Evaluation Of Jamun-Based Polyherbal Formulations for Wound Healing By In Vitro Assays

Herbal remedies and drugs have played a significant role in curing diseases throughout the history of mankind. Herbal medicines have the potential to treat and cure illnesses like ulcers, skin infections, inflammation, scabies, leprosy, venereal disease and healing of wounds.1  Wound healing is defined as a complex process occurring by the regeneration or reconstruction of damaged tissue.3 A wound is a disruption of living tissue's cellular, anatomical, and functional integrity caused by physical, chemical, electrical, or microbial threats to the tissue.3  Plant products were reported to contain growth factors, cell signalling molecules and cell adhesion molecules.4,5 It is found that the multiple combinations of plant extracts have shown higher phytotherapy efficacy compared with the products with single extracts. Likewise, extracts of Hypericum perforatum, Sabal serrulata, Hedera helix, and Boswellia serrata also showed better therapeutic efficacy than those of the synthetic drugs (standard) used in the clinical studies.6  Medicinally, the Jamun fruit (Syzygium cumin) is reported to have antidiabetic, antihyperlipidaemic, antioxidant, antiulcer, hepatoprotective, antiallergic, antiarthritic, antimicrobial, anti-inflammatory, antifertility, antipyretic, antiplaque, radioprotective,  nephroprotective, and antidiarrhoeal activities.7 Jamun honey is effective in wound closure, re-epithelialization, collagen deposition, and pro-angiogenic potential.8 Clove (Syzygium aromaticum) has high antimicrobial, antioxidant, and anti-inflammation potential.9 The effect of Clove Flower Extract on wound healing of Type 1 diabetic albino rats  indicated that the topical application of CFE hydrogel improved wound size, wound index, and mRNA expression of the wound healing markers.10     Clove oil's ability to inhibit the growth of MRSA and wound-healing effect was studied on the rats by excision wound model.11  Alcoholic extract of Triphala has shown in vitro antimicrobial activity against wound pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes. Triphala (a traditional ayurvedic herbal formulation), incorporated in collagen sponge was investigated for its healing potential on infected dermal wounds in albino rats. Wound reduction rate, collagen content, and matrix metalloproteinases in the granulation tissue, histology, and Fourier transform electron microscopy analysis were done to obtain the healing pattern. This dressing is an effective wound cover in the management of infected dermal wound.12 The betel leaf extract cream can accelerate the healing of wounds in male white rats which are characterized by the higher dosage of betel leaf extract will further accelerate the healing of incisions in male white rats.13  Water extract of stem bark of Azardirachta indica shows significant wound healing properties in excision as well as incision wound models hence supporting its traditional use. Therefore, a cream/paste formulation of water extract of neem bark can be used as an ideal dressing agent for the treatment of wounds.14 Subsequently, the neem bark has also been reported to possess antidiabetic, hypoglycemic, antioxidant, immunomodulatory, antiviral, and larvicide potential.  The antimicrobial activity of methanolic extract of bark of pipal (Ficus religiosa) against E.coli , Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger. It showed higher activity at 400mg/ml concentration against the tested bacteria.15  The present study involves the use of eight plant extracts as four combinations of plant extracts containing jamun fruit as a common ingredient of the formulations. The plants were grouped and different types of extracts viz, Hot Aqueous extract (Jamun, Lavang, Neem, Pipal), Cold Alcoholic extract (Jamun, Lavang, Neem, Betel),50% Cold Alcoholic extract (Jamun, Amla, Hirda, Beheda, Lavang) and Hot alcoholic extracts (Jamun, Amla, Beheda, Hirda, Lavang, Neem) were prepared. The polyherbal combinations were studied for their antibacterial and bactericidal activity by in vitro assays. The angiogenic potential, cell viability on the 3T3 cell line, and wound healing by scratch assay were assessed. The polyherbal formulation showed promising results.

MATERIALS AND METHODS

1. Collection and Processing of plant material for Extract preparation

Dry powders of fruits of Jamun (Syzygium cumini), Lavang (Syzygium aromaticum), Amla (Embelica officinalis), Hirda (Terminalia chebula), Beheda (Terminalia bellarica). Bark of Neem (Azardirachta indica), Pipal (Ficus religiosa), and Betel (Piper betle) leaves were obtained from authentic Ayurvedic shops of Mumbai.

The powders in the following combinations were extracted as different extracts as given below.

• Hot Aqueous extract: Jamun, Lavang, Neem, Pipal.
• Cold Alcoholic extract: Jamun, Lavang, Neem, Betel.
• 50% Cold Alcoholic extract: Jamun, Amla, Hirda, Beheda, Lavang
• Hot alcoholic extract: Jamun, Amla, Beheda, Hirda, Lavang, Neem

The resulting extracts were concentrated using a rotary vacuum evaporator under reduced pressure and at a temperature maintained below 4°C.

2. Extract preparation

2.1: Aqueous extract preparation.¹⁶

Aqueous extract of Jamun fruit, Lavang fruit, bark of Neem, and Pipal was prepared by soaking 10 grams of each finely ground powder with —— mL of distilled water. The mixture was boiled in a water bath, till the quantity was reduced to ¼th of the original volume. It was then filtered through muslin cloth and further heated till a honey-like consistency was obtained. Then the extract was kept at 37? till the water evaporated completely and it was then stored in the refrigerator at 4 degree C

2.2 Alcoholic extract preparation.¹⁶

Hot alcoholic extract:

Hot alcoholic extract preparation was carried out by continuous hot extraction by the Soxhlet apparatus. Crude powders of Jamun, Amla, Beheda, Hirda, Lavang, and Nee in equal amounts were placed into thimble using filter paper and introduced into the siphon tube of the extractor. Organic solvent 100% ethanol, was used for refluxing. The extract was evaporated to dryness and stored in refrigerator at 4 degree C

Cold alcoholic extract:

Ten grams of each powder in combination with Jamun, Lavang, Neem, and Betel powders were kept in a 200 ml conical flask and 100 ml of alcohol was added individually. The mouth of the conical flask was covered with aluminium foil and kept in shaker conditions for 24 hours for continuous agitation at 150 rev/min for thorough mixing and also complete elucidation of active materials to dissolve in the respective solvent. Then the extract was filtered by using muslin cloth followed by Whatman no 1 filter paper. The solvent from the extract was removed with the water bath temperature of 50°C. Finally, the residues were collected and used for the experiment.

50% cold alcoholic extract:

An equal amount of powders of Jamun, Amla, Hirda, Beheda, and Lavang,was mixed with 50% alcohol in distilled water and kept under shaker conditions for 24-48 hours. It was then filtered through muslin cloth and stored in the refrigerator.

3. Bacterial pathogens from burn wound infections

3.1 Standard Cultures

Standard ATCC strains- Escherichia coli 25922, Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, Klebsiella pneumoniae 700603 were used for the studies.

3.2 Clinical isolates

Resistant and Sensitive clinical isolates were obtained by collecting swab samples from burn wound patients. The isolates Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were selected for study.  The inoculum used was 24-hour old culture broth of 0.01 O. D at 620 nm.

4. Evaluation of Antibacterial activity against sepsis-causing organisms.

4.1 Antibacterial activity by Agar ditch method.¹⁷

The antibacterial efficacy of the combinations was assessed by the agar ditch method using both standard and clinical isolates. A concentration range of 5 mg/mL to 50mg/mL of extract combinations were individually mixed with molten agar butts. Subsequently, the mixture of a particular concentration of polyherbal extract and molten agar butt was poured into a ditch created on the nutrient agar plate, and isolates were streaked perpendicularly to the ditch.

4.2 Determination of Minimum Inhibitory Concentration (MIC) of polyherbal extract¹⁸

The lowest concentration of the antibacterial agent at which the organism is completely inhibited is called its Minimum Inhibitory Concentration. Minimum Inhibitory Concentration of the test compounds were determined by plate dilution method. The different concentration of extracts 5mg/mL to 20 mg/mL were mixed with agar and poured in the sterile empty Petri plates. Isolates were spot inoculated onto the plates and were incubated at 37°C for 24 hours.

5. Assessment of the Bactericidal Effect of the polyherbal extracts by Kill Time Studies ¹⁹

The four formulations were further assessed for the ability to have bactericidal and bacteriostatic effect on the ATCC, Resistant and Sensitive burn wound isolates. The method uses the principle of contact time period i.e., the time required by the test compound to exhibit bacteriostatic and bactericidal effects.

Method:

Standard ATCC cultures, Resistant & Sensitive clinical isolates were mixed with an effective concentration of Polyherbal extracts in a sterile tube containing sterile Mueller and Hinton agar. At an interval of 1 hour, a Loopful of the broth containing a mixture of extract and culture was spot inoculated on the St. Nutrient agar plate. The plates were incubated at 37^oC for 24 hours and the spot with the time showing no growth was recorded as the time required for the extract to exhibit antibacterial effect.

6. Assessment of Angiogenic Potential of the polyherbal extracts by CAM Assay (Chorioallantoic membrane assay)

Angiogenic potential of Polyherbal extract combinations was determined by Chicken embryo Chorioallantoic Membrane Assay. The assay relies on the principle of sprouting of blood vessels in response to Angiogenic agent. Variety of herbals has been reported to stimulate or inhibit angiogenesis in the CAM assay.²⁰ The quantification was carried out in chick embryo by counting the primary vessels and vessels sprouted from primary branches e.g. secondary, tertiary and quaternary branches manually in selected areas. Comparison for quantification of blood vessels in control-treated, test-treated embryos was carried out.

Method:

Fertilized Chicken eggs were collected from Poultry,wiped with alcohol, and incubated at 37^oC. The eggs were observed for the presence of embryo. Eggs showing blood rings or clumps were ruled out from the study. On day 8, under the laminar airflow hood, a small window was punctured carefully on the eggs for observation. After observation, windows were sealed with Parafilm. On day 9, Polyherbal extracts with different concentrations such as 2µg/ml, 5 µg/ml, 10µg/ml, 20µg/ml, control DMSO were loaded in the eggs.²¹

7. in vitro assessment for cytotoxicity and wound healing of polyherbal extracts

7.1 Cytotoxicity assessment by MTT Assay. ²²

The cytotoxicity of the Polyherbal formulation was assessed on mouse 3T3 fibroblast cells using the MTT assay. It is a colorimetric assay that monitors and records the reduction of yellow 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), functioning on the principle of assessing cell viability. The reduction of MTT served as an indicator of cytotoxicity. The experiment was carried out over a predefined incubation period and under meticulously controlled experimental conditions.

Maintenance of Cell Lines: Assessment of cytotoxicity of the Polyherbal formulation was studied on mouse 3T3 fibroblast cells using MTT assay. 3T3 cells were revived and 0.05 million cells were seeded in 96 well plates and were incubated overnight at 37°C in a CO₂ incubator with 5% CO₂. Post incubation, the plate was observed under a microscope to observe the fully confluent cells. A total of four Polyherbal extracts were assessed for toxicity at different concentrations ranging from 0.0625 µg/ml to 1 µg/ml for the extracts.²³ MTT Assay: The MTT assay is a colorimetric method that involves measuring the reduction of yellow 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The principle is based on the number of cells present and the assumption that those dead cells or their products do not reduce tetrazolium. The MTT enters the mitochondria in the cells and is reduced to purple-colored formazan crystals, which are insoluble. The cells were solubilized with dimethyl sulfoxide (DMSO) and then released; the solubilized formazan reagent was measured spectrophotometrically. Cell viability was evaluated by the MTT assay with 3T3 mouse fibroblast cells.The cells were incubated overnight in the presence of test samples in a CO₂ incubator at 37°C. After observing the cells under the microscope 10µL of  5mg/mL MTT reagent was added in the wells and incubated for 4 hours at 37 degree C. Following 4 hours of incubation, the media was discarded, and the formazan crystals were dissolved by adding 100 µL of DMSO and the absorbance was measured at 595 nm.

7.2 Evaluation of wound healing potential of polyherbal extracts by Scratch assay

in vitro Scratch assay mimics wound contracture in vivo. The scratch assay is for quantitative determination of fibroblast migration and proliferation into the wounded monolayer.  In this assay, an artificial gap, a so-called "scratch" is typically created in a cell monolayer with a sharp object such as a pipette tip or syringe needle. The assay is carried out in a multi-well plate. The monolayers recover and wound healing occurs in a process that can be observed over time. The wound heals in a patterned fashion cells polarize toward the wound, initiate protrusion, migrate, and close the wound. Progression of these events can be monitored by manually imaging samples fixed at different time points, or by time-lapse microscopy.

Methodology: 3T3 cells were used for the assessment of wound healing efficacy under the influence of polyherbal extracts. The cells were revived by incubating overnight in a CO₂ incubator at 37°C, and in presence of 5%CO₂. Once the confluent layer of cells was observed under the microscope, the scratch was made in the cells and the media was discarded from the cells followed by washing the wells with PBS. The cells were then replenished with fresh media and the scratch was observed under the microscope and the image was documented. Post-observation test samples were added to the respective labelled wells, and the plates were incubated in a CO₂ incubator at 37°C, 5%CO₂ for 3 days. The microscopic images were captured and documented at 24, 48, and 72 hrs. The wound healing properties of the samples were compared with the control well, where the scratch was replenished only with the media without the addition of any samples.²⁴

RESULTS & DISCUSSION

The extraction of the four combinations gave a yield in the range of 12-15% . The Standard ATCC bacterial strains, namely Escherichia coli 25922, Klebsiella pneumoniae 700603, Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, were selected for the study.  The burn wound sepsis causing Resistant & Sensitive clinical isolates of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa were selected based on the susceptibility test against the following antibiotics: Imipenem, Meropenem, Doripenem, Ertapenem, Cefepime, Ampicillin, Kanamycin, Nitrofurantoin, Gentamicin, Vancomycin, Oxacillin, Cefoxitin, Methicillin, Penicillin G, Amoxicillin, Piperacillin-Tazobactam, Amikacin, Nalidixic acid and Tetracycline.

Antibacterial activity & Minimum Inhibitory Concentration (MIC) of the polyherbal extracts

All four polyherbal combinations showed antibacterial activity against all the 12 selected microorganisms in the range of 5 mg/ml to 20mg/mL by Agar ditch method. Hence determination of Minimum inhibitory Concentration was carried out at the concentration range of 0.5% to 2% i.e., 5 mg/ml to 20 mg/ml, based on preliminary antibacterial efficacy screening.  Combination 1 and Combination 4 which are of the hot aqueous extract and hot alcoholic extract are effective at a lower concentration compared to other combinations of alcoholic extracts. Combination 1 and Combination 4 showed MIC at 1.5% for standard ATCC strains,1% for the Sensitive strains, and 2 % for the Resistant strains. MIC of Combination 2 and Combination 3 was 2% for the ATCC strains & Resistant strains of all four organisms. And 1.5% for the Sensitive strains.Combination 1 of Hot Aqueous Jamun, Lavang, Neem, Pipal showed the lowest MIC among all the four combinations followed by combination 4 of Hot alcoholic extract of Amla, Beheda, Hirda, Jamun, Lavang, and Neem. These results are of prime importance in deciding an effective concentration range, and the effective combinations, especially for combating infections caused by prevalent pathogens causing infections in burn wounds.

Assessment of the Bactericidal effect of the poly herbal extracts by Kill Time Studies on the selected bacterial isolates.

The four combinations were further assessed for their ability to have bactericidal and bacteriostatic effect on the ATCC, Resistant, and Sensitive burn wound isolates. The method uses the principle of contact period i.e., the time required by the test compound to exhibit bacteriostatic and bactericidal effects. Combination 1 containing Jamun, Lavang, Neem, and Pipal is effective in a shorter period followed by combination 4 containing Triphla, Jamun, Lavang & Neem. While combination 3 requires more contact time to have its bactericidal effect on the burn wound sepsis-causing organisms. Resistant strains of Pseudomonas, Klebsiella, and Escherichia demonstrated bactericidal effect after 5-6 hours of contact time whereas sensitive strains showed significant inhibition after contact time of 2 hours. Not much difference in kill time was observed in Resistant and Sensitive Staphylococcal and Klebsiella strains. Of all the isolates, Resistant strains of Pseudomonas required longer contact period for exhibiting a bactericidal effect while the Staphylococcus strains were inhibited at shorter contact time. The ATCC strains were inhibited in a shorter period of time. The below graph  1 shows the comparative bactericidal effect of the four polyherbal combinations against the selected ATCC, Resistant, and Sensitive burn wound isolates.

Graph 1: Kill time studies of all four combinations against ATCC, Resistant & Sensitive strains indicating contact inhibition of isolates in hours

Assessment of Angiogenic Potential of polyherbal extracts by CAM Assay (Chorioallantoic membrane)

The CAM assay is a widely used experimental model that relies on the principle of blood vessel sprouting in response to angiogenic agents.CAM assay involves the placement of a test substance onto the Chorioallantoic membrane of a developing chicken embryo. By observing the response of blood vessels in the CAM to the polyherbal extracts, the researchers could evaluate their angiogenic potential. Concentrations used were 2 µg/ml, 5 µg/ml, 10 µg/ml, and 20µg/ml. At 20 µg/ml concentration of formulation, a significant increase in tertiary and quaternary vessels(angiogenesis) was observed and compared with the control group.CAM without the treatment agent served as a reference control, while a negative control using DMSO (dimethyl sulfoxide) was also included. DMSO is often used as a vehicle for delivering test compounds and is expected to have no angiogenic activity on its own. Combination 3 shows angiogenesis at 2 µg/mL while the other three Combinations showed angiogenesis at 5 µg/mL. This suggests that the four polyherbal extracts have the potential to promote the formation of new capillaries adjacent to wounds, which is an important step in the wound healing process.

Table no 1: Determination of Angiogenesis of different concentrations of polyherbal extracts by CAM Assay

KEY+: Shows Angiogenesis, -: No Angiogenesis

5.1 Cytotoxicity assessment of polyherbal extracts by MTT Assay

The MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay relies on the principle of conversion of MTT into formazan crystals and the determination of mitochondrial activity and cell proliferation by living cells. This in vitro method was used to determine the percentage of cell viability, which is a relative measure of the Cytotoxicity of the test compounds. MTT assay represents the cell viability of 3T3 fibroblast cells after 24 hrs of incubation. As the concentration increases, the viability of cells tends to decrease.  The effect of increasing concentrations of polyherbal extracts is graphically presented in the Graph no 2  below, illustrating the relationship between the concentration of the extracts and the percentage cellular viability.  At the highest concentration, the polyherbal extracts exhibited a cell viability percentage of more than 60%, while at the lowest concentration, the viability percentage exceeded 80%. Across all concentrations, the poly herbal extracts displayed minimal cytotoxicity, as evidenced by the consistently high percent cell viability values. Combination 1 and 2 show viability above 100% at the lowest concentration, thus enhancing the cell proliferation. Combination 4 is in the range of 65-74 % viability. While combination 2 is above 88% for all the concentrations. All the combinations are showing viability above 50% at both the highest and lowest concentrations, ranging from 0.0625 µg/ml to 1 µg/ml. Thus, indicating all the combinations are non-cytotoxic to the cell line.

Graph no 2: Evaluation of the % Viability of Human dermal cell line when treated with different concentrations of Combination 1 to 4

in vitro Wound healing potential of the polyherbal extracts by Scratch assay

In this study, Murine 3T3 Fibroblast cells were used, and the time required to close the gap in the confluent cell monolayer in the presence of the Herbal extract combination was monitored. Proliferation and migration of fibroblasts are the primary steps in wound healing. Thus, Migration and proliferation of fibroblast cells to seal the gap were taken as a measure of wound healing and were recorded at a timely interval of 24 hrs, 48 hours, and 72 hours. It was evident from the images that the following four combinations exhibited faster wound contracture compared to the conrols. Photographs indicating comparative cell migration in control (non-treated) and formulation(treated) are shown in Table no 2.

Table no 2: Details of wound contracture and mobilization of fibroblast cells observed in vitro by Scratch assay