Immunostimulatory activity of Stachys mialhesi de Noé

Background Immunostimulatory therapy is now being recognized as an alternative to conventional chemotherapy for a variety of disease conditions, involving the impaired immune response of the host. In the present study, the immunostimulatory effect of the butanolic extract obtained from S. mialhesi aerial parts, was evaluated in vivo. Methods The immunostimulant potential of the plant extract on the phagocytic activity was measured by the carbon clearance rate test. Results Our research revealed that at different doses (50, 100 and 500 mg/kg), S. mialhesi extract increased the phagocytic activity in a dose dependant manner when compared with the control and thus the clearance rate of carbon was faster after the administration of the plant extract. Conclusion S. mialhesi extract exhibited a dose-dependent immunostimulant effect on the reticuloendothelial system, which could be attributed to the presence of active principles in this butanolic extract.


Background
Immunostimulators have been known to support T-cell function, activate macrophages, granulocytes, complement and natural killer cells apart from affecting the production of various effectors molecules generated by activated cells (Paraimmunity) [1].
It is expected that non-specific effects offer protection against different pathogens, including bacteria, fungi, and viruses and constitute an alternative to conventional chemotherapy [2].
Immunostimulatory therapy is now being recognized as an alternative to conventional chemotherapy for a variety of disease conditions, involving the impaired immune response of the host [3].
The genus Stachys (Lamiaceae) includes about 200 to 300 species in the world [4] and is considered to be one of the largest genera of this family. In Algeria, this genus is represented by 14 species including the endemic species S. mialhesi de Noé [5].
In Iran, the aerial parts of S. inflata Benth are used to treat infection, asthma, rheumatic and other inflammatory disorders [8]. S. lavandulifolia Vahl was used as an anxiolytic and sedative [9].
The study of [10] showed inhibitory effects of S. obtusicrena on both cellular and humoral immune responses and suggests that this effect may in part be due to the induction of apoptosis in proliferative lymphocytes.
The stimulation index of all cultures treated with different concentrations of the extract of S. obtusicrena was less than 1 [10].
The present investigation was undertaken to evaluate the immunostimulatory effect of the butanolic extract obtained from the aerial parts of S. mialhesi, using in vivo model.

Plant material
Aerial parts of S. mialhesi de Noé were collected in April 2005 at Djebel El-Ouahch Constantine (North Eastern Algerian). The voucher specimen was identified by Professor Gérard De Bélair (University Badji-Mokhtar, Annaba) and was deposited at the Musée botanique de la Ville d' Angers (France) under the reference MBAng2005.10.

Preparation of the extract
Air-dried and powdered aerial parts (1 kg) of S. mialhesi were extracted with 70% MeOH. The residue was suspended in water and extracted successively with petroleum ether, dichloromethane, ethylacetate and n-BuOH.

Animals
Adult male Mus Musculus mice (2.5-3 month old) from central pharmacy Algeria, weighing (25-36 g) were used for determination of the phagocytic activity. The animals were kept under standard laboratory conditions of humidity, temperature (25± 1°C) and light (12 h day: 12 h night), and allowed free access to food and water.

Phagocytic activity
The clearance rate of carbon was measured by the method of [11].
Animals were divided into four groups, consisting of 5 mice in GI, GII and GIV but the GIII consisting of 4 mice. Group I (Control) was given 0.9% NaCl (0.5 ml/ mouse, i.p), groups II, III and IV were administered by i.p injection with different concentrations of S. mialhesi extract (50, 100 and 500 mg/kg) respectively.
After 48 h of i.p injection, the mice were administered with carbon ink suspension at a dose of 0.1 ml/10 g through the tail vein; the mixture consisted of black carbon ink 3 ml, saline 4 ml and 3% gelatin solution 4 ml.
Blood samples were taken from the retro orbital vein by using glass capillaries, at 5 and 15 min. Blood sample drops (14) were mixed with 0.1% sodium carbonate solution (4 ml) for the lysis of erythrocytes and the absorbance measured at 675 nm using a spectrophotometer.
The phagocytic activity is expressed by the phagocytic index K which measures all the reticuloendothelial system function in the contact with the circulating blood. The clearance rate is expressed as the half-life period of the carbon in the blood (t 1/2 , min). These are calculated by means of the following equations: Where OD 1 and OD 2 are the optical densities at times t 1 and t 2 respectively.

Statistical analyses
Results were analyzed for differences between the groups across dietary treatments by one -way ANOVA test and Tukey's multiple comparison tests (SPSS version 9).

Results
Effect of S. mialhesi extract on the phagocytic activity and the carbon clearance rate is shown in Table 1.
The present data showed that there is a difference in the means for the phagocytic index (K) between groups (GI, GII, GIII and GIV) P=0,058 and the group IV is significantly different from GI at P= 0,041.
This indicates that S. mialhesi extract enhanced the phagocytic activity by stimulating the reticuloendothelial system in a dose dependant manner ( Figure 1).
As shown in Figure 2, the clearance rate of carbon was faster after 48 h but not significantly between (GI, GII, GIII and GIV) groups P=0,149.
This indicates that the extract shows carbon clearance enhancing activity and affirms that S. mialhesi extract enhanced the phagocytic activity.

Discussion
The reticuloendothelial system is clearing particulate substances, such as bacteria, and altered endogenous materials, such as fibrin aggregates [12]. Phagocytosis is the mechanism by which microorganisms and foreign bodies, dead or injured cells are removed.
Measurement of the activity of the reticuloendothelial system depends upon estimation of the rate of clearance from the blood of foreign materials, such as colloidal carbon [12].
Treatment by S. mialhesi extract enhanced the rate of carbon clearance from the blood when compared to the control group. This reflects the enhancement of the phagocytic activity of mononuclear macrophage and nonspecific immunity, which includes opsonisation of the foreign particulate matter with antibodies and complement C3b, leading to a more rapid clearance of foreign particulate matter from the blood [13]. [14] demonstrated that cocoa polyphenols are powerful stimulators of the innate immune system and the first reaction of the adaptive immunity. The immunostimulatory effect was showed following the isolation and culture of purified monocytes, CD4 and CD8 cells in the presence of cocoa polyphenols and after they were confronted with lipopolysaccharides.
Our results revealed that S. mialhesi extract appeared to stimulate the phagocytic activity by increasing the clearance rate of carbon by the cells of the reticuloendothelial system. This is due to the fact that it contains natural physiologically active substances such as Terpenoids, Flavanoids and phenolic compounds [15].

Conclusion
Qualitative phytochemical analysis of the S. mialhesi extract revealed that it contains natural physiologically active substances such as Terpenoids, Flavanoids and phenolic compounds [15], and it is already reported that naturally occurring phenolic compounds have immunomodulatory activity [16].
The present study established that S. mialhesi extract stimulated non specific immune response of the animals by increasing the phagocytic activity, measured in terms of phagocytic index and this could be attributed to its natural components.
This stimulation indicates the presence of active principles in the S. mialhesi extract. The active principles responsible for the stimulatory effect are yet to be identified.  Figure 1 Effect of S. mialhesi extract on phagocytic activity. Figure 2 Effect of S. mialhesi extract on the carbon clearance rate.