GC- MS, FTIR, UV Analysis and in vitro Antioxidant Activity of a Nigeria Poly Herbal Mixture: Pax Herbal Bitters

Elufioye and Mada: GC- MS, FTIR, UV Analysis and in vitro Antioxidant Activity of a Nigeria Poly Herbal Mixture: Pax Herbal Bitters

Authors

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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).

Table 1

Percentage (%) yield of various extracts/fractions of Paxherbal Bitters.

FractionWeight (g)(w/v) %
n-Hexane1.100.0579
Dichloromethane1.520.08
DCM residue1.490.0784
Methanol4.510.237
Aqueous0.670.0353

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.

MetaboliteTestObservationInference
AlkaloidsDragendorffFormation of reddish black precipitate indicates the presence of alkaloids.present
WagnerThe formation of brownish precipitate indicates the presence of alkaloids.present
MayerThe formation of a white creamy precipitate shows and indicates presence of alkaloids.present
FlavonoidsGeneral testThe presence of pinkish red colour that was developed within 2-3 minpresent
Lead acetate testFormation of yellow color precipitate indicates the presence of flavonoids.present
Alkaline Reagent test Formation of intense yellow colour, which becomes colourless on addition of dilute acid.Present
GlycosidesBorntragersFormation of rose-pink color in the ammoniacal layerPresent
Cardiac glycosidesKeller-Kiliani test Formation of a purple, reddish brown, brown ring at the interface and green colour in the acetic layer.present
SaponinsFoam testPersistence of the foam formed for about ten min.Present
Froth TestFormation of more than 1cm layer of foam which stood for more than 5 min.Present
Steroids/ TerpenesSalkowski’s TestThe presence of a golden yellow colouration.Present
Liebermann-burchard testThe appearance of brown ring at the interphase.Present
TanninsLead acetate testThe presence of white precipitate.present
PhenolsFerric chloride testFormation of bluish black colourPresent
AnthraquinonesBorntrager testFormation of a rose-pink colouration in the aqueous layer prior to the addition of 10% ammonia solution.present

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.

Figure 1

DPPH radical scavenging activity of various fractions of Pax herbal bitters.

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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.

Figure 2

Total phenolic content (TPC).

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Total Flavonoid content (TFC)

Total flavonoids content is represented in Figure 3.

Figure 3

Total Flavonoid Content (TFC).

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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.

Figure 4

GC-MS peaks of n-hexane fraction.

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Table 3

Names of chemicals, molecular formula and peak area of compounds from n-Hexane fraction.

PeakRetention time (RT) minName of compoundMolecular formulaMolecular weightArea of compound% peak area
15.570(9-Oxabicyclo[3.3.1]non-6-en-3-yl) Methanol CyclohexanolC8H12O124.181592460.327
25.948IsoborneolC10H18O154.252959904.540
36.200Terpinen-4-olC10H18O154.2522135681.576
46.457Bicyclo[4.1.0]heptan-3-ol, 4,7,7-trimethyl-, (1.alpha.,3.alpha.,4.be ta.,6.alpha.)C14H12O206.327684894.606
511.792,4-Di-tert-butylphenol2245823
612.334CyclohexanemethanolC7H14O114.1928922495.932
712.419(1R,7S,E)-7-Isopropyl-4,10-dimethylenecyclodec-5-enolC15H24204.3423153014.748
812.797(1aR,4S,7R,7aS,7bR)-1,1,7-Tetramethyl-1a,2,3,4,6,7,7a,7b-octahydro-1H Cycloprop[e]azulen-7-olC15H24O22013683792.086
913.318(1R,9R,E)-4,11,11 Trimethyl-8-methylenebicyclo[7.2.0]undec-4-eneC15H24204.3545483819.328
1013.569IsospathulenolC15H24O220.35977182620.041
1113.804(1S,4aR,7R)-1-4a-Dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,7-octahydronaphthaleneC15H24204.3510427072.138
1213.896AlloaromadendreneC15H24204.356900361.415
1314.411Ledene alcoholC15H25O220.3522671114.650
1415.921p-HeptylacetophenoneC15H22O218.3446003789.345
1518.0272,4-Decadienamide, N-isobutyl-, (E,E)-C14H25NO223.35619067612.696
1622.398ShogaolC17H24O3276.38738881215.154
Table 4

Correlation of identified compounds with reported biological activity.

S/NName of the compoundBiological activityReferences
1(9-Oxabicyclo[3.3.1]non-6-en-3-yl)Methanol CyclohexanolNo yet reported
2IsoborneolAntioxidant, neuroprotective15
3Terpinen-4-olAnti-inflammatory, antifungal, antibacterial16, 17
4Bicyclo[4.1.0]heptan-3-ol, 4,7,7-trimethyl-, (1.alpha.,3.alpha.,4.be ta.,6.alpha.)Antioxidant15
52,4-Di-tert-butylphenolAntioxidant, antifungal
6CyclohexanemethanolAnti-inflammatory, antiviral
7(1R,7S,E)-7-Isopropyl-4,10-dimethy lenecyclodec-5-enol
81H-Cycloprop[e]azulen-7-ol
9(1R,9R,E)-4,11,11-Trimethyl-8-methlenebicyclo[7.2.0]undec-4-eneAnti-inflammatory, neuroprotective, antidepressant, anti-alcoholism
10IsospathulenolImmunoinhibitory18
11Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2R(2.alpha.,4a.alpha.,8a.beta.)]-Antifungal19
12AlloaromadendreneAntioxidant,anti-aging, antimicrobial agent15,12
13Ledene alcoholAntioxidant, antifungal,20
14p-HeptylacetophenoneAntiallergic
152,4-Decadienamide,N-isobutyl-, (E,E)-Antioxidant and protection agent
16ShogaolAnticough, induce apoptosis, memory enhancer21, 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.

Figure 6

FTIR of Crude extract.

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Figure 7

FTIR peaks of methanolic fraction.

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Figure 8

FTIR peaks of n-Hexane fraction.

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Figure 9

FTIR from DCM Residue.

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Figure 10

FTIR peaks of DCM fraction.

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Table 5

FTIR peak values and functional groups of crude extract of Pax herbal bitters.

PeaksFunctional groups
3399.00Alcohols, phenols, carboxylic acid
2937.14Alkane, aldehyde
2371.42Unknown
1617.33Amine, amide, alkene
1446.76Alkane, Nitro
1364.45Nitro, alkane, fluoride
1269.91Esters, ethers, anhydride, alcohol, carboxylic acids
1066.29Esters, ethers, cyanide
592.00Alkyl halide
469.66Alkyl halide

UV Analysis

The UV spectra of the hexane, DCM and crude extracts are as shown in Figures 11-13.

Figure 11

UV Spectrum from crude extract.

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Figure 12

UV Spectrum of n-Hexane extract.

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Figure 13

UV Spectrum of DCM Extract.

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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

Figure 5

Mass spectra of some of the identified compounds from Paxherbal Bitter.

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Figure 14

Chemical structures of some of the identified compounds from paxherbal bitters.

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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.

Peak valuesFunctional groups
3407.00Amine, amide, carboxylic acid
2937.14Alkane stretch, aldehyde
2337.14Unknown
1629.00Alkene (C=C), amide (C=O)
1446.15Alkanes (-CH3)
1393.00Alkanes (-CH3)
1273.31Amine
1021.53Amine
694.61C-X, Aromatic alkane out of plane
570.59Alkyl halide {C-X (X= Br, I))
Table 7

FTIR peak values and functional groups of DCM extract of pax herbal bitters.

Peaks valuesFunctional groups
3423.00Amine, amide, alcohol including phenol
2930.92Alkane stretch
1705.91Ketone, C=O (COOH), carboxylic acid
1610.67Non- acid carbonyl
1511.91Nitro
1454.49Aromatic alkane, Nitro (R-NO2), N=O
1370.20Nitro, Alkane
1262.16C-X (X=F), S=O
1162.17Alcohol, ethers, esters, anhydride
1122.27Amines, alcohol, esters, ethers, anhydride
1035.39Alcohol
625.63Alkyl halide
566.50Alkyl halide
Table 8

FTIR peak values and functional groups of DCM residue of pax herbal bitters.

Peak valuesFunctional groups
3396.21Alcohols, phenols, carboxylic acids, primary and secondary amine
2931.14Alkane, aldehyde
1621.53Alkene, amide
1444.30Alkane, Nitro
1363.60Nitro, sulfones, sulfonyl chloride
1270.46Esters, ethers, anhydride, alcohol
1076.51Alcohol, amines, esters, ethers
601.63Alkyl halide
404.14Alkyl halide
Table 9

FTIR peak values and functional groups of n-Hexane fraction of pax herbal bitters.

Peak valuesFunctional groups
3375.00Alcohol, phenol, carboxylic acid
2933.00Alkane, aldehyde
2360.00Unknown
2045.71Alkyne, nitriles
1711.75Ketone, carboxylic acid
1504.14Nitro, aromatic
1454.93Alkane, Nitro
1373.05Nitro, alkane, fluoride
1246.60Esters, ether, alcohol, anhydride
1156.47Cyanide, esters, ether
1036.76Esters, ether
939.69Alkyl halide
606.60Alkyl halide

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.

CONCLUSION

The herbal bitters studied revealed the presence of several chemicals with reported biological activity which may be contributing to the pharmacological claims of the product.

ACKNOWLEDGEMENT

We would like to acknowledge the National Institute of Technology for providing the library data that enabled the identification of the chemical constituents in the samples.

CONFLICT OF INTEREST

Authors declare no conflict of interest.

ABBREVIATIONS

DCM

Dichloromethane

DPPH

2, 2-diphenyl-1-picrylhydrazyl hydrate

TPC

Total phenolic content

TFC

Total flavonoids content

GC–MS

Gas Chromatography-Mass Spectroscopy

FTIR

Fourier-transform infrared spectroscopy

UV

Ultra-violent.

REFERENCES

1. 

Karan V, Kumar V , authors. Trade and Production of Herbal Medicines and Natural Health Products, sponsored by ICN-UNIDO. AREA Sciences Park. Trieste, Italy: 2002;91:

2. 

Bannerman RH , author. Traditional Medicine in modern health care. World Health Forum. 1982;3(1):8–13

3. 

World Health Organization. Quality Control Methods for Medicinal Plant Materials. WHO Geneva: 1998

4. 

Tabassum N, Ahmad F , authors. Role of natural herbs in the treatment of hypertension. Pharmacog Rev. 2011;5(9):30

5. 

Oyewo EB, Adetutu A, Adebisi JA , authors. Immunomodulatory activities of Yoyo Bitters: recommended dose precipited inflammatory response in male Wistar Rats. Pakistan Journal of Biological Sciences. 2013. p. 1–9

6. 

Ogbonnia SO, Mbaka A, Igbokwe NH, Anyika EN, Alli P, Nwakakwa N , authors. Antimicrobial evaluation, acute and subchronic toxicity studies of Leone bitters, a Nigerian polyherbal formulation, in rodents. Agric Biol J N Am. 2010;1(3):366–76

7. 

Cousins D, Huffman MA , authors. Medicinal properties in the diet of gorillas: An ethno-pharmacological evaluation. Afr Study Monographs. 2002;23:65–89

8. 

Saganuwan AS , author. Some medicinal plants of Arabian Pennisula. J Med Plants Res. 2010;4(9):766–88

9. 

Trease GE, Evans WC , authors. A Text-book of Pharmacognosy. 1989a. Bailliere Tindall Ltd; London: p. 53

10. 

Brand-Williams W, Cuvelier M, Berset C , authors. Use of free radical method to evaluate antioxidant activity. LWT-Food Science and Technology. 1995;28(1):25–30

11. 

Singleton VL, Rossi JA , authors. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult. 1965;16(3):144–58

12. 

Miliauskas G, Venskutonis PR, Van Beek TA , authors. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry. 2004;85(2):231–7

13. 

Yousif E, Haddad R, El-Hiti GA, Yusop RM , authors. Spectroscopic and photochemical stability of polystyrene films in the presence of metal complexes. Journal of Taibah University for Science. 2017;11(6):997–1007

14. 

Loizzo MR, Tundis R, Conforti F, Menichini F, Bonesi M, Nadjafi F , authors. et al. Salvia leriifolia Benth (Lamiaceae) extract demonstrates in vitro antioxidant properties and cholinesterase inhibitory activity. Nutrition Research. 2010;30(12):823–30

15. 

Abdelhady MI, Hamdy AH , authors. Antioxidant and antimicrobial activities of Callistemon comboynesis essential oil. Free Rad Antioxidants. 2012;2(1):37

16. 

Carson CF, Hammer KA, Riley TV , authors. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev. 2006;19(1):50–62

17. 

Hammer KA, Carson CF, Riley TV , authors. Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J Antimicrob Chemother. 2004;53(6):1081–5

18. 

Bamoniri A, Mazoochi A , authors. Determination of bioactive and fragrant molecules from leaves and fruits of Ferula assa-foetida L. growing in central Iran by nano scale injection. Digest Journal of Nanomaterials and Biostructures (DJNB). 2009;4(2):323–8

19. 

Wang C, Wang Z, Qiao X, Li Z, Li F, Chen M , authors. et al. Antifungal activity of volatile organic compounds from Streptomyces alboflavus TD-1. FEMS Microbiology Letters. 2013;341(1):45–51

20. 

Chan-Wei Y, Wen-Hsuan L, Fu-Lan H, Pei-Ling Y, Shang-Tzen C, Vivian HL , authors. Essential Oil Alloaromadendrene from Mixed-Type Cinnamomum osmophloeum Leaves Prolongs the Lifespan in Caenorhabditis elegans. J Agric Food Chem. 2014;62(26):6159–65

21. 

Suekawa M, Ishige A, Yuasa K, Sudo K, Aburada M, Hosoya E , authors. Pharmacological studies on ginger. I. Pharmacological actions of constituents of pungent constituents, (6)-gingerol and (6)-shogaol. Journal of Pharmacobio-dynamics. 1984;7(11):836–48

22. 

Moon M , author. 6-Shogaol, an active consistent of ginger, attenuates neuroinflammation and cognitive deficits in animal models of dementia. Biochemical and Biophysical Research Communications. 2014;449(1):8–13

23. 

Aniagu SO, Nwinyi FC, Akumka DD, Ajoku GA, Dzarma S, Izebe KS , authors. et al. Toxicity studies in rats fed nature cure bitters. African Journal of Biotechnolog. 2005;4(1):72

24. 

Barnett R , author. Bitter medicine: gout and the birth of the cocktail. The Lancet. 2012;379(9824):1384–5

25. 

Elufioye TO, Awosika OA , authors. Quality evaluation of Poza bitters, a new poly herbal formulation in the Nigerian market. Afri J Trad Compl and Alter Med. 2015;12(1):17–22

26. 

Olumese EO, Adegbolagun OM , authors. Comparative Physicochemical and Microbial Evaluation of Six Herbal Bitters Distributed Within Southwestern Nigeria. Nig. J Pharm Res. 2017;11(1):132–9

27. 

Adeoye AO , author. Galenicals in modern medicine: Focus on Swedish bitters. Nig J Nat Pro Med. 1997;1(1):6–9

28. 

Adeyemi OS, Fambegbe M, Daniyan OR, Nwajei I , authors. Yoyo Bitters. A polyherbal formulation influenced some biochemical parameters in Wistar rats. J Basic and Cli Physio and Pharmacol. 2012;23(4):135–8

29. 

Onyeaghala AA, Omotosho IO, Shivashankara AR , authors. Chemical Isolation and Characterization of a Popular Detoxifying Herbal Remedy Yoyo Bitters (YYB) Using GC-MS, NMR and FTIR Analysis. Int Res J Pure and Applied Che. 2015;6(4):190–200

30. 

Madziga HA, Sanni S, Sandabe UK , authors. Phytochemical and Elemental Analysis of Acalypha wilkesiana Leaf. J Ame Sci. 2010;6(11):510–4

31. 

Rahman MM, Gray AI , authors. A benzoisofuranone derivative and carbazole alkaloids from Murraya koenigii and their antimicrobial activity. Phytochem. 2005;66(13):1601–6

32. 

Han S, Siegel DS, Morrison KC, Hergenrother PJ, Movassaghi M , authors. Synthesis and anticancer activity of all known (−)-Agelastatin alkaloids. The J Org Chem. 2013;78(23):11970–84

33. 

Nayak BS, Pereira LM , authors. Catharanthus roseus flower extract has wound-healing activity in Sprague Dawley rats. BMC Compl Alte Med. 2006;6(1):41

34. 

Kar A , author. Pharmaocgnosy and Pharmacobiotechnology (Revised-Expanded Second Edition). New Age International Limited Publishers; New Delhi: 2007. p. 332–600

35. 

Yalavarthi C , author. A review on identification strategy of phyto constituents present in herbal plants. Inter J Res Pharmaceut Sci. 2016;4(2):123–40

36. 

Edeoga HO, Okwu DE, Mbaebie BO , authors. Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotech. 2005;4(7):685–8

37. 

Sumner LW, Mendes P, Dixon RA , authors. Plant metabolomics: large-scale phytochemistry in the functional genomics era. Phytochemistry. 2003;62:817–36

38. 

Tighe S, Gao YY, Tseng SC , authors. Terpinen-4-ol is the most active ingredient of tea tree oil to kill Demodex mites. Translational Vis Sci Tech. 2013;2(7):2–8

39. 

Hammer KA, Carson CF, Riley TV , authors. Antifungal activity of the components of Melaleuca alternifloral (tea tree) oil. J Appl Microbiol. 2003;98:853–60

40. 

Harold M , author. On Food and Cooking: The Science and Lore of the Kitchen (2nd ed.). New York: Scribner; 2004. p. 425–6

41. 

Suekawa M, Ishige A, Yuasa K, Sudo K, Aburada M, Hosoya E , authors. Pharmacological studies on ginger. I. Pharmacological actions of constituents of pungent constituents, (6)-gingerol and (6)-shogaol. J Pharmacobio-dynamics. 1984;7(11):836–48

42. 

Moon M , author. 6-Shogaol, an active consistent of ginger, attenuates neuroinflammation and cognitive deficits in animal models of dementia. Biochem Biophy Res Comm. 2014;449(1):8–13

43. 

Brudler R, Rammelsberg R, Woo TT, Getzoff ED, Gerwert K , authors. Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy. Nat Struc Mol Bio. 2001;8(3):265–70

44. 

Torey A, Sasidharan S, Yeng C, Latha LY , authors. Standardization of Cassia spectabilis with respect to authenticity, assay and chemical constituent analysis. Molecule. 2010;15(5):3411–20

45. 

Tiainen E, Drakenberg T, Tamminen T, Kataja K, Hase A , authors. Determination of phenolic hydroxyl groups in lignin by combined use of 1H NMR and UV spectroscopy. Holzforschung. 1999;53(5):529–33

GRAPHICAL ABSTRACT

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SUMMARY

  • Pax herbal bitters contain several chemical compounds with previously proven biological activities that may account for the claimed pharmacological effects.

ABOUT AUTHORS

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Dr. Taiwo O. Elufioye: Is a senior lecturer in the Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Nigeria.