Biometals

https://doi.org/10.1007/s10534-019-00230-3 (0123456789().,-volV)( 0123456789().,-volV)

Investigation of total zinc contents and zinc-protein profile in medicinal traditionally used for diabetes treatment

Ammar M. Ebrahim . Ahmed O. Alnajjar . Maged E. Mohammed . Abubakr M. Idris . Mohammed E. A. Mohammed . Bernhard Michalke

Received: 3 November 2019 / Accepted: 29 November 2019 Ó Springer Nature B.V. 2019

Abstract This study aims at the assessment of total summed Zn concentrations from protein fractions zinc contents, water zinc extract contents and zinc- compared to total Zn was 103.0 ± 4.8%. Based on the protein profile in medicinal plants traditionally used FAO/WHO classification, Sesamum indicum, Nigella for diabetes treatment. While zinc-protein profile was sativa, Trigonella Foenum-graecum and Pennisetum screened in samples using the online coupling of glaucum are classified as highest Zn-content. For size exclusion chromatography-inductively coupled protein profile, zinc was quantified in 330–430 and plasma-mass spectrometry (SEC–ICP–MS), total zinc 50–60 kDa fractions of all examined plants while no contents and zinc water extract contents were deter- contents were quantified in the inorganic fraction mined using inductively coupled plasma-optical emis- 0.05–0.4 kDa of all plant species. Also, only three sion spectrometry (ICP-OES). The analysis of two plant species recorded Zn contents in the phytate certified reference materials with similar matrix for fraction (0.9–1.5 kDa fraction). The of the total zinc content revealed recovery values of 97.1% and the colocynthis and 100.5% while the average of recovery of the were with the highest extractable zinc concentration;

A. M. Ebrahim A. M. Idris (&) Á M. E. A. Mohammed Research and Consulting Institute, King Faisal University, Department of Chemistry, College of Science, King Hofuf, Saudi Arabia Khalid University, Abha 61321, Saudi Arabia e-mail: [email protected] A. M. Ebrahim Sudan Atomic Energy Commission, Khartoum, Sudan A. M. Idris Research Center for Advanced Materials Science A. O. Alnajjar (RCAMS), King Khalid University, Department of Chemistry, College of Science, King Faisal P.O. Box 9004, Abha 61413, Saudi Arabia University, Hofuf 31982, Saudi Arabia B. Michalke M. E. Mohammed Research Unit Analytical BioGeoChemistry, Helmholtz Department of Pharmaceutical Sciences, College of Center, Munich, Germany Clinical Pharmacy, King Faisal University, Hofuf 31982, Saudi Arabia B. Michalke Research Center for Environmental Health, Neuherberg, M. E. Mohammed Germany Department of Pharmacognosy, College of Pharmacy, Zagazig University, Zagazig 44519, 123 Biometals

13.55 ± 0.45 and 10.08 ± 0.63 mg/kg, respectively. (Karasinski et al. 2014; Silva et al. 2019). Zinc is also The highest Zinc capturing capacity was reported for a cofactor in more than 200 enzymes and it has also the 50–60 and 70–87 KDa protein fractions. paracellular and intracellular signalling functions (Ferna´ndez-Mene´ndez et al. 2016). Additionally, zinc Keywords Diabetes Á SEC–ICP–MS Á Trace is useful for the nervous system; whereas zinc element speciation Á Zinc deficiency has been associated with modified neu- rodevelopment and effects on infant behavior, and cognitive and movement abilities (Liu et al. 2015). Abbreviations Furthermore, close relationship between trace ele- CE Capillary electrophoresis ments and type 2 diabetes mellitus (T2DM) has DM Diabetes mellitus become more evident in the recent years (Asadi et al. GC Gas chromatography 2017; Atalay et al. 2017; Simic et al. 2017; Skalnaya HPLC High performance liquid chromatography et al. 2017; Zhang et al. 2017). Insulin mimicking ICP Inductively coupled plasma effect and additional signalling function of zinc in IEC Ion exchange chromatography counteracting diabetes were widely proven and MS Mass spectrometry described in a respective review article (Wang et al. SEC Size exclusion chromatography 2019). It also is known that insulin secretion from the SFC Supercritical fluid chromatography pancreas is zinc dependent (Li 2014; Rutter et al. 2016) and zinc is known for having a similar impact like insulin since it stimulates glucose uptake by adipocytes, lipogenesis and insulin action. Introduction On the other side, it has been well established that the bioavailability and biological activity of trace Trace elements, or secondary minerals, play serious elements in food strongly depend on their chemical roles (essential or toxic) in the functioning of life on species (form) (Ferna´ndez-Mene´ndez et al. 2016). our planet (Ovca et al. 2011; Oteef et al. 2015; Furthermore, determination of element species is also Ebrahim et al. 2017; Ashaiekh et al. 2019). The major informative in examining the action mechanism of function of trace elements in the cells of the living metal-drugs, besides the effects of exogenous metal organisms is that they act as cofactors for lots of exposures to biological systems (Bishop et al. 2018). enzymes (mostly proteins) and they are well known to Notably, Food and Agriculture Organization induce protein functions and to stabilize the different reported that 20% of the world’s population are at protein orders (Gupta 2015; Palm-Espling et al. 2012). risk of Zn deficiency based on food balance data from Concerning the zinc interaction with protein function more than 170 countries (Sandstead and Au 2007). and structure, it was reported by many studies that zinc This phenomena was mainly attributed to one or all of acts cofactor for many enzymes such as the carbonic three issues; food consumption with a low zinc anhydrase, alcohol dehydrogenase and aminopepti- content, unavailable species of zinc in food or the dases. In addition, zinc is well known to play major result of diseases that impair intestinal absorption and/ roles in protein function and ordering beside its role in or increase intestinal zinc losses (Ovca et al. 2011). regulation of gene expression and control of cell In other words, it has become a consensus that the activities such as the zinc finger proteins (Maret quantification of total element contents in food 2013, 2017). The role of the zinc ions in secretion and samples provides limited information. Accordingly, potentiation of insulin action was well studied and qualitative and quantitative speciation analysis of became a scientific fact that found its way to the trace elements in various food matrices has become of clinical practice (Li 2014). vital interest (Ferna´ndez-Mene´ndez et al. 2016). In particular, zinc is an essential trace element to all Despite the essential role of zinc in the functioning living organisms (Silva et al 2019) as it plays a vital of life, limited information is available on the bio- role in the metabolic activity of more than 300 of the available and bio-functional species of zinc in edible body’s proteins. Hence, zinc is considered vital for cell plants. In turn, it is accepted knowledge that metal- division and the synthesis of DNA and proteins phytates, such as zinc-phytate, are very common 123 Biometals species in edible plants or plant extracts, which tightly Materials and methods bind metals and by that way strongly reducing (up to inhibiting) bioavailability (Alloway 2008). Accord- Sample collection and sample pretreatment ingly, appropriate doses of a digestible and bio- available zinc species have not been examined yet. Eleven plant samples were collected from herbal In very early studies, traditionally, elemental spe- shops in Alahssa city, Saudi Arabia. Selection of plant ciation analysis has been accomplished through two species was based on the use for treatment and major protocols; (i) extraction of desired species adjuvant treatment of DM. The parts used as food were followed by element determination in extracts using extracted, washed by distilled deionized water, dried techniques such as ICP and AAS, or (ii) direct in an oven at 70 °C and homogenized in an agate mill separation by HPLC, GC, SFC, IEC, SEC, CE, (Spex Europe, Stanmore, UK). Table 1 shows the however, followed by unspecific UV-peak quantifica- scientific names, common names and the parts used. tion, but without element specific detection. Today, hyphenation techniques using species separation com- Sample treatment for total determination of zinc bined with element selective detection, such as SEC– content ICP–MS, IEC-ICP-MS, CE-ICP-MS, have proven to be the methods of choice for elemental speciation The considered parts of plant samples were split into analysis. Namely, the hyphenation of SEC–ICP–MS two parts for total and water extract analysis. For total provided accurate, sensitive and informative results analysis, the samples were treated by a wet digestion for speciation elemental analysis of various matrices procedure. 100 mg of sample were weighed into for different elements (Silva et al. 2019; Bishop et al. quartz vessels and mixed with 1 ml of HNO3 (analyt- 2018; Galvez et al. 2018; Wojcieszek et al. 2017; Wei ical reagent grade obtained from Sigma-Aldrich). The et al. 2017). digestion was carried out using a microwave system In another context, a couple of studies reported (CEM Discover SP-D). The microwave program was about positive, supporting effects of medicinal set at three steps: ramp 4 min with increasing plants with elevated zinc concentration in treatment temperature to 200 °C, hold temperature for 2 min of diabetes (Talukdar and Hossain 2014; Bnouham and cooling for 10 min. After digestion, samples were et al. 2010; Habicht 2012). These papers are in filtered using filter paper no. 42 and filled up to 8 mL general support practitioners who apply such medic- with Milli-Q water. inal plants as adjunct treatment for diabetes. In the current study, it has been proposed to carry out Sample treatment for zinc speciation analysis speciation analysis of Zn in eleven plants intensely and determination of zinc water extract used for diabetes treatment for shedding some light on how their supporting health effects could be For the extraction procedure, 200 mg of plant samples explained. For this purpose, SEC–ICP–MS was used were weighed and mixed with 5 mL of Milli-Q water in order to examine the association of zinc to in Falcon tubes and the pH was adjusted at 4 using proteins in water extract. On the other hand, the 0.1 M HCl. The mixture was shacked for 24 h at examined plants are consumed in a tea-preparation 80 °C. The mixture was then centrifuged at 3500 rpm form of the dried material. Hence, zinc in hot water for 30 min. The mixture was filtered using 45 lm extract provide the bioaccessible content. Accord- filter. Extractions were performed in duplicate per ingly, ICP-OES was used for direct determination of plant sample. The extracts were used for determina- Zn in the bulk samples and hot water extract. tions of Zn contents and Zn-protein profile.

Measurement by inductively coupled plasma optical emission spectrometry (ICP-OES)

Inductively coupled plasma-optical emission spec- trometry (ICP-OES) was used for the measurements of the total contents of zinc in bulk plant samples and hot 123 Biometals

Table1 The examined plant species and the parts used Toyopearl TSK HW 40S (TosoHaas, Stuttgart, Ger- Plant scientific name Common name Part used many) for the separation below 2 kDa. Isocratic elution (0.7 ml/min) was conducted using a 9:1-

Sesamum indicum Sesame mixture of 50 mM NH4Ac, pH 5.8 and 500 mM Olea Europaea Olive NH4Ac, 10 mM Tris, 5Vol% MeOH, pH 8.0. The Vaccinium myrtillus Bilberry injection volume was 20 ll. The effluent of the Nigella sativa black cumin Seeds columns passed a UV-detector (254 nm, 220 nm) Trigonella Foenum-graecum Fenugreek Seeds and was introduced through a Meinhard nebulizer and Pennisetum glaucum Pearl millet Seeds a cyclon spray-chamber to the ICP-MS. Dynamic

Calligonum comosum Carthage Whole plant Reaction Cell (DRC) mode with NH3 as reaction gas Citrullus colocynthis Colocynth Fruit eliminated isobaric interferences. Momordica Charantia Bitter Fruit ICP-MS parameters were RF power 1300 W, Opuntia Ficus-indica Barbary fig Fruit plasma gas flow 17 l Ar/min, nebulizer gas flow Haloxylon Salicornicum Ramth Whole plant 0.92 l Ar/min, daily optimized, cell gas flow 0.52 mL NH3/min, dwell time 1000 ms, monitored isotopes 52Cr, 55Mn, and 66Zn. Prior to sample measurement, a mass calibration water extracts. The ICP-OES system was ‘‘Ciros- was carried out for both SEC columns. The retention Vision’’ from Ametek-Spectro, Kleve, Germany. The times (tR) of the standards ferritin (440 kDa), c- measured spectral element line was 213.856 nm. globulin (190 kDa), arginase (107 kDa), transferrin Sample introduction was carried out by a peristaltic (78 kDa), HSA (66 kDa), b-lactoglobulin (37 kDa), pump connected to a micromist nebulizer with a oxidized and reduced glutathione (612 Da, 307 Da), cyclon spray chamber. The RF power was set to citrate (192 Da), inorganic Mn and Fe (55 Da, 56 Da) 1400 W, the plasma gas was 15 L Ar /min, whereas the were recorded and tR were correlated to respective nebulizer gas was 0.6 L Ar/min. molecular masses. The calibration equation of HMM- Regularly after ten measurements three blank and LMM-compounds are shown in Eqs. 1 and 2, determinations and a control determination of a respectively. The correlation coefficients of Eqs. 1 certified standard (CPI) were performed. The calcula- and 2 were 0.9971 and 0.9763, respectively. tion of results was carried out on a computerized lab- 3 2 logðMWÞ¼À0:0009tR þ 0:0977tR À 3:5058RT data management system, relating the sample mea- þ 46:998 surements to calibration curves, blank determinations ð1Þ and control standards. Certified reference material ERM-CD 281 (rye grass) and SRM 1547 (Peach logðÞ¼À MW 0:014tR þ 3:0747 ð2Þ leaves) were digested and analyzed. For Zn-quantification, post-column-flow-injection Separation of Zn fractions by SEC–ICP–MS (FI) was performed. Zinc-peak areas from samples were quantified according to respective FI-calibration. The species characterization of zinc was realized by The sum of quantified peaks was compared to total coupling an HPLC system (Knauer 1100 Smartline zinc measurement per sample to determine the recov- inert Series, Berlin, Germany) to an ICP-MS system ery rate during SEC–ICP–MS. (NexIon 300D, Perkin Elmer, Rodgau-Ju¨gesheim, Germany). The HPLC system was equipped with Risk assessment two columns to ensure the separation of high and low molecular mass (HMM/LMM) compounds. The first The conceivable adult and children risk of exposure to column (600 9 10 mm ID) was packed with Toy- Zn from the intake of plant sample species, in term of opearl TSK HW 55S (TosoHaas, Stuttgart, Germany) chronic daily intake (CDI), was calculated using with a separation range from 1 to 700 kDa. The second Eq. 3. (USEPA 2007); where Ci is the metal concen- PEEK column (250 9 8 mm ID) was packed with tration (mg/kg) in plant, IngR is the ingestion rate

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Results

Analytical quality control

The measured value of Zn in the CRM ‘‘ERM-CD 281 (rye grass)’’ was 29.6 ± 0.5 mg/kg while the certified value is 30.5 ± 1.1 mg/kg. For the CRM ‘‘SRM 1547 (peach leaves)’’, the measured value was 18.0 ± 0.6 mg/kg while the certified value was 17.9 mg/kg. These measurements resulted in recovery values of 97.1% for ERM-CD 281 and 100.5% for SRM 1547 indicating acceptable accuracy of the total Zn measurements. The limit of quantification was 0.6 mg/kg. On the other hand, Zn recovery during SEC–ICP–MS (as determined by comparing sum of peaks with total zinc determination (set 100%)) was 103 ± 4.8% (n = 6) indicating also acceptable accu- racy of Zn-protein measurements. For Zn-protein profile determination, mass bal- ances are an important means to check the reliability of plant species characterization with respect to absorption of element species to chromatographic stationary phases (losses) or, in turn, Zn-contamina- Fig. 1 Chromatogram of the extract of Momordica Charantia tion from mobile phases during separation. Therefore, (black line). Peak deconvolution and evaluation (colored lines) chromatographic recovery was examined. The recov- TM was performed with Peakfit Software. Based on retention ery values (%) were calculated as the ratio of the sum times of deconvoluted peaks and the mass calibration of the columns molecular masses are assigned to fractions. Phytate and of quantified Zn-peak elution from SEC–ICP–MS to inorganic Zn eluted at 65 min and 90 min (both not present in the total Zn concentration in hot water extracts of the this chromatogram) medicinal plants. The results revealed that in nine of the eleven plant extracts, the recovery was rather (200 mg/day for children and 100 mg/day for adult), consistent and ranged from 93.6 to 107.4%, averaging ExFr is the exposure frequency (350 day for both to 103.0 ± 4.8%, whereas Vaccinium myrtillus children and adults), ED is the exposure duration showed 84.2% and Trigonella foenum graecum (6 year for children and 24 for adult), BW is the body reached 118.3%. As a result, the accuracy of the weight (15 kg for children and 70 kg for adult) and AT measurements of Zn concentration in protein fraction is the averaging time (365 9 6 day for children and could be accepted. 365 9 24 for adult). To assess non-carcinogenic risk from the intake, the hazard index (HI) was calculated Total zinc contents using Eq. 4; where RfD is the reference dose of Zn (0.003). RfD features the health risk of non-carcino- Table 2 shows total Zn concentrations in the bulk plant genic adverse effects. HI is used to assess non- samples and in hot water extracts, in addition to the carcinogenic risk from the metal. percentage of the water extractable Zn fraction. Zn  concentrations were determined in hot water extract Ci  IngR  ExFr  ED CDI ¼  CF ð3Þ because for adjunct DM treatment a dried matter form BW  AT of the examined plants is used for tea-preparation. In CDI addition, the examined plants are also used as food in HI ¼ ð4Þ fornoncarcinogenicrisk RfD their bulk forms. On the other hand, the risk assessment factors from Zn exposure by intake to children and adult are 123 Biometals

Table 2 Total contents (mg/kg) of Zn in digested samples and water extracts Plant Total Zn concentration ± SD Zn concentration extract ± SD Extractable Zn-fraction

Sesamum indicum 54.90 ± 1.9 1.99 ± 0.19 3.62% Olea Europaea 7.79 ± 0.5 1.84 ± 0.02 23.62% Vaccinium myrtillus 3.34 ± 0.2 2.90 ± 0.04 86.68% Nigella sativa 58.80 ± 2.1 5.52 ± 1.01 9.39% Trigonella Foenum-graecum 29.80 ± 1.3 2.91 ± 0.40 9.75% Pennisetum glaucum 34.00 ± 1.5 6.07 ± 0.34 17.84% Calligonum comosum 11.80 ± 0.6 4.05 ± 0.33 34.32% Citrullus colocynthis 12.80 ± 0.6 10.08 ± 0.63 78.71% Momordica Charantia 21.20 ± 1.1 13.55 ± 0.45 63.92% Opuntia Ficus-indica 14.30 ± 0.7 1.26 ± 0.06 8.81% Haloxylon Salicornicum 6.67 ± 0.4 2.32 ± 0.00 34.71%

Table 3 Risk assessment Risk factor Children Adult factors from Zn exposure by intake to children and adult Sesamum indicum Nigella sativa Sesamum indicum Nigella sativa

Concentration 5.49E?01 5.88E?01 5.49E?01 5.88E?01 CDI ingestion 7.02E-04 7.52E-04 7.52E-05 8.06E-05 Rfd Ing 3.00E-01 3.00E-01 3.00E-01 3.00E-01 HI 2.34E-03 2.51E-03 2.51E-04 2.69E-04 compiled in Table 3. The USEPA guidelines (2007) species based on molecular weight using SEC–ICP– suggested that HI \ 1 indicates no health risk is MS. expected to occur while HI C 1 indicates moderate or high risk for adverse human health effects. Discussion Zinc-protein profile Total zinc contents In general, for elemental speciation it is important that the species of interest are extracted from the plant Irrespective of Zn speciation, FAO/WHO (2002) samples with an extractant that preserves species ranked Zn total contents in food into three categories: integrity, prevents structural changes and maintains (a) highest level (25–50 mg/kg) that is found in whole- proteins and protein-metal complexes (Ovca et al. grain cereals, lean red meat and legumes; (b) moderate 2011). The medicinal plants examined in the current level (10–25 mg/kg) that is found in polished rice, study are typically applied as hot water extracts like processed cereals with low extraction rates and meat tea. Hence, extraction by hot water was applied for with high content; (c) modest level (\ 10 mg/kg) getting likely the Zn-binding forms as in typical that is found in green leafy vegetables, and disease treatment. tubers, fruits and fish. Based on this classification, Figure 1 shows exemplarily the zinc SEC–ICP– Sesamum indicum, Nigella sativa, Trigonella Foe- MS-chromatogram of the extract from Momordica num-graecum and Pennisetum glaucum are classified Charantia and Table 4 summarizes the zinc-protein as highest Zn-content. Calligonum comosum, Citrul- profiles in hot water extracts of the examined plant lus colocynthis, Momordica Charantia and Opuntia Ficus-indica are classified as moderate Zn-content

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Table 4 Zn concentrations (mg/kg) in protein fractions of medicinal plants Plant species Protein fraction (kDa) 330–430 153–220 125–145 100–120 70–87 50–60 22–28 10–14 1.9–3.7 0.9–1.5 0.05–0.4c

Sesamum indicum 0.20 \ LOQb 0.52 \ LOQ \ LOQ 0.40 0.10 0.45 0.32 \ LOQ \ LOQ Olea Europaea 0.07 \ LOQ 0.37 \ LOQ \ LOQ 0.07 0.07 0.15 \ LOQ 1.12 \ LOQ Vaccinium myrtillus 0.09 0.26 0.17 \ LOQ 2.03 0.06 0.00 0.29 \ LOQ \ LOQ \ LOQ Nigella sativa 0.06 0.12 0.23 \ LOQ 1.00 3.52 0.59 n.d \ LOQ \ LOQ \ LOQ Trigonella Foenum- 0.07 0.09 0.11 \ LOQ 0.52 1.30 \ LOQ 0.52 0.29 \ LOQ \ LOQ graecum Pennisetum glaucum 0.25 \ LOQ 1.14 \ LOQ n.d 3.30 0.32 0.82 \ LOQ 0.23 \ LOQ Calligonum 0.59 0.09 0.09 \ LOQ 0.45 1.63 0.05 0.54 0.54 0.06 \ LOQ comosum Citrullus colocynthis 0.62 1.00 0.37 \ LOQ 3.98 0.62 \ LOQ 3.48 \ LOQ \ LOQ \ LOQ Momordica 0.40 \ LOQ \ LOQ 0.94 0.67 10.25 0.81 0.47 \ LOQ \ LOQ \ LOQ Charantia Opuntia Ficus-indica 0.03 \ LOQ 0.11 0.10 \ LOQ 0.93 0.05 0.04 \ LOQ \ LOQ \ LOQ Haloxylon 0.07 0.26 \ LOQ 0.14 0.14 1.36 0.05 0.26 0.04 \ LOQ \ LOQ Salicornicum aTotal concentration in water extract (mg/kg) bLimit of quantification cInorganic fraction while Olea Europaea, Vaccinium myrtillus and Zinc-protein profile Haloxylon Salicornicum are classified as modest Zn- content. In general, for elemental speciation it is important that Momordica Charantia reported 21.20 ± 1.1 mg/ the species of interest are extracted from the plant kg total zinc concentration in extract, which is within samples with an extractant that preserves species the range of what was reported in the species from integrity, prevents structural changes and maintains three states in Malaysia (17.2–46.8 mg/kg) (Yap et al. proteins and protein-metal complexes (Ovca et al. 2019). However, lower level was reported in the 2011). The medicinal plants examined in the current extract of same species from Pakistan (5 ± 1.2 mg/ study are typically applied as hot water extracts like kg) (Kosanovic et al. 2009). tea. Hence, extraction by hot water was applied for Notably, FAO/WHO (2002) recommended Zn getting likely the Zn-binding forms as in typical concentration of 50 mg/kg as the maximum allowable disease treatment. limit in food. Accordingly, Nigella sativa and Sesa- As shown in Table 4, the only fractions recorded mum indicum recorded higher concentrations than the quantifiable levels in all examined plants species were allowable limit. Hence, it is desirable to study the 330–430 kDa and 50–70 kDa. In contrast, Zn was not possible risk to human from Zn exposure. As shown in detected in the inorganic fraction 0.05–0.4 kDa of all Table 3, the HI values in Nigella sativa and Sesamum plant species. Also, only three plant species recorded indicum for both children and adult are less than unity Zn contents in the phytate fraction (0.9–1.5 kDa indicating no non-cancer risk to children and adult fraction). The high zinc concentrations (2–10 mg/kg) from consumption. Notably, the HI values depend on were observed in the following descending order: many factors, as described in Eqs. 3 and 4 (Said et al. Momordica Charantia (10.25 mg/kg) by the fraction 2014; Idris et al. 2015; Asiri et al. 2019; Idris et al. 50–70 kDa [ Citrullus colocynthis (3.98 mg/kg) by 2019). the fraction 70–90 kDa [ Nigella sativa (3.52 mg/kg) by the fraction 50–70 kDa [ Citrullus colocynthis (3.48 mg/kg) by the fraction 10–14 kDa [ Vaccinium myrtillus (2.03 mg/kg) by the fraction 70–90 kDa. 123 Biometals

The highest Zn concentration recorded in the Conclusion Momordica charantia extract is supposedly the proteic fraction; a finding that seems to be supported by This study demonstrates Zn-protein profiles, besides Yibchok-Anun et al. (2006), who investigated the the total Zn contents in bulk and hot water extract, of effect of a protein extract from Momordica on the eleven plants traditionally used for diabetes treatment glucose uptake by adipocytes. They found a protein in many countries. The literature survey indicates component of Momordica, which showed insulin-like Momordica charantia is the most popular plant that has effect being responsible for the hypoglycemic effect. anti-diabetic species. The current study revealed that The exact nature of the compound(s) associated with Citrullus colocynthis is the most similar plant to zinc in the 50–70 kDa fraction from this study is Momordica charantia in the term of total Zn concen- presently not clear. tration in hot extract as well as Zn-protein profiles. For Citrullus colocynthis, differences are seen in Hence, Citrullus colocynthis could also be recom- Zn-distribution. There is no one outstanding fraction mended as adjunct anti-diabetic treatment. The anti- with zinc in the 50–70 kDa fraction, but the fractions diabetic activity of Momordica charantia may be due 70–90 kDa and 22–28 kDa have similar Zn concen- to its high and available concentration of zinc. trations of nearly 4 mg/kg. For this medicinal plant, Despite both Nigella sativa and Sesamum indicum too, the zinc-carrying fractions are supposedly proteic, recorded total Zn contents higher than the permissible but not phytate fractions (around 1 kDa), explaining limit of FAO/WHO, the risk assessment through Zn-availability. dietary intake proposes no non-cancerous risk. The curative action of zinc in DM is proven since This study is limited as it is a simulative study. many years and its explanation on a molecular level is Determination of the extractable zinc concentration proceeding, including mimicking action of insulin or (bioaccessibility) is better to be determined in study acting in signaling (Wang et al. 2019). From that designs that involve animals or humans. background it can be explained that Momordica charantia and Citrullus colocynthis, both having the Acknowledgement The Authors would thank the Deanship of highest Zn-amounts in extracts and both having Scientific Research, King Faisal University (KFU) for its moral and financial support in funding this Project No ‘‘17122006’’. proteic Zn-binding forms, differently from phytates, show positive effects when applied as adjunct treatment in DM or even as sole treatment in References countries where pharmaceutical DM treatment is less available. Alloway BJ (2008) Zinc in soils and crop nutrition, 2nd edn. It should be noted that a couple of further metabo- IZA/IFA, Brussels, Belgium/Paris lites, such as saponines, Momordicoside T and others Asadi S, Moradi MN, Khyripour N, Goodarzi MT, Mahmoodi M (2017) Resveratrol attenuates copper and zinc home- are considered to influence positively glucose meta- ostasis and ameliorates oxidative stress in type 2 diabetic bolism and lower triglycerides (Habicht 2012; Tan rats. Biol Trace Elem Res 177:132–138. https://doi.org/10. et al. 2008). Aside zinc, these metabolites must be 1007/s12011-016-0861-6 considered as effective co-factors, which may play a Ashaiekh MA, Eltayeb MAH, Ali AH, Ebrahim AM, Salih I, Idris AM (2019) Spatial distribution of total and bioavail- more pronounced role in those medicinal plants where able heavy metal contents in soil from agricultural, resi- zinc concentration is lower. dential and industrial areas in Sudan. Toxin Rev Taken together, the combined treatment of DM 38:93–105. https://doi.org/10.1080/15569543.2017. with zinc and further metabolites from Momordica 1419491 Asiri HFM, Idris AM, Said TO, Sahlabji T, Alghamdi MM, El- charantia resulted in significant reduction of HbA1C Zahhar AA, El Nemr A (2019) Monitoring and health risk values, the long-term parameter for blood glucose assessment of some pesticides and organic pollutants in levels, caused by insulin-sensitizing effect Miura et al. fruit and vegetables consumed in Asir region. Saudi Ara- (2001). It also improved the glucose tolerance in high bia. Frsen Environ Bull 29:615–625 Atalay H, Boyuk B, Guzel S, Altay M, Kiziler AR, Aydemir B fat diet-induced insulin-resistant mice (Tan et al. (2017) Serum trace elements in type 2 diabetes mellitus. 2008). Acta Med Mediterranea 33:795–800. https://doi.org/10. 19193/0393-6384_2017_5_117

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