Antioxidant and Antidiabetic Potential of Endophytic Fungal Fractions Recovered from Tinospora Cordifolia (Willd.) Leaves

Introduction

Type 2 diabetes mellitus (T2DM) is a persistent disorder attacking the people globally and about 463 million are diagnosed with this disorder at present.¹ Although preliminary treatment for diabetes starts with food and exercise 90% of patients with diabetes cannot maintain long term glycemic control with diet and exercise alone. Hence, fluctuations in blood glucose levels can lead to many serious complications like neuropathy, nephropathy, retinopathy and cardiovascular diseases.² T2DM treatment involves administration of oral hypoglycemic agents like Sulfonylureas (Tolbutamide, Glibenclamide, Glipizide), Biguanides (Phenformin, Metformin), Thiazolidinediones (Troglitazone, Rosiglitazone) and a-Glucosidsase inhibitors (Acarbose, Miglitol). But the drawback of insulin and oral hypoglycemic agents is that none of them are ideal in correcting blood glucose levels round the clock, with single dose administration. T2DM also made a drastic increase in global health expenditure of about 760 USD which makes an economic and societal burden.² Thus, there is a demand to discover a potential antihyperglycemic agent from alternative sources.

The evidence of plant-fungus associations occurred in Paleozoic era, where a record of close physical association was noticed in roots of fossil tree Amyleonradicians. This record is considered as a stand point of original endophyte symbiosis.³ Fungi that colonize a plant without causing symptoms at any specific moment can be termed as endophytic fungi.⁴ In the past two decades, a great deal of information on the role of endophytic fungi in nature has been obtained including their role as an important source of medicinally useful constituents. The metabolites secreted by endophytes may be similar to or derivatives of those produced by the host plant. Many novel secondary metabolites like Mogroside-V (sweetener) from Siraitiagrosvenorii,⁵ Huperzine-A (AChE inhibitor) from Huperzia serrata,⁶ Graphislactone-A (antioxidant) from Trachelospermumjasminoide,⁷ Podophyllotoxin (anticancer) from Podophyllum hexandrum⁸ were isolated from predominant endophytic isolates.

Materials and Methods

Plant Material

Leaves of Tinospora cordifolia (Willd.) were collected from Dharwad, Karnataka, India and were authenticated by Dr. S S Hebbar, Department of Botany, Government Science College, Dharwad. A herbarium was retained in the PG Pharmacognosy Department (SETCPD/ Ph.cog/herb/13/05/2019), SET’s College of Pharmacy, Dharwad.

Isolation of Endophytes

Tinospora cordifolia leaves were cleaned thoroughly to remove any foreign particles adhering using running tap water and were dried. The leaves were submerged in 70% ethanol for 1 min, rinsed with running water 4-5 times and surface sterilized for 30 s with 1% NaOCl (sodium hypochlorite) and again rinsed 4-5 times with sterilized distilled water. The washings were tested and the last washing was used as fungi-free control. Pieces of sterile leaves 1 cm each were inoculated on sterilized potato dextrose agar (PDA) media with 150 mg/L streptomycin. The plates were incubated at 25 ± 2 °C for 7-14 days. Features of the colonies, mycelia and spores were observed for pure isolates. Two pure endophytic fungi namely TCLF-1 and TCLF-2 (endophytic fungi of Tinospora cordifolia) leaves were selected for fermentation.⁹ 

Fermentation and extraction

The purified isolates of TCLF-1 and TCLF-2 were inoculated and fermented separately into a 3000 mL Erlenmeyer flask containing 600 mL of potato dextrose broth. The flask was incubated at 25°C -27°C for 21 days. Later, after 21 days of incubation, 500 mL of chloroform was added to flask and left overnight. Mycelia from the broth were separated by homogenization at 4000 rpm for 30 min and filtered by Whatman filter paper under vacuum. Further, three equal volumes of ethyl acetate followed by three times with equal volumes of n-butanol were used to extract the aqueous phase obtained after chloroform. The chloroform, ethyl acetate and n-butanol fractions of TCLF-1 and TCLF-2 were dried with vacuum using rotary evaporator (Super fit Rotavap, PBU-6) and weighed.¹⁰ They were designated as T1EA, T1nB, T2EA and T2nB respectively and were used for in vitro antioxidant and antidiabetic studies.

Preliminary phytochemical investigation

Preliminary phytochemical analysis of T1EA, T1nB, T2EA, T2nB were carried out following standard procedures.¹¹

Phenotypic Identification

Colony Morphology

Morphology of colonies developed on the agar slant was studied by the Lacto phenol cotton blue staining. The characteristics were matched with the known organisms from the literature.¹²

Molecular characterization by PCR sequential analysis

Genomic DNA was isolated from the given organism using the genomic DNA extraction kit (Bhat Biotech Ltd. Bangalore, India).^13,14 Amplification of the 16s rRNA gene was performed using the primers, Forward primer, 5’-TCCGTAGGTGAACCTGCGG-3’ and Reverse Primer, 5’- TCCTCCGCTTATTGATATGC-3’. Polymerase chain reaction (PCR) was performed by using the following method: A total volume of 50 microliters was taken in a 0.2 mL thin-walled PCR tube. Amplification was done using a thermocycler (DNAAMP Bhat Biotech). Denaturation was conducted at 95°C initially for 10 minutes, followed by 35 cycles at 94°C for one minute. Annealing and extension steps were done at 56°C and 72°C respectively for one minute followed by final extension at 72°C for 10 minutes. The PCR products from ITS gene PCR reactions were purified using GENEASY GEL ELUTION KIT. Automated DNA sequence -3037xl DNA analyzer from Applied Biosystems using BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems) was used to sequence both strands of the rDNA region and amplified by PCR. Sequence analysis software version 5.2 from applied biosystems was used to study the sequence data and to align and generate dendrograms. Before phylogenetic analysis, sequences were aligned as plus and minus strands using appropriate software. Sequences were compared to the non-redundant National Center for Biotechnology Information (NCBI) database using BLASTN. For the most similar sequences, the expected value and e values were noted. Ten similar neighbors were aligned using CLUSTAL W2. A phylogram for the multiple alignment file thus obtained was created using MEGA5 software.^15,16

In Vitro Free Radical Scavenging Activity

Reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical

TCLF-1 and TCLF-2 fractions were screened for their ability to scavenge DPPH radical following established method.¹⁷

Reaction with hydroxyl radical

Deoxyribose method¹⁸ was used to study the hydroxyl radical scavenging effect of TCLF-1 and TCLF-2 fractions.

Determination of reducing power

Fractions of TCLF-1 and TCLF-2 were subjected to reducing power assay by the method as described by Oyaizu (1986).¹⁹

In Vitro Antidiabetic Activity of Endophytic Fractions

Determination of alpha amylase inhibitory activity

T1EA, T1nB, T2EA and T2nB at different concentrations (20-100 µg/mL) were used for the activity. Mixture of 200 microliters of 0.02 M sodium phosphate buffer, 20 microliters of enzyme and endophytic fractions (20-100 µg/mL) were incubated for 10 min at room temperature, followed by incorporation of 200 microliters of starch in all test tubes. The reaction was terminated with addition of 400 microliters 3,5- Dinitro salicylic acid (DNS) reagent and placed in boiling water bath for 5 min, cooled and diluted with 15 mL of distilled water and absorbance was measured at 540 nm. The control samples were prepared without any derivatives. The % inhibition was calculated according to the given formula.²⁰

Inhibition %=    Abs 540 (control)-Abs 540 (extract) X 100

                                      Abs 540 (control)

Plots of percent inhibition versus log inhibitor concentration gave the IC₅₀ values by non-linear regression analysis from the mean inhibitory values.

Acarbose was used as reference standard.

Determination of alpha glucosidase inhibitor activity

P-Nitrophenyl-α-D-glucopyranoside, acarbose and alpha glucosidase were purchased from Sigma (USA). Phosphate buffer (100 mM; pH 6.8) was used to dissolve alpha glucosidase and considered as enzyme extract. Substrate used was P-Nitrophenyl-α-D-glucopyranoside. Endophytic fractions ie., T1EA, T1nB, T2EA and T2nB at different concentrations (20-100 µg/mL) were mixed with 320 microliters of 100 mM phosphate buffer pH 6.8 at 30°C for 5 minutes. Three milliliters of 50 mM sodium hydroxide was added to the mixture and the absorbance was read at 410 nm. The control samples were prepared without any fraction. The % inhibition was calculated according to the given formula.²¹

Inhibition %=    Abs 410(control)-Abs 410 (extract) X 100

                                      Abs 410 (control)

Plots of percent inhibition versus log inhibitor concentration gave the IC₅₀ values by non-linear regression analysis from the mean inhibitory values.

Acarbose was used as the reference standard.

Determination of aldose reductase inhibitor activity

Inhibition of aldose reductase (AR) activity was measured according to the method described earlier.²² The reaction cocktail contained 1 mL of 1M potassium phosphate buffer (pH 6.2), 0.4 mM lithium sulfate, 5 l M 2-mercaptoethanol, 10 l M DL-glyceraldehyde, 0.1 ml of lM NADPH, and enzyme. The reaction mixture was incubated at 37°C for 10 min. The enzymatic reaction was initiated by addition of NADPH and activity was measured by recording the decrease in absorbance at 340 nm. Various concentrations of T1EA, T1nB, T2EA and T2nB (20-100 µg/mL) were added to assay mixture. AR activity in the absence of inhibitor was considered as 100%. For the determination of IC₅₀ values, the concentration of individual fraction required to achieve 50% AR inhibition was calculated. Quercetin was used as standard inhibitor.

Results

Two fungal endophytes TCLF-1 and TCLF-2 were isolated from leaves of Tinospora cordifolia (Figure 1). The percentage yield of the ethyl acetate and n-butanol fractions of TCLF-1 and TCLF-2 were 4.2%, 3.4%, 5.4% and 4.1% respectively.

Preliminary Phytochemical Analysis

Preliminary phytochemical analysis of ethyl acetate and n-butanol fraction of TCLF-1 and TCLF-2 showed the presence of alkaloids, flavonoids, carbohydrates, and tannins as chief secondary metabolites.

Molecular characterization of fungal endophytes by PCR sequential analysis

Two pure culturable fungal isolates from the leaves were collected and designated as TCLF-1(HSCP1-1) and TCLF-2 (HSCP1-2). The sequence of the ITS gene from TCLF-1 and TCLF-2 that of matching sequences from 10 nucleotide sequences were aligned by using the Maximum likelihood method based on the Tamura-Nei model (Figure 2). The tree with the highest log likelihood (-1060.9137) is shown for TCLF-1 and log likelihood (-1417.5227) is shown for TCLF-2. Initial tree(s) for the heuristic search were obtained. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. The analysis involved 11 nucleotide sequences. There were a total of 506 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. Based on these results, TCLF-1 and TCLF-2 were identified as Trichoderma longibrachiatum isolate TL10 and Aspergillus versicolor isolate BAB-6580.

In Vitro Free Radical Scavenging Activity

DPPH assay

The IC50 values for T1EA and T1nB were found to be 145.02 µg/mL and 143.72 µg/mL respectively; whereas for T2EA and T2nB, it was 177.68 µg/mL and 77.47 µg/mL respectively. For ascorbic acid, IC50 value was found to be 36.18 μg/mL. T1EA, T1nB, T2EA and T2nB fractions showed significant scavenging activity. T2nB exhibited potent DPPH scavenging effect among the fractions tested. The results are shown in Figure 2.

Hydroxyl Scavenging Assay

The IC50 values for T1EA and T1nB were found to be 196.43 µg/mL and 198.52 µg/mL respectively. The IC50 values for T2EA and T2nB were found to be 141.13 µg/ mL and 103.15 µg/mL respectively, where as IC50 value for mannitol was found to be 118.07 μg/mL. Among the fractions tested, T2nB exhibited potent OH radical scavenging effect as shown in Figure 3.

Reducing power Assay

Increase in the reducing power was observed with increase in the concentration for TCLF-1 and TCLF-2 fractions. The results showed more effective reductive ability.

In Vitro Antidiabetic Activity of Endophytic Fractions

Alpha amylase inhibitory activity T1EA, T1nB and acarbose exhibited inhibitory effects on the α-amylase activity with an IC50 value of 117.06 μg/mL, 71.1 μg/mL and 49.18 μg/mL respectively.

T2EA and T2nB inhibited the α-amylase activity with an IC50 value of 116.56 μg/mL and 77.36 µg/ mL respectively. T1EA, T1nB and acarbose showed appreciable α-amylase inhibitory effects. T1nB and T2nB showed better inhibitory activity as compared to T1EA and T2EA. The results are depicted in Figure 4.

Alpha glucosidase inhibitor activity

There was a dose dependent increase in percentage inhibitory activity against alpha glucosidase in TCLF1 and TCLF-2 fractions. T1EA, T1nB and acarbose showed an IC50 value of 73.98 µg/mL, 150.72 µg/ mL and 100.96 µg/mL respectively. T2EA, T2nB also exhibited good activity with IC50 value of 193.82 µg/mL and 73.11 µg/mL respectively. TCLF-1 fractions showed the greater % inhibition of the alpha glucosidase enzyme as compared to others (Figure 5).

Aldose reductase inhibitor activity

T1EA, T1nB and quercetin showed inhibitory effects on aldose reductase with an IC50 value of 70.83 μg/mL, 67.58 μg/mL and 50.53 μg/mL respectively. T2EA and T2nB inhibited the aldose reductase with IC50 value of 86.73 μg/mL and 104.9 µg/mL respectively. T1EA, T1nB showed appreciable aldose reductive inhibitory action as compared to other fractions. The results are depicted in Figure 6.

Discussion

Diabetes mellitus (DM) is caused by lack of insulin secretion. DM is also a disarray of carbohydrate, lipid and protein metabolisms characterized by a high blood glucose concentration, hyperglycemia (fasting plasma glucose > 7.0 mmol/L or plasma glucose > 11.1 mmol/L 2 hours after a meal) caused by insulin deficiency, often combined with insulin resistance.²³ Diabetes mellitus accounts for over and above 90% of total diabetes cases. Severe hyperglycemia has led to various diabetic complications such as retinopathy, cardiopathy, cataracts, nephropathy and neuropathy.²⁴

There is an increasing interest relating to plant microbe association, especially the structure and function of endophytes that occupy plants without apparently causing any damage. Endophytes are microorganisms having the potentiality in colonizing internal tissues of the plant. They are present abundantly in all types of plants. Studies have been reported that they protect plants from pathogens.²⁵ They form a wide range of relationships such as mutualistic, communalistic symbiotic, and trophobiotic. Endophytes produce secondary metabolites that can be used in modern medicine for various therapeutic activities.²⁶ Therefore, endophytes are potential sources of plant associated natural products.

In this study, two fungal endophytes TCLF-1 and TCLF2 were isolated from leaves of Tinospora cordifolia. By sequential analysis, TCLF-1 and TCLF-2 were identified as Trichoderma longibrachiatum isolate TL10 and Aspergillus versicolor isolate BAB-6580. Trichoderma longibrachiatum is a fast-growing fungus in the genus Trichoderma.

It produces off-white colonies changing to greyish green with age.²⁷ It is a soil fungus found all over the world mainly in warmer climates. It is usually found on, other fungi, dead wood building material and animals. These species can grow at varied temperatures, the optimal temperature for growth is ≥ 35°C.²⁸ Trichoderma longibrachiatum is a clonal species reproducing through one celled, smooth walled conidia. T. longibrachiatum also characterizes in one of several grades of Trichoderma, comprising twenty-one various species. Trichoderma longibrachiatum exhibits many secondary metabolites such as polyketides, pyrones, terpenes and diketopiperazine-like compounds.²⁹ Aspergillus versicolor is a slow-growing filamentous fungus commonly found in damp indoor environments and on food products.

Colonies differ in colour, surface attributes and growth rate which depended on growth characteristics showed musty odour with moldy homes. Hepatotoxic and carcinogenic mycotoxin sterigmatocystin were prodcued by them.^30,31 A. versicolor is an eye, nose, and throat irritant. Mycotoxins, such as aflatoxin B1 and nidulotoxins are produced in lower concentrations by A. versicolor. Various metabolites isolated from A. versicolor showed antibacterial, insecticidal, cytotoxic and fungicidal activity ³².

Preliminary phytochemical investigations of fractions of TCLF-1 and TCLF-2 found to contain flavonoids, alkaloids, triterpenoid and tannins. Antioxidant metabolites play an important role in prevention of various disorders associated with cancer, cardiovascular, respiratory diseases to increase the immunity.³³ DPPH is an easy, rapid and sensitive method to screen the antioxidant activity of a specific compound or plant extracts. T1EA, T1nB, T2EA and T2nB showed significant scavenging activity with DPPH radicals. Fenton reactions in mitochondria and endoplasmic reticulum produce hydroxyl radicals. The endophytic extracts showed hydroxyl radical scavenging activity in a dose dependent manner due to inhibition of deoxyribose degradation. T1EA, T1nB, T2EA and T2nB diminished the metal concentration in Fenton reaction showing the chelating effect on ferrous ions with a comparison to Ethylenediaminetetraacetic acid (EDTA). The reducing power of extracts of TCLF-1, TCLF-2 (50-450 μg/mL) and ascorbic acid increased steadily with the increase in concentration. All extracts showed more effective reductive ability by mono and dihydroxyl substitutions in the aromatic ring, possessing potent hydrogen donating abilities. Hence, the free radical scavenging effect of endophytic fractions may be attributed to the antioxidant principles secreted by the endophytic fungi.

The endophytic fractions were tested for their antidiabetic properties by inhibition of enzymes like α-amylase, α-glycosidase and aldose reductase. The alpha-amylase inhibition is responsible for the therapeutic targets due to which oligosaccharide digestion is delayed to absorbable monosaccharides in the intestine, resulting in decreased postprandial hyperglycemia.^34,35 Alpha-glucosidase inhibitors inhibit the absorption of carbohydrates from the gut and used in the treating DM or impaired glucose tolerance. Aldose reductase found in all mammalian cells is affected by diabetic complications. It minimizes glucose to sorbitol which further metabolizes to fructose by sorbitol dehydrogenase.  Cataract can be prevented by inhibition of aldose reductase activity. So, aldose reductase inhibition may be beneficial in secondary diabetic complications.³⁶

Previous studies reported the antidiabetic potential of endophytic fungi. Crude fractions of an endophytic Aspergillus sp. isolated from the stem and leaves of Ficusreligiosa showed in vitro antidiabetic activity.³⁷ In another study, fungal extract and the 4-des-hydroxyl altersolanol A isolated from Nigrosporaoryzae inhabited in leaf of Combretum dolichopetalum proved to have antidiabetic activity in vivo.³⁸ Seven endophytic fungi isolated from Indonesian medicinal plants gave inhibitory activity to α-glucosidase enzyme.³⁹ Further, xanthones isolated from an endophytic Penicillium canescens found to possess α-glucosidase inhibitory activity.⁴⁰ In our study, endophytic fractions of Trichoderma longibrachiatum TL10 and Aspergillus versicolor BAB6580, T1EA, T1nB, T2EA, T2nB potentially inhibited α- amylase, α-glucosidase and aldose reductase enzymes. This is the first report on exploitation of endophytic fungi of Tinospora cordifolia leaves for antidiabetic activity. This may be due to the secondary metabolites present in the fractions and probably working on antioxidant mechanism.

Conclusion

The results of the present study indicate that ethyl acetate and n-butanol fractions of TCLF-1 and TCLF-2 showed appreciable free radical scavenging and in vitro antidiabetic activity. Further in vivo work is needed to validate our outcome, along with the identification of marker compounds.