Studies on the combinations of some herbals with various chemical entities as a potent antifungal agents

A widespread increase in the prevalence of fungal infections has been documented in recent decades. Candida albicans infections, which are frequently refractory and linked with high morbidity and mortality, place a significant burden on public health, despite the fact that existing antifungal medicines are restricted and associated with toxicity. Fungi are one of the most underappreciated killers, as evidenced by the fact that Amphotericin B and other commercially available antifungal therapies are still recognized as gold standards. The majority of commonly used antifungal medications have toxicity, effectiveness, and cost disadvantages. As a result of these limitations, there is a growing demand for the development of a novel antifungal medication treatment that acts selectively on new targets while having the fewest adverse effects. Natural goods, whether as pure phytocompounds or regulated plant extracts, give prospects for the development of lead compounds that may subsequently be turned into diverse synthetic medications with the appropriate alterations. These herbs can also be used as a component of a herbal synthetic combination, lowering the minimum required dose of the synthetic medicine (when taken singly) and reducing the risk of adverse effects. The goal of this research is to reduce the minimum required concentrations of today's antifungal medications by mixing them with a few less well-known herbal extracts while maintaining their efficacy.


Introduction
A fungus causes a fungal infection, also referred as mycosis, which is a skin infection. Fungi are found in millions of different species. They can be found in the earth, on plants, on surfaces around the house, and on your skin. They often can cause skin issues like rashes or bumps. Fungi are eukaryotic, meaning they have nuclei, mitochondria, and cell membranes, much like human cells. Their membranes, however, take two main sterols, ergosterol and lanosterol. Antibacterial therapy's effectiveness has produced an environment conducive to Many such fungi live only in the topmost layer of the epidermis (stratum corneum) and do not penetrate deeper. Fungal infections are typically treated with antifungal drugs, usually with antifungal drugs that are applied directly to the affected area (called topical drugs). Topical drugs may include creams, gels, lotions, solutions, or shampoos. Antifungal drugs may also be taken by mouth. Antifungal creams, liquids or sprays (also called topical antifungals) these are used to treat fungal infections of the skin, scalp and nails, includes clotrimazole, econazole, ketoconazole, miconazole, tioconazole, terbinafine and amorolfine. But these antifungal agents may cause many serious side effects such as a bit of itch, burning or redness where the antifungal preparation has been applied, may cause blisters and some allergic reactions in few people. Some antifungal preparations cause liver problems or more serious side-effects in a small number of people when orally administered. 2 Candida albicans and Candida glabrata account for the majority of the fungus infected population, with C. albicans accounting for 49% and C. glabrata for 22%, respectively. As a result, the remainder of this article's research will focus solely on Candida albicans. Candida is a yeast species (fungus) generally lives in small amounts on your skin. They can multiply and cause infection under certain circumstances. Candida infections most commonly occur in the vaginal (vaginal thrush), oral (oral thrush), and skin. Terbinafine, Itraconazole, Clotrimazole, Econazole, and Amphotericin B are some of the medications that are accepted and used to treat candidiasis. 3 But considering the treatment which includes Terbinafine, it has many serious side effects and may cause stomach aches, loss of appetite, feeling sick (nausea), tummy upsets, diarrhea, headache, rash, taste disturbance and muscle or joint pains. 4 Renal insufficiency, hypokalemia, hypomagnesemia, metabolic academia, and polyuria related to nephrogenic diabetes insipidus are all symptoms of Amphotericin B nephrotoxicity. 5 As a result, Terbinafine and Amphotericin B are considered to be the most important drugs in this initiative. Terbinafine has a MIC of 12 g/ml, according to multiple publications, and the concentrations in marketed formulations are approximately identical to the MIC value. 6 The MIC of Amphotericin B has been reported to be 1.09g/ml in multiple journals, and the concentrations in commercially available preparations are virtually identical to the MIC. 7 But it is still unknown and undiscovered as to how the side effects associated with the Terbinafine and amphotericin B can be reduced without co-administration of other pharmaceuticals which thereby increase the chances of drug interactions. This project directs towards decreasing the concentration of the Terbinafine and Amphotericin B while associating it with the use of herbal plant extract. In a generalized manner the project is intended to focus on researching over use of combination of drug and herbals as antifungal treatment. The majorly caused and commonly found fungal infection includes Candidiasis. This project points at reducing the toxic effects of Terbinafine and Amphotericin B by decreasing its MIC and making combinations with three different herbs so as to get the additive effect or at least the equivalent effect. This guarantees that, even if the concentration of a drug that produces significant toxicity is reduced, an equivalent or additive effect can be reached using a few herbals with high antifungal activities. Terbinafine and amphotericin B's adverse effects are proportional to their concentration, therefore they can be reduced to some extent.

Experimental
The experiment is set up in such a way that finding the most reliable, intermediate combination concentration among all potential combinations and concentrations is possible. Assume a general experiment response when Terbinafine is used in conjunction with other medications. Each series is built by combining Terbinafine doses in ascending order with supporting Extract concentrations in descending order, with the goal of identifying the combination concentration that provides the best overall response. This design effectively emphasises solutions to issues such as, If the plant extract has any action against the fungus individually -As the highest dose of plant extract (F) is paired with blank dose of Terbinafine (A), the response shown by this combination will only be due to the action of plant extract.
Because the highest dose of Terbinafine will be combined with Blank concentration, the response in this plate will be comparable to the medium combination responses if any additive activity against fungus occurs as a result of the combination. So, if the previous plates show action near or equivalent to the highest dose of terbinafine, it's safe to believe that Terbinafine and extract have synergistic effects.
Selection of the best combination that can effectively replace Terbinafine as a single medicine -The combination pair that demonstrates action in the desired range with a lower dose of Terbinafine may be clearly recognized and chosen.
The experiment can also be categorized under 1. Correlation -The experiment intends to identify and produce a combination product, that is nearly as equal or better than marketed fungal creams. 2. Experimental -The experiment intends to find out whether the herbal products work against fungus.

Heliotropium indicum linn
The plant for this reports the plant was collected from the garden areas of Koperkhairane, Navi Mumbai. For the preparation of plant extract the leaves and the stem were separated. The collected plant parts were separated from undesirable materials or plants or plant parts. They were sun-dried for one week after cutting into small pieces. Once dried the leaves and the stems were grinded to form a moderately coarse powder with the help of a suitable mechanical grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. The weight of the powder obtained was obtained as 19.79gm and 20.36gm of stems and leaves respectively. For the extraction of plant material, the powder of dried leaves and stems were processed with the help of Soxhlet apparatus. Extract was prepared using 300ml of ethanol. Extract was filtered using Whatman No. 1 filter paper to remove all un-extractable matter, including cellular materials and other constitutions that are insoluble in the extraction solvent The extract obtained is then carried out in a suitable evaporator which will concentrate bulky solution down to small volumes. The further studies were done using this concentrated extract. 8

Extraction: Cold Maceration
Leaves of T. grandiflora were collected, washed and rinsed properly. They were dried in shade and powdered mechanically. About 24.16gm of the leaves powder was macerated with pet. ether, ethyl acetate and ethanol and stored for 72 hours in ice cold condition for the extraction of phytochemicals. At the end of the third day extract was filtered using Whatman No. 1 filter paper to remove all unextractable matter, including cellular materials and other constitutions that are insoluble in the extraction solvent. The entire extract was concentrated to dryness using electric water bath and stored in an air tight container free from any contamination until it was used.

Extraction: Cold maceration of stem
Dried stems of plant were grinded and powder was macerated using 250ml of Ethanol (95%) and kept for 72 hours in 2-8 degree Celsius. The extract was filtered using Whatman filter paper number 1 and concentrated using suitable evaporator.

Ash procedure
Dried flowers were grinded and converted to ash by heating for 1hr in a muffle furnace. Per batch has an average of 5.4grams of plant material converted to 1.12 gram of ash. Total weight of Ash obtained is 11.6 gram

For ash sample
1. The Prepared Ash was weighed and delivered to the zone of inhibition, initial weight being .02gm, with an increment of .01gm in successive combinations. 2. Quantities of 0.02gm, 0.03gm, 0.04gm, 0.05gm and 0.06gm will be measured on a sensitive weighing machine and pack separately on small butter papers. 3. While administering in combination, pour the desired amount of ash in the hole before adding liquid samples.

Procedure for stock solution (100ug/ml) Preparation for Terbinafine
1. Weigh 1 gm Terbinafine API powder 2. In a 100ml flash dissolve the measured powder in 50ml of water and 50ml of methanol, and shake for 5 minutes. (Concentration achieved -10mg/ml).   Weighed ash is to be administered before Terbinafine, in Ash + Terbinafine combination 7. Terbinafine is to be administered in ascending order, and the complimentary sample of the combination is to be administered in descending order.
Note -Streaking of microbes on plate and sampling into the plates is strictly to be done in aseptic conditions with proper protective measures (Gloves, Cap and Mask).

Coding
T -Terbinafine (Always first in coding, followed by extract) Combination of each extract with Terbinafine is done on 2 petri dishes with 6 holes total

Observation table (Amphotericin B + Extracts) Coding
Amphotericin B (Always first in coding, followed by extract) Combination of each extract with Amphotericin B is done on 2 petri dishes with 6 holes total

Chemical test for detection of inorganic constituents 8.6. Procedure
Prepare ash of drug material. Add 50% v/v HCl or 50% HNO 3 to ash. Keep for 1 hour or longer. Filter. With filtrate perform the following test.

Result and Discussion
Out of the five plant extracts tested against Candida Albicans in combinations with standard antifungal drug Terbinafine, the three combinations showed prominent response as additive and individual agents. Their overall response (in combination with Terbinafine and      Red fumes observed. Nitrates may be present. Amphotericin B) was in the order -Lepitagathis trinervis ash > Thunbergia grandiflora leaves > Heliotropium indicum leaves. Based on the observations above, it is clear that the selected Herbal species when combined with present day antifungal drugs Terbinafine and Amphotericin B, gave good or nearly as good actions as compared to higher concentration of Terbinafine and Amphotericin B given individually. Another Herbal species that showed no additive response but did show slight response as an individual drug is Heliotropium Indicum Linn stems and Lepidagathis Trinervis Stem. This suggests that on increasing the concentration of the extract, Heliotropium Indicum Linn stems and Lepidagathis Trinervis Stems may show better response as Individual or as a combination.

Conclusion and Outcomes
This project aimed to find and propose novel alternatives to present conventional antifungal drugs. To be able to compare together all the herbal species response on varying concentration and with different drugs, concentrations for herbal and drugs in each dose was standardized. Though standardization did help in comparing all the responses and also eliminating assumptions, this does not with certainty account if remaining Herbal extracts can show Antifungal action (Individually or in Combination) at much higher concertation, For example Even after decrease in response concentration in both Amphotericin B and Terbinafine (with rising extract concentration) Heliotropium Indicum Linn. stem did show slight rise in response as pure extract which suggests higher concentration of this drug have chance of showing adequate action. With obtaining positive results to many of the combinations, this project can be further be extended to research in multiple direction. As subject of our experiment is an ideal Fungal species (Candida albicans), possessing many similar features with other fungal species of same or other genus, we are very hopeful of future use of our research against other pathological fungus that may cause common diseases or fatal infections. To make the extracts more potent, separation and purification of all potential active substance in extract can be utilized. Purification will make the Herbal Formulation / Combination much more specific in its action and can also be used as parent moiety for molecular modelling which if successful can be developed into series of such new drugs (with necessary modification) with a main chemical base frame. Antifungal combinations used in our experiment can be commercialized into various dosage forms, for both topical (Gels, creams, ointments against topical fungal infections) and systemic use (after invitro test). In the end, we summarize by stating that the experiment proved three of the five extracts obtained from three different Species to be Novel antifungal agents in combinations with present antifungal agents like Terbinafine and Amphotericin B or individually.

Source of Funding
None.

Conflict of Interest
None.