Natural antioxidants from plants increase the antioxidant capacity of plasma and reduce the risk of certain diseases such as cancer, heart disease and stroke. Natural substances stemming from vegetables have multiple interests taken advantage in the industry: in food, in beauty care and pharmacy. Among these compounds we find in a big part (party) the secondary métabolites which especially became famous in therapeutics^[1]. Of numerous métabolites secondary sectors (high schools, Secondary) essentially polyphenols are antibiotics in the broad sense, because they protect plants against mushrooms, bacteria, animals and even other plants^[2]. Polyphenols are also known for their biological activities that are directly related to human health. They are used in chemotherapy and in the treatment of several types of cancer^[3], as well as the treatment of cellular aging, skin protection^[4] in general and non-communicable diseases^[5]. Polyphenols are known by their anti-oxidant^[6] and enzymatic activity^[7]. Currently, thanks to the development of extraction methods and techniques of physico-chemical and biological analyses, Investigations into plant extracts rich in high antioxidant molecules and the measurement of their antioxidant activity in different media remain important research areas. Previous studies by Said Omar S H have shown that Plectranthus aromaticus essential oil is antimicrobial^[8] and antioxidant^[9]. Our work falls within the framework of the valorization of the extracts of an aromatic and medicinal plant called Plectranthusaromaticus (Parovi in vernacular language) which belongs to the family of Lamiaceae more precisely to the genus Plectranthus, this genus contains about 300 species of herbs or annual or perennial sub-shrubs, often succulent. Plectranthus aromaticus is one of the most important aromatic and medicinal succulent plants that have distinctive scented leaves with short erect and tender hairs^[10]. This study aims at the phytochemical study of polyphenols, their estimation in the extracts obtained and the evaluation of their antioxidant activity.

Materials and Methods

Vegetable material and extraction

The Plectranthus aromaticus leaves used in this study were harvested at Ngnadomboeni in the Hamahamet region located northeast of the island of great Comoros at an altitude of about 750 m. After being well dried in the shade, these leaves are carefully stored until use. Delipidation is carried out after the plant has been milled and subjected to Soxhlet extraction using hexane, and the residue is then macerated with stirring in a water / acetone mixture (30/70v/v), for 2 hours at room temperature. The resulting mixture was filtered and then with the rotary evaporator at (50°C) the filtrate was evaporated and this operation was repeated twice to exhaust the residue. The product obtained was lyophilized and the water-acetone extract (WAE) was obtained. Subsequently, in order to achieve a separation of phenolic compounds, the resulting extract (WAE) was solubilized in distilled water, and then it underwent liquid-liquid extractions, using two solvents, ethyl acetate and butanol. Three extracts are obtained, ethyl acetateextract (EAE), butanol extract (BUE) and aqueous extract (AQE).

Phytochemical tests of polyphenols

Phytochemical tests are simple and rapid chemical characterization reactions that allow the detection of different families of polyphenols and are based on precipitation or staining phenomena by reagents specific to each family of compounds^[11]. These tests are carried out on the plant and the extracts in order to locate the different families of secondary metabolites.

Test for tannin

The presence of tannins in plant extracts is demonstrated by adding to 1 mL of the extract, 1 mL of distilled water and 1 to 2 drops of a ferric chloride solution (FeCl₃) diluted 10 times, the color turns blue in the presence of gallic tannins and greenish blue in the presence of catechetical tannins^[12]. The differentiation between gallic and catechetical tannins is made by the Stiasny reagent (formol/concentrated hydrochloric acid 2:1 v / v) according to the following operating methods^[13].

Catechetical tannins: 1 mL of the extract is collected, 1 mL of the Stiasny reagent is added, and the resulting mixture is heated in a water bath at 90°C for 15 min. The appearance of a red precipitate shows the presence of catechetic tannins.

Gallic tannins: The revelation of gallic tannins consists of filtering the previously heated and sampled 0.5 mL of filtrate. The filtrate is then saturated with sodium acetate; a few drops of 2% (m/v) FeCl₃ are added to this mixture. The appearance of a blue-black stain indicates the presence of gallic tannins not precipitated by the Stiasny reagent.

Test for flavonoids

The flavonoid detection reaction principle is to place 5 mL of the extract in a tube, to which 1 mL of concentrated hydrochloric acid (HCl) and some magnesium-turning fragments are added.The presence of flavonoids is evidenced by the appearance of a red or pink color^[14].

Test for glycosides

One mL of the solution of the extract is placed in a test tube, 1 mL of acetic acid and 1 mL of concentrated sulfuric acid are added, and then 2 to 3 drops of 2% FeCl₃. The appearance of a green blue or a brown ring indicates the presence of glycosides^[15].

Test of reducing compounds

The detection of the reducing compounds consists in treating 1 ml of each extract with 2 ml of distilled water and 20 drops of Fehling liquor and then heating in a water bath. A positive test is indicated by the formation of a red-brick precipitate^[16].

Dosage of polyphenols and flavonoids

The levels of secondary metabolites are calculated in Plectranthus aromaticus extracts by colorimetric and spectrophotometric assays following conventional protocols. The study of the quantitative estimate of total polyphenols of the various extracts was carried out using the Folin - Ciocalteu method adapted by^[17]. In test tubes, a volume of 0.2 mL of each sample dilution or the gallic acid (used as standard) is added to 1 mL Folin-Ciocalteu reagent diluted 10 fold, after 2 min, 0.8 mL of Na₂CO₃ (7, 5%) is added. A control is prepared in parallel, under the same conditions, with distilled water instead of the extract solution, then the entire is incubated for half an hour at 25°C and the absorbance is determinated at 765 nm. The determination of the quantity of total polyphenols was performed by means of the calibration line (y = ax + b) obtained by the reading of the optical density as a function of the concentrations of the gallic acid. The polyphenol content is expressed in milligrams equivalent of gallic acid per gram of dry matter (mgEGA / gDM)^[18].

The quantification of flavonoids was performed by a method based on the formation of complexes between flavonoid compounds and aluminum trichloride^[19]. 1 mL of each sample dilution or quercetin (used as standard) is blended with 3 mL of distilled water. After agitation, 0.3 mL of sodium nitrate (5%) is added and the mixture is agitated well. After 5 min, 0.2 mL of aluminum chloride are added to the mixture, then the whole is incubated in the darkness at ambient temperature for half an hour, afterward 0.5 mL of sodium hydroxide (1M) are added then the optical density is determined at 510 nm. It is expressed in milligrams of quercetin equivalent per gram of dry matter (mgEQ/gDM).

Evaluation of antioxidant activity

There are many methods of evaluating the antioxidant activity of plant extracts, and in this study we have chosen to evaluate the antioxidant activity of Plectranthus aromaticus extracts using two different methods: the method of trapping the free radical DPPH (2.2-diphenyl-1-picrylhydrazyl) and the method of iron reduction FRAP (Ferric Reducing Antioxidant Power).

DPPH free radical-scavenging activity

The antiradical activity of the polyphenolic compounds in the prepared extracts is evaluated by measuring their ability to trap the DPPH free radical. This radical of dark violet turns yellow when reduced. The principle is to prepare increasing concentrations by dilution of the stock solution of the various extracts and ascorbic acid taken as standard.The DPPH solution is prepared at a concentration of 30 µg / mL in ethanol. In test tubes, 0.1 mL of each solution is added to 2 mL of the freshly prepared DPPH solution. A negative control is prepared in parallel with methanol. Then the whole is incubated in the dark for 30 minutes. The absorbance is measured at 517 nm, three optical density measurements were determined for each solution.Percent inhibition was calculated from the following equation:

Abs (B): absorbance of white.

Abs (E): absorbance of the sample^[20,21].

This formula makes it possible to draw the line which represents the variation of the percentages of inhibition according to the different concentrations of each sample (y= ax+b). From this line, it is possible to deduce the concentration which allows to reduce 50% of the DPPH radical (IC50) for each sample studied as well as ascorbic acid. The tests were performed in triplicate.

FRAP ferric-reducing activity

The assessment of antioxidant potency is based on the ion reduction reaction (Fe⁺³) present in the potassium ferrocyanide ion complex (Fe⁺²) and is determined by the method described by^[22]. The reducing power of the phenolic compounds is determined by the FRAP method by means of a series of dilutions of the stock solutions of the various methanol extracts for WAE, EAE, BUE and aqueous extract AQE in distilled water.Ascorbic acid solutions (used as standard) are prepared in the same way.

Test tubes each containing 0.4 mL of sample solution, 1 mL of phosphate buffer (0.2 M; pH 6.6) is added and 1 mL of potassium hexacyanoferrate (K₃Fe(CN)₆) at 10 g / L. The set is heated to 50°C in a water bath for 20 minutes.Then 1 mL of trichloroacetic acid is added and the mixture is centrifuged at 3000 rpm for 10 minutes. Finally, 1 mL of supernatant is mixed with 1 mL of distilled water and 0.2 mL of ferric chloride (FeCl₃) at 0.1 %. A blank is prepared under the same operating conditions. The absorbances are read at 700 nm, three measurements of the optical density have been determined for each solution.

To explore the results obtained, the antioxidant activity of the extracts is evaluated by comparing the lines (y = ax+b) absorbances obtained as a function of the different concentrations used in the ascorbic acid calibration curve.The increase in absorbance corresponds to an increase in the reducing power of the tested fractions. From these lines it is possible to deduce the concentration equivalent to 0, 5 of the absorbance of each extract studied as well as for ascorbic acid^[23]. The tests were carried out in triplicate.

Results and Discussion

Extraction of extracts

The results of the quantitative study (table 1) show that from 35 g of dry matter after extraction with Soxhlet followed by maceration and liquid-liquid extraction, a total of 5.92 g of extractable products can be obtained from the solvents used (about 17% starting material). Water-acetone-extractable polar products account for 9.4% compared to 7.51% of hexane-extractable nonpolar compounds. The yield is expressed as a percentage of the mass of the extract in relation to the mass of the dry matter, we obtain the water-acetone extract (9.4%), followed by the hexanic extract (HexE) (7.51%) and butanolic extract (6.47%), and finally the aqueous extract (1.48%), and ethyl acetate extract (1.45%) were obtained with similar yields.

Table 1: Quantitative Results of Extracts.

*: Expressed in relation to dry matter

Results of phytochemical tests

Phytochemical tests on the plant leaves (table 2) showed the presence of gallic tannins, flavonoids, sterols, triterpenes, coumarins, reducing compounds, and glycosides. On the other hand, catechetical tannins, alkaloids, anthraquinones and saponins are absent.

Table 2: Results of phytochemical tests on Plectranthus aromaticus leaves.

The results of the phytochemical tests carried out on the extracts of Plectranthus aromaticus leaves are grouped in table 3. We note the presence of gallic tannins, flavonoids, reducing compounds and glycosides in the WAE extract. This is in agreement with the results obtained on the leaves of the plant. It is also noted that all these sought-after compounds have been revealed in the BUE extract and glycosides are present in the different extracts. In addition, flavonoids are detected in the EAE extract and reduction compounds in the AQE extract.

Table 3: Phytochemical test results on extracts.

(+) : presence and (-) : absence

Content of total phenols and flavonoids in extracts

The results of the total polyphenol contents obtained are presented in Table 4. These results clearly show that the extracts obtained are rich in polyphenols. The initial WAE extract contains the majority of the phenolic compounds extracted; its polyphenol content is in the order of 28 mgEAG/gMS. This confirms the results of the phytochemical tests carried out on the leaves of the plant and on the WAE extract. Extracts from the WAE separation contain about 14.8% for EAE extract, 16% for AQE extract and 21.8% for BUE extract. According to the phytochemical tests carried out, the BUE extract contains the different types of polyphenols which may explain the higher polyphenol content compared to the other two extracts. However, there is a decrease in the total polyphenol content after separation, which has not been elucidated to date. Previous work on Plectranthus aromaticus extracts from India^[24], recorded polyphenols in the order of 11.53 mgEAG/gMS in the acetone extract and 8.39 mgEAG/g MS in the butanolic extract.

The results of the flavonoid contents in Table 4 show that the WAE extract is rich in flavonoids with a content of 17.88 ± 0.39 mg EQ/g MS. This is more than three times the amount of flavonoids obtained in the EAE extract (5.39 ± 0.04 mgEQ /gMS) and the BUE extract (4.39 ± 0.57 mgEQ / gMS). Flavonoid contents reported by (C.T. Sulaiman., et al. 2018) for Plectranthus aromaticus are in the order of (8.85± 0.42 mg EQ / g MS) for acetone extract and (2.56 ± 0.28 mg EQ / g MS) for butanol extract.The aqueous extract did not reveal the presence of flavonoids according to the results of the phytochemical tests.

The distribution of secondary metabolites may change during plant development and the difference in phenolic content between plants depends on a number of intrinsic and extrinsic factors (climatic conditions, geographical area, cultural practices, harvest maturity and storage conditions), which stimulate the biosynthesis of secondary metabolites such as polyphenols^[24,25].

Table 4: Polyphenol content in the various extracts of Plectranthusaromaticus.

Antioxidant activity of extracts

The evaluation of the antioxidant activity of Plectranthus aromaticus extracts is carried out by the DPPH radical reduction test (2.2'-diphenyl-1-picrylhydrazyl) and the FRAP method (Ferric Reducing Antioxidant Power).The absorbance measurements of the solutions are obtained using a spectrophotometer following the reduction of this radical which is accompanied by the passage of its violet color (DPPH) to a yellow coloring (DPPH-H) measurable at 517 nm. These lines (figure 1) express the variation in the percentage inhibition of DPPH in a known concentration range.

Figure 1: Percentage of DPPH inhibition based on concentrations of Plectranthusaromaticus extracts

From these lines, we can deduce the concentration needed to reduce 50% of DPPH (IC50) for ascorbic acid and each extract. The IC50 is inversely proportional to theantioxidant capacity of a compound since it expresses the quantity required to decrease the concentration of the DPPH radical to 50%. It is noted that the lower the IC50 value, the greater the antiradical power of a compound. The (Table 5) shows the IC50 values obtained for each extract of Plectranthusaromaticus.

Table 5: Total antioxidant capacity of different leaf extracts of Plectranthusamboinicus.

These results show that ascorbic acid (AA) has a very potent anti-radical activity with IC50 of the order of 0.08 mg/mL, which is equal to that obtained by^[27] but lower than the 0.13879 mg/mL obtained by^[28] The IC50 values obtained for the four extracts show that EAE ethyl acetate extract is the most active (0.301 mg/mL) followed by WAE, BUE and AQE extracts respectively.

The antioxidant value of Plectranthusaromaticus extracts is then compared with ascorbic acid using the FRAP method. We have plotted the rights representing the variation in reducing power expressed as a function of mass concentration (figure 2) and (Table 5) shows the IC50 values obtained for each extract.

Figure 2: Comparison of the lines of each extract with that of ascorbic acid

The results obtained show that the antioxidant activity of ascorbic acid is higher than that of Plectranthusaromaticus extracts (figure 2). The ethyl acetate extract is the most active with an IC50 value equal to (0.240 mg/mL) followed by the EEA extract, confirming the results obtained by the DPPH radical trapping method. Based on these results it can be concluded that the original EEA extract, containing all phenolic compounds, is not necessarily the most active, and that its separation by liquid extraction-liquid has improved the antioxidant power and provide a more powerful extract.According to phytochemical tests on extracts, EAE contains only flavonoids and glycosides. The nature and types of these compounds may be responsible for these results^[29-31].

Conclusion

The present study has shown that Plectranthus aromaticus is still an important resource for new therapeutic compounds to fight non-communicable diseases. This work allowed us to perform a phytochemical analysis and to highlight the presence of secondary metabolites in our Plectranthus aromaticus extracts. The results revealed the presence of gallic tannins, flavonoids, sterols and triterpenes, coumarin, glycosides and reducing compounds.The total polyphenol content in the various extracts is estimated by spectrophotometric analyses using the Folin-Ciocalteu, the extracts contain a high content of total polyphenols in the order of 28 mg EAG / gDM and flavonoids, estimated at 17.88 ± 0.39 mg EQ / gDM using the aluminum trichloride method.

The evaluation of the antioxidant activity of these extracts is revealed by two different methods, first the DPPH free radical reduction test and the iron reduction test (FRAP). The calculation of the IC50 values obtained by both methods revealed that the EAE ethyl acetate extract is the most active extract followed by the WAE water-acetone extract. However, this activity remains low compared to ascorbic acid, which is a powerful antioxidant. This plant native to the Comoros can be considered as one of the essential medicinal plants for the reduction of non-communicable diseases.However, further research is needed to confirm the activities obtained and explain the mechanism of action of antioxidant activity for different NCDs. In addition, further studies are needed to isolate and elucidate the structure of antioxidant active components from this plant species.

References

1. 1. Gurib-Fakim, A., Medicinal plants: traditions of yesterday and drugs of tomorrow. (2006) Mol Aspects Med 27(1): 1-93.

   Pubmed| Crossref| Others

2. 2. Buchanan, B., Gruissem, W., Jones, R., (2000) American Society of Plant Physiologists. 24 1250-1318.

   Pubmed| Crossref| Others

3. 3. Manach, C., Regerat, F., Texier, O., et al. Bioavailability, metabolism and physiological impact of 4-oxo-flavonoids. (1996) Nutr Res 16(3): 517-544.

   Pubmed| Crossref| Others

4. 4. Menaa, F., Menna, A., Tréton, J., Polyphenols against akin aging in polyphenols in human health and disease. (2014) 1: 819-830.

   Pubmed| Crossref| Others

5. 5. Almehizia, A. A, Abuelizz, H. A, Taie, H. A. A, et al. Étudier l’activité antioxydante de benzo [g] triazoloquinazolines en corrélation avec une étude DFT. (2018) J pharmaceutique saoudien

   Pubmed| Crossref| Others

6. 6. Halliwell, B., Free radicals and antioxidants: a personal view. (1994) Nutr Rev 52(8): 253-265.

   Pubmed| Crossref| Others

7. 7. Heinrich, M., Teoh, H.L., Galanthamine from snowdrop – the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. (2004) J Ethnopharmacol 92(2-3): 147-162.

   Pubmed| Crossref| Others

8. 8. Said Omar, S. H.,  Satrani, B., Ghanmi, M., et al. Activité antimicrobienne et composition chimique de l’huile essentielle de PlectranthusaromaticusRobx de l’île de la Grande Comores. Biotechnol Agron Soc Environ 15(2): 251-258.

   Pubmed| Crossref| Others

9. 9. Said Omar, S. H. Contribution à la valorisation de cinq plantes aromatiques et médicinales des Comores par la cractérisation chimique et la bioactivité de leurs huiles essentielles. (2011) Université Ibn Tofail Kenitra Maroc.

   Pubmed| Crossref| Others

10. 10. Arumugam, G., Swamy, M. Sinniah, U. Plectranthusamboinicus (Lour.) Spreng: importance botanique, phytochimique, pharmacologique et nutritionnelle. (2016) Molecules, 21(4) : 369.

    Pubmed| Crossref| Others

11. 11. Hagerman, F.C., Walsh, S. J., Staron, R. S., et al.  (2000) J Gerontol Une Biol Sci Med Sci. juillet 55 (7): 336-346

    Pubmed| Crossref| Others

12. 12. Trease, G.E., Evans, W.C. Pharmacognosy. (1989) 13ème edition: Ballière-Tindall: 436-445.

    Pubmed| Crossref| Others

13. 13. Nguessan, K., Kadja, B., Zirihi, G. N., et al. Screening phytochimique de quelques plantes médicinales ivoiriennes utilisées en pays Krobou (Agboville, Côte-d’Ivoire). (2009) Sciences & Nature: 1-15.

    Pubmed| Crossref| Others

14. 14. Falleh, H., Ksouri, R., Chaieb, K., et al. Phenolic composition of Cynaracardunculus L. orangs, and their biological activities. (2008) C R Biol 331(5): 372-379.

    Pubmed| Crossref| Others

15. 15. Bruneton, J. Phytochemical Study and Evaluation of Cytotoxicity, Antioxidant and Hypolipidemic Properties of Launaea taraxacifolia Leaves Extracts on Cell Lines HepG2 and PLB985. (1993) Pharmacognosie : 915.

    Pubmed| Crossref| Others

16. 16. Trease, G.E., Evans, W.C., Pharmacognosy, 14th ed. London. (1996) ELSB, Baillire Tindal.

    Pubmed| Crossref| Others

17. 17. Singleton, V. L. Rossi, J.A. Colorimetry of total phenolic with phosphomolybdic-phosphotungstic acid reagents. (1965) J American Society Enol Viticul 16: 144-158.

    Pubmed| Crossref| Others

18. 18. Taurre, N.,  Andlauer, W. Influence de la vérité de fraise et du période de récolte sur les contenus en antioxydants et en anthocyanes, ainsi que sur la répartition de l’acide ellagique dans le fruit, les akènes, les feuilles et le rhizome. (2007) Diplôme de l’ingénieur Haute Ecole de Suisse occidentale: 11.

    Pubmed| Crossref| Others

19. 19. Harborne, J.B., Plant Phenolics: Encyclopedia of Plant Physiology. (1980) New series 8: 329-402.

    Pubmed| Crossref| Others

20. 20. Falleh, H., Ksouri, R., Chaieb, K., et al. Phenolic composition of Cynaracardunculus L. orangs, and their biological activities. (2008) C R Biol (331(5): 372-379.

    Pubmed| Crossref| Others

21. 21. Zeghad, N. Etude du contenu polyphénolique de deux plantes médicinales d’intérêt économique (Thymus vulgaris, Rosmarinusofficinalis) et évaluation de leur activité antibactérienne. (2009) Thèse de magister option Biotechnologie végétal à l’université Mentouri Constantine.

    Pubmed| Crossref| Others

22. 22. Ouis, N., Ahmed, H. Phytochemical analysis and antioxidant activity of the flavonoids extracts from pods of Ceratonia silique L. (2017) J Pharm Pharmaceutics 4(2): 159-165.

    Pubmed| Crossref| Others

23. 23. Oyaisu, M.. Studies on products of browning reaction Antioxidative activities of products browning reaction prepared from glucose amine. (1986) J Nut 44(6): 307-315.

    Pubmed| Crossref| Others

24. 24. Sulaiman, C.T., Deepak, M., Balachandran, I., Spectrophotometric and tandem mass spectroscopic analysis of Indian borage (Plectranthusamboinicus (Lour.) Spreng) for its polyphenolics characterization. (2018) University J Basic Applied Sci. 7(4): 471-473.

    Pubmed| Crossref| Others

25. 25. Estevinho, L., Pereira, A.P., Moreira, L., et al. Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey. (2008) Food Chem Toxicol 46(12): 3774–3779.

    Pubmed| Crossref| Others

26. 26. Podsedek, A., Natural antioxidants and antioxidant capacity of Brassica vegetables: A review. (2007) LWT 40(1): 1-11.

    Pubmed| Crossref| Others

27. 27. Bentabet, N., Boucherit-Otmani, Z., etBoucherit, K. Composition chimique et activité antioxydante d’extraits organiques des racines de Fredoliaaretioides de la région de Béchar en Algérie. (2014) Phytothérapie 12(6) : 364–371.

    Pubmed| Crossref| Others

28. 28. Talbi, H., Boumaza, A. K., El-mostafa, J. (2015) Mater. Environ. Sci 6 (4): 1111-1117.

    Pubmed| Crossref| Others

29. 29. Hamburger, H., K. Hostettmann, (1991) Phytochemistry 30(12): 3874.

    Pubmed| Crossref| Others

30. 30. Mojab, F., kamalinejab, M., Ghaderi, N., et al. Phytochemical screening of some species of Iranian plants. (2003) Iran J Pharma Research 2(2): 77-82.

    Pubmed| Crossref| Others

31. 31. Rizk. A.M. constituants of plants growing in Qatar. (1982) Fitoterapia 53(2): 35-42.

    Pubmed| Crossref| Others