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Table of Contents
ORIGINAL ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 4  |  Page : 7-14

Investigation on prescription screening of a polyherbal preparation based on splenocyte proliferation activity and its preparation method


1 Traditional Chinese Medicine and Natural Medicine Research Institute, School of Pharmacy, Lanzhou University, Lanzhou 730000, China
2 Institute of Integrated Traditional Chinese and Western Medicine, Gansu University of Traditional Chinese Medicine; Gansu Key Laboratory of Traditional Chinese Medicine for Prevention and Treatment of Chronic Diseases; Gansu Clinical Medical Center of Integrated Traditional Chinese and Western Medicine Treatment of Cardiovascular and Cerebrovascular, Lanzhou 730000, China

Date of Submission29-Dec-2016
Date of Acceptance22-May-2017
Date of Web Publication9-Jan-2018

Correspondence Address:
Prof. Fang-Di Hu
School of Pharmacy, Lanzhou University, 199 Donggang Road West, Lanzhou 730000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_14_17

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  Abstract 

Objective: The present study aims to optimize the extraction conditions of polysaccharides and flavonoids from a polyherbal preparation consisting of three kinds of Chinese medicinal herbs, Codonopsis pilosula, Crataegus pinnatifida, and Lycium barbarum, and evaluation of its immunomodulatory activity in immunosuppressed mice. Materials and Methods: An orthogonal design (L9 [3]4) was constructed to achieve the optimal extraction conditions. The immunomodulatory action of the polyherbal preparation was studied at three doses of 10, 20, and 40 mL/kg/day orally by measuring splenocyte proliferation in mice model of cyclophosphamide-induced immunosuppression. Results: The chosen parameters, including the ratio of solvent-to-raw material, duration of extraction, and extraction times, were the fundamental variables that influenced the extract yields. The highest yield of total polysaccharides content was 54.3 mg/mL when the ratio of solvent-to-raw material, duration of extraction, and number of extractions were 12:1, 1.5 h, and 3, respectively. The maximum extraction yield of the flavonoids was 3.5 mg/mL when the ratio of solvent-to-raw material was 12:1, the extraction time was 2 h, and the number of extractions was 3. The prescription screening showed that the impact of the polyherbal preparation on the splenocyte proliferation capacity was more pronounced than its disassembled components. Oral administration of the polyherbal preparation could significantly increase the concanavalin A-stimulated mouse spleen cells proliferation in a dose-dependent manner. Conclusion: These findings suggested that the polyherbal preparation possesses potential for augmenting the immune activity due to the polysaccharide and flavonoid content in these herbal medicines.

Keywords: Codonopsis pilosula, Crataegus pinnatifida, flavonoids, Lycium barbarum, polysaccharides


How to cite this article:
Ahmed Abdelmoaty AA, Ma YL, Bai R, Zheng XP, Hu FD, Li YD. Investigation on prescription screening of a polyherbal preparation based on splenocyte proliferation activity and its preparation method. World J Tradit Chin Med 2017;3:7-14

How to cite this URL:
Ahmed Abdelmoaty AA, Ma YL, Bai R, Zheng XP, Hu FD, Li YD. Investigation on prescription screening of a polyherbal preparation based on splenocyte proliferation activity and its preparation method. World J Tradit Chin Med [serial online] 2017 [cited 2018 Oct 19];3:7-14. Available from: http://www.wjtcm.net/text.asp?2017/3/4/7/222604


  Introduction Top


Immunomodulators are compounds that are fit for interacting with the immune system to stimulate or suppress particular parts of the host response. They have been of interest for a long time since it can be used as an alternative to classical antibiotics which are becoming ineffectual because of the expansion of microbial resistance.[1]

Most of the anticancer drugs currently used in chemotherapy have cytotoxic side effects to normal cells, which may seriously affect patients' quality of life, regardless of their great curative effects. Questions remain on how to best decrease the toxicity and improve the curative effect of chemotherapy. In recent years, many researchers got interested in the use of medicinal herbs and plant extracts used in traditional therapy for their chemopreventive purposes.[2]

Cyclophosphamide (CP) is a medication mainly used in chemotherapy with a high therapeutic index and broad spectrum of activity against a variety of cancers such as lymphoma, myeloma, and chronic lymphocytic leukemia.[3] Immunosuppression is a major side effect of long-term CP therapy in cancer patients, which is a major limiting factor in clinical chemotherapy without efficacious remedies. As a tumor grows progressively, the immune system of tumor-bearing hosts is frequently impaired.[4]

Nowadays, Chinese medicinal herbs have attracted a great deal of attention due to its low toxicity and curative effects.[5] In fact, combining chemotherapy drugs and immunomodulatory agents such as many traditional medicinal herbs are used to improve immunity and reduce the toxicity of chemotherapy.[6] Dangshen (the root of Codonopsis pilosula of the Campanulaceae family), Shanzha (the fruit of Crataegus pinnatifida of the Rosaceae family), and Goji (the fruit of Lycium barbarum of the Solanaceae family), of which the polyherbal preparation is composed, are extensively used in traditional Chinese medicine. These medicinal plants have shown immunomodulatory activity.[7],[8],[9]

Radix C. pilosula is a traditional Chinese medicinal herb distributed in the Northeast Provinces of China, which belongs to the family Campanulaceae. In some cases, it is used as a substitute of the much more expensive Panax ginseng. It has been used commonly in China folk for strengthen the middle warmer, invigorate the spleen, and support the lung.[10] Moreover, it has the functions of antitumor, antimicrobial, antioxidant and enhancing cellular immunity.[11],[12],[13]

C. pinnatifida (hawthorn) is a member of family Rosaceae. This species is broadly distributed in Asia, Europe, and North America.[14] More than 1000 species of genus Crataegus have been recognized around the world. However, C. pinnatifida and C. pinnatifida Bge. var. major N. E. Br. are common in China.[15] Modern investigations have exhibited that C. pinnatifida has various pharmacological impacts, for instance, it's effect on the cardiovascular, digestive, and endocrine systems, and also on pathogenic microorganisms.[16]

L. barbarum fruit, generally called Goji berry or wolfberry, is an outstanding herb in traditional Chinese medicine. Recently, it is being used not only in China but also worldwide as a popular health food ingredient in numerous forms such as soups, drinks, and a variety of solid foods.[17] Goji berry has demonstrated an extensive variety of health benefits, for example, the functions and activities associated with the kidney, liver, eyesight, sex, circulation, immune system, and longevity.[18] Besides, it has different health effects such as immunomodulation,[19] anticancer,[20] and antioxidant activities.[21]

Modern pharmacology research has shown that these herbal medicines derived compounds, including polysaccharides and flavonoids which represent the major classes of bioactive ingredients of them, have immunostimulatory action.[22],[23],[24] On the other hand, few studies have reported the immunomodulatory activity of other components in these herbal medicines, for example, saponins (triterpenoid glycosides) in C. pilosula which have a minor effect on the immune system functions compared to the immunomodulatory effect of the polysaccharides.[25]

Polysaccharides isolated from botanical sources represent an important class of compounds because a diversity of biological activities have been associated with them, including antitumor,[26] antioxidant,[27] anti-inflammatory,[28] and immunomodulating agent.[29],[30] They can modulate the immune responses via their stimulatory activity on macrophages which enhance phagocytosis, antigen processing ability, production of reactive oxygen species and nitric oxide (NO), and secretion of cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL-8, IL-12, and IFN-β2.[31],[32]

Flavonoids are the most common group of secondary plant metabolites that are widely distributed throughout the plant kingdom. They have a variety of health benefits such as antioxidant,[33] anti-atherosclerotic,[34] anti-inflammatory,[35] and improve immunity.[36],[37] They are reported to have a role in cardiovascular disease prevention and anti-platelet aggregation.[38] A variety of in vitro and in vivo experiments have shown that flavonoids possess anti-allergic and anti-inflammatory activities. Their influences on a variety of inflammatory processes can be assessed by their immunomodulatory effects on neutrophils, basophils, eosinophils, T- and B-lymphocytes, macrophages, and platelets.[39] In addition, flavonoids can inhibit expression of inducible NO synthase, cyclooxygenase, and lipoxygenase. These enzymes are responsible for the production of a great amount of NO, prostanoids, leukotrienes, and other mediators of the inflammatory pathway such as cytokines, chemokines, or adhesion molecules. Moreover, flavonoids inhibit mitogen-induced immunoglobulin secretion of IgG, IgM, and IgA isotypes in vitro.[40]

Hence, the present study goes for exploring the optimization of the extraction parameters of polysaccharides and flavonoids from a polyherbal preparation by employing an orthogonal L9 (3)4 test design. The major constituents are C. pilosula, C. pinnatifida, and L. barbarum. The immunomodulatory activity of the polyherbal preparation by concanavalin A (Con A)-induced splenocyte proliferation in CP-treated mice was evaluated.


  Materials and Methods Top


Drugs and chemicals

The roots of C. pilosula and the fruits of C. pinnatifida and L. barbarum were purchased from the Yellow River Herbal Medicine Market in Lanzhou city (Lanzhou, PR China). Zhen Qi Fuzheng Capsules (batch number: J20150504) were purchased from (Gansu Fuzheng Pharmaceutical Technology Co., Ltd.). CP (Lot No. 5K086A, Baxter International Limited) was purchased from the First Hospital of Lanzhou University. Complete RPMI 1640 medium, 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), Con A, and red cell lysate were purchased from Beijing Solarbio Science and Technology Co., Ltd. Fetal calf serum (FCS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. Standard glucose (batch number: 1304070) and standard rutin (batch number: 05-1001) were purchased from Shanghai Traditional Chinese Medicine Standardization Research Center. All other chemicals used in the experiment were of analytical grade.

Experimental animals

Kunming female mice (20 ± 2 g) were used for this study, and these animals were obtained from Experimental Animal Center of Lanzhou University (Lanzhou, PR China). Throughout the experiment, the animals were housed 10 animals per cage and kept up at ambient temperature of 25°C ± 2°C, 30%–60% humidity, under 12:12 h light-dark cycle. All mice were allowed to be free access to water and food. The animals were habituated to laboratory conditions for 72 h before the experimental protocol to minimize any nonspecific stress. All animal experiments complied with the approval of Lanzhou University Animal Ethics (approval number: SCXK Gan 2013-0002).

Prescription screening of the polyherbal preparation and its disassembled components

The animals were divided into ten common groups comprising ten animals each. Group-I was given distilled water (20 mL/kg/day p.o) and served as normal control. Group-II served as model group treated with distilled water (20 mL/kg/day p.o). Group-III animals were marked as positive control by treating them with Zhen Qi Fuzheng preparation (ZFP) which suspended in distilled water (1000 mg/kg/day p.o). ZFP, composed of Radix Astragali (Huang qi in Chinese) and Fructus Ligustri Lucidi (Nüzhenzi in Chinese), which has been recorded in the “Ministry of Health Drug Standards” of Chinese medicine formulated prescription, is generally utilized as a part of clinical practice for the treatment to improve immunity, increase leukocytes, protect bone marrow and adrenal cortex, and promote the recovery of normal functions as an accessory of surgical operation, radiotherapy, or chemotherapy, and it could also be used as an adjuvant therapy of cancer.[41] Group-IV was treated with 20 mL/kg/day p.o of the polyherbal preparation containing C. pilosula, C. pinnatifida, and L. barbarum. Group-V, Group-VI, and Group-VII were received 20 mL/kg/day p.o of the extract derived from C. pilosula with C. pinnatifida, C. pilosula with L. barbarum, and C. pinnatifida with L. barbarum, respectively. Group-VIII, Group-IX, and Group-X were administered 20 mL/kg/day p.o of the extract of C. pilosula, C. pinnatifida, and L. barbarum separately. The animals of all groups were treated with their respective drugs orally for 4 weeks. Following 3 progressive weeks of oral treatment, the mice of all groups except Group-I (normal control) were injected with CP (40 mg/kg, i.p.) for 2 consecutive days.

Preparation of polysaccharide and flavonoid from the polyherbal preparation

The plant materials of the polyherbal preparation (3 g of C. pilosula, 3 g of C. pinnatifida, and 1.5 g of L. barbarum) were extracted with distilled water (water-plant materials mixtures [mL/g] ranging from 8:1 to 12:1). The duration of the extraction ranging from 1 to 2 h and the given number of extractions extended from 1 to 3 times. The temperature ranged from 65°C to 95°C and was kept constant through the whole extraction process. The mixture was centrifuged (5 min, 3000 rpm, 25°C), and afterward, the supernatant was isolated from insoluble deposit with filter paper. The extracts were defatted by the method of Sevag to remove proteins, carbohydrates, nucleic acids, pigments, and so on. The extracts were then concentrated using a rotary evaporator under reduced pressure at 70°C to a concentration of 0.1875 g/mL.

Optimization of polysaccharide and flavonoid extraction

The extraction process of the polyherbal preparation rich in polysaccharides and flavonoids was optimized through an orthogonal (L9 [3]4) test design. Nine extractions were carried out to study the effects of ratio of solvent to raw materials (a), extraction time (b), and number of extractions (c) on the extraction yield of polysaccharides and flavonoids through a single-factor exploration. The ratios of solvent to raw material 8:1, 10:1, and 12:1, duration of extraction of 1, 1.5, and 2 h, and number of extractions 1, 2, and 3. [Table 1] shows the experimental conditions for the extraction of polysaccharide and flavonoid from the polyherbal preparation.
Table 1: Factors and levels of the orthogonal analysis

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Determination of polysaccharide content in the polyherbal preparation

The content of polysaccharide extracted from the polyherbal preparation was determined by phenol-sulfuric acid method using glucose as standard.[42] The standard glucose was dried at 105°C for 30 min. For making the calibration curve, a standard solution of glucose was prepared by dissolving 3 mg of glucose in a 25 mL volumetric flask to obtain a standard solution of 0.1204 mg/mL. Aliquots of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the prepared glucose standard solution were added to separate tubes and diluted with water till 2.0 mL. Exactly 1.0 mL of the diluted solution was taken out and mixed with 1 mL 5% phenol and 5 mL concentrated sulfuric acid followed by shaking for 10 min. The absorbance was measured at 490 nm. All measurements were performed in triplicate. The content of polysaccharide was determined according to the equation of linear regression (y = 23.892x − 0.00038, R2 = 0.9990).

Determination of the flavonoid content in the polyherbal preparation

The concentration of total flavonoids was determined using the aluminum nitrate colorimetric method (NaNO2-Al(NO3)3-NaOH spectrophotometric method).[43] For making the calibration curve, a standard solution of rutin was prepared by dissolving 5.07 mg of rutin in 25 mL of 70% methanol. Aliquots of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the prepared rutin standard solution were added to 25 mL volumetric flasks. 1 mL of 5% NaNO2 was added to each flask followed by shaking for 6 min. 1 mL of 10% Al (NO3)3 was also added followed by shaking for 6 min and then the addition of 10 mL of 4% sodium hydroxide solution. Water was added to the mark and the solutions were shaken and left for 15 min before the measurement. The absorbance was measured at 505 nm, and all the measurements were performed in triplicate. The content of flavonoid was determined according to the equation of linear regression (y = 5.5341x − 0.00173, R2 = 0.99975).

Pharmacodynamic validation of the polyherbal preparation

Mice were randomly divided into six common groups comprising ten animals each. Group-I was given distilled water (20 mL/kg/day p.o) and served as normal control. Group-II served as model group treated with distilled water (20 mL/kg/day p.o). Group-III animals were marked as positive control by treating them with ZFP which suspended in distilled water (1000 mg/kg/day p.o). Group-IV, Group-V, and Group-VI were treated with low (10 mL/kg/day p.o), medium (20 mL/kg/day p.o), and high dose (40 mL/kg/day p.o) of the polyherbal preparation. The animals of all groups were treated with their respective drugs orally for 4 weeks. Following 3 progressive weeks of oral treatment, the mice of all groups, except Group-I (normal control), were injected with CP (40 mg/kg, i.p.) for 2 consecutive days.

Concanavalin A -induced splenocyte proliferation assay/3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

After 4 successive weeks of oral treatment, the animals were sacrificed and their spleens were aseptically removed with scissors and forceps. The spleens were passed through a 200-mesh nylon film to obtain a homogeneous cell suspension and the erythrocytes were lysed with RBC lysis solution. After centrifugation for 5 min (1000 rpm/min), the cells were washed three times with Hank's liquid and suspended in RPMI-1640 medium containing 10% FCS.

To carry out the splenocyte proliferation reaction, spleen cell suspension (1 × 104 cell/mL) was pipetted into 96 well plates (50 μL/well) and cultured at 37°C for 72 h in a humid saturated atmosphere containing 5% CO2 in the presence of Con A (final concentration: 0.325 μg/mL). Con A was added to five holes, and the other five holes were added with the same volume of the blank medium. After 68 h, MTT solution at 5 mg/mL (25 μL) was added to each well and incubated for 4 h. At the end of the incubation, the cells were lysed with acidified isopropanol and the untransformed MTT was removed carefully by pipetting. To each well, 100 μL sodium dodecyl sulfate was added and homogenized for at least 10 min to fully dissolve the colored material. The optical density value (absorbance) of the dissolved mitochondrial formazan precipitates was evaluated at 570 nm wavelength. The value of plus Con A holes minus the others without Con A represents the splenocyte proliferation capacity.

Statistics

The results were expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance using SPSS/20 software generated by IBM (International Business Machines Corporation) which is an American multinational technology company located in Armonk, New York, United States. Values of P < 0.05, <0.01 were considered to be a statistically significant.


  Results and Analysis Top


The effects of the polyherbal preparation and its disassembled components on concanavalin A-induced splenocyte proliferation

The effects of the polyherbal preparation and its disassembled components on Con A-induced splenocyte proliferation in immunosuppressed mice are shown in [Figure 1].
Figure 1: The effects of the polyherbal preparation on and its disassembled components on concanavalin A-induced proliferation activity of mouse splenocytes Results are represented as mean ± standard deviation (n = 10), *P < 0.05, **P < 0.01 when compared to Group-II, #P < 0.05, ##P < 0.01 when compared to Group-IV. Group-I: Normal control, Group-II: Model group, Group-III: Positive control group, Group-IV: Polyherbal preparation, Group-V: Codonopsis pilosula with Crataegus pinnatifida, Group-VI: Codonopsis pilosula with Lycium barbarum, Group-VII: Crataegus pinnatifida with Lycium barbarum, Group-VIII: Codonopsis pilosula, Group-IX: Crataegus pinnatifida, Group-X: Lycium barbarum

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As observed in [Figure 1], a significant decrease (P < 0.01) in the splenocyte proliferation capacity was observed in Group-II (model group) when compared with Group-I (normal control group). The splenocyte proliferation capacity of Group-III (positive control group), Group-IV (polyherbal preparation-treated group), and Group-VI (C. pilosula with L. barbarum-treated group) was found to be significant or highly significant (P < 0.05 or P < 0.01) when compared to Group-II (model group). Group-X (L. barbarum-treated group) showed nonsignificant difference in the splenocyte proliferation capacity when compared to Group-II and Group-IV (P > 0.05). The other groups had the tendency to promote the proliferation of splenocyte in immunosuppressed mice compared to Group-II, but the splenocyte proliferation capacity was significantly higher for the group treated with the polyherbal preparation (P < 0.05, P < 0.01). Therefore, optimization of the extraction parameters of polysaccharides and flavonoids is needed to prepare the polyherbal preparation of C. pilosula with C. pinnatifida and L. barbarum.

Optimization of the extraction parameters of polysaccharides and flavonoids from the polyherbal preparation

The initial phase in the extraction method of polysaccharide and flavonoid from the polyherbal preparation is to optimize the operating conditions to get an effective extraction of the target compounds and to avoid the co-extraction of the undesired compounds. Since different parameters potentially influence the extraction process, the optimization of the experimental conditions is a crucial step in the development of a solvent extraction method. As observed in [Table 2], the proportion of solvent-to-raw material, duration of extraction, and extraction times were chosen as the orthogonal factors. Optimization of the appropriate extraction conditions in the polysaccharide and flavonoid extraction can be achieved by utilizing an experimental design. In the present study, all the selected factors were examined using an orthogonal L9 (3)4 test design.
Table 2: Results of the orthogonal test

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Range analysis and variance analysis results of the polysaccharide yield

As shown in [Table 3], it can be found that the effect of the different factors on the extraction yield of polysaccharides decreases in the order: C > A > B according to the range values (R). Thus, the optimum conditions were considered as A3B2C3 according to the K values of each level, namely, the ratio of water-to-raw material, the extraction time, and the number of extractions were 12:1, 1.5 h, and 3, respectively; the extraction yield was the highest. The analysis of variance results [Table 4] showed that the number of extractions (factor C) had the highest effect, while the extraction time (factor B) had the least effect on the extraction yield of polysaccharides from the polyherbal preparation.
Table 3: The range analysis results of the polysaccharide yield

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Table 4: Variance analysis results of the polysaccharide yield

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Range analysis and variance analysis results of the flavonoid yield

According to the range values (R) represented in [Table 5], the influence of the different factors on the extraction yield of flavonoids was C > B > A. Thus, the optimum conditions were a combination of A3B3C3 according to the K values of each level, namely, the ratio of water-to raw-materials, the extraction time, and the number of extractions were, 12:1, 2 h, and 3, respectively; the extraction yield was the highest. The analysis of variance results [Table 6] indicated that the number of extractions (factor C) had the greatest effect, while the ratio of water-to-raw materials (factor A) had the lowest effect on the extraction yield of flavonoids from the polyherbal preparation.
Table 5: The range analysis results of the flavonoid yield

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Table 6: Variance analysis results of the flavonoid yield

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The effect of the polyherbal preparation on concanavalin A-induced splenocyte proliferation

The effect of the polyherbal preparation on Con A-stimulated splenocyte proliferation in immunosuppressed mice is shown in [Figure 2].
Figure 2: The effect of the polyherbal preparation on concanavalin A-induced proliferation activity of mouse splenocytes. Results are represented as mean ± standard deviation (n = 10), *P < 0.05, **P < 0.01 when compared to model group. Group-I: Normal control, Group-II: Model group, Group-III: Positive control group, Group-IV: Low dose of polyherbal preparation, Group-V: Medium dose of Polyherbal preparation, Group-VI: High dose of polyherbal preparation

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As observed in [Figure 2], the splenocyte proliferation activity in model group was significantly lower than the normal control group (P < 0.01). In comparison with the model group, when Con A was added as mitogen for splenocytes, the polyherbal preparation could significantly enhance the cell proliferation activity (P < 0.05 or P < 0.01) in a dose-dependent manner.


  Discussion Top


Mathematical modeling is an efficient statistical method; it includes mathematical and statistical techniques for investigating the effects of different factors on the extraction process, as well as optimizing the extraction conditions.[44] Among the current modeling methodologies, an orthogonal experimental design was used to optimize the extraction parameters of polysaccharides and flavonoids from a polyherbal preparation consisting of C. pilosula, C. pinnatifida, and L. barbarum. The ratio of water-to-raw materials, extraction time, and number of extractions were considered as the most important factors during the extraction process of the samples. This statistical method has the advantages of decreasing the number of experimental trials and understanding the interactions between factors by utilizing the orthogonal design table and statistical analysis.[45] Range analysis can be utilized to get the parameters optimized, to accomplish the predetermined features, and to uncover the statistic principle based on the hidden or equivocal factors.[46] Variance analysis is one of the most important statistical techniques, which is used to uncover the main factor and interaction effects of variables. It is also used to identify the procedure parameters that are statistically significant. In this method, F ratio is employed to recognize the significant parameters from others. F value is the ratio of the variance estimation of the treatment effect to the variance estimation of the error. The high value of F ratio means that the selected parameter has a significant effect on the evaluation index compared with the error variation. The sum of squares and degrees of freedom corresponding to the eliminated terms are added into the residual sum of squares and degree of freedom.[47]

Immunostimulatory effects of a drug or nourishing medicine are difficult to evaluate in healthy people or animals. Therefore, CP is used as immunosuppression inducer to Kunming mice. CP itself is a prodrug and must first undergo metabolic activation, catalyzed by the hepatic cytochrome P450-4-hydroxy-CP and later to phosphoramide mustard and acrolein. The mustard component produces a cytotoxic effect by preventing cell replication, while acrolein is linked with its toxic side effect.[48] CP administration causes hematopoietic depression (leukemia, lymphoma, and pancytopenia), nausea, vomiting, gonadal atrophy, and liver, renal, and bladder injury.[49]

The polyherbal preparation is made from C. pilosula, C. pinnatifida, and L. barbarum. These three kinds of Chinese medicinal herbs have shown a good immunomodulatory activity and have been used in China and some other Asia countries as herbal medicines for many years. In mice with CP-induced immunosuppression, oral administration of the polysaccharide from C. pilosula increases the thymus and spleen index and the phagocytic activity of peritoneal macrophages and recovers the activity of α-naphthyl acetate esterase in peripheral lymphocytes.[50],[51]C. pinnatifida can increase the leucocyte count and enhance the phagocytic activity of macrophages and had effects on spleen and lymphocytes in in the same model.[52] In one study, L. barbarum polysaccharides (LBPs) antagonized the suppressive effect of CP on T-lymphocyte proliferation. In addition, administration of LBP restored the reduced natural killer cell activity caused by CP administration in mice.[53] The polyherbal preparation is developed from C. pilosula, C. pinnatifida, and L. barbarum, which suggests that its effect in the treatment may be related with the above pharmacological activities of the three Chinese medicinal herbs. In the present study, the immunomodulatory activity of the polyherbal preparation was investigated by assessing its impact on splenocyte proliferation activity in mice model of CP-induced immunosuppression.

Splenocyte proliferation is a marker of immunoenhancement. Splenocytes synthesize and release mediators, including TNF-α, NO, and IL-2, which can activate immune response, inflammation, and tissue regeneration.[54] In this work, immunosuppression in CP-treated mice is manifested by the marked reduction of the splenocytes proliferation activity. After 4 weeks of the oral treatment with plant extracts, the splenocytes of the treated mice displayed a heightened response to the mitogen Con A in a dose-dependent pattern. These results are consistent with previously published studies.[55],[56],[57]

This study is the first to demonstrate the immunostimulatory activity of low, intermediate, and high oral doses of a polyherbal preparation consisting of C. pilosula, C. pinnatifida, and L. barbarum in immunosuppressed mice.


  Conclusion Top


The study has illustrated a statistical method based on orthogonal L9 (3)4 test design to determine the optimum extraction conditions that give high extraction yield of polysaccharides and flavonoids from a polyherbal preparation comprising C. pilosula in combination with C. pinnatifida and L. barbarum. The results showed that the ratio of solvent-to-raw material, number of extractions, and duration of extraction were the main variables that influenced the yields of the extracts. The immunostimulatory activity of low, intermediate, and high oral doses of the polyherbal preparation was elucidated by Con A-induced splenocyte proliferation in mice model of CP-induced immunosuppression. This speculates that these Chinese medicinal herbs and their active ingredients are good candidates as an efficacious adjuvant therapy for immunomodulation. Nonetheless, more studies are required for the specific immunological mechanism of the extracts and the interaction with other medicines to support these conclusions. The findings of the present study may provide experimental evidence for further researches and clinical application of the polyherbal preparation.

Financial support and sponsorship

The Project of Science and Technology Agency of Gansu (1504FKCA010) and Science and Technology Agency of Lanzhou (2014-2-30) and the item of scientific and technological researches from Gansu Province Administration Bureau of Traditional Chinese Medicine (GZK-2015-19).

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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