Abstract: A study on the effect of a local medicinal mushroom, Laetiporus sp. (Polyporaceae) extract on total blood cholesterol level has been conducted using Wistar rats with high cholesterol feed and prophylthiouracil (PTU) as hypercholesterolemia inducing agent. The study was aimed to evaluate the inhibition and reduction effect of the dried (hot water) extract of grain grown mycelia on hypercholesterolemia of the rat model. The rats were randomly classified into tested group and control group, each group consists of six rats. After a preliminary study, two doses level of the extract, i.e., 55 and 110 mg kg-1 b.wt. were tested, respectively. Commercial lovastatin tablet at a dose of 1.8 mg kg-1 b.wt. was also incorporated in the test as reference medicine. Total blood cholesterol level was observed once a week over 4 weeks. The trial was also conducted on human by involving 19 volunteers. They were asked to consume one Laetiporus sp. capsule per day after meal before going to bed over 30 days. The fungal extract at dose 55 mg kg-1 b.wt. could inhibit the blood cholesterol level by an average of 11.0% and dose 110 mg kg-1 b.wt. could inhibit by an average of 19.0% compared to commercial lovastatin at dose 1.8 mg kg-1 b.wt. which could inhibit at an average of 14.6% over 4 weeks. On the other hand, dose 110 mg kg-1 b.wt. could reduce blood cholesterol level by an average of 11.4%, compared to commercial lovastatin tablet which could reduce by an average of 5.5% over 4 weeks. In addition, blood cholesterol level was found to be reduced in 14 out of 19 (73.6%) of human volunteers while 5 out of 19 (26.4%) were increased. It is concluded that the mycelial extract of Laetiporus sp. is potential to be used as an anti hypercholesterolemia agent.
INTRODUCTION
Laetiporus sp. is one species of brown rot fungi. It is a wild macro fungi (Basidiomycete) growing around tropical-subtropical area including Indonesia. There is very little study on local (Indonesian) medicinal mushroom including Laetiporus sp. has been done. Other species of Laetiporus such as Laetiporus sulphureus has been well known for their medicinal properties. For example, methanol and dichloromethane extracts of Laetiporus sulphureus was reported to exhibit high pancreatic lipase inhibitory activity (83±5%) (Slanc et al., 2004). A new lanostanoid triterpene, 3-oxosulfurenic acid (1), together with three known triterpenes (3, 4 and 7) were isolated from the fruit bodies of Laetiporus sulphureus which show cytotoxicity effect on human myeloid leukemia HL-60 cells (Leon et al., 2004).
Our previous findings demonstrated that local Laetiporus sp. isolated from West Java-Indonesia, contained lovastatin (Aryantha and Sidharta, 2007). Lovastatin which has been reported as an anti hypercholesterolemia substance was initially isolated from microfungi (Penicillium citrinum) (Endo et al., 1976). Recently, some edible mushrooms have been identified to posses anti hypercholesterolemia. Inonotus obliquus extract was found to be significantly reduced total cholesterol and triglyceride while increased high-density lipoprotein cholesterol level in serum of diabetic mice (Lu et al., 2010). Commercial button mushroom (Agaricus bisporus) was also reported to reduce plasma total cholesterol (TC) and low-density lipoprotein (LDL) of rat by 22.8 and 33.1%, respectively after 4 weeks of treatment (Jeong et al., 2010). Furthermore, Zhang et al. (2009) reported that Tremella aurantialba extracts has strong abilities to reduce the levels of total cholesterol and total triglyceride in serum of rats. In addition, Hypsizygus marmoreus (Bunashimeji), Pleurotus eryngii (Eringi) and Grifola frondosa (Maitake) were also reported to be significantly lower serum total cholesterol of mice at week 8, 10, 12, 14 and 16 after treatment (Mori et al., 2008).
The hypercholesterolemia is generally defined as a condition in which the concentration of cholesterol carrying lipoproteins in plasma exceeds an arbitrary normal limit. Clinical concern arises because an elevated concentration of blood lipid content (cholesterol and triglycerides) can accelerate the development of atherosclerosis, with its dual sequelae of thrombosis and infraction. The normal blood cholesterol level is less or equal to 200 mg dL-1. The blood cholesterol level higher than 265 mg dL-1 increasing heart attack risk by 5 times (Gilman et al., 1990). This study was to find out the ability of the mycelial (hot water) extract of local Laetiporus sp. to inhibit and to reduce the hypercholesterolemia in Wistar female rats.
MATERIALS AND METHODS
This research was carried out during January 2008 to December 2008.
Anti-hypercholesterolemia material: Pure culture of Laetiporus sp. was sub cultured on Potato Dextrose Agar (PDA) plate and incubated over 5 days at room temperature. This agar culture was then cut into 1x1 cm2 with scalpel aseptically before being inoculated into sterile cereal based medium consisting of broken corn (25%), red rice (15%), sawdust (50%), rice bran (10%) on dry weight based. After 1 month incubation at cereal based medium, the mycelial culture was ready to be extracted. The whole growing mycelia along the substrate were collected and extracted in boiling water for 30 min and then filtered. The water extract were then evaporated and dried. Brown dry extract with specific taste and odor was made into powder. Commercial lovastatin tablet was used as a reference medicine. The suspension of lovastatin was prepared in 1% solution of tragacanth with dose 1.8 mg kg-1 b.wt.
Hypercholesterolemia inducing agent consists of high cholesterol feed and prophylthiouracil (PTU). The high cholesterol feed was made of 15% of duck egg yolk, 10% of chicken liver, 15% of lamb fat and 60% of standard feed for rat. The solution of 0.1% PTU was also fed to the rat ad libitum.
Assay animal: Wistar female rats with 160-200 g of body weight prepared by Center for Life Science Institut Teknologi Bandung, Indonesia.
Toxicity analysis: Five animals both male and female were randomly chosen to do the toxicity test. Five doses of Laetiporus sp. extracts i.e., 1,250 (D1250); 2,500 (D2500); 5,000 (D5000); 7,500 (D7500) and 10,000 (D10000) (mg kg-1 b.wt.) were examined on assay animal and compared with control (no extract added). The animals were first observed for the growth pattern by weighing the body weight daily over 14 days. In addition, standard pharmacology activities including: visit up, visit down, motoric normal, motoric up, motoric down, straub, piloerection, ptosis, corneal reflex, pineal reflex, lacrimation, vasodilatation, catalpesy, hanging, retablishment, walking backward, walking forward, flexy, harfner respond, wriggling, grooming, tremor, body trembling, vocalization, urination and defecation of the animal were observed over 3 min per period of observation. After 14 days, the animals were killed for analyzing the internal organs including lung, heart, kidney, testis, spleen, liver and vesica seminalis. The organ index was calculated based on the ratio between the weight of the organ and the body.
Anti-cholesterol experimental method
There were two experimental methods: Cholesterol inhibition method
and cholesterol reducing method. Preliminary study was done to find out reasonable
doses of Laetiporus sp. extracts before determining the working doses
both for inhibition and reducing methods.
Inhibition method, the animals were randomly classified into 4 groups: (1) control, (2) Laetiporus sp. mycelial extract at dose 55 mg kg-1 b.wt. (3) Laetiporus sp. mycelial extract at dose 110 mg kg-1 b.wt. (4) lovastatin at dose 1.8 mg kg-1 b.wt. Each group consists of 6 rats. The high cholesterol feed and PTU were administered simultaneously with the testing medicine orally once a day over 28 days.
Reducing method, the animals were randomly classified into 3 groups : (1) control (2) Laetiporus sp. extract at dose 110 mg kg-1 b.wt. (3) lovastatin at dose 1.8 mg kg-1 b.wt. Each group consists of 6 rats. The high cholesterol feed and PTU were given over 4 weeks (28 days) to make hypercholesterolemia. At day 29, the blood samples were collected from the tail vein to determine the starting high cholesterol level and immediately followed by administration of the testing medicine. The testing medicine was given orally once a day over 28 days without high cholesterol diet and PTU anymore.
In both methods, before the experiment was started, the total blood cholesterol level of animals was measured as starting normal level. Total blood cholesterol level was observed once a week over 4 weeks. The blood samples were collected from the tail vein. The cholesterol level was measured by colorimetric method with standard enzymatic reagents. One way analysis of variance was used to evaluate the data.
Human trial: Water extract of Laetiporus sp. mycelia was made up as capsules for human trial. Each capsule was filled with 500 mg of dried extract by using a manual capsule maker. Nineteen volunteers were involved for the test over 30 days. Each person was asked to consume one capsule per day after having meal before going to bed. Their total blood cholesterol was measured before having Laetiporus sp. capsule. After 30 days of treatment once again their blood cholesterol was measured at a local professional public clinic laboratory. The result of the test was then analysed.
RESULTS
Toxicity effects: Laetiporus sp. extract did not cause any toxicity effect on rat as evidenced by the growth pattern and organ index values of the assay animals. Figure 1 shows that the male rat body weight on all doses is not significantly different compared to the body weight on control animal. Like wise, female rat body weight, as shown by Fig. 2 also does not differ significantly between all doses treatment compared to control. In addition, Fig. 3 shows that organ index values of male rat do not differ significantly between all doses and control. The same phenomena also occurred on organ index values of female rat as can be seen on Fig. 4, there is no significant different between all doses and control. Growth pattern of the animal tended to fluctuate, increase during 3-5 days and then decrease on the day 6th then again increased for the following days until reach the peak at day 11th and 12th. Organ index values of male and female animals were ranging as follows: heart 0.34-0.42; lung 0.54-0.65; liver 3.89-5.1; kidney 0.98-1.56; spleen 0.2-0.54; testis 0.55-0.71; ovarium 0.04-0.08; vesica seminalis 0.25-0.5 dan uterus 0.26 and 0.65. All organ indexes are not significantly different between doses treatment compared to control.
Cholesterol inhibition effect on rat: Both doses level of mycelial extracts (55 and 110 mg kg-1 b.wt.) and commercial lovastatin at dose 1.8 mg kg-1 b.wt. were able to inhibit the increase of cholesterol level in the blood. On the other hand, the control group gave a sharp increase of cholesterol level since the first week from 101.99 to 110.73 mg dL-1.
As a hypercholesterolemic inhibitor, Laetiporus sp. mycelial water extract at dose 55 mg kg-1 b.wt. could inhibit the increase of cholesterol level by 9.1, 8.2, 18.8 and 7.8% on week 1, 2, 3 and 4, respectively or in average of 11% over 4 weeks. On the other hand, fungal extract at dose 110 mg kg-1 b.wt. could inhibit the increasing of cholesterol level by 10.5, 19.8, 28.6 and 17.0% on 1, 2, 3 and 4 week, respectively or in average of 19.0% during 4 weeks.
| Fig. 1: | The effect on growth pattern of male rat after treatment with Laetiporus sp. extracts (letters in bracket indicate significance) |
| Fig. 2: | The effect on growth pattern of female rat after treatment with Laetiporus sp. extract (letters in bracket indicate significance) |
Compared to commercial lovastatin tablet it only could inhibit the blood cholesterol level by 11.1, 12.1, 25.4, 10.0% on 1, 2, 3 and 4 week, respectively or in average of 14.6% over 4 weeks (Table 1).
Cholesterol lowering effect on rat: The result shows the reducing effect of Laetiporus sp. extract and commercial lovastatin over 4 weeks. After one week period of time, a significant reduction of blood cholesterol level was achieved by the treatment of fungal extract.
| Fig. 3: | The effect on male rat organs after treatment with Laetiporus sp. extract (letters above the bar indicate significance of data among groups) |
| Fig. 4: | The effect on female rat organs after treatment with Laetiporus sp. extract (letters above the bar indicate significance of data among groups) |
The same pattern was also achieved by the commercial lovastatin treatment. The cholesterol level was remain constant for the following 7 days and sharply decreased after 14 days. As a hypercholesterolemic reducer, dose of 110 mg kg-1 b.wt. of the mycelial water extract could reduce the blood cholesterol level by 9.4, 15.9, 14.7 and 5.6% on week 1, 2, 3 and 4, respectively or in average of 11.4% over 4 weeks. Compared to commercial lovastatin tablet, it can only reduce the blood cholesterol level average by 5.5% over 4 weeks (Table 2).
Cholesterol lowering effect on human: As can be seen on Fig. 5, fourteen out of nineteen (73.6%) volunteers were discovered to have their total blood cholesterol to decrease and 5 out of 19 (26.4%) were increased. The decrease of blood cholesterol varies from 1 up to 45 mg dL-1 while the increase varies from 2-66 mg dL-1.
| Table 1: | Percentage inhibition of blood cholesterol level by Laetiporus sp. extracts and |
| Table 2: | Percentage reduction of blood cholesterol level by Laetiporus sp. extracts and commercial lovastatin product compared to control |
| Fig. 5: | The effect of Laetiporus sp. extract on blood cholesterol level in human volunteers |
Overall results indicate that water mycelial extract of local Indonesian Laetiporus sp. is quite potential for both inhibiting and reducing blood cholesterol level which is almost as effective as commercial anti cholesterol medicine (lovastatin).
DISCUSSION
Anticholesterol agent has been produced commercially from Aspergillus terreus (Manzoni et al., 1998), Penicillium citrinum (Endo et al., 1976) and Monascus purpureus (Endo, 1979). Unfortunately, A. terreus, P. citrinum and M. purpureus also produce secondary metabolites as mycotoxin which may have side effects to human health. Riba et al. (2008) reported that 50% of Aspergillus terreus isolated from 85 samples of wheat destined for human consumption in Algeria were Ochratoxin A (OTA) producer (0.01 to 9.35 μg-1. A study in Spain also showed that fourteen isolates (10 P. verrucosum and 4 of P. citrinum) from total 155 isolates of Pencillium sp. were citrinin (CIT) producers (Bragulat et al., 2008). In addition, the most frequently isolated genera of moulds from 50 samples of poultry feed in Venezuela, were Aspergillus (36%) and Penicillium (20%). Of these genera, the most frequently isolated species were A. flavus, A. terreus and P. citrinum (Figueroa et al., 2009).
Both OTA and CIT are reported to be commonly found in moldy foods in endemic areas of nephropathy, which is associated with urinary tract cancers (Knasmuller et al., 2004). Meanwhile, Mayura et al. (1984) reported that OTA and CIT, when occur simultaneously, may interact to enhance prenatal toxicity and teratogenicity. Furthermore, extract from submerged Monascus cultures was reported to produce mycotoxin which possess embryotoxic and teratogenic activities (Martinkova et al., 1995). These informations suggests that micro fungi (Aspergillus, Penicillium and Monascus) may have negative impacts when used as anti hypercholesterolemial agent. Therefore, safer sources of antihypercholesterolemial agents are required.
Fruiting bodies of edible oyster mushroom (Pleurotus ostreatus), were found to contain a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase which was known as lovastatin (Gunde-Cimerman and Cimerman, 1995). In addition, crude methanol extract of other Pleurotus (P. sapidus and P. saca) were also shown to produce the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor known as mevinolin (Gunde-Cimerman et al., 1993).
Our previous results also confirmed the cholesterol lowering and inhibition effects by mycelial hot water extract of Pleurotus ostreatus in Wistar rat (Yuli et al., 2006). Both mycelial and fruiting body hot water extracts of this mushroom were confirmed to contain lovastatin in other study (Aryantha and Widayanti, 2004). Recently, a local species of Laetiporus was examined and confirmed to produce lovastatin from our previous investigation (Aryantha and Sidharta, 2007). The lowering and inhibition effects of blood cholesterol by this local mushroom apparently were caused by the same substances (lovastatin). In order to prove this hypothesis, further research by using pure extract of this mushroom needs to be conducted. Up to this stage, the hot water extract of this mushroom was proven to inhibit and to lower blood cholesterol in this study.
Lovastatin was found to be selectively can inhibit hydroxymethyl glutaryl-coenzyme-A (HMG-CoA) reductase, the first enzyme in cholesterol biosynthesis (Endo, 1992). The mechanism involved in controlling plasma cholesterol levels is the reversible inhibition of HMG-CoA reductase by statins due to similarity of the acid form of the statins to HMG-CoA, the natural substrate of the enzymatic reaction (Manzoni and Rollini, 2002). The hypocholesterolemic effect of statins was known to reduce the Very Low-Density Lipoproteins (VLDL) and Low-Density Lipoproteins (LDL) as well as to increase the High-Density Lipoproteins (HDL), with a subsequent reduction of the LDL:HDL-cholesterol ratio. The use of natural statins therefore, can lead to a reduction in hypercholesterolemia related diseases, but the possible interaction of natural statins with other medicine must be taken into account.
There are five types of statin in clinical use i.e. Lovastatin and pravastatin (mevastatin derived) are natural statins of fungal origin, while symvastatin is a semi synthetic lovastatin derivative. Atorvastatin and fluvastatin are fully synthetic statins, derived from mevalonate and pyridine, respectively. Other natural statins which are also fungal origin such as monacolins and dihydromonacolins have also been characterized (Manzoni and Rollini, 2002).
Until recently, there is no information available about anti hypercholesterolemia obtained from Laetiporus sp. The most well known species of Laetiporus is L. sulphureus. Four strains of L. sulphureus are known from nature based on growth cultural conditions and fruit body formation. All strains demonstrated antimicrobial activity against a wide spectrum of Gram-positive and Gram-negative bacteria including methicillin-resistant strain of Staphylococcus aureus (MRSA) and glycopeptide-resistant strain of Leuconostoc mesenteroides. (Ershova et al., 2003). In addition, a new lanostanoid triterpene, 3-oxosulfurenic acid together with three known triterpenes were also isolated from the fruit bodies of Laetiporus sulphureus. Cytotoxicity of these compounds and their derivatives was evaluated on HL-60 cells. Further studies revealed that a compound called acetyl eburicoic acid of this mushroom was the most potent apoptosis inducer (Leon et al., 2004). This pro-apoptotic stimulus implies possible therapeutic potential and may guide feasibility for more potent statins in anti-cancer strategies (Minichsdorfer and Hohenegger, 2009).
Laetiporus extracts seems to be more effective for inhibiting the cholesterol synthesis rather than reducing cholesterol level in the blood. Nevertheless, cholesterol reducing effect of Laetiporus extracts seems to be effective after 2 weeks of treatment. Reducing effect on human blood cholesterol is quite variative among human volunteers. Higher reducing effects occur on some volunteers and low reduction also happened to some volunteers. In contrast, 5 volunteers are in fact their blood cholesterol level increased. This phenomena may occur due to the life style and daily diet of these people may not be appropriate since their diet were not restricted to certain type of food.
To anticipate the negative effects of the Laetiporus sp. extract on the health aspects, toxicity evaluation was also conducted on this research. Almost all aspect toxicity being evaluated did not give any different results compared to control treatment. Therefore, Laetiporus sp. extract can be considered to be safe as hypercholesterolemia treatment agents.
ACKNOWLEDGMENT
We thank Ministry of Higher Education-Republic Indonesia for funding this research. Our appreciation and thanks also go to Mr. Slamet Sutambah for taking care for the animal, Mr. Afif and Muslih for their as asistance during live blood aalysis as well as Ms. Safitri and Mr. Tedy Dermawan for their assistance during experiment.