Amino acids, often called “the building blocks of life”, are primary metabolites which play a vital role in nutrition and health maintenance. They are used as ingredients in cosmetic and pharmaceutical products and as special nutrients in the medical field. They are commonly employed in transfusions, in the manufacture of artificial sweeteners such as aspartame and as intermediate precursors for the production of antibiotics. Amino acids, individually or in group, are also very important for the treatment of many disease conditions.
Free amino acids (FAAs) are also indispensable for healthy skin. They constitute the largest component (~ 40%) of the so-called natural moisturizing factor (NMF) and are very important in maintaining the moisture balance of skin. The most abundant FAAs within the NMF are l-serine (Ser) (~ 36%), l-glycine (Gly) (~ 22%) and l-alanine (Ala) (~ 13%). Citrulline (Cit), ornithine (Orn), histidine (His) and arginine (Arg) all account for 6-8%. In another study Ser, Gly, Cit, Ala, His, and threonine (Thr), in that order, are the dominant FAAs in the horny layer of human skin and account for as much as 80%. Methionine (Met), cysteine (Cys), and tryptophan (Try) are present in smallest concentrations; and proline (Pro) is obscured because of the large amount of Cit masking its presence.
Twenty-three (23) FAAs were detected in human corneocytes of 4 different study groups in a study by Hussain et al. (2019) at different concentrations. The level of NMFs including FAAs can decline in dry skin conditions due to many disease conditions such as atopic dermatitis, ichthyosis vulgaris, psoriasis, and age in addition to environmental conditions. The best way to overcome such disease condition seems to be the delivery of the major components of the NMF to the human skin in the form of moisturizers.
FAAs for food, cosmeceutical and pharmaceutical applications can be obtained in four different methods, namely extraction (from natural resources), chemical synthesis, enzymatic synthesis, and fermentation. Despite the advancements in chemical synthesis and biotechnology, the need for herbal medicines is still at the top indicating the extraordinary relation between human beings and herbs, an almost mystical interdependence. As reported by the world health organization (WHO), about 80% of the total world population uses herbal medicines as their first-line primary health-care.
This resurgence of public interest in herbal remedies has been attributed to several factors including but not limited to the following:
- herbal medicines are practically a mixture of many bioactive chemicals that can act synergistically;
- they might be more effective as compared to similar substances obtained through chemical synthesis;
- they are economically feasible and can be used by people at all economic levels;
- they are preferable in terms of safety (side effects), contraindications and interactions with other substances;
- they have superior structural diversity, complex structure and multiple stereo centers.
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Understanding their benefits, the WHO has been working toward increasing the use of herbal medicines and currently about 25% of new drugs approved by Food and Drug Administration (FDA) and/or the European Medical Agency (EMA) are directly or indirectly plant based. Hence, research on plant-based bioactive molecules is among the top research topics in the pharmaceutical sector. Due to such reasons, many countries have been extensively using their plants for pharmaceutical and cosmeceutical applications.
Ethiopia, a country endowed with a diverse biological resources and home of about 6500-7000 species of higher plants of which 12% are endemic, is one of the six regions rich in plant biodiversity and these can be of course alternative sources of FAAs. Use of such sustainable and natural ingredients for cosmeceutical applications has a lot of benefits to the population in fostering sustainability and natural remedial approaches.
Free amino acids (FAAs), the major constituents of the natural moisturizing factor (NMF), are very important for maintaining the moisture balance of human skin and their deficiency results in dry skin conditions. There is a great interest in the identification and use of nature-based sources of these molecules for such cosmeceutical applications. The objective of the present study was, therefore, to investigate the FAA contents of selected Ethiopian plant and fungi species; and select the best sources so as to use them for the stated purpose.
A total of 60 plant and fungi species were collected from Gullele Botanical Garden and local supermarkets found in Addis Ababa, Ethiopia. All the plant and mushroom species were authenticated by the Ethiopian Biodiversity Institute, Addis Ababa, Ethiopia. Plant varieties such as cereals, leguminous plants, vegetables, fruits, spices and tea plants were included. In addition to the commonly used food items, attention was given to some indigenous aloe plants. One of the most common mushrooms found in Ethiopia, the Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm., was also included in the study. Moreover, residuals from food processing such as the peals of fruits were also included.
All the l-amino acid standards (Ala, Cys, Ser, Pro, His, Cit, Gly, Orn, Thr, Trp, Arg, Met, aspartate (Asp), glutamate (Glu), asparagine (Asn), glutamine (Gln), γ-aminobutyric acid (GABA), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), lysine (Lys), O-acetylserine, oxyproline, methionine oxide, taurine (Tau), and tyrosine (Tyr)) and the internal standard, norvaline, were purchased from Sigma-Aldrich. Reagent grade n-pentane, sodium borate, and 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) were also commercial products from Sigma-Aldrich. HPLC grade methanol and acetonitrile were used and these were purchased from Roth (Karlsruhe, Germany). Ultrapure water (resistivity 18.2 MΩ) purified by TKA X-CAD ultrapure water purification system (Thermo Fisher Scientific, Waltham, MA, USA) was used at all steps where water was required. Chromabond® Multi 96 filter plates and Chromabond® Sorbent HR-X were from Macherey-Nagel (Düren, Germany).
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The analysis of FAAs was conducted as per the method reported elsewhere (Ziegler et al. 2019). Each sample was collected in triplicate and the collected samples were freeze dried (Alpha 2-4-LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). Five milligrams (5 mg) of each of the lyophilized samples was weighed using dual range analytical balance (Model XA105, Mettler Toledo, USA) and transferred to a 2 mL Eppendorf tube. A steal bead of 5 mm in diameter was inserted to each Eppendorf tube and the samples were pulverized in a mixer mill (Model MM 301, Retsch GmbH, Germany) at 25 s−1 for 50 s. Two hundred microliters (200 µL) of extraction solvent [a mixture of water and 10 mM norvaline (1 mL: 5 µL)] was added to each sample and the samples were mixed thoroughly for 20 min on a vortex (JK Janke and Kunkel IKA, model IKA VIBRAX-VXR). The samples were then centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 5 min and the supernatant was transferred to a 1.5 mL Eppendorf tube. This solution was again centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 5 min and the supernatant was transferred to a new 1.5 mL Eppendorf tube.
Twenty millimolar (20 mM) stock solution of each amino acid standard was prepared in ultrapure water. Five microliters (5 μL) of each of the resulting solutions was transferred to a 2 mL Eppendorf tube and the resulting mixture was diluted to 500 μL with the same solvent. Finally, serial standard solutions were prepared for each amino acids and the internal standard to get a final concentration of 0, 2, 4, 8, 16, 64 and 128 pmol/µL after derivatization. After thawing at room temperature, 25 µL of the standard and sample solutions were transferred to 1.5 mL Eppendorf tubes. Fifty microliters (50 μL) of 0.5 M sodium borate buffer pH 7.9 and 100 μL 6 mM Fmoc-Cl solution (in acetone) were added to each solution and the resulting mixture was incubated for at least 5 min after mixing. Five hundred microliters (500 μL) of n-pentane was added to each solution, mixed thoroughly, centrifuged (Model 5415C, Eppendorf®, Germany) at 10,000×g for 1 min and the upper (organic phase) was discarded. This step was repeated two more times.
Solid-phase extraction (SPE) was conducted using Chromabond Multi 96-well plate (Macherey-Nagel, Düren, Germany) containing 50 mg/well HR-X-resin (Macherey-Nagel, Düren, Germany). First, the SPE plate was conditioned by 1 mL of methanol followed by 1 mL of water. In this and all subsequent steps, the liquid was passed through the resin by centrifugation at 500×g for 5 min using JS5.3 swingout rotor in an Avanti J-26XP centrifuge (Beckman Coulter, Fullerton, CA, USA). 500 µL of 5% (v/v) acetonitrile was added to the sample and standard solutions mentioned in “Sample derivatization and processing”. Then, the resulting solutions were quantitatively loaded onto the SPE plate, washed with 1 mL of water and the flow through was discarded after centrifugation. In the next step, 1 mL of methanol was added into the 96-deep well plate and eluted to a new block by centrifugation. Finally, the eluates were transferred from the 96-deep well block to 2 mL Eppendorf tubes, and allowed to evaporate under vacuum in an Eppendorf Concentrator (Model 5301, Eppendorf, Hamburg, Germany) at 45 °C for 45 min.
Chromatographic separation was achieved using Agilent 1290 liquid chromatography system equipped with Zorbax Eclipse Plus C18 Rapid Resolution HD column (2.1 × 50 mm, 1.8 µm, Agilent). The column temperature was maintained at 30 °C. Gradient elution with solvent A (0.2% v/v acetic acid in water) and solvent B (0.2% v/v acetic acid in acetonitrile) was used as mobile phase at a flow rate of 700 µL/min. Solvent A was held constant at 75% for 0.3 min and decreased to 50% over the next 6.7 min. Then, it was held at 2% over the next 0.7 min and increased to 75% for the next 0.4 min. Detection was done using API 3200 Triple Quadrupole LC-MS/MS system equipped with an ESI Turbo Ion Spray interface, operated in the negative ion mode (AB Sciex). The ion source parameters were set as follows: curtain gas was used at a pressure of 30 psi. The ion spray voltage was − 4500 V and the ion source temperature was set at 350 °C. Both the nebulizing and drying gas pressure were set at 50 psi. Triple quadrupole scans were acquired in the multiple reaction monitoring (MRM) mode with Q1 and Q3 set at “unit” resolution. Scheduled MRM was performed with a window of 90 s and a target scan time of 0.5 s. The mass spectrum (MS) parameters describing the MRMs for each FMOC-Cl derivatized amino acid were as reported by Ziegler et al. The data analysis was done by automatic integration using Analyst software. A calibration curve was constructed using the standard solutions and from the graph the slope of the regression line was determined.
The FAA contents of the different plant and mushroom species included in the present study are shown in Table 1. Evidently, the concentrations are significantly different and the total FAAs found in the water extracts of the different species tested ranged from 0.86 mg/g (peal of mango, Mangifera indica L.) to 400.01 mg/g (oyster mushroom, Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm.) as calculated on dry basis.
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| Sample | Total FAAs (mg/g) |
|---|---|
| Mango Peel (Mangifera indica L.) | 0.86 |
| Oyster Mushroom (Pleurotus ostreatus) | 400.01 |
Natural Moisturizing Factor (NMF) Composition
About 59 different plant species and oyster mushroom were included in the study and the concentrations of 27 FAAs were analyzed. Each sample was collected, lyophilized, extracted using aqueous solvent, derivatized with Fluorenylmethoxycarbonyl chloride (Fmoc-Cl) prior to solid-phase extraction and quantified using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) system. All the 27 FAAs were detected in most of the samples. The dominant FAAs that are part of the NMF were found at sufficiently high concentration in the mushroom and some of the plants.
The study included the following plant species:
Allium sativum L., Allium spathaceum Steud., Ananas comosus (L.) Merr., Arachis hypogaea L., Avena abyssinica Hochst., Brassica nigra L., Brassica oleracea var., Cannabis sativa L., Capsicum annuum L., Carica papaya L., Citrullus lanatus (Thunb.) Matsum., Citrullus lanatus (Thunb.) Matsum., Coriandrum sativum L., Cucumis sativus L., Cucurbita pepo subsp. pepo convar., Cuminum cyminum L., Daucus carota subsp. sativus (Hoffm.) Arcang., Glycine max (L.) Merr., Helianthus annuus L., Lactuca sativa L., Linum usitatissimum L., Malus domestica Borkh., Mangifera indica L., Mangifera indica L., Matricaria chamomilla L., Mentha × piperita L., Moringa oleifera Lam., Nigella sativa L., Ocimum basilicum L., Persea americana Mill., Phaseolus vulgaris L., Piper nigrum L., Pisum sativum var. Abyssinicum (A., Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm., Ruta chalepensis L., Rumex abyssinicus Jacq., Salvia rosmarinus Schleid., Sesamum indicum L., Solanum lycopersicum L., Syzygium aromaticum (L.) Merr., Thymus vulgaris L., Trigonella foenum-graecum L., Allium sativum L., Allium spathaceum Steud., Ananas comosus (L.) Merr., Arachis hypogaea L., Avena abyssinica Hochst., Brassica nigra L., Brassica oleracea var., Cannabis sativa L., Capsicum annuum L., Carica papaya L., Citrullus lanatus (Thunb.) Matsum., Citrullus lanatus (Thunb.) Matsum., Coriandrum sativum L., Cucumis sativus L., Cucurbita pepo subsp. pepo convar., Cuminum cyminum L., Daucus carota subsp. sativus (Hoffm.) Arcang., Glycine max (L.) Merr., Helianthus annuus L., Lactuca sativa L., Linum usitatissimum L., Malus domestica Borkh., Mangifera indica L., Mangifera indica L., Matricaria chamomilla L., Mentha × piperita L., Moringa oleifera Lam., Nigella sativa L., Ocimum basilicum L., Persea americana Mill., Phaseolus vulgaris L., Piper nigrum L., Pisum sat…
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