INTRODUCTION:
Hyperpigmentation can be treated through the inhibition of the enzyme tyrosinase. Melanin is a chemical found in the skin for the darkening of the skin. Therefore cosmetics treat hyperpigmentation through the inhibition of the enzyme tyrosinase. Tryosinase catalyzes the addition of -OH group on tyrosine to form 3-(3,4- dihydroxyphenyl)-L- alanine (DOPA). It further converts DOPA into dopaquinone. Known cosmetics for hypopigmentation includes adverse agents like corticosteroids and hydroquinone that inhibits melanocyte maturation, Kojic acid and hydroxyquinone are also used for the inhibition of tyrosinase, but they possess some levels of toxicity1-2. Therefore, the search for local plants with less toxicity is a welcome development.
It is a known fact that diet, lifestyle and stress are major causes of modern-day diseases. When the rate of production of free radicals outweighs the rate of cellular antioxidant defense it is termed oxidative stress. This delicate system when tilted towards more production of reactive oxygen species leads to many chronic, cardiovascular, hyperpigmentation, neurodegenerative diseases etc. Antioxidants in the body protect cells from oxidative damage from radicals3-5. However, in most disease state and stressed situations, these protective antioxidants become very low or absent. Therefore, there is the need for supplement from plants.
Acmella caulirhiza is a flowering herbaceous plant in the family asteraceae. The plant produces gold/yellowish flowers. Locally the plant is employed in the management of analgesics, toothaches and sores. This suggests that Acmella caulirhiza may be bacteriocidal or fungicidal6-7. Acmella caulirhiza is active against anopheles larvae8-9. Acmella caulirhiza is rich in Medicinal constituents. Many parts of the plant contain spilanthol responsible for its soothing properties10. Literatures searched indicated that there are few articles on HPLC profiling of phenolics in Acmella caulirhiza, antioxidants and antityrosinase activity. That is why this research was carried out.
MATERIALS AND METHOD:
Reagents:
Methanol, sodium hydroxide, distilled water, DPPH, Dipotassium hydrogen phosphate, Dihydrogen potassium phosphate,sulfuric acid, sodium phosphate, ammonium molybdate, ascorbic acid, phosphoric acid, histidine, sodium hypochlorite, potassium nitrite, N-N-dimethyl-P-nitrosaniline, hydrochloric acid, Evans blue, hydrogen peroxide, nicotinamide adenine dinucleotide reduce (NADH), Nitroblue tetrazolium (NBT), manganese oxide, taurine, 2,4,6-tripyridyltriazine (TPTZ), ferric chloride, sodium acetate, acetic acid, N-methylphenazine methosulphate (PMS), rutin, ascorbic acid, gallic acid and quercetin.
Sample collection:
The plant Acmella caulirhiza (fresh flower) was harvested from Ebeni Community, Southern Ijaw Local Government Area, Bayelsa State. The plant was botanically confirmed in the Department of Pharmacognosy, Niger Delta University, Bayelsa State. May, 2023. Herbarium number: NDU0127.
Preparation of Methanolic extracts of Acmella caulirhiza flower:
The Acmella caulirhiza flower were harvested and kept at room temperature to be dried for 14 days (two weeks). It was crushed to form particles. A total of 6.48g of crushed flower was soaked with 400ml of methanol for 72 hours (3 days). It was later centrifuged at 5000xg for 10 min and supernatant collected. The supernatant was concentrated. The extract was weighed and it was 1.05g. It was then kept safely for further use.
Total Flavonol Content:
The method of Milliauskas11 was adopted in a reaction mixture consisting of AlCl3 (2g/100ml) and sodium acetate (5g/100ml) incubated at 20 0C for 2hrs 30min in the presence of Acmella caulirhiza (5mg/ml) or different concentrations of rutin as reference. Total flavonol was calculated as mg rutin equivalent per g extract.
Total Anthocyanin Content:
The pH difference method of Lee12, was utilized. The absorbance at 510nm and 700nm for a mixture of Acmella caulirhiza and hydrochloric acid potassium buffer pH 1 were used for calculation. The absorbance at 510nm and 700nm for a mixture of Acmella caulirhiza and acetic acid sodium acetate buffer pH 4.5, were used for calculation, of total anthocyanidin 3-glycoside equivalent/g extract.
B-Carotene and Lycopene Content:
B-carotene and lycopene were extracted from Acmella caulirhiza flower using acetone-hexane according to Nagata and Yamashita13. The absorbances were calculated and expressed as mg lycopene/g extract and mg B-carotene/g extract.
Total Antioxidant Capacity (TAC):
The total antioxidant capacity of Acmella caulirhiza was evaluated based on the transfer of electron on ammonium molybdate (VI) blue to greenish molybdate (V) which absorbs at 695nm by the plant extract as described by Prieto14. The (TAC) was calculated based on the reference antioxidant ascorbic acid as µg ascorbic acid equivalent/gram extract (µg AAE/g).
Ferric Reducing Antioxidant Power (FRAP):
Benzie and Strain15 showed that plant extracts antioxidant potentials can be measured through the reduction of 2, 4, 6 tri-(2 pyridyl)-1, 3, 5 triazine at low pH. The FRAP reagent was mixed with different concentrations of Acmella caulirhiza at (0 – 250 µg/ml), ascorbic acid serves as reference and results were presented as percentage.
1, 1-Diphenyl -2 Picrylhydrazyl Radical Scavenging
DPPH radical prepared in absolute ethanol at 0.4 mM was added to Acmella caulirhiza extract. The decrease in absorbance of different concentrations of extract measured spectrophotometrically and was compared with ascorbic acid as standard Gyamfi16.
Hypochlorous Scavenging Assay:
The reaction mixture consisted of Acmella caulirhiza extract at 0 – 250 µg/ml, phosphate buffer, hypochlorous acid, taurine and potassium iodide as described by Weiss17. The yellowish solution was measured at 350 nm and ascorbic acid served as standard.
Singlet Oxygen Scavenging Assay:
Singlet oxygen is produced by NaOCl/H2O2 reaction as described by Chakraborty18, which react with N,N dimethyl-p-nitrosoaniline to discolor it. N,N dimethyl-p-nitrosoaniline absorbs at 440nm, therefore the addition of Acmella caulirhiza in the reaction mixture at 0 – 250 µg/ml prevents the bleaching of N,N dimethyl-p-nitrosoaniline in the reaction mixture measured through absorbances and results calculated as percentages rutin served as standard.
Superoxide Anion Radical Scavenging Assay:
The reaction mixture 1ml contained phosphate buffer, N-methylphenazine methosulphate (PMS), NADH (nicotineamide-adenine-dinucleotide), nitroblue tetrazolium chloride (NBT) and Acmella caulirhiza. The bleaching of NBT was measured at 562 nm described by Robak19.
Peroxynitrite Scavenging Assay:
In a 1 ml reaction mixture containing peroxynitrite, Acmella caulirhiza and Evans blue as reported by Hazra20. The peroxynitrite scavenging was determined at 305 nm and ascorbic acid used as standard.
In Vitro Antityrosinase Activity:
The reaction mixture for antityrosinase included L-DOPA, phosphate buffer, Acmella caulirhiza or kojic acid as refence and 165 unit/ml mushroom tyrosinase. The absorbance was detected at 475 nm as described by Zhang21.
High performance liquid chromatography of methanolic extract of Acmella caulirhiza:
Methanolic extract of Acmella caulirhiza was dissolved in methanol (50mg/ml) and was loaded in Agilent Phenyl column (10 µL) and heated at 250C, the elution was 1ml/min of water and acetonitrile. phenolics were detected and standard was also prepared as sample22.
Statistical analysis:
The data are mean ± SEM, n = 3. The data was processed using SPSS version 17.0 operated on windows systems New York, USA. Significant values are considered at p < 0.05.
RESULTS:
Percentage yield of plant
Percentage yield was 41.02%
Table 1. Showing result of total antioxidant and phytochemicals in Acmella caulirhiza methanolic extract
|
Antioxidant/Phytochemicals |
Acmella caulirhiza |
|
Total Antioxidant content |
103.65 ± 3.11 (μgAAE/g) Extract |
|
Anthocyanin |
19.20 ± 1.3 (mg C-3-GE/g) |
|
Flavonol |
466.13 ± 3.79 (mg RE/g) |
|
b-carotene |
2.25 ± 0.17 mg/100ml |
|
Lycopene |
0.16 ± 0.04 mg/100ml |
Values are mean ± SD of triplicate samples.
AAE = Ascorbic Acid Equivalent
C-3-GE = cyanidin-3-glucoside equivalent
RE = rutin equivalent
Figure 1: percentage inhibition of FRAP by Acmella caulirhiza flower extract and the standard reductant ascorbic acid, values are mean±S.D of triplicate values p<0.05 as compared with the 0µg/ml
Figure 2: percentage inhibition of DPPH by Acmella caulirhiza flower extract and the standard reductant ascorbic acid, values are mean±S.D of triplicate values p < 0.05 as compared with the 0µg/ml
Figure 3 percentage inhibition of Hypochlorous acid (HOCl) by Acmella caulirhiza flower extract and the standard reductant ascorbic acid, values are mean±S.D of triplicate values p<0.05 as compared with the 0µg/ml
Figure 4 percentage inhibition of singlet oxygen by Acmella caulirhiza flower extract and the standard rutin, values are mean±S.D of triplicate values p < 0.05 as compared with the 0µg/ml
Figure 5 percentage inhibition of superoxide anionby Acmella caulirhiza flower extract and the standard quercetin, values are mean ± S.D of triplicate values p < 0.05 as compared with the 0µg/ml
Figure 6: percentage inhibition of peroxynitriteby Acmella caulirhiza flower extract and the standard gallic acid, values are mean±S.D of triplicate values p< 0.05 as compared with the 0µg/ml
Fig. 7: Showing the HPLC chromatogram of phenolics in Acmella caulirhiza extract
Table 2 showing retention time (RT), amount (mg/100g) and name of phenolic compound
|
Retention time (min) |
Amount (mg/100g) |
Phenolic compound |
|
8.072 |
1.57 x 10-6 |
Phloretic acid |
|
8.524 |
3.11 x 10-6 |
Vanillic acid |
|
9.468 |
10.58 |
p-hydroxybenzoic acid |
|
9.857 |
2.60 x 10-6 |
Cinnamic acid |
|
10.043 |
8.35 x 10-6 |
Protohatechuic acid |
|
11.352 |
8.30 |
p-coumaric acid |
|
11.446 |
2.49 x 10-5 |
o-coumaric acid |
|
11.954 |
1.84 x 10-5 |
Gallic acid |
|
14.151 |
9.94 x 10-3 |
Ferulic acid |
|
14.434 |
2.65 |
Syringic acid |
|
15.398 |
8.25 |
Ellagic acid |
|
15.614 |
2.92 x 10-6 |
Piperic acid |
|
15.884 |
3.09 |
Luteolin |
|
18.052 |
239.74 |
Caffeic acid |
|
18.840 |
2.92 x 10-5 |
Sinapinic acid |
|
22.600 |
51.61 |
Chlorogenic acid |
|
22.853 |
1.12 x 10-4 |
Chloric acid |
DISCUSSION:
The skin is beautiful without excessive pigmentation, it is most valued by ladies. Due to stress and lifestyle the skin can become hyperpigmented. Antioxidants from plant extract can help. Anthocyanins are a class of flavonoid found in flowers, fruits and vegetables. Anthocyanins displayed different antioxidant properties such as superoxide scavenging and inhibition of lipid peroxidation23. Therefore in the present study the amount of total anthocyanin was 19.20±1.34mg cynanidin-3-glucoside equivalent per gram extract. This amount contributed to the antiradical activity of Acmella caulirhiza.
Flavonols like quercetin, kaempferol and isorhamnetin are flavonoids with triple phenolic rings. The flavonols are also effective in eliminating radicals due to their hydroxylated phenols24. The study investigated total flavonol in Acmella caulirhiza and was 466.13±3.79mg rutin equivalent per gram extract.
Lycopene with multiple double bonds is an efficient singlet oxygen scavenger25. Apart from quenching singlet oxygen it can trap peroxyl radical25. The values of lycopene content in the present study revealed 0.16± 0.04mg/100ml in the extract of Acmella caulirhiza. B-carotene is an ingredient of retinal it is an antioxidant that can scavenge singlet oxygen and lipid peroxidation26. The amount of b-carotene in Acmella caulirhiza was 2.25±0.17mg/ml extract. These contents are partly responsible for the antiradical scavenging properties of Acmella caulirhiza.
Phenolics detected from the HPLC were phloretic acid, vanillic acid, gallic acid, cinnamic acid, ferulic acid, syringic acid, ellagic acid, piperic acid, luteolin, caffeic acid, sinapinic acid and chicoric acid. The phenolics in order of abundance found in Acmella caulirhiza flower extract were caffeic acid 239mg/100g, chlorogenic acid 51.61mg/100g, p-hydroxybenzoic acid 10.58mg/100g, p-coumaric acid 8.30mg/100g, ellagic acid 8.25mg/100g, luteolin 3.09mg/100g and syringic acid 2.65mg/100g. Most phenolics with -OH group have the ability to contribute hydrogen atom, thus acting as good radical scavengers27.
Total antioxidants capacity of Acmella caulirhiza was assessed based on the conversion of Mo (VI) to Mo (V) by bioactives in Acmella caulirhiza extract. The ability of the extract to act as a very strong reducing agent was calculated and expressed as µg AAE/gram extract. This value is far higher than that reported by Atere28 on costus afer. This shown in table 1 suggest that Acmella caulirhiza possess more reductive tendency than Costus afer.
Ferric reducing antioxidant power (FRAP) is another reductive assay that utilizes TPTZ. The ability of Acmella caulirhiza to reduce TPTZ was also assessed. The result presented in fig 1 as percentages indicates that Acmella caulirhiza showed higher reductant tendency of the extract was high but not as that of ascorbic acid.
DPPH is a stable purple radical that is reduced by acmella caulirhiza to a light yellow colour. The result of this assay is depicted in fig 2. The results showed higher percentage inhibition by Acmella caulirhiza as compared to standard ascorbic acid. This report is similar to the work of Atere28 who also reported scavenging potential of Costus afer leaves against DPPH.
Hypochlorous acid is formed at sites of inflammation by myeloperoxidase. Hypochlorous acid can add chlorine to chloresterol and disrupt membrane29. Acmella caulirhiza and ascorbic acid independently scavenge HOCl, based on the inhibition of the chlorination of taurine. Although ascorbic acid showed higher parentages of inhibition as reference, the results are in line with the report of Hazra20.
A powerful oxidizing agent. is the excited state of oxygen called singlet oxygen30 In this study Acmella caulirhiza was showed to be an efficient scavenger of singlet oxygen but not as powerful as rutin. This study is in accordance with the report of Hazra20, who showed also the singlet oxygen scavenging potential of Spondias pinnata extract.
The superoxide redical is formed by the electron transport chain during metabolism. Acmella caulirhiza and quercetin at different concentration (0 – 250µg/ml) protect NBT oxidation from superoxide. The rate of protection expressed as percentage depicted in fig 5 showed that Acmella caulirhiza is a potent scavenger of superoxide.
Peroxynitrite produced in vivo from nitric oxide and superoxide can damage DNA to form nitrated DNA product29. Perioxynitrite oxidizes Evans’s blue as described by Beckman31 – Acmella caulirhiza quenched peroxinitrites these percentage results were compared to gallic acid and the results showed higher percentages of inhibition of peroxynitrite.
Acmella caulirhiza extract showed a range of inhibition at 25-250µg/ml, as the concentration of Acmella caulirhiza increases, the percentage inhibition of tyrosinase also increases at 25µg/ml Acmella caulirhiza inhibition of tyrosinase was 7.29±1.52 and that of kojic acid at same concentration was 47.19±1.02%. Again, at the maximum concentration of 250µg/ml the inhibition by Acmella caulirhiza was 65.42±1.15% and kojic acid at same concentration was 93.86±1.42%. Although the potency of kojic acid in tyrosinase inhibition was higher, Acmella caulirhiza still possesses inhibitory properties against tyrosinase. Flavonoids and phenolics have been implicated with the inhibition of tyrosinase21,32,27. Acmella caulirhiza possesses high levels of phenols and flavonoids. This was showed from the reactive oxygen species scavenging properties of Acmella caulirhiza.
Therefore, Acmella caulirhiza possesses antiradical potential, a very good source of phenolics and antityrosinase activity; it is valuable in the cosmetic and pharmaceutical industries. Although more research is needed in the elucidation of the mechanism of action and the ingredients present in Acmella caulirhiza flower extract that are behind this mechanism.
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Received on 09.08.2024 Revised on 04.10.2024 Accepted on 20.11.2024 Published on 05.12.2024 Available online on December 28, 2024 Research J. Topical and Cosmetic Sci. 2024; 15(2):85-90. DOI: 10.52711/2321-5844.2024.00015 ©A and V Publications All right reserved
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