Phytochemical, Antioxidant, hair growth and wound healing property of Juniperus excelsa, Olea oleaster and Olea europaea

2.1 Plant material collection and extraction

The leaves of. Juniperus excelsa sample,Olea oleaster (wild olive) and Olea europaea (Cultivar olive) samples were collected from Al Jabel AL Akhadar, sultanate of Oman during January 2017. The collected leaves were cleaned, dried under the shade and made fine powder by using heavy blender. 20 g of each plant sample was weighed and dissolved in 200 ml of 80 % methanol and subjected for extraction through maceration (Reddy et al., 2018). The samples were placed in rotary shaker under RT at 200 rpm for 24 h. The filtrate was collected and to the residue the process of maceration was repeated and the final filtrates were evaporated under constant pressure and temperature through Rota evaporator. While the aqueous extract was evaporated in boiling water bath at 100⁰c. The final crude extracts were collected and stored in refrigerator till further use.

2.2 Phytochemical screening

The prepared extracts were subjected to standard method (Trease and Evans, 1989) of phytochemical analysis, viz.tannins, alkaloids, saponins, cardiac glycosides, terpenes, flavonoids, phenol, carbohydrate and protein. 1 % of individual extract from crude extracts were dissolved in 10 ml DMSO and final volume was adjusted with distilled water for phytochemical analysis.

2.3 DPPH assay

The antioxidant activity of each samples was determined through free radical scavenging by DPPH method(Goggi and Malpathak, 2017). Four different concentrations 50, 100,150,200 µg/ml were tested for each extract (water, methanol and oil). 0.1 mM DPPH was prepared in 100 ml pure Methanol. Each concentration of individual extract was placed in a reaction mixture containing DPPH solution (2.050, 2.00,1.950, 1.900 ml) at a final concentration to become 50, 100,150,200 µg/ml, and shaken vigorously for 1 min. The reaction mixtures were incubated at room temperature under dark condition for 10 min, later incubation the samples were measured at 517 nm in UV spectrophotometer. 100 % Methanol was used as the blank. The scavenging activity of each extract was estimated based on the percentage of inhibition of DPPH using the following formula, $$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\frac{Acontrol-Aextract}{Acontrol}\mathrm{x}100$$

2.4 Effect of various extract on wound healing and hair growth

Twelve healthy male mice from animal house facility, University of technology and Applied sciences- Higher College of Technology, prior to experiment all the ethical approval was taken and were segregated into 4 groups and each group consists of three mice as follows.

Group1: treated with aqueous extract of J. excelsa.

Group 2: treated with wild olive extract.

Group 3: cultivar olive extract and.

Group 4: positive control.

Briefly, the wound was made longitudinal measuring 2 cm on the dorsal side of the mouse was made by using sterile scissor and razor. The experimental groups received the respective treatment using swaps around the wound continuously for 2 weeks.

To study the hair growth, the hair on each mice measuring a distance of 2 cm length and 2 cm width at abdomen side was removed using sterile blade. For both plant material different extracts were applied on all the experimental animals respectively. While for control cage, mice were separated, two mice were treated as positive control by applying commercial medicine (Betadine-wound care), whereas coconut oil was used as positive control for hair growth study. The treatment was continued for 10 days and the observations were recorded by measuring the distance recovered (±cm) /density of hair growth.

3.1 Phytochemical screening

The water, methanol and oil extracts of Juniperus excelsa, Olea oleaster (wild olive) and Olea europaea (Cultivar olive) showed the presence of saponin, cardiac glycosides, terpenes, phenol and carbohydrates. The water extract of wild /cultivar olives leaves junipers leaves revealed the presence of all the tested phytochemicals except tannins and alkaloids in methanol extract, terpenes and flavonoids in J excelsa aqueous extract represented in Table 1.

The methanol extract of tested plant materials revealed the presence of all above mentioned phytochemical except the tannins (-), protein and flavonoids (-). All the oil extract showed the presence of all tested phytochemicals (+++) except flavonoids (-) and alkaloids (-). In addition, the methanol and oil extract of both the tested plant samples do not show any positive result for flavonoids (-). Saponins (+++) and cardiac glycosides (+++) are found abundant in all tested plant materials with respect to the solvents. While phenols also found abundant (+++) in all the tested plant material except oil extract of J excelsa (-) and cultivar olive (-). Terpenes are also showed the presence at high quantity (+++) in all the tested plant materials except J excelsa leaf aqueous extract (-). Alkaloid are found at high concentration (++++) in aqueous and methanol extract of both the plant materials except oil extract. Tannins showed (+++) in all the plant extracts with respect to solvents except methanolic extract.

Several reports revealed that phytochemicals are responsible for protection from several chronic diseases like polygenic disorder, cancer, cardiovascular disease and Alzheimer’s. Phytochemicals are able to scale back the aerophilic injury to our cells that causes varied diseases like cancer because they tend to inhibit cell proliferation and angiogenesis (i.e. the growth of new blood vessels) which are both trademarks of cancer. In addition, phytochemicals also play a role in regulation of nitric oxide, important in relaxing blood vessels and therefore increasing blood flow (Oliveira 2015). Differences in the ingredients in leaves grown in different part of world results the composition of individual plant may vary widely due to the climate, geographical location and time of collection. Nabi et al., 2012a, 2012b Reported that alkaloids, flavonoids, saponins, phenols and terpenes are present in the leaves of crude extracts whereas terpenoids and cardiac glycoside were absent in the leaves of crude extract of Junipers excelsa. The exploration of various phytoconstituents and their importance is an interesting and valuable field in the modern era to treat many human ailments with less side effects. Among the search of plants for saponins has been stimulated by the need for readily accessible sources for saponins, which can be converted in the laboratory to animal sterols of therapeutic importance (Reddy et al., 2012). Presence of some of the phytochemicals like terpenoids, alkaloids, phenolic, flavonoids, saponins and cardiac glycosides are involved in hypoglycemic activity (Reddy et al., 2016).

3.2 DPPH assay

Free radical scavenging activity of Juniperus excelsa,Olea oleaster (wild olive) and Olea europaea (Cultivar olive) extracts by DPPH method and the results are presented in Graph 1A,B&C.

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Graph 1

DPPH assay A. Water extract, B. Methanolic extract and C. Oil. All the values are the mean of triplicates.

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The antioxidant Property is depending on the interaction of active substances with oxidative free radicals. The assumption of DPPH method is that the antioxidants react with the stable free radical and gradually leads discoloration. The degree of discoloration indicates the scavenging potential of samples. In this study, the three extract (water, methanol and oil) from Juniperus excelsa, Olea oleaster (wild olive) and Olea europaea (Cultivar olive) were capable to decolorize the DPPH at varied levels. DPPH free radical scavenging potential of water extract of junipers (50 µg/ml = 83.30 %) found to be more effective while at 200 µg/ml the scavenging percentage was found to be 64.62 %. whereas the water extract of cultivar olives found to be 22.92 % at 150 µg/ml. Since the junipers and cultivar extract does not show the presence of flavonoids and phenols therefore, it showed the lowest percentage of antioxidant compared with wild olives that has rich content of flavonoids and phenols. The presence of flavonoids and phenols in the sample extract convert to reactive non-stable DPPH free radical into stable nonreactive stable DPPH from by donating electron or hydrogen radical.

The antioxidant property of the respective plant extracts is relevant the presence of phenols and flavonoids. The strong free radical scavenging activity is directly proportional with the different extract that contain high quantity of phenols and flavonoids. This indicates that the tested plant extracts possess a good source for natural antioxidant to prevent free radical oxidative damage. The antioxidants of plant act as inhibitor of oxidative damage even at minute concentration, which have diverse physiological role in the body (Weli et al., 2013). During oxidative stress, this property helps the plants to survive through such conditions. Especially in Mediterranean diet rich in natural antioxidants, limit the incidence of cardio and cerebrovascular diseases (Sudhir et al.,1986). The specific antioxidants belonging to several phytochemical components like phenols, flavonoids, and carotenoids are responsible to scavenge free radicals such as O₂, OH, or lipid proxy radicals in plasma (Banerjee et al., 1993).

3.3 Wound healing study

Wound healing effects of water extract obtained from Junipers excelsa,Olea oleaster (wild olive) and Olea europaea (Cultivar olive) by maceration method of extract was studied on mice, the results are placed in Table 2 and Fig. 1. It was noticed that the J excelsa extract showed the best effect on the recovery of linear wound which is 1.6 cm followed by cultivar (1.467 cm) and wild olive (1.433 cm) respectively, the initial size of the wound was 2.0 cm. Several reports suggested that the J excelsa extract is enriched with natural bioactive substances which provides antimicrobial activity, moisturizing activity and building of the skin to help in healing the wound and cuts, also reduction of inflammation at the wound site.

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Fig. 1

Effect of Juniperus excelsa, Olea oleaster (wild olive) and Olea europaea (Cultivar olive) on wound healing.

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3.4 Hair growth

On the hair growth study, we found that the wild and cultivar olive water extract have the best effect on hair growth which are 1 cm and 1.05 cm respectively, the results are placed in Table 3 and Fig. 2. The J excelsa has the least distance covered than the other extract (wild, cultivar olive and control (coconut oil) which is 0.5 cm. J excelsa and olive extract will simply penetrate the scalp and its cleansing result helps in detoxifying the hair follicles and promotes blood circulation within the scalp. It will increase nutrients to the hair follicles that facilitate in up hair growth. Continuous use of J excelsa oil and olive oil can restrict the assembly of DTH that successively forestall hair fall.

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Fig. 2

Effect of Juniperus excelsa, Olea oleaster Effect of Junipers excelsa, Olea oleaster(wild olive) and Olea europaea (Cultivar olive) on hair growth.

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Most of the living organisms specifically plants and animals keep huge amounts of antioxidants on it, due to their role in counteracting cell damage, as well as their ability to repair damaged cells. Many experts revealed that increased antioxidants in the body can also help slow the process of ageing, and may even increase longevity (Edward 2009). Many factors can adversely affect this process and lead to improper and impaired wound healing such as improper diet, infection at the wound site with microorganism, drugs, elderly age, diabetes and other disease conditions (Kerstein 2007). In addition, oil could be a natural source of antioxidants that fight with free radicals.