Inhibitory Effect of Major Terpenoids of Essential Oil from Agathosma betulina Leaves on Collagenase and Elastase Enzymes Involved in Skin Aging

Abstract

Collagenase and elastase enzymes that cause the degradation of collagen and elastase have both been implicated in skin aging. Agathosma betulina is a popular plant in South Africa used in aromatherapy and folk medicine but the scientific knowledge regarding its anti-aging activity is yet to be explored. This study aimed to investigate the roles of the essential oil of A. betulina leaves on skin aging and its inhibitory effect on collagenase and elastase activities. In this work, the chemical profiling of essential oils of A. betulina leaves was explored. The antioxidant activity of essential oil of A. betulina leaves at different concentrations (0.07–10 µg/mL) was assessed using standard assay methods for 1,1-dihenyl-2-picryhydrayl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS). Standard procedures for elastase and collagenase assays were also employed to determine the anti-aging skin activity of the essential oil at the same concentrations. Gas chromatography-mass spectrometric analysis showed the presence of 43 constituents with menthone (29.2%), limonene (23.7%), and pulegone (8.4%) as the major compounds. The oil expressed strong radical scavenging activity against DPPH and ABTS radicals at higher concentrations, with over 90% inhibition, particularly at 2.5, 5, and 10 µg/mL compared to the control (butylated hydroxytoluene). The oil also demonstrated stronger inhibitory activity against elastase at higher concentrations (5 and 10 µg/mL) and concentration-dependent inhibitory activity against collagenase compared to the control (ursolic acid). The essential oil of A. betulina leaves could be a potential candidate for the cosmetic industry to retard skin wrinkles and other manifestations of skin aging particularly associated with extrinsic factors.

Keywords

anti-aging - antioxidant - essential oil - collagenase - elastase - Agathosma betulina

Introduction

The skin is the body's largest sense organ, involved in osmoregulation and performing other vital functions. Normal physiological processes within the cells of the body produce certain molecules known as free radicals, which upon accumulation penetrate the skin tissues, altering the skin's elasticity and structural integrity, leading to skin deterioration such as aging and wrinkling. Skin aging can be defined as a biological process that induces changes in the structural integrity and physiology of the skin.[1] Although skin aging is a natural phenomenon that usually occurs alongside the natural aging of the entire organism, extrinsically, exposure of skin to excess ultraviolet (UV) radiation also influences the skin's aging process. UV radiation generates peroxyl and other radicals that break down into more damaging radical products including malondialdehyde, which cross-links and polymerizes the elastin protein of the skin, reducing the skin's elasticity, affecting the skin's water permeability, and accelerating skin aging.[2] This process ultimately leads to wrinkling of the skin, an obvious symptom of aging.[3] In addition, nitric oxide (NO) is a reactive nitrogen species whose concentration is increased by photo-radiation.[4] As a bio-regulatory metabolite, NO is mainly involved in neuromuscular signal transmission, immune response, vasodilatation, blood pressure regulation, and several other physiological processes of the body. However, with regard to skin aging, NO, as a free radical, stimulates collagenase and elastase and induces the loss of collagen and elastin through oxidative stress, as a result, changing skin structure and leading to wrinkling.[3] [5] Collagen and elastin are important protein components that promote skin elasticity. Therefore, inhibiting the activity of elastase and collagenase plays a key role in preventing skin wrinkles and aging.

In general, essential oils are rich sources of terpenoids and phenolic compounds, which act as antioxidants and scavengers of free radicals. As volatile substances, essential oils contain a mixture of terpenoid compounds, which are secondary metabolites in plants synthesized from the mevalonic pathway of plant primary metabolites, and function in defense and other signaling processes within the plant.[6] The term aromatic plants is used to describe plants that contain essential oils because of the aroma they give to plants. For centuries, essential oils have been used in traditional medicines to promote and maintain health. By the 20th century, essential oils became an integral part of aromatherapy[7] due to their potential to ameliorate disease and heal. In addition, due to their unique aroma and fragrance, essential oils have also become an essential component in cosmetics and perfumery.

Agathosma, commonly called Buchu, belongs to the Rutaceae family and is an aromatic plant also known for its ability to produce essential oil and multiple healing properties.[8] There are over 150 known species of Agathosma plant across all regions of South Africa, particularly in the Cape Floral Kingdom.[9] Among them, Agathosma betulina and A. crenulata are the most popularly utilized species. The plant has become a common and widely consumed medicine in most households and has been commercialized in South Africa due to the large number of products obtained from this plant such as tea and vinegar. Other cultivars have been reported in Europe and Asia where they are locally used for the treatment of urinary tract diseases, stomach aches, fever, coughs, flu, and rheumatism.[10] In South African folk medicine, essential oils extracted from Agathosma are used in aromatherapy and body massage, and they are topically applied to the skin by the natives of the country to prevent wrinkling, moisturize the skin, and address skin-related infections.[11]

Several researchers have analyzed the chemical profiling of the Agathosma genus and showed that the plants are rich in volatile compounds including limonene, menthone, linalool, L-pulegone, etc., called terpenes.[12] [13] [14] These terpenes have been reported to be the major bioactive constituents, predominantly found in the oil of most cultivars of Agathosma, and are known to be responsible for the characteristic aroma of the plant as well as many of the phytopharmacological effects.[12] [15] Other compounds include hydrocarbons, phenolics, sulfur, and nitrogen-containing compounds.[14] [16] Essential oils extracted from Agathosma have been shown to have pharmacological effects including anti-inflammatory activity and antibacterial activity against gram-positive and gram-negative bacteria.[8] Despite the extensive use of Agathosma in South African folk medicine for aromatherapy, there is a dearth of scientific information substantiating the skin anti-aging activity of the plant purported by folk medicine. Also, to the best of our knowledge, this is the first study to report the anti-aging activity of the plant. In view of this gap, the present study was conducted to investigate the antioxidant and skin anti-aging activity of essential oil extracted from A. betulina leaves.

Materials and Methods

Chemicals and Reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH), Tris-HCL buffer, elastase (porcine pancrease), N-succinyl-(Ala) 3-p-nitroanilide (SANA), ursolic acid, collagenase from Clostridium histolyticum, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium persulfate (K₂S₂O₈) were products of Sigma Chemical Company, St Louis, MO, United States while butylated hydroxytoluene (BHT) was a product of Sihauli Chemicals Company, Mumbai, India, and were purchased from KINGDAD suppliers, Abuja, Nigeria. All other reagents used were of analytical grade.

Collection of Plant Material

A. betulina leaf and oil (undiluted) were commercially purchased from soil aromatherapy Windermere Farm, Gingindlovu, South Africa, at an early morning hour (8 a.m.). The leaf was authenticated at the herbarium unit of the Faculty of Pharmacy, Cairo University. Then, the oil was taken to the laboratory for the experimental study.

Chemical Profiling of Essential Oils of Agathosma betulina

Chemical constituents of essential oil extracted from A. betulina were evaluated using a gas chromatography-mass spectrometry (GC-MS) analysis according to a reported study.[17] The analyzer was operated on a 7890A model Agilent gas chromatographic system equipped with a 5975C mass selective detector (MSD) and 7693 series autosampler loaded with data system (Santa Clara, CA, United States). The GC column was an HP-5ms fused silica capillary with a stationary phase of 5% phenyl-methyl polysiloxane and a film thickness of 30 m × 0.25 µm. Helium was the carrier gas, with a column head pressure of 7.07 psi and a flow rate of 1 mL/min. Inlet temperature was 200°C and the MSD temperature was 280°C. GC oven temperature program was initially used at 40°C for 10 minutes, then increased to 200°C at a rate of 3°C/min and then to 220°C at a rate of 2°C/min. The oil was dissolved in chloroform, 1 µL of which was injected for analysis. The essential oil components were profiled and identified by the mass-to-charge ratio (m/z) based on the individual retention index (RI) and compared to their mass spectrum using the Chem Station data system, the National Institute of Standards and Technology, and literature. The relative percentage of each compound identified by the GC-MS was determined using the formula: % composition = (peak area of a compound)/total peak area of all compounds × 100.

In vitro Antioxidant Assay

1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay

The procedure was adopted according to a reported study[18] but with a slight modification. A. betulina oil was dissolved in ethanol to reach the final concentrations (0.07, 0.15, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL). The oil at different concentrations (0.1 mL) was mixed with 0.2 mL of DPPH solution (0.15 mmol/L in 80% methanol solution). The solution was incubated for 30 minutes at room temperature (25°C) in the dark, with shaking at intervals. Upon mixing, the solution began to bleach, indicating antioxidant activity. The absorbance at 517 nm was measured using a UV spectrophotometer (Model SP-UV52, Wincom Company Limited, China). BHT was used as the reference control, while water together with other reacting substances except the sample was used as a blank for calculation. The experiment was carried out in triplicate. DPPH inhibitory activity was calculated in percentage using the expression: % inhibition = (A_blank − A_sample)/A_blank × 100%. Where A_blank is the absorbance of the blank experiment and A_sample is the absorbance of the sample.

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Radical Scavenging Assay

The procedure was adopted according to a reported study[19] with a slight modification. The principle is based on the antioxidant ability to stabilize the green-blue ABTS chromophore formed by the reaction of ABTS and K₂S₂O₈. A. betulina oil was dissolved in ethanol to reach the final concentrations (0.07, 0.15, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL). ABTS^•+ solution was formed by mixing 7 mmol/L ABTS aqueous solution and 2.45 mmol/L K₂S₂O₈ aqueous solution (1:1, v:v) and incubated in the dark at 25°C for 16 hours to yield a completely oxidized ABTS. Prior to usage, the ABTS^•+ solution was diluted with water until an absorbance of 0.7 to 0.8 (not less or higher) was achieved. Note: At higher concentrations, water was added, and at lower concentrations less than 0.7, ABTS was added. The oil at different concentrations (30 μL) and ABTS^•+ solution (1 mL) were pipetted into a microtiter plate, mixed thoroughly, and incubated at 25°C for 6 minutes. Absorbance at 745 nm was then recorded by a UV spectrophotometer (Model SP-UV52, Wincom Company Limited, China). BHT was used as the reference control, while water containing the reacting substances except the sample was used as the blank. The experiment was carried out in triplicate. ABTS inhibitory activity was calculated in percentage using the expression: % inhibition = (A_blank − A_sample)/A_blank × 100%. Where A_blank is the absorbance of the blank experiment and A_sample is the absorbance of the sample.

In vitro Elastase and Collagenase Inhibition Assay

Anti-Elastase Activity

The anti-elastase activity was carried out according to a reported study.[2] The inhibitory activity was based on the intensity of the color released during cleavage of SANA, which is the substrate of elastase. A. betulina oil was dissolved in ethanol to reach the final concentrations (0.07, 0.15, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL). SANA was dissolved in 0.1 mol/L Tris-HCl buffer (pH = 8.0) at a concentration of 1 mmol/L. SANA solution (200 μL) and the oil sample (20 μL) were mixed and incubated at 25°C for 10 minutes; then elastase (0.03 U/mL, 20 μL) was added and re-incubated at 25°C for 10 minutes. The solution was vortexed, and absorbance at 410 nm was determined. Ursolic acid has anti-aging activity[20] and was used as a standard anti-elastase control. The experiment was carried out in triplicate. The percentage enzyme inhibition was calculated as (Abs_control – Abs_sample)/Abs_control × 100%. Where Abs_control is the absorbance of the control (without the oil sample) and Abs_sample is the absorbance of the control (with the oil sample).

Anti-Collagenase Activity

The anti-collagenase activity was conducted according to a reported study[21] with a slight modification. N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) is a synthetic substrate, which was hydrolyzed in the presence of collagenase.[22] FALGPA solution at 1 mmol/L was prepared in tricine buffer (50 mmol/L) containing 400 mmol/L NaCl and 10 mmol/L CaCl₂ (pH = 7.5). Clostridium histolyticum collagenase (1 U/mL) in tricine buffer was freshly prepared as a stock solution. The oil extract was diluted with tricine buffer at different concentrations (0.07–10 µg/mL). Then 50 µL of each was transferred into a 96-well microplate, then, 50 μL of the stock solution was added. The mixture was incubated at 37°C for 20 minutes. Afterward, FALGPA (50 μL) was added to start the reaction. The total reaction mixture (150 μL) was further incubated at the same temperature and for the time. Absorbance at 340 nm was determined, and the percentage inhibition was calculated using the expression: % Inhibition = [A_control − (A_sample − A_sb)]/A_control × 100%. Where A_control is the absorbance of the control (tricine buffer + substrate + enzyme only), A_sample is the absorbance of the sample with enzyme, and A_sb is the absorbance of control without enzyme.

Statistical Analysis

All the data are presented as means ± standard error of the mean (SEM). Each experiment was repeated at least three replicates. Data were subjected to t-test analysis for comparison using GraphPad Prism version 8.0.2 with a p-value less than 5 (p < 0.05) being significant.

Results

The chromatogram of chemical constituents of essential oil of A. betulina leaf and their respective retention times is presented in [Fig. 1]. The chemical profile is shown in [Table 1]. The GC-MS analyzer identified a total of 43 chemical compounds, mainly belonging to terpene hydrocarbons in the form of monoterpenes, oxygenated terpenes, and sesquiterpenes. The most abundant chemical components were pulegone, limonene, and menthone constituted with relative abundances of 8.4, 23.7, and 29.2%, respectively. The relative abundance of other chemical constituents ranged between 0.1 and 2.9% ([Table 1]).

Table 1 Chemical profile of essential oils from Agathosma betulin a leaf

S/N	RI	Compound	Relative abundance (%)	Type of compound
1	1032	α-Pinene	2.4	Monoterpenes
2	1076	Camphene	0.1	Monoterpenes
3	1118	β-Pinene	0.3	Monoterpenes
4	1174	Myroene	1.8	Monoterpenes
5	1188	α-Terpinene	0.3	Monoterpenes
6	1203	Limonene	23.7	Monoterpenes
7	1213	1,8-Cineole	2.7	Monoterpenes
8	1215	p-Mentha-1,3,6-triene	0.3	Monoterpenes
9	1225	(Z)-2-hexanal	0.1	Aldehyde
10	1250	Menthatriene	0.7	Monoterpenes
11	1266	(E)-β-Ocimene	0.7	Monoterpenes
12	1280	p-Cymene	0.8	Monoterpenes
13	1290	Terpindene	0.2	−
14	1329	3-Methylcyclohexanone	0.1	Cyclic ketones (hydrocarbon)
15	1437	α-Thujone	0.1	Monoterpenes
16	1475	Menthone	29.2	Monoterpenes
17	1553	Linalool	0.5	Oxygenated monoterpenes
18	1576	cis-Isopulegone	0.2	Monoterpenes
19	1611	Terpinen-4-ol	0.7	Oxygenated monoterpenes
20	1614	trans-Dihydrocarvone	0.2	Monoterpenes
21	1634	Necisomenthol	0.1	Monoterpenes
22	1641	Menthol	0.1	Monoterpenes
23	1654	Pulegone	8.4	Monoterpenes
24	1682	δ-Terpineol	0.1	Monoterpenes
25	1704	Myrtenylacetate	0.3	Monoterpenes
26	1706	α-Terpineol	0.3	Monoterpenes
27	1719	Borneol	0.1	Monoterpenes
28	1733	Neodihydrocarveol	0.1	Monoterpenes
29	1748	Piperitone	0.4	Monoterpenes
30	1751	psi-Diosphenol	2.9	Monoterpenes
31	1776	8-Hydroxyisomenthone	0.6	Monoterpenes
32	1789	2-Hydroxyisomenthone	0.3	Monoterpenes
33	1823	p-Menthan-1(7),5-dien-2-ol	2.5	Sesquiterpene
34	1845	2-Dodecyl acetate	0.1	Oxygenated monoterpenes
35	1864	p-Cymen-8-ol	0.1	Oxygenated monoterpenes
36	1867	Menthan-3-one	0.1	Monoterpenes
37	1882	cis-Carveol	0.1	Monoterpenes
38	1900	cis-8-Mercapto-p-menthan-3-one	0.1	Monoterpenes
39	1984	trans-2-Acetoxyisomenthone	0.2	Monoterpenes
40	2029	Methyleugenol	0.4	Phenylpropanoid
41	2104	1-Hydroxy-pseudo-diosphenol	0.7	Monoterpenes
42	2140	4-Hydroxy-diosphenol	0.6	Monoterpenes
43	2179	trans-p-Menthon-8-thiol acetate	0.1	Oxygenated monoterpenes

Abbreviation: RI, retention index.

Fig. 1 Gas chromatogram of essential oil from Agathosma betulina leaf.

The antioxidant activity of the essential oil of A. betulina leaves against DPPH and ABTS radicals was assessed. The oil and BHT demonstrated scavenging activity for DPPH ([Fig. 2]) and ABTS ([Fig. 3]), with the radical scavenging activity of essential oil of A. betulina leaves for DPPH and ABTS significantly superior to BHT at higher concentrations of 0.625, 1.25, 2.5, 5, and 10 µg/mL (all p < 0.05). In particular, the oil demonstrated over 90% inhibition of DPPH and ABTS radicals at 2.5, 5, and 10 µg/mL, with the percentage inhibition being 95.01, 95.17, and 95.65% for DPPH radicals, and 94.51, 94.90, and 95.08% for ABTS.

Fig. 2 DPPH radical scavenging activity of Agathosma betulina leaf essential oil at different concentrations. *p < 0.05 versus the control (BHT) at the same concentration. BHT, butylated hydroxytoluene; DPPH, 1,1-dihenyl-2-picryhydrayl.

Fig. 3 ABTS radical scavenging activity of Agathosma betulina leaf essential oil evaluated at different concentrations. *p < 0.05 versus the control (BHT) at the same concentration. ABTS, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; BHT, butylated hydroxytoluene.

The inhibitory effect of essential oil of A. betulina leaves on elastase and collagenase was assessed. The oil and ursolic acid demonstrated inhibitory activity for elastase ([Fig. 4]) and collagenase ([Fig. 5]). However, the inhibitory effect of the oil on elastase activity was significantly lower compared to ursolic acid, particularly at concentrations below 2.5 µg/mL (p < 0.05), but at high concentrations of 5 and 10 µg/mL, the inhibitory effect (91.67 and 92.65% respectively) was close to that of the control (95.48 and 95.81%, respectively) (all p < 0.05). A similar change in trend was observed in the inhibitory effect of the oil on collagenase activity. At higher concentrations, especially at 5 and 10 µg/mL, the oil had inhibition rates of approximately 91 and 92%, close to the control's inhibition value (95%) at the same concentrations. Overall, the essential oil of A. betulina leaves showed a significant inhibitory effect on elastase and collagenase activities at a higher concentration range of 2.5 to 10 µg/mL (all p < 0.05).

Fig. 4 Inhibition of elastase activity by Agathosma betulina leaf essential oil. *p < 0.05 versus the control (ursolic acid) at the same concentration.

Fig. 5 Inhibition of collagenase activity by Agathosma betulina leaf essential oil. *p < 0.05 versus the control (ursolic acid) at the same concentration.

Discussion

GC-MS is one of the most effective and frequent methods to identify chemical constituents. As shown in [Table 1], the majority of A. betulina chemotypes were terpenoids in the form of monoterpenes, constituting the main type of terpenes identified. Oxygenated terpenes and sesquiterpenes were also present. This is consistent with the report that terpenes are mostly present in essential oil form in higher medicinal plants and exist in many families, including Rutaceae[23] to which A. betulina under the current investigation belongs. A study of Agathosma plants using GC-MS revealed that the predominant chemotypes found among most cultivars of the plant were limonene, menthone, and pulegone.[15] Viljeon et al performed qualitative and quantitative analysis of the chemical constituents of the essential oils from 18 different species of Agathosma grown commercially in South Africa, which were chemically profiled with GC-MS. The study reported limonene is the most common and dominant chemical constituent found in many species. The percentage abundance of limonene, menthone, and pulegone were 23.7, 29.2, and 8.4%, respectively, in A. betulina while 34.9, 16.6, and 13.4%, respectively, in A. crenulata.[15] Collins et al carried out similar work and revealed that essential oils extracted from A. betulina were also characterized by large amounts of limonene and menthone.[24] Other studies have also identified limonene, menthone, and pulegone as the main constituents of the essential oil of some plants.[25] [26] [27]

In literature, the biological activities of limonene, menthone, and pulegone are well documented. For instance, over 15 pharmacological efficacies have been attributed to limonene.[28] Ibrahim et al's preclinical work showed that limonene, a major bioactive principle of Citrus aurantifolia essential oil, was able to regulate fasting blood sugar by mimicking the mechanism of metformin, a reference anti-diabetic drug, thereby expressing anti-diabetic activity and also demonstrating anti-dyslipidemic activity in experimental rats.[6] Also, according to Baylac et al,[29] the essential oils from A. cullina, a species similar to A. betulina, demonstrated anti-inflammatory activity by inhibiting 5-lipoxygenase activity in vitro due to its high composition of limonene. Limonene has also been shown to be an active terpene against gram-positive bacteria.[27] Menthone and pulegone are also terpenes reported to be good antimicrobial candidates.[25] The antimicrobial mechanism of these terpenes is predicted to be through membrane disruption.[30] In this work, the antioxidant activity of A. betulina essential oil was revealed by inhibiting DPPH and ABTS radicals, particularly at its higher concentrations (5 and 10 µg/mL).

In DPPH assays, DPPH radicals are usually characterized by a purple coloration which upon contact with an antioxidant agent acting as a proton donor easily decolorizes to yellow, causing it to appear as non-radical in the form of DPPH-H.[31] Therefore, agents capable of decolorizing DPPH radicals are considered to be good antioxidants. Although the antioxidant mechanisms of limonene and other monoterpenes are yet to be fully understood, it has been suggested that limonene is a good source of proton (hydrogen) donors capable of donating proton (hydrogen) to pro-oxidants to mop their radical nature. Moreso, limonene, and menthone from the essential oils of many plants have been reported to act as antioxidant agents through hydrogen-donation mechanisms to scavenge free radicals.[26] [32] [33] Thus, the antioxidant activity of A. betulina essential oil may be attributed to the presence of limonene that scavenged DPPH and ABTS radicals at the investigated doses, as evident in this study, converting them to DPPH + H and ABTS* non-radical forms, respectively, and may act synergistically with other monoterpenes that are relatively highly abundant in the oil such as menthone and pulegone. The antioxidant activity of essential oils from other plants that contain limonene as a major chemical constituent has also been confirmed using DPPH and ABTS assays.[33] [34] [35]

Collagenase and elastase are metalloenzymes responsible for the degradation of collagen and elastin, which are key protein promoters of skin elasticity.[36] [37] However, elastin is a greater marker of skin wrinkling and aging than collagen. These protein enzymes (collagenase and elastase) become highly expressed when the skin loses its elasticity, which may arise from high exposure of the skin to UV light of the sun or due to oxidative stress induced on the skin, thereby leading to wrinkling and aging. Therefore, agents capable of inhibiting both the enzymes are considered good candidates to ameliorate skin wrinkling, aging, and tumors. The anti-collagenase and anti-elastase activities of the essential oil extracted from Curcuma aromatica, Helichrynum italicum, and Syzygium cumini have also been documented.[38] [39] [40] This work further demonstrated the anti-collagenase and anti-elastase activities of essential oil obtained from A. betulina leaf by inhibiting the activities of these enzymes.

Although the actual inhibitory mechanism by which limonene and other terpenes inhibit collagenase and elastase enzymes is unclear, some studies suggest that it may be unconnected with its anti-inflammatory and antioxidant activities.[35] Collagenase, as a metalloprotein, requires a metal for its catalytic reaction to degrade collagen and cause wrinkling and aging; therefore, removing the metal from this enzyme will reduce its catalytic action. Likely, one of the mechanisms by which the oil inhibits enzyme activity may be through the chelation of metal ions of the major terpenes in the oil, resulting in the loss of enzyme activity as observed at higher concentrations. The metal-chelating capacity of monoterpene-rich essential oil from lemon (Citrus limoni) containing limonene has been confirmed according to a reported study.[41] Elastase is a proteolytic enzyme that degrades the release of elastin, a protein that contributes to skin elasticity. It cleaves protein at the carboxyl side of small hydrophobic amino acids and possesses a unique proficiency in digesting elastin. In this work, the essential oil of A. betulina significantly reduced the activity of the elastase enzyme, indicating that the major monoterpenes of the oil interfere with the catalytic activity of the enzyme at the active site, which is a major mechanism of the inhibitors of enzymes. This inhibition can reduce tissue degradation and inflammation processes commonly associated with aging.

The inhibitory effect of the essential oil of A. betulina leaves on collagenase and elastase activities may be due to the synergistic or additive effect of the major chemical constituents of the oil, particularly limonene, menthone, and pulegone. Fraternale et al identified limonene and α-pinene as major chemical constituents of the essential oil from Helichrynum italicum. They investigated the anti-collagenase and anti-elastase activities of limonene and α-pinene and found that the combined administration of limonene and α-pinene demonstrated stronger inhibitory activity on collagenase and elastase, whereas, in the aforementioned study, limonene and α-pinene alone had lower inhibitory activity.[39] Mori et al conducted similar studies on the essential oils from lemon (C. limon), grapefruit (C. paradisi), and juniper (Juniperus communis), all of which contained limonene and α-pinene as their major chemical constituents.[42] They demonstrated a stronger inhibitory effect on elastase activity when compared to separate studies of limonene and α-pinene, in which low activity was noticed.[42] Limonene has been previously reported to enhance the biological activity of other terpenes.[43] Given the above, limonene may produce better biological activity, particularly as an inhibitor of collagenase and elastase activities, when used in combination with other monoterpenes through a synergistic or additive effect than when used alone. Furthermore, due to the presence of limonene, various Citrus species, viz., Citrus hystrix, C. reticolata, C. aurantium, C. limon, and C. clementina, have also been reported to express interesting anti-collagenase and anti-elastase activities.[35] [44] [45] [46] [47] In this work, essential oils obtained from A. betulina leaf demonstrated significantly stronger inhibitory action over elastase and collagenase, most especially at higher concentrations of 5 and 10 µg/mL, which was consistent with the facts discovered by Mori et al that the essential oils from three different Citrus species contain limonene as part of the major chemical constituents, and demonstrated a strongest inhibitory activity on elastase also at a higher concentration of 250 µg/mL.[42] Mangena et al attributed the antimicrobial activity of essential oils of Pteronia incana to β-pinene and α-pinene, which were the major chemotypes found in the plant.[48] Additionally, the essential oil of Salvia ringens revealed 1,8-cineole and pinene as the predominant chemical compounds, which were responsible for antibacterial activity against gram-negative bacteria.[49] Based on these assertions, limonene, menthone, and pulegone, as the most abundant chemotypes of the essential oil of A. betulina, could be responsible for the antioxidant activity and collagenase and elastase inhibitory activity by acting in synergy, particularly at higher concentrations where stronger enzyme inhibition was observed.

Nonetheless, other chemotypes in the essential oil of A. betulina were present in smaller amounts, and their involvement may not be jettisoned as they may also contribute to the observed activities since essential oils are composed of heterogeneous mixtures of different constituents, and each constituent contributes to biological activities either synergistically or additively. In silico studies are usually employed to determine protein–ligand interactions; therefore, in view of this, conducting an in silico study will help to infer which of the three chemical components has a stronger binding affinity for enzymes. We also recommend an in vivo study of the essential oil of A. betulina, more specifically, the major chemotypes from this study at the tissue level to further substantiate the anti-aging activity. We also suggest that D-galactose-induced aging in rats and senescence-accelerated aging in mice may likely be the most appropriate in vivo model, as these rodents share similar genetic and physiological characteristics; thus, biochemical parameters and genes relating to aging can be determined. Additionally, these models are cost-effective when studying the anti-aging effects of an agent or drug.

Conclusion

The study showed that the essential oil of A. betulina leaves contained limonene, menthone, and pulegone as major chemotypes and are responsible for the inhibition of collagenase and elastase enzymes involved in skin wrinkling and aging. The study therefore concludes that the essential oil of A. betulina contains potential inhibitors that may be employed as anti-wrinkling and anti-aging candidates in cosmetics, perfumery, and pharmaceutical industries.

Conflict of Interest

None declared.

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Publication History

Received: 27 September 2024

Accepted: 14 March 2025

Article published online:
28 April 2025