TLC-Based Metabolite Profiling and Bioactivity-Based Scientific Validation for Use of Water Extracts in AYUSH Formulations

We aimed to develop a chromatographic method for scientific validation of water extract of some important Indian traditional plants used in AYUSH-based formulation as immunomodulator and to evaluate their bioactive potential. Fruits of Phyllanthus emblica L. and Piper nigrum L., stem of Tinospora cordifolia (Willd.) Miers, rhizome of Curcuma longa L., leaves of Ocimum sanctum L. and Achillea millefolium L., roots of Withania somnifera L., and stem bark of Azadirachta indica A. Juss. were coarsely powdered and extracted in three different solvents (water, ethanol, and hydroethanol). The antioxidant potential was determined through 1, 1-diphenyl-2-picrylhydrazyl and ferric reducing capacity methods. Thin-layer chromatography (TLC) was carried out for the comparative metabolite profiling of the extracts using toluene, ethyl acetate, and formic acid (5 : 4 : 1, v/v/v) as a solvent system. In vitro immunomodulatory activity of the extracts has been tested on splenocyte proliferation and pinocytic assay. Hydroethanolic extract (HEE) of most of the plant materials has the highest phenolic and flavonoid contents, followed by water extract (WE) and ethanolic extract (EE), whereas the water extracts of most of the plant material showed better antioxidant activity. Almost all extract exhibited splenocyte proliferation and pinocytic activity in a dose-dependent manner. But water extract showed significantly higher splenocyte proliferation and pinocytic activity as compared to the other two extracts. TLC analysis resulted in detection of totally 63 and 56 metabolites at 254 nm and 366 nm, respectively. Through principal component analysis (PCA), it was observed that metabolite pattern of different extracts from same plant materials may be different or similar. This preliminary result can be used for quality evaluation and to develop a synergy-based polyherbal combination of water extracts of selected plant materials.

M-CSF-Stimulated CD11b+ Myeloid Cells Induce Alopecia Areata in C3H/HeJ Mice

Alopecia areata (AA) is a T cell-mediated autoimmune skin disease with clinical features of hair loss and skin inflammation. It is unclear whether other immune cells except T lymphocytes are also involved in the development of AA. Here, our results reveal that dermal injection of either CD11b+ myeloid cells isolated from AA-affected skin or non-AA splenocyte-derived CD11b+ cells treated with macrophage colony-stimulating factor (M-CSF) induces AA in C3H/HeJ mice. The functional similarity of these cells in induction of AA seems to be due to a higher expression of M-CSF in AA affected skin. To explore the mechanism by which dermal injection of CD11b+ cells induce AA, we co-culture either AA derived skin cells or M-CSF-stimulated CD11b+ cells with naïve splenocytes. The results of splenocyte proliferation assay and immunoglobulin release in conditioned medium show a significant increase of splenocyte proliferation and IgG level in conditioned medium under both conditions as compared to controls. Most activated splenocytes induced by M-CSF-stimulated myeloid cells are B lymphocytes. B cell activation are further confirmed in AA-affected skin and skin draining lymph nodes of AA mice. In conclusion, in this study, we have provided evidence that M-CSF stimulated CD11b+ cells are able to induce AA in C3H/HeJ mice through a possible mechanism by activating B lymphocytes. This finding may provide insight for understanding the pathogenesis of AA.

Macrophage immunomodulatory activity of Acanthopanax senticousus polysaccharide nanoemulsion via activation of P65/JNK/ikkαsignaling pathway and regulation of Th1/Th2 Cytokines

Nanoemulsions (NE) are used widely in pharmaceutical drug formulations and vaccine preparation, and Acanthopanax senticousus polysaccharide (ASPS) is a natural bioactive compound with immunostimulatory activity. Therefore, NE-loaded ASPS is expected to provide immunological enhancement for effective treatment. In the present study, Acanthopanax senticousus polysaccharide (ASPS was encapsulated into nanoemulsions, the resultant ASPS–NE were coated with a negative charge, and the immune enhancement mechanism of these ASPS-NE formulations was analyzed. The immunosuppressive animal models (70 ICR mice, male) for the study were established using cyclophosphamide. In addition, the activation of splenocyte proliferation, phagocytosis of the macrophages, the ratio of CD4+ to CD8+, the concentrations of the cytokines in serum, Western blot analysis was used for the analysis of the P65/JNK/ikk α signaling pathway in the peritoneal macrophage s. The results revealed that the ASPS-NE could stimulated the proliferation of splenocytes and enhance immunity. The ASPS-NE induced the expression of different cytokines (TNF-α, IFN-γ, IL-2, and IL-6), could activate the expressions of P65, JNK, and ikkα, and regulated the Th1/Th2 cytokines. These findings demonstrated the potential of ASPS-NE formulations for drug delivery and to induce potent and sustained immune responses.

Echinacea purpurea Alleviates Cyclophosphamide-Induced Immunosuppression in Mice

Echinacea purpurea (EP) has been widely used to treat upper respiratory infections, influenza, and the common cold. It can also exert various pharmacological activities, such as anti-inflammatory and anti-allergic effects. However, the potential of EP to modulate immune reactions remains unclear. Therefore, we evaluated the immunostimulatory effects of EP in cyclophosphamide (CP)-induced immunosuppressed mice. In this study, EP extract (12.5, 25, or 50 mg/kg) was orally administered to cyclophosphamide-induced immunosuppressed BALB/c mice. Then, indexes of immune organs, including the spleen and thymus, were recorded. Splenocyte proliferation and natural killer (NK) cell activities were measured by lactate dehydrogenase assay. Subsets of T cells, such as CD4+ and CD8+, were measured by flow cytometry, and immuno-cytokines, such as interleukin (IL)-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, were measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The immunosuppressed mice showed decreased thymus and spleen indexes and immune cell activities. Treatment of EP elevated the indexes of immune organs, splenocyte proliferation, and NK cell activities in CP-induced immunosuppressed mice. Simultaneously, administration of EP reversed the CP-induced decrease in T-lymphocyte subsets (CD4+ and CD8+) and immunocytokines (IL-2, TNF-α, and IFN-γ). Taken together, these findings suggest that EP could be used to enhance health and immunity in immunosuppressed conditions.

Silk Sericin Activates Mild Immune Response and Increases Antibody Production

To clarify whether nanoparticles of silk sericin (SS) and silk fibroin (SF) can induce inflammation and immune responses, we analyzed splenocyte proliferation, apoptosis and cytokine release to identify the effects of SS and SF on mouse splenocytes in vitro. We implanted mice with SS and SF through intraperitoneal, intramuscular, and subcutaneous routes to evaluate the innate and adaptive immune response to SS and SF in vivo. Cytokines in the serum and spleen were analyzed by Luminex and antibody array. Antigen-specific antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) at week 1 and 5 after implantation. Distinct cell populations in the spleen and bone marrow were analyzed by flow cytometry. SS suppressed the proliferation of splenocytes and CD11b+CD27− NK cells, induced splenocyte apoptosis, and increased interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in the culture supernatant. SF suppressed splenocyte proliferation, induced splenocyte apoptosis, and increased the titer of TNF-α in culture supernatants. At both week 1 and 5 after implantation with SS, mouse serum interleukin-1 α (IL-1 α) and keratinocyte chemoattractant (KC) were decreased, SS-specific antibody was increased, the proportion of bone marrow CD4+ T cells was increased, and the proportion of splenic neutrophils was decreased. At week 5 after subcutaneous implantation with SF, mouse serum IL-1α, and splenic IL-6, TIMP-1, IL-4, MCP-1, IFN-γ, TCA-3, TNF-α, and IL-17 were decreased. SS was able to induce a mild immune response, as evidenced by CD4+ T cell activation, splenocyte apoptosis, and antigen-specific antibody secretion. Comparatively, SF had low immunogenicity and anti-inflammatory properties.

Immunomodulation Effect of Kaempferia galanga Ethanolic Extract On In Vitro Lymphocyte Cell Proliferation

<p><em>Kaempferia galanga</em> L. is a traditional medicine with antitumor properties, as indicated by its immunomodulatory activities. This study aimed to determine the effect of <em>K. galanga</em> on lymphocyte cell proliferation activity as an indicator of immunomodulatory properties. This study was conducted at the Indonesian Research Center for Veterinary Science (February to April 2018). The immunomodulatory activity of the extract was evaluated with an <em>in vitro</em> splenocyte proliferation assay. The assay was based on cellular enzymatic synthesis to transform the XTT from formazan tetrazolium as an indicator. The <em>K. galanga</em> extract was obtained by 96% ethanol extraction. The test was conducted in an aseptic condition, consisted of five treatment groups with three replications each. Three groups of splenocyte cell culture, each with extract concentration of 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1, as well as a positive (Concanavalin A/Con A) and negative (cell only) control. The cell suspension (10x10⁴cells/ml) was distributed on 96-well plates and cultured following the treatment groups. The same five plates were made for five days of observation and retrieved daily by observing an Elisa reader at 450 nm. The extract of <em>K. galanga </em>at 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1 significantly (P &lt;0.05) promoted splenocyte proliferation compared to control. Therefore, it was expected that <em>K. galanga </em>has a high potential to be used as immunomodulators. Hence, further investigations should be done to clarify the mechanisms of the immunomodulatory effect of <em>K. galanga</em> as an antitumor <em>in vivo</em>.</p>

Structural Characterization and Immunomodulatory Activity of a Novel Polysaccharide From Lycopi Herba

Lycopi Herba has been broadly used as a traditional medicinal herb in Asia due to its ability to strengthen immunity. However, it is still obscure for its material basis and underlying mechanisms. Polysaccharide, as one of the most important components of most natural herbs, usually contributes to the immunomodulatory ability of herbs. Here, we aimed to detect polysaccharides from Lycopi Herba and examine their potential immunomodulatory activity. A novel polysaccharide (LHPW) was extracted from Lycopi Herba and purified by DEAE-52 cellulose chromatography and G-100 sephadex. According to physicochemical methods and monosaccharide composition analysis, LHPW was mainly composed of galactose, glucose, fructose, and arabinose. NMR and methylation analyses indicated that LHPW was a neutral polysaccharide with a backbone containing →3,6)-β-D-Galp-(1→, →4)-β-D-Galp-(1→ and →4)-α-D-Glcp-(1→, with the branches of →1)-β-D-Fruf-(2→ and →6)-α-D-Galp-(1→. Immunological tests indicated that LHPW could activate macrophage RAW264.7 and promote splenocyte proliferation. This study discovered a novel polysaccharide from Lycopi Herba and showed it was a potential immunomodulator.

Synthesis, Physicochemical Characteristics and Plausible Mechanism of Action of an Immunosuppressive Isoxazolo[5,4-e]-1,2,4-Triazepine Derivative (RM33)

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 μg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

Result of splenocyte proliferation assay of preparation of Pteridium aquilinum

Bracken (Pteridium aquilinum, L. Kuhn), which is used widely in the Eastern and Western medicine and food, has attracted researchers interest. It is related with norsesquiterpene glucoside ptaquiloside, which is carcinogen, contained in the bracken. We quantitatively determined ptaquiloside in the subdued bracken, which is a way of decreasing or inactivating toxic effect and increasing the treatment activity in raw material of drug. The results showed the ptaquiloside was present in both samples, with sample 1 of 10.06 ug/g and with a minimum of 0,41ug/g in subdued sample 2. Therefore, the aim of the present study was to evaluate subdued sample 2 splenocyte proliferation assay using by MTT kit and splenocyte from Balb/c mice. MTT assay showed cell viability index of group of Соn A was 231,4 and cell viability index of preparation group was 233,7. Also, the group of Preparation with Con A was showed the high ability stimulate. Эгэл бавран (pteridium aquilinum (l.) Kuhn)-гийн бэлдмэлийн дэлүүний эсийн хуваагдлыг олшруулах идэвхийг судалсан дүн Өрнө, Дорнын анагаах ухаан болон хүнсэнд хэрэглэгдэж ирсэн Эгэл бавран (Pteridium aquilinum (L.) Kuhn) нь судлаачдын сонирхлыг ихээхэн татаж ирсэн билээ. Энэ нь түүнд агуулагдах норсесквитерпений бүлгийн гликозид болох птаквиклозид нь хорт хавдар үүсгэдэг (канцероген)-тэй холбоотой юм. Бид Эгэл бавранг уламжлалт аргаар номхотгол хийж, Өндөр мэдрэмжит шингэний хроматографиар птаквиклозидын хэмжээг тодорхойлоход номхотгосон дээжинд 0.41 мкг/г, номхотгоогүй дээжинд 10.06 мкг/г хэмжээтэй птаквиклозид агуулагдаж байсан юм. Тиймээс номхотгол хийсэн дээжний дархлаанд үзүүлэх нөлөөг in vitro орчинд Balb/c үүлдрийн цагаан хулганы дэлүүний эсийг олшруулах идэвхээр МТТ өнгөт урвалын аргаар судлан тогтоов. Митоген буюу Соn A-тэй үүрний эсийн амьдрах чадварын индекс нь 231,4, загвар бэлдмэлтэй үүрний эсийн амьдрах чадварын индекс 233,7 байгаа нь хяналтын бүлэгтэй ойролцоо дүн үзүүлсэн байна. Харин бэлдмэлийг митогентэй хамт хэрэглэхэд эсийн амьдрах чадварын индексийг хамгийн их нэмэгдүүлж байна. Түлхүүр үг: птаквиклозид, дэлүүний эс, MTT өнгөт урвалын арга, номхотгол

Aqueous Extract of Kan-Lu-Hsiao-Tu-Tan Ameliorates Collagen-Induced Arthritis in Mice by Inhibiting Oxidative Stress and Inflammatory Responses

Background: Kan-Lu-Hsiao-Tu-Tan (KLHTT) exhibits anti-psoriatic effects through anti-inflammatory activity in mice. However, the therapeutic effects of KLHTT on rheumatoid arthritis (RA), another significant autoimmune inflammatory disorder, have not been elucidated. Herein, we explored the anti-arthritic effects of KLHTT on collagen-induced arthritis (CIA) in mice. Methods: KLHTT was extracted by boiling water and subjected to spectroscopic analysis. Chicken collagen type II (CII) with complete Freund’s adjuvant was intradermally injected to induce CIA in DBA/1J mice. Anti-CII antibody, cytokines, malondialdehyde (MDA), and hydrogen peroxide (H2O2) were measured using ELISA, thiobarbituric acid reactive substances, and a hydrogen peroxide assay kit. Splenocyte proliferation was tested using thymidine incorporation. Th1 and Th17 cells were analyzed by flow cytometry. Results: Oral KLHTT treatment (50 and 100 mg/kg) ameliorated mouse CIA by decreasing the levels of interleukin (IL)-1β, IL-6, IL-17A, and tumour necrosis factor-α in the paw homogenates and serum. KLHTT also suppressed anti-CII antibody formation, splenocyte proliferation, and splenic Th1 and Th17 cell numbers. Additionally, KLHTT showed antioxidant activity by reducing the concentrations of MDA and H2O2 in paw tissues. Conclusions: The therapeutic effects of KLHTT in CIA mice were through regulating oxidative stress and inflammatory responses. Our results suggest that KLHTT has potential to treat RA.