Immunomodulatory Effect of Gymnema sylvestre (R.Br.) Leaf Extract: An In Vitro Study in Rat Model

Gymnema sylvestre Wild R.Br (family: Asclepidaceae) is a valuable medicinal plant used in folk medicine to treat diabetes, obesity, asthma etc. in India for antiquity. Diabetes mellitus is a syndrome characterized immunologically by lymphocyte apoptosis and reduced cell-mediated and humoral immunity. Modulation of immune responses to alleviate diseases has been of interest, and traditional herbal medicines may play an important role in this regard. In this study, we aim to evaluate the immunomodulatory potential of methanolic extract of G. sylvestre leaf using rat model. HPLC analysis of leaf extract was carried out for gymnemic acid. The method involves the initial hydrolysis of gymnemic acids, the active ingredients, to a common aglycone followed by the quantitative estimation of gymnemagenin, using gymnemagenin as reference standard. Gymnemic acid content was 2.40% (w/w) in G. sylvestre leaf extract. In vitro immunomodulatory activity of the methanolic extract of G. sylvestre leaf (1–200μg/ml) was evaluated by gauging its effects on nitroblue tetrazolium reduction and nitrite release in rat peritoneal macrophages and on mitogen (ConA, PHA and LPS) induced splenic lymphocyte proliferation. G. sylvestre leaf extract showed significant (<0.05) enhancement in NO and ROS generation in macrophages and in proliferation of lymphocytes in dose dependent manner. EC50 value was 3.10, 3.75 and 2.68μg/ml for NBT reduction, nitrite release and lymphoproliferation, respectively. Potential effect was observed at 100 μg/ml in NO and ROS generation in macrophages and 20 μg/ml in lymphocyte proliferation. G. sylvestre leaf extract stimulates macrophage reactivity, increasing the level of activity even higher when combined with PMA or LPS. These findings suggest the presence of active compounds, gymnemic acid, in methanolic extract of G. sylvestre leaf that stimulates both myeloid and lymphoid components of immune system, and therefore can restore the innate immune function. Through this study, the traditional knowledge of anti-diabetic property of G. sylvestre is scientifically supplemented with its immunomodulatory properties.


INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.
Objectives 4 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

Ethical statement 5
Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Study design 6
For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when). c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.

7
For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example: a. How (e.g. drug formulation and dose, site and route of administration, anaesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s). b. When (e.g. time of day). c. Where (e.g. home cage, laboratory, water maze). d. Why (e.g. rationale for choice of specific anaesthetic, route of administration, drug dose used).  Allocating animals to experimental groups 11 a. Give full details of how animals were allocated to experimental groups, including randomisation or matching if done. b. Describe the order in which the animals in the different experimental groups were treated and assessed.

12
Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).
Statistical methods 13 a. Provide details of the statistical methods used for each analysis. b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

Baseline data 14
For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).
Numbers analysed 15 a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50% 2 ).
b. If any animals or data were not included in the analysis, explain why.

Outcomes and estimation
16 Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval).
Adverse events 17 a. Give details of all important adverse events in each experimental group. b. Describe any modifications to the experimental protocols made to reduce adverse events. c. Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research.

Generalisability/ translation
19 Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology.
Funding 20 List all funding sources (including grant number) and the role of the funder(s) in the study.