GC-FID guided Identification and Quantification of detectable Phytochemicals in selected Commercial Chamomile Herbal Tea

Kenneth C. Ugoeze¹, Nora Amadi¹, Ngozi A. Okoronkwo², Sunday O. Abali¹, Kennedy E. Oluigbo³, Bruno C. Chinko^{*, 4}

¹Department of Pharmaceutics & Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Port Harcourt, Nigeria.

²Department of Zoology & Environmental Biology, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria.

³Department of Clinical Pharmacy and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Agbani City, Enugu, Nigeria.  

⁴Department of Human Physiology, Faculty of Basic Medical Sciences, University of Port Harcourt, Port Harcourt, Nigeria.

*Corresponding author: Bruno C. Chinko, Department of Human Physiology, Faculty of Basic Medical Sciences, University of Port Harcourt, Port Harcourt, Nigeria

Received: 23 December 2022; Accepted: 05 January 2023; Published: 14 January 2023

1. Introduction

Phytochemicals are considered plant-based bioactive chemicals with various therapeutic and nutritious benefits [1-3]. They are classified as primary (PM) or secondary metabolites (SM). The PM comprises the common sugars, amino acids, proteins, purines and pyrimidines of nucleic acids, chlorophylls, etc. whereas the SM are the plant chemicals such as alkaloids, terpenes, flavonoids, lignans, plant steroids, saponins, phenolics, glycosides, etc. [1].   Due to their antimicrobial, anti-inflammatory, anthelmintic, anti-carcinogenic, anti-genotoxic, anti-proliferative, anti-mutagenic, anti-allergic, immune-modulatory and anti-oxidative properties, the SM could play protective roles against pathogens or damaging disorders [4-9]. There are over 8000 natural flavonoids [5] with their health-based biotic actions ascribed to their antioxidant influence [6, 7]. The antioxidant and anti-inflammatory actions of the flavonoids aid in toxin-mediated stress and protracted infection inhibition [10]. Flavonoid-rich foods include all nourishments of plant basis, predominantly tea, fruit, vegetables, grains, legumes, nuts, and wine [11, 12].

Tea is processed from the vegetative parts of Camellia sinensis [13]. They are used as a medicinal drink by two-thirds of the globe [14] and have been designated as “safe” by the US Food and Drug Administration's (FDA) list of compounds generally recognized as safe (GRAS) [15]. Normally, Tea refers to dried leaves processed from leaves of Camellia sinensis (true tea).  Other infusions got from parts of other suitable plants are also noted as tea, but, are called tisanes or herbal tea, hence, there are true or traditional tea and herbal tea [12, 13]. True tea is grouped as green, black/dark, white, yellow and oolong which is established on the extent of oxidation of the leaves [16]. Green tea is a non-fermented tea [17], they are processed from freshly harvested vegetation of the tea plant without fermentation after withering, steaming or pan firing, drying and grading to retain its content of polyphenols [15, 16, 18]. On the other hand, black tea is wholly fermented tea, processed with polyphenol oxidase converting its polyphenols into a series of new products like theaflavins, theaflagallins and thearubigens obtainable in black tea  [16, 18]. Oolong tea is a semi-fermented tea gotten when leaves are wilted in the sun and moderately battered to attain brief partial oxidation resulting in an intermediate product, the oolong tea [18-20].

Herbal tea or tisanes implies any infusions taken as a drink derived from other parts of vegetation other than Camellia sinensis [6, 15]. They are often caffeine free compared to traditional teas. It could be gotten from plants like chamomile, rose hip, ginger, turmeric, valerian, hibiscus, peppermint, etc. [21]. Chamomile is one of the early therapeutic herbs with standardized forms prepared from dried flowers of Matricaria species and of the daisy family, Asteraceae/ Compositae [22]. Two common varieties are the German Chamomile (Matricaria chamomilla) and Roman Chamomile (Chamaemelum nobile) [23]. Their preparations are used to manage several ailments like hay fever, inflammation, muscle spasms, menstrual disorders, insomnia, ulcers, wounds, gastrointestinal disorders, rheumatic pain and haemorrhoids while their essential oils are applied widely in cosmetics and aromatherapy [24, 25]. Their therapeutic influence is largely based on their dried flowers' terpenoid and flavonoid content [24]. Chamomile herbal tea is essentially caffeine-free and is believed to be a calming and soothing effect which promotes sleep and reduces anxiety [26, 27]. They have also been found to be effective in treating injuries, ulcers, eczema, gout, skin irritations, bruises, burns, canker sores, neuralgia, sciatica, rheumatic pain, haemorrhoids and mastitis [25, 28]. Aside from their use as tea, and tincture, they are used as poultices and tincture [29]. Some of the flavonoids found in chamomile are apigenin, quercetin, and patuletin [30, 31]. Apigenin is known to bind to benzodiazepine receptors in the brain to enhance sleep and diminish insomnia [32, 33]. Similarly, their essential oils are used in aromatherapy to enhance sleep and relieve anxiety [34].

Despite the aforementioned benefits and safety margin of chamomile, there has been no confirmation of their reaction to other medications or their safety among children and pregnant women. Therefore, the present study aims to identify and quantify the phytochemicals in selected commercial chamomile herbal tea using Gas Chromatography with flame ionization detection (GC-FID) methods.

2. Materials and Methods

2.1 Procurement of Samples

Five brands of chamomile herbal tea (CHT) were randomly sourced from retail outlets in Port Harcourt, Nigeria and coded CHT-A, CHT-B, CHT-C, CHT-D and CHT-E. Also, a crude unpackaged dry chamomile flower was sourced from a retail outlet in Abuja and coded as CHT-F while its oil extract was coded as CHT-G.

2.1 Preparation of samples and extraction of phytoconstituents

A teabag of each CHT was macerated in 200 ml of ethanol (Honeywell, Germany) for 48 hours. It was filtered and evaporated to obtain the extract. A 0.1 g of the extract was re-extracted in 25 ml of ethanol in a test tube immersed in a water bath (60 ^o C) for 90 mins. It was transferred to a separatory funnel. The test tube was washed in steps with 20 ml of ethanol, 10 ml of cold water, 10 ml of hot water and 3 ml of n-hexane (BDH, England) respectively and transferred into the separatory funnel. The extracts were pooled and washed thrice with 10 ml of 10 % v/v ethanol-aqueous solution. The solution was dried with anhydrous sodium sulphate (Sigma-Aldrich, USA) and the solvent was evaporated. The sample was solubilized in 1000 ul of pyridine (Sigma-Aldrich, USA), of which 200 ul was transferred to a vial for analysis [35, 36].

2.2 Quantification of the phytoconstituents by GC-FID

The analysis of phytoconstituents was carried out on a BUCK M910 Gas Chromatography fitted with an HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm in thickness of film). Spectroscopic detection by GC–FID involved an electron ionization system which used high-energy electrons (70 eV). Pure helium gas (99.995 %) was employed as the carrier gas with a flow rate of 1 mL/min. The early temperature was set at 50 –150 °C with an increasing rate of 3 °C/min and a holding time of about 10 min. Later the temperature was amplified to 300 °C at 10 °C/min. A 1µl of the prepared 1% of the extracts diluted with respective solvents was injected in a splitless mode. The relative quantity of the chemical compounds present in each of the extracts was expressed as a percentage based on the peak area produced in the chromatogram [36, 37]. Bioactive compounds extracted from the respective batches of extracts were identified based on the GC retention time on the HP-5MS column and matching the spectra with the computer software standards data (Replib and Mainlab data of GC–FID systems).

3. Results

3.1 GC-FID Quantified Phytoconstituents of selected Chamomile Herbal Tea

Table 1: Composition of the phytochemical constituents detected in CHT-A

Table 2: Composition of the phytochemical constituents detected in CHT-B

Table 3: Composition of the phytochemical constituents detected in CHT – C

Table 4: Composition of the phytochemical constituents detected in CHT-D

Table 5: Composition of phytochemical constituents detected in CHT-E

Table 6: Composition of the phytochemical constituents detected in CHT-F

Table 7: Composition of the phytochemical constituents detected in CHT-G

4. Discussions

The results obtained from the GC-FID of selected chamomile herbal tea and dry chamomile flower and essential oils are summarised in Tables 1 – 7. These results are discussed in this section under flavonoids, alkaloids, glycosides, saponins, tannins, steroids, anti-nutrients and other phenols such as resveratrol.

4.1 Flavonoids

The level of flavonoids detected in the various CHT were as CHT-B > CHT-E > CHT-G > CHT-A > CHT-C > CHT-C > CHT-D > CHT-F. The types of flavonoids identified include proanthocyanin, anthocyanin, flavan-3-ol, naringenin, rutin, flavanones, kaempferol, flavone, catechin, epicatechin, etc. (Tables 1-7). The variation in the composition of these flavonoids from one CHT to the other may be attributed to differences in environment or influences like latitude, longitude, rainfall, temperature and soil quality [38]. Given these differences, and with CHT-B presenting the highest concentration of flavonoids displayed its subgroup of flavonoids as Rutin   > flavonone > flavone > anthocyanin > epicatechin > kaempferol > naringenin > proanthocyanin, etc. (Table 2). Flavonoids have been known to possess immense pharmacological benefits such as anti-oxidative, anti-mutagenic, anti-inflammatory, anti-carcinogenic, antitumour, anti-HIV, antidiarrhoeal, antihepatotoxic, antifungal, antilipolytic, vasodilator, immunostimulant and anti-ulcerogenic properties and enzyme modulatory functions [39, 40]. Rutins have been found in fresh leaves, red wine and tea [12, 41, 42]

4.2 Alkaloids

The composition of alkaloids detected was CHT-A > CHT-D > CHT-G > CHT-E > CHT-B > CHT-F > CHT- C. The various forms of alkaloids detected were ribalinidine, lunamarin, spartein, ephedrine, etc., though, ephedrine was not detected in CHT-A and CHT-G.  Alkaloids are natural products that have heterocyclic nitrogen atoms [43-45]. They found use in the ancient preparation of spices, drugs and poisons. Lunamarin retains anticancer, immunomodulatory, anti-estrogenic and anti-amoebic activities [46] while ribalinidine possesses a radical scavenging influence.

4.3 Glycosides

The composition of glycosides identified in the various CHT were CHT-F > CHT-B > CHT-C > CHT-D > CHT-E > CHT-G > CHT-A with cardiac and cyanogenic glycosides prevailing (Tables 1-7). With the highest level of glycosides detected in CHT-F, the extent of cardiac and cyanogenic glycosides was 4.37 and 14.33 % respectively.  In CHT-B, they were 3.15 and 15.34 % correspondingly whereas it was 3.69 and 11.72 % in CHT-C. Further, it was 3.59 and 9.81% in CHT-D while in CHT-E it was 4.07 and 8.63 %. Only cardiac glycoside was detected in CHT-A and CHT-G at 2.11 and 2.34 % respectively. The cyanogenic glycosides retained higher concentrations where it was detected. Glycosides are plant-based substances comprising of a glucose unit confined to an aglycone like alcohol, phenol or steroid nucleus through a glycosidic bond [47] with potent antibacterial, antifungal, anti-inflammatory, antioxidant, antiviral and anticancer activities [48, 49]. Cardiac glycosides are used in the treatment of cardiac insufficiency [48, 50] by increasing the output force of the heart and decreasing its rate of contractions by inhibiting the cellular Sodium-Potassium-ATPase pump [50]. However, their relative toxicity prevents their extensive application [51]. On the other hand, cyanogenic glycosides which are mostly found in foods including linamarin, amygdalin and prunasin [52] are known to release hydrogen cyanide when chewed or digested [53] resulting in significant cyanide poisoning. However, processing methods, such as peeling, drying, grinding, soaking and fermentation, boiling or cooking have been reported to cause a significant reduction in the cyanogenic glycosides of processed foods [54].

4.4 Saponins

The saponin detected was sapogenin which occurred in these CHTs as CHT-C (4.90 %) > CHT-D (4.17 %) > CHT-E (2.45 %) > CHT-B (2.16 %) (Tables 1-7). Saponins constitute a vast group of glycosides occurring in many plants and are characterized by their surfactant properties. They are grouped as triterpenoid and steroid saponins [55].  The steroidal saponins are essential precursors for steroid drugs, comprising anti-inflammatory agents, androgens, oestrogens and progestins [56]  while triterpene saponins exhibit various pharmacological activities, including anti-inflammatory, molluscicidal, antitussive, expectorant, analgesic and cytotoxic influences and include the ginsenosides, which are responsible for some of the pharmacological activity of ginseng and the active triterpenoid saponins from liquorice [57, 58].

4.5 Tannins

Tannins were detected only in these CHTs as CHT-A (2.54 %) > CHT-G (2.48 %) > CHT-F (2.22 %). The existence of tannins in tea leaves accounts for the bitter and dry sensation felt when tea is tasted. Tannins are higher in black tea than in oolong, green and white teas [59]. Tannins also occur in red wine, coffee, grapes, apple juice, strawberry, raspberry, blackberry, pomegranate, plums, walnuts, olives, chickpeas, lentils, chocolate and cocoa [60].  Foods rich in tannins have been considered to be of low nutritional value since tannins have been reflected as an anti-nutrient, due to their ability to decrease the efficiency in converting the absorbed nutrients to relevant substances [61, 62]. Tea polyphenols and several components of tannin have been suggested as anti-carcinogenic and many tannin molecules have also been shown to reduce the mutagenic activity of several mutagens. These properties have been attributed to their anti-oxidative properties which enable them to defend against oxidative impairment [61].

4.6 Anti-nutrients

The ‘anti-nutrients’, comprise lectins, oxalates, phytates, phytoestrogens and tannins [63]. Phytates and oxalates were detected in the CHTs as CHT-F > CHT-A > CHT-G > CHT-C > CHT-D > CHT-E (Tables 1, 3-7). Oxalate was higher than phytate in CHT-F (16.77 %), CHT-A (12.76 %) and CHT-G (12.71 %) whereas phytate was higher than oxalate in CHT-C (13.51 %), CHT-D (11.53 %) and CHT-E (4.57 %). Anti-nutrients limit the bioavailability of vital nutrients by binding to vital micronutrients which prevents the body from absorbing them or hindering the peak effects of some digestive enzymes, thereby, inhibiting the appropriate breakdown of food [63]. For example, oxalates are known to affect calcium absorption and use by forming calcium oxalate crystals which could lead to kidney stones. They also irritate and cause swelling in the mouth and throat, and are capable of forming tissue crystals leading to indications of arthritis [64]. Some of the health benefits derivable from dietary phytate include anti-cancer, anti-calcification, antioxidant, antihyperglycaemic and hypolipidaemic activities [63, 65]. They can bind to harmful trace elements like lead and cadmium thereby reducing their bioavailability. It has been associated with certain health benefits, including blood glucose – and lipid-lowering effects, anticancer activity, antioxidant properties, and anti-calcification. The ability of phytate to bind toxic trace elements such as cadmium and lead and reduce their bioavailability has been documented [66].

4.7 Steroids

Steroids were detected in the CHTs, but, due to the limitations of the study, the type of steroid identified was not identified. However, steroids were detected in the order CHT-G (13.12 %) > CHT-A (12.95 %) > CHT-C (10.05 %) > CHT-D (8.93 %) > CHT-F (7.83 %) > CHT-E (6.27 %) > CHT-B (6.23 %) (Tables 1-7). Amongst the plant-based steroids, phytosterols are the most abundant [67]. They are known to reduce blood cholesterol by inhibiting intestinal absorption of cholesterol thereby reducing the risk of heart attack and stroke [67]. They have also been demonstrated to slow the in vitro development and progression of various cancers [68].

4.8 Resveratrol

Resveratrol, a non-flavonoid polyphenol was detected in low levels in CHT-D (2.28 %) > CHT-E (1.70 %) > CHT-B (1.57 %). It is a polyphenolic phytoalexin formed by plants like grapes, peanuts and berries and retains anti-inflammatory, antioxidant, antiplatelet, anticancer and anti-diabetic activities [69, 70]. In vitro investigations have also revealed its ability to prevent all phases of carcinogenesis comprising initiation, promotion and progression at lower doses [71]. However, at higher doses, resveratrol acts as a pro-apoptotic compound which signals the death of cancer cells. They are also able to depress cardiac function [71].

5. Conclusion

The GC-FID-guided phytochemical identification and quantification of selected CHTs showed that they contain mostly detectable flavonoids, alkaloids, glycosides, saponins, tannins, steroids, anti-nutrients and other phenols such as resveratrol. This present study corroborates the literature on the abundant phytochemical constituents of chamomile which serves as the basis for the numerous health benefits ascribed to chamomile herbal tea.

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