INTRODUCTION
Herbal medicine predates the other forms of health care used by humans and it has evolved alongside development of modern civilization.1 Herbal medicines in most developing countries have played a central role in health care since time immemorial.2 In most developing countries, herbs rather than drugs are often used in health care. Herbal medicines are ‘finished, labeled medicinal products that contain as active ingredients, above ground or underground parts of plants or other plant materials, or combinations thereof, whether in the crude state or as plant preparations.3 Plant materials in this case include juices, gums, resins, fatty oils, essential oils and any other substances of this nature. Herbal formulations in several dosage forms have been claimed to be very beneficial to human health. They are known to be effective in improving blood circulation, purifying the kidney and reducing the development of kidney stones.4 Other benefits include improving digestion, reducing blood pressure, assisting in the elimination of bad cholesterol, preventing the development of diabetes and improving the immune system and memory.4-5
Bitters are traditionally alcoholic preparations flavoured with botanical matter such that the end result is characterized by a bitter, sour, or bittersweet flavor. Medicinal herbal bitters contain blended ingredients in a water or alcohol (tincture) base. Originally sold as a digestive aid because of their ability to increase the production of saliva and digestive juices, bitters became popular in Europe in the 1600s. They generally have been reported to prevent kidney and bladder infections, help to regulate blood pressure and dilate arteries, facilitate digestion, prevent disorders like ulcers, gastritis, insomnia, stress and depression and prevent overweight and excess body fat.6 Phytochemical analysis has shown that bitters contain complex carbohydrate, alkaloids, vitamins and minerals that have antioxidant, antiviral and antispasmodic properties. It has also been shown that these ingredients work together to reduce inflammation, control pain, relax muscles and improve digestion and elimination.7 Bitters can also be effective as appetite stimulant.8
Pax herbal bitters® is a tincture of different herbal ingredients and has net volume of 190 mL, 6.42FL; characterized by dark brownish colour with strong bitter taste, aromatic odour and 100% moisture content. The bitter, according to manufacturer’s claim, was formulated to promote blood circulation, prevents kidney stones associated to digestion, activates bile flow, and increases immunity of the body against bacteria and fungi infections. This product, though widely accepted and used has no report on its chemical characterization. In this study, we profiled the metabolites present in different extracts of Pax herbal with the view to correlating them with its claimed pharmacological actions.
MATERIALS AND METHODS
Extraction
The Pax Herbal bitters® syrup (10 x 190 mL) were exhaustively extracted with n-hexane, dichloromethane (DCM) and methanol separately for 24 h. The different extracts were concentrated at 40oC using Rotary evaporator and stored for subsequent analysis.
Qualitative Phytochemical study
Analysis for various phytoconstituents in the formulation was carried using standard method.9 The presence of alkaloids, saponins, flavonoids, cardiac glycosides, and tannins were evaluated.
DPPH Antioxidant Assay
The radical scavenging ability of the bitters (crude) as well as DCM and methanolic extracts were determined using the stable radical DPPH (2, 2-diphenyl-1-picrylhydrazyl hydrate).10
Determination of Total phenolic content (TPC)
The total phenolic content of the bitters, DCM and methanolic extracts were determined using the folin-ciocalteu’s phenol reagent.11
Determination of Total flavonoids content (TFC)
This was carried out based on the aluminium chloride colorimetric assay method.12 Quercetin at varying concentrations was used as standard.
GC-MS analysis of n-Hexane fraction
GC – MS analysis was carried out using GC – MSD 5975 Agilent instrument. Column thickness, length, and internal diameter were 0.25 μm, 30 meters and 0.32 mm respectively. Helium was used as carrier gas at a flow rate of 10 mL/min. The column temperature was initially kept at 80ºC and increased to 290ºC at a rate of 10ºC /min. The injector temperature was 250ºC and split ratio was adjusted at 1:100. The injection volume was 2 μL in ethyl acetate and detector was Mass Selective Detector. The relative percentage peak area of each compound was calculated by dividing its average peak area with the total area of all compounds present. Detected peaks were interpreted by comparing with the National Institute of Standards and Technology (NIST) library data (Ver.2.0-Year 2005) to ascertain the names and molecular weights of the components of the test samples.13
FTIR analysis
FTIR analysis of the various extracts was done on Perkin Elmer Spectrophotometer system in the mid IR region of 400-4000 cm-1 with 16 scan speeds.14
UV-VIS spectroscopy analysis
UV-Visible spectra for various extracts was performed on a PerkinElmer lambda 25 UV-VIS spectrophotometer equipped with 1.0 cm quartz cells.14
RESULTS
Percentage yield
The solvent-solvent extraction gave a yield of 0.0579 % (w/v) for n-Hexane extract, 0.08 % (w/v) for DCM, 0.0784 % (w/v) for DCM residue, 0.237 % (w/v) for methanol extract and 0.0353 % (w/v) for aqueous (Table 1).
Phytochemical screening
The result from the qualitative phytochemical screening carried out on the crude bitter is as presented in Table 2.
Table 2
Phytochemical screening of crude bitters.
DPPH radical scavenging antioxidant assay
The DPPH radical scavenging antioxidant activity result indicating the highest activity in the DCM extract is represented in Figure 1.
Total Phenolic content (TPC)
The Total phenolic composition of the extracts is shown in Figure 2. Methanol extract had highest phenolic content of 20 mg/g followed by DCM extract (5.39 mg/g) and crude extract (4.43 mg/g) using Garlic as the standard.
GC-MS
The GC-MS analysis of the hexane fraction showing various peaks is presented in Figure 4 with the interpretation when compared with a standard library data shown in Table 3. The correlation of some of the identified compounds with reported biological activities is also presented in Table 4.
Table 3
Names of chemicals, molecular formula and peak area of compounds from n-Hexane fraction.
Table 4
Correlation of identified compounds with reported biological activity.
| S/N | Name of the compound | Biological activity | References |
|---|---|---|---|
| 1 | (9-Oxabicyclo[3.3.1]non-6-en-3-yl)Methanol Cyclohexanol | No yet reported | |
| 2 | Isoborneol | Antioxidant, neuroprotective | 15 |
| 3 | Terpinen-4-ol | Anti-inflammatory, antifungal, antibacterial | 16, 17 |
| 4 | Bicyclo[4.1.0]heptan-3-ol, 4,7,7-trimethyl-, (1.alpha.,3.alpha.,4.be ta.,6.alpha.) | Antioxidant | 15 |
| 5 | 2,4-Di-tert-butylphenol | Antioxidant, antifungal | |
| 6 | Cyclohexanemethanol | Anti-inflammatory, antiviral | |
| 7 | (1R,7S,E)-7-Isopropyl-4,10-dimethy lenecyclodec-5-enol | ||
| 8 | 1H-Cycloprop[e]azulen-7-ol | ||
| 9 | (1R,9R,E)-4,11,11-Trimethyl-8-methlenebicyclo[7.2.0]undec-4-ene | Anti-inflammatory, neuroprotective, antidepressant, anti-alcoholism | |
| 10 | Isospathulenol | Immunoinhibitory | 18 |
| 11 | Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2R(2.alpha.,4a.alpha.,8a.beta.)]- | Antifungal | 19 |
| 12 | Alloaromadendrene | Antioxidant,anti-aging, antimicrobial agent | 15,12 |
| 13 | Ledene alcohol | Antioxidant, antifungal, | 20 |
| 14 | p-Heptylacetophenone | Antiallergic | |
| 15 | 2,4-Decadienamide,N-isobutyl-, (E,E)- | Antioxidant and protection agent | |
| 16 | Shogaol | Anticough, induce apoptosis, memory enhancer | 21, 22 |
FTIR Analysis
The FTIR peaks of the various samples including the crude drug, methanol, hexane, DCM extracts and DCM residue are presented in Figures 6-10 while the identified functional groups are reported in Table 5.
Table 5
FTIR peak values and functional groups of crude extract of Pax herbal bitters.
DISCUSSION
Bitters are composed of complex mixture of compounds with a wide range of molecular structures. This is because bitters are usually made as aqueous or alcoholic extracts of several medicinal plants. This complexity makes complete characterization of the chemical composition difficult. Notwithstanding, the compositional data of bitters is important for a better understanding of the very many pharmacological claims attributed to them. Few studies have contributed toward the current knowledge of medicinal bitters. Some of these involve assessment of toxicity,23 efficacy,24 quality evaluation,25 physicochemical analysis,26 formulation studies,27 and effect on biochemical parameters.28 There are only few characterization studies for instance Yoyo bitters.29
In this research, we profiled the metabolites in Pax herbal bitters using three different spectroscopic methods. Samples used for analysis were purchased from pharmacies in Ibadan, South western Nigeria.
Qualitative phytochemical screening of crude Paxherbal bitters indicated the presence flavonoids, saponins, alkaloids and steroids while phenols, tannins, anthraquinone, glycosides and terpenoids were mederately present. Alkaloids have pharmacological applications as anesthetics and CNS stimulants,30 antimicrobial,31 anticancer,32 memory enhancing,33 and many others. Antioxidants and free radical scavengers have been attributed to flavonoids.34 Phenolics essentially represent a host of natural antioxidants, used as nutraceuticals to combat cancer, as an anti-inflammatory agent and to prevent heart ailments to an appreciable degree.35 Terpenoids and steroids are a large and diverse class of naturally occurring chemicals found in all classes of living organisms. Glycosides are drugs used in the treatment of heart diseases and are found as secondary metabolites in several medicinal plants.36
Qualitative analysis of the samples revealed highest phenolic in the methanol (20 mg/g) followed by DCM extract (5.39 mg/g) while flavonoids were found to be in almost equal amount DCM and methanolic extract (2.548 mg/g). This result supports previous report that bitters contain flavonoids, phenols and polyphenols which are believed to be responsible for antioxidants activity. Flavonoids are polyphenols found mostly in fruits, vegetables and certain beverages that have diverse beneficial effects. DPPH radical scavenging antioxidant activity determined was also found to be higher in the DCM extract compare to methanolic and the crude extracts thus corroborating the antioxidant potentials of flavonoids in bitters.
A combination of GC-MS, FTIR and UV spectroscopic methods was used to characterize the various fractions of the bitters. Over the past years, many highly accurate and sensitive methods for the analysis of complex mixtures of compounds have been developed.37 However, GC-MS covers relatively larger classes of compounds.
GC-MS analysis of n-Hexane fraction revealed sixteen compounds thirteen of which has been reported with biological activities while three have not been linked to any biological activity as shown in Tables 3 and 4. The GC-MS spectra of compounds identified from n-Hexane fraction showing the retention time and peak area of the various compounds are shown in Figure 4. A typical mass spectrum of some of the chemicals obtained after matching with data in the NIST library is also shown in Figure 5 and some of their structures are as shown in Figure 14. Terpinen-4-ol has been reported to be the most active ingredient in tea tree oil with antibacterial38 and antifungal39 effects. Shogaol is a pungent constituent of ginger similar in chemical structure to gingerol.40 It is a strong antitussive and it has been reported to reduce blood pressure and gastric contraction.41 It has also been linked also to memory and cognitive enhancing properties.42
FTIR spectroscopy is a useful method for obtaining information on the chemical nature of natural product mixture. It detects the vibrational frequencies and intensities of individual functional groups of the components in the mixture with high sensitivity and time resolution and permit quantification of specific classes of dissolved organic matters including aromatic and aliphatic organic compound containing oxygen, nitrogen, and sulfur functional groups.43 The use of FTIR spectral fingerprinting for herbal preparation tends to focus on the identification and assessment of the stability of the functional groups in chemical constituents. The results of the FTIR spectrum of n-Hexane extract of Pax Herbal Bitters is shown in Tables 5-9. About fourteen areas were identified in the mid infrared (MID) domain and the fingerprint region. The FTIR confirmed the presence of alcohols, phenols, alkanes, alkynes, alkyl halide, aldehyde, esters, carboxylic acids, aromatics, nitro compounds and amines in all the extracts. FTIR spectroscopy has been proven to be reliable and sensitive method for detection of biomolecular composition and can assist the manufacturer in controlling and ensuring the consistency and quality standard of products.44
Table 6
FTIR peak values and functional groups of methanolic extract of pax herbal bitters.
Table 7
FTIR peak values and functional groups of DCM extract of pax herbal bitters.
Table 8
FTIR peak values and functional groups of DCM residue of pax herbal bitters.
Table 9
FTIR peak values and functional groups of n-Hexane fraction of pax herbal bitters.
UV absorbance has been shown to be useful in estimating dissolved aromatic carbon content for instance the phenolic hydroxyl groups in a sample.45 The qualitative UV spectrum profile of crude, n-Hexane and DCM extracts of PaxHerbal Bitters in Figures 11 to 13 was selected from wavelength 190 to 900 nm of both UV and VIS region. The various peaks as seen above also contributed to structural elucidation of compounds which may be present in the polyherbal mixture.
