Biotransformation in Combination of Traditional Medicinal Plants: The Effect of Wickerhamomyces Anomalus on the Antidiabetic Potential and Metabolite Proling

Background: Biochemical transformation is a green and ecient strategy that can be of help in case of potentiation of herbal treatments. Diabetes requires daily therapeutic interventions and the use of herbals can be safe and effective alternatives however higher doses of herbs can cause much inconvenience. The potentiation of traditional medicinal plants based polyherbal combinations to reduce the dosage can be an attractive approach. Methods: The present study explored the biochemical transformation of a polyherbal extract derived from Syzygium cumini seed, Mangifera indica seed kernel, Momordica charantia fruit and Gymnema sylvestre leaf using W.anomalus. The polyherbal extract was chromatographed and evaluated for antidiabetic potential, Pre and Post fermentation. Also, the fermentation kinetics was assessed as W. anomalus can produce ethanol which can be self-limiting. Results: The extract produced no behavioral or physical changes in animals, conrming the safety in acute toxic exposure. The chromatographic analysis showed good presence of Hesperidin, Mangiferin and Corilagin in non-fermented extract which was signicantly reduced and content of Apigenin, Luteolin, Quercetin, kaempferol and Isorhamnetin were increased after fermentation. Conclusions: The polyherbal extract showed good antidiabetic activity however fermented polyherbal extract showed a similar effect at half the dosage. The effects are also corroborated by histological studies.


Introduction
Fermented traditional medicine represent an important part of the worldwide. Whole-cell biotransformation has been reported as an important tool that can be used to modify the chemical composition of the substrate medium. The chemical analysis of the substrate and product gives an idea about the enzyme structure as well as the metabolic infrastructure of the microorganism. Also, the use of microbes can yield a speci c set of a substrate with the use of milder conditions which further enables simple downstream processing to afford a usable concoction. The successful use of a combinatorial fraction also validates the use of a systems biology approach in herbal-based preparation as it provides a holistic response [1][2][3][4][5].
Biotransformation using pure microbial cells is the perfect choice because of speci c reasons like good surface-volume ratio, high growth rate, the higher rate of metabolism leading to an e cient transformation of substrates under sterile conditions [3]. Microbial metabolism comprises Primary metabolism, which is in control of cell capacity, and secondary metabolism, which utilizing existing metabolic pathways produces substances for the acclimatization of the organism to its environment.
Secondary metabolism generates small molecular weight metabolites that are not necessary for growth but that offer various advantages and may occasionally have medical/industrial signi cance [4,5].
Diabetes is speedily emerging as the most common ailment in the world. Being a chronic disorder, Diabetes needs lifelong interventions. Currently available oral hypoglycemic agents need extreme caution and regularity to maintain the sugar level. The use of herbal therapy can help in maintaining the sugar level however the dose constraints affect its use in the long term. Antidiabetic activity of polyherbal formulations has been evaluated to discover more effective and safe therapeutic agents. Flavonoids being an omnipresent category have the widest circulation in the plant kingdom. Further various avonoids have been established to possess the antidiabetic activity along with antioxidant, free radical scavenging, anti-in ammatory, anti-carcinogenic and antimutagenic properties combined with their ability to modify cellular metabolism. They are known inhibitors of COX (Cyclooxygenase), LOX (Lipoxygenase), XO (Xanthine oxidase) and phosphoinositide 3-kinase [1].
Gino et al evaluated the combinatorial effect of a poly-herbal combination having a hypoglycemic and hypolipidemic agent. The antidiabetic activity of combination of Salacia oblonga, Tinospora cordifolia, Emblica o cinalis, Curcuma longa and Gymnema sylvestre was determined in streptozotocin (55 mg/kg, i.p.)-induced diabetic rats, at a dose of 100 mg/kg [6].
Nagja et al determined the anti-diabetic activity of a polyherbal formulation (containing Eugenia jambolana, Gymnema sylvestre, Momordica charantia and Andrographis paniculata and sodhana processed extract of Myristica fragrans) in streptozotocin-induced type 2 diabetic rats [7]. Mazlan et al studied biotransformation of M. charantia fruit juice via lactic acid bacterium fermentation and observed reduced bitterness, reduced sugar content and production of aglycones and other metabolites as well as improvement in its α-glucosidase inhibition compared with the fresh, non-fermented juice [8]. Petchi et al. evaluated the synergistic effect of Glycosmis pentaphylla, Tridax procumbens, and Mangifera indica in diabetes [9]. Vidya et al (2013) demonstrated the hypoglycemic and antidiabetic activity of a polyherbal formulation, in both normal and experimentally induced hyperglycemic (Streptozotocin) rats [2].
Fermentative biotransformation is an ecofriendly and e cient strategy of chemical modi cations that have the potential to alter the therapeutic outcomes of the substrates. Wickerhamomyces anomalus is a biocontrol yeast and contains several enzymes e.g. invertase, phytase, glycosidase etc. Diabetes needing lifelong interventions must have effective, safe and sustainable therapeutic options i.e. polyherbal extract fermented with W. anomalus.
The present study explores the phytochemical and therapeutic outcome of fermentative biotransformation in polyherbal extract of a composite extract derived from Syzygium cumini seed; Momordica charantia fruits, Mangifera indica seed kernel, Gymnema sylvestre leaves. Wickerhamomyzes anomalus (Strain no. MTCC-4133) was utilized for fermentative biotransformation. The fermentation process was further documented for kinetics and the avonoid pro le of fermented extract was quantitatively compared with non-fermented extract using HPTLC pro ling. Fermented extract (FE) and non-fermented extract (NFE) were assessed for the acute toxicity in rats and antidiabetic potential in Streptozotocin-induced diabetes in rats, with Glibenclamide as reference standard. Also, the histopathological changes in the vital organs i.e. kidney, pancreas and liver are studied to understand the cellular changes during treatment.

Preparation of polyherbal Extract:
All the dried crude materials were physically cleaned, washed with water and then dried in an oven at 40 C for 3 h. It was then coarsely ground and sieved over 10# sieve. The retained material was utilized for extraction purposes [3].
All 4 ingredients were mixed in equal ratio (500 g each) in a polybag and then packed in a soxhlet apparatus. It was extracted for 72 h using water. The extract so obtained was concentrated utilizing a vacuum evaporator and nally dried to yield brown powder (396 g).

Fermentation of polyherbal Extract:
Wickerhamomyzes anomalus (Strain no. MTCC-4133) was utilized for fermentation. The lyophilized culture ampoule was revived on MYPG medium at 28 ± 0.5 °C for 72 h. The seed culture media was prepared and inoculated with slant media containing the fungal biomass and incubated at 28 ± 0.5 °C for 48 h. The dried polyherbal extract was dissolved in water to obtain 50% dispersion and employed as a substrate in the fermentation. The autoclaved 50% aqueous dispersion was inoculated with 5% v/v seed culture and placed in an incubator at 28 ± 0.5 °C. The duration for fermentation was selected as 72 h [5,8].

2.4 Phytochemical screening
The non-fermented and fermented extracts were subjected to initial phytochemical screening for determining their chemical composition. The absence or presence of various phytoconstituents like. carbohydrates, alkaloids, steroids, saponins, tannins, avonoids and terpenoids were identi ed by recommended test methods [10,11].

Physicochemical characterization (Fermentation Kinetic Pro le)
The fermentation kinetics was examined through the determination of pH, speci c gravity, optical density, phenolic content, avonoid content, reducing sugar and alcohol production at every 6 h up to 72 h. pH was taken as such whereas for reducing sugar and speci c gravity, a ltered sample was used. For the determination of Optical density, the ltered medium was analyzed against 50% aq. dispersion as a blank. TPC was determined in relations of gallic acid equivalents. The avonoid content was estimated in terms of Quercetin content. The ethanol content was assessed utilizing Gas Chromatography (GC) [12,13] .
The total phenolics were estimated utilizing the Folin and Ciocalteu method. A calibration curve was prepared using graded concentration (5-500 µg/ml) of Gallic acid. Test and standard readings were made utilizing a UV spectrophotometer at 765 nm together with the blank reagent. The phenolic content was expressed as gallic acid equivalents(GAE/g of dry plant material) based on a standard curve of gallic acid (5-500 µg/ml) [12].
Total avonoid content was estimated by utilizing the aluminum chloride colorimetric technique. A standard calibration curve of quercetin was prepared using its graded concentration. The absorbance was estimated against blank at 420 nm wavelength with UV-spectrophotometer. The total avonoid content was expressed as mg quercetin equivalent (QE)/g of dried plant material.
Aqueous decoction and its fermented product were partitioned successively with hexane, chloroform, and ethyl acetate. The extracts were dried, and 50 mg ethyl acetate extract was dissolved in methanol to make the sample solution (10 mg/ml)

Acute toxicity study
Acute toxicity of the fermented extract (FE) and non-fermented extract (NFE) was determined to evaluate its toxicity potential. Rats were divided into a control and four test groups (n = 6). The test groups received FE and NFE orally at the doses of 1000, and 2000 mg/kg body weight respectively. After administration, the animals were kept in separate cages and were allowed to food and water ad libitum. The animals were then observed for possible behavioral changes, allergic reaction (skin rash, itching) and mortality for the next 72 h[2].

Antidiabetic activity
The protocol of the study was reviewed and approved by the Institutional Animal Ethical Committee (Registration Number: 1044/PO/Re/S/07/CPCSEA and Project Proposal no-ITS/10/IAEC/2018). All the animal activities were performed on male albino rats weighing 100-150 g. The rats were maintained on a normal fat diet and were fasted overnight with free access to water before any observation. During the experiment, the animals were divided into ve groups of six animals in each group. Treatment was administered with a glass syringe and micro-suction canula no. 18.

Oral Glucose tolerance test
The blood glucose concentration of animals was measured at the beginning of the study. Then the rats were orally treated with 3 g/kg body weight glucose solution. The measurements were repeated after 30, 60, 120 and 180 minutes after the glucose load [2].

Induction of diabetes:
Animals have fasted for 24 hrs then a single intraperitoneal injection of freshly prepared Streptozotocin (65 mg/kg dissolved in 0.1 M citrate buffer, pH 4.5) was injected. The animals were left aside for 30 minutes and then a 10% glucose solution was placed in the cages for 48 hrs. The diabetes was con rmed by estimation of blood glucose level on the third day. Rats having Blood Glucose Level > 250 mg/dl were used for the study. For the antidiabetic activity, the animals were divided into ve groups of six animals in each group. Treatment with FE and NFE started after the last Streptozotocin injection. Blood samples were collected at 0 day, 7 days, 14 days, 21 days and 28 days till the end of the study.

Histopathological Studies:
Rats were euthanized according to the CPCSEA guidelines. Pancreas, liver and kidney tissue were macroscopically examined and slices of each organ were xed in 10% neutral buffered formalin with PBS. After the processes of dehydration, cleaning and in ltration, the organs were embedded in para n wax and section through a Micro-tome. The tissues were stained with hematoxylin and eosin. The slides were examined and captured with a digital microscope camera.

Statistical analysis
The data were expressed as mean ± SEM. The data of hypoglycemic activity, oral glucose tolerance test (OGTT), and antidiabetic activity were analyzed by one-way analysis of variance (ANOVA) followed by

Results & Discussion
Diabetic burden is taking a heavy toll on human health as well as the World economy. Since it's not curable, its management using oral hypoglycemic is very much essential for enhancing the patient life's quality. The use of herbal options can lower the dose of oral hypoglycemic providing better e cacy however the herbal therapeutics have a higher dosage so their potentiation will greatly help in reducing the dosage and enhancing the patient compliance.
Biotransformation is a process by which organic compounds are transformed from one form to another to reduce the persistence and toxicity of the chemical compounds. These transformations can be grouped under the categories of hydrolysis, reduction, oxidation, condensation, isomerization, introduction of functional groups and development of new carbon bonds.
3.1 Phytochemical Screening: Both the extracts (NFE and FE) were phytochemically screened for the presence of alkaloids, glycosides, steroids, terpenoids, phenolics etc. The observations are given in Table 1. Steroids were absent in both the extracts whereas alkaloids, carbohydrates, saponins, terpenoids, phenolics and avonoids were present in both the extracts. In various tests for different categories of avonoids, FE showed good presence of diverse avonoids. give an idea about the sequence of fermentation without any tedious analytical processing, e.g., the alcohol content ensures that the fermentation has taken place. The other tests like pH, solid content, and speci c gravity indicate the physical nature of the extract. Throughout the fermentation process, pH, speci c gravity, optical density, phenolic content, reducing sugar and alcohol production were measured at every 6 h up to 72 h. Table 2 shows the changes in various parameters as the time of fermentation progresses.  During fermentation, speci c gravity decreases due to ethanol generation and utilization of free sugar content. pH becomes acidic due to the liberation of free acids. The optical density increases throughout the study period indicating exponential growth of the microbial culture. Also, the strain produced a good quantity of ethanol by consuming a major part of sugar content. W. anomalus is an ethanol tolerant yeast and can grow under high ethanol content. The content of phenolics shows great enhancement as well as the contents of avonoids. Total phenolic content increases by 5.86 times and total avonoid content increases by 3.61 times during fermentation (Fig. 1). Figure 1: Physicochemical changes as a function of time 3.3 HPTLC Fingerprinting: The study employed 3 fruit materials that are rich in complex phenolics which yielded simple phenolics upon fermentative biotransformation thereby giving higher total phenolic content. Also, it has good avonoid content along with avonoid glycosides which gets hydrolyzed during the study yielding free avonoid aglycones, estimated as Quercetin equivalents. The structures of various avonoid compounds analyzed in the study are shown in Fig. 2.  Figure 4 shows the HPTLC densitogram for NFE and FE.  Table 3 shows the quantitation of various compounds in NFE and FE. The quantitation was performed by the preparation of calibration curves. All 8 compounds showed good linearity under selected analytical conditions. The quantitation also corroborates the hydrolysis process during fermentation. The content of free aglycones increased in FE. Validation was performed to establish the validity of the method. All 8 compounds were quantitated in non-fermented and fermented extract.  Table 4 showed the blood glucose level during the oral glucose tolerance test. FE showed good control over glucose levels at a lower dose level. The data is expressed as mean ± S.D. The observations were taken on 6 animals. The values having *p < 0.005 were signi cant and **p < 0.001 were highly signi cant.

Antidiabetic activity
Antidiabetic activity was determined in Streptozotocin-induced diabetes of rats. As indicated by Table 5, FE restores the normal glucose level within 2-3 h. Also, the overall effect of 200 mg/Kg dose is similar to 400 mg/Kg. Based on correlation, free avonoid content must be responsible for increased antidiabetic activity. Free aglycones are less bulky and more lipophilic thereby easy to absorb through the small intestine. This increases the effective bioavailability and in turn, increases the antidiabetic activity. All values were expressed as mean ± SD (n = 6). *p < 0.005 when compared to control group **p < 0.001 when compared to standard (One way ANOVA followed by Dunnett's test).
Glibenclamide showed a gradual decrease in 21 days, the effects plateau further. Non-fermented extract showed a similar pattern till 28 days whereas fermented extract showed almost similar reduction in blood glucose within 7 days.

3.7.
Histopathological studies: Figure 5 shows the histological structures of Kidney, pancreas and liver of Normal, diabetic and treated (Non-fermented ex. & fermented ex.) animals. As is evident, the fermented as well as non-fermented extract showed a great extent of restoration in vital organs. However, it is pertinent to note that fermented extract has been more effective in the restoration of normal histological infrastructure.

Conclusion
The present study summarizes the physical, chromatographic and therapeutic transformation in polyherbal extract upon fermentation. W. anomalus, being an ethanol-producing strain, generates a substantial quantity of ethanol thereby decreasing the speci c gravity, however, the microbial strain also affected the pH of the extract. Further, the strain grows steadily as indicated by enhanced optical density. The effects on chemical pro le initially re ected an increase in TPC and TFC. In chromatographic analysis of NFE and FE, the main difference was the content of aglycones. In NFE, several glycosides were present but after fermentation, the content of glycosides decreased indicating the occurrence of glycolysis and hydrolysis during fermentation. During fermentation, various acids are released, decreasing the pH and catalyzing the acidic hydrolysis of glycosidic bonds. Most oligomeric avonoids are converted to monomers and dimmers under acidic conditions. Further, W. anomalus can produce glycosidases which can directly attack the glycosidic bonds to release the aglycones. Based on physicochemical and chromatographic observations, it can be said that the microbial cells grew and feed off the free sugar to generate ethanol however to maintain the osmotic stress, glycosides are hydrolyzed and free sugars generated. The fermentation makes the chemical composition of FE more lipophilic and free aglycones are directly absorbed by the small intestine. The pharmacological effects are further corroborated by histological observations. The enhanced content of aglycones might be the main reason behind the enhanced antidiabetic activity of FE.  Structures of avonoid compounds analyzed in the study Apigenin and Luteolin belong to Flavone category, quercetin, kaempferol and Isorhamnetin belong to avonols. Mangiferin is a xanthone glycoside, hesperidin is avanone glycoside, Corilagin is phenolic glycoside.   Histopathological comparison of normal, diabetic, Fermented extract-treated animals and Non-fermented extract-treated animals.