Phytochemical analysis, antioxidant and anti-inflammatory activities of Chassalia kolly leaves extract, a plant used in Benin to treat skin illness

In the last decades, it has been intensively studied natural alternatives to synthetic products. Chassalia kolly is one of the medicinal plants used traditionally for treatment of skin infections. This study aimed at identifying the diversity secondary metabolites, to investigate the antioxidant and anti-inflammatory activities of ethanolic extract of Chassalia kolly leaves. Total phenols were determined by Folin Ciocalteu method. The aluminum trichloride method has been used to quantify total flavonoids, while the determination of condensed tannins was carried out by the hydrochloric vanillin method. The antioxidant capacity was evaluated by 2, 2-diphenyl-l-picrylhydrazyl (DPPH), 2, 2'-Azino-bis(3ethylbenzothiazolin-6-sulfonic acid) (ABTS) and phosphomolybdenum methods. In vitro anti-inflammatory activity of ethanolic extract of Chassalia kolly leaves was evaluated by method membrane stabilization. In this results, Chassalia kolly leaves contain flavonoid, mucilages, anthocyans, sterols and terpenes, saponosides, alkaloids and reducing compounds. The contents of total phenols, total flavonoids and total tannins are respectively (20.41±2.11) μgGAE/mgDM; (30.29±2.18) μgQE/mgDM and (1.211±0.122)μgCE/mgDM. The results obtained during this work allowed us to assert that all extracts of the studied plant have very good antioxidant properties (IC50=0.05μg/μL). The ethanolic extract of the leaves of Chassalia kolly showed an anti-inflammatory activity more interesting than aspirin which is the reference compound used in this study.


Introduction
Infectious diseases are one of the major causes of death in tropical countries. For a long time, chemotherapy based on the use of antibiotics remained the fastest and most effective way to treat infections. Unfortunately, the upsurge of microbial resistance to antibiotics and the spread rate of resistant bacterial species became major public health concern. In recent years, research has turned to therapeutic alternatives to prevent the upsurge from bacterial resistance to existing molecules. African flora in general and Benin in particular, abounds in an important reserve of medicinal plants. Thus, medicinal plants occupy an important place in the African pharmacopoeia. Even nowadays, medicinal plants have been used for treatments of various diseases since antiquity and still play an important role to cover the basic health needs in the developing countries [1]. Skin is larger than any other organ in humans. Like other organs, various bacterial, viral, and inflammatory diseases, as well as cancer, affect the skin. Skin diseases like acne, atopic dermatitis, and psoriasis often reduce the quality of life seriously [2]. Skin diseases are often not considered in priority areas of health systems planning, due in part to the infrequent mortality, and also to the lack of awareness of the burden of skin diseases in public health terms [3]. Skin diseases are abundant and diverse, accounting for about one third of medical consultations of Africa [4]. Skin diseases affect 21% to 87 % of children in African developing countries and constitute up to a third of outpatient visits to Pediatricians and Dermatologists [5], [6]. Despite their common occurrence, skin diseases receive less attention as compared with diseases such as malaria, pneumonia and HIV/AIDS, which cause significant mortality [7], [8]. However, the spectrum of the skin diseases varies from region to region due to several factors such as genetical, socioeconomical and environmental field [8]. Most of the people in rural and urban areas of the developing world depended on the medicinal plants for the treatment of infectious diseases, in particular the infections of skin. Therefore, effective treatment of skin disorders is important. Chassalia kolly the family Rubiaceae, is a soft-stemmed shrub native to West Africa [9]. Chassalia species have been prescribed for wound dressing, inflammation and liver disease, as an insect repellant and skin diseases in traditional medicine in Africa [10]. The studies on biological activities of medicinal plants and search for source of new therapeutic drugs, we now report on insecticidal, antimicrobial, phyto-and cytotoxicity of Chassalia kolly, whole plant extract [11]. The aim of this study is to promote Chassalia kolly, a plant used in Benin to treat skin infections by identifying secondary metabolites and then evaluating antioxidant and anti-inflammatory activities.

Plant material
Chassalia kolly leaves used in this study were collected from Abomey-Calavi in Benin.

Chassalia kolly leaves pretreatment
After harvesting, the samples were dried at laboratory temperature until their plant mass stabilized and then reduced to powder.

Plant extracts
The extraction was made with ethanol and hydroethanolic under ultrasounds. Briefly, 10 g of powdered biomass were mixed with 100 mL solvent and sonicated for two hours at 50℃ with Bandelin (Sonorex Digitech device). Further, all the extracts were filtered through Whatman No.1 filter paper and concentrated under vacuum (Buchi R215, heating bath B-491, rotation 280 rpm, vacuum controller V-850 of 290 mbar) at (50±1) ℃. The residues were dried to constant weights and stored in the darkness at 4℃ to avoid the degradations until use [12], [13], [14].

Preliminary phytochemical screening
Secondary metabolites were carried out by coloration and precipitation reactions specific to each family of metabolites [15][16][17].

Determination of polyphenolic compounds
Total phenol content Total phenolic content was determined using the Folin-Ciocalteu colorimetric method. This method consisted of using a mixture of phosphotungstic and phosphomolybdic acids, which were reduced during the oxidation of phenols into a mixture of tungsten blue oxide and molybdenum. Finally, the absorbance was measured at 760 nm using a spectrophotometer (with Infinite 200 PRO-Tecan microplate) and the total phenol content are expressed in micrograms of gallic acid equivalence per milligram of dry matter (µgGAE/mgDM) [12], [18], [19].

Total flavonoids content
The method of aluminum trichloride (AlCl3) was used to quantify the total flavonoids. This technique was based on the formation of the aluminum complex flavonoids. The absorbance was read at 415 nm using a spectrophotometer (Infinite 200 PRO-Tecan microplate) and the Total flavonoid content are expressed in micrograms quercetin equivalence per milligram of dry matter (µgQE/mgDM) [20].

Condensed tannin content
Vanillin and hydrochloric acid method was used to determine the total condensed tannins content. The absorbance were measured at 500 nm using the spectrophotometer (Infinite 200 PRO-Tecan microplate) and the tannin content was expressed in micrograms catechin equivalence per milligram dry matter (µgEC/mgDM) [21].

Antioxidant activity
The antioxidant activity of the ethanolic extract of Chassalia kolly leaves evaluated by ABTS, DPPH and ammonium molybdate tests.

DPPH free radical scavenging assay
100 μL of ethanolic extract of Chassalia kolly leaves were added to the five wells of the first two 96-well microplate lines. Then a gradual dilution of a ratio of two was carried out starting with the second line with methanol until the last line (8th line) where 100 µL of dilution was discarded at each well. In the first three wells of each row, 100 μL of the methanolic solution of DPPH (0.1mg/mL) were added and 100μL of methanol in the other, two wells of each row to prepare the negative control. The positive control is prepared in parallel by mixing 100μL of methanol with 100μL of the DPPH solution. After incubation in the dark room the temperature, the absorbance is measured after 15 minutes for one hour at 517 nm using the spectrophotometer (microplaque Infinite 200 PRO-Tecan) [12], [19], [22].
Ap: Positive control absorbance; An: Negative control absorbance; As: sample absorbance

Phosphomolybdenum assay
100µl of ethanolic extract of Chassalia kolly at different concentrations were added to 1000µL of a reagent composed of sulfuric acid (0.6M), sodium phosphate (28mM) and ammonium molybdate (4mM). The tube was incubated at 95°C for 90 minutes and after cooling, the absorbance was measured at 695nm. The control consists of 100μL of dissolution solvent mixed with 1000μL of the reagent mentioned above. Samples and controls are incubated under the same conditions and then the absorbance is measured using a spectrophotometer (Infinite 200 PRO-Tecan). The results obtained are expressed in micrograms ascorbic acid equivalent per milligram of dry matter of the extract (µgAAE/mgDM) [23].

ABTS free radical scavenging assay
The potential of the extracts to reduce the ABTS .+ radical was evaluated in using the method described by Miller et al [23]. The principle is based on the ability of an antioxidant to stabilize the blue-green cationic radical ABTS .+ by transforming it into colorless ABTS .+ by trapping a proton. The cationic radical ABTS .+ was obtained from 10 ml of ABTS (2mM) and 100μl of potassium persulfate (70 mM). The mixture was stored in the darkness for 6 hours before performing the test. 100 µL of extract was added to 100 µL of ABTS + and the absorbance was measured at 734 nm after 15 min for 1 hour. The blank was prepared by mixing 100µL of extract with 100µL of ethanol with a control or 100 µL of methanol was mixed with 100µL of the ABTS .+ radical. The potential of the extracts to reduce the ABTS .+ radical was expressed in microgram equivalence of Trolox per milligram of dry matter (µgETx/mgDM) from the calibration line of the Trolox [12], [19], [22].

Anti-inflammation activity
Anti-inflammatory activity was assessed by determining the stabilizing potential of the membrane of red blood cells. For this, 100μL of fresh human blood were mixed with 900 μL of sodium chloride solution (0.9%) and then centrifuged at 8000 rev/min for 10min. Then 300µL of supernatant was added to 300µL of extract and stirred for 30 min. For the negative control, 300µL of sodium chloride solution was mixed with 300 µL of supernatant. Aspirin was used as a reference compound by mixing 300 µL of aspirin (8µg/µL) and 300 µL of methanol. The samples were incubated at 56 °C for 30 min, centrifuged at 2500 rpm for 5min and the absorbance of the supernatant was measured at 560 nm. The experiment was carried out in triplicating. The percentage of stabilization of the red blood cell membrane was calculated [24], [25] [26].

Secondary metabolites in the Chassalia kolly leaves
The secondary metabolites identified in the leaves of Chassalia kolly are listed in Table 2. The results of the preliminary phytochemical screening revealed the presence of glycosides, alkaloids, flavonoids, anthocynes, reducing compound, mucilages and saponosids in the Chassalia kolly leaves. In the Chassalia kolly leaves collected from the Ibadan (Nigeria) glycosides, alkaloids and flavonoids have been identified [27]. The variation of secondary metabolites observed at the level of our samples compared to previous work could be related to the harvest period, the nature of the soil or climatic factors [28], [29]. The use of Chassalia kolly in traditional medicine to treat skin infections could be explained in particular by the presence of mucilages, saponosides and flavonoi ds in the leaves of this plant [30], [31].   The curves in Figure 2 show the percentage of DPPH radical scavenging as a function of the concentrations of the ethanolic extract from the leaves of Chassalia kolly. At the concentrations tested, there is a gradual increase in the percentage of scavenging of the DPPH radical by the ethanolic extract of Chassalia kolly leaves up to more than 95% before becoming practically constant. It is noted that the reaction of this extract is simultaneous. After 15 min, the reaction is almost complete. For this extract, the concentration making it possible to trap 50% of the DPPH radical is 0.05 μg/μL. This interesting antioxidant activity of Chassalia kolly is linked to the content of phenolic compounds in this plant. This extract could be used to fight against free radical attacks on the skin on the one hand and for the preservation of perishable food products on the other hand instead of these synthetic products.

Total antioxidant capacity of the ethanolic extract of Chassalia kolly leaves
The total antioxidant capacity of the ethanolic extract of Chassalia kolly is expressed in microgram equivalent of ascorbic acid per milligram of dry matter (µgEAA/mgDM) using the calibration curve plotted with ascorbic acid.
Calibration curve for measuring the total antioxidant capacity The total antioxidant capacity of the extracts studied is expressed in micrograms equivalents of ascorbic acid per milligram of dry matter from the calibration curve (y=4.0916x-0.0629; R 2 = 0.9929) Figure 3. The results obtained are expressed in μg equivalent of ascorbic acid per mg of dry matter (μgEAA/mgDM).

Figure 3
Calibration curve for the evaluation of the total antioxidant capacity This total antioxidant capacity is (0,058±0,002) µgEAA/mgDM for the ethanolic extract of this plant. The interesting activity of this extract is linked to its content of phenolic compounds.

Figure 4
Calibration curve for the potential of the extracts to reduce the ABTS .+ radical The potential of the extracts to reduce the ABTS .+ radical as a function of the reaction time expressed in micrograms Equivalence of trolox per milligram of dry matter is given in Table 3. The reduction potential of ABTS .+ varies from (33.163±3.255) µgEqTx/mgMS after 15 min of reaction at (16.908±3.019)µgEqTx/mgMS after one hour of reaction. Therefore, the potential of the ethanolic extract of Chassalia kolly leaf to reduce the radical cation ABTS .+ gradually decreases over time. From the analysis of this table, it emerges that the ethanolic extract of Chassalia kolly leaf showed interesting antioxidant activity. This noted activity would be due to the content of phenolic compounds in this plant. In view of the interesting antioxidant activity of the ethanolic extract Chassalia kolly, it could be used to fight against free radical attacks suffered by the skin.

Anti-inflammatory activity
The percentages of membrane stabilization on red blood cells by ethanolic extract of Chassalia kolly leaves and aspirin are given in table 4.
The ethanolic extract of Chassalia kolly leaves showed a percentage of limb stabilization of (75.916±0.142) % while for aspirin which is used as a reference compound is (70.125±0.11) %. So the ethanolic extract of Chassalia kolly showed more pronounced anti-inflammatory activity than aspirin. This noted activity could be explained by the presence of flavonoids and mucilages in the leaf of Chassalia kolly [31], [32]. The interesting anti-inflammatory activity of Chassalia kolly could justify its use in traditional medicine to treat skin infections.

Conclusion
Natural substances extracted from vegetal biomass have multiple benefits exploited in industrial biotechnology of food, cosmetics and pharmaceuticals as well. Plant secondary metabolites are a source of potential novel therapeutic. Skin infections still remain the leading cause of morbidity among skin diseases. The present study focused on phytochemical, antioxidant, antimicrobial, and anti-inflammatory properties of Chassalia kolly leaves. From the results obtained, it emerges that Chassalia kolly leaves have many secondary metabolites including glycosides, alkaloids, flavonoids, anthocynes, reducing compound, mucilages and saponosids.
Regarding antioxidant activity, the ethanolic extract showed interesting activity (CI50=0.05μg/μL). The ethanolic extract of Chassalia kolly showed more pronounced anti-inflammatory activity (P=75.916±0.142) % than aspirin (P=70.125±0.114) % which is a reference compound. The results of this study justify the use of Chassalia kolly in traditional medicine. Ethanolic extract of Chassalia kolly leaves could be used to fight against free radical attacks on the skin, to treat skin conditions and for the preservation of perishable food products instead of using synthetic products to avoid undesirable and toxic side effects on humans being.