Pharmacological Evaluation of an Optimized Polyherbal Suspension for Anti- Inflammatory Activity in Experimental Models

Inflammation is a normal reaction of the body to tissue damage, infection by microorganisms, chemical irritants, or harmful substances 1. The inflammatory process is vital to the protection of tissues and healing from injury, but chronic or abnormally high levels of inflammation can be a factor in the development of a range of chronic pathological conditions such as rheumatoid arthritis, cardiovascular diseases, diabetes mellitus, inflammatory bowel diseases, and neurodegenerative diseases. So, inflammation control is still a big challenge in healthcare today Anti-inflammatory agents used in the treatment of inflammatory disorders include synthetic anti- inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. These drugs relieve symptoms, but can cause gastric ulceration, gastrointestinal irritation, renal failure, hepatotoxicity and immunosuppression with prolonged use. Due to such restrictions, efforts have been focused on the search of safer therapeutic alternatives from natural sources 3. For centuries, medicinal plants have been an important source of therapeutic agents in traditional medicines. A significant anti-inflammatory activity has been reported for numerous secondary metabolites that are found in plants such as flavonoids, phenolic compounds, tannins, terpenoids, alkaloids, glycosides, and steroids 4. They are pharmacologically active due to their inhibitory activity on the cyclooxygenase and lipoxygenase pathways, inhibition of the release of inflammatory mediators, and action as a scavenger of reactive oxygen species and as a modulator of inflammatory signaling pathways 5. The notion of polyherbalism is a crucial part of Ayurvedic and traditional medicine systems, which involve the formulation of medicinal plant mixtures for the purpose of creating a synergistic effect between the bioactive constituents 6. Polyherbal formulations are thought to be more effective than single plant preparations because they have multi-target pharmacological action and have reduced dose related toxicity 7. In recent years, with the increasing interest in herbal therapeutics, scientific validation and pharmacological standardization of formulations have become of great importance. The medicinal plants chosen in the current study such as Commiphora mukul, Moringa oleifera, Curcuma longa, Cissus quadrangularis, Zingiber officinale, Cinnamomum verum and other traditionally important plants have been individually reported to have anti-inflammatory, antioxidant and protective pharmacological properties. However, little scientific data are available on optimized combinations of these medicinal plants in vivo pharmacological evaluation 8. Thus, the present study was conducted to assess the anti-inflammatory activity of the optimized polyherbal suspension on the basis of the known experimental animal models. The study was aimed to scientifically validate the developed formulation by carrageenan induced paw edema model for its traditional medicinal application.

MATERIALS AND METHODS

2.1 Collection and Authentication of Plant Materials

The plants selected for the present investigation were those with medicinal importance that have been reported to possess anti-inflammatory properties. The plants selected for use as medicine were Commiphora mukul (gum resin), Moringa oleifera (stem bark), Cinnamomum verum (bark), Elettaria cardamomum (fruit), Eucalyptus globulus (leaf), Aloe vera (leaf), Ricinus communis (seed), Cissus quadrangularis (stem), Linum usitatissimum (seed), and Capsicum annuum (fruit). The plant materials were gathered from the local herbal sources and natural habitat of Uttar Pradesh, India in February 2024. All plant materials collected were thoroughly cleaned, shade dried at room temperature and stored in appropriate condition for further processing 9. All the medicinal plants used in this study were taxonomically authenticated at the Institute of Environment and Sustainable Development, Banaras Hindu University, Mirzapur (U.P.), India, by Dr. Rajani Srivastava (Assistant Professor). Voucher specimens were deposited in the herbarium of the department for future reference.

2.2 Chemicals and Reagents

Analytical grade chemicals/reagents were used throughout the present investigation. Ethanol and methanol were used for extraction procedures. The polyherbal suspension was prepared by using the following ingredients, such as carboxymethyl cellulose, polysorbate-80, potassium sorbate, and citric acid. The standard anti-inflammatory drugs used were Diclofenac sodium and indomethacin 10.

2.3 Experimental Animals

Healthy Wistar albino rats weighing 150–200 g of either sex were used for the experimental studies. The animals were bought from the registered animal house facility of United Institute of Pharmacy, Prayagraj, Uttar Pradesh, India 11. The animals were kept at standard laboratory conditions of 25 ± 2°C temperature, 55 ± 5% relative humidity and 12 h light/dark cycle. Pellets were fed and water was provided ad libitum during the entire study. For experimentation, animals were acclimatized to laboratory condition. All experimental protocols carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA). The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. UIP/IAEC/Nov. 2025/18) of United Institute of Pharmacy, Prayagraj, Uttar Pradesh, India.

2.4 Preparation of Hydroalcoholic Extracts

The plants material collected was shade dried and coarsely ground using mechanical grinder. The size of the powders was uniformed by passing through sieve no. 40 12. Cold maceration method with ethanol: water (70:30 v/v) extraction solvent was used for the extraction of individual plant materials. About 100 g of the plant powder was macerated in 1000 mL of hydroalcoholic solvent with shaking at intervals at room temperature for seven days. The extracts were filtered through muslin cloth followed by Whatman filter paper No.1 and concentrated under reduced pressure with a rotary vacuum evaporator for the preparation of dried extracts. The dried extracts were kept in airtight containers at 4°C for further use 13.

2.5 Preparation of Polyherbal Formulations

Three polyherbal formulations designated as F1, F2, and F3 were prepared by mixing hydroalcoholic extracts of selected medicinal plants in predetermined ratios using geometric dilution method to ensure uniform blending and homogeneity 14.

2.6 Preparation of Optimized Polyherbal Suspension

Based on the preliminary phytochemical screening and antioxidant evaluation results, the optimized formulation (F3) was used for the preparation of polyherbal suspension. The amounts of each extract required were dispersed in distilled water with the addition of carboxymethyl cellulose as a suspending agent and polysorbate-80 as a wetting agent. Potassium sorbate was used as preservative and citric acid as pH adjuster. Final volume was adjusted with distilled water, and formulation was thoroughly mixed to obtain a homogeneous suspension. Prior to pharmacological testing, the appearance and homogeneity of the prepared suspension, sedimentation characteristics and ability to re-disperse were assessed visually 15.

2.7 Acute Oral Toxicity Study

Optimized polyherbal suspension was subjected to acute oral toxicity study as per guidelines laid down by OECD (423 Acute Toxic Class Method). Experimental animals were monitored continuously for behavior, toxicity, mortality, food consumption, water consumption and autonomic after oral application of the formulation. Animals were periodically observed for 14 days to identify delayed toxic symptoms. Based on the toxicity study, suitable dose levels of the formulation were selected for anti-inflammatory evaluations 16.

2.8 Evaluation of Anti-inflammatory Activity

Optimized polyherbal suspension was tested for its anti-inflammatory activity in various experimental models of inflammation in Wistar albino rats. The details of the experimental models employed in the present study are summarized in Table 1 17.

Group	Day 0 (g)	Day 7 (g)	Day 14 (g)
Control	192 ± 5.4	198 ± 6.2	205 ± 7.1
F1	190 ± 4.8	197 ± 5.6	203 ± 6.4
F2	195 ± 6.1	201 ± 6.7	208 ± 7.5
F3	193 ± 5.2	200 ± 5.8	207 ± 6.9

Figure 1: Effect of polyherbal formulations on body weight during acute oral toxicity study

Figure 2: Effect of polyherbal formulations on hematological parameters during acute oral toxicity study.

Figure 3: Effect of polyherbal formulations on biochemical parameters during acute oral toxicity study.

Figure 4: Effect of polyherbal formulations on body weight during 28-day oral toxicity study.

Figure 5: Effect of polyherbal formulations on hematological parameters following repeated oral administration.

Figure 6: Effect of polyherbal formulations on biochemical parameters following repeated oral administration.

Figure 7: Histopathological sections of liver, kidney, and heart tissues showing normal cellular architecture (H&E stain, 400×).

Group	% Inhibition
Indomethacin	50.58%
F1 Low	15.29%
F1 High	27.05%
F2 Low	23.52%
F2 High	31.76%
F3 Low	29.41%
F3 High	41.17%

Figure 8: Effect of high dose polyherbal formulations (F1, F2, and F3) on carrageenan- induced paw edema in rats.

Group	% Inhibition
Indomethacin	30.67%
F1 Low	6.67%
F1 High	12.00%
F2 Low	9.33%
F2 High	14.67%
F3 Low	13.33%
F3 High	20.00%

Figure 9: Effect of high dose polyherbal formulations (F1, F2 and F3) on egg albumin- induced paw edema in rats.

Group	% Inhibition
Indomethacin	50.00%
F1 Low	18.18%
F1 High	26.14%
F2 Low	22.73%
F2 High	29.55%
F3 Low	28.41%
F3 High	37.50%

Figure 10: Effect of high dose polyherbal formulations (F1, F2 and F3) on formalin- induced paw edema in rats.

The produced formulations were effective in significantly decreasing the paw edema caused by formalin when compared with the disease control group. The maximum inhibition was shown by F3 high dose with highly significant activity (p < 0.001) among all the tested groups.

3.3.4 Combined Discussion of Anti-inflammatory Activity

All of the developed formulations, F3 proved to be more effective in the anti-inflammatory activity in the carrageenan-induced, egg albumin-induced and formalin-induced inflammation models. The synergistic effect of F3 may be due to the presence of flavonoids, phenolic compounds, terpenoids and glycosides as observed during the phytochemical screening and TLC Finger print analysis. These phytoconstituents have been reported to have anti-inflammatory and anti-oxidative properties against inflammatory diseases.

CONCLUSION

In the present investigation, anti-inflammatory efficacy and safety profile of the optimized polyherbal suspension prepared using selected medicinal plants traditionally used for inflammatory disorders was successfully proved. The developed formulations were subjected to acute and sub-acute oral toxicity studies and in experimentally induced inflammatory models in Wistar albino rats. The toxicity testing in accordance with OECD guidelines showed good safety, non-toxic and well tolerated formulations, no mortality, no behavioural abnormalities and no significant toxic manifestations throughout the experimental period. Hematological and biochemical parameters remained in normal physiological limits and histopathological examination did not reveal any tissue damage caused by the treatment of any vital organs like liver, kidney and heart. In all the models of paw edema tested, F3 has the greatest anti-inflammatory activity in carrageenan-induced, egg albumin-induced and formalin-induced paw edema. The formulation was found to be dose-dependent in inhibiting inflammation and showed the pharmacological activity similar to indomethacin, which is the standard anti-inflammatory. The optimized formulation may be responsible for the increased therapeutic activity, which could be explained from the synergic activity of the different bioactive phytoconstituents such as flavonoids, phenolics, terpenoids and glycosides of the selected medicinal plants. The overall results of the present study support scientifically the traditional medicinal usage of the developed polyherbal formulation and indicate that the optimized polyherbal suspension exhibit good anti-inflammatory activity with acceptable safety. Hence, developed formulation can be considered a potential herbal therapeutic approach for inflammation related diseases and should be explored in clinical and mechanistic studies.

ACKNOWLEDGEMENT

The authors are thankful to the School of Health & Allied Sciences, Career Point University, Kota, Rajasthan, and United College of Pharmacy, Prayagraj, Uttar Pradesh, for providing necessary facilities and support to carry out the present research work. The authors also express sincere gratitude to Dr. Rajani Srivastava, Banaras Hindu University, for authentication of the plant materials used in the study.

REFERENCES

1. Harvanová G, Duranková S. Inflammatory process: Factors inducing inflammation, forms and manifestations of inflammation, immunological significance of the inflammatory reaction. Alergologia Polska-Polish Journal of Allergology. 2025;12(1):54- 61.
2. Bennett JM, Reeves G, Billman GE, Sturmberg JP. Inflammation–nature's way to efficiently respond to all types of challenges: implications for understanding and managing ―the epidemic‖ of chronic diseases. Frontiers in medicine. 2018 Nov 27; 5:316.
3. Kasturi J, Palla PR, Bakshi V, Bogg N. Non-steroidal anti-inflammatory drugs: an overview. Journal of Drug Delivery & Therapeutics. 2019 Jan 2.
4. Napagoda M, Wijesundara D. Medicinal plants as sources of novel therapeutics: The history, present, and future. Chem. Nat. Prod. Phytochem. Pharm. Med. Plants. 2022 Apr 19;3:4-18.
5. Liu J, Han X, Zhang T, Tian K, Li Z, Luo F. Reactive oxygen species (ROS) scavenging biomaterials for anti-inflammatory diseases: from mechanism to therapy. Journal of hematology & oncology. 2023 Nov 30;16(1):116.
6. Karole S, Shrivastava S, Thomas S, Soni B, Khan S, Dubey J, Dubey SP, Khan N, Jain DK. Polyherbal formulation concept for synergic action: A review. Journal of Drug Delivery & Therapeutics. 2019 Jan 2.
7. Rajini PS, Muralidhara M. Therapeutic efficacy of ayurvedic polyherbal formulations (PHF): Interactive mechanisms and broad-spectrum activities against neurological disorders. InAyurvedic herbal preparations in neurological disorders 2023 Jan 1 (pp. 89- 111). Academic Press.
8. Mukherjee PK, Banerjee S, Kar A. Molecular combination networks in medicinal plants: understanding synergy by network pharmacology in Indian traditional medicine. Phytochemistry reviews. 2021 Aug;20(4):693-703.
9. Korus A. Effect of preliminary processing, method of drying and storage temperature on the level of antioxidants in kale (Brassica oleracea L. var. acephala) leaves. LWT-Food Science and Technology. 2011 Oct 1;44(8):1711-6.
10. Usin S, Omolara DO. Phytochemical analysis and evaluation of ethanol and aqueous extracts of Piliostigma thonningii leaf for in vitro antioxidant activities. Nigerian Journal of Biochemistry and Molecular Biology. 2022 Mar 31;37(1):72-81.
11. Lord LK, Ingwersen W, Gray JL, Wintz DJ. Characterization of animals with microchips entering animal shelters. Journal of the American Veterinary Medical Association. 2009 Jul 15;235(2):160-7.
12. Senawong K, Katekaew S, Juntahum S, Laloon K. Impact of grinding and sorting particle size on phytochemical yield in Dipterocarpus alatus leaf extract. International journal of biomaterials. 2023;2023(1):4512665.
13. Chuah PN, Nyanasegaram D, Yu KX, Mohamed Razik R, Al-Dhalli S, Kue CS, Shaari K, Ng CH. Comparative conventional extraction methods of ethanolic extracts of Clinacanthus nutans leaves on antioxidant activity and toxicity. British Food Journal. 2020 Oct 5;122(10):3139-49.
14. Anwar S, Kausar MA, Parveen K, Zahra A, Ali A, Badraoui R, Snoussi M, Siddiqui WA, Saeed M. Polyherbal formulation: The studies towards identification of composition and their biological activities. Journal of King Saud University-Science. 2022 Oct 1;34(7):102256.
15. Yap VL, Tan LF, Rajagopal M, Wiart C, Selvaraja M, Leong MY, Tan PL. Evaluation of phytochemicals and antioxidant potential of a new polyherbal formulation TC-16: additive, synergistic or antagonistic? BMC Complementary Medicine and Therapies. 2023 Mar 28;23(1):93.
16. Paule MG, Meck WH, McMillan DE, McClure GY, Bateson ME, Popke EJ, Chelonis JJ, Hinton SC. The use of timing behaviors in animals and humans to detect drug and/or toxicant effects. Neurotoxicology and teratology. 1999 Sep 1;21(5):491-502.
17. Joshi P, Yadaw GS, Joshi S, Semwal RB, Semwal DK. Antioxidant and anti-inflammatory activities of selected medicinal herbs and their polyherbal formulation. South African journal of botany. 2020 May 1; 130:440-7.
18. Hajhashemi V, Minaiyan M, Banafshe HR, Mesdaghinia A, Abed A. The anti- inflammatory effects of venlafaxine in the rat model of carrageenan-induced paw edema. Iranian journal of basic medical sciences. 2015 Jul;18(7):654.
19. Najim SM, Abd MR, Fadhil AA, Hassan AF. Fimasartan attenuates edema and systemic changes in egg albumin-induced paw inflammation in rats. Journal of Advanced Pharmacy Education & Research| Jan-Mar. 2023;13(1).
20. Soyocak A, Kurt HÜ, Cosan DT, Saydam FA, Calis IU, Kolaç UK, Koroglu ZO, Degirmenci I, Mutlu FS, Gunes HV. Tannic acid exhibits anti-inflammatory effects on formalin-induced paw edema model of inflammation in rats. Human & Experimental Toxicology. 2019 Nov;38(11):1296-301.
21. Russell DW, Kahn JH, Spoth R, Altmaier EM. Analyzing data from experimental studies: A latent variable structural equation modeling approach. Journal of counseling psychology. 1998 Jan;45(1):18.