Phytocompounds of Bistorta macrophylla (D. Don) Sojak. as bioavailability enhancers of uconazole and amphotericin B to better manage Candida species infections

Bistorta macrophylla (D. Don) Sojak. is a medicinal plant of high altitude and so far, not been scientically explored? Since prehistoric times, B. macrophylla has been used to cure stomach pain, pyretic fever, u, lungs infections, diarrhea, vomiting. The present research was aimed to examine the phytochemicals, antifungal, and synergistic potential of methanolic extracts of B. macrophylla. Methanolic extract of B. macrophylla was found to have high phenolic (191.18 ± 29.18 mg g −1 GAE) and avonoid (26.71 ± 3.21 mg g −1 RE) content. Methanolic extract also demonstrate strong antifungal action with diameter of zone of inhibition of 17.5±0.5 mm (fungicidal) against both the strains of C. albicans (MTCC277 and ATCC90028). The minimal inhibitory concentration (MIC) of methanolic extract was found to be 62.5 µg ml −1 against C. albicans (MTCC277 and ATCC90028). In addition, the combination of methanolic extract of B. macrophylla with antifungal antibiotics (uconazole and amphotericin B) showed synergistic interaction with MIC reduction from 4-128 folds against both candida strains. GC-MS analysis of methanolic extract revealed the presence of 15 major phytocompounds with area more than 1%. Molecular docking showed that sucrose and 9,9-Dimethoxybicyclo [ 3.3.1] nona-2,4-dione has highest binding energy of -6.3 and -5.1 KJ/mol against Cytochrome P450 14 alpha-sterol Demethylase (PDB ID: 1EA1) protein respectively. Combination of methanolic extract of B. macrophylla with antifungal antibiotics (uconazole, amphotericin B) can be used to treat drug-resistant candida. Methanolic extract of rhizome of B. macrophylla showed synergistic potential with uconazole and amphotericin B against fungal strains. To analyze the synergistic potential between antifungal antibiotics (uconazole and amphotericin B) and methanolic extract of rhizome of B. macrophylla, nine combinations based on MIC of antibiotics and methanolic extract of rhizome of B. macrophylla were prepared. Based on the minimum FICI value, the combination of extract equivalent to MIC (62.5 µg ml −1 ) and uconazole equivalent to ½MIC (15.625 µg ml −1 ) and ½ MIC of extract (31.25 µg ml −1 ) and ½ MIC of amphotericin B (15.625 µg ml −1 ) showed best synergistic potential against C. albicans (MTCC277). The synergistic combination of methanolic extract and uconazole enhanced the potency of the uconazole and methanolic extract by 32 folds against C. albicans (MTCC277). The combination of amphotericin B with methanolic extract enhanced the potency of amphotericin B and methanolic extract by 128 folds. Combination of ½ MIC of extract (31.25 µg ml −1 ) and MIC of uconazole (15.625 µg ml −1 ); ½ MIC of extract (31.25 µg ml −1 ) and ½ MIC of amphotericin B (15.625 µg ml −1 ) showed best combinations against C. albicans (ATCC90028) as shown in Table 3. The synergistic potential of uconazole in combination with methanolic extract was enhanced by 116 folds and 16 folds for


Introduction
Since prehistoric time, medicinal plants and herbs have been discovered and used in folk medicine (Stojanoski 1999). Now phytocompounds are at the forefront of drug discovery. According to World Health Organization (WHO), "Any plant whose one or more part, contain substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs" is classi ed as medicinal plant. WHO has estimated that about 80% of the World population rely almost exclusively on traditional medicine for their primary healthcare needs (Farnsworth 1988). Medicinal plants are the "backbone" of traditional medicine system, and most of the people in the less developed countries utilized these traditional medicinal plants on a regular basis (Davidson-Hunt 2000) for their well-being. Plants produce or secrete an array of chemical compounds and use them for various biological functions including defense against insects, fungi and bacteria. Scientists are now exploring these phytocompounds as novel therapeutics approach to solve the emerging issues of multi-drug resistance (Rolta et al. 2020b; ; Salaria et al. 2021). Antimicrobial resistance is a major threat to animal and human health development, affecting our ability to treat a range of infections. Treatments for a growing number of infections have become less effective in many parts of the world due to emergence of drug resistance (WHO 2016). Among many other diseases, candidiasis caused by Candida albicans is one of the major concerns (López-Martínez 2010). Fluconazole and amphotericin B are the most commonly and widely used antibiotics to treat Candidiasis. Some strains of Candia have developed resistance (antifungal resistance) to these antibiotics and are di cult to treat (Helmerhorst et al. 1999). The rise of antifungal resistance is very high and discovery of new antibiotics is very slow. Centres for disease control and prevention (CDC) has reported that some strains of Candida are highly resistant to even rst line and second line of antifungal medications, such as uconazole and the echinocandins (anidulafungin, caspofungin, and micafungin). Candida auris has been reported to be resistant to uconazole and amphotericin B and now it has become a serious health concern (Satoh et al. 2009; Lockhart et al. 2016). The individual with drug resistant infection has to consume higher dosage of antibiotics, which in turn leads to emergence of drug resistance and also responsible for many side effects and ultimately life expectancy (Gould et al. 2013). The mechanisms of antibiotic resistance in mainly due to mutation in gene encoading the target site of FQs (DNA gyrase and topoisomerase IV), over expression of e ux pumps and protection of the FQ target site by a protein designated Qnr (Munita and Arias 2016). The combination therapy of two or more antibiotics (synergism) has been in practice for some, but this has also resulted in selection pressure on pathogens and drug toxicity (Dudhatra et al. 2012).
Therefore, the current need is to search for molecules that are not antibiotics itself, but enhance the potency /availability of the antibiotics. Such molecules should be non-toxic and enhance the bioavailability of antibiotics in combination and act synergistically. Potent bio-enhancers could potentially lower the dosage of antibiotics and therefore reduced toxicity to the host. Phytochemicals of medicinal plants have huge potential to be developed as bio-enhancer of antibiotics and feed the drug discovery pipeline to develop new therapies to manage candidiasis. India has huge diversity of medicinal plants and rich system of traditional medicines and ethno pharmacology. Especially, Himalayan region houses 1748 species of unique medicinal plants with various traditional and modern therapeutic uses (Samant et al. 1998), including 675 species of wild edible plants (Samant and Dhar 1997) and 118 species of medicinal plants yielding essential oils. High altitude of Himalayas is a unique habitat that resulted in the accumulation of unique phytoconstituents in medicinal plants and warrants their exploitation in a holistic manner to counter the problem of multidrug resistance and for the well-being of people. The Indian sub-continent is suitable for cultivation of large number of medicinal and aromatic plants, which can be used as raw materials for pharmaceuticals, perfumery, cosmetics, avors and food. The Chanshal valley of Himachal Pradesh is regarded as hub of medicinal plants and trees. It contains several species of medicinal plants and trees e.g. Rheum emodi, Juniperus communis, Bistorta macrophylla, Jurinea macrocephala, Picrorhiza kurroa, Pleurospermum bruninis and Betula utilis etc. and people of Dodra Quar and Rohru region utilize these medicinal plants or trees for the cure and preventions of different diseases (Jaundice, wound healing, fever, cough, respiratory problems, boils, nerve disorder, dysentery ulcers etc.) since ancient time.
B. macrophylla is one of the important and unexplored medicinal plant species of alpine region of Himalayas and is commonly known as Kukhri, Chhotaninayin, Kande-re-ninai in Hindi and Snakeweed in English. B. macrophylla belongs to family polygonaceae. B. macrophylla is a stout perennial herb and arise from a woody root stock (rhizome), reticulate. It is native to mountain regions of West-China, Pakistan, Bhutan, North India (Himachal Pradesh, Uttarakhand), West India (Western Ghats) and Nepal (Chauhan 1999). In India, it is distributed at altitudes ranging from 3300 to 3800 meters height in the temperate and subtropical regions of Himalayas (Chauhan 1999). Rhizomes of B. macrophylla are widely used in Ayurvedic and traditional medicine as antidiarrheal, antidysenteric, alleviates stomach pain, anti-in ammatory, and anti-pyretic (Gaur 1999). Traditionally, B. macrophylla is used to cure the stomach problems and paste of B. macrophylla rhizome is given to children and infants for stomach problems. Older patients chew the roots for the same problem (Chauhan 1999). Paste and powder of B. macrophylla roots was taken orally to cure fever, ulcer and toothache. Paste of B. macrophylla roots by prepared by rubbing roots on hard surface of stone with few drops water. It is also used to cure tuberculosis, in ammation, pyretic fever, u, lungs disorders, diarrhea, vomiting, arthritis, gout, kidney stones or hyperacidity and hypertension (Wangchuk et al. 2016). B. macrophylla is known for its traditional medicinal values, but not explored scienti cally, except one report by Chandra et al. (2016). Therefore, the current study was designed to study the potential of methanolic extract of rhizome of unexplored B. macrophylla as bioavailability enhancer of uconazole and amphotericin B against to manage candidiasis and identify the major phytocompounds by GC-MS analysis. The rhizome of B. macrophylla was thoroughly washed with running tap water to remove the soil particles and surface sterilized using 70% ethanol for 2 minutes, followed by washing with sterilized distilled water. The rhizomes were cut into small pieces and dried in hot air oven at 40 ºC (until no further reduction in weight for 6 h) and then ground to ne powder with the help of electric grinder.

Materials And
About 50 grams of dried powder of rhizome of B. macrophylla was subjected to methanol (500 ml) extraction using hot continuous method in a Soxhlet apparatus. The extract was ltered through Whatmann lter paper no. 1 and the collected ltrate was dried at 40 ºC. The dried methanolic extract was stored at 4 ºC in air tight bottles until further use.

Qualitative analysis of phytochemicals present in methanolic extract of B. macrophylla
The rhizome extract of B. macrophylla was tested for the presence of major phytocompounds such as phenolics, avonoids, tannins, saponins, alkaloids, glycosides, phytosteroids and carbohydrate by protocols described earlier (Khandelwal 2008). For the detection of alkaloids and glycosides, 50 mg of methanolic extract was dissolved in 5 ml of diluted HCl (1%, w/w) and ltered through Whatman lter no1. The resultant ltrate was used for the detection of alkaloids and glycosides. On the other hand, 50 mg of methanolic extract was dissolved in 5 ml of sterilized water and then ltered. The ltrate was used for the detection of phenolics, tannins, phytosterols, phytosteroids, carbohydrate, avonoids, proteins and amino acids. The total phenolic content of methanolic extract of B. macrophylla rhizome was determined by using Folinciocalteau reagent (Singleton 1999) total phenolic content was calculated from the calibration curve of gallic acid (5-100 µg ml −1 ) and expressed in terms of gallic acid equivalents (GAE) per gram of the extract and calculated using the following equation: Where 'C' is total content of phenolic compounds in mg g-1 plant extract in GAE, 'c' is the concentration of Gallic acid estimated from the calibration curve (mg ml-1), 'V' is the volume of extract in milliliter and "m" is the weight of crude plant extract in grams.

Quanti cation of total avonoid content
The total avonoid content of ethanolic extract of rhizome of B. macrophylla was quanti ed using aluminium chloride (AlCl3) method (Zhishen et al. 1999). The avonoid content was calculated from standard curve of rutin (5-100 µg ml −1 ) and expressed as rutin equivalents (RE) per gram of the extract and was calculated by the following equation: where 'C' is total content of avonoid compounds in mg g-1 plant extract in rutin equivalent; 'c' is the concentration of rutin calculated from the calibration curve in mg ml −1 , 'V' is the volume of extract in ml, and 'm' is the weight of crude plant extract in grams. ml of YPD agar medium was poured in sterile 100 mm petri-dish and allowed to solidify. Then, fungal culture of optical density of 0.12~0.15 at 530 nm equivalent to 0.5 McFarland standard was uniformly spread on the surface of the YPD agar medium using sterile cotton swabs and plated were allowed to dry under aseptic conditions. The wells were punched with the cork borer (6 mm) in the agar and 50 µg (1 µg ml −1 ) rhizome extract was loaded in the wells and allowed to diffuse. Assay plates were incubated of 48 h at 30 ºC and the zone of inhibition was measured using Hi Antibiotic Zone scale-C (Himedia Biosciences, Mumbai (India). Fluconazole lter disk (10 µg) purchased from Himedia Biosciences, Mumbai (India) was used as a positive control and dimethyl sulphoxide (DMSO) and methanol alone were used as solvent alone as a control in the antifungal assay. The tests were performed in triplicate and results were recorded as mean ± SD.

Broth dilution assay to determine the minimum inhibitory concentration (MIC) of methanolic extract of B. macrophylla rhizome
The minimum inhibitory concentration (MIC) of the methanolic extract of rhizome of B. macrophylla was measured by broth dilution method described under CLSI guidelines using 5-tripheny tetrazolium chloride (CLSI 2012). The methanolic extract was dissolved in DMSO and geometric dilutions ranging from 500-0.025 µg ml −1 were prepared in a 96-well microtiter plate, including one growth control (YPD broth containing DMSO) and a positive control (YPD broth inoculated with fungal culture and containing amphotericin B (25 µg) or uconazole (25 µg). Assay plates were incubated for 48 hours at 30 ºC. Following incubation, 5-tripheny tetrazolium chloride (5 µg) was added to each well and incubation was continued for another 2 h. Change in colour from purple to pink or colourless was observed and used as a measure to calculate the MIC. Helium was used as a carrier gas, at a ow rate of 1.0 ml minutes −1 and mass spectra were recorded in the scan mode. The ionization voltage was 70 eV. The split ratio was 10:1. The ion source temperature was 230 ºC, Interface temperature was 280 ºC. Solvent cut time was 3 minutes. For the analysis, 1 µl of 1mg ml −1 of the sample was used. The constituents of extract were identi ed based on their retention time (Rt) with respect to the reference. The scan range was 45-450 m/z. The identi cation of compounds was based on matching unknown peaks with MS-data bank (NIST 2.0 electronic Library).

Drug likeness calculations of B. macrophylla compounds
The purpose of the drug scan was to see if certain phytochemicals met the drug-likeness criteria. For assessing drug similarity properties such as amount of hydrogen acceptors, Lipinski's lters were used. Drug likeness prediction was done by using online server Molinspiration (http://www.molinspiration.com) were applied for examining drug likeness attributes as including quantity of hydrogen acceptors (should not be more than 10), quantity of hydrogen donors (should not be more than 5), molecular weight (mass should be more than 500 daltons) and partition coe cient log P (should not be less than 5). The smiles format of each of the phytochemical was uploaded for the analysis (Rosell and Crino 2002).

Analysis of antifungal activity of methanolic extract of B. macrophylla rhizome by agar well diffusion and broth dilution method
An antifungal activity assay was performed by using agar well diffusion method and observed as zone of inhibition against the tested fungal strains. Methanolic extract of B. macrophylla showed inhibition to growth of fungal strains. Antifungal activity was fungicidal (cell death) against C. albicans (MTCC27 and ATCC90028); whereas uconazole and amphotericin B were fungi static (growth arrest) against C. albicans (MTCC277 and ATCC90028) shown in Table 2 and gure 3a.   Table 3. The synergistic potential of uconazole in combination with methanolic extract was enhanced by 116 folds and 16 folds for methanolic extract. On the other hand, the synergistic potential of amphotericin B in combination with methanolic extract was enhanced by 128 folds for amphotericin B and methanolic extract.   Table 4. The list of phytocompounds identi ed in the methanolic extract of B. macrophylla is summarized in Table 4.  Docking results showed that among all the selected compounds of B. macrophylla sucrose showed the highest binding energy (-6.3 KJ/mol) followed by 9,9- Interactive amino acids are shown in Table 5 and Figure 5. 3D structures of protein ligand complexes are shown in Figure 6. Furthermore, all the selected phytocompounds were screened for drug likeness and ADME/T.  Whereas Sucrose, Linoleic acid ethyl ester and 1,1,1,3,5,5,5-Heptamethyltrisiloxane showed one violation which is acceptable (Table 6).

Conclusion
This is the rst report on systematic study of methanolic extract of B. macrophylla and showed antifungal activity. Methanolic extract also increased potency of uconazole and amphotericin B by 8-128 folds. Hence

Figure 2
Schematic diagram to measure the synergistic activity between methanolic extract of rhizome of B. macrophylla and antibiotics ( uconazole or amphotericin B) using different dilutions. Assay was done using 96 wells micro titer plates. Purple color indicates dead cells, whereas pink color represents viable cells.