INTRODUCTION:
In fact, the use of medicinal plants for the treatment of diseases dates back to the history of human life, that is, since human beings have sought a tool in their environment to recover from a disease, the use of plants was their only choice of treatment. More than a tenth of the plant species (over 50 000 species) are used in pharmaceutical and cosmetic products. However, the distribution of medicinal plants across the world is not uniform.
Today, according to the WHO, more than 80% of the world’s population rely more often on traditional drugs, mainly plants, serving as the main source of health care1. One of the most attractive thing is the sun and its radiation specially UV radiation which causes damage to skin, inducing changes in collagen and elastic fibers. The UV radiation (UVR) is divided into three ranges: UVA (320–400nm), UVB (290–320nm), and UVC (100–290nm)2,3.
Despite the fact that the atmospheric O2 and the ozone layer absorb, UVR still can produce several deleterious effects in human skin including damage in DNA, RNA, and proteins4. The associated photocarcinogenic injuries directly promote DNA damage, DNA is an important macro molecule that absorbs UVR and hence can mutate 5,6, which is associated with acceleration of skin aging and risk of skin cancer7.
Herbal extracts and oils have complex compositions, resulting in the exhibition of different effects, such as antioxidant, sun blocking, anti-inflammatory, and immunomodulatory. Moreover, the efficacy of herbal extracts in improving skin appearance and treatment of various skin diseases is very well understood8. Medicinal plants also contain some organic compounds which provide definite physiological action on the human body due to their antioxidant potential which known as an attractive option to be used in sunscreen formulations for the prevention of skin damage due to solar radiation9,10.
The olive tree, botanically-classified as Olea europaea L., is one of the most important fruit trees in Mediterranean region11. Olive leaf extracts are of special interest for their therapeutic effects.
Several studies have reported that olive leaves extract and their contents possess a wide range of biological properties including anti-diabetic, anti-carcinogenic, anti-atherosclerotic, anti-inflammatory, antimicrobial, and antifungal activities, antiarrhythmic, spasmolytic, immune-stimulant, anti-atherosclerotic, hypotensive, anti-inflammatory, antioxidant, anti-thrombic and hypoglycemic effects were represented12,13.
A study has indicated that extract of olive leaves had a capacity to lower blood pressure in animals and increase blood flow in coronary arteries, relieved arrhythmia and prevented intestinal muscle spasms, diarrhea, to treat respiratory and urinary tract infections14. Besides, polyphenols and other contents in olive leaves have several beneficial effects on health: These extracts have different classes of bio phenols including phenolic acids, phenolic alcohols (hydroxytyrosol and tyrosol), flavonoids (luteolin 7-O-glucoside, rutin, apigenin 7-O-glucoside, luteolin 4-O-glucoside), and secoiridoids (oleuropein and oleuropein-aglycone)15,16. Oleuropein (whose structure is shown in Figure 1), which is a secoiridoid, is the major and most abundant phenolic compound in olive leaves. The concentration of oleuropein can reach up to 140 mg/g (14%) on a dry matter basis in young olives and 60-90 mg/g of dry matter in the leaves17.
Besides oleuropein, flavonoids are a class of natural products present in fruits, vegetables, and beverages; synthesized by plants; and exhibiting many important effects such as protection against pathogens and UVB radiation7.
Olive trees are widespread in Syria and the leaves consider as a waste part. So, this research was designed in order to evaluate the way of extraction and the sun protective potential of leaf extracts.
MATERIALS AND METHODS:
Chemicals:
Ethanol and acetonitrile (HPLC grade) were obtained from PanreacQuimica SLU, oleuropein standard (HPLC grade) was purchased from Sigma- Aldrich company.
Preparation of the leaves extracts:
The leaves of different cultivars of olive tree which include Nipali and Mousaabi from Homs, khoderi and zaity from Tartous in Syria. The collection was directly from the trees in September 2021.
Leaves were washed with tap water and dried at ambient temperature then ground to a fine powder in mill. The plant powder was stored at room temperature in the dark until extraction then grounded material was extracted by several ways:
5g of olive leaves sample were placed in a Soxhlet thimble in a Soxhlet apparatus and extracted with 250ml ethanol/water (80:20 v/v) for 4 hours at 60˚C. (extract 1)
5g was extracted with hot water (250ml) for 30 minutes (extract 2)
5g were defatted using petroleum ether (40-60°C). The defatted powder was soaked in dark flasks separately for 30 min using ethanol/ water/ HCl (80: 19: 1 v/v/v) (extract 3)
5g were extracted with ethanol/ water (80:20) on water bath at 60˚C for an hour (extract 4)
Then, the extracts were cooled to room temperature, and filtered. The filtered extracts were then evaporated in rotary evaporator at 50˚C under vacuum for 2 hours. The concentrated extracts were stored in a refrigerator at 2˚C to 8˚C until analyzed for oleuropein content by HPLC18. Then we applied the best method of extraction according to HPLC results on the four different cultivars of olive leaves and repeated determination of oleuropein content by HPLC with the same conditions.
Determination of Oleuropein in Olive Leaf Extracts by HPLC:
The identification and quantitative determination of oleuropein from the extracts (1,2,3,4) were performed using HPLC systemwith silica based C18 bonded phase column (C18, 250mm × 4.6mm ID, 5μm) (Agilent Technologies) with mobile phase consisting of a mixture of water and acetonitrile (80/20 volume ratio) containing 1% acetic acid at a flow rate of 1.0mL/min. UV detector at 240nm was used for oleuropein determination. The injection volume used is 20.0 μl for both standard and sample solutions17. Identification of oleuropein in olive leaf extracts was based on retention times in comparison with standard of oleuropein. The concentration of oleuropein in the extracts was calculated using peak area and the calibration curves obtained from oleuropein standard solution.
Analysis extracts by Infrared (IR) Spectroscopy:
The samples were analyzed by IR Shimadzu. The wavenumber range set is 500-4000 cm-1 which is under mid infrared region.
Determination of SPF:
The ethanolic extract was dissolved in ethanol to a final concentration of 50, 100,150,300,500μg/ml and 1 mg/l. The SPF model used in this study was according to the methodology described by Mansur et al. (1986). The sample absorbances were measured in UVB wavelength range (290-320nm), with 5-nm increments and quadruplicate determinations were made at each point using ethanol as a blank. The SPF was calculated by applying the Mansur equation:
SPF Spectrophotometric = CF*
Mansur et al. (1986) developed a very simple mathematical equation which is as follows:
Where EE: erythemal effect spectrum; I: solar intensity spectrum; Abs: absorbance of sunscreen product; CF: correction factor (= 10)19,20,21,22.
The values of EE x I are constants and are showed in Table
Table 1. Values of EE x I
|
Wavelength(nm) |
EExI(normalized) |
|
290 |
0.0150 |
|
295 |
0.0817 |
|
300 |
0. 2874 |
|
305 |
0.3278 |
|
310 |
0.1864 |
|
315 |
0.0839 |
|
320 |
0.0180 |
RESULTS AND DISCUSSION:
Determination of Oleuropein in Olive Leaf Extracts by HPLC:
After extraction by four different methods and drying the extract till the constant weight. Identification and quantitative determination of oleuropein from analyzed sample were done by comparing the chromatogram of the samples. The sample chromatograms are presented in Figure 2.
The extract (1) chromatogram indicated high concentration of oleuropein and the Soxhlet apparatus gives the best result.
Figure 1. HPLC chromograph for olive leaf extract:
(1) Extract 1. (2) Extract 2. (3)Extract 3. (4) Extract 4
The next step was to analysis four different cultivars of olive leaves from different location in Syria and oleuropein determination of samples was done by comparing the chromatogram of standard with sample chromatograms using HPLC. Figure 3,4.
Figure 2. HPLC chromograph for oleuropein standard
Figure 3. HPLC chromograph for different cultivars of olive leaf extract
(1) Khoderi . (2) Mousaabi. (3) Zaity. (4) Nipali
The average of oleuropein content in olive leaf extracts for the four cultivars (Nipali, Mousaabi, khoderi, zaity) was 2.9%, 7%, 3.5%, 1.6% respectively. As the amount of oleuropein, which is the most abundant phenolic compound in olive leaves, ranges between 1 and 14%23.
Analysis extracts by Infrared (IR) Spectroscopy:
Absorption band were observed in regions around 3500 cm-1 with wide shoulder shape represents O-H (phenolic, hydrogen bonding), in the region around 3000 cm-1 for C-H aliphatic stretch, in the region around 1620 cm-1 for double band C-O stretch, band around 1100 cm-1 corresponding to C-O phenols, and the band in region between 650-950 cm-1 for aromatic C-H bending figure 524.
The IR spectra Emphasizes structure of some compounds found in olive leaves and specially oleuropein structure.
Figure 4. IR spectra of olive leaves
Determination of SPF:
SPF numbers has become a worldwide standard for measuring the effectiveness of sunscreen products. It gives an idea about how long one can stay in the sun without getting burn by the sun rays. The ethanolic extract of olive leaves had the sunscreen activity evaluated with the method developed by Mansur et al. (1986). The results of invitro SPF values are shown in table 2. The SPF values of the extracts tested were concentration-dependent, the increase in concentration resulted in an increment of the SPF. This activity can be attributed to the phenolic compound found in olive leaf extracts. The calculated values of SPF were fallen in the range of 0.6–29.9. the highest SPF value was showed at 1mg/ml of ethanolic extract concentration for Mosaabi.
Table 2. SPF value calculation of ethanolic olive extract for four different cultivars
|
50 µg/ml |
100 µg/ml |
150 µg/ml |
300 µg/ml |
500 µg/ml |
1mg/ml |
|
|
Zaiti |
0.95 |
1.22 |
3.16 |
4.29 |
4.29 |
14.48 |
|
Khodeiri |
0.71 |
1.95 |
4.60 |
12.43 |
16.57 |
19.65 |
|
Nipali |
2.65 |
7.21 |
10.54 |
20.95 |
27.17 |
27.91 |
|
Mosaabi |
0.67 |
0.88 |
2.20 |
5.37 |
8.90 |
29.96 |
Figure 5. Graphical representation of SPF values of olive leaf extract at different concentrations
Although there is availability of various synthetic sunscreens, their application is limited because of their harmful effects on the skin. Consumer acceptance is more when the sunscreens are prepared with plant extracts. Apart from filtering UV radiation, herbal combinations have several beneficial effects to the skin 21.
CONCLUSION:
our study showed that olive leaves which are waste materials from cultivation are a rich source of natural phenolic compounds and also highlighted the effective solvents to extract the bioactive polyphenolic content from olive leaves by an easily available extraction method, low cost and simple technique. The olive leaves contain phenolic compounds; the oleuropein, hydroxytyrosol, verbascoside, apigenin-7-glucoside and luteolin-7-glucoside. The large number of phenolic compounds present in olive leaves aroused the interest of researchers around the world and the studies with animals and humans have reported beneficial health effects such as the capacity of antioxidant, anti-hypertensive, hypoglycemia, hypocholesterolemia, cardioprotective, anti-inflammatory.
The sample chromatogram indicated high concentration of oleuropein in the ethanolic extract, which is the main compound in the olive leaves. And Mosaabi specie has the highest amount of oleuropein among four cultivars. The rich content of olive leaf extract in oleuropein indicates to be a truthful source with photoprotective effect. The SPF values were calculated for the four cultivars of olive leaves and the results showed good SPF value which could become a good, cheap and easily available formulation ingredients in sunscreen products.
ACKNOWLEDGMENTS:
The authors are thankful for this financial support
DECLARATION OF CONFLICTING INTERESTS:
The author (s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article
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Received on 01.09.2022 Modified on 03.10.2022
Accepted on 14.11.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(5):2187-2191.
DOI: 10.52711/0974-360X.2023.00359