Evaluation of Synergism and Carbon clearance model (Macrophage phagocytosis) by using Ethanolic extracts of Convolvulus pluricaulis, Michelia champaca and Chromolaena odorata in Rats
1Assistant Professor, Department of Pharmaceutical Chemistry,
LSHGCT’s Gahlot Institute of Pharmacy, Koparkhairane, Navi Mumbai - 400709, Maharashtra, India.
1Professor and Head of Department (Pharmaceutics),
Pacific Academy of Higher Education and Research University, Udaipur - 313024, Rajasthan, India.
2Professor, Department of Pharmacognosy,
LSHGCT’s Gahlot Institute of Pharmacy, Koparkhairane, Navi Mumbai -400709, India.
2Principal, LSHGCT’s Gahlot Institute of Pharmacy, Koparkhairane - 400709, Navi Mumbai,
Maharashtra, India.
2Senior Specialist, Rashid Hospital ED, Dubai, United Arab Emirates.
*Corresponding Author E-mail: mkpawar230@gmail.com
ABSTRACT:
Background: Evaluation of preclinical immunomodulatory models are very crucial in drug discovery and development. Dysfunctional immune system is associated with disorders like autoimmune diseases, inflammatory diseases, cancer and as a treatment immunosuppressive or immunostimulant drugs are given aiming at immune system disease. Literature states that plants carry phytochemicals which exerts various biological activity in humans, mainly anti-inflammatory and anti-cancer properties which are likely to overcome the complications or adverse effects caused by synthetic drug treatments. Objective: To evaluate immunomodulatory effects of selected three medicinal plant extracts of Convolvulus pluricaulis (CP), Michelia champaca (MC), and Chromolaena odorata (CO) individually and to evaluate synergistic effects by combination index (CI) from herbal combination (COMB) prepared by combination of above three herbal extracts. Materials and Methods: The ethanolic extract of whole part of selected plants at low and high dose levels were subjected for macrophage phagocytosis by in-vivo Carbon clearance method based on acute oral toxicity studies. Results: The extracts showed increase in phagocytic index in comparison to control groups. Significant response was seen at both the dose levels for CP extract, at high dose for MC and CO extract and at low dose for COMB extract. Combination index: The combination index was found to be <1 for herbal combination, indicative of synergism among the drugs. Conclusion: The extracts has shown effects on cellular immune responses and has therapeutic potential to modulate the immune system and could be employed as an effective immunomodulatory plant in future. The combination of selected three herbal extracts was exerting synergistic effects to modulate immune system.
KEYWORDS: Macrophage phagocytosis, Carbon clearance method, Synergism, Combination index.
INTRODUCTION:
Vertebrates may encounter millions of microorganisms like pathogenic bacteria, viruses, fungi, and protozoa that can interfere with immune functions and cause damage to the host. Immunology comprises of evolutions that has better understanding of many diseases like cancer, immunological behavior of autoimmune disease, organ transplantation etc. Our immune system has complex biological structures and processes comprising of collection of cells and organs which protects us from disease. These cells play important role in sustaining immunity within our body and it is performed by either phagocytosis against microorganisms or by attacking non-self-cells by release of natural killer cells (NKC).1,2 When a phagocyte examines a cell, including your cells or bacteria and viruses that have entered the body, it tests them to determine whether the cell is a pathogen. Pathogens have pathogen-associated molecular patterns (PAMPs) and phagocytes have pattern recognition receptors (PRRs). So when a phagocyte examines a cell, including your cells or bacteria and viruses that have entered the body, it tests them to determine whether the cell is a pathogen by looking out for these features – PAMPs that human cells do not have. Once the PRR recognizes PAMP, it binds the cells (adherence) and initiates the phagocytosis process.
Macrophages are white blood cell type which engulf and digest microorganisms, clear our debris and dead cells thus playing an important role in human immune system. They present antigens to immune cells such as T cells for further destruction. 2-4 Figure 1 shows the detail functions of macrophage.5
Figure 1 Macrophage functions5
Immunodeficiency diseases are the conditions in which body’s defense mechanisms are weakened or damaged, leading to severe microbial infections recurring again and again. This type of infections may intensify the susceptibility to malignancies (Figure 2).6 Specific body’s defense by host cells either by humoral or cell mediated immunity (macrophage phagocytosis) is stimulated by pathogen acting as an antigen.2 Allopathic medicines used in the treatment have certain risk factors and may have severe side effects thus impacting life.7 Since ancient times, plants have been extensively used for treating various disorders and several important chemical constituents having therapeutic efficacy have been identified (Figure 3).8,9 Level of side effects and toxicity associated with herb related drugs are rare and can benefit populations requiring herbal remedies to treat certain immune disorders 8-14
Multiple herbs are used in combination or polyherbal formulation (more than one herb) for treating illness as per traditional medicinal systems. As compared to single herbal formulation, polyherbal formulation have greater benefits due to synergism.14-16 Also, active phytoconstituents in individual extract are not adequate to achieve noteworthy therapeutic benefits. Hence, combining multiple herbs in a particular ratio delivers better therapeutic effect and may reduce toxicity.17-19 Hence combined extracts of plants are preferred than individual extracts. Different approaches are used to determine the synergistic, additive effect and antagonistic effect of plants.20,21 In this research we have adopted Loewe additivity model for calculating combination index and evaluating synergism in combination extract. Three medicinal plants were selected – Convolvulus pluricaulis, Michelia champaca and Chromolaena odorata for in-vivo immunomodulatory study - as the plants are well known ethnobotanical herbs.
Figure 2 Immunodeficiency disease - a Cause of cancer
Figure 3 Phytochemicals involved in Immunomodulation
The plants were collected from Sanjay Gandhi National Park, India and authentication was performed at Agharkar Research Institute, Pune, India by Dr. R.K. Chaudhary. The extract of whole plant was obtained by using solvent ethanol in soxhlet extractor (50 g in 500 ml). The whole plant extract was prepared by using ethanol as a solvent (50 g in 500 ml) in soxhlet extractor. Storage of extracts was done at 8-15ºC.
The extracts were tested for presence of phytochemicals as per the procedure mentioned in C.K. Kokate.22,23 The GC-MS and LC-MS studies were carried on all three extracts at IIT Bombay for structural elucidation of chemical constituents present in extracts.
The protocol (CRY/2021/028) for animal studies was approved by Institutional Animal Ethics Committee (IAEC) of Crystal Biological Solutions Laboratory, Pune, Maharashtra, India (Reg No. 2030/PO/RcBiBt/S/ 18/CPCSEA) and was in accordance with the committee for the purpose of control and supervision of experiments on animals (CPCSEA, 1960).
Total 60 rats were used for carbon clearance model, six rats of wistar strain in each group of either sex having 150 to 200 g body weight. For acute oral toxicity – total 9 female rats were used. Acclimatization period: 7 days, environmental conditions: Room temperature between 22±3°C, relative humidity 55±5% and 12 hours’ light and 12 hours’ dark cycle was maintained. Accommodation: three rats in each cage with clean paddy husk. Diet: Pelleted feed supplied by Nutrivet Pvt Ltd. ad libitum during the study. Water: RO filtered water was provided ad libitum. Food deprivation protocol was followed for 16 h prior to study with free access to water. Dosing was given according to body weight of rats.
CP, MC, and CO ethanolic extracts at low and high dose – 100 and 200mg/kg.
Combination (COMB) of above three ethanolic extracts at single low dose of 100 mg/kg.
Standard drug cyclophosphamide at 50mg/kg was used.
Suspending agent - 0.5% carboxymethyl cellulose (CMC) was used. Test items were suspended in 0.5% CMC before dosing
Six rats in each group were used for this study. Extracts were dosed as per the standard protocol and animals were examined for signs and symptoms. Body weights were recorded on weekly basis.
Rats for study were observed clinically for salivation, lacrimation, urination, palpitation, heart rate tremors and convulsions on daily basis and recorded in daily observation chart.
|
Group |
Type of groups |
Route of administration |
|
1 |
Control (0.5% sodium CMC solution) as vehicle |
Oral |
|
2 |
Disease control (Induction of disease) - Animal Indian ink (colloidal carbon) |
Intravenous ink dispersion through tail vein |
|
3 |
CP – 100 mg/kg |
Oral |
|
4 |
CP – 200 mg/kg |
Oral |
|
5 |
MC – 100 mg/kg |
Oral |
|
6 |
MC – 200 mg/kg |
Oral |
|
7 |
CO – 100 mg/kg |
Oral |
|
8 |
CO – 200 mg/kg |
Oral |
|
9 |
COMB – 100 mg/kg |
Oral |
|
10 |
Standard (cyclophosphamide) – 50 mg/kg |
Oral |
Acute oral toxicity:
OECD guideline 423 was followed for acute oral toxicity study in female rats.24,25 The study was conducted at two dose levels – 300 mg/kg and 2000 mg/kg. Interpretation of results (LD50) were done as per the globally harmonized system (GHS) classification of chemicals. The following conditions were followed:
The rats were observed for first 30 mins, 1 hour, 2 hours, 3 hours and 4 hours after dosing for any clinical signs and symptoms like changes in skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic, central nervous systems, somatomotor activity, behavior pattern. Afterwards, for once a day animals were observed for total 14 days. Rats were also observed for any tremor, convulsions, salivation, diarrhea, lethargy, sleep and coma. All the observations were recorded in daily observation chart.
Body weights:
Body weights of rats were observed and recorded on test day 0 (before treatment) and on days 7 and 14 after treatment or at death.
Necropsy:
Rats were subjected for gross necropsy and all body openings were observed with naked eye to study if there are any alteration in normal body organs like brain, heart, liver, lungs, kidneys, spleen, pancreas, adrenal glands, ovaries etc.
Rats were orally administered with vehicle, extracts and standard drug for 10 days. After 48 hours of last oral drug administration, intravenous injection (0.3 ml per 30 g) of animal Indian ink (colloidal carbon) through rat’s tail vein was administered. After the administration of ink injection, blood samples of each rat at an interval of 0 and 15 mins were collected in micro-centrifuge tubes by retro-orbital puncture method. 50µl blood was lysed with 4ml of 0.1% Na2CO3 solution. Measurement of absorbance was done at 660nm on UV visible spectrophotometer. Phagocytic index was calculated as below:
K=(LogeOD1-LogeOD2)/15; OD1 is optical density at 0 min and OD2 is optical density at 15 mins.
D1 D2
CI = --------------- + --------------
(Dx)1 (Dx)2
Where, (Dx)1 and (Dx)2 defines dose of drug D1 and D2 individually.
Interpretation of results were accomplished using one-way ANOVA method (dunnett’s test) and the graph data is expressed as mean ± SEM. P value < 0.05*, 0.01**, 0.001***, 0.0001**** indicates significant effect of treatment in rats when compared to control groups.
Results for phytochemical screening are as per table 1. In our previous research, various phytoconstituents belonging to phenols/flavonoids, terpenoids, alkaloids, glucosinolate etc. were identified in the extracts of CP, MC and CO through GC-MS and LC-MS analysis.31-33
Table 1: Phytochemical screening of extracts
|
Test |
CP |
MC |
CO |
|
Alkaloids |
+ |
+ |
+ |
|
Carbohydrates |
+ |
+ |
+ |
|
Phenolic compounds and tannins |
+ |
+ |
+ |
|
Flavonoids |
+ |
+ |
+ |
|
Proteins and amino acids |
- |
- |
+ |
|
Gums and mucilages |
+ |
- |
+ |
|
Steroids |
- |
+ |
- |
Plant extracts (ethanolic) of CP, MC and CO were found to be therapeutically safe and LD50 value was found to be >2000 – 5000mg/kg (category 5) when treated at dose 300mg/kg and 2000mg/kg. Female rats appeared normal throughout the study. Normal body weight gain was observed during 14 days’ observation period and there were not any signs of toxicity considering weight gain. Gross pathological alterations were not seen in any female rats in any group.
The CP extract was found to be significant at 100 and 200mg/kg. The extract of MC and CO were not significant at 100mg/kg. However, significant results were obtained when dose was administered at 200mg/kg for both MC and CO extract. The combination extract (COMB) displayed promising results which were significant at lower dose of 100 mg/kg. Thus, the selected plants showed convincing immunomodulatory activity when compared to control group in this study model.
Table 2: Evaluation of results for carbon clearance (phagocytic index) model
|
Types of groups |
Treatment groups (mg/kg body weight per oral – b. wt. p. o.) |
Phagocytic index |
|
Group I (Vehicle control) |
0.5% sodium CMC |
0.0012±0.000213 |
|
Group II (Disease control) |
Colloidal carbon ink |
0.0014±0.000222 |
|
Group III |
CP (100) |
0.0024±0.000069* |
|
Group IV |
CP (200) |
0.0032±0.000247**** |
|
Group V |
MC (100) |
0.0028±0.000078 |
|
Group VI |
MC (200) |
0.0030±0.000213*** |
|
Group VII |
CO (100) |
0.0021±0.000128 |
|
Group VIII |
CO (200) |
0.0029±0.000144*** |
|
Group IX |
COMB (100) |
0.0036±0.000152**** |
|
Group X |
Standard (50) |
0.0033±0.000195**** |
Figure 4 Carbon clearance response (Phagocytic index, K)
The synergistic effect from the herbal combination (COMB) was obtained by combination index, where CI was found to be <1, indicative of synergism among the drugs.
The colloidal carbon preparation – Indian ink, when injected intravenously, phagocytes in liver and spleen eliminate carbon particles. The carbon clearance involves macrophage activity and rapid elimination of carbon particles from blood circulation is ascribed to reticuloendothelial (RET) cells. Statistical data shows linear growth which suggests that extracts have shown stimulation of macrophage. This is confirmed by results which indicates the dose related increase in carbon clearance rate by RET cells. The extracts have found to support B and T cells concepts of immunity. 26,29,34 The combination at 100 mg/kg showed significant increase in phagocytic index as 0.0036±0.000152****. This indicates synergism in the combination extract. The standard cyclophosphamide showed phagocytic index of 0.0033±0.000195****.
Considering this research work, we could conclude that the selected plant extracts (ethanolic) possess the immunostimulatory potentials when studied for in-vivo immunomodulatory model of carbon clearance. The combination of the above three plant extracts was exerting synergistic effects to modulate the immune system, and the synergistic effect obtained by combination index was found to be CI<1, indicative of synergism among the drugs. This research data could be referred and considered in the future, when finding new drug molecules to treat immunodeficiency diseases especially cancer or inflammation. The immunomodulatory potential of these plants can be attributed to the phenols/ flavonoids found in these plants through GC-MS and LC-MS analysis in our previous work.31-33 To enhance the medicinal effects of the plant, researchers can turn into novel drug delivery systems such as nanoemulsions, metallic nanoparticles, magnetic microspheres, liposomes.35,36
We would like to thank Agharkar Research Institute, IIT Bombay, Crystal Biological Solutions Lab, Gahlot Institute of Pharmacy, Kashibai Navale College of Pharmacy for providing laboratory facility to conduct and study this work.
CONFLICT OF INTEREST:
None.
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Graphical Abstract:
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Received on 01.02.2024 Revised on 14.06.2024 Accepted on 05.09.2024 Published on 12.06.2025 Available online from June 14, 2025 Research J. Pharmacy and Technology. 2025;18(6):2441-2446. DOI: 10.52711/0974-360X.2025.00348 © RJPT All right reserved
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