Bangladesh J Pharmacol. 2012; 7: 33-35.


| Research | Article |

Trichoderma harzianum: A new fungal source for the production of cyclosporin A

Asma Azam, Tehmina Anjum and Wajiha Irum

Institute of Plant Pathology, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan.

Principal Contact


Pure cultures of Trichoderma harzianum, T. aureoviride, T. reesei, T. koningii and T. hemantum were  checked for  their  potential to produce  cyclosporin A. Production medium used for drug production was consisted of: glucose, 5%; peptone, 1%; KH2PO4, 0.5%; KCL, 0.25% (w/v). Whereas, butyl acetate was used to extract the fermentation medium for cyclosporine A. This was then analyzed through High Performance Liquid Chromatography and the chromatograms obtained were compared with that of cyclosporine and with the external standard cyclosporin A 98.5% pure. Only chromatogram of T. harzianum showed a peak at 2.779, which was comparable with both the standards. Mass spectroscopy of this peak showed [CsA + H] +  ion of m/z 1203. The amount of drug calculated was 44.06 µg/mL.


Cyclosporin A is a member of group of cyclic undecapeptides with anti-inflammatory, immunosuppressive, antifungal and antiparasitic properties (Sallam et al., 2005). It is used extensively in the prevention and treatment of graft-versus-host reactions in bone marrow transplantation and for the prevention of rejection of kidney, heart and liver transplants. Cyclosporin A is a major secondary metabolite usually produced by an aerobic filamentous fungus, Tolypocladium niveum (Park et al., 2006). Although cyclosporin A was initially developed as an antifungal  antibiotic (Deo et al., 1984), it is currently prescribed as one of the most important immunosuppressive drugs for the treatment of organ transplants, as well as patients with autoimmune diseases, including AIDS, owing to its superior T-cell specificity and low levels of myelotoxicity (Borel, 1986). It consists of 11 amino acids with a molecular weight of 1202.6 and occurs as a white solid with a melting point of 148°C to 151°C (natural) and 149°C to 150°C (synthetic). It is stable in solution at temperatures below 30°C but is sensitive to light, cold, and oxidization (IARC, 1990).

The  drug was first isolated from T. inflatum (Gams,1971) and after that very few studies were planned to explore  other  sources  for  its  production. Until  now most  of  the  research work related to cyclosporin A deals with T. inflatum and Aspergillus terreus. The present study was designed to explore Trichoderma species for this drug production.

Materials and Methods

Acquisition and maintenance of T. harzianum

Pure cultures T. harzianum (FCBP 140), T. aureoviride (FCBP 234), T. reesei (FCBP 271), T. koningii (FCBP 585) and  T. hemantum (FCBP  907)  were  obtained  from  the First Fungal  Culture  Bank  of  Pakistan (FCBP)  and maintained on Malt extract agar (MEA) medium and preserved at 4ºC.

Seed inoculum preparation

Malt Yeast extract (MY) medium was selected as seed medium for inoculum preparation of selected fungi. MY medium is composed of malt extract 2%, yeast extract 0.4% and initial pH was adjusted to 5.7 using 1.0 M HCl. 50 mL of MY medium was prepared and poured in Erlenmeyer flasks (250 mL capacity) plugged with  cotton  plugs.  These prepared medium flasks were sterilized by autoclaving at 121ºC and 15 lb/inch2  pressure for 15-20 min. By using cork borer, 0.8 cm disk of five days old cultures on MEA of Trichoderma species were inoculated in sterilized medium in flasks. The inoculated flasks were incubated on orbital shaker at 200 rpm for 72 hours at 30ºC (Borel et  al.,  1977).  The  inoculum  of  all  the  strains  was prepared by the same procedure.


For cultivation 50 mL production medium specially design for cyclosporin A production, was prepared with following composition: glucose, 5%; peptone, 1%; KH2PO4, 0.5%; KCL, 0.25% (w/v), at pH 5.3. According to the methodology of Agathos et al., (1986) 5 mL of seed inoculum from each isolate was introduced into 250 mL Erlenmeyer flasks containing 50 mL of production medium. The fermentation was continued at 28 ± 1ºC for 10 days, at 200 rpm.

Cyclosporin  A  extraction 

Harvested  fermentation medium was mixed with 30 mL of butyl acetate and stirred at 200 rpm for 24 hours at 30ºC. Organic layer was  formed after  24  hours, which  was  separated  by separating funnel and evaporated under vacuum till dryness. Dried extract was weighed and dissolved in 30 mL methanol.

Measurement of fungal biomass

The aqueous layer of cultivation medium containing fungal pellets was filtered  for  harvest  of  biomass. The filtration was performed by using Whatman filter paper No. 1.  The fresh biomass was collected on the filter paper by using a conical funnel. Filters were slightly rinsed with water 1-2 times for the washing of media and weighed on digital  weighing machine. The filters were dried in oven at 40ºC and re-weighed after cooling. Following formula was used to calculate the dry biomass:

Fungal dry biomass = weight of filter paper-weight of dry filtrate

Cyclosporin A analysis

Level  of  cyclosporin  A  in  the crude extract was analyzed by High Performance liquid chromatography (HPLC). HPLC was carried out using Hitachi system consisting of L-2100/2130 pump, L-2420 UV-VIS detector with a detection span from 190 to 900 nm. Analysis was done using a C18   column  with a 5  µm particle size, and acetonitrile:methanol:water (42.5:20:37.5, v/v) as mobile phase at a flow rate of 0.8 mL/min with UV detection at 215 nm.

Cyclosporin A confirmation

For  initial  confirmation  of cyclosporin A, Sandimmun Neoral® capsule (Novartis) containing 100  mg of cyclosporin  was used. For  this purpose 100 µL from capsule was taken with the help of micropippet and dissolved in 10 mL of methanol. Then, HPLC of this solution was carried out according to the conditions entioned above.

The retention time and peak area of the samples were compared  for  final  confirmation  and  quantification, with the external standard cyclosporin A 98.5% pure (Sigma-Aldrich, Fluka). The standard was analyzed under same conditions as described.

Estimation of cyclosporin A

The cyclosporin A level in crude extracts was determined by the following formula:

% cyclosporin A by weight  = As (Wr Vs ÷ArWsVr)×  purity of reference ArWsVr

Where, As is area of sample peak; Ar is area of reference peak; Wr is weight of reference material in grams; Ws is weight of sample in grams; Vs is volume of sample; Vr   is volume of reference material .T he area of sample peaks and of reference peak was calculated from the chromatograms obtained by HPLC program LaChrom Elite.

Mass spectroscopy

The ESI/MS spectrum was obtained from   single-quadrupole   mass   spectrometer. Sample was introduced using a silica capillary at a flow rate of 4.0 µL/min. The nebulizer gas was optimized and set at a rate of 1.6 L/min, and an electrospray potential of 4200 V was applied in the interface sprayer. A curtain gas of ultrapure nitrogen was pumped into the interface at the rate of 1.2 L/min.

Results and Discussion

Trichoderma as antagonists controlling wide range of pathogens are well documented (Ajith and Lakshmidevi, 2010). As cyclosporin A is also known for its antifungal properties, five species of Trichoderma were checked for their potential to produce this drug. The drug extracted in butyl acetate was confirmed as cyclosporin A by comparing chromatograms with Sandimmun Neoral capsules containing cyclosporin. A as active ingredient and pure cyclosporin A. Sandimmun Neoral capsules showed a clear peak at 2.768 whereas peak at 2.81 was recorded by authentic compound (Figure 1, Figure 2). In a previous studies Sallam et al., (2003) recorded cyclosporin A peak at 3.033 which was produced by Aspergillus terreus following almost similar experimental plan as is described in this study.

Among five tested Trichoderma species only T. harzianum (FCBP 140) isolated from air showed a comparable peak at 2.779 (Figure 3). The ESI-MS analysis showed the ion [CsA + H]+  at m/z 1203. The estimated yield of cyclosporine A was 44.06 µg/mL with a 0.262 g/50 mL of fungal biomass. No such peak was recorded in other tested species. However this quantity was found lower to that recorded by earlier workers in the most studied fungal strain i.e., T. inflatum. As Dreyfuss et al., (1976) and Agathos et al., (1986) found a very good yield of 105.5 mg/L and Balakrishnan and Pandey (1996) showed that T. inflatum can produce higher levels of cyclosporin A up to 183 mg/L.

Beside T. inflatum so far this drug has been reported to be produced from Aspergillus terreus, Fusarium solani and Fusarium oxysporum (Sallam et al., 2003). This study confirms T. harzianum as a new fungal source of cyclosporine A production.


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