytical targets for these groups is needed. Thus, (at 25 &.30 ◦C), developing chamber (10 × 10 & 20 × 10 cm), appli- geraniin, which is reportedly the most abundant bioactive hydrolysable cation instrument (instruments 1 & 2) and saturation times (20, 25 & 30 tannin in the Phyllanthus genus (Agyare et al., 2011), rutin and iso- min) were made. Finally, the fingerprint and the content of the marker quercitrin, which are also bioactive flavonoids and phyllanthin, another compounds were established to be stable over 48 h for the analytes’ prominent bioactive lignan, were selected (Fig. S2). A wavelength of λ = solutions, over 60 min for the underivatized and derivatized spots on the 270 nm was observed to be optimum in detecting the peaks of tannins, plate pre-and post-profile development. The method could then be said flavonoids and lignans in the chromatogram, thus providing a repre- to be fit for purpose for both identification and content assessment of sentative fingerprint of the plant’s major phytochemistry. Fig. 4 shows a PU. typical UPLC chromatogram developed for the quality assessment of PU. This UPLC method was similarly validated following the recom- mendations in the ICH Q2(R1) guidelines (ICH, 2005) (Table 3). The method was initially investigated for specificity, where the presence of 5 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Fig. 3. Typical thin layer chromatograms of Phyllanthus urinaria aerial parts (herein referred to as PUAP) under different modes of detection with other Phyllanthus species with their fingerprint analysis. A: Replicate HPTLC profiles of PUAP extracts showing repeatability of the fingerprint. B: TLC identification of analytical marker compounds in PUAP extract by use of reference standards, rutin, isoquercitrin, and gallic acid. C: 2D development of PUAP profile using similar conditions in both cases. This forms part of validation investigations. D: Simultaneous development of HPTLC profiles of PUAP and the reference compound, phyllanthin using the mobile system (toluene: ethylacetate: formic acid (69:30:1, v/v/v). E: Representative profiles of different Phyllanthus species (1–3: P. fraternus; 4–6: P. niruri; 7–9: P. emblica; 10–12: P. urinaria; 13–15: P. amarus; 16–18: P. muellerianus; and 19–21: unknown Phyllanthus sp. A, B, and C) used in the test for specificity. F: Score plot of fingerprints of different Phyllanthus species performed by rTLC web application (Fichou et al., 2016) using data from grayscale video-densitometric like channel. Observed differences in the fingerprints for different species, especially for P. urinaria. Differences seen in their respective scores. the marker compounds was confirmed in the PU extract from LC-MS/MS 2.5. Optimization of sample extraction conditions analysis. Also, the retention times, UV spectra and m/z values for the respective peaks in the chromatograms from the UPLC and MS analysis To ensure the adequate recovery of analytes during the sample were found to be consistent with analytical data of the corresponding extraction for quantitative assessment of PU, the extraction conditions reference standards. When the UV and mass spectra were again assessed for the herbal material were investigated and optimized according to the for peak purity analysis at the respective retention times, it was found BBD model. The procedure, as described previously (Orman et al., 2023) that the spectra were comparable at the beginning, middle, and end of involved the investigation of the effects of parameters such as extraction the peaks. Linearity (r2 > 0.99) was established within the concentration time (A), sample-to-solvent ratio (B), number of extractions (C) and ranges for the marker compounds (Table 3) and their respective peak method of extraction (D) on quality attributes including extraction yield, areas, thus enabling the quantitative assay of the compounds, a process and concentrations of rutin (% wt/wt), isoquercitrin (% wt/wt), geraniin which was also shown to be accurate, with a recovery ranging between (% wt/wt) and phyllanthin (% wt/wt) (Table S2). Statistical evaluation 100.2 and 101.3%. In the test for precision (repeatability and interme- of the effects with the input variables with ANOVA as well as modelling diate precision), the RSD for the peak areas ranged between 0.17 and with multiple linear regression, resulted in a combination of linear and 0.61%. This showed that the peak area calculations were precise and second-order polynomial models (Eqns (2)–(6)) which adequately esti- reliable for quantitative purposes. With deliberate changes to the mates the contribution of the input variables on each of the outcomes. respective column temperature and the flow rate of the method, the The results from the ANOVA are also shown in Table 4. With the models RSDs were <2.0%, demonstrating a robust method with respect to having p < 0.05 and large F-values (range: 5.71–7.87), the predictive variable conditions. Finally, PU test solution used for analysis was models were considered to be too significant to have resulted from noise shown to be stable >48 h, as the change in response over the period did signals (Orman et al., 2023). not change significantly. The outcomes from the validation of the UPLC method are summarized in Table 3. Yield (%w /w)= 9.60 + 0.44A − 0.63B+ 3.16C + 0.23D1 − 0.70D2 (2) 6 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Sqrt(Rutin) (%w /w)= 0.16 + 0.009A+ 0.007B+ 0.03C − 0.02D1 + 0.01D2 + 0.0007AB − 0.01AC − 0.003AD1 + 0.008AD2 + 0.02BC − 0.02BD1 + 0.02BD2 − 0.006CD1 + 0.004CD − 0.01A2 + 0.0006B22 − 0.03C2 (3) ISQ (%w /w)= 0.04 + 0.002A − 0.003B+ 0.01C − 0.002D1 + 0.002D2 + 0.0008AB − 0.003AC − 0.001AD1 + 0.003AD2 + 0.009BC − 0.008BD1 + 0.008BD2 − 0.003CD + 0.001CD − 0.002A21 2 + 0.001B2 − 0.008C2 (4) respectively, when sonication was held constant. The extraction parameters, C and D were considered to be the keys as Geraniin (%w /w)= − 0.86 + 0.08A+ 0.09B+ 0.20C − 0.24D1 + 0.14D2 their effects were significant on almost all the quality attributes or (5) outcomes. In effect, the extraction yield, and the concentrations of almost all the marker compounds were much impacted by the number of times the extraction was carried out as well as the method used: As the log10Phyllanthin(%w/w)= − 1.50+0.08A+0.05B+0.22C − 0.30D1+0.13D2 number of extractions increase, the concentrations and yield also in- (6) crease and vice versa. Also, when emphasis was placed on the concen- trations of geraniin and phyllanthin (considered to be representative Where the coefficient estimates of the experimental factors A, B, and C markers for two important classes of compounds in the plants (hydro- represent the expected changes in responses per unit changes in factor lysable tannins and lignans), using mechanical shaking approach (D1) value when all remaining factors are held constant. In the same stead, D1 produced better outcomes than cold maceration (D2) and sonication and D2 indicate mechanical shaking and cold maceration methods (reference method). The model graphs describing the relationship Table 2 Summary of validation data for HPTLC analysis of Phyllanthus urinaria aerial parts for both qualitative and quantitative purposes. Rutin Isoquercitrin Gallic acid Phyllanthinα Identification Retardation factor (% RSD) 0.35 (2.09) 0.64 (0.98) 0.96 (0.92) 0.44 (1.50) System suitability Relative retardation (Rref) (% RSD)* 1.00 (0.00) 1.83 (1.73) 2.74 (1.99) 1.26 (2.68) Specificity Marker spots confirmed in PU profile. PU profile is different from other Phyllanthus species Accuracy Percentage Recovery (mean ± SD)a 98.5 ± 3 101.7 ± 4 100.1 ± 4 99.8 ± 4 Linearity Range (ng/band) 100–5000 100–5000 500–5000 10–500 Regression equation y = 419.2 + 5.152x - y = 636.7 + 8.149x - y = 0.3944x - y = 174.2 + 11.02x - 0.0005248x2 0.0007675x2 6.096 0.002977x2 (Adjusted) Correlation coefficient (R2) 0.9940 0.9976 0.9992 0.9980 Precision Instrumental precision (% RSD for RF, Peak Area) 1.50, 1.70 0.65, 1.90 0.43, 1.03 0.92, 1.94 Repeatability 1b (% RSD) 1.55 1.55 1.18 1.19 Repeatability 2c (% RSD) 0.90 1.64 1.11 1.47 Intermediate precision 1d (% RSD) 1.95 0.65 1.31 1.68 Intermediate precision 2e (% RSD) 1.45 1.62 1.31 1.68 Robustness Δ saturation time (% RSD for RF, Peak Area) 1.82, 1.61 1.14, 1.33 0.70, 1.44 1.77, 1.85 Δ development temperature (% RSD for RF, Peak 1.65, 1.60 0.90, 1.34 0.30, 1.22 1.37, 1.14 Area) Δ developing chamber (% RSD for RF, Peak Area) 1.64, 1.80 0.90, 0.42 0.60, 1.34 1.31, 1.56 Δ application instrument (% RSD for RF, Peak 1.63, 1.23 0.91, 1.07 0.60, 1.34 0.65, 1.86 Area) Δ mode of application (% RSD for RF, Peak Area) 1.55, 1.12 1.33, 2.37 0.30, 2.60 1.29, 1.30 Stability Analyte solution (%RSD) 1.56 2.96 2.91 2.76 During development Marker compounds are stable throughout 2D development. No artefacts detected. Analytes on plate No difference in densitometric profiles within 2 h of sample stay on plate before development Derivatized analytes No difference in densitometric profiles of spots within 1 h after derivatization. SD – Standard deviation; RSD – Relative standard deviation; α Mobile system for developed for the assessment of this marker (toluene: ethylacetate: formic acid (69:30:1, v/v/v)) was different from the other three. *Relative retardation (Rref) was calculated as a ratio of RF of any marker compound to RF of isoorientin (adopted as an internal reference marker) in every developed fingerprint. a Average of triplicate determinations from three different concentrations (50, 100, 150% of the respective working concentrations) (n = 9) on the same plate. b Repeatability for qualitative purpose, average of RF determinations from three different plates on the same days and 4 tracks per plate (n = 12). c Repeatability for quantitative purpose, average of triplicate determinations of three concentration levels on same plate at the same day (n = 9). d Intermediate precision of the fingerprint, average of four developments on a plate for three days (n = 12). e Intermediate precision for quantitative purpose, average of triplicate developments of marker compounds on the same plate in three different days. 7 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 the following extraction conditions as predicted from the design space modelled with a desirability of 0.637: PU sample extracted in a 2% sample-to-solvent ratio each for 30 min and repeated three times using mechanical shaking method. The predictive ability of the design space was tested, and the responses were found to be within ±5% deviation (Fig. S4). 2.6. QAMS approach to quality assessment of Phyllanthus urinaria samples In resource-challenged settings, the lack of reference standards for markers described for a developed method could serve as a barrier to its adoption for quality control purposes (Yan et al., 2015). In such in- stances, the QAMS approach has been proposed, in which case one of the markers is used as an internal reference marker with its content deter- mined by external calibration method (ECM), and the quantitative as- says of other marker compounds (irrespective of their number) carried out using their corresponding RCFs as determined with reference to the internal reference from a method development process (Yan et al., Fig. 4. UPLC chromatograms of acetone: water (7 : 3 v/v) extract of Phyllanthus 2015). The quantitation of geraniin, isoquercitrin, and phyllanthin were urinaria aerial parts (PU) (top) and the analytical markers considered for assay (bottom). Detection at λ 270 nm [1] Geraniin (t 6.28 min; m/z 975.0 similarly carried out by determining their RCFs using rutin as the in-= R = = [M+Na]+); [2] rutin (tR = 9.61 min; m/z 633.1 [M Na]+); [3] isoquercitrin ternal reference standard. The estimation of the RCFs was then subjected = + (t = 9.91 min; m/z = 487.0 [M+Na]+); [4] phyllanthin (t = 18.08 min; m/z = to validation to assess precision, accuracy, and robustness of the QAMS R R 441.1 [M+Na]+). procedure. The results of these investigations are summarized in Table 5. between the extraction conditions and the outcomes are shown in the The validation outcomes show that the approach could provide a Supplementary Data (Fig. S3). These observations were confirmed by reliable alternative to the use of reference standards for all the four the predictive models, where the coefficients for the C and D variables markers for PU. Therefore, by determining the content of rutin from the were comparatively bigger than the coefficients for the other variables use of its reference compound in an ECM approach, the contents of the (with the exception of the constants). Between the two variables, the other markers could be determined by using their respective RCFs and coefficients for C were comparatively bigger than that of D, and this the peak areas as determined from the chromatogram of the sample. showed that the effect of changing the number of extractions was carried out is a bit greater than the effect of changing the method of extraction. 2.7. Effects of spatio-temporal variations on quality of Phyllanthus Additionally, the results of the experiments indicated that extraction urinaria aerial parts and specification setting time and ratio of sample-to-solvent were not very critical. As per these observations, it may be proposed that instead of extracting a sample for The effects of spatio-temporal variations on the content of the marker a longer time (for example, 1–5 h), it may be preferable to rather extract compounds in PU were evaluated using a batch of 36 samples collected the sample multiple times within the same duration. This assertion could from five different regions in Ghana (Ashanti, Central, Eastern, Volta also hold for the preparation of extracts for herbal formulations. and Western) which were distributed over 3 agroecological zones For the purpose of this study, the optimization led to the adoption of (coastal savannah, deciduous and evergreen). The samples were also Table 3 Summary of validation results for the UPLC method for identity testing and assay of Phyllanthus urinaria aerial parts extract. Parameter Geraniin Rutin Isoquercitrin Phyllanthin Identification Retention time (% RSD) 6.28 (0.15) 9.61 (0.06) 9.91 (0.11) 18.08 (0.01) System suitability Relative retention time (RRT)a (% RSD) 0.65 (0.13) 1.00 (0.00) 1.03 (0.09) 1.88 (0.06) Specificity Marker peaks confirmed in PU from PDA and QDa analysis of chromatograms. Accuracy Percentage Recovery (mean ± SD)b 100.4 ± 1.2 101.3 ± 1.0 100.5 ± 1.5 100.2 ± 1.7 Linearity Range (μg/mL) 10–100 5–30 20–50 5–20 Regression equation y = 7588x – 14690 y = 7355x + 858.4 y = 9009x – 14040 y = 2555x – 93.67 Correlation coefficient (R2) 0.9960 0.9999 0.9994 0.9946 Precision Repeatability (% RSD) 0.61 0.47 0.52 0.36 (n = 6) Intermediate Precision (% RSD)c 0.30 0.46 0.17 0.27 Robustness Δ column temperature (% RSD) 1.86 0.60 0.52 0.85 Δ flow rate (% RSD) 1.59 0.36 0.31 0.76 Stability of analyte solution (% RSD) 1.24 0.69 0.66 1.49 SD – Standard deviation; RSD – Relative standard deviation. a Relative retention time (RRT) was calculated as a ratio of retention time of any other marker compound to that of geraniin (adopted as an internal reference marker). b Average of triplicate determinations from three different concentrations (that is, 50%, 100% and 150% of working concentrations) (n = 9). c Average of triplicate determinations in three different days. 8 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Table 4 Summary of ANOVA results from Box-Behnken experimental model for optimization of sample extraction conditions. Parameter Yield Conc. of rutin(% wt/wt) Conc. of ISQ(% wt/wt) Conc. of GER(% wt/wt) Conc. of PHY(% wt/wt) F-value p-value F-value p-value F-value p-value F-value p-value F-value p-value Model 7.65 < 0.0001 5.71 <0.0001 7.3 <0.0001 7.01 <0.0001 7.87 <0.0001 A-Extraction time 0.67 0.4168 3.12 0.0885 1.39 0.2494 1.94 0.1714 1.95 0.1709 B-Extraction ratio 1.36 0.2512 1.82 0.1889 2.93 0.0987 2.39 0.1306 0.5674 0.4558 C–No. of extractions 34.58 < 0.0001 41.91 < 0.0001 62.26 < 0.0001 12.7 0.001 13.44 0.0007 D-Extraction type 0.8331 0.4423 5.03 0.0139 1.14 0.3355 9 0.0006 11.7 0.0001 AB 0.0011 0.917 0.1533 0.6985 AC 1.76 0.1952 2.5 0.1257 AD 0.6088 0.5513 0.7586 0.478 BC 11.65 0.002 16.03 0.0004 BD 6.07 0.0067 9.14 0.0009 CD 0.3663 0.6967 0.7613 0.4768 A2 1.79 0.1924 0.4676 0.4999 B2 0.007 0.9342 0.3406 0.5643 C2 11.31 0.0023 14.26 0.0008 p-values in bold formatting were considered significant. collected over the two major seasons (rainy and dry) in the country. This therefore calls for stringent quality control measures to ensure the Among the markers assessed, geraniin recorded the highest con- quality of starting materials (in this case, PU) used in the formulation of centration in most of the samples while the least was isoquercitrin. The herbal medicinal products as for example, used in Ayuverdic medicine. contents of the markers as determined from the samples collected For that reason, there is a need to adhere to established acceptance ranged as follows: geraniin = 0.42–3.22 % wt/wt; rutin = 0.12–0.50 % criteria for the control of these markers. As part of the study, we wt/wt; isoquercitrin = 0.02–0.10% wt/wt; and phyllanthin = 0.07–1.05 considered setting specifications for these markers by first considering % wt/wt (Fig. 5A and B). The predominance of geraniin in PU (as seen in the distribution of the individual contents in the samples. Irrespective of the chromatogram – Fig. 4), and confirmed with its high content is the spatio-temporal effects on the contents, normality tests (including consistent with the results from the study on P. muellerianus by Agyare Shapiro-Wilk, Kolmogorov-Smirnov and Anderson-Darling tests) et al., where together with furosin, they were shown to possess signifi- showed that the data were significantly drawn from normally distrib- cant wound healing effects (Agyare et al., 2011). uted populations. Thus, assuming a maximum of 10% substandard level When the contents of the marker compounds were further investi- for each marker content, the acceptance limits for the marker com- gated, some variations were seen, and this could partly be attributed to pounds were established as follows: geraniin ≥0.50% (wt/wt); rutin spatio-temporal variations. For instance, samples originating from the ≥0.14% (wt/wt); isoquercitrin ≥0.03% (wt/wt); and phyllanthin evergreen zone contained significantly lower rutin content than those ≥0.10% (wt/wt), related to the dried mass of the plant material, from coastal savannah and deciduous zones (p = 0.0274) (Fig. 5C), for respectively. Considering for example the use of the plant as an which the rutin content was comparable. In terms of the regional dis- anthelmintic, the acceptance limit estimated for geraniin far exceeds the tribution, it was also evident that the contents of geraniin (p = 0.0299) concentration required to exhibit inhibitory effects against C. elegans in a and rutin (p = 0.0157) varied greatly from one region to the other mortality assay (Jato et al., 2021). Thus, it could be assumed that the (Fig. 5D). It was further observed that the contents of all four markers estimated limits may be useful to control the quality of the plant for were greatly affected by the month of sample collection (p < 0.05) medicinal use. Applying these limits to the PU plant materials collected, (Fig. 5E). Samples generally collected in October recorded the least of one sample (ID: PU009) could be said to be of a poor quality because the the contents for each marker (Fig. S5). Additionally, the content of concentrations of all four markers were lower than limits proposed. Few phyllanthin was observed to be higher in samples collected later in the of them had the concentrations of one or two of the markers below the year than those collected earlier. The final factor to be considered was estimated limits. Generally, the compliance rate ranged between 89 and the potential effect of seasonal variations (dry and rainy seasons). The 94%. data indicated that the contents of three of the markers, including ger- aniin, rutin and isoquercitrin across the two seasons were comparable (p > 0.05) (Fig. 5F). For phyllanthin however, the content was significantly 2.8. Purity assessment of the Phyllanthus urinaria samples higher in the dry season than in the rainy season (p = 0.0004). The differences in the contents of the marker compounds in PU may likely By way of testing for the purity of the plant materials, a pooled affect the therapeutic potential of the same plant from different places. sample from the 36 samples was screened for the presence of pesticides residues. It has become necessary to consider the pesticides residues Table 5 Summary of results of the QAMS approach for Phyllanthus urinaria aerial parts. Geraniin Rutin Isoquercitrin Phyllanthin RCF 0.9489 – 1.1205 0.3359 Precision Same day (% RSD) 0.34 – 0.39 0.29 Different days (% RSD) 1.02 – 0.17 0.26 Robustness Δ column temperature (% RSD) 0.46 – 0.50 0.29 Δ flow rate (% RSD) 2.03 – 0.41 0.83 Accuracy (%), % RSD 100.1, 0.19 – 93.4, 3.58 95.7, 0.04 QAMS: Quantitative assessment of multicomponents by single markers; RCF: Relative correction factor; RSD: Relative standard deviation; RCF: relative correction factors calculated relative to rutin; RSD: relative standard deviation; ECM: external calibration method. 9 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Fig. 5. Batch analysis of 36 samples of Phyllanthus urinaria aerial part materials from different places and times of collection in Ghana. A: Box plot of the marker contents. Data represented as mean with interquartile range. B: Circular stacked plot of the contents of the marker compounds in each of the 36 PU samples. Each content is a representation of the mean from triplicate determinations. C: One-way ANOVA to evaluate the effect of agroecological zones on contents of markers. D: One-way ANOVA to evaluate the effect of regional origin of sample on contents of markers; E: One-way ANOVA to evaluate the effect of different months of collection; F: One-way ANOVA to evaluate the effect of different climatic season of sampling on contents of markers. screening because of recent reports of the detection of several pesticides material adheres to the restrictions on particularly hazardous pesticides in food and plant-based medicinal products beyond their maximum while maintaining appropriate levels of other less toxic pesticides. residue limits (MRLs) (Agbeve et al., 2013, 2014; Donkor et al., 2016; Analysis of the pooled PU sample revealed the presence of chlor- Opuni et al., 2021). The public therefore gets exposed to these harmful pyrifos (0.100 ± 0.000 mg/kg), and folpet (sum of folpet and phthali- and potentially harmful chemicals at levels that could be of concern mide, expressed as folpet, 0.221 ± 0.001 mg/kg) which may be because of the current inadequate regulatory framework regarding the exceeding the MRLs of non-dried, fresh herbs (European Parliament, rational use of pesticides. For safety reasons, it is crucial that the plant 2005). The rest of the 349-pesticides panel were either absent or below 10 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 their detection limits. Chlorpyrifos, a moderately toxic pesticide, for Where Y represents the measured response associated with each factor which the use has been linked to neurodevelopmental problems, a high level combination, βo is a constant, βi is the slope or linear effect of the risk of developing cancer, and even mortality (Ubaid ur Rahman et al., input factor xi, βii is the quadratic effect of input factor xi, and βij is the 2021), was present in a considerable residual level that is about 10 times linear-by-linear interaction effect between the input factor xi and xj. the MRL of 0.01 mg/kg. This situation is deemed very disturbing considering the fact that PU is widely used for different medicinal pur- poses in Ghana. On the other hand, folpet is considered to be less 4.4. Extract preparation for subsequent analytical investigations harmful (World Health Organization WHO, 2020b). Taking a cue from the results of the pesticides analysis, the regulatory structure currently The plant material was cleaned, air-dried at room temperature for in place to regulate their use needs to be thoroughly reviewed. three weeks, and pulverized in a mortar after freezing with liquid ni- trogen. An amount of 0.5 g of the pulverized plant material was 3. Conclusions extracted 3 times, each for 30 min with 25 mL of acetone: water (7:3, v/ v) mixture by use of a mechanical shaker (GFL 3018 model, Germany). The current study on the analytical investigations of Phyllanthus The suspensions were centrifuged for 10 min at 3000×g and the clear urinaria aerial parts provides validated analytical methods for quality supernatant was evaporated and dried in-vacuo. The residue was resus- control, with specifications and a draft monograph. The draft mono- pended in a small amount of water (Aqua Millipore) and lyophilized. graph has been written according to the format of the European Phar- The lyophilized samples were stored at – 20 ◦C prior to analysis. For macopoeia and is displayed as Monograph 1 in the Additional Data File analysis, 10 mg/mL methanol (MeOH) solution of the extract was pre- for future consideration and discussion by regulatory officials and in- pared, centrifuged at 6020×g (Mikro 120, Hettich Zentrifugen) for 5 min dustry participants in the herbal medicine sector. and 2 mL of the supernatant was transferred into HPLC vials. 4. Experimental 4.5. LC-MS/MS characterization of P. urinaria aerial part extract 4.1. Solvents, reagents, and reference standards The phytochemical characterization of the acetone: water extract (7:3) of P. urinaria was performed using an LC-MS/MS analytical All solvents and reagents used were of analytical quality and were approach and instrumentation system described previously (Orman obtained from VWR International (Darmstadt). The purified water was et al., 2023). The elution system comprised of a binary solvent compo- made in-house by Millipore simplicity 185 system (Merck, Darmstadt). sition with solvent A: water with 0.1% formic acid, and solvent B: 2-Aminoethyl-diphenylborinate (natural product reagent, NP) and acetonitrile with 0.1% formic acid. Eluting was carried out at 0.4 polyethylene glycol 400 (PEG) were from Merck (Darmstadt). The mL/min, using the following gradient system: t0min: 13% B, t5min: 36% B, reference standards including rutin (purity >95%), isoquercitrin (purity t8min: 100% B, t15min: 100% B, t15.1min: 5% B, t20min: 5% B. The injection >95%), gallic acid (purity >95%), and phyllanthin (purity >95%), were volume was 2 μL. Metlin (Smith et al., 2005), MassBank (Horai et al., obtained from Sigma–Aldrich (Deisenhofen, Germany) and Phytolab 2010), MMCD (Cui et al., 2008) and KNApSAcK (Afendi et al., 2012) (Vestensbergsgreuth). Geraniin (purity >98%) was purchased from mass spectral databases as well as Reaxys enabled the identification of Atkin Chemicals, Inc., China. the eluted compounds. A typical chromatogram is displayed in Fig. 1 and compounds identified are summarized in Table 1. 4.2. Plant material 4.6. Phytochemical differences between P. urinaria and closely related The leaves of PU and other related species, including P. amarus, P. Phyllanthus species fraternus, P. nuriri, P. muellerianus were collected from different parts of Ghana between March 2020 and December 2021. Commercial extracts The composition of PU was comparatively studied with that of of P. emblica were donated by an anonymized herbal manufacturer in authenticated closely related Phyllanthus species including P. fraternus, Germany. The collection sites of the samples are indicated in the Sup- P. niruri, P. amarus, P. muellerianus, and P emblica in an untargeted LC-MS plementary Data (Table S1). The plants were identified and authenti- metabolomic analysis using an analytical procedure previously cated by botanists, Dr. George Henry Sam (Department of Herbal described in literature (Orman et al., 2023). After pre-preprocessing the Medicine, KNUST, Kumasi, Ghana) and Mr. Tonny Asafo Agyei (Center mass spectral data, the resulting bucket table consisted of 378 buckets for Plant Medicine Research, Mampong, Ghana). Voucher specimens are which was subsequently used as data matrix for the statistical modelling deposited in the herbarium of the University of Münster, Institute of using the MetaboAnalyst 5.0 server (https://www.metaboanalyst.ca). Pharmaceutical Biology and Phytochemistry, Germany. The respective Prior to the modelling, the data were further pre-processed by log voucher identification numbers (#IPBP728 to 812) are displayed in transformation and pareto scaling. Table S1 of the Supplementary Data File. 4.7. HPTLC analysis of PU extract 4.3. Optimization of sample extraction procedure for chromatographic analysis The HPTLC procedure used for both qualitative and quantitative analyses of PU was developed with conditions previously described in The procedure used to extract the Phyllanthus samples for analytical literature (Orman et al., 2023). Where applicable, the different condi- investigations was optimized using the Box-Behnken Design (BBD) tions are highlighted. model from Design Expert software (version 11, Stat Ease Inc., 2017) in Standard solutions: 1 mg/mL stock solution of each of the reference an experimental design approach reported in literature (Orman et al., compounds, rutin, isoquercitrin, phyllanthin and gallic acid, was sepa- 2023). The quality attributes monitored were extraction yield (% ◦rately prepared in MeOH and stored at - 20 C for use. The stock solu- wt/wt), and concentrations (% wt/wt) of the marker compounds, ger- tions were then serially diluted with MeOH to obtain the following aniin, rutin, isoquercitrin, and phyllanthin. A second-order polynomial working concentrations for validation and further analysis: rutin and function (Eqn. (1)) was used to describe the interaction between the isoquercitrin (10–500 μg/mL equivalent to 100–5000 ng/band); phyl- extraction parameters and the quality attributes. lanthin (1–50 μg/mL equivalent to 10–500 ng/band); and gallic acid ∑ ∑ ∑ 2 (50–500 μg/mL equivalent to 500–5000 ng/band). Y = βo + βixi + βiixii + βijxixj + ε (1) Chromatographic conditions: The analysis was performed by use of a 11 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 CAMAG HPTLC system (Muttenz, Switzerland). This set up comprised an LC-MS/MS and GC-MS/MS techniques. Depending on the polarities of the Automatic TLC sampler ATS 4, a Twin Trough Chamber with steel lid pesticides targeted, two sample preparation approaches were used, (20 × 10 cm and 10 × 10 cm), TLC scanner 3 in combination with including the modified Quick, Easy, Cheap, Effective, Rugged and Safe winCATS software (version: 1.April 4, 6337) and TLC Visualizer 2 (QuEChERS) method (European Committee for Standardization, 2018) controlled by visionCATS software (version: 3.0). Test solutions were and the Quick Polar Pesticide (QuPPe) method (Anastassiades et al., applied at 10 μL as 6 mm bands using a 25 μL Hamilton syringe 2020). The details of the procedures carried out are as described previ- (Bonaduz) on silica gel 60 F254 coated HPTLC plates (10 × 10 cm and 20 ously in literature (Orman et al., 2023). The list of pesticides screened × 10 cm; Merck, Germany). A saturation time of 25 min at room tem- with their corresponding analytical data are reproduced in Table S3. perature was ensured. Two different mobile phase systems were used: í. toluene: ethylacetate: formic acid (69:30:1, v/v/v), which has been 4.11. Statistical analysis optimized for the detection and quantitation of phyllanthin; ii. ethyl- acetate: water: formic acid (75:15:10, v/v/v) has been optimized for the To analyse the data, the study used both univariate and multivariate detection and quantitation of rutin, isoquercitrin, and gallic acid. data analysis techniques, determining the mean, standard deviation, and Detection: λ = 254 nm, and 366 nm pre- and post-derivatization with relative standard deviation. Within method validation and batch PU Natural Product Reagent and polyethylene glycol (NP-PEG). Densito- samples analyses, statistical tests such as the t-test, and ANOVA with metric scanning of the underivatized plate was performed at λ = 254 nm, post-hoc tests were used to compare analytical results. Multiple linear and derivatized plates at λ = 360 nm in the absorbance mode. regression analysis and principal component analysis (PCA) were used Validation of the HPTLC Method (ICH Q2(R1)): The HPTLC method as to optimize the sample extraction procedure and assess marker content developed for both qualitative and quantitative purposes, was validated in batch samples, respectively. To authenticate PU samples, the LC-MS according to International Council of Harmonization (ICH) Q2(R1) metabolomics study in several Phyllanthus species employed tech- guidelines (ICH, 2005) using procedures previously described in litera- niques such as volcano plot analysis, clustered heatmap analysis, and ture (Orman et al., 2023). The following parameters were assessed: Partial Least-Squares Discriminant Analysis (PLS-DA). Statistical sig- specificity, accuracy, linearity, precision, reproducibility, and stability nificance was assessed using p < 0.05 (*), p < 0.01 (**), and p < 0.001 of analytes. The outcomes of the validation are summarized in Table 2. (***) 4.8. UPLC analysis of PU extract Ethical approval Standard solutions: Stock solutions of the reference compounds (each This article does not contain studies with human participants per- 1 mg/mL in MeOH) were prepared. The working standard solutions formed by any of the authors. were prepared by diluting the stock standard solutions with MeOH to the following series of concentrations: geraniin (10–100 μg/mL), rutin (5–30 μg/mL), isoquercitrin (20–50 μg/mL) and phyllanthin (5–20 μg/ Funding mL). Test solutions:10 mg/mL in MeOH of the lyophilized extracts. The project was funded by the Deutsche Forschungsgemeinschaft UPLC analysis was performed with an Acquity UPLC™ system (DFG, German Research Foundation) – Project 423277515 (HE1642/12- (Waters) equipped with PDA eλ detector (λ = 210–400 nm), QDa de- 1). tector (ESI, positive and negative modes, single quadrupole, 100–1250 Da), RP-18 stationary phase (Acquity UPLC HSS T3 column; 1.8 μm, 2.1 Contributors statement × 100 mm), Autosampler, and binary solvent manager. The mobile phase was made of solvent A (water 0.1% formic acid) and solvent B EO, EOB and AH conceptualized the study. EO, IK carried out the + (acetonitrile + 0.1% formic acid). The gradient elution system employed experimental work and evaluated the data; EO wrote the manuscript was as follows: t0min: 2% B, t2min: 8% B, t11min: 18% B, t15min: 35% B, draft; JJ, VS, EOB, AH revised the MS; VS, EOB, AH, CA applied for the t20min: 100% B, t22min: 100% B, t24min: 2% B, t25min: 2% B. A flow rate of research grants, SOB, SAN commented on the MS. The study was ◦ 0.5 mL/min, injection volume of 2 μL, and column temperature of 40 C designed by EO, EOB and AH. were adopted. Data acquisition and processing were performed using Waters Empower 3 (Waters, Milford, Milwaukee, USA). Declaration of competing interest Validation of the UPLC Method (ICH Q2(R1)): Similarly, the validation of the UPLC method for PU was carried out in accordance with ICH Q2 The authors declare that they have no known competing financial (R1) (ICH, 2005) guidelines, using procedures previously described in interests or personal relationships that could have appeared to influence literature (Orman et al., 2023). The parameters investigated included the work reported in this paper. specificity, accuracy, linearity, precision, reproducibility, and stability of analytes. Data availability 4.9. Quantitative assessment of multicomponents by single markers Data will be made available on request. (QAMS) approach to content assay in Phyllanthus urinaria Acknowledgements The QAMS approach to content assay was investigated by deter- mining the relative correction factors (RCFs) of geraniin, isoquercitrin, We are grateful to Ben Adzelekey, Gladys Schwinger, Clifford Asare, and phyllanthin from their respective peak areas, in relation to rutin Alfred Ofori Agyemang, Kwadwo Kwegir, John Botwe, Rahman Tikrom, used as an internal reference marker (Zhu et al., 2017). The procedures Dominic Mensah, Stephen Amankwah, Samuel Kyei and Abdul Nasir for adopted are previously reported in literature (Orman et al., 2023). the collection of the plant samples from across the country. We are also grateful to Dr. George Henry Sam of Department of Herbal Medicine, 4.10. Pesticides residues analysis KNUST, Kumasi, Ghana, and Mr. Tonny Asafo Agyei of Center for Plant Medicine Research, Mampong, Ghana for authenticating the samples Similarly, a pooled PU sample was screened for the presence of con- used in the study. We also acknowledge Dr. Jandirk Sendker for taminants from a panel of 349 pesticides and related contaminants using measuring LC-MS samples. 12 E. Orman et al. P h y t o c h e m i s t r y 215 (2023) 113854 Appendix A. Supplementary data Govindaraghavan, S., Sucher, N.J., 2015. Quality assessment of medicinal herbs and their extracts: criteria and prerequisites for consistent safety and efficacy of herbal medicines. Epilepsy Behav. 52, 363–371. https://doi.org/10.1016/j. Supplementary data to this article can be found online at https://doi. yebeh.2015.03.004. org/10.1016/j.phytochem.2023.113854. Hassarajani, S.A., Mulchandani, N.B., 1990. Securinine type of alkaloids from Phyllanthus niruri. Indian J. Chem. Sect. B-Organic Chem. Incl. Med. 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