- Email: [email protected]
c mice
International Immunopharmacology 48 (2017) 203–210
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Immunostimulatory activity of plumieride an iridoid in augmenting immune system by targeting Th-1 pathway in balb/c mice
MARK
Jasvinder Singha,b, Arem Qayuma,b, Rachna D. Singhd, Mytre Koula,b, Anpurna Kaula,b,⁎, N.K. Sattic, Prabhu Duttc, Abid Hamida,b, Shashank Singha,b,⁎ a
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India AcSIR (Academy of Science and Innovative Research), New Delhi, India c Natural Product Chemistry Division, CSIR - Indian Institute of Integrative Medicine, Jammu 180001, India d Department of Conservative Dentistry & Endodontics, Indira Gandhi Government Dental College and Hospital, Jammu, India b
A R T I C L E I N F O
A B S T R A C T
Keywords: Plumieride Plumieria acutifolia Cell mediated immunity Humoral immunity Immunosuppressive Immunorestorative Cytokines
Plumieride, an iridoid glucoside isolated from Plumieria acutifolia leaves was investigated for its immunostimulatory activity on humoral, cell mediated and intracellular cytokine levels in sensitized and unsensitised balb/c mice. Plumieride restores the suppressed cell mediated, humoral immune response and also enhances the release of TNF- α, IFN-γ, and IL-2 (Th-1) in immune compromised cyclosporine and cyclophosphamide treated balb/c mice. It does not stimulate the IL-4 (Th-2) expression. Plumieride demonstrates significant augmentation of Th-1 response in immunosuppressed balb/c mice. Results of the present study suggested that plumieride can be developed as an immunostimulatory adjuvant to treat the immune suppression in various disease condition(s).
1. Introduction Plants which are used as “tonic” in folk medicine are believed to possess immunostimulating bioactive natural products [1,2] which enhance positive health and protect against infections by conditioning the body's immune system [3]. Hence, the drugs of plant origin are gaining popularity and are being investigated for improvement of immunological disorders [3,4]. From the last few decades, significant researches have been conducted to search a plant based novel adjuvant which can boost the immune system of the body against invading pathogens. To counter the foreign infiltration, immune system needs a well organized action that includes awakening of T and B cells response and increased release of cytokines. The activation of T and B cells movement destroy the pathogens. Therefore, for a healthy body, healthy immune system is imperative. The current research trends focus on the identification and development of better therapeutically active immunomodulators which are directed towards activities related to stimulation or inhibition of immune factors along with their integrated functions [5]. The comprehension of immune-modulators had given birth to new immunomodulation strategies to overcome incurable diseases involving cancer, AIDS, arthritis, and allergies. Thus, it becomes the most desirable therapy for extending our understanding for treatment of immune disorders. In Indian medicine system, such as ayurveda, the medicinal and aromatic plants having adaptogeneic,
⁎
rejuvenating and immunomodulating properties are under continuous process of study [6,7]. Iridoids are, bitter tasting monoterpenoid lactones which are known for immunostimulatory [8,9], hepatoprotective [10], anti-inflammatory [11,12], antifungal [13] and antibacterial [14] properties. From many centuries, the iridoid containing plants Ajuga bracteosa, Boschniakia rossica, Enicostema axillare [11] are being used in folk medicine as a bitter tonic, sedative, febrifuge, cough medicine, the remedy for wound healing and insecticidal properties [15]. It was also reported that an endophytic colletotrichum gloeosporioides isolated from the plumieria acutifolia was set up to produce a diterpenoid compound namely taxol an anticancer drug [16]. Preliminary study revealed that plumieride isolated from Plumeria acutifolia possess the immunostimulatory property. Moreover, it was also found to be effective against Human Immune Deficiency Virus-1(HIV-1) and antipsychotic disorders [17]. The study was conducted to determine the effect of plumieride on the humoral and cellular response in animals (normal and immunosupressed with chemotherapy agents) to ascertain its usefulness in immune compromised state. Most importantly, cytokines always remain the key regulator during the imbalance of immune system [18]. Therefore, the influence of plumieride on the regulation of cytokines such as Tumor Necrosis Factor-α (TNF-α) and Interferon γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4) have been studied. An in-depth study of the immune system is believed to provide both the theoretical and therapeutic background of many chronic disorders. The
Corresponding authors at: Cancer Pharmacology Division, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India. E-mail addresses: [email protected] (A. Kaul), [email protected] (S. Singh).
http://dx.doi.org/10.1016/j.intimp.2017.05.009 Received 9 January 2017; Received in revised form 20 April 2017; Accepted 8 May 2017 1567-5769/ © 2017 Published by Elsevier B.V.
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
present study hypothesis that plumieride possesses immunomodulatory potential which can be used in immune suppressed conditions like chemotherapy associated immune suppression or HIV. Development of a safe and effective immunomodulator for clinical use has become a grail for many pharmaceutical investigators world over. Keeping these factors in mind, the efforts have been executed towards the formulation of new strategies, to modulate the immune response, for the effective treatment of various maladies associated with the immune system. 2. Materials and methods 2.1. Chemicals and assay kits Plumieride (purity 96.40%) a white crystalline substance was procured from Natural Product Chemistry Division of CSIR-Indian Institute of Integerative Medicine (IIIM), Jammu. Murine enzyme linked immunosorbent assay (ELISA) kits for IL-1α, IL-2, TNF-α, and IFN-γ were obtained from the BD Biosciences, Concanavalin A (Con-A), Lipopolysaccharide (LPS), Cyclosporine, Cyclophosphamide and Levamisole were purchased from Sigma, Colloidal carbon (Indian ink) and RPMI 1640 medium were obtained from the local market and Gibco BRL Life Technologies Inc. (Grand Island, N.Y., USA) respectively. Fig. 1. (a) HPLC chromatogram of Plumieride at 225 nm. (b) Structure of Plumieride.
2.2. Preparation of test material this plant might be due to its influence on the immune system of the organism which has not been reported in the literature so far (Fig. 1).
Plumeria acutifolia leaves were dried in shade and grounded to coarse powder. 500 g powdered leaves were extracted by dipping in ethanol: distilled water in the ratio of (1:1) in a glass separator. The material was allowed to remain in contact with a solvent for overnight. The extract was filtered and marc was extracted four times under similar conditions. Pooled extract was centrifuge and particle free extract was concentrated under vacuum to obtain a residue (125 g).The residue was fractionated with CHCl3, EtOAc, and MeOH. The MeOH fraction was loaded into glass column packed with diaion hp.-20 resin and eluted sequentially with deionised water, water: methanol mixture (1:1) followed by methanol. Methanol fraction containing the target compound was subjected to further purification over Sephadex LH-20. Column was initially eluted with deionised water followed by a mixture of water and methanol (1:1) and finally with 100% methanol. The fraction eluted with MeOH: H2O (1:1), on crystallization from EtOAc yielded pure plumieride. Purity profile of the isolated plumieride was tested on Waters 2996 HPLC system equipped with a binary pump, an auto sampler, an electronic degasser, thermostatic column oven, a diode array detector. The data was entered in a computer and was analysed with Empower software for analysis. The LC separations were optimized using RP-18, Merck column (4 × 250 mm, 5 μm) where; mobile phase consisted of a mixture of methanol and water in the ratio of 2:3. Elution was achieved with a flow rate of 0.6 ml/min. Before use, the mobile phase was filtered through 0.45 mm filter (Millipore). The temperature of the column was maintained at 30 °C to provide sharpness to the eluting peak. The UV chromatogram was recorded at 225 nm. The compound showed a peak at a retention time of 5.689 in the HPLC-UV (DAD) chromatogram and the percentage of plumieride in the extract was found to be 1.25%. Plumieride was obtained as colorless amorphous powder with a melting point of 229 °C, [α]21D-114 [c,0.5% MeOH], MS: FABMS [M + Na] + m/z 493. The compound was identified as iridoid i.e.,plumieride{1S-[1a,4aa,7a(R)7aa]}-1-(b-D-glucopyranosyl)-4a,7a-dihydro-4′-(1 hydroxymethyl)-5′-oxospiro[cyclopenta (C) pyrano (7H), 2′ (5′H)-furan]4-carboxylic acid methyl ester on the basis of comparison of its spectral data (1H, 13C NMR, IR) with n the literature. Keeping in view the high reputation of plumieride-{1S-[1a,4aα,7a(R)7aa]}-1-(β-D-glucopyranosyl)-4a,7a-dihydro-4′-(1-hydroxymethyl)-5′oxospiro[cyclo-penta (C) pyrano (7H), 2′ (5′H)-furan]-4-carboxylic acid methyl ester, obtained from Plumeria acutifolia in traditional system of medicine, we were tempted to contemplate that the restorative and revitalizing ability of
2.3. Experimental animals Female balb/c mice (18–22 g) were, procured from the CSIR-Indian Institute of Integrative Medicine, Jammu (India) with prior approval from the Institutional Animals Ethics committee (IAEC (85/2/16). The animals were reared under standard laboratory conditions, i.e. temperature (25 ± 2 °C), relative humidity 65–75% and a photoperiod of 12:12 h (light: dark). The animals were fed on a pellet diet and water was given ad libitum. 2.4. Acute safety study An initial acute safety study was carried out as per OECD guidelines No. 423. The compound was administered at different doses to determine no observe adverse effect level (NOAEL) in female balb/c mice. The animals were observed for any change in general behavior/or untoward symptom and mortality over a two week period. On day 14th, biochemical and hematological parameters were evaluated to determine any end organ toxicity. 2.5. Antigen (SRBC) Fresh sheep red blood cells (SRBC) agglomerated aseptically from the jugular vein of sheep and was stored in cold, sterile alsever's solution [19] then washed three times with pyrogen free, sterile normal saline (0.9% NaCl w/v) and cell count was adjusted to 1 × 107 cells/ml for immunization and challenge at the required time schedule. 2.6. Immunization Six female balb/c mice were grouped. Plumieride was administered orally at different doses 1.56–25.0 mg/kg p.o. daily for six days and control received normal saline. Levamisole was used to elicit the immune system at a dose of 2.5 mg/kg p.o. [20]. Cyclosporine and Cyclophosphamide at a dose of 5 mg/kg and 200 mg/kg p.o. respectively was used to suppress the immune system of animals. All groups of animals were immunized with 200 μl of SRBC (1 × 107) intraperitoneal (i.p) on day 1 of the experiment. Moreover, 20 μl of SRBC was injected 204
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
keep them moist and was incubated for 60 to 90 min at 37 °C in the incubator. By using inverted microscope plaques in SRBC lawn were calculated. Each plaque represented a product of an individual antibody forming cell [25].
on the hind foot of mice for DTH assay. 2.7. Delayed type hypersensitivity (DTH) response The immune system was suppressed by cyclosporine and antigen was injected intraperitoneal (i.p.).Oral drugging was given for six days. The thickness of the left hind footpad was measured on 7th day with a spheromicrometer (pitch, 0.01 mm) and was considered as the control. Mice were vaccinated by injecting 20 μl of 1 × 107 SRBC/ml subcutaneously into the right hind footpad. The foot thickness was measured again at 24 h after challenge [21].
2.13. Carbon clearance The Phagocytic function of the RES (reticulo-endothelial system) was assayed in groups of six mice each by injecting i.v 160 mg/kg b.w of 1.6% suspension of gelatin stabilized carbon particles of 20–25 μM size [23]. Blood samples were collected immediately before and at intervals varying between 2 and 120 min. After intravenous injection of carbon particles. An aliquot (10 μl) of blood samples were lysed with 2 ml of 0.1% acetic acid and transparency determined spectrophotometrically at wavelength of 675 nm till transparency was equivalent to standard (levamisole) was obtained as per described by Hudson hay carbon clearance assay to assess the effect of the drugs on phagocytic capacity of reticuloendothelial system (RES) of the animals which constitutes initial defense mechanism against infectious microorganisms [26].
2.8. Skin allograft rejection The homologous grafts of skin were transplanted in groups of 6 inbred mice, as per the method explained by Billingham [22]. Graft retention time was determined by daily observation the criterion being followed is to access the survival of epithelium. Plumieride at 3.12–25 mg/kg p.o. concentration was fed orally daily for six days and graft rejection time was recorded. Two parallel controls, one given vehicle and other standard cyclosporine at 5 mg/kg p.o. were run for comparison of the activity.
2.14. Measurement of CD4+ and CD8+ T cells
2.9. Lymphocyte proliferation assay
Blood was collected from retro-orbital plexus of sensitized mice in 0.1% of EDTA. Then add monoclonal antibodies (CD4+ and CD8+) and incubate for 30 min. After that, add FACS lysing solution to the samples and incubate for 10 min. Then centrifuge it and discard the supernatant and dissolve the pellet in washing buffer and centrifuge it again and add 1 ml PBS to the pellet after staining with antibodies, cells were washed and re-suspended in PBS for flow cytometric analysis, which was performed on a FACS Calibur flow cytometer [27].
Mice were sacrificed under aseptic conditions and spleen was removed. The splenocytes were isolated by crushing the spleen under a pair of frosted glass slides and erythrocytes were lysed with ammonium chloride (0.08% w/v). The cells are then centrifuged at 1400 rpm for 10 min and the cell pellet was washed three times with RPMI media. 2 × 106 cells/ml in 100 μl complete RPMI media is seeded in 96 well plates. Con-A (0.5 μg/ml) and LPS (1 μg/ml) were added to wells separately for stimulation of B and T cells respectively. Plumieride was also added to the wells and the plates were incubated for 72 h. 20ul MTT dye [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (2.5 mg/ml) was added 4 h prior to termination of the experiment. Then, the absorbance was measured by ELISA at 570 nm [23].
The serum level of cytokines i.e. IL – 2, IFN-γ, TNF-α, IL-4 in immunesuppressed animals treated with plumieride was estimated by ELISA method. The protocols were followed as mentioned in the commercially available kits of BD Biosciences.
2.10. Evaluation of hematological parameters
2.16. In-vitro phagocytic activity of peritoneal macrophages
Blood was collected from the retro-orbital plexus of immune suppressed balb/c mice on 7th day of the experiment and analysed for hematological imbalance.
The phagocytic function of macrophages isolated from peritoneal cavity was analysed by method described by Lehrer [28]. In this method the peritoneal macrophages were collected by flushing the peritoneal cavity with 5 ml sterile ice cold 0.15 M PBS containing 2 U/ ml preservative free heparin. After that cells were allowed to adhere to glass cover slip (22 × 22 mm) for 2 h in incubator at 37 °C, 5% co2 and 95% relative humidity in presence and absence of different conc. of plumieride. Then the cover-slip was thoroughly washed with phosphate buffer saline (PBS) to remove non adherent cells. During this period heat killed Candida albicans cells were opsonized for 90 min with 20% mouse serum, washed three times and resuspended with final conc. of 2 × 109 cells/ml. Then 100 μl of opsonized C. albicans cells was then spread over monolayer and mixture was incubated for 15 min. After that cover slip was washed with PBS and stained with trypan-eosin and then samples were immediately evaluated microscopically to determine for phagocytosis.
2.15. Cytokines measurement
2.11. Humoral antibody titre Six animals were put in each group and each animals immune system was suppressed by cyclophosphamide and was immunized by injecting 200 μl of 1 × 107 SRBC/ml intraperitoneal (i.p.) on day 1 followed by oral drugging as described above. Blood was collected on day 7 for primary antibody titre. Hemagglutination antibody titers were determined following the modified micro titration technique described by Nelson and Midenhall [24]. 2.12. Plaque forming cell (PFC) assay The IgM reaction to an antigen (SRBC) was observed in mice treated with Plumieride. Mice were injected with 0.2 ml of 10% (v/v) suspension of SRBC in normal saline. Animals were sacrificed and their spleen was removed aseptically. The spleen was crushed and suspension of splenocytes was made in RPMI supplemented with FBS. Cell count was adjusted to 2 × 107 cells/ml. Take 0.1 ml of this cell suspension, 0.3 ml of 5% SRBC, 0.1 ml of guinea pig serum and 0.5 ml of RPMI 1640 medium and total 150 μl quantity of this mixture and add to Cunningham's chamber. The chamber was sealed with paraffin wax to
2.17. Mycobacterium tuberculosis H37Rv infection The balb/c mice were infected intranasally with Mycobacterium tuberculosis H37Rv (103 CFU/mouse). The infected mice were divided in groups of 6 mice each. Treatment started one week after the infection. Mice from early control group were sacrificed, left lung was aseptically removed, homogenized and dilutions were placed on Middlebrook 7H10 agar to determine the CFU (Colony forming unit). Mice from 205
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
significantly increased which indicated that plumierides stimulated the immune system. An increase of 1.27 fold was seen in WBC count. Levamisole used as the control increased all the blood parameters (Table 1).
late control and treatment group were scarified one week after the infection and lung CFU was determined. 2.18. Statistical assessment For statistical analysis, the data was entered and analysed on MS Office 2007 (Microsoft Crop. Redmond, WA). The data was expressed in Mean ± SEM (Standard error of measurement). For the purpose of comparison One Way ANNOVA was used to evaluate the potential difference between the experiments were done in triplicates. Dunnett's test was used to analyse different variables (GraphPad Prism 5.01, GraphPad Software, USA). A value of p < 0.05 was set as statistically significant.
3.6. Humoral antibody titre Serum was isolated from the blood collected from the treated animals on 7th day of the experiment and antibody titre reading was increased in all the five concentrations i.e. 1.56, 3.12, 6.25, 12.50, 25.00 mg/kg p.o. The maximum effect was seen at 12.5 mg/kg p.o. with 30.76% stimulation. A four fold increases in stimulation from 1.56 mg/kg to 12.5 mg/kg have been observed. Moreover, Plumieride at conc. of 25 mg/ml showed a stimulation of 23.07% which was almost 1.33 fold decrease at this concentration when compared with 12.5 mg/ kg. Levamisole used as positive control showed 38.94% stimulation. The response was found to be dose-dependent and significant. Cyclophosphamide suppressed the immune system by 31.39% (p < 0.05) (Fig. 3(a)).
3. Results 3.1. Acute toxicity study Plumieride was found to be safe at a single dose of 2000 mg/kg p.o. No change was observed in behavior, biochemical or hematological parameters (Data not Shown). Therefore, NOAEL was found to be 2000 mg/kg p.o.
3.7. Plaque forming cell assay The SRBC response was also evaluated by the primary antibody IgM. Plumieride was administered orally at five concentrations between 1.56 and 25.0 mg/kg p.o. for five days from the day of sensitization. There was increase in a number of antibodies secreting cells by 5.89 fold from lower to higher concentration. The number of plaque forming cells was 58.92% at 25 mg/kg and 56.30% at 12.50%. The control group was levamisole with 74.30%.The results were significant and dose dependent (p < 0.05) (Fig. 3(b)).
3.2. Cell mediated immune response In order to examine the basic mechanism(s) of action on immunomodulation, plumieride was administered and produced a dose related to increase in SRBC induced DTH reaction (cell mediated immunity) which shows the stimulatory effect on ‘T’ lymphocytes. Cyclosporine at 200 mg/kg p.o. suppressed the immune system by 35.42%. Plumieride elicited the cell mediated immunity by 15.78% at a concentration of 1.56 mg/kg and at 12.5 mg/kg the stimulation was 36.84% (p < 0.05). There was a 2.33 fold increase i.e. from 1.56 mg/kg to 12.5 mg/kg. Plumieride at a concentration of 25 mg/kg showed 30.52% stimulation. Levamisole (2.5 mg/kg p.o.) was used as a positive control with 38.94% stimulation (Fig. 2(a)).
3.8. Carbon clearance The oral administration of plumieride for seven days increased the clearance of carbon particles from the circulation of mice. Plumieride at a concentration of 25 mg/kg showed 23.80% efficacy and at 12.5 mg/ kg showed 17.46% effective in clearing the particles. There was a 4.13 fold increase from lower to higher concentration. Levamisole at 2.5 mg/ kg showed 33.33% increase and was used as the control. The results were found to be dose dependent (Fig. 3(c)).
3.3. Skin allograft rejection The graft of skin was transplanted between the mice of the same group and the animals were monitored daily to determine increase/ decrease in rejection time. Plumieride, when administered orally at four different concentrations (3.12 to 25. mg/kg p.o.), had hastened the rejection time by 38.61% at 25 mg/kg. There was concentration dependent increase in rejection time. At conc. of 12.50 mg/kg, the rejection time was decreased by 34.61%. This experiment supported our B (Humoral) and T (Cell mediated) cell experiment that plumieride is an immunostimulant. Cyclosporine was used as the positive control and found to increase the rejection time by 39.69% (Fig 2(b)).
3.9. Effect of Plumieride on CD4 and CD8 The expression of CD4 T and CD8 T in immunesuppressed control mice was 24.04% and 14.36% respectively while in case of levamisole the expression of CD4 T and CD8 T had increased to 30.60% and 16.90% respectively and at concentration of 12.5 mg/kg. The CD4 and CD8 expression levels increased to 42% and 24% respectively. These results indicated that plumieride had activated the Th-1 pathway significantly (Fig. 3(d)).
3.4. Lymphocyte proliferation assay 3.10. Estimation of cytokines (Th-1 and Th-2) by ELISA In Lymphocyte proliferation assay, the plumieride was found to stimulate the proliferation of T and B cells. Plumieride in all the three concentrations (20 μM,10 μM, and 1 μM) elicited the proliferation of T cells. The proliferation of T cell at a concentration of 20 μM was higher than the con-A control. B cells proliferation was highest at 20 μM and with lower concentrations, the effect was also less. The effect in both T and B cells was dose dependent (Fig. 2(c & d)).
The effect of plumieride on the release of different cytokines at four concentrations was studied (3.12, 6.25, 12.50, 25.0 mg/kg). Plumieride stimulated both Th-1(IFN-γ, IL-2, TNF-α) and Th-2 (IL-4) cytokines. Plumieride significantly stimulated IFN-γ, IL-2, TNF-α.The IFN-γ secretion was enhanced by 9.95% at 12.50 mg/kg oral dose. The sensitized control group showed 5.35% and non-sensitized control showed 2.70% of IFN-γ expression (Fig. 4). The increase was observed at all the concentrations but maximum effect was seen at 12.50 mg/kg. The IL-2 expression was 19.44% at 12.5 mg/kg oral dose. In a sensitized and non-sensitized group, the expression was 4.15% and 6.06% respectively. TNF-α expression was increased by 2.19% at 12.5 mg/kg oral dose. In the case of Th-2 (IL-4), expression was found to be increased non-significantly by 6.49% at conc. of 12.5 mg/kg oral (Fig. 4(a,b,c,d)).
3.5. Plumieride effect on hematological parameters Blood was collected from immunosuppressed mice and showed that hemoglobin level increased in all the concentrations and it was maximum at 12.5 mg/kg p.o. There was a 1.15 fold increase from concentration 6.25 mg/kg p.o. to 12.5 mg/kg p.o. The WBC count was 206
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 2. (a) Plumieride was administered orally (1.56–25 mg/kg) for seven days post-immunization, an increase of 15.78 to 36.84% in the expression of DTH reaction was observed in immune suppressed mice. The effect was dose dependent and highly significant (P < 0.001) at 12.5 mg/kg dose. (b) Plumieride showed statistically significant effect (decrease in rejection time) that was 34.61 and 38.61% at 12.50 and 25 mg/kg p.o. dose, respectively. Cyclosporine a standard control at 5 mg/kg increased the rejection time by 39.69%. (c) The proliferation of B cells was maximum at concentration of 20 μM when cells were stimulated with L.P.S and with the lower concentration the effect was down. (d) T cell proliferation was higher in all the concentration. The stimulation of T cells was done by Con-A. Table 1 Blood samples were collected from immune-suppressed balb/c mice that were treated with different conc. of Plumieride i.e. 6.25, 12.5 and 25 mg/kg p.o. Samples were immediately analysed for hematological parameters like white blood cell count (WBC), hemoglobin (HB), Hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), lymphocyte, monocyte and granulocytes by using automatic hematology analyzer. Effect of plumieride on blood cells of immune suppressed balb/c mice Treatment mg/kg p.o.
Control Control immune suppressed Levamisole Plumieride (25.0) Plumieride (12.5) Plumieride (6.25)
Hemoglobin conc.
RBC count in million/ cmm
Platelet count in thousand/ cmm
WBC count in thousand/ cmm
Differential count Lymphocyte
Monocytes
Granulocyte
9.20 ± 0.37 6.00 ± 0.27
6.55 ± 0.37 5.00 ± 1.15
528.20 ± 18.29 500 ± 16.66
13.20 ± 0.50 9.00 ± 0.55
74.18 ± 1.11 42.00 ± 1.56
2.10 ± 0.0 1.00 ± 0.11
23.72 ± 0.58 17.55 ± 0.55
8.23 6.98 7.60 6.59
6.43 5.59 6.31 5.39
1162.20 ± 51.22 842.60 ± 1.07 877.80 ± 3.90 587.60 ± 18.09
14.00 13.70 13.72 10.76
70.74 55.56 65.34 49.52
2.36 2.35 3.36 4.32
27.90 42.09 31.30 46.24
± ± ± ±
0.24 0.07 0.15 0.16
± ± ± ±
0.22 0.23 0.14 0.06
± ± ± ±
0.37 0.65 1.06 0.51
± ± ± ±
0.51 0.40 0.09 1.38
± ± ± ±
0.36 0.11 0.07 0.14
± ± ± ±
0.82 0.37 0.91 1.92
control with 40.33% phagocytosis (Fig. 5).
3.11. In-vitro phagocytic activity of peritoneal macrophages The population of polymorphonuclear leukocyte (PMNL) is usually seen during the early hours of antigen processing, whereas macrophages occur at later stages. Plumieride increased the phagocytic function of macrophages that were isolated from peritoneal cavity of mouse. There 1.42% increase in % phagocytosis from lower conc. (1 μg/ml) to higher conc. (30 μg/ml). Levamisole is used as the positive
3.12. Mycobacterium tuberculosis H37Rv infection The mice infected with Mycobacterium tuberculosis H37Rv. One week after the infection the CFU load in the early control reached 4.7 log while in the late control was 6.8 log. The early control indicates the CFU load at the start of the treatment, whereas late control indicates the 207
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 3. (a) Plumieride (1.56–25 mg/kg p.o.) produced a dose dependent antibody synthesis in immune-suppressed balb/c mice. The immunestimulatory effect of plumieride was highly significant at 12.5 mg/kg p.o. dose levels. (b) Administration of plumieride (3.12–25 mg/kg) orally for 5 consecutive day's begining from day of immunization produced an increase in the number of antibody secreting cells (PFC) by 10.15–58.92% in mouse spleen. The effect was highly significant. (p < 0.05) (c) Oral administration of plumieride (3.12–25 mg/kg) for consecutive seven days and 30 min prior to carbon injection enhanced the clearance rate of carbon particles from circulation in normal animals at dose of 25 mg/kg p.o. (d) The values of the (unsensitised group) were 24.04% of CD4+ T cells and 14.36% % of CD8+ cells and in the (sensitized group) the value of CD4+ and CD8+ T cells was 30.60% and 16.90% respectively. This shows an increase in CD4+ and CD8+ T cells in experimental animals treated with different doses of Plumieride.
with scaling down in the skin graft rejection time in mice treated with plumieride. The basic mechanism involved is a slowdown in graft rejection time by the stimulation of T-lymphocytes i.e., CD4 and CD8 positive T cells. Activation of macrophages is important for the enhancement of an immune system which is majorly accomplished by CD4 T cells. The Complete Blood Count (CBC) results showed an increase in leukocyte counts in dose dependent manner in immune suppressed balb/c mice treated with plumieride indicate it as an immunostimulant. The proliferation of T cells was also confirmed by the lymphocyte proliferation assay in which the con-A stimulated T cells showed maximum proliferation. The major function of CD4 T cells is to activate macrophages and activation of macrophages are important for enhancement of immune system [29]. Present study showed that plumieride activates the CD4 T cells and this increases the functioning of macrophages. The increased number of phagocytes is evident by clearance of carbon particles from reticuloendothelial cells moreover, it also hastens the T & B lymphocytes involved in phagocytosis in-vitro by murine macrophages indicating an increase in the functioning of macrophages (innate response) by causing stimulation of non-specific immune response. The upregulation of the humoral immune response against the antigen (SRB) also signals towards the response of macrophages in antibody synthesis. Immune homeostasis needs the differential and controlled expression of cytokines and their receptors [30]. There are a number of examples in
bacterial load of the untreated group. Treatment with plumieride at 12.5 mg/kg did not allow the bacteria to grow and the bacterial load was close to the early control. The combination of plumieride with rifampicin (anti-tuberculosis drug) was synergistic. The combination could reduce the CFU load to 3.0 log as compared to rifampicin alone that stood at 5.0 log. This combination reduced the initial CFU load (early control) by 1.7 log. 4. Discussion Immunomodulators are a class of natural or synthetic agents which alter the immune system. They have the ability to augment, restore and inhibit to produce crave immune responses. Immunomodulatory drugs constitute thalidomide and its analogues, lenalidomide, pomalidomide apremilast and many more. An iridoid glucoside plumieride obtained from Plumieria acutifolia was investigated for its immunomodulatory potential in the present study. Iridoids are known to possess a large number of biological activities and the immune potentiating activity of iridoids has also been reported in the literature [27]. Similarly, the present work describes the ability of plumieride to act as a significant immunopotentiating agent in response to antigen (SRB), plumieride produced a dose related increased in delayed type hypersensitivity reaction which is a cell mediated response that shows its specific stimulatory effect on ‘T’ lymphocytes. However this is corroborated 208
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 4. (a) The IFN-γ (Th1 signature cytokine) was enhanced in the groups treated with plumieride showing maximum expression (9.95%) at 12.50 mg/kg oral dose. The sensitized control group showed 5.35% and non-sensitized control showed 2.70% of IFN-γ expression. There was an increase in all the test drug treated groups with maximum effect being observed at 12.50 mg/kg per oral dose. (b) Animals treated with plumieride at graded oral doses showed increase in Intracellular IL - 2 expressions. The percentage of IL-2 secreted was 19.44% at 12.50 mg/kg per oral test drug dose (maximum effect).The percentage of IL-2 in the normal non-sensitized control group and in sensitized control group was 4.15% and 6.06% respectively. (c) TNF-α (Th-1cytokine) were increased in dose dependent manner and were significant at 12.5 mg/kg.The percentage of TNF-α at 12.5 mg/kg is 2.19%. (d) The IL-4 (Th2 signature cytokine) expression in sensitized control and non-sensitized control groups was 4.83% and 3.80% respectively .The maximum expression of IL-4 in treated groups was 6.49% at the dose of 12.50 mg/kg per oral dose. However, this was not statistically significant.
cells. It, however, gave some expression of IL-4 that was not significant and may be possibly due to cross effects of CD4 T cells secreted cytokines, thus expressing specific Th1 response. Plumieride did not show any cytotoxicity and both T and B cell proliferation was within normal range. The mechanistic studies of botanical isolated molecule i.e. plumieride suggests that it is an immunoactive compound which concurrently or simultaneously modulates immune matrix and restores homeostatic conditions which can play important role in immunotherapy [34].
experimental models where alteration of the Th1/Th2 balance by the administration of cytokines or cytokine antagonists control the outcome of the diseases. The function of CD4 T cells is the activation of macrophages and that plays a key role in escalating the activity [31]. Also, on the basis of present study, CD-4 T cell activation by plumieride may be one of the factors that is attributing towards the increase in the functioning of the macrophages. The combination of plumieride with rifampicin showed synergism in being effective. The combination could reduce the CFU load to 3.0 log as compared to rifampicin alone that stood at 5.0 log. Plumieride at 12.5 mg/kg oral dose did not allow the bacteria (Mycobacterium Tuberculli) to grow. T helper (Th1) lymphocytes homeostasis is a pivotal role in arranging the suitable cytokine responses and hence remains as one of the targets for Immunomodulation. Th1-type cytokines (IFN-γ, IL-2, TNF-α) exhibit cell-mediated immunity and targets intracellular parasites while Th2-type cytokines (interleukins 4) are affiliated with humoral immunity. Cytokines play an important role in the regulation of hematopoiesis, mediating the differentiating migration, activation, and proliferation of phenotypically diverse cells [32].The analysis and expression of cytokines in biological liquids had become a commonly used technique in research and clinical laboratories and is clearly important in expanding our comprehension of many immunological functions [33].The results suggest that oral administration of plumieride induced a dose related increased expression of interferon gamma (IFNγ), IL-2 (interleukin-2), TNF-α (Tumor Necrosis Factor) in CD4 T
5. Conclusions Our pragmatic study establishes plumieride as an immunostimulant that up-regulates Th1 pathway. Such agents are being looked for the treatment of infectious diseases, immunodeficient diseases, or for generalized immunosuppression induced by drug treatment; for combination therapy with antibiotics; and as adjuncts for vaccines. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgements Author thanks, Director of CSIR-IIIM for providing the facility. Also 209
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
obstruction in guinea pigs, Int. Arch. Allergy Immunol. 95 (2–3) (1991) 128–133. [6] N. Farnsworth, The role of medicinal plants in drug development, Natural Products and Drug Development, 1984, pp. 17–30. [7] R.P. Labadie, Steven M. Colegate, Russell J. Molyneux, Immunomodulatory compounds, Bioactive Natural Products, CRC PRESS Inc., 1993, pp. 279–317. [8] H. Baxter, J.B. Harborne, G.P. Moss, Phytochemical Dictionary: A Handbook of Bioactive Compounds From Plants, CRC press, 1998. [9] S.K. Sharma, N. Kumar, Antimicrobial potential of Plumeria rubra Syn Plumeria acutifolia bark, Der Pharm Chemica 4 (4) (2012) 1591–1593. [10] R. Ansari, B. Aswal, R. Chander, B. Dhawan, N. Garg, N. Kapoor, D. Kulshreshtha, H. Mehdi, B. Mehrotra, G. Patnaik, Hepatoprotective activity of kutkin–the iridoid glycoside mixture of Picrorhiza kurrooa, Indian J. Med. Res. 87 (1988) 401–404. [11] A. Viljoen, N. Mncwangi, I. Vermaak, Anti-inflammatory iridoids of botanical origin, Curr. Med. Chem. 19 (14) (2012) 2104–2127. [12] B. Dinda, S. Debnath, R. Banik, Naturally occurring iridoids and secoiridoids. An updated review, part 4, Chem. Pharm. Bull. 59 (7) (2011) 803–833. [13] B. Dinda, S. Debnath, Y. Harigaya, Naturally occurring secoiridoids and bioactivity of naturally occurring iridoids and secoiridoids. A review, part 2, Chem. Pharm. Bull. 55 (5) (2007) 689–728. [14] R. Tundis, M.R. Loizzo, F. Menichini, G.A. Statti, F. Menichini, Biological and pharmacological activities of iridoids: recent developments, Mini-Rev. Med. Chem. 8 (4) (2008) 399. [15] G. Singh, S. Bani, S. Singh, A. Khajuria, M. Sharma, B. Gupta, S. Banerjee, Antiinflammatory activity of the iridoids kutkin, picroside-1 and kutkoside from Picrorhiza kurrooa, Phytother. Res. 7 (6) (1993) 402–407. [16] K. Nithya, J. Muthumary, Growth studies of Colletotrichum gloeosporioides (Penz.) Sacc.-a taxol producing endophytic fungus from Plumeria acutifolia, Indian J. Sci. Technol. 2 (11) (2009) 14–19. [17] M.P. Dobhal, G. Li, A. Gryshuk, A. Graham, A.K. Bhatanager, S.D. Khaja, Y.C. Joshi, M.C. Sharma, A. Oseroff, R.K. Pandey, Structural modifications of Plumieride isolated from Plumeria bicolor and the effect of these modifications on in vitro anticancer activity, J. Org. Chem. 69 (19) (2004) 6165–6172. [18] V.S. Georgiev, J.F. Albright, Cytokines and their role as growth factors and in regulation of immune responses, Ann. N. Y. Acad. Sci. 685 (1) (1993) 584–602. [19] J.B. Alsever, R.B. Ainslie, A new method for the preparation of dilute blood plasma and the operation of a complete transfusion service, N. Y. State J. Med. 41 (1941) 126–131. [20] M.A. Tempero, Y. Haga, C. Sivinski, D. Birt, L. Klassen, G. Thiele, Immunologic effects of levamisole in mice and humans: evidence for augmented antibody response without modulation of cellular cytotoxicity, J. Immunother. 17 (1) (1995) 47–57. [21] N. Doherty, Selective effects of immunosuppressive agents against the delayed hypersensitivity response and humoral response to sheep red blood cells in mice, Agents Actions 11 (3) (1981) 237–242. [22] R. Billingham, P.B. Medawar, The technique of free skin grafting in mammals, J. Exp. Biol. 28 (3) (1951) 385–402. [23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983) 55–63. [24] D. Nelson, P. Mildenhall, Studies on cytophilic antibodies. II. The production by guinea-pigs of macrophage cytophilic antibodies to sheep erythrocytes and human serum albumin: relationship to the production of other antibodies and the development of delayed-type hypersensitivity, Aust. J. Exp. Biol. Med. Sci. 46 (1) (1968) 33. [25] A.T. Cunningham, A. Szenberg, Further improvements in the plaque technique for detecting single antibody-forming cells, Immunology 14 (4) (1968) 599. [26] C. Atal, M. Sharma, A. Kaul, A. Khajuria, Immunomodulating agents of plant origin. I: Preliminary screening, J. Ethnopharmacol. 18 (2) (1986) 133–141. [27] B. Ghule, P. Yeole, In vitro and in vivo immunomodulatory activities of iridoids fraction from Barleria prionitis Linn, J. Ethnopharmacol. 141 (1) (2012) 424–431. [28] R. Lehrer, Ingestion and destruction of Candida albicans, Methods for Studying Mononuclear Phagocytes, 1981, pp. 693–708. [29] M. Whelan, J. Whelan, N. Russell, A. Dalgleish, Cancer immunotherapy: an embarrassment of riches? Drug Discov. Today 8 (6) (2003) 253–258. [30] W.W. Overwijk, K.S. Schluns, Functions of γC cytokines in immune homeostasis: current and potential clinical applications, Clin. Immunol. 132 (2) (2009) 153–165. [31] J. Zhu, W.E. Paul, CD4 T cells: fates, functions, and faults, Blood 112 (5) (2008) 1557–1569. [32] B. Patwardhan, Ayurveda: the designer medicine, Indian Drugs 37 (2000) 213–227. [33] A. Kelso, Cytokines: principles and prospects, Immunol. Cell Biol. 76 (4) (1998) 300–317. [34] R. Verpoorte, Y. Choi, H. Kim, Ethnopharmacology and systems biology: a perfect holistic match, J. Ethnopharmacol. 100 (1) (2005) 53–56.
Fig. 5. (a) Mice infected with Mycobacterium tuberculosis H37Rv. After one week of infection early control CFU load reached 4.7 log and late control CFU load 6.8 log. Plumieride at a conc. of 12.5 mg/kg reduced the CFU load 5.1 whereas rifampicin alone reached 5.3 log. The combination of rifampicin + plumieride CFU load was 3.0 log with p < 0.05 value.(b) Plumieride increased the phagocytosis of heat killed Candida albicans by murine macrophages at 30 μg/ml with 38.50 and p < 0.01 and at lower conc. 1 μg/ ml the phagocytosis is 27.00. Levamisole was used as positive control and phagocytosis value was 40.23 with p < 0.001.
author thanks, funding agency CSIR-New Delhi for granting fund through project (BSC-0205).We are thankful to Dr. M.M Baskar, Lead Reviewer, Clinical Pharmacology, New Drug Submissions, Health Canada, Santé Canada for checking/correcting the manuscript for English language. References [1] H. Wagner, P.M. Wolff, New Natural Products and Plant Drugs with Pharmacological, Biological Or Therapeutical Activity, Proceedings of the First International Congress on Medicinal Plant Research, Section A, Held at the University of Munich, Germany, September 6–10, 1976, Springer Science & Business Media, 2012. [2] O. Sticher, Plant mono-, di-and sesquiterpenoids with pharmacological or therapeutical activity, New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical Activity, Springer, 1977, pp. 137–176. [3] P. Das, R. Tripathi, V. Agarwal, A. Sanyal, Pharmacology of kutkin and its two organic acid constituents cinnamic acid and vanilic acid, Indian J. Exp. Biol. 14 (4) (1976) 456. [4] M. Sharma, C. Rao, P. Duda, Immunostimulatory activity of Picrorhiza kurroa leaf extract, J. Ethnopharmacol. 41 (3) (1994) 185–192. [5] W. Dorsch, H. Stuppner, H. Wagner, M. Gropp, S. Demoulin, J. Ring, Antiasthmatic effects of Picrorhiza kurroa: androsin prevents allergen-and PAF-induced bronchial
210
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Immunostimulatory activity of plumieride an iridoid in augmenting immune system by targeting Th-1 pathway in balb/c mice
MARK
Jasvinder Singha,b, Arem Qayuma,b, Rachna D. Singhd, Mytre Koula,b, Anpurna Kaula,b,⁎, N.K. Sattic, Prabhu Duttc, Abid Hamida,b, Shashank Singha,b,⁎ a
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India AcSIR (Academy of Science and Innovative Research), New Delhi, India c Natural Product Chemistry Division, CSIR - Indian Institute of Integrative Medicine, Jammu 180001, India d Department of Conservative Dentistry & Endodontics, Indira Gandhi Government Dental College and Hospital, Jammu, India b
A R T I C L E I N F O
A B S T R A C T
Keywords: Plumieride Plumieria acutifolia Cell mediated immunity Humoral immunity Immunosuppressive Immunorestorative Cytokines
Plumieride, an iridoid glucoside isolated from Plumieria acutifolia leaves was investigated for its immunostimulatory activity on humoral, cell mediated and intracellular cytokine levels in sensitized and unsensitised balb/c mice. Plumieride restores the suppressed cell mediated, humoral immune response and also enhances the release of TNF- α, IFN-γ, and IL-2 (Th-1) in immune compromised cyclosporine and cyclophosphamide treated balb/c mice. It does not stimulate the IL-4 (Th-2) expression. Plumieride demonstrates significant augmentation of Th-1 response in immunosuppressed balb/c mice. Results of the present study suggested that plumieride can be developed as an immunostimulatory adjuvant to treat the immune suppression in various disease condition(s).
1. Introduction Plants which are used as “tonic” in folk medicine are believed to possess immunostimulating bioactive natural products [1,2] which enhance positive health and protect against infections by conditioning the body's immune system [3]. Hence, the drugs of plant origin are gaining popularity and are being investigated for improvement of immunological disorders [3,4]. From the last few decades, significant researches have been conducted to search a plant based novel adjuvant which can boost the immune system of the body against invading pathogens. To counter the foreign infiltration, immune system needs a well organized action that includes awakening of T and B cells response and increased release of cytokines. The activation of T and B cells movement destroy the pathogens. Therefore, for a healthy body, healthy immune system is imperative. The current research trends focus on the identification and development of better therapeutically active immunomodulators which are directed towards activities related to stimulation or inhibition of immune factors along with their integrated functions [5]. The comprehension of immune-modulators had given birth to new immunomodulation strategies to overcome incurable diseases involving cancer, AIDS, arthritis, and allergies. Thus, it becomes the most desirable therapy for extending our understanding for treatment of immune disorders. In Indian medicine system, such as ayurveda, the medicinal and aromatic plants having adaptogeneic,
⁎
rejuvenating and immunomodulating properties are under continuous process of study [6,7]. Iridoids are, bitter tasting monoterpenoid lactones which are known for immunostimulatory [8,9], hepatoprotective [10], anti-inflammatory [11,12], antifungal [13] and antibacterial [14] properties. From many centuries, the iridoid containing plants Ajuga bracteosa, Boschniakia rossica, Enicostema axillare [11] are being used in folk medicine as a bitter tonic, sedative, febrifuge, cough medicine, the remedy for wound healing and insecticidal properties [15]. It was also reported that an endophytic colletotrichum gloeosporioides isolated from the plumieria acutifolia was set up to produce a diterpenoid compound namely taxol an anticancer drug [16]. Preliminary study revealed that plumieride isolated from Plumeria acutifolia possess the immunostimulatory property. Moreover, it was also found to be effective against Human Immune Deficiency Virus-1(HIV-1) and antipsychotic disorders [17]. The study was conducted to determine the effect of plumieride on the humoral and cellular response in animals (normal and immunosupressed with chemotherapy agents) to ascertain its usefulness in immune compromised state. Most importantly, cytokines always remain the key regulator during the imbalance of immune system [18]. Therefore, the influence of plumieride on the regulation of cytokines such as Tumor Necrosis Factor-α (TNF-α) and Interferon γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4) have been studied. An in-depth study of the immune system is believed to provide both the theoretical and therapeutic background of many chronic disorders. The
Corresponding authors at: Cancer Pharmacology Division, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India. E-mail addresses: [email protected] (A. Kaul), [email protected] (S. Singh).
http://dx.doi.org/10.1016/j.intimp.2017.05.009 Received 9 January 2017; Received in revised form 20 April 2017; Accepted 8 May 2017 1567-5769/ © 2017 Published by Elsevier B.V.
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
present study hypothesis that plumieride possesses immunomodulatory potential which can be used in immune suppressed conditions like chemotherapy associated immune suppression or HIV. Development of a safe and effective immunomodulator for clinical use has become a grail for many pharmaceutical investigators world over. Keeping these factors in mind, the efforts have been executed towards the formulation of new strategies, to modulate the immune response, for the effective treatment of various maladies associated with the immune system. 2. Materials and methods 2.1. Chemicals and assay kits Plumieride (purity 96.40%) a white crystalline substance was procured from Natural Product Chemistry Division of CSIR-Indian Institute of Integerative Medicine (IIIM), Jammu. Murine enzyme linked immunosorbent assay (ELISA) kits for IL-1α, IL-2, TNF-α, and IFN-γ were obtained from the BD Biosciences, Concanavalin A (Con-A), Lipopolysaccharide (LPS), Cyclosporine, Cyclophosphamide and Levamisole were purchased from Sigma, Colloidal carbon (Indian ink) and RPMI 1640 medium were obtained from the local market and Gibco BRL Life Technologies Inc. (Grand Island, N.Y., USA) respectively. Fig. 1. (a) HPLC chromatogram of Plumieride at 225 nm. (b) Structure of Plumieride.
2.2. Preparation of test material this plant might be due to its influence on the immune system of the organism which has not been reported in the literature so far (Fig. 1).
Plumeria acutifolia leaves were dried in shade and grounded to coarse powder. 500 g powdered leaves were extracted by dipping in ethanol: distilled water in the ratio of (1:1) in a glass separator. The material was allowed to remain in contact with a solvent for overnight. The extract was filtered and marc was extracted four times under similar conditions. Pooled extract was centrifuge and particle free extract was concentrated under vacuum to obtain a residue (125 g).The residue was fractionated with CHCl3, EtOAc, and MeOH. The MeOH fraction was loaded into glass column packed with diaion hp.-20 resin and eluted sequentially with deionised water, water: methanol mixture (1:1) followed by methanol. Methanol fraction containing the target compound was subjected to further purification over Sephadex LH-20. Column was initially eluted with deionised water followed by a mixture of water and methanol (1:1) and finally with 100% methanol. The fraction eluted with MeOH: H2O (1:1), on crystallization from EtOAc yielded pure plumieride. Purity profile of the isolated plumieride was tested on Waters 2996 HPLC system equipped with a binary pump, an auto sampler, an electronic degasser, thermostatic column oven, a diode array detector. The data was entered in a computer and was analysed with Empower software for analysis. The LC separations were optimized using RP-18, Merck column (4 × 250 mm, 5 μm) where; mobile phase consisted of a mixture of methanol and water in the ratio of 2:3. Elution was achieved with a flow rate of 0.6 ml/min. Before use, the mobile phase was filtered through 0.45 mm filter (Millipore). The temperature of the column was maintained at 30 °C to provide sharpness to the eluting peak. The UV chromatogram was recorded at 225 nm. The compound showed a peak at a retention time of 5.689 in the HPLC-UV (DAD) chromatogram and the percentage of plumieride in the extract was found to be 1.25%. Plumieride was obtained as colorless amorphous powder with a melting point of 229 °C, [α]21D-114 [c,0.5% MeOH], MS: FABMS [M + Na] + m/z 493. The compound was identified as iridoid i.e.,plumieride{1S-[1a,4aa,7a(R)7aa]}-1-(b-D-glucopyranosyl)-4a,7a-dihydro-4′-(1 hydroxymethyl)-5′-oxospiro[cyclopenta (C) pyrano (7H), 2′ (5′H)-furan]4-carboxylic acid methyl ester on the basis of comparison of its spectral data (1H, 13C NMR, IR) with n the literature. Keeping in view the high reputation of plumieride-{1S-[1a,4aα,7a(R)7aa]}-1-(β-D-glucopyranosyl)-4a,7a-dihydro-4′-(1-hydroxymethyl)-5′oxospiro[cyclo-penta (C) pyrano (7H), 2′ (5′H)-furan]-4-carboxylic acid methyl ester, obtained from Plumeria acutifolia in traditional system of medicine, we were tempted to contemplate that the restorative and revitalizing ability of
2.3. Experimental animals Female balb/c mice (18–22 g) were, procured from the CSIR-Indian Institute of Integrative Medicine, Jammu (India) with prior approval from the Institutional Animals Ethics committee (IAEC (85/2/16). The animals were reared under standard laboratory conditions, i.e. temperature (25 ± 2 °C), relative humidity 65–75% and a photoperiod of 12:12 h (light: dark). The animals were fed on a pellet diet and water was given ad libitum. 2.4. Acute safety study An initial acute safety study was carried out as per OECD guidelines No. 423. The compound was administered at different doses to determine no observe adverse effect level (NOAEL) in female balb/c mice. The animals were observed for any change in general behavior/or untoward symptom and mortality over a two week period. On day 14th, biochemical and hematological parameters were evaluated to determine any end organ toxicity. 2.5. Antigen (SRBC) Fresh sheep red blood cells (SRBC) agglomerated aseptically from the jugular vein of sheep and was stored in cold, sterile alsever's solution [19] then washed three times with pyrogen free, sterile normal saline (0.9% NaCl w/v) and cell count was adjusted to 1 × 107 cells/ml for immunization and challenge at the required time schedule. 2.6. Immunization Six female balb/c mice were grouped. Plumieride was administered orally at different doses 1.56–25.0 mg/kg p.o. daily for six days and control received normal saline. Levamisole was used to elicit the immune system at a dose of 2.5 mg/kg p.o. [20]. Cyclosporine and Cyclophosphamide at a dose of 5 mg/kg and 200 mg/kg p.o. respectively was used to suppress the immune system of animals. All groups of animals were immunized with 200 μl of SRBC (1 × 107) intraperitoneal (i.p) on day 1 of the experiment. Moreover, 20 μl of SRBC was injected 204
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
keep them moist and was incubated for 60 to 90 min at 37 °C in the incubator. By using inverted microscope plaques in SRBC lawn were calculated. Each plaque represented a product of an individual antibody forming cell [25].
on the hind foot of mice for DTH assay. 2.7. Delayed type hypersensitivity (DTH) response The immune system was suppressed by cyclosporine and antigen was injected intraperitoneal (i.p.).Oral drugging was given for six days. The thickness of the left hind footpad was measured on 7th day with a spheromicrometer (pitch, 0.01 mm) and was considered as the control. Mice were vaccinated by injecting 20 μl of 1 × 107 SRBC/ml subcutaneously into the right hind footpad. The foot thickness was measured again at 24 h after challenge [21].
2.13. Carbon clearance The Phagocytic function of the RES (reticulo-endothelial system) was assayed in groups of six mice each by injecting i.v 160 mg/kg b.w of 1.6% suspension of gelatin stabilized carbon particles of 20–25 μM size [23]. Blood samples were collected immediately before and at intervals varying between 2 and 120 min. After intravenous injection of carbon particles. An aliquot (10 μl) of blood samples were lysed with 2 ml of 0.1% acetic acid and transparency determined spectrophotometrically at wavelength of 675 nm till transparency was equivalent to standard (levamisole) was obtained as per described by Hudson hay carbon clearance assay to assess the effect of the drugs on phagocytic capacity of reticuloendothelial system (RES) of the animals which constitutes initial defense mechanism against infectious microorganisms [26].
2.8. Skin allograft rejection The homologous grafts of skin were transplanted in groups of 6 inbred mice, as per the method explained by Billingham [22]. Graft retention time was determined by daily observation the criterion being followed is to access the survival of epithelium. Plumieride at 3.12–25 mg/kg p.o. concentration was fed orally daily for six days and graft rejection time was recorded. Two parallel controls, one given vehicle and other standard cyclosporine at 5 mg/kg p.o. were run for comparison of the activity.
2.14. Measurement of CD4+ and CD8+ T cells
2.9. Lymphocyte proliferation assay
Blood was collected from retro-orbital plexus of sensitized mice in 0.1% of EDTA. Then add monoclonal antibodies (CD4+ and CD8+) and incubate for 30 min. After that, add FACS lysing solution to the samples and incubate for 10 min. Then centrifuge it and discard the supernatant and dissolve the pellet in washing buffer and centrifuge it again and add 1 ml PBS to the pellet after staining with antibodies, cells were washed and re-suspended in PBS for flow cytometric analysis, which was performed on a FACS Calibur flow cytometer [27].
Mice were sacrificed under aseptic conditions and spleen was removed. The splenocytes were isolated by crushing the spleen under a pair of frosted glass slides and erythrocytes were lysed with ammonium chloride (0.08% w/v). The cells are then centrifuged at 1400 rpm for 10 min and the cell pellet was washed three times with RPMI media. 2 × 106 cells/ml in 100 μl complete RPMI media is seeded in 96 well plates. Con-A (0.5 μg/ml) and LPS (1 μg/ml) were added to wells separately for stimulation of B and T cells respectively. Plumieride was also added to the wells and the plates were incubated for 72 h. 20ul MTT dye [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (2.5 mg/ml) was added 4 h prior to termination of the experiment. Then, the absorbance was measured by ELISA at 570 nm [23].
The serum level of cytokines i.e. IL – 2, IFN-γ, TNF-α, IL-4 in immunesuppressed animals treated with plumieride was estimated by ELISA method. The protocols were followed as mentioned in the commercially available kits of BD Biosciences.
2.10. Evaluation of hematological parameters
2.16. In-vitro phagocytic activity of peritoneal macrophages
Blood was collected from the retro-orbital plexus of immune suppressed balb/c mice on 7th day of the experiment and analysed for hematological imbalance.
The phagocytic function of macrophages isolated from peritoneal cavity was analysed by method described by Lehrer [28]. In this method the peritoneal macrophages were collected by flushing the peritoneal cavity with 5 ml sterile ice cold 0.15 M PBS containing 2 U/ ml preservative free heparin. After that cells were allowed to adhere to glass cover slip (22 × 22 mm) for 2 h in incubator at 37 °C, 5% co2 and 95% relative humidity in presence and absence of different conc. of plumieride. Then the cover-slip was thoroughly washed with phosphate buffer saline (PBS) to remove non adherent cells. During this period heat killed Candida albicans cells were opsonized for 90 min with 20% mouse serum, washed three times and resuspended with final conc. of 2 × 109 cells/ml. Then 100 μl of opsonized C. albicans cells was then spread over monolayer and mixture was incubated for 15 min. After that cover slip was washed with PBS and stained with trypan-eosin and then samples were immediately evaluated microscopically to determine for phagocytosis.
2.15. Cytokines measurement
2.11. Humoral antibody titre Six animals were put in each group and each animals immune system was suppressed by cyclophosphamide and was immunized by injecting 200 μl of 1 × 107 SRBC/ml intraperitoneal (i.p.) on day 1 followed by oral drugging as described above. Blood was collected on day 7 for primary antibody titre. Hemagglutination antibody titers were determined following the modified micro titration technique described by Nelson and Midenhall [24]. 2.12. Plaque forming cell (PFC) assay The IgM reaction to an antigen (SRBC) was observed in mice treated with Plumieride. Mice were injected with 0.2 ml of 10% (v/v) suspension of SRBC in normal saline. Animals were sacrificed and their spleen was removed aseptically. The spleen was crushed and suspension of splenocytes was made in RPMI supplemented with FBS. Cell count was adjusted to 2 × 107 cells/ml. Take 0.1 ml of this cell suspension, 0.3 ml of 5% SRBC, 0.1 ml of guinea pig serum and 0.5 ml of RPMI 1640 medium and total 150 μl quantity of this mixture and add to Cunningham's chamber. The chamber was sealed with paraffin wax to
2.17. Mycobacterium tuberculosis H37Rv infection The balb/c mice were infected intranasally with Mycobacterium tuberculosis H37Rv (103 CFU/mouse). The infected mice were divided in groups of 6 mice each. Treatment started one week after the infection. Mice from early control group were sacrificed, left lung was aseptically removed, homogenized and dilutions were placed on Middlebrook 7H10 agar to determine the CFU (Colony forming unit). Mice from 205
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
significantly increased which indicated that plumierides stimulated the immune system. An increase of 1.27 fold was seen in WBC count. Levamisole used as the control increased all the blood parameters (Table 1).
late control and treatment group were scarified one week after the infection and lung CFU was determined. 2.18. Statistical assessment For statistical analysis, the data was entered and analysed on MS Office 2007 (Microsoft Crop. Redmond, WA). The data was expressed in Mean ± SEM (Standard error of measurement). For the purpose of comparison One Way ANNOVA was used to evaluate the potential difference between the experiments were done in triplicates. Dunnett's test was used to analyse different variables (GraphPad Prism 5.01, GraphPad Software, USA). A value of p < 0.05 was set as statistically significant.
3.6. Humoral antibody titre Serum was isolated from the blood collected from the treated animals on 7th day of the experiment and antibody titre reading was increased in all the five concentrations i.e. 1.56, 3.12, 6.25, 12.50, 25.00 mg/kg p.o. The maximum effect was seen at 12.5 mg/kg p.o. with 30.76% stimulation. A four fold increases in stimulation from 1.56 mg/kg to 12.5 mg/kg have been observed. Moreover, Plumieride at conc. of 25 mg/ml showed a stimulation of 23.07% which was almost 1.33 fold decrease at this concentration when compared with 12.5 mg/ kg. Levamisole used as positive control showed 38.94% stimulation. The response was found to be dose-dependent and significant. Cyclophosphamide suppressed the immune system by 31.39% (p < 0.05) (Fig. 3(a)).
3. Results 3.1. Acute toxicity study Plumieride was found to be safe at a single dose of 2000 mg/kg p.o. No change was observed in behavior, biochemical or hematological parameters (Data not Shown). Therefore, NOAEL was found to be 2000 mg/kg p.o.
3.7. Plaque forming cell assay The SRBC response was also evaluated by the primary antibody IgM. Plumieride was administered orally at five concentrations between 1.56 and 25.0 mg/kg p.o. for five days from the day of sensitization. There was increase in a number of antibodies secreting cells by 5.89 fold from lower to higher concentration. The number of plaque forming cells was 58.92% at 25 mg/kg and 56.30% at 12.50%. The control group was levamisole with 74.30%.The results were significant and dose dependent (p < 0.05) (Fig. 3(b)).
3.2. Cell mediated immune response In order to examine the basic mechanism(s) of action on immunomodulation, plumieride was administered and produced a dose related to increase in SRBC induced DTH reaction (cell mediated immunity) which shows the stimulatory effect on ‘T’ lymphocytes. Cyclosporine at 200 mg/kg p.o. suppressed the immune system by 35.42%. Plumieride elicited the cell mediated immunity by 15.78% at a concentration of 1.56 mg/kg and at 12.5 mg/kg the stimulation was 36.84% (p < 0.05). There was a 2.33 fold increase i.e. from 1.56 mg/kg to 12.5 mg/kg. Plumieride at a concentration of 25 mg/kg showed 30.52% stimulation. Levamisole (2.5 mg/kg p.o.) was used as a positive control with 38.94% stimulation (Fig. 2(a)).
3.8. Carbon clearance The oral administration of plumieride for seven days increased the clearance of carbon particles from the circulation of mice. Plumieride at a concentration of 25 mg/kg showed 23.80% efficacy and at 12.5 mg/ kg showed 17.46% effective in clearing the particles. There was a 4.13 fold increase from lower to higher concentration. Levamisole at 2.5 mg/ kg showed 33.33% increase and was used as the control. The results were found to be dose dependent (Fig. 3(c)).
3.3. Skin allograft rejection The graft of skin was transplanted between the mice of the same group and the animals were monitored daily to determine increase/ decrease in rejection time. Plumieride, when administered orally at four different concentrations (3.12 to 25. mg/kg p.o.), had hastened the rejection time by 38.61% at 25 mg/kg. There was concentration dependent increase in rejection time. At conc. of 12.50 mg/kg, the rejection time was decreased by 34.61%. This experiment supported our B (Humoral) and T (Cell mediated) cell experiment that plumieride is an immunostimulant. Cyclosporine was used as the positive control and found to increase the rejection time by 39.69% (Fig 2(b)).
3.9. Effect of Plumieride on CD4 and CD8 The expression of CD4 T and CD8 T in immunesuppressed control mice was 24.04% and 14.36% respectively while in case of levamisole the expression of CD4 T and CD8 T had increased to 30.60% and 16.90% respectively and at concentration of 12.5 mg/kg. The CD4 and CD8 expression levels increased to 42% and 24% respectively. These results indicated that plumieride had activated the Th-1 pathway significantly (Fig. 3(d)).
3.4. Lymphocyte proliferation assay 3.10. Estimation of cytokines (Th-1 and Th-2) by ELISA In Lymphocyte proliferation assay, the plumieride was found to stimulate the proliferation of T and B cells. Plumieride in all the three concentrations (20 μM,10 μM, and 1 μM) elicited the proliferation of T cells. The proliferation of T cell at a concentration of 20 μM was higher than the con-A control. B cells proliferation was highest at 20 μM and with lower concentrations, the effect was also less. The effect in both T and B cells was dose dependent (Fig. 2(c & d)).
The effect of plumieride on the release of different cytokines at four concentrations was studied (3.12, 6.25, 12.50, 25.0 mg/kg). Plumieride stimulated both Th-1(IFN-γ, IL-2, TNF-α) and Th-2 (IL-4) cytokines. Plumieride significantly stimulated IFN-γ, IL-2, TNF-α.The IFN-γ secretion was enhanced by 9.95% at 12.50 mg/kg oral dose. The sensitized control group showed 5.35% and non-sensitized control showed 2.70% of IFN-γ expression (Fig. 4). The increase was observed at all the concentrations but maximum effect was seen at 12.50 mg/kg. The IL-2 expression was 19.44% at 12.5 mg/kg oral dose. In a sensitized and non-sensitized group, the expression was 4.15% and 6.06% respectively. TNF-α expression was increased by 2.19% at 12.5 mg/kg oral dose. In the case of Th-2 (IL-4), expression was found to be increased non-significantly by 6.49% at conc. of 12.5 mg/kg oral (Fig. 4(a,b,c,d)).
3.5. Plumieride effect on hematological parameters Blood was collected from immunosuppressed mice and showed that hemoglobin level increased in all the concentrations and it was maximum at 12.5 mg/kg p.o. There was a 1.15 fold increase from concentration 6.25 mg/kg p.o. to 12.5 mg/kg p.o. The WBC count was 206
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 2. (a) Plumieride was administered orally (1.56–25 mg/kg) for seven days post-immunization, an increase of 15.78 to 36.84% in the expression of DTH reaction was observed in immune suppressed mice. The effect was dose dependent and highly significant (P < 0.001) at 12.5 mg/kg dose. (b) Plumieride showed statistically significant effect (decrease in rejection time) that was 34.61 and 38.61% at 12.50 and 25 mg/kg p.o. dose, respectively. Cyclosporine a standard control at 5 mg/kg increased the rejection time by 39.69%. (c) The proliferation of B cells was maximum at concentration of 20 μM when cells were stimulated with L.P.S and with the lower concentration the effect was down. (d) T cell proliferation was higher in all the concentration. The stimulation of T cells was done by Con-A. Table 1 Blood samples were collected from immune-suppressed balb/c mice that were treated with different conc. of Plumieride i.e. 6.25, 12.5 and 25 mg/kg p.o. Samples were immediately analysed for hematological parameters like white blood cell count (WBC), hemoglobin (HB), Hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), lymphocyte, monocyte and granulocytes by using automatic hematology analyzer. Effect of plumieride on blood cells of immune suppressed balb/c mice Treatment mg/kg p.o.
Control Control immune suppressed Levamisole Plumieride (25.0) Plumieride (12.5) Plumieride (6.25)
Hemoglobin conc.
RBC count in million/ cmm
Platelet count in thousand/ cmm
WBC count in thousand/ cmm
Differential count Lymphocyte
Monocytes
Granulocyte
9.20 ± 0.37 6.00 ± 0.27
6.55 ± 0.37 5.00 ± 1.15
528.20 ± 18.29 500 ± 16.66
13.20 ± 0.50 9.00 ± 0.55
74.18 ± 1.11 42.00 ± 1.56
2.10 ± 0.0 1.00 ± 0.11
23.72 ± 0.58 17.55 ± 0.55
8.23 6.98 7.60 6.59
6.43 5.59 6.31 5.39
1162.20 ± 51.22 842.60 ± 1.07 877.80 ± 3.90 587.60 ± 18.09
14.00 13.70 13.72 10.76
70.74 55.56 65.34 49.52
2.36 2.35 3.36 4.32
27.90 42.09 31.30 46.24
± ± ± ±
0.24 0.07 0.15 0.16
± ± ± ±
0.22 0.23 0.14 0.06
± ± ± ±
0.37 0.65 1.06 0.51
± ± ± ±
0.51 0.40 0.09 1.38
± ± ± ±
0.36 0.11 0.07 0.14
± ± ± ±
0.82 0.37 0.91 1.92
control with 40.33% phagocytosis (Fig. 5).
3.11. In-vitro phagocytic activity of peritoneal macrophages The population of polymorphonuclear leukocyte (PMNL) is usually seen during the early hours of antigen processing, whereas macrophages occur at later stages. Plumieride increased the phagocytic function of macrophages that were isolated from peritoneal cavity of mouse. There 1.42% increase in % phagocytosis from lower conc. (1 μg/ml) to higher conc. (30 μg/ml). Levamisole is used as the positive
3.12. Mycobacterium tuberculosis H37Rv infection The mice infected with Mycobacterium tuberculosis H37Rv. One week after the infection the CFU load in the early control reached 4.7 log while in the late control was 6.8 log. The early control indicates the CFU load at the start of the treatment, whereas late control indicates the 207
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 3. (a) Plumieride (1.56–25 mg/kg p.o.) produced a dose dependent antibody synthesis in immune-suppressed balb/c mice. The immunestimulatory effect of plumieride was highly significant at 12.5 mg/kg p.o. dose levels. (b) Administration of plumieride (3.12–25 mg/kg) orally for 5 consecutive day's begining from day of immunization produced an increase in the number of antibody secreting cells (PFC) by 10.15–58.92% in mouse spleen. The effect was highly significant. (p < 0.05) (c) Oral administration of plumieride (3.12–25 mg/kg) for consecutive seven days and 30 min prior to carbon injection enhanced the clearance rate of carbon particles from circulation in normal animals at dose of 25 mg/kg p.o. (d) The values of the (unsensitised group) were 24.04% of CD4+ T cells and 14.36% % of CD8+ cells and in the (sensitized group) the value of CD4+ and CD8+ T cells was 30.60% and 16.90% respectively. This shows an increase in CD4+ and CD8+ T cells in experimental animals treated with different doses of Plumieride.
with scaling down in the skin graft rejection time in mice treated with plumieride. The basic mechanism involved is a slowdown in graft rejection time by the stimulation of T-lymphocytes i.e., CD4 and CD8 positive T cells. Activation of macrophages is important for the enhancement of an immune system which is majorly accomplished by CD4 T cells. The Complete Blood Count (CBC) results showed an increase in leukocyte counts in dose dependent manner in immune suppressed balb/c mice treated with plumieride indicate it as an immunostimulant. The proliferation of T cells was also confirmed by the lymphocyte proliferation assay in which the con-A stimulated T cells showed maximum proliferation. The major function of CD4 T cells is to activate macrophages and activation of macrophages are important for enhancement of immune system [29]. Present study showed that plumieride activates the CD4 T cells and this increases the functioning of macrophages. The increased number of phagocytes is evident by clearance of carbon particles from reticuloendothelial cells moreover, it also hastens the T & B lymphocytes involved in phagocytosis in-vitro by murine macrophages indicating an increase in the functioning of macrophages (innate response) by causing stimulation of non-specific immune response. The upregulation of the humoral immune response against the antigen (SRB) also signals towards the response of macrophages in antibody synthesis. Immune homeostasis needs the differential and controlled expression of cytokines and their receptors [30]. There are a number of examples in
bacterial load of the untreated group. Treatment with plumieride at 12.5 mg/kg did not allow the bacteria to grow and the bacterial load was close to the early control. The combination of plumieride with rifampicin (anti-tuberculosis drug) was synergistic. The combination could reduce the CFU load to 3.0 log as compared to rifampicin alone that stood at 5.0 log. This combination reduced the initial CFU load (early control) by 1.7 log. 4. Discussion Immunomodulators are a class of natural or synthetic agents which alter the immune system. They have the ability to augment, restore and inhibit to produce crave immune responses. Immunomodulatory drugs constitute thalidomide and its analogues, lenalidomide, pomalidomide apremilast and many more. An iridoid glucoside plumieride obtained from Plumieria acutifolia was investigated for its immunomodulatory potential in the present study. Iridoids are known to possess a large number of biological activities and the immune potentiating activity of iridoids has also been reported in the literature [27]. Similarly, the present work describes the ability of plumieride to act as a significant immunopotentiating agent in response to antigen (SRB), plumieride produced a dose related increased in delayed type hypersensitivity reaction which is a cell mediated response that shows its specific stimulatory effect on ‘T’ lymphocytes. However this is corroborated 208
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
Fig. 4. (a) The IFN-γ (Th1 signature cytokine) was enhanced in the groups treated with plumieride showing maximum expression (9.95%) at 12.50 mg/kg oral dose. The sensitized control group showed 5.35% and non-sensitized control showed 2.70% of IFN-γ expression. There was an increase in all the test drug treated groups with maximum effect being observed at 12.50 mg/kg per oral dose. (b) Animals treated with plumieride at graded oral doses showed increase in Intracellular IL - 2 expressions. The percentage of IL-2 secreted was 19.44% at 12.50 mg/kg per oral test drug dose (maximum effect).The percentage of IL-2 in the normal non-sensitized control group and in sensitized control group was 4.15% and 6.06% respectively. (c) TNF-α (Th-1cytokine) were increased in dose dependent manner and were significant at 12.5 mg/kg.The percentage of TNF-α at 12.5 mg/kg is 2.19%. (d) The IL-4 (Th2 signature cytokine) expression in sensitized control and non-sensitized control groups was 4.83% and 3.80% respectively .The maximum expression of IL-4 in treated groups was 6.49% at the dose of 12.50 mg/kg per oral dose. However, this was not statistically significant.
cells. It, however, gave some expression of IL-4 that was not significant and may be possibly due to cross effects of CD4 T cells secreted cytokines, thus expressing specific Th1 response. Plumieride did not show any cytotoxicity and both T and B cell proliferation was within normal range. The mechanistic studies of botanical isolated molecule i.e. plumieride suggests that it is an immunoactive compound which concurrently or simultaneously modulates immune matrix and restores homeostatic conditions which can play important role in immunotherapy [34].
experimental models where alteration of the Th1/Th2 balance by the administration of cytokines or cytokine antagonists control the outcome of the diseases. The function of CD4 T cells is the activation of macrophages and that plays a key role in escalating the activity [31]. Also, on the basis of present study, CD-4 T cell activation by plumieride may be one of the factors that is attributing towards the increase in the functioning of the macrophages. The combination of plumieride with rifampicin showed synergism in being effective. The combination could reduce the CFU load to 3.0 log as compared to rifampicin alone that stood at 5.0 log. Plumieride at 12.5 mg/kg oral dose did not allow the bacteria (Mycobacterium Tuberculli) to grow. T helper (Th1) lymphocytes homeostasis is a pivotal role in arranging the suitable cytokine responses and hence remains as one of the targets for Immunomodulation. Th1-type cytokines (IFN-γ, IL-2, TNF-α) exhibit cell-mediated immunity and targets intracellular parasites while Th2-type cytokines (interleukins 4) are affiliated with humoral immunity. Cytokines play an important role in the regulation of hematopoiesis, mediating the differentiating migration, activation, and proliferation of phenotypically diverse cells [32].The analysis and expression of cytokines in biological liquids had become a commonly used technique in research and clinical laboratories and is clearly important in expanding our comprehension of many immunological functions [33].The results suggest that oral administration of plumieride induced a dose related increased expression of interferon gamma (IFNγ), IL-2 (interleukin-2), TNF-α (Tumor Necrosis Factor) in CD4 T
5. Conclusions Our pragmatic study establishes plumieride as an immunostimulant that up-regulates Th1 pathway. Such agents are being looked for the treatment of infectious diseases, immunodeficient diseases, or for generalized immunosuppression induced by drug treatment; for combination therapy with antibiotics; and as adjuncts for vaccines. Conflict of interest statement The authors declare that they have no conflict of interest. Acknowledgements Author thanks, Director of CSIR-IIIM for providing the facility. Also 209
International Immunopharmacology 48 (2017) 203–210
J. Singh et al.
obstruction in guinea pigs, Int. Arch. Allergy Immunol. 95 (2–3) (1991) 128–133. [6] N. Farnsworth, The role of medicinal plants in drug development, Natural Products and Drug Development, 1984, pp. 17–30. [7] R.P. Labadie, Steven M. Colegate, Russell J. Molyneux, Immunomodulatory compounds, Bioactive Natural Products, CRC PRESS Inc., 1993, pp. 279–317. [8] H. Baxter, J.B. Harborne, G.P. Moss, Phytochemical Dictionary: A Handbook of Bioactive Compounds From Plants, CRC press, 1998. [9] S.K. Sharma, N. Kumar, Antimicrobial potential of Plumeria rubra Syn Plumeria acutifolia bark, Der Pharm Chemica 4 (4) (2012) 1591–1593. [10] R. Ansari, B. Aswal, R. Chander, B. Dhawan, N. Garg, N. Kapoor, D. Kulshreshtha, H. Mehdi, B. Mehrotra, G. Patnaik, Hepatoprotective activity of kutkin–the iridoid glycoside mixture of Picrorhiza kurrooa, Indian J. Med. Res. 87 (1988) 401–404. [11] A. Viljoen, N. Mncwangi, I. Vermaak, Anti-inflammatory iridoids of botanical origin, Curr. Med. Chem. 19 (14) (2012) 2104–2127. [12] B. Dinda, S. Debnath, R. Banik, Naturally occurring iridoids and secoiridoids. An updated review, part 4, Chem. Pharm. Bull. 59 (7) (2011) 803–833. [13] B. Dinda, S. Debnath, Y. Harigaya, Naturally occurring secoiridoids and bioactivity of naturally occurring iridoids and secoiridoids. A review, part 2, Chem. Pharm. Bull. 55 (5) (2007) 689–728. [14] R. Tundis, M.R. Loizzo, F. Menichini, G.A. Statti, F. Menichini, Biological and pharmacological activities of iridoids: recent developments, Mini-Rev. Med. Chem. 8 (4) (2008) 399. [15] G. Singh, S. Bani, S. Singh, A. Khajuria, M. Sharma, B. Gupta, S. Banerjee, Antiinflammatory activity of the iridoids kutkin, picroside-1 and kutkoside from Picrorhiza kurrooa, Phytother. Res. 7 (6) (1993) 402–407. [16] K. Nithya, J. Muthumary, Growth studies of Colletotrichum gloeosporioides (Penz.) Sacc.-a taxol producing endophytic fungus from Plumeria acutifolia, Indian J. Sci. Technol. 2 (11) (2009) 14–19. [17] M.P. Dobhal, G. Li, A. Gryshuk, A. Graham, A.K. Bhatanager, S.D. Khaja, Y.C. Joshi, M.C. Sharma, A. Oseroff, R.K. Pandey, Structural modifications of Plumieride isolated from Plumeria bicolor and the effect of these modifications on in vitro anticancer activity, J. Org. Chem. 69 (19) (2004) 6165–6172. [18] V.S. Georgiev, J.F. Albright, Cytokines and their role as growth factors and in regulation of immune responses, Ann. N. Y. Acad. Sci. 685 (1) (1993) 584–602. [19] J.B. Alsever, R.B. Ainslie, A new method for the preparation of dilute blood plasma and the operation of a complete transfusion service, N. Y. State J. Med. 41 (1941) 126–131. [20] M.A. Tempero, Y. Haga, C. Sivinski, D. Birt, L. Klassen, G. Thiele, Immunologic effects of levamisole in mice and humans: evidence for augmented antibody response without modulation of cellular cytotoxicity, J. Immunother. 17 (1) (1995) 47–57. [21] N. Doherty, Selective effects of immunosuppressive agents against the delayed hypersensitivity response and humoral response to sheep red blood cells in mice, Agents Actions 11 (3) (1981) 237–242. [22] R. Billingham, P.B. Medawar, The technique of free skin grafting in mammals, J. Exp. Biol. 28 (3) (1951) 385–402. [23] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983) 55–63. [24] D. Nelson, P. Mildenhall, Studies on cytophilic antibodies. II. The production by guinea-pigs of macrophage cytophilic antibodies to sheep erythrocytes and human serum albumin: relationship to the production of other antibodies and the development of delayed-type hypersensitivity, Aust. J. Exp. Biol. Med. Sci. 46 (1) (1968) 33. [25] A.T. Cunningham, A. Szenberg, Further improvements in the plaque technique for detecting single antibody-forming cells, Immunology 14 (4) (1968) 599. [26] C. Atal, M. Sharma, A. Kaul, A. Khajuria, Immunomodulating agents of plant origin. I: Preliminary screening, J. Ethnopharmacol. 18 (2) (1986) 133–141. [27] B. Ghule, P. Yeole, In vitro and in vivo immunomodulatory activities of iridoids fraction from Barleria prionitis Linn, J. Ethnopharmacol. 141 (1) (2012) 424–431. [28] R. Lehrer, Ingestion and destruction of Candida albicans, Methods for Studying Mononuclear Phagocytes, 1981, pp. 693–708. [29] M. Whelan, J. Whelan, N. Russell, A. Dalgleish, Cancer immunotherapy: an embarrassment of riches? Drug Discov. Today 8 (6) (2003) 253–258. [30] W.W. Overwijk, K.S. Schluns, Functions of γC cytokines in immune homeostasis: current and potential clinical applications, Clin. Immunol. 132 (2) (2009) 153–165. [31] J. Zhu, W.E. Paul, CD4 T cells: fates, functions, and faults, Blood 112 (5) (2008) 1557–1569. [32] B. Patwardhan, Ayurveda: the designer medicine, Indian Drugs 37 (2000) 213–227. [33] A. Kelso, Cytokines: principles and prospects, Immunol. Cell Biol. 76 (4) (1998) 300–317. [34] R. Verpoorte, Y. Choi, H. Kim, Ethnopharmacology and systems biology: a perfect holistic match, J. Ethnopharmacol. 100 (1) (2005) 53–56.
Fig. 5. (a) Mice infected with Mycobacterium tuberculosis H37Rv. After one week of infection early control CFU load reached 4.7 log and late control CFU load 6.8 log. Plumieride at a conc. of 12.5 mg/kg reduced the CFU load 5.1 whereas rifampicin alone reached 5.3 log. The combination of rifampicin + plumieride CFU load was 3.0 log with p < 0.05 value.(b) Plumieride increased the phagocytosis of heat killed Candida albicans by murine macrophages at 30 μg/ml with 38.50 and p < 0.01 and at lower conc. 1 μg/ ml the phagocytosis is 27.00. Levamisole was used as positive control and phagocytosis value was 40.23 with p < 0.001.
author thanks, funding agency CSIR-New Delhi for granting fund through project (BSC-0205).We are thankful to Dr. M.M Baskar, Lead Reviewer, Clinical Pharmacology, New Drug Submissions, Health Canada, Santé Canada for checking/correcting the manuscript for English language. References [1] H. Wagner, P.M. Wolff, New Natural Products and Plant Drugs with Pharmacological, Biological Or Therapeutical Activity, Proceedings of the First International Congress on Medicinal Plant Research, Section A, Held at the University of Munich, Germany, September 6–10, 1976, Springer Science & Business Media, 2012. [2] O. Sticher, Plant mono-, di-and sesquiterpenoids with pharmacological or therapeutical activity, New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutical Activity, Springer, 1977, pp. 137–176. [3] P. Das, R. Tripathi, V. Agarwal, A. Sanyal, Pharmacology of kutkin and its two organic acid constituents cinnamic acid and vanilic acid, Indian J. Exp. Biol. 14 (4) (1976) 456. [4] M. Sharma, C. Rao, P. Duda, Immunostimulatory activity of Picrorhiza kurroa leaf extract, J. Ethnopharmacol. 41 (3) (1994) 185–192. [5] W. Dorsch, H. Stuppner, H. Wagner, M. Gropp, S. Demoulin, J. Ring, Antiasthmatic effects of Picrorhiza kurroa: androsin prevents allergen-and PAF-induced bronchial
210