Biological Properties of Polyherbal Formulations: A Review of their Antimicrobial, Anti-Inflammatory, Antioxidant, and Toxicological Activities

The use of traditional and folklore systems of medicine has been an important part of healthcare worldwide


INTRODUCTION
Throughout history, natural remedies made from herbs have been used to treat various physiological disorders, and are generally considered safe.According to the World Health Organization, around 80% of the global population relies on herbal-based products to treat different ailments. 1he global market for herbal formulations is currently estimated at $1.5 billion and it is expected to expand significantly as demand for natural remedies used for preventing and curing different diseases increases. 1rbs/herbal products are vital sources of novel pharmaceuticals because they contain a significant amount of secondary metabolites, such as alkaloids, flavonoids, isoflavonoids, lignans, quinones, catechols, coumarins, polyphenols, lectins, monoterpenes, and triterpenes which are accountable for the positive effects of the herbal products. 1,2The quality and quantity of these bioactive compounds determine the therapeutic effectiveness of herbal formulations. 1For instance, due to the abundance and diversity of the flavonoid and phenolic content of Datura stramonium, it has been reported to exhibit significant antiinflammatory and antimicrobial activities. 3rgemone mexicana, on the other hand, contains a lot of alkaloids and has traditionally been used to treat various dermatological disorders, warts, and inflammatory complications. 3 When monotherapy is inadequate to combat a clinical condition, traditional medicine employs polyherbal approaches. 4lyherbal medicines, which contain two or more herbal ingredients are often more effective than single drugs because of their complementary and/ or potentiating activities.The combination of two or more herbal extracts brings about increased therapeutic efficiency, enhanced pharmacological actions, faster relief, and reduced adverse effects as compared to conventional medicine due to a lower dose of administration. 1,2,4,5Polyherbal medicines are now widely preferred and used around the world because of their high effectiveness, ready availability, low toxicity, and environmentally friendly nature, and it reduces the time of treatment or the individual cost of anti-inflammatory and antimicrobial drugs, resulting in lower prescription costs. 3,5The concept of polyherbal combination has been well established and has achieved remarkable success in allopathic medicine, providing patients with new hope. 6esistant to antimicrobials.Therefore, plant-derived products can regulate microbial growth in a variety of circumstances.Despite the development of new antibiotics over the last three decades, the problem of microbial resistance to these drugs has increased.Bacteria are known to have the genetic ability to transmit and acquire resistance to drugs used as therapeutic agents, 8 which underscores the importance of discovering and developing novel pharmaceuticals.
Inflammation refers to the response of tissue to harmful stimuli such as physical injury, irritants, and pathogens.This response is identified by an increase in vascular permeability, alterations in blood flow, and migration of leukocytes to the affected regions. 9e five distinguishing characteristics of inflammation are tumour (swelling/oedema), colour (redness), dolour (pain), fever (warmth), and functio laesa (organ/tissue dysfunction). 10The body responds to inflammation by releasing proinflammatory cytokines such as interleukins (IL-1β and IL-6), interferons (IFN), and tumour necrosis factor-alpha (TNF-α).These cytokines induce cyclooxygenase-2 (COX-2) which produces prostaglandins (PGs) leading to inflammation, swelling, and pain.Another inflammatory mediator, leukotrienes, is produced by the metabolism of arachidonic acids by lipoxygenase (LOX).Activation of macrophages results in the production of reactive oxygen species (ROS) which causes oxidative stress. 11idative stress stimulates the release of proinflammatory cytokines and nitric oxide (NO) which exacerbate inflammatory diseases.Nitric oxide helps in the inhibition of mitochondrial enzymes and the activation of COXs in the production of PGs.Therefore, any substance or drug that inhibits COX-2, LOX, NO, and ROS can be useful in the prevention and treatment of inflammatory diseases. 11To manage inflammation, there are numerous drugs available, however, the most commonly used are non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, indomethacin, naproxen, ketoprofen, and ibuprofen. 12These drugs have limitations such as being palliative, inaccessible, unaffordable and having low efficacy, along with causing undesirable effects that may even be life-threatening. 9,13Therefore, more research is needed to explore medicinal plants or polyherbal medicines as potentially effective therapeutic agents for inflammatory diseases.
Antioxidants play a crucial role in reducing cellular components' oxidation by molecular oxygen, preventing the chain reactions that result in the production of harmful free radicals like reactive oxygen species (ROS) and reactive nitrogen species (RNS).By removing free radical intermediates and inhibiting further oxidative reactions while being oxidized themselves, antioxidants are considered as important as nutraceuticals due to their many health benefits.They protect the body against oxidative stress linked to various chronic diseases and disorders.Antioxidants are any substance that, even in minute concentrations, can inhibit or delay the oxidation of a substrate or other molecules. 14herefore, antioxidants due to their ability to scavenge free radicals could potential be useful in the prevention and treatment of diseases linked to oxidants or free radicals.Phenolic acids, which are found in plants, have been shown to have a variety of biological effects, including antioxidant activity. 15Antioxidant enzymes such as superoxide dismutase (SOD) and catalase act as defensive mechanisms by providing NADPH, which is required for the regeneration of glutathione (GSH) and protection against oxidative damage.Additionally, oxidative stress can lead to a decrease in glutathione S-transferase (GST) activity and a reduction in GSH content. 16e 2,2 diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay is a commonly employed in vitro antioxidant assay used to determine the antioxidant potential of polyherbal extracts and in measuring the radical scavenging capability.The biological agent's ability to scavenge free radicals can be used to calculate antioxidant activity. 17DPPH is a free radical having the strong ability to accept electrons to become a stable diamagnetic molecule.The potent DPPH radical scavenging activity of polyherbal formulation (PHF) has been attributed to its hydrogen-donating ability.The decrease in DPPH solution absorbance following the addition of PHF can be attributed to the strong reaction between the antioxidant-rich formulation and the DPPH solution.This reaction leads to the scavenging of the DPPH radical, which can be visualized by the colour change from purple to yellow in the DPPH solution upon the addition of PHF.The significant results of PHF in the DPPH in vitro antioxidant test indicate that it has a high reducing potential and can effectively scavenge free radicals. 18rthermore, ABTS assay involves the generation of a blue/green chromophore resulting from the reaction between ABTS and potassium persulfate.This assay measures the relative ability of antioxidant compounds in plant extracts to scavenge the ABTS radical cation (ABTS), compared to standard quantities of synthetic antioxidant Trolox and the water-soluble vitamin E analogue. 5Thus, antioxidants present in plants have potent properties for degenerating and scavenging harmful free radicals, such as ABTS. 5 This study has reviewed all the research work conducted on antimicrobial, anti-inflammatory and antioxidant properties of polyherbal formulations in the past twelve year around the world.This is to validate and promote the use of polyherbal formulations, identify gaps in current knowledge and highlights area which more research is needed as well as to identified the herbal combination which can be used in the development of new pharmaceuticals.
Even though polyherbal remedies have reportedly been used for the management and treatment of various illnesses, concerns regarding their safety have been raised. 19It is important to note that the quality, safety, and efficacy of polyherbal formulations can vary depending on the source and preparation method.Therefore, standardized formulations and proper quality control measures are necessary to ensure their safety and efficacy.

MATERIALS AND METHODS
The literature search was conducted from September 2022 to December 2022 and the study is based on a mixed-method review approach, which includes combining quantitative and qualitative research.A systematic comprehensive literature search was conducted on the following databases Google scholar, publishing sites such as Elsevier, JStor, Scopus, Science Direct, Cab Direct and BioMed Central (BMC), and PubMed.The databases and literature sources were chosen based on the topic covered and the main search and key terms included "polyherbal formulation", "antimicrobial", "anti-inflammatory", "toxicity" and "antioxidant activities".Search terms were set to be in the title, keywords, and abstract.The review focuses on work done on polyherbal medicine all over the world and the flow of study is shown in Figure 1.

RESULTS AND DISCUSSION
The therapeutic use of non-toxic conventional medicinal plants is gaining increasing attention as an alternative and cost-effective approach for the prevention and treatment of various diseases. 20ccording to the World Health Organization, about 60-80% of the population in developing countries relies primarily on plants for their primary healthcare needs.The rise of herbal medicines has significantly increased foreign trade, and the global herbal medicine market is projected to hit approximately USD 117 billion by 2024. 21For centuries, natural herbs and extracts have been utilized in both developed and developing countries to cure different ailments.Natural products have a distinct chemical richness, which results in a diversity of biological activities and drug-like properties; as a result, they have emerged as one of the most valuable tools for creating new lead compounds and scaffolds.These products will continue to be used in order to meet the urgent need for effective drugs development, and they will play a pivotal role in the discovery of drugs for the treatment of human diseases. 22Moreover, synthetic antimicrobial agents available in the market exhibit certain drawbacks such as toxicity, low efficacy, the emergence of antimicrobial resistance, and high cost, which render them ineffective.Therefore, the development of a potent cure derived from plants would be a significant step forward in the treatment of microbial diseases. 23,24lyherbal preparations, which are made from a combination of different medicinal plants, contain multiple bioactive constituents that interact with one another to achieve increased therapeutic effectiveness in the maintenance of human health. 25,268][29][30] This review reports that the polyherbal formulations exhibit significant antimicrobial, anti-inflammatory, and antioxidant activities (Table 1), and play a major role in the treatment and management of various diseases.The article lists some commercial and non-commercial polyherbal formulations all around the world and found that India has topped the number of publications on polyherbal formulations followed by Nigeria, and Pakistan (Figure 2).

Antimicrobial activity of polyherbal formulations
Based on ethnobotanical research, plants have been identified as a potential natural source of antimicrobial drugs that will provide novel or lead compounds that can provide unique or primary compounds to combat infections globally. 313][34] Tannins, for instance, are water-soluble polyphenols found in many plant foods and have been recognized as effective antibacterial agents against various pathogens.They have been reported to prevent the growth of microorganisms (fungi, yeasts, bacteria, and viruses) by precipitating microbial protein. 35,36annins can deactivate enzymes that cause microbial adhesion, as well as hinder the movement of certain proteins.They also combine with polysaccharides to form a complex. 37Similarly, flavonoids form a complex with either soluble or extracellular proteins and eventually bind to the bacterial cell wall.On the other hand, lipophilic flavonoids can considerably disturb the microbial cell membrane.Alkaloids are known to possess anti-tumour, analgesic, antispasmodic, and bactericidal potentials; Saponins are prominent for their anti-cancer, antioxidant, antimicrobial, and anti-inflammatory properties, 35,36 and steroids contain anti-inflammatory activity. 35The antibacterial activity of these plant constituents is due to their ability to either block the production of the bacterial cell wall or disrupt the permeability of the cell membrane, ultimately resulting in the death of the microorganisms. 3,5tibacterial agents work by inhibiting or regulating enzymes involved in cell wall biosynthesis, nucleic acid metabolism, and protein synthesis, a process known as translation inhibition.Another mechanism is membrane structure disruption, which causes changes in cellular functions.The majority of antibiotics are designed to reduce multiplication, thereby killing the organism.Some phytoconstituents in herbal formulations may bind to Gram-negative bacteria's membrane phospholipids and disrupt membrane integrity.Furthermore, it may inhibit peptidoglycan synthesis by blocking specific enzymes, thereby reducing the multiplication of bacteria and killing the organisms. 38e review findings revealed that the antimicrobial effectiveness of polyherbal formulations varied depending on the concentration and type of microorganisms tested.Some of the herbal remedies, such as THR-SK004, THR-SK010, and THR-SK011 used for the treatment of wounds demonstrated potent antibacterial activity against methicillinresistant S. aureus, methicillin-susceptible S. aureus and S. aureus with MIC 90 values of 4, 8 and 4 µg/mL respectively.This indicates that these herbal remedies meet the criteria proposed by Cos et al. (2006), which consider extracts with a selective activity and MIC values below 1-50 µg/mL as noteworthy.Also, polyherbal remedies such as Ambrex (8.7-23.4μg/mL), 4 Ya-Sa-Marn-Phlae (4-32 µg/mL) 20 , and KWTa, KWTb, KWTc, HBfs, HBts, AL, and FB (≤ 25 µg/mL) 39 had antimicrobial activities lower than 50 µg/mL, hence, they possess strong antimicrobial activity.
The majority of the polyherbal formulations demonstrated synergistic broad-spectrum antibacterial and antifungal activity, with MIC values ranging between 0.625-10 mg/mL.Some of the formulations, such as Ambrex, Chyawanprash, Plashbijadi churna, Vranahitkara ghruta, NOQ19, and Polyherbal nano colloids, etc exhibited antimicrobial activity comparable to the standard drugs.This suggests that these polyherbal remedies have the potential to be used as alternative treatments for bacterial and fungal infections.However, some polyherbal remedies such as Leone Bitters, Chandra Kalka, Sharkaradi Kalka, and Diakure did not possess any antimicrobial activity.This  The nitric oxide (NO) inhibitory results revealed that THR-SK010 ethanol extract exhibited NO production inhibition activity with IC50 value of 71.06, while the water extract was apparently inactive (IC50 > 100 µg/mL).
Inhibition of lipopolysaccharide induced nitric oxide production.

Antimicrobial:
The ethanol extracts of the herbal remedies had strong antibacterial potency against methicillin-resistant S. aureus, methicillinsusceptible S. aureus, and S. aureus at the MIC 90 values of 4, 8, and 4 µg/mL respectively.However, none of the water extracts of the formulations displayed anti-staphylococcal activity In vitro antibacterial assay using broth microdilution method.

Thailand 50 Antiinflammatory:
The nitric oxide (NO) inhibitory results revealed that THRSK011 ethanol extract exhibited NO production inhibition activity with IC50 value of 72.67 µg/mL, while the water extract was apparently inactive (IC50 > 100 µg/mL).
Inhibition of lipopolysaccharide induced nitric oxide production.

5.
Polyherbal used for the treatment of vaginal infection.

Antimicrobial:
The hydroalcoholic extract of the remedy demonstrated synergistic broad-spectrum antibacterial and antifungal activity against S. aureus, S. agalactiae, E. coli, C. albicans, and A. fumigatus with the MIC values ranged between 5-7 mg/mL.
In vitro antibacterial assay using agar well diffusion method, India 21

Antimicrobial:
The methanolic extracts of the remedy were significantly effective against S. aureus, B. subtilis, E. coli, and P. aeruginosa in a concentration-dependent manner.
Agar well diffusion and broth dilution method. India.

Antiinflammatory:
When compared to normal and negative control animals, the polyherbal (2% and 5%) had a strong and maximal antiinflammatory effect after 180 minutes of carrageenan injection.
In vivo experiment using Adult Wistar rats. Toxicological: The polyherbal was found to be safe up to a dosage of 2000 mg/kg, with no changes in the hair, skin, or behavior of treated animals.Also, there was no signs of skin irritation or adverse reaction in the rats.
In vivo assay using Adult Wistar rats. 12.
Prepared from 37 different herbs Antimicrobial: The result depicted that all the polyherbal remedies had anti-tubercular activity, with some showing more activity at concentrations less than 25 µg/mL.Also, the MIC values exhibited inhibitory activity against M. tuberculosis at 1.562 µg/mL.However, the control isoniazid exhibited more inhibitory activity at 0.05 µg/mL.In vitro antibacterial assay by agar well diffusion method.
In vitro antiinflammatory activity by membrane stabilization method. Anti-oxidant: In the DPPH assay, the maximum scavenging activity in terms of percentage of inhibition was observed in methanol extract (98.63%) and ethyl acetate extract (98.55%), whereas the minimum scavenging activity was observed in water extract (84.21%) and hexane extract (83.34%).The extracts showed higher activity in comparison with the natural antioxidant rutin (60.32), and the synthetic BHT (64.40) which probably marks the potential antioxidant capacity.The FRAP assay showed that the methanol extract had the highest reducing power activity of 117.67 ± 1.17 mM Fe (II) E/g extract followed by hexane extract (84.

Albezzia lebbeck Cyprus rotandus Solanum xanthocarpum
Antimicrobial: Shrishadi possesses strong antimicrobial activity with the highest activity against P. aeruginosa and C. parapsilosis at 16 mg/mL and 15 mg/mL respectively.
In vitro analysis using disc diffusion method. India.33 23.

PHF used for the treatment of wounds
Manilkara zapota (leaf), The result revealed that 0.5 mL of the preparation had activity against P. acnes, S. aureus, S. epidermidis with ZOI of 18.33 mm, 19.20 mm, and 26.30 mm respectively, while the standard drug (clindamycin) was at 26.80 mm.
In vitro assay by agar well diffusion method. India.

Antiinflammatory:
A significant reduction in paw oedema was observed in AHPL/AYCAP/0413 and Diclofenac groups as compared to the control group.The percentage inhibition of paw oedema for the AHPL/AYCAP/0413 and for standard Diclofenac sodium groups was 51% and 58% respectively.
In vivo experiment using carrageenan-induced paw oedema in rats.

Antimicrobial:
The ethanol extract of choornam inhibited the growth of S. aureus and P. aeruginosa.
The inhibitory zone of the extract was 2.1 cm against P. aeruginosa and 1.9 cm against S. aureus while ciprofloxacin showed an inhibitory zone of 3.1 cm against P. aeruginosa and 3 cm against S. aureus.
In vitro antibacterial assay by agar well diffusion method.India. Anti-oxidant: The extract also showed a dose-dependent ability to scavenge DPPH radicals thus establishing its antioxidant potential.
The antioxidant potential of the choornam was analyzed by DPPH assay.

27.
DAS-77® effective in the treatment of piles, dysentery, cholera, menstrual pain, stomach disorder, ulcer, constipation, diarrhoea, and sexually transmitted infections Mangifera indica (bark), and Carica papaya (root). Antimicrobial: The MIC of the extract against S. aureus and E. coli was at 100 mg/mL and 50 mg/mL respectively, while that of ciprofloxacin and tetracycline were at 50 µg/mL and 62.5 µg/ mL, respectively.The extract did not show any inhibitory effect against E. faecalis and P. aeruginosa.
The antimicrobial activity using the agar-well diffusion method.

Nigeria
Toxicological: The result showed that DAS-77 was not toxic as no mortality and visible signs of toxicity were observed I the mice upon administration of DAS-77 up to 20 mg/kg.Writhing, grooming, increased locomotor activity, and convulsion were the behavioral manifestations observed with the intraperitoneally route.
In vivo assay using Mice.

Antimicrobial:
The methanolic extract inhibited the growth of one or more test pathogens than the aqueous extract. -India.

Antimicrobial:
The result depicted that DiaKure fails both the antibacterial and anti-fungal activity tests.
In vitro antimicrobial by cup plate method and filter paper disk method. India.

30.
Ativishadi churna used in the treatment of diarrhea.
In vitro antibacterial by using disk diffusion method. India.

Ficus benghalensis, Ficus racemose, Ficus religiosa and Ficus microcarpa
Antimicrobial: The remedy was found to possess activity against S. aureus with the ZOI of 20.0 mm.
Aladejana EB, et al.Biological Properties of Polyherbal Formulations: A Review of their Antimicrobial, Anti-Inflammatory, Antioxidant, and Toxicological Activities 32.
PHF used against gastrointestinal diseases.
Made from a total of 25 plants species to form 14 polyherbal recipes. Antimicrobial: The polyherbal remedies showed activity against both tested fungal and bacterial isolates with the inhibition zone ranging from 19.67 mm -30 mm.Some of the polyherbal recipes have enhanced antimicrobial potential with better efficacy than tested antibiotics.

PH combination
Withania somnifera, Bacopa monnieri, and cinnamomum zeylanicum Antimicrobial: The Polyherbal formulation showed moderate to mild antibacterial activity against most of the tested bacteria.
In vitro antibacterial by the cup and plate method.Australia 99 Anti-oxidant: The reducing power of the extract increased with the concentration.
Antioxidant activity was determined by Ferric ion-reducing antioxidant power assay. 34.
Agar disc diffusion and agar well diffusion assay.

Sri Lanka 100
Anti-oxidant: The antioxidant activity of the 20% ointment samples was higher than the 10% ointment samples.The reducing power increased with the increased concentration.

Antioxidant activity was determined by
Ferric ion-reducing antioxidant power assay.

Antimicrobial:
The cream inhibited the growth of P. acnes, S. epidermidis and S. aureus with the ZOI of 20.68, 28.20, and 21.40 mm respectively, which was comparable to clindamycin.The overall MIC of the cream was observed at 2.5 mg/mL.
Agar well diffusion method.

India. 101 Antiinflammatory:
The result revealed that there was a significant reduction in rat paw oedema (43%) with AHPL/AYTOP/0213 which was also comparable to diclofenac sodium cream (56.09%).
In vivo experiment using carrageenan-induced paw oedema in rats.

Toxicological:
The findings showed that the rat's skin where AHPL/AYTOP/0213 cream was applied showed no erythema or edema, and the primary skin irritation index of the cream was calculated as 0.00.
In vivo assay using Wistar albino rats.

36.
Chandra Kalka used for the treatment of respiratory disorders of paraplegia disease and congestion.Sharkaradi Kalka used for the treatment of fever in children as well as, cough, asthma conditions and constipation.
A total of 31 medicinal plants are used to prepare the Chandra Kalka and 18 are used in the preparation of the Sharkaradi Kalka Antimicrobial: Both PHF do not possess antibacterial activity at the concentration of 1 mg/mL against E. coli, S. aureus, B. subtilis, and S. epidermidis.
Agar well diffusion method.

Sri Lanka 51
Antiinflammatory: Both formulations showed moderate antiinflammatory activity as they were able to stabilize the erythrocyte membranes.
Anti-inflammatory activity was determined using a human red blood cell membrane stabilization assay.
Antioxidant activity was evaluated using the DPPH radical scavenging assay.

Antimicrobial:
The results showed that the formulation possesses antibacterial activity at the concentration of 5 mg/mL against E. faecalis, S. aureus, K. pneumonia, and P. aeruginosa.
In vitro antibacterial activity using agar well diffusion method.

India. 102
Anti-oxidant: The results showed that the formulation possesses a better scavenging effect at lower concentrations (100 μg/mL).
The FRAP assay reveal that the mixture showed dose depended on an increase in concentration with maximum absorption of 0.767 at 300 μg/mL.Antimicrobial:

DPPH
The PHF had significantly higher antibacterial activity than the control group.
Agar well diffusion method.

India 40
Anti-oxidant: The PHF exhibited the antioxidant activity of 90% at 100 ug/mL, whereas ascorbic acid was found to be 75.16  In vitro antibacterial by disc diffusion method.

Toxicological:
The methanol extract of polyherbal fraction showed cytotoxicity against the two cell lines with the LC 50 value 467 ± 2.9 mg/ml against HeLa cell line and >800 mg/ml against MCF-7 cell lines.
The extract was tested against HeLa and MCF cell lines.

41.
Entoban syrup used for the treatment of acute gastrointestinal infections.

Antimicrobial:
The syrup inhibited the growth of S. aureus, S. enterica, E. coli, S. dysenteriae, P. aeruginosa, and V. cholerae with the ZOI measured to be 18. 25   29.03 μg/mL) which was comparable with ascorbic acid (94%) IC 50  15.52 μg/mL.The hydrogen peroxide scavenging ability of the extract was found to be higher (IC 50 20.88μg/mL) than the ascorbic acid (19.81 μg/mL).
DPPH and hydrogen peroxide scavenging methods.

Toxicological:
The result showed that the formulation helps in the proliferation of fibroblast cells without affecting the viability.Cytocompatibility of the formulation was studied on mouse 3T3 fibroblast cells using MTT assay.

Aloe vera and Curcuma longa
Antimicrobial: The extracts exhibited potential antimicrobial activity against the tested microorganisms.
Agar well diffusion method.India.Antimicrobial: The remedy exhibited antimicrobial activity against E. coli, S. typhi, V. cholerae and K. pneumoniae with MICs between 0.8 -12.5 mg/mL.
In vitro antibacterial assay using the microdilution method.
Tanzania 106 Toxicological: The formulation showed very low acute toxicity in mice and in the brine shrimp lethality test.
The ethanol extracts of the remedy gave an LC 50 value of 66.12μg/ml.The formulation was well tolerated during the 14-day observation period and at doses up to 5000 mg/kg body wt.
In vitro assay using brine shrimp lethality test and in vivo using Sprague Dawley rats.

46.
Jatyadi Thailam (JT YG ) used for the treatment of chronic wound.

India. 108
Antiinflammatory: Both formulations exhibited significant dose-dependent anti-denaturation and antiproteinase activities with AFI exhibiting maximum inhibition of 91.05% and YG (52.24%) at the concentration of 2500 μg/ mL while the standard drug aspirin showed 95.53% inhibition of protein denaturation at its lowest concentration of 500 μg/mL.The thailam also showed marked stabilization of the human red blood cell membrane against thermal hemolysis and inhibited heat-induced hemolysis more effectively in a dose-dependent manner.Leone Bitters used as an appetite stimulant and stomachic.
Antimicrobial: Leone Bitters do not possess antimicrobial activity at the concentration used.
In vitro antimicrobial using the cup diffusion method.

Nigeria. 86
Toxicological: The acute toxicity study showed 80% death for all the animals that received 20.0 g/kg bwt of the extract and 42.8 % and 14.3 % for animals that received 15.0 g and 10.0 g respectively, while there was no death in the animals that received 5.0 g/kg bwt and less.Decrease in the body weight was observed in rats that received higher doses, no morphological changes in the colour of the organs however, the histological result revealed that at the dose of 500 mg/ kg bwt, the drug may have affected the sperm production rate in the testis.Also, no abnormality was observed in the tissue morphology of the liver, kidney and heart.However, there was significant changes in the various organs' weights especially in kidney, liver and brain in the animals.
In vivo assay using Swiss albino mice and Wistar rats.

Antimicrobial:
The findings revealed that PHF-M1 extract possesses considerable amount of antimicrobial inhibition against mastitis causing pathogens with the MIC values ranging from 24±0.58 to 12.33±0.33.
In vitro antimicrobial using agar diffusion method.

India. 77
Toxicological: The results of the acute oral toxicity test showed that PHF-M1 did not exhibit any toxicity at dose rates of 5, 50, 300, and 2000 mg/kg.The animals in the study showed no signs of ill health and their food and water intake remained normal.No behavioral changes were observed during the study period.
In vivo assay using Wistar rats.

51.
Forty-six herbal branded products -Antimicrobial: The result depicted that only nine of the herbal preparations exhibited better antimicrobial activities, while the rest of the formulations had no activity.
In The ZOI of the antimicrobial efficacy of the formulation was found to be 24 mm, 25 mm, and 25 mm; and the MIC values of 1.5 mg/ mL, 1.5 mg/mL, and 2.0 mg/mL against S. flexneri, S. enterica, and E. coli respectively.Also, the results show that the polyherbal formulation significantly reduced biofilm formation by 67.94%, 65.56%, and 51.94% in the presence of 0.8 mg/mL formulation against S. flexneri, S. enterica, and 0.9 mg/mL against E. coli when compared to the control.

In vitro assay by well-diffusion method and broth dilution method
The biofilm inhibition activity was determined on 96 well-polystyrene plates India 75 Toxicological: The result depicted that different concentrations (5 mg/kg, 50 mg/kg, 300 mg/ kg, and 2000 mg/kg) of PHF administered did not produce any significant changes in behavior, postural abnormalities, impairment in food and water intake, and loss or yellowing of hair.No mortality of animals was also observed.
In vivo assay using albino rats

Viola tricolor, Echinacea purpurea, Allium sativum, Matricaria chamomilla, Triticum repens
Antiinflammatory: The result showed that the remedy exerted significant (P < .05)anti-inflammatory effects in carrageenan-induced paw oedema mouse model in a time-dependent manner, with significantly higher efficacy at 250 mg/ Kg BW, than indomethacin (4 mg/Kg BW) In vivo experiment using Swiss albino mice Kenya 9 Toxicological: The result depicts that phytexponent was not toxic to Vero E6 cells (CC 50 >1000 μg/ml) compared to the control cyclophosphamide (CC 50 =2.48μg/ml).
In vitro assay in Vero E6 cell line

Bhāraṅgyādi Clerodendrum serratum, Hedychium spicatum and Inula racemosa
Anti-oxidant: The result suggests that the polyherbal formulation is a good potential for antioxidant activity as the reducing power showed a dose-depended increase in concentration with maximum absorption of 0. Toxicological: The result showed a progressive increase in mortality of brine shrimp with an increase in the concentration of the solution.At a concentration of 10 μg/mL, the test drug showed low mortality of 16.66% while at 1000 μg/mL it had a mortality of 86.66%.
In the acute toxicity study, at the dose of 5000 mg/kg, no treatment related toxic manifestations, behavioral changes, or mortality was observed.
In the sub-acute study, no sign of toxicity and lethality was observed, however, there was some hematological and biochemical variations observed at 3 different doses of 2000, 300, and 50 mg/kg bw.

Brine Shrimp and
In vivo assay using Wistar Rats

Leaves of Boerhavia diffusa, Solidago virgaurea, Vitex negundo, and thymoquinone
Anti-oxidant: The study evaluated the antioxidant activity of BSVT in CCl 4 induced hepatorenal toxicity in rats.The results showed that BSVT at a dose of 100 mg/kg was able to recover the reduced levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as the increased levels of malondialdehyde (MDA), in both liver and kidney tissues in a dose-dependent manner.
The results were similar to those of the standard drug Cystone®.The study concludes that BSVT ameliorates the hepatorenal toxicity in a dose-dependent manner.
In vivo (rats The results show that DRHM® has significant antioxidant activity in treating hydrogen peroxide-induced oxidative stress in rats.
The study found that H 2 O 2 intoxication significantly decreased serum activities of SOD, CAT, and GPx, while DRHM® treatment significantly increased the activities of these enzymes.In addition, DRHM® treatment significantly decreased the MDA concentration and increased the GSH concentration compared to the H 2 O 2 control.The findings suggest that the phytoconstituents in DRHM® are responsible for the ameliorative effects of boosting the antioxidant defense system.

Nigeria 114
Toxicological: The polyherbal formulation was tolerable up to 10 mL/kg b.w since there was no significant behavioural and body weight change as well as mortality within the study period.This finding indicates that the lethal dose is higher than 10 mL/kg b.w.The polyherbal formulation was tolerable in mice up to 10 mL/kg b.w, showing that the lethal dose is above 10 mL/kg b.w and hence, DRHM® is relatively safe.However, further studies are warranted to evaluate the long-term effects of using this polyherbal formulation especially at the molecular level.
In vivo assay using male Swiss albino mice Anti-oxidant: The results indicated that CCl4-induced liver damage significantly decreased the activities of hepatic antioxidant enzymes such as catalase (CAT), peroxidase (Px), glutathione-S-transferase (GST), and superoxide dismutase (SOD), as well as the level of vitamin C, while elevating the lipid peroxidation level.However, the administration of "Livshis" resulted in a significant recovery in the levels of these biosensors toward the control level.In comparison, the distilled water-pretreated cum CCl 4 -injected animals did not recover significantly from the liver damage induced by CCl 4 .
In vivo study using male albino rats India 115 Toxicological: The study depicted that treatment with Livshis up to 3200 mg/kg did not produce any lethal or toxic symptoms in the rats.
During the 14-day period after the oral administration, no mortality or morbidity, no tremors, salivation, diarrhoea, sleep, coma, death, or unusual behaviours were observed in the rats.
In vivo study on male albino rats The results showed that administration of ethanol to rats increased the levels of lipid peroxidation products such as thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HP) and decreased the levels of antioxidants.Co-administration of Livomap with ethanol significantly reduced the levels of these lipid peroxidative products and increased the levels of nonenzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione to near normal as compared to ethanol-induced rats.Livomap was found to be beneficial in alleviating ethanol-induced oxidative damage.The study suggests that Livomap could reduce the generation of free radicals and increase the scavenging mechanism of free radicals.The results also showed that Livomap significantly increased the activities of antioxidant enzymes such as SOD, CAT and GPx, which are important components of the cellular defense system against ROS and RNS, and are defense against oxidative damage by supplying NADPH, which is needed for the regeneration of GSH Anti-oxidant: The results showed that GOV possessed significant antioxidant activity.It dosedependently increased the activity of CAT, GPx, GSH, GST, SOD, and total protein in the serum, kidney, and liver of the rats with acetaminophen (APAP)-induced damage compared to the toxin control groups.Also, GOV significantly reduced the Malondialdehyde (MDA) contents in the liver of rats compared to the toxin-induced control group.The serum levels of CAT, GPx, GSH, GST, and SOD were significantly decreased in the APAP-treated toxin control group compared to the groups that were administered different doses (2, 4, and 8 g/ kg) of GOV.However, the activities of these enzymes were restored to normal by GOV administration.At 8 g/kg, GOV significantly increased the activity of GSH compared to all the groups, while at 4 g/kg, GOV increased the levels of GSH, GST, and total protein compared to Liv 52 and Silymarin groups.
In vivo study using Wistar albino rats Nigeria 116 Toxicological: The study depicted that the oral (16,000 mg/kg) and intraperitoneal (2,500 mg/ kg) administration of GOV did not result in deleterious effect or mortality 24 hr after and within seven days post treatment irrespective of the extract doses tested.
In vivo study using Wistar albino rats

Anti-oxidant
The IC 50 value of the formulation was found to be 62.45, indicating that Livomyn has significant antioxidant activity.Its antioxidant activity was also compared to ascorbic acid (ASC) as a standard, and the results showed that Livomyn was effective in action on free radicals.The results showed that the polyherbal formulation has the potential to scavenge free radicals, which are known to be the main cause of oxidative damage.
In vitro by 1, Anti-oxidant: The study found that paracetamol induced oxidative stress in the liver of mice, as well as a decrease in the activity of antioxidant enzymes such as glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-tranferase (GST), catalase (CAT), and glutathione (GSH) contents.However, Habbe-Asgand (250 mg/kg) showed significant antioxidative potential against paracetamol toxicity.It increases the activity of GR, GPx, GST, and CAT.These findings suggest that the antioxidant constituents in Habb-e-Asgand may be mediating the attenuation of liver injury caused by paracetamol metabolites.The study concludes that Habbe-Asgand may be used as a prophylaxis for ROS related liver injury.
In vivo assay using Swiss albino male mice India 61 The results demonstrated that PNK (100 and 500 mg/kg) has significant antioxidant activity as it significantly increased the levels of glutathione, superoxide dismutase, and catalase compared to the CCl 4 -treated group.Additionally, the decreased level of TBARS indicates that PNK has an antilipid peroxidative and/or adaptive nature against the damaging effects of free radicals produced by carbon tetrachloride.
In vivo (Wistar rats) India 64 Toxicological: The acute toxicity result showed that there was no mortality at any of the tested doses (up to 3000 mg/kg) at the end of 14 days.
In vivo (Wistar rats)
In particular, Karisalai Karpam at 100 and 200 mg/kg doses significantly produced in vivo antioxidant activity by restoring liver GSH, blood GSH, SOD, CAT, and GPx levels.Karisalai Karpam (200mg/kg) was found to be effective in increasing liver and blood GSH, SOD, and CAT levels.
In vivo using Wistar rats India 66

Solanum nigrum and Cichorium intybus
Anti-oxidant: The results showed that CCl4 administration caused a significant increase in TBARS levels and a decrease in GSH, SOD, and CAT levels in hepatic tissues.However, treatment with the polyherbal formulation (4.5 ml/ kg) significantly reduced TBARS levels and increased GSH, SOD, and CAT levels, indicating its antioxidant potential.
In Anti-oxidant: The study depicted that Virgoliv syrup (1 ml/ kg) showed significant antioxidant activity against CCl 4 -induced hepatic damage as the levels of antioxidant enzymes such as CAT, GSH, and SOD were significantly increased in liver tissue of rats treated with Virgoliv syrup and silymarin when compared to CCl 4 -treated rats.The increased MDA levels observed in CCl4-treated rats were also reduced after treatment with Virgoliv syrup.
In vivo assay using Sprague-Dawley rats India 68 Toxicological: The result of acute toxicity study revealed that VLS was safe up to 2000 mg/kg.The rats were alive, active and healthy during the period of observation.
In vivo assay using Sprague-Dawley rats Anti-oxidant: The polyherbal formulation HP-4 (100mg) showed significant antioxidant activity, as evidenced by increasing the levels of SOD, GPx, GR, and GSH, while decreasing the levels of TBARS in the liver homogenates of mice.These findings suggest that HP-4 has the potential to prevent oxidative stressinduced liver damage caused by free radicals.
In vivo (mice) India 65 Anti-oxidant: The study showed that the levels of endogenous anti-peroxidative enzymes such as SOD and catalase were increased, and the glutathione contents were increased in rats treated with Livplus, indicating its antioxidant activity.Therefore, it can be inferred that Livplus may have a significant impact on reducing oxidative stress in the liver, making it an effective antioxidant

In vivo Albino Wistar rats
India 119 Toxicological: The oral administration of Livplus in rats up to the dose 2000 mg/kg did not show any sign of toxicity and no mortality for 14 days, indicating that Livplus up to oral dose of 2000 mg/kg of body weight is safe.
In vivo Albino Wistar rats

Withania somnifera, amber, Pistacia lentiscus, Orchis mascula and Cycas circinalis
Anti-oxidant: The result revealed that Ambrex (250 and 500 mg/kg) and showed significant antioxidant activity as it was able to restore the levels of SOD, catalase, and glutathione, to near-normal levels.The study also showed that Ambrex inhibited the formation of hepatic malondialdehyde induced by MTX.These findings suggest that Ambrex may protect the liver against oxidative stressinduced damage, possibly by enhancing the levels of endogenous antioxidants and inhibiting the expression of pro-apoptotic genes In vivo assay using Swiss albino mice.India 120 Aladejana EB, et al.Biological Properties of Polyherbal Formulations: A Review of their Antimicrobial, Anti-Inflammatory, Antioxidant, and Toxicological Activities 83.

Costus speciosus, Fumaria indica, and Cichorium intybus
Anti-oxidant: The study found that cisplatin-induced hepatorenal toxicity resulted in a significant increase in oxidative stress markers such as MDA levels and a decrease in antioxidant parameters such as SOD, GPx, GST, and CAT activities.However, the administration of the CFCT formulation significantly restored the levels of these antioxidant parameters, which could be attributed to the formulation's contribution to the antioxidant defense system by scavenging free radicals and reducing oxidative stress and inflammatory responses In vivo study using male Wistar rats Saudi Arabia 121

Berberis vulgaris, Rosa damascene, Cichorium intybus, Cucumis sativus, Portulaca oleracea, Rheum palmatum, and Nardostachys jatamansi
Anti-oxidant: The polyherbal formulation decreased lipid peroxidation and maintained the levels of glutathione and total antioxidant capacity.These findings suggest that ZES has a significant antioxidant effect, which protects against liver damage.Compared to the control group, the study found that the polyherbal formulation had a better antioxidant effect on the liver.
In vivo assay in a rat model Iran 122 Toxicological: The acute toxicity result showed that there was no mortality or morbidity observed in the rats after the oral administration of single doses of 1000 and 2000 mg/kg.
In vivo assay in a rat model

Cajanus cajan, Lawsonia inermis Mimosa pudica, Uraria picta and Operculina turpethum
Anti-oxidant: The results demonstrated that PHF prevents the depletion level of GSH and decrease in the activity of SOD in CCl4-induced liver injury in rats.In addition, PHF also showed a significant decrease in the LPO levels signifying the potent antioxidant activity.
In vivo using albino rats.

India 123
Toxicological: In the acute toxicity study, the doses of 1000, 2000 and 4000 mg/kg showed no mortality or any behavioral changes in the rats.Thus, the PHF was considered to be safe up to 4000 mg/kg.
In vivo using albino rats.

Tritone (Livosone)
Eclipta alba, Tinospora cardifolia, Curcuma longa and Picrorrhiza kurroa Anti-oxidant: The results showed that Tritone (Livosone) had free radical scavenging activity comparable to that of the standard antioxidant ascorbic acid.In addition, the study found that as the concentration of Tritone increased, the free radical scavenging activity also increased.
In Toxicological: The result showed that no mortality was observed at 2000mg/kg dose.
In vivo method using Adult male Albino Wistar rats 1ndia 125 88.

Anti-oxidant:
The oral toxicity result showed the extract to be safe for use.

Toxicological:
The result showed that after 28 days administration of Daouri (300-1200 mg/kg), the body weight of the rats was not affected.However, compared with the control group, low body variation was observed.The serum concentration of alanine transaminases, creatinine and urea were not affected, but the serum concentration of alkaline phosphatase was increased.In addition, abnormality was observed in the livers of the rats.
In vivo assay using Wistar rats Togo 79 90.
PHF used in the treatment for various ailments such as malaria, typhoid, waist pain, back pain

Hibiscus sabdariffa and Aloe barbadensis
Toxicological: No notable behavioral changes or mortality was observed in rats during the acute toxicity testing.The sub chronic administration remedy did not cause any significant changes in the serum activities of liver-function enzymes, hematological markers, serum electrolytes and other evaluated blood-chemistry indices.
The liver photomicrographs showed that treatment of animals with the polyherbal mixture did not induce any visible lesions or derangements in the examined organ.
In Toxicological: In the acute toxicity study, no morbidity and mortality were observed with single dose administration 2000 mg/kg/day BW) in the mice.
In the sub-acute toxicity study, no significant changes with 50 mg/kg/day, but at doses of 100 and and 200 mg/kg/day, morphological changes with some damage in liver and kidney tissues of male and female animals was observed.Hence, prolonged use at higher dose i.e. 200 mg/kg/day of this polyherbal formulation should be avoided.
In vivo assay using Swiss mice and Albino Wistar rats Pakistan 85 93.

Toxicological:
The result of acute toxicity after oral administration reveals that the herbal formulation has LD 50 greater than 5000 mg/ kg In the sub-chronic toxicity, no significant changes in biochemical, hematological and histopathological parameters was observed.However, some indicators such as urea, creatinine, hemoglobin, and RBC count were altered.
In vivo assay using Albino mice and white rabbits Pakistan 80 94.

Yagari used for the management and treatment of prostate disorder
Nauclea latifolia, and Erythrophleum suaveolens.Toxicological: In the acute assay, no mortality or uncoordinated movement was observed in the rats treated with doses up to 5000 mg/kg body weight.The sub-chronic toxicity revealed that at concentrations of 250, 500 and 1000 mg/kg, no significant changes in the hematological and histopathological parameters was observed, however, the body weight of the rats significantly increased.Toxicological: Acute toxicity results showed that the LD 50 of HB Cleanser Bitters was greater than 5000 mg/kg with no mortality.There was a significant increase in body weight.In addition, at 1 ml/kg, there was significant increase in total bilirubin, conjugated bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and urea more than the other doses compared to negative control.A significant decrease was observed in the levels of total protein and albumin compared to negative control.Hence.HB cleanser Bitters may adversely affect hepatic and renal indices and therefore should be used with caution.
In vivo assay using adult Wistar rats Nigeria 87 The pathological examination of the animals showed that the heart of the group treated with Jedi had chronic inflammatory cell infiltrates, while the hearts of the other groups showed no abnormalities.The kidney tissue showed congested vessels and the lung tissue showed a reduction in air-filled alveolar spaces and infiltration of alveoli and interstitium by aggregates of inflammatory cells, indicating moderate to severe pulmonary inflammation.The lung tissue of rats treated with herbal liquors showed congestion of pulmonary vessels and interstitial hemorrhages.The genotoxic evaluation of rat lymphocytes showed that the rats administered with the herbal liquors developed significant DNA strand breaks, indicating oxidative DNA damage.These results suggest that the herbal liquors contain substances that produce reactive oxygen species that have pathological effects on certain organs and can lead to DNA damage and mutations.
In vivo assay using Wistar rats Nigeria 127 98.

Drepanoalpha® hard capsules used for the management of sickle cell disease
Justicia secunda (leaves) and Moringa oleifera (leaves) Toxicological: In the acute toxicity test, no mortality occurred at the limit dose of 5000 mg/kg of B.W. during the observation period of 14 days.However, slight changes in behavior, respiration and hypoactivity were observed in treated Guinea pigs for a few minutes after dosing.
No sign of toxicity or organ damage was observed on the tested organs (heart, stomach, kidneys, liver).The results of haematological (red and white blood cells counts, haemoglobin, haematocrit) and biochemical (ALT, AST, albumin, total protein) tests did not show significant differences between control and test groups.
In vivo experiment using Guinea pigs Congo 84 99.
Ade & Ade used for systemic detoxifiers and as diuretic agent -Toxicological: At the different doses administered in the acute toxicity study, no mortality, no changes in their feeding habits, stool, urine and the animals were active throughout the study.In the sub-chronic toxicity study, the formulation reduced cholesterol level at low and high doses in animals, it may be beneficial on cardiovascular risk factor.In addition, the formulation had no side effect on the liver, heart and the kidney in all the doses.
In vivo assay using Swiss mice and Albino Wistar rats.Bobwell® used for the management of diabetes mellitus.
Gongronema latifolium (leaves), Garcinia kola (leaves), Vernonia amgydalina (leaves), Sphenocentrum jollyanum (leaves) and Kigelia africana (leaves) Toxicological: In the acute toxicity study, no changes in the behavior, no adverse gastrointestinal effects and no changes in the sensory nervous system responses were observed in the animals.
In the animals that received the highest dose, there was significant increase in their body weight, alanine aminotransferase (ALT), decrease in the plasma glucose level, and increase in creatinine level while aspartate aminotransferase (AST) decreased significantly.
The photomicrograph of hepatic tissue showed focal necro-inflammation around the portal hepatics.There was marked increase in the haemoglobin level and in the red blood cell count and white blood cells.
In vivo assay using Swiss albino mice Nigeria 128 101.
YoyoBitters™, Ogidiga™ and BabyOku™) -Toxicological: The result showed a dose dependent decrease in the hemoglobin concentration and percentage packed cell volume; red blood cell, white blood cell, lymphocytes, neutrophil and platelets counts (2 mL/kg bwt).Also, a dose-and tissue-dependent increase in induction of apoptotic DNA fragmentation was observed in the triherbal groups relative to control groups.Also, an increase in micronucleated polychromatic erythrocytes was formed in a dosedependent manner in the multi-herbal groups when compared with the control groups.
In vivo assay using Wistar rats Nigeria 129 The remedies showed a dose-and tissuedependent increase in induction of apoptotic DNA fragmentation in the polyherbal groups relative to control groups.Also, an increase in micronucleated polychromatic erythrocytes were formed in a dose-dependent manner in the polyherbal groups when compared with the control groups.
The haematological parameters showed dose dependent decrease in the hemoglobin concentration and percentage packed cell volume; red blood cell, white blood cell, lymphocytes, neutrophil and platelets counts No abnormality and sign of toxicity produced at a dose of 2000 mg/kg body weight.The HAF was found to be safe up to a dose of 2000 mg/kg body weight.
In vivo assay using Sprague Dawley female rats India 131 highlights the importance of thoroughly researching and evaluating the effectiveness of polyherbal remedies before using them to treat infections.
Interestingly, Triple bark and some formulations showed antibacterial activity that was significantly higher than the control drugs, 40,41 while NOQ19 showed excellent antiviral efficacy, eliminating 100% of the virus at a concentration of 0.9 mg/mL, with an IC 50 of 0.2 mg/mL. 24hese findings suggest that certain polyherbal formulations have the potential to be used as effective treatments for bacterial, fungal, and viral infections.Additionally, the study found that some of the polyherbal remedies exhibited anti-tubercular activity, with the most active remedies being KWTa, HBfs, and HBts, which exhibited inhibitory activity against M. tuberculosis at 1.562 µg/mL. 39This highlights the potential of polyherbal remedies as an alternative or complementary treatment for tuberculosis, which is a major global health concern.According to Famewo et al. 42 some of these polyherbal formulations contain the essential mineral nutrients and vitamins that could probably be boosting the immune system of tuberculosis patients.Thus, they can enhance the immune system and help the host fight against microbial infections by increasing the activity of immune cells, such as macrophages, neutrophils, and natural killer cells.
The differences observed in the MIC values of the reviewed polyherbal formulations against various microorganisms could be attributed to several factors, including the mode of action of the formulations by direct inhibition of the growth and proliferation of microorganisms or disruption of microbial cell membranes, the differences in cell wall composition and/or genetic makeup of the microorganisms as well as the synergistic effects of different phytoconstituents present in the herbal formulations.These findings are important as they highlight the potential benefits of using polyherbal formulations.However, further research is needed to determine the effectiveness of these remedies on a larger scale and against a wider range of microorganisms.Additionally, studies should be conducted to determine the safety and potential side effects of these formulations before they can be recommended for widespread use.Overall, this review provides valuable insight into the potential of polyherbal remedies as a natural and effective means of treating bacterial infections.

Anti-inflammatory activity of polyherbal formulations
4][45] The upregulated synthesis of inflammatory mediators results from the activation and enhanced activity of inducible nitric oxide synthase (iNOS), COX-2 enzymes, and pyrogenic cytokines such as tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin (IL-6), among others. 9,46herefore, to qualify as an anti-inflammatory agent in an in vivo assay, it should alter the effects of carrageenan-induced inflammation, leading to the alleviation of typical features such as redness, oedema, pyrexia, algesia, and tissue dysfunction. 46,47e results of the studies indicate that the polyherbal medicines investigated possess anti-inflammatory activities, with some of these remedies used in wound and diabetes management.Phytexponent, a polyherbal medicine used for pain and inflammation treatment, was examined for its anti-inflammatory activity using carrageenan-induced paw oedema mice.The study found that Phytexponent demonstrated significant anti-inflammatory effects in a time-dependent manner, with greater efficacy at 250 mg/Kg BW than the positive control indomethacin (4 mg/Kg BW).The polyherbal medicine also significantly reduced oedema in a dose-and time-dependent manner, and it effectively inhibited both the early and late phases of inflammation.
The study suggests that Phytexponent's anti-inflammatory effects could be attributed to the inhibition of the COX enzyme.The timedependent increase in percentage inhibition of oedema may be due to a higher bioavailability of active molecules, following metabolism and distribution to target sites. 9Overall, these findings suggest that polyherbal medicines such as Phytexponent could be considered potential alternatives for the management of inflammation, as they demonstrate promising anti-inflammatory effects.Further studies are necessary to explore the mechanisms of action of this remedy.
In addition, IBS-20 used for the treatment of irritable bowel syndrome, showed potent anti-inflammatory effects on innate immune cells in vitro as well as on the murine model of colitis in vivo, by suppressing the up-regulation of key inflammatory cytokines. 48Furthermore, Dev et al. reported that PHGs (2% and 5%) used for wound healing showed significant and maximal anti-inflammatory activity after 180 min in carrageenan-induced paw oedema as compared to normal control and negative control animal groups. 3The inflammation induced by the carrageenan lasts approximately up to 5 h after injection.Also, the AHPL/AYCAP/0413 Capsule used in the treatment of Acne exhibited anti-inflammatory activity with a significant reduction in rat paw oedema (51% inhibition) which was comparable to that of the control, Diclofenac (58% inhibition). 49The ethanolic extracts of THR-SK010 and THR-SK011 polyherbal inhibited nitric oxide (NO) production, suggesting its potential in the treatment of inflammation and wounds. 50he findings suggest that polyherbal medicines can be effective in managing inflammation and offer a safer alternative to non-steroidal anti-inflammatory drugs with their known adverse effects.
During inflammation, lysosomal hydrolytic enzymes can be released into the surrounding tissues, causing damage to organelles and leading to a variety of disorders.Researchers such as Chamara et al.
(2018) 51 have investigated the potential anti-inflammatory effects of polyherbal formulations using erythrocyte membrane stabilization methods.This method helps to determine if the formulations have the ability to stabilize lysosomal membranes and prevent the release of lysosomal components that can cause inflammation and tissue damage.Chamara et al. 51 found that both formulations exhibited moderate anti-inflammatory activities, which could be attributed to the phytoconstituents of the herbal formulations, which exert intense stabilizing effects on lysosomal enzymes.Lysosomal stabilization is important in reducing inflammation by preventing the liberation of lysosomal components of activated neutrophils which generally tend to cause tissue damage and inflammation. 52milarly, Abbas et al., 5 investigated the in vitro anti-inflammatory activity of different extracts of polyherbal formulations used for treating urinary tract infections using the membrane stabilization method.The findings showed that the water extract (1 mg/mL) exhibit maximum inhibition of inflammation (36.54%), followed by ethyl acetate (33.27%), methanol (25.98%), and hexane (9.44%) extracts.The water, ethyl acetate, and methanol extracts exhibited more effective activity than the standard anti-inflammatory drug Diclofenac sodium (15.42%).It can be deduced that the phytoconstituents present in water extract may stabilize the lysosomal membranes and prevent the release of enzymes that can cause inflammation and tissue damage.Hence, the findings of these studies suggest that polyherbal formulations have the potential to prevent and cure inflammation and tissue damage that may be an emerging effect of various disorders.The stabilizing effects of phytoconstituents present in these formulations on lysosomal enzymes may play a critical role in reducing inflammation and promoting healing.
In conclusion, polyherbal medicines can exert anti-inflammatory effects through various mechanisms of action, depending on the specific herbs and their constitutent bioactive compounds.Polyherbal medicines may help prevent inflammation by inhibiting pro-inflammatory enzymes involved in the production of inflammatory molecules, such as cyclooxygenase (COX) and lipoxygenase (LOX); 53,54 by using antioxidants to scavenge free radicals and reactive oxygen species that are generated during inflammatory processes, thereby reducing oxidative stress and inflammation; 55 by modulating the immune system and reducing inflammation through regulation of the immune cells' (such as macrophages, T cells, and B cells) activities; 56 by stabilization of cell membranes and prevention of the release of pro-inflammatory molecules, such as cytokines and chemokines. 57Overall, polyherbal medicines can provide a comprehensive approach to preventing inflammation by targeting multiple pathways and molecules involved in the inflammatory response.However, it's important to note that the specific effects and mechanisms of individual herbs may vary, and the safety and efficacy of polyherbal formulations should be evaluated through rigorous scientific studies.

Antioxidant activity of polyherbal formulations
Antioxidants are molecules that can prevent, delay or remove oxidative damage caused by free radicals.They work by reacting with free radicals, which reduces the harmful effects of oxidative stress. 51he primary function of antioxidants is to neutralize the excess free radicals and safeguard cells against their toxic effects, thereby helping to prevent diseases. 15When produced in excess, reactive oxygen species (ROS) can cause oxidative damage by destroying antioxidant enzymes, damaging biological macromolecules such as DNA and RNA, and impairing cell signalling pathways, which can lead to apoptosis or cell death.Different antioxidant compounds can decrease oxidative stress by chelating free radicals, scavenging free radicals, or modulating the activities and levels of antioxidant enzymes and their reducing potential. 18In addition, phenolic compounds such as phenols, tannins, saponins, flavonoids, and steroids are also known to exhibit antioxidant properties. 58,59Therefore, phytocompounds are powerful antioxidants that can neutralize and eliminate harmful free radicals. 5ee radicals, especially ROS, are involved in carcinogenesis' initiation, promotion, and progression.Oxidative damage to DNA and cellular components caused by ROS can lead to cancer-related mutations. 60onsequently, antioxidants help in protecting the human body from ROS-induced damage, and consumption of natural antioxidants has been linked to a lower risk of cancer and other diseases associated with oxidative damage, which is linked to phenolic compounds and the phenolic hydroxyl group. 60e use of polyherbal formulations for their antioxidant properties has gained attention in recent years due to their potential to protect the human body from free radicals.Various methods, such as DPPH radical scavenging activity, FRAP assay, ABTS radical cation scavenging activity, hydroxyl free radical-scavenging activity, scavenging of superoxide radical, nitric oxide radical scavenging capacity, lipid peroxidation assay, and in vivo antioxidants in albino rats, have been used to confirm the antioxidant activity of these polyherbal formulations.
The results have shown that many of the polyherbal formulations exhibited strong antioxidant activities in a concentration-dependent manner, with some showing even more activity than standard drugs.For instance, the polyherbal formulation used for treating urinary tract infections showed the highest scavenging activity of 98.63% which was higher than the natural antioxidant rutin (60.32) and the synthetic BHT (64.40). 5Similarly, the Thalipathiri Chooranam used for the treatment of cough showed a gradual increase in nitric oxide radical scavenging activity in a concentration-dependent manner, with a maximum reduction of 79.6 (100 μl), which was comparable to the positive control gallic acid (78.9).The reducing power of the remedy was highest at 100 μl (83.29), resulting in the reduction of Fe 3+ to Fe 2+ by donating an electron.This indicates that the reduced power capacity of the extract can serve as a significant indicator of its potential antioxidant activity. 35e potent antioxidant activity of both THR-SK010 and THR-SK011 against free radicals such as DPPH and hydroxyl radicals may be due to the potent activity of active constituents present in the herbal formulations. 50The antioxidant capacity of most herbal sources is usually associated with their phenolic contents.Antioxidants that react quickly with free radicals may react slowly or may be inert to the DPPH radical. 5The higher DPPH radical scavenging activity observed in the polyherbal formulations indicates that the extracts can scavenge other free radicals in the human body.Therefore, natural antioxidant agents have gained attention because they can protect the human body from free radicals.
One study found that Habb-e-Asgand exhibited significant antioxidative potential against paracetamol toxicity by increasing the activity of antioxidant enzymes such as glutathione reductase, glutathione peroxidase, glutathione S-tranferase, catalase, and glutathione contents. 61Similarly, another study showed that rats treated with isoniazid and rifampicin experienced severe oxidative stress-induced lipid peroxidation, but supplementation with heptoplus and Liv 52 prevented oxidative damage and restored normal serum liver biochemical markers. 624][65][66] BV-7310, a combination of plant extracts, also demonstrated potent antioxidant activity, suggesting a synergistic effect on antioxidant activity. 67rthermore, the study showed that Virgoliv syrup exhibited significant antioxidant activity against CCl4-induced hepatic damage, as evidenced by the increased levels of antioxidant enzymes such as CAT, GSH, and SOD. 68These findings suggest that polyherbal formulations have potential therapeutic value in protecting against oxidative stressrelated diseases.
However, some PHFs such as Evans healthy bitter, Yoyo bitter, Fidson bitter, Swedish bitter, Oroki herbal mixture, Pax herbal mixture, Asheitu Adams blood purifier, Asheitu Adams formula for diabetes, e.t.c cause depletion of hepatic antioxidant enzymes.The administration of Fidson bitters and Asheitu Adams blood purifier significantly decreased superoxide oxidase and glutathione-S-transferase concentrations in comparison with the control, while Yoyo bitter and Asheitu Adams formula mostly decreased reduced glutathione in a non-significant manner.Additionally, the PHFs caused a decrease in the CAT levels of the experimental animals, 14 which means that the continuous mobilization of these enzymes due to the daily introduction of free radical-generating polyherbal then led to the depletion of antioxidant enzymes as a result of exhaustion in the course of scavenging the overproduced free radicals.Also, the decrease in GSH concentrations, a naturally occurring antioxidant that prevents free radical damage to cells and helps in the detoxification process by conjugating with oxidants, must have resulted from the free radical scavenging activity of GST. 14 It is important to note that while polyherbal formulations have potential benefits, they may also have adverse effects if not used correctly.Therefore, it is necessary to conduct further studies to determine the appropriate dosage and duration of use to ensure maximum benefits and minimal side effects.Additionally, caution should be exercised when selecting and using polyherbal formulations, and it is recommended to consult with a healthcare professional before use.
In conclusion, polyherbal medicines may help prevent oxidative stress by scavenging free radicals and other reactive oxygen species that cause oxidative damage to cells and tissues; by upregulating the expression and activity of endogenous antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), which help to neutralize free radicals and prevent oxidative damage; by chelation of metal ions, such as iron and copper, which can generate free radicals and promote oxidative stress; and by reducing oxidative stress by suppressing the production of pro-inflammatory molecules, such as cytokines and chemokines.

Toxicological activity of polyherbal formulations
Currently scientists are trying to explore development of new polyherbal therapy.Phytochemcials such as, polyphenols and flavonoids have potent anticancer activities by involving regulation of various signal transduction pathways of cancer cell growth and suppression and proliferation of oncogenes and formation of tumor, modulation of enzyme activity, induction of apoptosis, reduction, oxidation, regulation of hormone metabolism and stimulation of the immune system and DNA repair. 69ladejana EB, et al.Biological Properties of Polyherbal Formulations: A Review of their Antimicrobial, Anti-Inflammatory, Antioxidant, and Toxicological Activities According to the National Cancer Institute (NCI) criteria, plant extracts with CC 50 < 30 μg/ml are considered to be cytotoxic after 48-72-hour exposure to cells. 70In the cytotoxicity assay, the therapeutic indexes of the ethanol extract of THR-SK010 were more than 10 (45.68 µg/ mL), indicating that the extracts exhibited no cytotoxicity as compared to the positive control ellipticine which had the value of 1.48 µg/mL. 50lso, phytexponent was found not toxic to Vero E6 cells (CC50 >1000 μg/ml) compared to the control cyclophosphamide (CC 50 =2.48μg/ mL). 9 The study of Devanathadesikan Seshadri et al. 69 revealed that the methanol extract of polyherbal fraction showed cytotoxicity against the two cell lines with the LC 50 value 467 ± 2.9 mg/ml against HeLa cell line and >800 mg/ml against MCF-7 cell lines.Thus, showing promising anticancer activity against HeLa and MCF cell lines.Phytochemcials such as, polyphenols and flavonoids have potent anticancer activities by involving regulation of various signal transduction pathways of cancer cell growth and suppression and proliferation of oncogenes and formation of tumor, modulation of enzyme activity, induction of apoptosis, reduction, oxidation, regulation of hormone metabolism and stimulation of the immune system and DNA repair.Furthermore, in vitro cytotoxicity test of a PHF1 using normal fibroblast cell line NIH3T3 and human malignant melanoma cell line A375 revealed that the formulation exhibited dose dependent inhibitory activity in the two tested cell lines.It exhibited dose-dependent toxicity (66.57% inhibition) against fibroblasts cells at the highest dose (320 μg/mL), and exhibited higher inhibitory activity (61.88%) against human malignant melanoma A375 cell line.Hence, it had cytotoxic effect against melanoma cancer cell line. 71 addition, Ramamoorthy et al. 41 rated cytotoxicity of the polyherbal formulation on the basis of the percentage viability relative to control group.Samples are considered non-toxic, if the viability is > 90%; the value between 60-90% viability indicates a slight toxicity; 30-59% means a moderate toxicity; and the values < 30% indicates that the sample is severely toxic to the cells. 72Hence, the result showed that the increasing concentration of the extract decreases the viability of cells and the IC 50 value of the extract is found to be 274.30μg/mL.PHF5 displayed anticancer activities via apoptosis.After 48 h, PHF5 showed a promising inhibitory effect against all cancer cells.The IC 50 values of PHF5 were 71.8, 64.8, 45.3, and 47.3 μg/mL against LoVo, HepG2, MCF-7, and MDA-MB 231 cells, respectively.The PHF5 extract reduced the viability of all cell lines in a dose dependent manner and the results confirmed that the inhibition of MCF-7 cell proliferation by the PHF5 extract was capable of inducing apoptosis. 73 the acute and sub chronic assays, most of the polyherbal formulations were found to be highly effective at low dose and highly safe even at high dose.For instance, DAS-77® used for the treatment of piles, dysentery, cholera, menstrual pain, stomach disorder, ulcer, constipation, diarrhoea and sexually transmitted infections did not induce lethality in mice when administered orally up to 20 g/kg in divided doses.
According to the assertion of Clarke and Clarke, 74 a substance that does not produce lethality up to 10 g/kg orally is relatively non-toxic, hence the herbal preparation can be said to be safe when administered orally.
In the study of Singh et al., 75 PHF used for the treatment of dysentery was reported to be safe as no mortality of animals was observed, no significant changes in behavior, postural abnormalities, impairment in food and water intake, and loss or yellowing of hair were also observed in the rats.In addition, Tabsaab used for the cure of tuberculosis was observed for 8-weeks and a 100% recovery rate in females and a 98% recovery rate in males, with no toxicity or side effects was observed in the patients. 76e sub chronic administration some remedies did not cause any significant changes in the serum activities of liver-function enzymes, hematological and histopathological parameters, serum electrolytes and other evaluated blood-chemistry indices.[79][80][81][82][83][84] However, in the sub-acute toxicity study of Hab-e-Kabad Noshadri tablets used for the treatment of hepatitis, enlargement of liver at doses of 100 and and 200 mg/kg/day, morphological changes with some damage in liver and kidney tissues of male and female animals was observed.Hence, prolonged use at higher dose i.e. 200 mg/kg/ day of this polyherbal formulation should be avoided. 85Also, decrease in the body weight was observed in rats that received higher doses of Leone Bitters, no morphological changes in the colour of the organs however, the histological result revealed that at the dose of 500 mg/ kg bwt, the drug may have affected the sperm production rate in the testis. 86In addition, at 1 ml/kg, there was a significant increase in total bilirubin, conjugated bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and urea in the rats that received HB cleanser Bitters.A significant decrease was observed in the levels of total protein and albumin compared to negative control.Hence.HB cleanser Bitters may adversely affect hepatic and renal indices and therefore should be used with caution. 87

CONCLUSION
The use of herbal medicine is gaining popularity due to the increasing prevalence of drug resistance in bacterial populations.While plantderived medicine is believed to be safer than synthetic drugs and provides profound therapeutic benefits, polyherbal formulations should be seen as adjunctive therapies that can boost immunity against diseases, rather than as replacements for conventional antibiotic treatments.This comprehensive review has shed light on the various benefits of polyherbal formulations in terms of their antimicrobial, anti-inflammatory, antioxidant and toxicological properties.It is worth knowing that the formulations have been traditionally used in the treatment and management of various diseases.The reviewed studies have demonstrated the potential of polyherbal formulations as an alternative approach to modern medicine.They have shown great potential as sources of nutraceuticals with high therapeutic importance, and they could serve as a blueprint for future developments in the pharmaceutical sector.However, there is a need for further research to validate the efficacy and safety of these formulations, carry out more clinical studies, identify the active constituents responsible for their therapeutic properties, and standardize their preparation and dosage.With the increasing demand for natural remedies, this review provides useful insights into the potential use of polyherbal formulations in the development of new pharmaceuticals.The folklore system of medicine has led to a scientific revolution in nutraceuticals and phytopharmacotherapy, and the use of polyherbal formulations is likely to increase in the future.This review serves as a valuable resource for researchers, healthcare professionals, and the general public interested in the potential benefits of polyherbal formulations.

Figure 1 :Figure 2 :
Figure 1: Flow of the study

Table 1 : Antimicrobial, anti-inflammatory, antioxidant, and toxicological activities of polyherbal formulations.
The results revealed that all the formulations viz.A, B, and C showed better ZOI ranging from 11.45 mm -19.22 mm against S. aureus, B. subtilis, A. niger, and E. coli as compared with the control (8.98 mm -9.76 mm).
Tecoma stans (leaf and flower), Cassia fistula (leaf and flower) Antimicrobial: At the concentration of 1000 μg/mL, the combination of C. fistula flowers and C. fistula leaf; C. fistula flowers and M. zapota leaf showed maximum ZOI of 23 mm and 21 mm for S. aureus; and 22 mm and 23 mm for A. niger respectively.The C. fistula flowers, M. zapota leaf and C. fistula leaf enhanced the antimicrobial nature more than the individual plants.Pharmacognosy Journal, Vol 15, Issue 5, Sep-Oct, 2023 24.
105Aladejana EB, et al.Biological Properties of Polyherbal Formulations: A Review of their Antimicrobial, Anti-Inflammatory, Antioxidant, and Toxicological Activities 45.