Home | About PM | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |  Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 68  |  Page : 13-21  

Immunomodulatory effect of Kaempferia parviflora against cyclophosphamide-induced immunosuppression in Swiss albino mice


1 Department of Plantation Crops and Spices, COH, Kerala Agricultural University, Thrissur, Kerala, India
2 Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur, Kerala, India
3 Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Thrissur, Kerala, India

Date of Submission29-May-2019
Date of Decision19-Jun-2019
Date of Web Publication31-Mar-2020

Correspondence Address:
Bibu John Kariyil
Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_233_19

Rights and Permissions
   Abstract 


Background: Kaempferia parviflora Wall. Ex. Baker (Black ginger) is an important medicinal plant used as a health-promoting tonic i.e., both a stimulant and a vitalizing agent in Thailand. Research findings are available on its aphrodisiac and anti-inflammatory activities. So far, there is no research work conducted on immunomodulatory effect of K. parviflora. Objectives: The present study was carried out to evaluate the immunomodulatory effect of ethanolic extract of rhizomes of K. parviflora in cyclophosphamide-induced immunosuppression in Swiss albino mice. Materials and Methods: Immunomodulatory status was assessed by physiological, hematological, biochemical, and histopathological observations. The weight of organs such as liver and spleen was recorded at the time of sacrifice. Gas chromatography mass spectrophotometry (GC-MS) analysis was performed for profiling compounds present in the extract. Results: Significant increase in body weight was observed on 12th day in K. parviflora-treated immunosuppressed mice. In hematological parameters, there was significantly higher lymphocyte count for K. parviflora-treated immunosuppressed mice. In hemagglutination test, conducted for the evaluation of humoral immune response, both K. parviflora alone and K. parviflora-treated immunosuppressed mice showed significant increase in titer value compared with cyclophosphamide control. Bone marrow cellularity test performed for evaluation of cellular immune response showed cyclophosphamide control group with significant lower bone marrow cellularity on 12th and 19th day while K. parviflora alone-treated and K. parviflora-treated immunosuppressed mice showed a significant increase in the bone marrow cellularity. The result of histopathology of spleen revealed to prevent the depletion of red pulp and white pulp on 12th day, and this prevention was marked on 19th day. GC-MS profiling showed that the extract contained eight compounds. Majority of the compounds belong to flavonoids class which might have helped in immunomodulation. Conclusion: The results of the present study revealed that the test extract possessed promising immunomodulatory activity.

Keywords: Biochemical, cellular response, gas chromatography mass spectrophotometry, hematological, humoral response, Kaempferia parviflora


How to cite this article:
Devi AR, Kariyil BJ, Raj N M, Akhil G H, Balakrishnan-Nair DK. Immunomodulatory effect of Kaempferia parviflora against cyclophosphamide-induced immunosuppression in Swiss albino mice. Phcog Mag 2020;16, Suppl S1:13-21

How to cite this URL:
Devi AR, Kariyil BJ, Raj N M, Akhil G H, Balakrishnan-Nair DK. Immunomodulatory effect of Kaempferia parviflora against cyclophosphamide-induced immunosuppression in Swiss albino mice. Phcog Mag [serial online] 2020 [cited 2020 Aug 6];16, Suppl S1:13-21. Available from: http://www.phcog.com/text.asp?2020/16/68/13/281686



SUMMARY

  • The present study revealed the administration of ethanolic extract of Kaempferia parviflora boosted the immune response in in vivo experiment in Swiss albino mice
  • In terms of increase in body weight and increase in relative organ weight
  • The plant extract increased body weight, relative organ weight and increased hematological parameters, namely total leukocyte and differential leukocyte counts, increase in titer value in hemagglutination test
  • The plant extract increased the number of bone marrow cells count, serum protein and globulin
  • Attenuation of cyclophosphamide-induced depletion of red pulp and white pulp in histopathology of spleen and the significant increase in footpad thickness of K. parviflora alone-treated group and K. parviflora-treated cyclophosphamide immunosuppressed group when compared with cyclophosphamide control was also observed.




Abbreviations used: GC-MS: Gas chromatography mass spectrophotometry; SRBC: Sheep Red Blood Cells; PBS: Phosphate buffer saline; HA: Hemagglutination assay; DTH: Delayed-type hypersensitivity; HSC - hematopoietic stem cells.


   Introduction Top


The medicinal Kaempferia species are the rhizomatous herbs belonging to the family Zingiberaceae. Kaempferia parviflora Wall. Ex Baker, popularly known as black ginger or Thai ginger, is indigenous to the northeastern part of Thailand. The herb is locally known as Krachaidum in Thailand and as Khongban Takhellei in Manipur.[1] The plant is about 30–40 cm tall. The number of leaves varies from 1 to several; blades are ovate or oblong shape and slightly unequal sided, upper surface are yellow green and the lower surface are green in colour. The rhizome is subglobose with several succulent roots in a fascicle. It has brownish skin with purple color interior flesh. Its inflorescence is enclosed by two innermost leaf sheaths.[2] Rhizomes of K. parviflora have been used as traditional medicine for rectifying male impotence, body pains, and gastrointestinal disorders among local people in the Northeast of Thailand.[3] In India, it has been reported to occur in the tropical evergreen forest of Imphal East district of Manipur.[1]

In Thailand, K. parviflora is well known as an energy enhancer with exceptional tonic effect. Fresh or dried rhizomes and dried powder in tea bag and wine are various products used by inhabitants of Thailand. Alcoholic infusion of K. parviflora rhizome has been used as a tonic for body pains and gastrointestinal disorders.[4] Rhizome is reported to have antimicrobial, aphrodisiac, antigastric ulcer, antidepressant, anticholinesterase activity, antiobesity, vasodilation, and antioxidant effect. Traditional medicines using K. parviflora are permitted by Thai Food and Drug Administration which include capsules, pills, tablets, powders, and essence tincture.[5] Various in vivo experiments in the test animals using K. parviflora extract showed reduction in obesity, diabetes Type II, cardiovascular disease, and inflammatory activity.[6],[7]

There are reports available on the effect of K. parviflora in strengthening the body in general and as sexual stimulant by the consumption of rhizome extracted with alcohol.[8] Increased whole-body energy expenditure in healthy men by the ethanolic extract may be useful as an antiobesity regimen.[9] Wattanathorn et al. also reported about the enhanced male sexual behaviors in aging rats by the administration of crude extract of this plant.[10] Another study in male mice showed that K. parviflora improved physical fitness performance and muscular endurance.[11] It also acts as modulator of multidrug resistance in cancer cells.[12] Adaptogenic activities of K. parviflora has been reported in mice.[13] All these studies indicate the general health-promoting effect of K. parviflora.

Many in vitro studies have shown anticancer activities of K. parviflora extract, i.e., K. parviflora extract was cytotoxic to SKOV3 cells,[14] 5a-reductase (5aR),[15] and human cholangiocarcinoma cell lines (HuCCA-1 and RMCCA-1).[16] Recent findings showed that K. parviflora rhizomes extract contained numerous flavonoids,[17] which was previously reported to possess antioxidant activity.[18],[19] Plants which possessed anticancer and antioxidant properties are reported to have immunomodulatory property.[20],[21]

The major components of K. parviflora volatile oil are α-copaene, dauca-5, 8-diene, camphene, β-pinene, borneol, and linalool while the hexane extract showed germacrene D, β-elemene, α-copaene, and E-caryophyllene as major constituents.[21],[22]

Even though cyclophosphamide is a widely used as an alkylating drug for the treatment of various types of cancers such as lymphoma, myeloma, and chronic lymphocytic leukemia, it is having effective immunosuppressive action which cross links the DNA of actively dividing cells thereby inhibiting the both cellular and humoral response immunity.[23] Cyclophosphamide can be used as immunosuppressive agent to study the immunomodulatory effects of plant extracts by various researchers.[24],[25]

Precise information on the immunomodulatory properties are lacking for K. parviflora. Hence the present study was carried out to evaluate the immunomodulatory effect of ethanolic extract of the rhizome of K. parviflora in cyclophosphamide-induced immunosuppression model in Swiss albino mice.


   Materials and Methods Top


Plant material

The rhizomes were collected from the field experiments conducted at field of the Department of Plantation Crops and Spices, Kerala Agricultural University, Vellanikkara. Identification was done by Dr. A. A. Mao (Scientist E), Botanical Survey of India, Eastern Regional Centre, Shillong. A herbarium was prepared, and voucher specimen with accession no. HERB/VPT/CVASMTY/3/2019 was deposited at Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Mannuthy.

Preparation of extracts

The rhizomes were shade dried followed by course pulverization using mechanical pulverizer. Plant extract was obtained using 95% ethanol by soxhlet extraction. The extract was then dried using rotary evaporator.

Experimental design

Experiment was conducted at Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Mannuthy. The experiment protocol was approved by Institutional Animal Ethics Committee of College of Veterinary and Animal Sciences, Mannuthy (Order no. IAEC/CVASMTY 4/17-18). Swiss albino mice, procured from Small Animal Breeding Station, Mannuthy were randomly divided in two sets of 48 each. First set of animals (Group A) were used for physiological, hematological, biochemical, and bone marrow cellularity tests with four subgroups (AI, AII, AIII, and AIV) of 12 animals each. Second set of animals (Group B) were used for delayed hypersensitivity with four subgroups (BI, BII, BIII, and BIV) of 12 animals each. Sheep Red Blood Cells (SRBC) antigen (1 × 108 cells/mL/100 Kg BW) were injected i. p. to mice of all the groups on 5th day except AI and BI. The experiment protocol is illustrated in [Table 1].
Table 1: Experiment protocol

Click here to view


Immunization

Blood was collected from the sheep maintained within the University Sheep and Goat farm, Mannuthy, in equal volume of Alsever's solution following sterile procedure. This was used for antigen preparation and stored at 4°C until used.

Measurement of physiological parameters

The weight of individual mouse was recorded before, during (on 12th day), and at the end (19th day) of the experiment. The weight of the organs such as spleen and liver were conjointly recorded at the time of sacrifice.

Measurement of hematological parameters

Blood samples were collected from submaxillary vein of all the mice from group AI to AIV on zero, 12th, and 19th day of the experiment. To prevent variations, blood samples were placed in ethylene diamine tetraacetic acid tubes for analysis.[26] Hematology analyser, model-Mythic 18 Vet (Orphee, Switzerland) was used for the analysis of total and differential leukocyte count.

Measurement of biochemical parameters

Blood collected from submaxillary vein of mice from group AI to AIV was taken test tube without anticoagulant, centrifuged at 2500 rpm for 10 min. Serum was separated for the estimation of protein, albumin, and hemagglutination test. Biochemical analysis was done using Semi-Automated Biochemical Analyser, model-Master T (Hospitex, Italy).

Measurement of immunological parameters

Hemagglutination test

Hemagglutination test was performed for the evaluation of cell-mediated immune response. The blood was collected from mice in the groups AI to AIV on zero, 12th, and 19th day of the experiment. Two-fold dilutions of sera were prepared in 0.15 M phosphate buffer saline (PBS) (PH 7.2), and 50 μl of each dilution was transferred into 96 well microtiter plates. Twenty-five microliter quantity of fresh one per cent Sheep SRBC suspension in PBS was added into each well and mixed thoroughly. Thereafter, they were incubated at 37°C for 1 h. The reciprocal of highest dilution of the test serum giving 50% agglutination had been expressed as hemagglutination assay titer.[27]

Bone marrow cellularity

Bone marrow cellularity was done for the evaluation of humoral immune response. Femurs of both the hind legs of the mice were dissected, and the condyles of the femurs were removed using sharp scissors.[28] Then, the bone marrow was flushed with 5 mL of 10% fetal bovine serum. The number of cells was counted hemocytometrically using invitrogen countess automated cell counter on 12th day and 19th day after sacrificing the animals.

Histopathology of spleen

On 12th and 19th days, the spleen of the animal was removed for histopathology. The tissue samples were fixed at 10% formalin and embedded in paraffin and the sections were stained using hematoxylin and eosin.[29] It was observed for histopathological lesions.

Delayed type hypersensitivity

Six mice from each groups of BI, BII, BIII, and BIV were primed with SRBC antigen i. p. on day 5 and was then challenged on day 12 with SRBC antigen s. c. on the right hind footpad. The left hind foot pad received 0.025 mL of saline alone. The footpad swelling was measured at three different dimensions using Vernier calipers after 24 h of challenge. The difference in footpad thickness was taken as a measure of delayed-type hypersensitivity. The test was repeated on 19th day in the remaining six mice of each group.[30]

Gas chromatography mass spectrophotometry analysis

The active phytochemical principles of K. parviflora was analyzed using Shimadzu gas chromatography mass spectrophotometry (GC-MS) (Model Number: QP2010S), Kerala Forest Research Institute, Peechi, Thrissur, Kerala. The oven temperature was maintained at 70°C for 2 min and then increased to 200°C in 5 min. The injector temperature was 260°C, and total analysis time was 50 min. One microliter aliquots of extracts were injected into the chromatographic capillary column of length 30 m, inner diameter 0.25 mm, and film thickness 0.25 μm after a clear baseline had been obtained. Major constituents were identified using mass spectrum NIST 11 and WILEY.

Statistical analysis

Analysis of covariance was done for hematological parameters and two factor analysis for organ weight and biochemical parameters. P < 0.05 was considered to be significant.


   Results Top


Body weight

The relative change in body weight from 0th to 12th day and 12th to 19th days are given in [Table 2]. There was significant increase in body weights on 12th day of experiment in group AI, AIII, and AIV. There were significant reduction in body in group AII in both the first six set of animals and second six set of animals. The body weights of mice in K. parviflora- and cyclophosphamide-treated group (AIV) showed a significant (P < 0.05) increase on 12th day of the experiment compared with cyclophosphamide control (AII). The highest relative increase in body weight was recorded on AIII with mean value of 9.074 ± 0.66 g while the relative decrease in body weight was lowest in AII with mean value of −6.276 ± 0.41 g.
Table 2: Effect of ethanol extract of Kaempferia parviflora in relative change in body weight in cyclophosphamide immunosuppressed Swiss albino mice, g

Click here to view


Organ weight

The weight of internal organs such as spleen and liver were taken on 12th and 19th day of the experiment and expressed as relative organ weights [Table 3]. There was no significant difference in liver weight on 12th and 19th day. On 12th day, both K. parviflora alone treated (AIII) and K. parviflora treated immunosuppressed animals (AIV) showed significant increase in spleen weight compared with normal control (AI) and cyclophosphamide control (AII). K. parviflora treated immune suppressed animals (AIV) showed significantly higher spleen weight on 19th day.
Table 3: Effect of ethanol extract of Kaempferia parviflora on the liver and spleen weight in cyclophosphamide immunosuppressed Swiss albino mice, g/100

Click here to view


Hematological parameters

Total leukocyte count recorded on zero, 12th, and 19th day of the experiment is presented in the [Table 4]. On 12th day, K. parviflora-treated immunosuppressed group showed significantly higher leukocyte count compared with cyclophosphamide- and normal-treated groups. On 19th day, significant increase in leukocyte count was observed in AIII and AIV. The lymphocyte count showed significant difference on 12th and 19th day of the experiment [Table 5]. On 12th day, AIV showed significant higher value compared with AI and AII. On 19th day, AIII and AIV showed significant increase in lymphocyte count compared with AII.
Table 4: Effect of ethanol extract of Kaempferia parviflora on the total leukocyte count in cyclophosphamide immunosuppressed Swiss albino mice, 103/μl

Click here to view
Table 5: Effect of ethanol extract of Kaempferia parviflora on the lymphocyte, monocyte and neutrophil in cyclophosphamide immunosuppressed Swiss albino mice

Click here to view


There was no significant difference in monocyte count on 12th day of the experiment. K. parviflora-treated immunosuppressed group showed significant increase in monocyte count on 19th day of the experiment as compared with cyclophosphamide control [Table 5]. There was no significant difference in neutrophil count on 12th day of the experiment. Neutrophil was found significantly highest in AII on 19th day [Table 5].

Immunological parameters

Hemagglutination titer was taken on 0th, 12th, and 19th day [Table 6]. Both AIII and AIV showed significant increase in titer value on 12th day with titer value of 938.667 and 938.667 respectively.
Table 6: Effect of ethanol extract of Kaempferia parviflora on the hemagglutination titer in cyclophosphamide immunosuppressed Swiss albino mice

Click here to view


Cyclophosphamide control group (AII) showed significant lower bone marrow cellularity on 12th and 19th day compared with normal control (AI). Both AIII and AIV showed significantly higher value compared with AI and AII on 12th and 19th day [Table 7].
Table 7: Effect of ethanol extract of Kaempferia parviflora on the bone marrow cellularity in cyclophosphamide immunosuppressed Swiss albino mice, millions

Click here to view


Biochemical parameters

Total protein and globulin

Total protein was observed lowest in AII on 12th and 19th day [Table 8]. There were significant increase in total protein in K. parviflora alone-treated (AIII) and K. parviflora-treated immune suppressed mice (AIV) when compared with cyclophosphamide control (AII). There was no significant difference in globulin content. On 12th day, globulin content was highest in AIV.
Table 8: Effect of ethanol extract of Kaempferia parviflora on the total protein and globulin in cyclophosphamide immunosuppressed Swiss albino mice, g/dl

Click here to view


Histopathology of spleen

Marked lymphoid depletion was observed in white pulp and marginal zone of spleen in cyclophosphamide-induced mice as compared with normal control animals [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Administration of K. parviflora induced hyperplasia in white pulp region [Figure 5] and [Figure 6]. However, administration of K. parviflora along with cyclophosphamide-induced mice revealed attenuated lymphocyte depletion on 12th day and marked attenuation of lymphocyte depletion on 19th day [Figure 7] and [Figure 8].
Figure 1: Histopathology of spleen of Swiss albino mice in control group receiving vehicle at 12th day. Light microscopic image of H and E-stained sections of spleen revealed red pulp and white pulp packed with lymphocytes (×100)

Click here to view
Figure 2: Histopathology of spleen of Swiss albino mice in cyclophosphamide alone-treated group at 12th day. Light microscopic image of H and E-stained sections of spleen. Depletion of lymphocytes in white pulp and marginal zone was observed (×100)

Click here to view
Figure 3: Histopathology of spleen of Swiss albino mice in control group receiving vehicle at 19th day. Light microscopic image of H and E stained sections of spleen. Red pulp and white pulp packed with lymphocytes (×100)

Click here to view
Figure 4: Histopathology of spleen of Swiss albino mice in cyclophosphamide alone-treated group at 19th day. Light microscopic image of H and E-stained sections of spleen. Severe depletion of lymphocytes in white pulp and marginal Zone was noted (×100)

Click here to view
Figure 5: Histopathology of spleen of Swiss albino mice in Kaempferia parviflora alone-treated group at 12th day. Light microscopic image of H and E-stained sections of spleen revealed white pulp and red pulp with proliferation of lymphocytes (×100)

Click here to view
Figure 6: Histopathology of spleen of Swiss albino mice in Kaempferia parviflora alone-treated group at 19th day. Light microscopic image of H and E stained sections of spleen. White pulp revealed hyperplasia of lymphocytes with new germinal centre development (×100)

Click here to view
Figure 7: Histopathology of spleen of Swiss albino mice in Kaempferia parviflora-treated cyclophosphamide immunosuppressed group at 12th day. Light microscopic image of H and E-stained sections of spleen. The plant extract attenuated the cyclophosphamide-induced lymphocyte depletions in white pulp and red pulp region (×100)

Click here to view
Figure 8: Histopathology of spleen of Swiss albino mice in Kaempferia parviflora-treated cyclophosphamide-immunosuppressed group at 19th day. Light microscopic image of H and E stained sections of spleen. Attenuation of cyclophosphamide-induced lymphocyte depletion was noted (×100)

Click here to view


Delayed type hypersensitivity

The increased in foot pad thickness of all the experimental animals were observed on 12th and 19th day [Table 9]. Both BIII and BIV showed significant increase in footpad thickness on 12th and 19th day.
Table 9: Effect of ethanol extract of Kaempferia parviflora on the foot pad thickness, mm

Click here to view


Gas chromatography mass spectrophotometry analysis

Phytoconstituents obtained on GC-MS analysis of K. parviflora is listed in [Table 10] and [Figure 9]. A total of eight compounds in ethanolic extract were identified. The majority of the compounds detected were belong to flavonoids and fatty acids. The major compound found were dimethylchrysin (74.43%) and techtochrysin (8.79%).
Table 10: Gas chromatography mass spectrophotometry analysis of ethanolic extract of Kaempferia parviflora

Click here to view
Figure 9: Chromatogram of gas chromatography mass spectrophotometry analysis of ethanolic extract of Kaempferia parviflora

Click here to view



   Discussion Top


In the present study, SRBC antigen (1 × 108 cells/mL/100 Kg BW) were injected i. p. to mice of all the groups on 5th day except AI and BI. The dose was selected as per Tizard.[31] Two hundred milligram per kilogram of ethanolic extract was administered to mice for 19 days. The dose was selected as per the toxicity study (unpublished data). The toxicity studies of K. parviflora was conducted and found that the plant extract was not toxic at 2000 mg/Kg. Again, reports are available on the administration of dose of 200 mg/Kg body weight to Swiss albino mice in in vivo studies of the immunomodulatory effects of methanol leaf extract of Gymnema sylvestre,[32] aqueous and ethanolic extract of dried tuber of Eulophia nuda.[33] Acute and chronic toxicity studies of, acute and chronic toxicity study of Kaempferia parviflora powder was reported by Chivapat et al.[34] There is a thumb rule that a safe upper limit for selecting a dose is 10% of LD50, when no experimental data existed.[35] Considering the cited reports and thumb rule, we have selected the dose of 200 mg/Kg.

Swiss albino mice, used in the study, are immunocompromised mice. Hence, to study the immunomodulatory effect of plant extracts, Swiss albino mice are the best animal models. Many researchers have used Swiss albino mice to study the immunomodulatory effect.[24],[25]

The first six animals in each group were sacrificed on 12th day and the remaining six were sacrificed on 19th day. The study design which included the sacrifice at day 12 and 19 was to assess the humoral immune response by hemagglutination test and cellular immune response by evaluation of bone marrow cellularity. For the evaluation of humoral and cellular immune response, the sacrifice at day 12 and day 19, respectively, is required.

The body weight of the animals and blood parameters like lymphocyte, neutrophils and monocytes may be influenced by external factors such as feed and water. Hence, to know the effect of the drug alone on body weight and blood parameters excluding the influence of external factors, the initial body weight and initial day blood parameters were used as a covariate to eliminate the variations due to influence of initial body weight and blood parameters.[36] Standard error (SE) were calculated separately for each group. We used SE for our data because we are comparing the different groups using mean value and the variation in the mean values is expressed as SE The sample size that we have taken is an estimate of population and hence the estimated variation in mean is expressed as SE. Standard deviation (SD) represents variations in the sample observations while SE represents the variations in the estimate of the population mean. Here, in the study analysis, we have compared the estimate of the population mean, and hence, we have used SE instead of SD.[36]

Body weight and organ weight

In the present study, there were significant increase in body weight on 12th day and 19th day for all the groups except compared with AII. The increase in body weight was noticed in plant extract-treated cyclophosphamide-induced immunosuppressed mice.[37] The increased in body weight may be due to better feed utilization.[38] Reduction in body weight was observed in cyclophosphamide-treated group in Swiss albino mice.[23]

K. parviflora alone and K. parviflora-administered cyclophosphamide-treated immunosuppressed group showed higher relative liver weight when compared with cyclophosphamide control group. Liver is the important organ that responds immediately to any antigen. Relatively higher liver weight when compared with cyclophosphamide control, and similar to that of normal control showed the restoration of normal activity of the liver.[38] Immunocompetency can be viewed as a slight increase in spleen weight,[39],[40],[41] and this increase in spleen weight was recorded in K. parviflora alone, and K. parviflora administered cyclophosphamide-treated immunosuppressed group.[38],[42]

Hematological parameters

In the study, a significant increase in leukocyte count was observed in K. parviflora alone and K. parviflora administered cyclophosphamide treated immunosuppressed group. Similar works have also shown that there is significant increase in leukocyte count of plant treated cyclophosphamide induced immunosuppressed mice.[43],[44] Increased in white blood cell count could be viewed as an important contributing factor in reducing the risk of various diseases.[45]

The major innate of immune cells are phagocytes and lymphocytes and increase in lymphocytes show immunostimulatory effect.[23],[46] In the present study, an increased lymphocyte count was observed in K. parviflora alone and K. parviflora administered cyclophosphamide-treated immunosuppressed mice.[23],[46]

Monocyte count was significantly higher in K. parviflora-treated immunosuppressed mice when compared with cyclophosphamide control. The innate immune response is strengthened by increase in monocytes.[47] Thus, it can be concluded that K. parviflora may have direct stimulating effect on myeloid progenitor cells.

In the present experiment, neutrophil count was found significantly highest in cyclophosphamide control group. The neutrophilia in the cyclophosphamide control group might be a compensatory mechanism to the drop in lymphocyte count observed. Concomitant administration of cyclophosphamide along with plant extract showed a significant decrease in neutrophil count comparable with that of normal group indicating the enhanced efficacy of plant extract in preventing cyclophosphamide induced neutrophilia. Increase in neutrophil count is attributed to marginalization of phagocytic cells, i.e. improved defensive response under normal circumstances as explained by Sultana et al.[44] It can be concluded that the plant extract stimulated hematopoietic system by increasing the lymphocytes and decreasing the neutrophil counts.[38]

Immunological parameters

Hemagglutination test

Both AIII and AIV showed significant increase in titer value while the lowest value was recorded recorded in cyclophosphamide treated group. Increase in proliferation and transformation of B lymphocytes in plasmocytes might increase in antibody titer.[48] The result of the present study suggested the increased immune response when treated with K. parviflora.

Bone marrow cellularity

The increase in number of bone marrow cells shows the effect of K. parviflora on enhancing immunological response. Bone marrow is a foremost hematopoietic organ. Bone marrow hematopoietic stem cells (HSC) can self-duplicate, proliferate and differentiate to the subordinate cells of positive lineage. Drugs and radiation could damage all the systems, organs and hematopoietic tissues of the whole body. Thus, there was depletion in HSC leading to depletion of mature hematopoietic cells due to administration of cyclophosphamide. The marked increase in bone marrow cell count on administration of K. parviflora is an indication of its immunomodulatory property. The administration of chyawanprash and brahma rasayana showed the enhanced level of bone marrow cellularity indicating that hematopoietic cells was stimulated and differentiated.[41] Cyclophosphamide potentially affect the bone marrow cellular production. However, K. parviflora extract was found to have a protective effect in cyclophosphamide induced immunosuppression in Swiss albino mice used in the present study.[49]

Biochemical parameters

Serum proteins and globulins are one of the indicators for altered immune status of the individual. So also, serum proteins have role in maintaining homeostasis and resistance to infections.[50] The result on the significantly higher serum protein in K. parviflora alone-treated and K. parviflora-treated immunosuppressed mice showed the higher immune response of the plant which might have been contributed by the higher values in terms of immunoglobulins and other humoral factors.[51]

Globulins are principally responsible for both the natural and acquired immunity that an individual has against invading organism.[45] The increase in the level of globulin on K. parviflora alone-treated mice indicates the antimicrobial action of this plant.

Histopathology of spleen

Depletion of lymphoid cells was observed in white pulp and marginal zone region in cyclophosphamide group. Similar works were observed in experiments conducted by other authors showing immunosuppression.[52] The proliferation of lymphocytes in the white pulp and red pulp in K. parviflora alone-treated group and attenuation of cyclophosphamide-induced lymphoid depletion in K. parviflora-treated cyclophosphamide immunosuppressed group confirm the immunomodulatory effect of K. parviflora.[52],[53]

Delayed type hypersensitivity

K. parviflora alone treated and K. parviflora treated immunosuppressed mice showed significant increase in foot pad thickness when compared with cyclophosphamide control group. One of the parameter to measure cell mediated immune response is to study the delayed type hypersensitivity in animal model, in which foot pad thickness is measured. Various experiments conducted in animal models had revealed that increase in foot pad thickness indicated cell mediated immunity.[54],[55] In the present study, the increased food pad thickness indicated that K. parviflora boosted the cell mediated immune response.[56]

Gas chromatography mass spectrophotometry analysis

The major compounds present in the extract were dimethylchrysin, techtochrysin, benzaldehyde, (diphenylmethylidene) hydrazine and tri-o-methylapigenin. The same compounds were previous reported by various authors.[3],[17] Tri-o-methylapigenin (synonym: 4', 5, 7-trimethoxyflavone) was reported to have antiplasmodial, antifungal, and antimycobacterial activity.[3] Techtochrysin, a methoxyflavone, was found to induce skeleton muscle hypertrophy.[57] Flavonoids isolated from different plant sources showed immunomodulatory effect.[58],[59] Thus, it could be concluded that the flavonoids such as dimethylchrysin, techtochrysin, tri-O-methylapigenin, 2-(4-hydroxy-3-methoxyphenyl)-3,7-dimethoxy-4 h-chromen-4-one might be responsible for the immunomodulatory activity.


   Conclusion Top


The study showed that administration of ethanolic extract of K. parviflora boosted the immune response in in vivo experiment conducted in Swiss albino mice. Further studies need to be explored to elucidate the exact mechanism of immunomodulatory effect of K. parviflora.

Acknowledgements

The authors would like to thank the authorities of College of Horticulture, Kerala Agricultural University, Thrissur for providing research grant for the conduct of this experiment. Authors would also like to thank the authorities of College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur for providing all facilities to carry out the research.

Financial support and sponsorship

Ph.D research grant to the first author with order No. R7/63695/17 dt. 15.07.2017 of Director of Research, Kerala Agricultural University. There is no sponsorship.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Devi NB, Das AK, Singh PK. Kaempferia parviflora (Zingiberaceae): A new record in the flora of Manipur. Int J Innov Sci Eng Technol 2016;3:661-5.  Back to cited text no. 1
    
2.
Catherine DL, Thohirah LA, Johnson S, Nur Ashikin PA, Maheran AA. Morphological description for kunyit hitam (Kaempferia parviflora) and breaking bud dormancy with BAP and ethephon treatments. Trans Malays Soc Plant Physiol 2014;22:139-41.  Back to cited text no. 2
    
3.
Yenjai C, Prasanphen K, Daodee S, Wongpanich V, Kittakoop P. Bioactive flavonoids from Kaempferia parviflora. Fitoterapia 2004;75:89-92.  Back to cited text no. 3
    
4.
Banjerdpongchai R, Suwannachot K, Rattanapanone V, Sripanidkulchai B. Ethanolic rhizome extract from Kaempferia parviflora wall. Ex. Baker induces apoptosis in HL-60 cells. Asian Pac J Cancer Prev 2008;9:595-600.  Back to cited text no. 4
    
5.
Wattanasri P. Development of Microemulsions, Microemulgels and Organogels for Transdermal Delivery of. Kaempferia parviflora Extract. Master of Pharmacy Program in Pharmaceutical Sciences. Silpakorn University; 2016.  Back to cited text no. 5
    
6.
Yorsin S, Kanokwiroon K, Radenahmad N, Jansakul C. Effects of Kaempferia parviflora rhizomes dichloromethane extract on vascular functions in middle-aged male rat. J Ethnopharmacol 2014;156:162-74.  Back to cited text no. 6
    
7.
Sae-wong C, Tansakul P, Tewtrakul S. Anti-inflammatory mechanism of Kaempferia parviflora in murine macrophage cells (RAW 264.7) and in experimental animals. J Ethnopharmacol 2009;124:576-80.  Back to cited text no. 7
    
8.
Maneenoon K, Khuniad C, Teanuan Y, Saedan N, Prom-In S, Rukleng N, et al. Ethnomedicinal plants used by traditional healers in Phatthalung province, Peninsular Thailand. J Ethnobiol Ethnomed 2015;11:43.  Back to cited text no. 8
    
9.
Matsushita M, Yoneshiro T, Aita S, Kamiya T, Kusaba N, Yamaguchi K, et al. Kaempferia parviflora extract increases whole-body energy expenditure in humans: Roles of brown adipose tissue. J Nutr Sci Vitaminol (Tokyo) 2015;61:79-83.  Back to cited text no. 9
    
10.
Wattanathorn J, Pangphukiew P, Muchimapura S, Sripanidkulchai K, Sripanidkulchai B. Aphrodisiac activity of Kaempferia parviflora. AmJ Agric Biol Sci 2012;7:114-20.  Back to cited text no. 10
    
11.
Toda K, Hitoe S, Takeda S, Shimoda H. Black ginger extract increases physical fitness performance and muscular endurance by improving inflammation and energy metabolism. Heliyon 2016;2:e00115.  Back to cited text no. 11
    
12.
Patanasethanont D, Nagai J, Yumoto R, Murakami T, Sutthanut K, Sripanidkulchai BO, et al. Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function. J Pharm Sci 2007;96:223-33.  Back to cited text no. 12
    
13.
Pripdeevech P, Pitija K, Rujjanawate C, Pojanagaroon S, Kittakoop P, Wongpornchai S. Adaptogenic-active components from Kaempferia parviflora rhizomes. Food Chem 2012;132:1150-5.  Back to cited text no. 13
    
14.
Paramee S, Sookkhee S, Sakonwasun C, Na Takuathung M, Mungkornasawakul P, Nimlamool W, et al. Anti-cancer effects of Kaempferia parviflora on ovarian cancer SKOV3 cells. BMC Complement Altern Med 2018;18:178.  Back to cited text no. 14
    
15.
Murata K, Hayashi H, Matsumura S, Matsuda H. Suppression of benign prostate hyperplasia by Kaempferia parviflora rhizome. Pharmacognosy Res 2013;5:309-14.  Back to cited text no. 15
    
16.
Leardkamolkarn V, Tiamyuyen S, Sripanidkulchai BO. Pharmacological activity of Kaempferia parviflora extract against human bile duct cancer cell lines. Asian Pac J Cancer Prev 2009;10:695-8.  Back to cited text no. 16
    
17.
Sutthanut K, Sripanidkulchai B, Yenjai C, Jay M. Simultaneous identification and quantitation of 11 flavonoid constituents in Kaempferia parviflora by gas chromatography. J Chromatogr A 2007;1143:227-33.  Back to cited text no. 17
    
18.
Vichitphan K, Vichitphan S, Sirikhansaeng P. Flavonoid content and antioxidant activity of Krachai-dum (Kaempferia parviflora). KMITL Sci Tech J 2007;52:97-105.  Back to cited text no. 18
    
19.
Thao NP, Luyen BT, Lee SH, Jang HD, Kim YH. Anti-osteoporotic and antioxidant activities by rhizomes of Kaempferia parviflora Wall. Ex. Baker. Nat Prod Sci 2016;22:13-9.  Back to cited text no. 19
    
20.
Zhang L, Khoo C, Koyyalamudi SR, Pedro ND, Reddy N. Immunostimulatory and anticancer activities of polysaccharides extracted from traditional anticancer Chinese medicinal herbs. Pharmacologia 2018;9:18-29.  Back to cited text no. 20
    
21.
Boudjeko T, Megnekou R, Woguia AL, Kegne FM, Ngomoyogoli JE, Tchapoum CD, et al. Antioxidant and immunomodulatory properties of polysaccharides from Allanblackia floribunda Oliv stem bark and Chromolaena odorata (L.) king and H.E. Robins leaves. BMC Res Notes 2015;8:759.  Back to cited text no. 21
    
22.
Pitakpawasutthi Y, Palanuvej C, Ruangrungsi N. Quality evaluation of Kaempferia parviflora rhizome with reference to 5,7-dimethoxyflavone. J Adv Pharm Technol Res 2018;9:26-31.  Back to cited text no. 22
[PUBMED]  [Full text]  
23.
Shruthi S, Vijayalaxmi KK, Shenoy KB. Immunomodulatory effects of gallic acid against cyclophosphamide-and cisplatin-induced immunosuppression in Swiss Albino Mice. Indian J Pharmaceut Sci 2018;80:150-60.  Back to cited text no. 23
    
24.
Raj S, Gothandam KM. Immunomodulatory activity of methanolic extract of Amorphophallus commutatus var. Wayanadensis under normal and cyclophosphamide induced immunosuppressive conditions in mice models. Food Chem Toxicol 2015;81:151-9.  Back to cited text no. 24
    
25.
Yadav SK, Nagarathna PK, Yadav CK. Research article of evaluation of immunomodulatory activity of Dalbergia latifolia on Swiss albino mice. J Pharm Biol Sci 2015;10:58-64.  Back to cited text no. 25
    
26.
Montejo JF, Mondonedo JA, Lee MG, Ples MB, Vitor II RJ. Hematological effects of Ipomoea batatas (camote) and Phyllanthus niruri (sampa-sampalukan) from Philippines in the ICR mice (Mus musculus). Asian Pac J Trop Biomed 2015;5:29-33.  Back to cited text no. 26
    
27.
Ray A, Mediratta PK, Puri S, Sen P. Effects of stress on immune responsiveness, gastric ulcerogenesis and plasma corticosterone in rats: Modulation by diazepam and naltrexone. Indian J Exp Biol 1991;29:233-6.  Back to cited text no. 27
    
28.
Mehra E, Vaidya MC. A Handbook of Practical and Clinical Immunology. New Delhi: C.B.S. Publishers; 1985. p. 44.  Back to cited text no. 28
    
29.
Saraf MN, Ghooi RB, Patwardhan BK. Studies on the mechanism of action of Semecarpus anacardium in rheumatoid arthritis. J Ethnopharmacol 1989;25:159-64.  Back to cited text no. 29
    
30.
Bancroft JD, Stevens A. Theory and Practice of Histological Technique. 3rd ed. New York: Churchill, Livingstone; 1990. p. 276.  Back to cited text no. 30
    
31.
Tizard IR. Veterinary Immunology: An Introduction. 7th ed. Philadelphia: WB Saunders Company; 2004. p. 496.  Back to cited text no. 31
    
32.
Ahirwal L, Singh S, Dubey MK, Bharti V, Mehta A, Shukla S. In vivo immunomodulatory effects of the methanolic leaf extract of Gymnema sylvestre in Swiss albino mice. Arch Biol Sci Belgrade 2015;67:561-70.  Back to cited text no. 32
    
33.
Kanase V, Patil DT. Evaluation of in vitro immunomodulatory activity of aqueous and ethanolic extract of Eulophia nuda Lindl. Asian J Pharm Clin Res 2018;11:252-6.  Back to cited text no. 33
    
34.
Chivapat S, Chavalittumrog P, Phadungpat S, Punyamong S. Kumar PK, Chansuvanich N. Acute and chronic toxicity study of Kaempferia parviflora Wall ex. Bak powder. J Thai Tradit Alternat Med. 2004;2:3-16.  Back to cited text no. 34
    
35.
Ghosh MN. Fundamentals of Experimental Pharmacology. 6th ed. Kolkatta, India: Hilton and Co.; 2008. p. 287.  Back to cited text no. 35
    
36.
Snedecor GW, Cochran WG. Statistical Methods. 8th ed. Kolkatta, India: Oxford and IBH Publishing Co.; 1994. p. 503.  Back to cited text no. 36
    
37.
Manu KA, Kuttan G. Immunomodulatory activities of punarnavine, an alkaloid from Boerhaavia diffusa. Immunopharmacol Immunotoxicol 2009;31:377-87.  Back to cited text no. 37
    
38.
Kumar SP. Immunomodulatory Effect of Fractions of Ethanolic Extract of Emblica officinalis (Amla) Fruit Pulp in Mice. M. V Sc. Thesis, Thrissur: Kerala Agricultural University; 2005.  Back to cited text no. 38
    
39.
Sumalatha RBP, Rama BP, Shwetha RB, Sadananda A. Studies on immunomodulatory effects of Salacia chinensis L. on albino rats. J Appl Pharm Sci 2012;2:98-107.  Back to cited text no. 39
    
40.
Lydyard PM, Whelan A, Fanger MW. Instant Notes in Immunology. New Delhi: Viva Books Private Limited; 2003.  Back to cited text no. 40
    
41.
Gnanasekaran S, Sakthivel KM, Chandrasekaran G. Immunostimulant and chemoprotective effect of Vivartana, a polyherbal formulation against cyclophosphamide induced toxicity in Swiss albino mice. J Exp Ther Oncol 2015;11:51-61.  Back to cited text no. 41
    
42.
Naseema KT. Evaluation of Immunomodulatory and Antibacterial Effects of Urine of Venchur and Crossbred Cows. M. V Sc. Thesis. Thrissur: Kerala Agricultural University; 2014.  Back to cited text no. 42
    
43.
Kajaria D, Tripathi JS, Tiwari SK, Pandey BL. Immunomodulatory effect of ethanolic extract of Shirishadi compound. Ayu 2013;34:322-6.  Back to cited text no. 43
[PUBMED]  [Full text]  
44.
Sultana R, Khanam S, Devi K. Immunomodulatory effect of methanol extract of Solanum xanthocarpum fruits. Int J Pharma Sci Res 2011;2:93-7.  Back to cited text no. 44
    
45.
Lawrence AK, Amado JP. Clinical Chemistry: Theory, Analyses and Correlation. 1st ed. Philadelphia: The Mosby Company Publishing Co.; 1984. p. 1476.  Back to cited text no. 45
    
46.
Nfambi J, Bbosa GS, Sembajwe LF, Gakunga J, Kasolo JN. Immunomodulatory activity of methanolic leaf extract of Moringa oleifera in wistar albino rats. J Basic Clin Physiol Pharmacol 2015;26:603-11.  Back to cited text no. 46
    
47.
Umair M, Javeed A, Ghafoor A, Ashraf M. Immunomodulatory activities of gemifloxacin in mice. Iran J Basic Med Sci 2016;19:985-92.  Back to cited text no. 47
    
48.
Mungantiwar AA, Nair AM, Shinde UA, Dikshit VJ, Saraf MN, Thakur VS, et al. Studies on the immunomodulatory effects of Boerhaavia diffusa alkaloidal fraction. J Ethnopharmacol 1999;65:125-31.  Back to cited text no. 48
    
49.
Morris HJ, Llaurado G, Gutiérrez A, Lebeque Y, Fontaine R, Beltrán Y, et al. Immunomodulating Properties of Pleurotus sp. Fruiting Bodies Powder on Cyclophosphamide Treated Mice. Proceedings of the 7th International Conference on Mushroom Biology and Mushroom Products; 2011.  Back to cited text no. 49
    
50.
Tothova C, Nagy O, Kovac G. Serum proteins and their diagnostic utility in veterinary medicine: A review. Vet Med 2016;61:475-96.  Back to cited text no. 50
    
51.
Shwetha R, Ballal B, Sumalatha PR, Acharya S. Studies on immunomodulatory effect of Pajanelia longifolia (Willd.) Schumann on albino rats. Int J Res Pharm Biomed Sci 2012;3:1642-51.  Back to cited text no. 51
    
52.
Yoon HS, Kim JW, Cho HR, Moon SB, Shin HD, Yang KJ, et al. Immunomodulatory effects of Aureobasidium pullulans SM-2001 exopolymers on the cyclophosphamide-treated mice. J Microbiol Biotechnol 2010;20:438-45.  Back to cited text no. 52
    
53.
Kim JW, Choi JS, Seol DJ, Choung JJ, Ku SK. Immunomodulatory effects of Kuseonwangdogo-based mixed herbal formula extracts on a cyclophosphamide-induced immunosuppressed mouse model. Evid Based Complement Alernat Med 2018;2018:1-8.  Back to cited text no. 53
    
54.
Shabbir A, Butt H, Shahzad M, Arshad HM, Waheed I. Immunostimulatory effect of methanolic leaves extract of Psidium guajava (guava) on humoral and cell-mediated immunity in mice. J Anim Plant Sci 2016;26:1492-1500.  Back to cited text no. 54
    
55.
Manure JY, Naikwade NS. Immunomodulatory activity of leaves of Rumex vesicarius Linn. and Symplocos racemosa Roxb. Int J Pharm Sci Res 2018;9:1537-44.  Back to cited text no. 55
    
56.
Shukla SH, Saluja AK, Pandya SS. Modulating effect of Gmelina arborea Linn. On immunosuppressed albino rats. Pharmacognosy Res 2010;2:359-63.  Back to cited text no. 56
    
57.
Ono S, Yoshida N, Maekawa D, Kitakaze T, Kobayashi Y, Kitano T, et al. 5-hydroxy-7-methoxyflavone derivatives from Kaempferia parviflora induce skeletal muscle hypertrophy. Food Sci Nutr 2019;7:312-21.  Back to cited text no. 57
    
58.
Jose J, Sudhakaran S, Sumesh Kumar TM, Jayaram S, Jayadevi V E. Study of in vitro immunomodulatory effect of flavonoid isolated from Phyllanthus niruri on human blood lymphocytes and evaluation of its antioxidant potential. Int J Phcogn Phytochem Res 2014;6:284-9.  Back to cited text no. 58
    
59.
Liu T, Zhao J, Ma L, Ding Y, Su D. Hepatoprotective effects of total triterpenoids and total flavonoids from Vitis vinifera L against immunological liver injury in mice. Evid Based Complement Alternat Med [Journal on the internet] 2012; Available from: https://doi.org/10.1155/2012/969386. [Last accessed on 2019 May 23].  Back to cited text no. 59
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]



 

Top
   
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed740    
    Printed40    
    Emailed0    
    PDF Downloaded265    
    Comments [Add]    

Recommend this journal