Md.Moklesur Rahman Sarker，Long Chiau Ming，Md.Zaidul Islam Sarker，M Shahabuddin Kabir ChoudhuriDepartment of Immunochemistry，Division of Pharmaceutical Sciences，Okayama University Graduate School of Medicine，Dentistry and Pharmaceutical Sciences，Tsushima-naka，Kita-ku，Okyama，700-80，JapanDepartment of Pharmacology，F(xiàn)aculty of Pharmacy，Lincoln University College，No.，Jalan Stadium SS 7/1，Kalana Jaya，701，Petaling Jaya，Selangor，MalaysiaFaculty of Pharmacy，Brain Degeneration and Therapeutics Group，Pharmaceutical and Life Sciences CoRe，Universiti Teknologi MARA，Puncak Alam，Selangor，MalaysiaDepartment of Pharmaceutical Technology，F(xiàn)aculty of Pharmacy，International Islamic University Malaysia，00，Kuantan，Pahang，MalaysiaDepartment of Pharmacy，F(xiàn)aculty of Biological Science，Jahangirnagar University，Savar，Dhaka，1，Bangladesh

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Immunopotentiality of Ayurvedic polyherbal formulations“Saribadi”and“Anantamul Salsa”withaugmentationofIgMproductionandlymphocytesproliferation: A preliminary study

Md.Moklesur Rahman Sarker1，2*，Long Chiau Ming3，Md.Zaidul Islam Sarker4，M Shahabuddin Kabir Choudhuri51Department of Immunochemistry，Division of Pharmaceutical Sciences，Okayama University Graduate School of Medicine，Dentistry and Pharmaceutical Sciences，Tsushima-naka，Kita-ku，Okyama，700-8530，Japan
2Department of Pharmacology，F(xiàn)aculty of Pharmacy，Lincoln University College，No.2，Jalan Stadium SS 7/15，Kalana Jaya，47301，Petaling Jaya，Selangor，Malaysia
3Faculty of Pharmacy，Brain Degeneration and Therapeutics Group，Pharmaceutical and Life Sciences CoRe，Universiti Teknologi MARA，Puncak Alam，Selangor，Malaysia
4Department of Pharmaceutical Technology，F(xiàn)aculty of Pharmacy，International Islamic University Malaysia，25200，Kuantan，Pahang，Malaysia
5Department of Pharmacy，F(xiàn)aculty of Biological Science，Jahangirnagar University，Savar，Dhaka，1342，Bangladesh

ARTICLE INFO

Article history: Received 6 Feb 2016 Received in revised form 1 Mar，2nd revised form 16 Mar 2016 Accepted 1 Apr 2016 Available online 30 May 2016

“Saribadi”“Anantamul Salsa”Ayurvedic medicine Immunostimulant Lymphocytes Splenocytes Immunoglobulin M antibody Proliferation Differentiation

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2016.05.004

ABSTRACT

Objective:To assess the immunopotentiality of Ayurvedic polyherbal preparations，“Saribadi”and“Anantamul Salsa”.

Methods:Freshly prepared BALB/c mice splenocytes were cultured with“Saribadi”or “Anantamul Salsa”treatment［doses of 0.25%，0.50%，0.75%，1.00%，1.50%，2.00%，3.00%and 4.00%（v/v）］at 37°C for 5 days.The immunoglobulin M（IgM）production and lymphocytes proliferation were determined by ELISA and MTT methods，respectively. Endotoxin contamination was assessed by treating the preparations with polymyxin B.

Results:The doses of“Saribadi”［0.25%，0.50%，0.75%and 1.00%（v/v）］significantly increased IgMproductions（0.966，0.728，0.695and0.615μg/mLvs.control 0.265μg/mL）and lymphocytes proliferation［absorbance 0.311，0.394，0.372 and 0.334 optical density （OD）vs.control 0.162 OD］.Similarly，the doses of“Anantamul Salsa”［0.50%，0.75%，1.00%and 1.50%（v/v）］promoted IgM productions（0.933，0.919，0.917 and 0.892μg/mL vs.control 0.502μg/mL）and the doses of“Anantamul Salsa”［0.50%，0.75%，1.00%，1.50%，2.00%，and 3.00%（v/v）］stimulated lymphocytes proliferation（absorbance 0.395，0.326，0.440，0.398，0.452 and 0.355 OD vs.control 0.199 OD）.The activity of“Saribadi”and“AnantamulSalsa”wasnotretardedbythetreatmentofpreparationswithpolymyxinB.

Conclusions:Immunomodulatory activity of“Saribadi”and“Anantamul Salsa”was unveiled for the first time.“Saribadi”and“Anantamul Salsa”possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production. These preparations may be useful in strengthening immune responses.However，further cellular and in vivo studies are required.

1.Introduction

The defensive system of human body，composed of innate and adaptive immunological arms，protects it from possible infections from microorganisms and prevents the formation of cancerous cells and/or eliminates them as soon as they appear as an antigen［1，2］.In certain conditions，such as，diabetes，obesity，burns，sepsis，stress，insomnia，AIDS，lackof exercise，poor nutrition and aging，the immunity is greatly suppressed［2-5］.Immunity is also suppressed in case ofreceiving radiotherapy，chemotherapy，and antibiotics［2，6，7］. The impaired immune system is greatly susceptible to several diseasesincludinginfections，cancers，allergy，asthma，ulcerative colitis and asthma［8，9］.Immunopotentiators play a vital role to upgrade the immunity to protect the body frompossibleinfectionandcancersunderthose immunocompromised conditions.

The popularity of herbal medicine is increasing due to the perceived tremendous side-effects of synthetic or allopathic medicines［10-12］.Herbal medicines are considered to be safe as it contains complementary ingredients that could neutralize the side-effects of one chemical from one to another.Although Ayurvedic medicines are one of the most popular forms of alternative medicines，scientific reports on the evaluation of those medicines are very negligible comparing to allopathic medicines［13］.Ayurvedic medicines are prepared from the multiple combination of a number of medicinal plants which are believed to act as an immunomodulating agent.Therefore，herbalmedicinesrecentlyhavegainedattentionforthe evaluation of immunopotentiality and the treatment of immune disorders［8］.Thisinvestigationwasdesignedforthe preliminarilyevaluationoftheimmunopotentialityof20 Ayurvedicpreparations， including“AmalakiRasayan”，“AnantamulSalsa”， “Ashokarista”， “Aswagandharista”，“Basakarista”，“Bolarista”，“Kalomeghasav”，“Kutajarista”，“Rohitakarista”，“Shirisharista”，“Ushisharista”，“Abhyarista”，“Arabindasav”，“Arjunarista”，“Saribadi”，“Dasamularista”，“Debdarbadyarista”，“Khadirarista”，“Moha Draksharista”and “Amritarista”.Among the 20 Ayurvedic preparations，only twopreparations（“Saribadi” and“AnantamulSalsa”）exhibited prospective immunopotentiating activities in vitro.

“Saribadi”is a polyherbal Ayurvedic preparation indicated for the treatment of gonorrhoea，syphilis，skin diseases and blood impurities［14］.No scientific literature has been reported on the pharmacological or therapeutic activity of“Saribadi”. “AnantamulSalsa” isindicatedforthetreatmentof gonorrhoea，syphilis，such as blood poisoning/purifier，all types of skin diseases including acne，skin-allergies dermatitis，boils，skin rashes and other skin disorders including gout and leprosy［14，15］.Furthermore，no scientific report is available on the pharmacological or therapeutic activities of“Anantamul Salsa”.Hemidesmus indicus is the main plant of both herbal preparations，“Saribadi”and“Anantamul Salsa”.This plant and its constituents were reported to possess antioxidant，anticancer，antidiabetic，antibacterial，antihyperlipidemic and cardioprotective effects［16-21］.

Scientific report is completely absent for the evaluation of “Saribadi”and“Anantamul Salsa”on the immunomodulating activities.Therefore，the present investigation was designed for the determination of immunopotentiating activity of those preparations measuring the production of immunoglobulin M （IgM）antibody responsible for humoral immunity and the proliferation of splenic lymphocytes.

2.Materials and methods

2.1.Preparation of drug sample

All the drugs used in this study including“Saribadi”and “Anantamul Salsa”were prepared according to the formulation and procedure mentioned in Bangladesh National Formulary of Ayurvedic Medicine and the Ayurvedic Pharmacopoeia of India［22，23］.All the ingredients were purchased from the reputed herbal medical ingredients’suppliers in Dhaka，Bangladesh. Thematerialswereconfirmedwiththeirmorphological features by a senior taxonomist at the Botany Department of Jahangirnagar University，Bangladesh.The preparations of the drugsweredonestrictlyunderthesupervisionofan Ayurvedic physician in the Ayurvedic Research Laboratory of PharmacyDepartmentinJahangirnagarUniversity Bangladesh.The samples were filled in centrifuge tubes and filtered using 0.22μm micro-filters just before its use.All the experiments were carried out in the Immunochemistry Laboratory of the Faculty of Pharmaceutical Sciences，Okayama University Japan.

2.2.Chemicals and reagents

RPMI 1640 was purchased from ICN Biomedicals，USA，and Eagle's minimum essential medium（MEM）was purchased from Nissui Pharmaceutical Co.，Ltd.，Japan.Lipopolysaccharide （LPS）from Escherichia coli 055:B5，bovine serum albumin，Sigma-Fast o-phenylenediamine，Tween-20，fetal calf serum （FCS）and MTT were purchased from Sigma Aldrich，Japan. Mouse IgM （purified），goat anti-mouse IgM antibody and horseradish peroxidase-conjugated goat anti-mouse IgM antibody were purchased from Zymed Laboratories Inc.，USA，Organon Teknika Corporation，USA，and Kirkegaad and Perry Laboratories，USA，respectively.

2.3.Study animal

Female BALB/c mice were obtained from Charles River，Japan.The animals were housed at pathogen-free rooms，12-light/12-dark cycle and at 20-25°C temperature in the animal facility of Okayama University，Japan.The animals were used at the age of 8-12 weeks.The experiments concerned with the use of mice were approved by the ethical approval of Animal Research Control Committee of Okayama University.

2.4.Preparation of murine whole spleen cells

Splenocytes（depleted erythrocytes）were prepared from BALB/c mice by the process as described by Sarker et al.［24，25］. Mice were sacrificed to collect spleens aseptically.The spleens were mashed using spatula through the strainer to pass the cells into MEM in a Petri-dish（Iwaki，Japan）.Cells were then suspended by Pasteur pipette and screened by passing through a mesh into the centrifuge tube.The cell suspensions were centrifuged for 5 min at 4°C temperature and speed at 2000 r/ min.Cells supernatants were removed and ammonium chloride potassium lysis buffer（0.15 mol/L NH4Cl，10 mmol/L KHCO3，0.1 mmol/Lethylenediaminetetraaceticacid disodiumsalt dihydrate；pH 7.2）was added to the cells inside the centrifuge tube for 5 min at room temperature for lysing the erythrocytes. With the addition of MEM into the centrifuge tube，cells were suspended and centrifuged at 2000 r/min at 4°C for 5 min.Cell pellets were washed two-times with the MEM after removal of the supernatants.Re-suspension of cells in MEM was performed and screened through mass to pass into another centrifuge tube to collect the splenocytes.Freshly prepared splenocytes viability was determined by trypan-blue exclusion method［26］.Cells that had viability more than 70%were used for the experiment purposes.

2.5.In vitro culture of lymphocytes

Lymphocytes were cultured according to the procedures described previously［26］.Freshly prepared spleen cells（mainly lymphocytes）weresuspendedinRPMI1640medium，supplemented with 10%heat-inactivated FCS，2 mmol/LL-glutamine，100 IU/mL penicillin G and 100μg/mL streptomycin. Lymphocytes（2.5×105cells/well）were plated in 96-well U-bottom plates（Nunc，Denmark）and cultured for 30 min at 37°C and 5%CO2containing airflow with humidified atmosphere. With the addition of 50μL 2-mercaptoethanol（concentration of 0.2 mmol/L）per well，the plate was incubated for 120 h at 37°C in the CO2incubator with/without the treatment with“Saribadi”or“Anantamul Salsa”and LPS.The plates were centrifuged，and supernatants were collected from each well and frozen at-30°C to perform IgM ELISA.After collecting the supernatants，the sediment cells pellets were used to perform MTT assay.

2.6.Measurement of lymphocytes proliferation by MTT method

The proliferation of lymphocytes in culture was measured by MTT method as mentioned earlier［27，28］.In brief，lymphocytes were cultured for 5 days as the condition mentioned above and 160μL supernatant was removed from each well.Freshly prepared RPMI/FCS medium （60μL）and MTT solution （25μL）were incorporated into each well and again incubated for 2 h.With the addition of stop solution（100μL/well），plates were again incubated overnight at 37°C in the dark environment.The absorbance was measured at 570 nm by using a plate reader（Bio-Rad Laboratories，USA）.

2.7.ELISA for the determination of IgM production

The IgM antibody production level was determined by a sandwich ELISA［29，30］.Briefly，96-well microtiter plate（Maxisorp，Nunc，Denmark）was coated with goat anti-mouse IgM （10μg/mL）forovernightincubationoftheplateinthedarkat4°C temperature.The plate was washed thrice with phosphate buffer saline containing 0.05%Tween 20（wash-buffer）.Blocking the plate wells with 1%bovine serum albumin for 2 h at room temperature，the platewas washed 3 times and 100μL/well of cultural supernatantsorstandard mouse-IgMwas added intoeach welland theplatewasincubatedatroomtemperaturefor2h.Afterwashing theplatesthricewithwashbuffer，50μLofhorseradishperoxidaseconjugated goat anti-mouse IgM（0.2μg/mL）was added into each well and the plate was incubated at room temperature for 1 h.The platewasagainwashedand 100μL of0.1mol/L citratebuffer（pH 4.0）containing 2.5 mmol/L 2，2′-azino-bis（3-ethylbenzthiazoline-6-sulfonic acid）and 0.17%H2O2was added into each well. Finally，theplatewasincubatedfor10minatroomtemperatureand the optical density（405 nm）was measured using an automatic plate reader（Bio-Rad Laboratories，USA）.

2.8.Statistical analysis of data

The experimental data were expressed as mean±SEM of three independent experiments.The differences between the control and treated groups were analyzed by Tukey honest significant difference test，Student's t-test and Dunnett's T3 test.P values less than 0.05 were considered as significant.

3.Results

3.1.PotentiationofpolyclonalIgMproductionby“Saribadi”and“Anantamul Salsa”

Figure 1.Effect of“Saribadi”and“Anantamul Salsa”on the production of IgM in cultural supernatants of murine lymphocytes.BALB/c female mice whole spleen cells（2.5×105cells/well）were cultured with the indicated concentrations of“Saribadi”（A）or“Anantamul Salsa”（B）at 37°C in the incubator having 5%CO2airflow for 5 days.The level of IgM produced in the cultured supernatants was determined by an ELISA. The data were mean±SEM of three independent experiments.*:P＜0.05；**:P＜0.01，as compared with the control（Dunnett's T3 test）.

Murine splenic cells were cultured with or without“Saribadi”or“Anantamul Salsa”at 37°C in a cell culture incubator for 5 days.The quantity of polyclonal IgM production in cultured supernatant was determined by an IgM-ELISA.LPS was used as a positive control.Our data（Figure 1A）showed that“Saribadi”at the doses of 0.25%，0.50%，0.75%，and 1.00%significantly enhanced the amount of polyclonal IgM production in treated lymphocytes compared to untreated cells.Although the 1.50% dose of“Saribadi”enhanced the production of IgM，the data were not statistically significant，and then declined.The highest enhancement of IgM production by“Saribadi”at a dose of 0.25%was found to be 3.65 times greater than that of control，whereas LPS（0.1μg/mL）caused 10.5 folds increment of IgM production.The concentration higher than 1.50%of“Saribadi”could not increase IgM level or those concentrations were toxic to the cells.Similarly，“Anantamul Salsa”at the doses of 0.50%，0.75%，1.00%and 1.50%significantly augmented the level of IgM production compared to untreated cells（Figure 1B）. “Anantamul Salsa”at the doses of 0.25%and 2.00%increased statistically insignificant levels of antibody productions but the doses 3.00%and 4.00%resulted in reduced production of IgM. The highest level of IgM production was observed by 0.50%of “Anantamul Salsa”which was found to be 1.86 times higher than the control.LPS（0.1μg/mL）promoted 8.4 folds IgM production comparing the untreated cells.

3.2.Inductionoflymphocytesproliferationby“Saribadi”and“Anantamul Salsa”

The lymphocytes collected from BALB/c female mice were cultured with/without different doses of“Saribadi”or “Anantamul Salsa”for 120 h and lymphocytes proliferation was determined by MTT method［25］.As shown in Figure 2A，“Saribadi”at concentrations of 0.25%，0.50%，0.75%and 1.00% significantly stimulated the proliferation of murine lymphocytes and the highest enhancement of proliferation was 2.43 times，exhibited by 0.50%dose of“Saribadi”.Although 1.50%dose of “Saribadi”slightly increased lymphocytes proliferation，2.00%，3.00%and 4.00%doses of the preparation were found to be cytotoxic as these doses remarkably reduced the number of lymphocytes in culture（Figure 2A）.On the other hand，lymphocytes proliferation was significantly increased by 0.50%，0.75%，1.00%，2.00%and 3.00%concentrations of“Anantamul Salsa”（Figure 2B）.Only the highest used dose of“Anantamul Salsa”（4%v/v）was found to be cytotoxic which drastically reduced the number of lymphocytes in culture.

3.3.Immunopotentialabilityof“Saribadi”and“Anantamul Salsa”was not deteriorated with polymyxin B treatment

We evaluated the endotoxin contamination（LPS incorporation in the preparations）possibility in“Saribadi”and“Anantamul Salsa”during the processing of the ingredients，in fermentation stage of manufacturing，etc.In order to exclude the endotoxin contamination possibility in“Saribadi”and“Anantamul Salsa”，the preparations were treated with polymyxin B （PMB）antibiotic during its incorporation in the culture of murine splenic cells.The investigation also resulted in a significant promotion of IgM production and lymphocytes proliferation by both“Saribadi”and“Anantamul Salsa”preparations in PMB-treated spleen cells as well（Figure 3A，B）.The amount of IgMproductionstimulatedbyPMB-untreated“Saribadi”（0.25%）was 1.1μg/mL，whereas PMB-treated“Saribadi”produced 0.8μg/mL of IgM.Both the PMB-untreated and-treated “Saribadi”produced significantly high levels of IgM comparing to the control（“Saribadi”-untreated）group.Production of IgM by“Saribadi”was reduced by 20.96%in PMB-treated group compared to PMB-untreated group and this reduction was statistically insignificant.Besides，the IgM production was also reduced（25.34%）in PMB-treated control group comparing to PMB-untreated control group（Figure 3A）.This comparison indicated that the slight reduction（20.96%）of IgM in PMB-treated“Saribadi”stimulated by cells was not due to the effect of PMB on“Saribadi”for the reduction of IgM production and it was only the effect of PMB itself on the cells.

In case of“Anantamul Salsa”，the production of IgM in PMB-treated“Anantamul Salsa”group was increased by 7.80%，instead of decreasing，comparing to PMB-untreated“Anantamul Salsa”group（Figure 3A）.This showed that the PMB treatment did not have any effect on the stimulating activity of“Anantamul Salsa”for the production of IgM.

Similarly，there was no significant difference in the activities of“Saribadi”and“Anantamul Salsa”for the proliferations of lymphocytesinPMB-untreatedandPMB-treatedgroups （Figure 3B）.The proliferation of lymphocytes was reduced by 20.00%，21.98%，and 9.38%in PMB-treated control，“Saribadi”and“Anantamul Salsa”groups，respectively.Thus，the data also clearlyindicatedthatthetreatmentof“Saribadi”and“Anantamul Salsa”by PMB had no influence on the activity of “Saribadi”and“Anantamul Salsa”for the proliferation of lymphocytes in culture.

Figure 2.Effect of“Saribadi”and“Anantamul Salsa”on the proliferation of murine lymphocytes in culture.BALB/c mice splenic lymphocytes（2.5×105cells/well）were incubated with the indicated concentrations of“Saribadi”（A）or“Anantamul Salsa”（B）at 37°C in the incubator having 5%CO2airflow for 5 days.The proliferations of cells were measured by MTT assay.The data were expressed as mean±SEM of three independent experiments.**:P＜0.01（increased level）；***:P＜0.001（increased level）；###:P＜0.001，as compared with the control（Tukey honest significant difference test）.

Figure 3.Evaluation of the contamination of“Saribadi”and“Anantamul Salsa”by bacterial endotoxin with the measurement of IgM production（A）and lymphocytes proliferation in the presence and absence of PMB.

BALB/c female mice lymphocytes（2.5×105cells/well）were treated with the optimum concentration of“Saribadi”（0.25%）and“Anantamul Salsa”（0.5%）with or without PMB at 37°C in the 5%CO2incubator for 5 days.A:Production of IgM in cultured supernatants was measured by an ELISA；B:The proliferations of cells were determined by MTT assay.The data were expressed as mean±SEM of three independent experiments（Student's t-test）.

4.Discussion

The increase in the quantity of antibody production is an indicator of the differentiation of B cells to antibody secreting plasma cells［1，2］.Thus，the present investigation demonstrated that“Saribadi” and“AnantamulSalsa” promotedthe differentiation of B cells to plasma B cells.Although the production of antibody was promoted by“Saribadi”and “Anantamul Salsa”at lower concentrations（0.25%-2.00%），the higher doses（3.00%and 4.00%）of the preparations were found to suppress the productions of IgM，suggesting that the preparations may contain an inhibitory substance which is effective at higher concentrations.

Increasing proliferation of cells is an indicator of immunostimulation［1］.Similartoantibodyproductionmanner，“Saribadi”at higher doses（＞2.00%v/v）were observed to be cytotoxic，whereas“Anantamul Salsa”dose（4.00%）was found to be cytotoxic，which suppressed the proliferation of lymphocytes.In both the cases of the evaluation of“Saribadi”and“Anantamul Salsa”，the LPS at the concentration of 0.1μg/mL could not significantly stimulate the proliferation of lymphocytes.

PMB binds to the lipid portion of bacterial LPS and thus inhibits the activity of LPS［26，31］.Thus，our results demonstrate that“Saribadi”and“Anantamul Salsa”did not contain bacterial LPS as contaminant.Therefore，it can be concluded that the immunostimulatingabilityof“Saribadi”and“Anantamul Salsa”on murine lymphocytes was not due to the presence of LPS，rather because of the active chemical（s）existing in the preparations.

Thus，the immunopotential property of“Saribadi”and “Anantamul Salsa”was supposed due to the presence of active constituent（s）from any of the plant materials having immunostimulatory or immunomodulatory properties or may be due to the combined immunostimulatory activities or synergistic effect of the immunostimulatory plant ingredients used in the preparations of“Saribadi”and“Anantamul Salsa”.

A strong immune system is very much essential to protect our body from the generation of several diseases such as infections and cancer.An attenuated immune system is highly susceptible to infections and cancers.Natural immunopotentiating agents are believed to be promising means for the prevention and treatment of many diseases that occur due to insufficient immunity［8，32］.Therefore，immunostimulants have very good demand to improve the impaired immunity.

“Saribadi”and“Anantamul Salsa”are“Asava”（fermented infusion）products which are prepared through fermentation process mediated by microorganisms.Classical literature indicates that microorganisms involved in“Asava”fermentation not only mediate the process but also involve microbial biotransformation of the initial“Asava”ingredients into more effectivetherapeuticend-products［25］.Bettertherapeutic properties can also be obtained due to the improvement of the extraction of drug molecules from the herbs by alcoholaqueous milieu which is also produced by microbes［25］.In addition，microbial metabolic products may be included in the final product prepared by“Asava”.Such kind of microbial chemical contribution may result in the therapeutic activity of theproduct.Hence，theimmunostimulatingactionof “Saribadi”and“Anantamul Salsa”found in this investigation may also be due to the metabolic products of microbes involvedinthefermentation， orunknownchemical substance（s）formedbecauseoftheintimatecontactof microbeswiththeherbalingredients.However，more exhaustive investigations are needed to substantiate the claim.

From our preliminary findings，it can be concluded that Ayurvedic herbal preparations，“Saribadi”and“Anantamul Salsa”possess immunopotentiating activity due to its capability on enhancing antibody production and lymphocytes proliferation.These two preparations may be useful for the up-regulation of immune responses and/or in case of impaired immunity to combat infections.However，as this is a preliminary study report，further details in vitro studies on the effects of those two herbal preparations on macrophage，natural killer cells，T-cells，cytokines［tumor necrosis factor-α，interferon-γ，interleukin （IL）-1β，IL-2，IL-4，IL-6，IL-12］productions as well as in vivo humoral and cellular immunological investigations are required to measure its actual immunomodulating potential and molecular mechanism of actions.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors are grateful to Prof.Eiichi Gohda，Department of Immunochemistry，F(xiàn)acultyofPharmaceuticalSciences，Okayama University Japan，for his valuable suggestions，guidance as well as arrangement of laboratory facilities to conduct this research.The authors would like to express their gratitude to Ministry of Higher Education and Universiti Teknologi MARA，Malaysia for the financial support［Grant No.LESTARI:600-RMI/DANA 5/3/LESTARI（52/2015）］.

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*Correspondingauthor:Md.MoklesurRahmanSarker，DepartmentofPharmacology，F(xiàn)acultyofPharmacy，LincolnUniversityCollege，47301，PetalingJaya，Selangor，Malaysia.
Tel:+601133432919
Fax:+603 7806 3479
E-mail:moklesur2002@yahoo.com
The experiments concerned with the use of mice were approved by the ethical approval of Animal Research Control Committee of Okayama University.
Foundation Project:Partially Supported by Ministry of Higher Education and Universiti Teknologi MARA，Malaysia（Grant No.LESTARI:600-RMI/DANA 5/3/ LESTARI（52/2015））.
Peer review under responsibility of Hainan Medical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.