Chemical Fingerprint and Anti-Sickling Activity of Rosmarinic Acid and Methanolic Extracts from Three Species of Ocimum from DR Congo

The aim of this study was to characterize the polyphenolic composition by determination of chemical fingerprints of Methanolic extracts of Ocimum canum Sims, Ocimum basilicum L. and Ocimum gratissimum L. from Democratic Republic of Congo and to compare their antisickling activity of that of rosmarinic acid, the major compound to those of methanolic extracts. Phytochemical analysis performed by TLC and HPLC analysis, showed that rosmarinic acid is the most abundant phenolic acid in these Ocimum species according to the following order O. basilicum L., O. gratissimum L. and O. canum Sims. Methanolic extracts of these three species and pure rosmarinic acid showed significant antisickling activities with minimal concentration of normalization values of 0.18 ± 0.03, 0.23 ± 0.04, 0.26 ± 0.04 and 0.31 ± 0.05 mg/mL for rosmarinic acid, O. basilicum L., O. gratissimum L. and O. canum Sims methanolic extracts respectively. The antisickling activity order is the same as that of the rosmarinic acid content, indicating that this polyphenolic acid would be among the main active molecules in these extracts. Corresponding author. D. D. Tshilanda et al.


Introduction
Sickle cell disease (SCD) or sickle anemia is a hereditary blood disorder due to an abnormal hemoglobin, hemoglobin S (HbS).In low oxygen pressure condition, HbS aggregate into intracellular polymers, that confer the shape called sickle form to erythrocytes.This Red blood cells (RBCs) shape modification makes them fragile and less flexible; what causes many complications of sicklers [1] [2].
This chronic disease affects mainly sub-Saharan African.In Democratic Republic of the Congo (RDC) more than one and half millions of people are affected [3]- [5].
Some proposed therapeutic options seem inappropriate for low-income countries population, mainly in Africa.The World Health Organization (WHO) reported that 80% of the African population relies on herbal medicine for therapy.The extensive use of herbal medicine in Africa, has been argued to be linked to cultural and economic reasons.This is why the WHO encourages African member states to promote and integrate traditional medical practices in their health system [6].
Surveys undertaken by our research team in DRC conducted to more than 115 medicinal plants used in Congolese traditional medicine to manage sickle cell disease among which was O. basilicum [5] [7]- [15].In fact, in our previous works, our research team has shown that O. basilicum like some of medicinal plants used in Congolese herbal medicine in the management of sickle cell disease have antisickling activity in vitro.This biological activity is mainly displayed by polar fractions among which methanolic one and anthocyanins, organic acids and their derivatives were among the active chemical groups [3]- [5] [12]- [20].O. canum and O. gratissimum, two other species of Ocimum genus found in DRC, even if they are not cited by traditional healers in the management of sickle disease, showed also in vitro antisickling activity.This, confirming similar results already obtain for some species of Justicia and Zanthoxylum (Fagara) genera [11] [21].
O. basilicum L., O. canum Sims and O. gratissimum L. are aromatic herbs which are used extensively to add a distinctive aroma and flavor to food.The leaves can be used fresh or dried when they are used as spice.These aromatic herbs have been used as medicinal plants in the treatment of several sickness [22]- [25].
Generally, the aromatic plants and spices of the Lamiaceae family, among which Ocimum species are rich in polyphenolic compounds and a large number of them are well known for medicinal properties [26]- [28].
Recently, our research team have tested antioxidant activity of different extracts from the three species and showed that methanolic extracts exhibited the highest activity [17] [19] [20].Furthermore, ursolic acid isolated from O. gratissimum [18] and butyl stearate isolated from O. basilicum leaves [17] showed antisickling activity in vitro.
The aim of this work was to analyze the chemical fingerprints of the methanolic extracts of the three quoted species of Ocimum from RDC, to correlate their antisickling activity for a better characterization and understanding of the three methanolic extracts activities.And, to screen the presence of rosmarinic acid (RA), a naturally occurring phenylpropanoid known for its antioxidant and anti-inflammatory activity [29]- [31] in the extracts and to test the antisckling activity of a pure RA.

Plant Material
Leaves of Ocimum species were collected in the vicinity of Mbuji-Mayi (O.canum) and of the University of Kinshasa (O.basilicum and O. gratissimum) in DRC on May 2013.The collected plants were identified by comparison with reference specimen available at Biology department of University of Kinshasa, DRC.Voucher specimens (Ocimum basilicum: 425/Devred, Ocimum canum: 686/A.Thieband, Ocimum gratissimum: 8016/R.Dechamps) were deposited at the herbarium of the same department.Leaves of plants were dried at the room temperature and finely grounded in a high speed mill (Retsch ZM 100 Model) to 0.02 inches size.The powder leaves were stored in the dark at the room temperature and used for solvent extraction.

Biological Material
The blood samples used for the bioassays in this study were taken from adolescent sickle cell disease patients attending the "Centre de Medicine Mixteet d'Anemie SS" (CMMASS), located in Kinshasa, DRC.A written consent for each patient was approved by the national ethic committee (N˚ BE117).Ethical clearance on the use of SS blood was strictly observed according to international rules [32].
In order to confirm their SS nature, the above mentioned blood samples were first characterized by hemoglobin electrophoresis on cellulose acetate gel at pH 8.5 and then stored at 4˚C in a refrigerator.

Chemicals
All solvents were of analytical grade and purchased from Merck VWR (Leuven, Belgium).
Rosmarinic acid, luteolin, vitexin were HPLC grade from Sigma Aldrich.Water was treated in a Milli-Q water ultra-purification system (Easy Pure Purification System).

Preparation of Samples
The samples were extracted from 1 g plant powder sonicated for 15 min in 10 mL methanol and filtered on a what man filter paper.One milligram of RA of HPLC analytical grade was dissolved in 10 mL methanol and gently shaken.

High Performance Liquid Chromatography Analysis
The High performance liquid chromatography (HPLC) analysis was carried out at 25˚C by an "Agilent 1100" HPLC chain connected to a diode array detector (DAD).All samples and standards were filtered through a 0.45 μm pore size syringe-driven filter before 20 μL of each one were injected into the HPLC-UV/DAD system.The separation was carried out using an Hypersil ODS column (4 mm × 250 mm) with a nonlinear gradient of acetonitrile (solvent A) and 0.05% trifluoroacetic acid in ultra-pure water (solvent B) in the following composition: T0: 0% A, 100% B; T1: 3% A, 97% B; T45: 40% A, 60% B; T46: 0% A, 100% B and T60 stop.The time (T) is expressed in minutes.The compounds were eluted at a flow rate of 1 mL/min and detected with UV-DAD.The UV spectra of elution peaks were recorded in the range from 250 to 340 nm and the chromatograms were monitored at 280 and 340 nm.The identification was based on the retention time and the absorption spectra in comparison to the references and the data available in the database of the system [33].The presence of RA was confirmed by HPLC fingerprint of control pure RA in superimposing this last to the fingerprint of each methanolic extract of the three Ocimum.

High Performance Liquid Chromatography (HPLC) Analysis
Figure 2 shows the structure and the HPLC fingerprint of pure RA used as control by reason of comparison to the three HPLC fingerprints of the three methanolic extracts of three Ocimum.
As it can be clearly seen, the pure RA (a) appears as a single peak to the retention time equal to 32.408 minutes.As above noticed the profile of methanolic extract of O. canum (b) shows two intense peaks.The most intense and consequently major compound appears to the retention time equal to 58.719 minutes, followed by the one at 32.494 minutes.This last has approximately equal RA retention time.These results are near to what found by Berhow et al. [35] who has found a retention time of 30.07 min.
The presence of RA in the methanolic extract of O. canum is confirmed by the overlay of both the pure RA peak and the characterized fingerprint of methanolic extract, at the same retention time.The available data in the database used as references of the system did not allow us to identify the compound to the retention time of 58.719 minutes.
It can also be seen in the fingerprint of O. basilicum (b) the most intense peak to the retention time of 32.300 min.Approximately this last value is equal to the one above of RA.These results are alike to that of Vlase et al. [36] in methanolic extract of O. basilicum from Romania.
The presence of RA is demonstrated by the superimposition of the peak of the HPLC fingerprint of pure rosmarinic acid with the corresponding peak of the HPLC fingerprint of the methanolic extract of O. basilicum, to the identical retention time.By comparing the two intensities identified above peaks between O. canum and O. basilicum, the peak of this last (b) appears to be more intense.Thus, that result confirms that O. basilicum has more important amount of RA than O. canum.
Ultimately, it is noticed in Figure 2 in the fingerprint of O. gratissimum (d) the most intense peak to the retention time of 32.310 min.This seems to be approximately to RA founded value.This result is comparable to that found by Costa et al. [37] in methanolic extract of O. gratissimum from Brazil.
As for the two first methanolic extracts, the presence of RA is shown by the overlay of the two peaks of pure RA and the one of the methanolic extract of O. gratissimum to the almost equal time of retention.Once more    As it can be seen from these micrographies, the negative control (Figure 3(a)) contains the majority of sickleshaped erythrocytes, confirming the Sickle cell nature of the blood.When betulinic acid, well known for its antisickling activity [17] [18] [38], is added as positive control (Figure 3(b)), Red blood cells show their normal circular (biconcave) shape.In the presence methanolic extracts of the three species of Ocimum (Figures 3(c)-(e and RA (Figure 3(f)), the majority of erythrocytes are reversed normal-shape.This fact indicates that the methanolic extracts have an antisickling activity and show also a good normalization effect of drepanocytes in hypoxic condition.The same behavior was already observed form active extracts from some plants used in the management of sickle disease in Congolese traditional medicine, justifying their traditional use.Some molecules contained in these extracts would interact with HbS and compete with its polymerization reaction.This would prevent the sickling of red blood cells.In fact, according to Russu et al. [39] when investigating on molecular basis for the antisickling activity of aromatic amino acids and related compounds by NMR, phenolic acids would interact with HbS on a binding site located at or near the beta 6 position (the site containing the mutation in beta 6Glu to Val).This binding would induce conformational changes in the amino-terminal domains of the beta chains.

Normalization Rate
To compare the antisickling activity of different drugs, it is necessary to determine the minimal concentration of normalization (MCN) or the concentration that normalizes 50% of drepanocytes (ED50) [5] [9] [10] [15] [16].Figure 4 gives the dose dependent antisickling activity of methanolic extracts of three Ocimum leaves and RA.
Figure 4 shows that the sickle cells normalization rate increases with the methanolic extracts doses until reaching the maximum threshold of which the normalization rate remains constant despite the increase of the extracts concentration.The determined minimal concentrations of normalization of each extract and RA i.e. the weakest concentration of extracts or RA for which the normalization rate is maximum are listed in the table.
As it can be noticed from Table 1, RA shows higher antisickling effect than all methanolic extracts.This indicates that the main secondary metabolites responsible of the antisikling effect in the three methanolic extracts have to be found as RA.This is confirmed by the fact that O. basilicum has highest amount of RA and exhibited   [18] [38]- [41].These results indicate that rosmarinic acid is among these antisickling active organic acids.The kwon antioxidant activity of RA [29] [35] [37] may also contribute to its antisickling activity, because sickle disease is in relation with the oxidative stress [42] [43].

Conclusion
The present study fingerprinted methanolic and tested the antisickling activity of extracts of three species of Ocimum from DR Congo: O. basilicum L., O. gratissimum L. and O. canum S. The TLC and HPLC analysis confirmed the presence of Flavonoids like kampferol derivates, phenolic acids and RA as phenol acid.This last is in high amount in the three Ocimum species extracts.The O. basilicum specie has a higher quantity of RA acid, followed by O. gratissimum and then O. canum.This order is also respected for the antisickling activity since pure RA has the highest antisickling activity and would be among the phenolic acids responsible of antisickling activity of these Ocimum species.RA could constitute a phyto-marker for the quality control of herbal medicines from these three plants without its isolation as pure compound.

Figure 1 (Figure 1 .
Figure 1(a) and Figure 1(b) show the TLC fingerprint of methanolic extracts of the three species of Ocimum firstlyalone and then with phenols controls, respectively.As above noticed, TLC analysis show that the different extracts of Ocimum contain polyphenols, including: Chromatogram (a): flavonoids of Kampférolkind (yellow-orange fluorescent spots) and phenolic acids (blue

Figure 2 .
Figure 2. HPLC-DAD chromatograms of Rosmarinic acid (a) and methanolic extracts from O. basilicum (b), O.canum (c), O. gratissimum (d) using a nonlinear gradient of acetonitril (A) and 0.05% trifluoroacetic acid in ultra pure water (B): 0 min, 0:100 (A:B), 1 min, 3:97(A:B); 45 min, 40:60 (A:B); 46min, 0:100 (A:B) and 60 min, stop at 1 mL/min on Hypersil ODS column (4 mm × 250 mm) with detection at 340 nm.The elution time is given in minutes (horizontal axis).On the y axis, the height of the elution peak, expressed in arbitrary milli-unities (mAU) corresponds to the concentration of the eluted compound in the sample analysed.again comparing the three fingerprints of the methanolic extracts of the three species according to their intensity, O. basilicum contains the highest amount of RA, followed by O. gratissimum and then O. canum. mAU

Figures 3 (
Figures 3(a)-(f) show respectively micrographies of SS blood alone in a NaCl 0.9% solution (negative control), betulinic acid (positive control) and the sickle cells blood incubated with the methanolic extract of O. canum, O. gratissimum and O. basilicum, and RA finally.As it can be seen from these micrographies, the negative control (Figure3(a)) contains the majority of sickleshaped erythrocytes, confirming the Sickle cell nature of the blood.When betulinic acid, well known for its antisickling activity[17] [18][38], is added as positive control (Figure3(b)), Red blood cells show their normal circular (biconcave) shape.In the presence methanolic extracts of the three species of Ocimum (Figures3(c)-(e))

Figure 3 .
Figures 3(a)-(f) show respectively micrographies of SS blood alone in a NaCl 0.9% solution (negative control), betulinic acid (positive control) and the sickle cells blood incubated with the methanolic extract of O. canum, O. gratissimum and O. basilicum, and RA finally.As it can be seen from these micrographies, the negative control (Figure3(a)) contains the majority of sickleshaped erythrocytes, confirming the Sickle cell nature of the blood.When betulinic acid, well known for its antisickling activity[17] [18][38], is added as positive control (Figure3(b)), Red blood cells show their normal circular (biconcave) shape.In the presence methanolic extracts of the three species of Ocimum (Figures3(c)-(e))

Figure 4 .
Figure 4. Evolution of the normalization rate of the drepanocytes form with the methanolic extracts of Ocimum and rosmarinic acid (RA).

Table 1 .
Minimal concentration of normalization (MCN) of different methanolic extracts and rosmarinic acid.activity, follow by O. gratissimum and then O. canum.In fact, most isolated antisickling active molecules are phenolic acids