TINOSPORA CORDIFOLIA AQUEOUS EXTRACT AMELIORATES THE SYSTEMIC INFECTION OF ASPERGILLUS FUMIGATUS IN BALB/C MICE

Objective: The present study was aimed to assess the antifungal activity of Tinospora cordifolia aqueous extract (TCAE) against Aspergillus fumigatus infection. Methods: TCAE was tested for in vitro antifungal activity against the isolates of A. fumigatus, Aspergillus flavus, and Aspergillus niger. To evaluate in vivo activity, various doses (10, 25, and 50 mg/kg) of TCAE were orally administered in A. fumigatus-infected mice for 7 days. The combination of prophylactic and therapeutic effect of TCAE was assessed by pre-treating the mice with 10 mg/kg of TCAE for 3 consecutive days before exposing them to A. fumigatus. Mice were treated with 10, 25, and 50 mg/kg doses of TCAE for 7 consecutive days’ post-A. fumigatus infection. The effectiveness of TCAE was evaluated by monitoring the survival rate and assessing the fungal burden in the kidney of the treated mice. Results: A. fumigatus-infected mice treated with TCAE at the doses of 25 and 50 mg/kg exhibited 50% and 20% survival rate, respectively, observed on day 40 post-treatment. Like to the survival data, the fungal burden was also found to be the lowest in the kidney of mice treated with TCAE at a dose of 50 mg/kg. The results showed that pre-treatment with TCAE (10 mg/kg) followed by post-infection treatment with 10, 25, and 50 mg/kg of TCAE for 7 days resulted in 40%, 50%, and 70% survival rate, respectively. Conclusions: These results suggest that TCAE may potentially be considered for its possible use in the treatment of the systemic infection of A. fumigatus.


INTRODUCTION
The therapeutic use of antibiotics has played a tremendous role in combating various infectious diseases [1]. Although the indiscriminate and extensive use of antifungals has resulted in the emergence of multidrug-resistant isolates of fungal pathogens, which are posing serious challenges to the clinicians [2]. Moreover, some antifungal agents, particularly polyene antibiotics, have been shown to exert serious untoward effects [3,4]. As there are limited numbers of antifungals available in the market, it is important to find suitable replacements for some of the currently used antifungals [5].
Giloy, Tinospora cordifolia, has been used for centuries in the Ayurvedic and Unani Systems of the Medicine for the treatment of ailments. It has been demonstrated to have antidiabetic, antioxidant, antihepatotoxic, and immunomodulatory properties [6][7][8]. The aqueous extract of T. cordifolia has been shown to protect against Escherichia coli and Staphylococcus aureus infections [9,10]. The active ingredient, G 1-4A, of T. cordifolia stem protected mice against lipopolysaccharideinduced endotoxic shock [11]. It also controlled the drug-resistance Mycobacterium tuberculosis by modulating the immune responses [12]. Recently, we showed that aqueous and methanolic extracts of T. cordifolia alleviated the Salmonella typhimurium infection in a mouse model [13].
Invasive aspergillosis (IA) caused by Aspergillus fumigatus is a major cause of mortality in immunocompromised persons, including patients with leukemia, undergoing bone marrow, and solid organ transplantation [14]. Treatment of fungal infections has been a challenge for clinicians due to the limited number of antifungals currently available in the market. Recent upsurge in the frequency of azole-resistant A. fumigatus isolates has further limited our antifungal armory. On the other hand, polyene antifungals have been shown to exert an acute renal toxicity in the treated subjects. Thus, it is important to find a plant-derived broad-spectrum antifungal agents that can be used to treat infectious fungal diseases. In the present study, we used T. cordifolia aqueous extract (TCAE) against A. fumigatus both in vitro and in a mouse model. The results of the present study demonstrated that TCAE effectively alleviated the systemic infection of A. fumigatus in the mouse model.

Mice
BALB/C mice of 10-12 weeks of age were used in this study. Mice were purchased from the animal house facility of King Saud University, Riyadh, Saudi Arabia. The techniques used for bleeding, injection, and sacrifice of animals were approved by an Animal Ethics Committee of the College of Applied Medical Sciences, Qassim University, Buraydah.

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agar. TCAE was dispensed in different wells (20 mg/ml) and incubated at 37°C for 24 h. The antifungal activity of TCAE was assessed by measuring the zone of inhibition. The wells containing amphotericin B (10 mg/ml) and saline were considered as positive and negative controls, respectively.

Preparation of A. fumigatus for infection
A. fumigatus was cultured as described in the previous section. Each mouse was infected through the intravenous route with a lethal dose of 7 × 10 5 viable A. fumigatus spores [14].

Quantitative analysis of A. fumigatus in the kidney
The severity of A. fumigatus infection was assessed by determining the fungal load in the kidney of mice untreated or treated with T. cordifolia extract. Three mice from each group were sacrificed on day 3 post-A. fumigatus infection, and their kidneys were taken out aseptically as described earlier [14]. Briefly, weighed portions of the kidney tissues were homogenized in 5 ml of sterile normal saline, and different dilutions of the suspension were plated on SDA plates. The plates were incubated at 37°C for 48 h. The numbers of viable A. fumigatus colonies were counted and the fungal load was determined by multiplying by the dilution factor.

Statistical analyses
Analysis of the survival of mice was performed using Kaplan-Meier curve, and various groups were compared by the log-rank test. Fungal burden (colony-forming units [CFU]) in the kidney was analyzed by one-way ANOVA using GraphPad Prism software version 5.0.

The aqueous extract of T. cordifolia shows in vitro antifungal activity
The aqueous extract of T. cordifolia showed potent activity against A. fumigatus, Aspergillus Flavus, and Aspergillus niger used in the study. A zone of inhibition was calculated as the percentage antifungal activity of amphotericin B (100%). Table 1 shows that T. cordifolia extracts showed the antifungal activity against all three strains of Aspergillus.

Treatment with TCAE results in increased survival of A. fumigatus-infected mice
The therapeutic effect of TCAE was determined by treating A. fumigatus-infected mice with the doses of 10, 25, and 50 mg/kg of TCAE for 7 consecutive days. Mice were observed for 40 days for their survival. All A. fumigatus-infected mice in the untreated group died by day 10. A. fumigatus-infected mice in the groups treated with 25 and 50 mg/kg of TCAE showed 30% and 40% survival, respectively (Fig. 1). The survival rate of mice in the group treated with TCAE at a dose of 50 mg/kg was found to be significantly greater as compared to that of mice in the untreated group (p<0.01).

Mice treated with TCAE showed more resistance against A. fumigatus infection
The severity of A. fumigatus infection was determined by culturing the tissue homogenates of kidneys of the A. fumigatus-infected mice treated or untreated with TCAE. There were higher CFUs of A. fumigatus in the kidneys of untreated mice (Fig. 2). Mice treated with 50 mg/kg of TCAE showed least CFUs in their kidney tissue homogenates (Fig. 2).

Prophylactic use of TCAE increases the therapeutic efficacy of the same formulation in combating A. fumigatus infection in mice
The combination of prophylactic and therapeutic effect of TCAE was assessed by pre-treating the mice with 10 mg/kg of TCAE for 3 consecutive days before challenging them with A. fumigatus. Mice were treated with three doses (10, 25, and 50 mg/kg) of TCAE for 7 consecutive days' post-A. fumigatus infection as described in the methods section. The results showed that pre-treatment with TCAE (10 mg/kg) followed by post-infection treatment with 10, 25, and 50 mg/kg of TCAE for 7 days resulted in 40%, 50%, and 70% survival, respectively, on day 40 (Fig. 3). The mice that received only TCAE pretreatment also showed increased survival compared to untreated mice (Fig. 3).
The assessment of the severity of Aspergillosis in the group of immunocompetent mice pre-treated with TCAE showed much reduced fungal loads in their kidneys compared to mice in other groups. Mice that were only PT with TCAE also resisted A. fumigatus infection as shown by lower fungal load in their kidneys compared to mice not receiving any TCAE pretreatment (Fig. 4). The group of immunocompetent mice pre-treated with TCAE followed by treatment with 50 mg/kg of TCAE showed the least CFU counts in their kidneys (Fig. 4). The data represent mean of three different experiments±S.D, TCAE: Tinospora cordifolia aqueous extract

DISCUSSION
In the recent years, the antibiotic resistance ability of A. fumigatus has contributed to increased frequency of Aspergillosis in human population [15]. The use of plants and their bioactive constituents has emerged as a promising alternative to traditional drugs in the treatment of fungal infections [16]. We have earlier shown that thymoquinone, a major bioactive constituent of Nigella sativa seeds, possesses a strong activity against Candida albicans in a mouse model [16]. Due to the problem of antibiotic resistance and prevalence of immune suppression, the treatment of Aspergillosis is posing a big threat to human society. Plant-derived antifungal agents can selectively act on various targets with fewer side effects. Since there are less chances of fungal resistance against herbal preparations due to their multiple mechanisms of action, the use of medicinal plants may be proved a better option for the treatment of drug-resistant Aspergillosis.
In the present study, the aqueous extract of T. cordifolia showed its activity against different Aspergillus spp. including A. fumigatus, A. flavus, and A. niger. TCAE was tested against A. fumigatus, A. flavus, and A. niger in in vitro studies and was effective in inhibiting the growth of these fungi. This encouraged us to use TCAE against A. fumigatus in a murine model. Interestingly, the result of in vivo studies confirmed the activity of TCAE against A. fumigatus. A. fumigatus-infected mice showed increased survival and less fungal load in their kidney tissues after treatment with TCAE, particularly at higher doses.
T. cordifolia and its constituents have been shown to possess immunostimulating activities [13]. An α-D-glucan, a constituent found in T. cordifolia, has shown its efficacy in stimulating NK cells, B cells, and T cells with simultaneous production of various immune-stimulatory cytokines. This stimulation of immune cells can play a very crucial role in combating the fungal infectious diseases. We have recently shown that both aqueous and methanolic extracts of T. cordifolia stimulate the secretion of important cytokines by macrophages that contribute to the elimination of S. typhimurium infection from mice [13]. The immunestimulating effect of T. cordifolia is also evident from the results of

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our in vivo studies as the combination of prophylactic and therapeutic use of TCAE was most effective against A. fumigatus. Thus, the use of T. cordifolia extracts for the treatment of Aspergillosis seems to have superiority over commonly used antibiotics as earlier possesses both immunostimulatory and antifungal activities.
Extensive use of antibiotics causes systemic toxicity and immune suppression in the treated patients and predisposes them to opportunistic bacterial and fungal infections. The use of herbal medicine minimizes the chances of toxicity, which may support its use for extended periods. Although more extensive studies are needed before considering T. cordifolia as an attractive and safe option in the treatment for Aspergillosis. Furthermore, this preparation may also be studied for its implications to treat other opportunistic infections in immunocompromised persons due to its immunopotentiating properties.

CONCLUSIONS
The results of the present study suggest that some bioactive constituents present in TCAE may be responsible for its activity against A. fumigatus.
It is essential that further study should be continued to isolate and purify the bioactive components of T. cordifolia that are responsible for antifungal activity.