Phytochemical Evaluation of Some Herbal Prebiotics and Their Potential Use in Food Supplements

CIUPERCĂ Oana Teodora1, ŢEBRENCU Carmen Elena1, RĂDUCANU Adina2, POP Anca Lucia3*, ONISEI Tatiana2

1The Research and Processing Center for Medicinal Plants PLANTAVOREL, Piatra Neamt, Romania, 

2 National Institute R&D for Food Bioresources, National Service for Medicinal, Aromatic Plants and Bee Products, Bucharest, Romania

3 Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, (ROMANIA)

E-mails: oana.ciuperca@plantavorel.ro, carmen@plantavorel.ro, adina.raducanu@bioresurse.ro, floarea.serbancea@bioresurse.ro, anca.pop@umfcd.ro, tatiana.onisei@bioresurse.ro 

* corresponding author: ancapop@hotmail.com 

Abstract

Herbal prebiotics have a remarkable influence on intestinal health, making them attractive agents for improving the quality of human life against various diseases. By determining the phytochemical composition of herbal prebiotics, could formulate food supplements to improve human health. Herbal prebiotics can be associated with probiotics which function can be significantly increased. The present study evaluates the phytochemical composition of some herbal prebiotics (topinambur – Helianthus tuberosus L. and psyllium – Plantago ovata) and their potential use in food supplements. The vegetal species were investigated in order to demonstrate their prebiotic potential by highlighting the presence of phytochemicals responsible for the effect. The composition of the vegetal species was evidenced using specific chemical tests, High Performance Thin Layer Chromatography (HPTLC) and Ultraviolet-Visible (UV-VIS) spectrophotometry. There were identified saccharides in both vegetal species using the reaction with thymol, mono and oligosaccarides such as D-fructose – Rf 0.69, D-lactose – Rf 0.67, D-galactose – Rf 0.53, D-sucrose – Rf 0.71 and D-glucose – Rf 0.64 and inulin – Rf 0.01  in topinambur tubers using chromatographic analysis. The quantitative evaluation of topinambur tubers using spectrophotometric determination revealed a content of total sugars expressed in fructose of 15.62 % g/g. These results are relevant for the obtaining of innovative formulas between topinambur tubers and psyllium husk in order to use them in food supplements with prebiotic effects. The action of these herbal prebiotics in association with probiotics ensure the maintaining of a normal intestinal function and health.

Keywords: herbal prebiotics, Helianthus tuberosus L. (tubers), Plantago ovata (husk), mono and oligosaccharides

Introduction

Herbal prebiotics are undigested ingredients that are selectively fermented in the human intestine and lead specific changes in the gut microbiota, thus improving the hosts’ health. In order to assess the potential of a food component to be considered as a prebiotic ingredient, several experimentations need to be performed to provide scientific confirmation. Herbal prebiotics boost the growth and activity of beneficial bacteria from the intestine [1, 2, 3]. The activity of the most commonly used bacteria (genus Bifidobacterium and Lactobacillus) that are good for our health is amplified by prebiotics, which control the growth of harmful bacteria. Most commonly used prebiotics are mannan-oligosaccharides, galactoglucomannans, lactose, inulin and oligofructose. Short chain carbohydrates used as prebiotics consist of three to ten sugar units derived from the cell wall of yeast and plants [4, 5, 6]. Topinambur (Helianthus tuberosus L.) (tubers) and psyllium (Plantago ovata) (husk) can be considered herbal prebiotics due to the important phytocompounds such as mono and oligosaccharides resposable for the effect.

Topinambur (Helianthus tuberosus L.) produces sugars in the plant organs of the soil and stores them in roots and tubers. Topinambur tubers have a water content (75–79%), carbohydrates, proteins and fats (2–3%). In addition, they also contain microelements such as phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and iron (Fe) in a proportion of 0.5% by fresh weight. Tubers also contain vitamins such as C and A. Among the carbohydrates in the tubers there are inulin (80%) and sugar residues such as fructose, sucrose and glucose. Inulin is one of the most common natural prebiotic fiber used in food supplements, and topinambur tubers are one of the main sources of inulin, along with chicory root. Due to its high inulin content, topinambur is also a valuable alternative source of prebiotic compounds [7, 8]. Psyllium, husk is the common name for the seed coat of the plant species Plantago ovata. The plant is cultivated or harvested to obtain seeds for therapeutic use. Psyllium is used in food supplements as husk obtained by removal of the seeds coat. Psyllium can be used to treat common constipation (constipation that is not caused by a physical change in intestine) and in the case of anal fissures, hemorrhoids and after surgerys on the anus or rectum. It can also be used at people who may be advised to increase their daily dietary fiber intake, such as patients with irritable bowel syndrome who suffer from constipation, or in combination with a diet at patients with hypercholesterolemia (high cholesterol levels in blood). It must be administered with a large amount of liquid. It can be used at adults and children older than 6 years to treat constipation, but should only be used at adults and children older than 12 years when it is used to increase fiber intake. The use in hypercholesterolemia always requires medical advice [4, 9, 10, 11, 12]. The beneficial effects of topinambur and psyllium are presented in  Fig. 1. 

Fig. 1 The beneficial effects of topinambur and psyllium [4, 7, 8, 9, 10, 11, 12].

Materials and methods

Plant material. The prebiotic herbs used in experimental research were topinambur – Helianthus tuberosus L. and psyllium – Plantago ovata and there are presented in Tab. 1.

Table. 1 Plant material used for study

Name	TOPINAMBUR	PSYLLIUM
Botanical name	Helianthus tuberosus L.	Plantago ovata
Family	Asteraceae	Plantaginacee
Part used	Tubers	Husk

The plant material was purchased from authorized distributors, being delivered with a quality certificate, in dried form. The dried material was grounded using a laboratory mill Microtron MB550 to particles with 0.8 mm medium size. The samples were stored in a clean desiccator until were used for phytochemical study, chromatographic and spectrophotometric evaluation. 

This study consisted in qualitative and quantitative evaluation of the bioactive compounds with prebiotic activity from the selected vegetal species and was conducted using the experimental diagram presented in Fig. 2. All the reference standards and chemicals were of analytical grade or pure (Sigma Aldrich, Merck, Roth, Extrasynthese).

Fig. 2 Experimental diagram of this study

Qualitative evaluation

Phytochemical study. In order to detect the presence of saccharides the phytochemical tests were performed according to the methods described by [13]. For both vegetal species (topinambur and psyllium) were applied the specific test for saccharides – reaction with thymol. The results of the tests were based on the visual observation of colour change, respectively the apparition of a carmine-red colour. HPTLC analysis: the study was done according to [14] using the equipment CAMAG LINOMAT IV, TLC 3 Scanner and as software WINCATS Planar Chromatography Manager. HPTLC plates G60 F254, 200x100mm, 100x100mm (Merck, Darmstadt, Germany)  were used as stationary phase for HPTLC identification.

HPTLC identification of mono and oligosaccharidesin topinambur tubers. Reference solutions: the solution of mono and oligosaccharides (D-fructose, D-glucose, sucrose, D-galactose, D-lactose, D-rafinose*5H2O, D-maltose) were prepared with a concentration of 0.2 mg/mL in distilled water and stored at 4°C until use. Sample preparation: the powdered vegetal material was extracted with distilled water by heat reflux extraction for 1 hour. The resulted extract was filtered, concentrated at ½ and applied on the chromatoplate. Chromatographic procedure: on the starting line of the chromatographic plate, the reference solutions and the topinambur tubers extract were applied. The chromatographic plate is inserted into the development tank, using as mobile phase a mixture of n-buthanol-acetic acid-distilled water (48:60:9) and left to migrate over a distance of 5 cm. The plate is dried in air and then is sprayed with identification reagent (anisaldehyde – sulphuric acid); the plate was heated in the oven at 115 ° C for 15 minutes. The derivatized plate is analyzed in visible light. It was used a CAMAG REPROSTAR III DIGISTORE II documentation system for making and storing the chromatogram images. The appearance of the characteristic spots for mono and oligosaccharides (greenish-gray or brown-yellow spots) indicated their presence.

HPTLC identification of inulin in topinambur tubers extract. Reference solutions: aqueous solution (0,02%) of inulin. Sample preparation: there were prepared two topinambur tubers extracts using distilled water, using heat reflux extraction, a 1:10 solid to liquid ratio and 15 minutes extraction time. Chromatographic procedure: on the starting line of the chromatographic plate, the reference solutions and samples were applied. The chromatographic plate is inserted into the development tank, using as mobile phase a mixture of anisaldehyde:acetic acid: sulphuric acid (10:5:85) and left to migrate over a distance of 5 cm. The plate is dried in air and then is sprayed with identification reagent (aniline – diphenylamine – phosphoric acid in methanol); the plate was heated in the oven at 110 ° C for 20 minutes. The derivatized plate is analyzed in visible light. The appearance of the characteristic bands (greenish-gray spots) for inulin indicated its presence.

Quantitative evaluation. Determination of total sugars expressed in fructose: is a own validated method that uses as reagents: resorcinol (1g/l) and hydrochloric acid (300g/l). A quantity of 0.4 g of powdered vegetal material is extracted with 25 ml of distilled water, by stirring at room temperature, for 15 minutes. The extractive solution is filtered through cotton filter in a 50 ml volumetric flask and brought to volume by repeated wash of the filter with portions of the extraction solvent. In a 25 ml volumetric flask bring 0.1 ml of extractive solution, 3 ml of alcoholic solution of 1 g/l resorcinol and 3 ml of 300 g/l hydrochloric acid. The content is mixed. The closed bottle with a stopper is heated for exactly 8 minutes in the boiling water bath. The solution turns red. Cool the flask immediately under cold water. Make up to 25 ml with distilled water. The absorbance of the resulted solution is determined by reading on a UV-VIS spectrophotometer at 480 nm.

RESULTS AND DISCUSSION

Qualitative evaluation

Phytochemical study. The results of phytochemical identification of saccharides are presented in Tab. 2. These revealed that the class of specific phyto-compounds for the analysed vegetal species were present. Therefore the test with thymol showed the presence of saccharides in tubers of topinambur and psyllium husk. 

Tab. 2 The results of qualitative tests applied on vegetal material

Specific test	TOPINAMBUR, tubers	PSYLLIUM, husk
Test for saccharides identification (the reaction with thymol)	++  (high intensity of colour)	+  (medium intensity of colour)

HPTLC identification of mono and oligosaccharidesin topinambur tubers

The HPTLC image was obtained using a development system represented by a mixture of solvents n-butanol: acetic acid: water in a ratio of 48: 60: 9, and after derivatization with the identification reagent (anisaldehyde-sulfuric acid) and indicated the separation of mono and oligosaccharides from the analysed samples. The reference substances used were identified at different values of Rf, which are shown in Tab. 3.

Tab. 3 Rf values of the reference substances (mono and oligosaccharides)

Reference substance	Rf Value
D-rafinose*5H2O	0,46
D-galactose	0,53
D-maltose	0,61
D-glucose	0,65
β-D-lactose	0,67
D-fructose	0,69
sucrose	0,71

The topinambur extract was applied to the plate on two tracks in a volume of 10 μl each, while the reference substances were applied to the plate in volumes of 5 μl. By comparison to the reference substances, the specific constituents (mono and oligosaccharides) of the analysed vegetal material were identified, following visualization after spraying with the identification reagent. The appearance of greenish-gray or brown-yellow spots in the analysed samples on the chromatogram examined under visible light indicated the presence of mono and oligosaccharides in the topinambur extract (Fig. 3). Thus, D-fructose (Rf 0.69), D-lactose (Rf 0.67), D-galactose (Rf 0.53), D-sucrose (Rf 0.71)and D-glucose (Rf 0.64) were identified in the topinambur extract.

Fig. 3 Chromatographic identification of mono and oligosaccharides in topinambur extract

Legend: E1: D-fructose, D-raffinose * 5H2O; E2: β-D-lactose, D-maltose, D-galactose; E3: sucrose, D-glucose; P1: concentrated topinambur extract

HPTLC identification of inulin in topinambur tubers 

The HPTLC image for inulin identification was obtained using a Merck G60F254 silica gel plate (100 x 100 mm) and indicated the separation of inulin from the analysed samples. The reference substance (inulin) was identified at an Rf value of 0.01.The topinambur extract was applied to the plate on two tracks in a volume of 10 μl (track 1) and 15 μl (track 2), and the reference substance – aqueous inulin solution – was applied to the plate in a volume of 5 μl. By comparison to the reference substance, inulin was identified in the analysed vegetal material, after visualization in visible light after spraying with the identification reagent. The appearance of greenish-grey bands in the samples analysed on the chromatogram evidenced the presence of inulin in the topinambur extract (Fig. 4).

Fig. 4 Chromatographic identification of inulin in topinambur extract

Legend: P1: concentrated topinambur extract (10μl), P2: concentrated topinambur extract (15μl) I: inulin (reference substance) (5μl)

Quantitative evaluation. Quantitative evaluation of topinambur extract was done by applying the UV-VIS spectrophotometric dosing method of total sugars expressed in fructose. The spectrophotometric method was applied for determining the concentration of an extract, based on the recorded absorbance. The maximum absorption of fructose was recorded at a wavelength of 480 nm. The absorbance measurement was performed with the UV-VIS CARY 50 spectrophotometer, using quartz cuvettes. The results of the quantitative determination of the total sugars expressed in fructose are presented in Tab. 4.

Tab. 4 The results of quantitative determination in topinambur extract 

Parameter/ Vegetal material	TOPINAMBUR, tubers
Total sugars expressed in fructose (%g/g)	15,62

The prebiotic potential of the selected vegetal species

The topinambur tubers and psyllium husk can represent an important source of vegetal fibers with prebiotic action due to the specific phyto-compounds that were evidenced in this study. The presence of some mono and oligosaccharides such as D-fructose, D-lactose, D-galactose, D-sucrose D-glucose and inulin in their composition suggests the possibility of use of these vegetal species in food supplements in different proportions to ensure the prebiotic effect. The elaboration of the association formulas from the selected vegetal species can represent an important step in obtaining food supplements with important prebiotic action (Fig. 5).

Fig. 5 The potential of the selected vegetal species for use in food supplements with prebiotic effects

Thus, the prebiotic profile of the association between topinambur tubers and psyllium husk is predominantly based on the beneficial action on intestinal health, efficient weight control, laxative action, support of normal intestinal function and emolying and protective action in intestine induced by the specific phytochemical compounds.  To complete the prebiotic effect, the association with probiotic bacteria from Lactobacillus and Bifidobacterium species is also recommended.

CONCLUSIONS

The phytochemical evaluation of the selected medicinal species – topinambur (Helianthus tuberosus L.) and psyllium (Plantago ovata) aim the identification and quantification of the bioactive principles with prebiotic potential using specific qualitative test for saccharides, High Performance Thin layer Chromatography and Ultraviolet-Visible spectrophotometry. There were identified saccharides in both vegetal species using the reaction with thymol, mono and oligosaccharides such as D-fructose – Rf 0.69, D-lactose – Rf 0.67, D-galactose – Rf 0.53, D-sucrose – Rf 0.71 and D-glucose – Rf 0.64 and inulin – Rf 0.01 in topinambur tubers using chromatographic analysis. The quantitative evaluation using spectrophotometric determination in topinambur tubers revealed a content of total sugars expressed in fructose of 15.62 % g/g.

The presence of this important phyto-compounds indicated their prebiotic effects due to their important role in gut health. These results are relevant for the obtaining of innovative formulas between topinambur tubers and psyllium husk in order to use them in food supplements with prebiotic effects. The action of these herbal prebiotics in association with probiotics ensure the maintaining of a normal intestinal function and health. The results of this study bring significant scientific information regarding the exploitation of the prebiotic potential of the two medicinal species by their association and their use in phytotherapy.

ACKNOWLEDGMENTS 

The study was supported by the subsidiary contract no.13 / 21.12.2018 (POC-A1-A 1.2.3.-2016), Contract no.57/ 05.09.2016/ MySmis 105509 – ”Valorificarea expertizei în cercetarea agroalimentară prin transfer de cunoștințe către mediul privat în vederea obținerii de produse alimentare sigure și optimizate nutrițional.”

REFERENCES

1. Gibson, G.R., Roberfroid, M.B. (1995). Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J Nutr., 125:1401–1412.
2. Slavin, J. (2013). Review Fiber and Prebiotics: Mechanisms and Health Benefits,  Nutrients, 5:1417-1435, doi:10.3390/nu5041417
3.  Peterson, C. T.,  Sharma, V.,  Uchitel,S., Denniston,  K., Chopra, D., Mills,P. J., . Peterson, S. N. (2018). Prebiotic Potential of Herbal Medicines Used in Digestive Health and Disease, J Altern Complement Med.,24(7): 656–665.
4. Grajek W., Olejnik A., Sip A. (2005). Probiotics, prebiotics and antioxidants as functional foods. Acta Biochimica Polonica, 52:665-671.
5. Toward, R., Montandon, S., Walton, G., Gibson, G.R. (2012). Effect of prebiotics on the human gut microbiota of elderly persons. Gut Microbes 3:57–60. 
6. Hutkins, R.W., Krumbeck, J.A., Bindels, L.B. (2016). Prebiotics: Why definitions matter, Curr. Opin. Biotechnol, 37:1–7.
7. Kolida, S., Tuohy, K., Gibson, G.R. (2002). Prebiotic effects of inulin and oligofructose. Br J Nutr, 87 Suppl 2:S193–S197.
8. Szewczyk A., Zagaja M., Bryda J., Kosikowska U., Stępień-Pyśniak D., Winiarczyk S., Andres-Mach M. (2019).Topinambur – new possibilities for use in a supplementation diet, Annals of Agricultural and Environmental Medicine 2019, Vol 26, No 1, 24–28
9. Jalanka, J., Major, G. , Murray, K., Singh, G., Nowak, A., Kurtz, C., Silos-Santiago, I., Johnston, J.M., de Vos,W.M., Spiller, R., (2019). The Effect of Psyllium Husk on Intestinal Microbiota in Constipated Patients and Healthy Controls, International Journal of Molecular Sciences, 20: 433; Doi:10.3390/ijms20020433.
10. ***EMA/HMPC/313600/2013 – Tegument de ispagul Plantago ovata Forssk., seminis tegumentum, Comitetul pentru medicamente din plante (HMPC).
11. ***EMEA (2013). Assesment report on Plantago ovate forssk., semen.
12. ***Assessment report on Plantago ovata Forssk., seminis tegumentum, 14 May 2013 EMA/HMPC/199775/2012 Committee on Herbal Medicinal Products (HMPC), European Medicines Agency.
13. Ciulei, I., Istudor, V., Palade, M., Albulescu, D., Gird, C.E. (1995). Analiza farmacognostica si fitochimica a produselor vegetale, Ed. Tehnoplast Company S.R.L., Bucuresti, 1995, vol. I.
14. Wagner, H., Baldt, S., Zgainski, E.M. (1996) Plant drug analysis. New York,: Springer.