NZ526800A - Formula for preparation of complex fungal polysaccharides - Google Patents

Abstract

An active fungal polysaccharide preparation is disclosed, which comprises a water soluble polysaccharide obtained from a fungus such as Ganodermal Lucidum and an acid soluble polysaccharide obtained from the same or a different fungus, wherein the water soluble polysaccharide is neutral and the acid soluble polysaccharide is alkaline. This active fungal polysaccharide preparation is suitable for providing beneficial effect in the treatment of cancerous tumor cells.

Description

526800 NEW ZEALAND PATENTS ACT, 1953 No: 526800/532459 Date: 3 July 2003/20 April 2004 COMPLETE SPECIFICATION POLYSACCHARIDE PREPARATION We, YI HUAI GAO a New Zealand Citizen of 9A Crowther Street, Avondale, Auckland, New Zealand, JIN LAN a citizen of the Peoples Republic of China and New Zealand resident of 9A Crowther Street, Avondale, Auckland, New Zealand, HE GAO a citizen of the Peoples Republic of China and New Zealand resident of 9A Crowther Street, Avondale, Auckland, New Zealand, WEI GAO a New Zealand Citizen of 9A Crowther Street, Avondale, Auckland, New Zealand. to be described in the following statement: Intellectual Property Office of N.Z. 01 JUL 20M RECEIVED Field Of The Invention The present invention relates to novel preparations of active fungal polysaccharides and a method of producing these preparations. More particularly but not exclusively the invention relates to a novel preparation having two principal components - a neutral water soluble and an alkaline, acid soluble polysaccharide component.

Background To The Invention Fungal polysaccharides (polysaccharides derived from fungi) have long been known to have a beneficial effect on a human or mammalian recipient. For example they have been shown to contain anti-tumour, anti-radiation, anti-oxidation qualities. They can also enhance the immunity system and lower blood sugars. A particular example of a fungus having such activity is Ganoderma Lucidum. This fungus has been known to have such properties, particularly to the Chinese, for thousands of years.

Fungal polysaccharides are traditionally extracted through slides of dried fruiting bodies of fungi; However this method is crude and relatively ineffective.

At present when people refer to the active fungi polysaccharides they meant water-soluble neutral polysaccharides. The feature of its molecular structure is triple helix structure of 13-D-glucan. The main monomers are glucose, galactose, arabinose, mannose and xylose. In some monomers there is glucuronic and galacturonic acid.

The presence of these traces of acids will hinder the effects of the polysaccharides.

Object of the Invention It is an object of the invention to provide a polysaccharide preparation from a fungus, and a method of preparing the preparation which shows an increased effectiveness over the prior art or which at least provides the public with a useful alternative. 3 Summary of the Invention According to a first aspect of the invention there is provided an active fungal polysaccharide preparation, comprising or including both: a) a water soluble polysaccharide obtained from a fungus, and b) an acid soluble polysaccharide obtained from a fungus.

Preferably both a) and b) are extracted from the same fungal species.

Preferably both a) and b) are extracted from the same fruiting body of the fungal species.

Alternatively a) and b) are obtained from different fungal species.

Preferably the water soluble polysaccharide is a neutral species, and the acid soluble polysaccharide is an alkaline species.

Preferably the water soluble polysaccharide has a pH in the range 6.5-7.5.

Preferably the acid soluble polysaccharide has a pH in the range 2.0-4.0 Preferably a) and/or b) are obtained from the Ganoderma Lucidum fungus.

Preferably both a) and b), in isolation, are active polysaccharides. Alternatively only one of a) or b), in isolation, is an active polysaccharide.

Preferably a) comprises 70-80% w/w of the preparation and b) comprises 20-30% w/w of the preparation.

More preferably a) comprises substantially 75% and b) comprises substantially 25% of the preparation.

Intellectual Property Office of N.Z. 1 1 m 2005 \J C H: V h sj j' V QftgS. tux- • Preferably the combination of a) and b) is a synergistic combination.

Preferably the preparation may take the form of a dosage unit, which may be a measured dose in powered form, a tablet, or capsule form.

Preferably the dosage unit is a capsule comprising or including: a) water soluble polysaccharide 105-120 mg per capsule, and b) Acid soluble polysaccharide 30-45 mg per capsule.

According to a second aspect of the invention there is provided a method of preparing an active fungal polysaccharide preparation comprising or including the steps of: a) extracting a water soluble polysaccharide from a first fungus, and b) extracting an acid soluble polysaccharide from a second fungus (which may or may not be of the same species and the first, and which even may be the same fruiting fungal body as the first), c) combining the two extracts.

Preferably the water soluble polysaccharide is a neutral species, and the acid soluble polysaccharide is an alkaline species.

Preferably the water soluble polysaccharide has a pH in the range 6.5-7.5.

Preferably the acid soluble polysaccharide has a pH in the range 2.0-5.0.

Preferably the first and/or second fungus is Ganoderma Lucidum.

Preferably the step c) of combining the two extracts, comprises or includes combining the two extracts in the following proportions: i) water soluble polysaccharide: 70-80% % w/w; more preferably substantially 75%. ii) acid soluble polysaccharide: 20-30% w/w; more preferably substantially 25%.

Preferably the step a) of extracting the water soluble polysaccharide comprises or includes the steps of: (i) adjustment of the pH to substantially 9 (+1), and lowering of the pressure; and (ii) recovery of the product using water.

Preferably the step b) of extracting the acid soluble polysaccharide comprises or includes (i) application of low pH (preferably substantially pH = 1.5-2.5) to extract the acid soluble polysaccharide, and (ii) application of high pH (preferably substantially pH = 11-12) to precipitate the acid soluble polysaccharide.

According to a third aspect of the invention there is provided an active fungal polysaccharide preparation prepared substantially according to the above method.

According to a fourth aspect of the invention there is provided use of an active fungal polysaccharide preparationof the invention, in the preparation of a medicament for providing a beneficial effect in a mammalian recipient.

Preferably the beneficial effect is the treatment of tumor cells.

Preferably the preparation is administered orally.

Preferably the preparation is administered on one occasion only. Alternatively the preparation is administered on a regular basis.

In one embodiment the preparation is administered to a recipient prior to and/or during and/or following a radiation and/or chemical cancer treatment regime.

Intellectual Property Office of N.Z. 1 8 APR 2005 Definitions As used herein, the following terms have the following specified meanings: Active Polysaccharide by active polysaccharide (or active flrngal polysaccharide) we mean a polysaccharide capable of producing a beneficial effect in a recipient.

Beneficial Effect by beneficial effect we mean a health enhancing effect in a recipient including treatment of a disease, illness or injury, or minimisation of side effects eg the side effects of chemical and radiation cancer therapies, or enhancing of the immunity system of the recipient.

Brief Description of the Figures The invention is illustrated with reference to the examples and the following Figures in which: Figure 1 The effect of FPC with different ratios of WSNP and ASAP on the growth of Sigo tumor in mice (n=10, x±S).

Figure 2 The effect of FPC with different ratios of WSNP and ASAP on the growth of Sigo tumor in mice (n=10, x±S).

Figure 3 The effect of FPC with different ratios of WSAP: ASAP on the growth of s180 tumor in mice.

Figure 4 The effects of serum induced by FPC with different ratios on the growth of Si go tumor cells in vitro. (n=10, x±S).

Figure 5 The effects of serum induced by FPC with different ratio on inhibition of tumor cells in vitro (N=10, x±S).

Figure 6 The effects of serums induced by FPC with different ratios on apoptosis of Si go tumor cells (n=10).

Detailed Description of the Invention The present invention deals with a novel fungal polysaccharide preparation. Conventional preparations of active polysaccharides extracted from fungi have involved only administering to a recipient, a water soluble fungal extract.

We have surprisingly found that the combination of a neutral water soluble polysaccharide together with an acid soluble alkaline polysaccharide provides a preparation having a far greater positive effect on the recipient than the water soluble extract alone.

As is discussed below, without being bound by any particular theory, we believe the neutral, water soluble polysaccharide primarily has effect in respect of humoral immunity in the recipient, whilst the acid soluble alkaline polysaccharide has principal effect in respect of the cellular immunity of the recipient. When combined, the two have a surprisingly positive effect on the recipient.

Experimental The following experiments outline our comparative studies involving the effectiveness of our fungal polysaccharide preparation comprising only a water soluble polysaccharide or only an acid soluble polysaccharide, along with a fungal polysaccharide preparation according to the invention having both a water soluble polysaccharide component and an acid soluble polysaccharide component. 1. Materials and methods 1.1 Samples The water soluble neutral polysaccharides (WSNP) and acid soluble alkali polysaccharides (ASAP) were extracted from a fungus (Ganoderma Lusidum) provided by Alpha Health Product Company (NZ) Ltd.

Office of H.Z. 1 1 m 2005 1.2 Reagents Cyclophosphamide (CY.): Hua-Lian pharmaceutical company, Shan] 8 Etoposide, VP-16: Lianyungang pharmaceutical company Jiangsu, China.

Sigo cells: Tumor Institute of Beijing. (Approval number: J.D.A 99001) 1.3 Test animals Animal Test Centre of Military Medical Academy Beijing China 2. Method 2.1 Growth test of tumor cells in vivo Test 1: We took BALB/C mice 80, body weight 20- 22g, half male and half female and, after implanting Sigo, divided than randomly into 8 groups of 10.

The normal control group used normal saline, and the positive control group used cyclophosphamide (CY). The 6 test groups used different ratios of WSNP and ASAP: 100:0 90:10 80:20 70:30 60:40 0:100.

Each were given the same dose: 1.5g.kg"' per day ig. This was continued for 10 days, 11th day killed the animals without pain (dislocation of neck vertebrae), then anatomized and the tumors examined.

Test 2: We took 60 BALB/C mice, body weight 20- 22g, half male and half female, and after implanting Sigo cells, divided them randomly into 6 groups of 10.

The normal control group used normal saline, and the positive control group used cyclophosphamide (CY).

The 4 test groups used different ratios of WSNP and ASAP: 95:5 85:15 9 75:25 65:35, Each were administered the same dose: l.Sg.kg'Vd"1, ig., and continued for 10 days. On the 11th day the animals were killed without pain (dislocation of neck vertebrae), then anatomized and the tumors examined. 2.2 Growth test of tumor cells in vitro We used MTT method as is known in the art. MTT method: is an animal test for investigating the effects of immunity by using Methyl Thiazolyl Tetrazolinm(MTT). 2.3Apoptosis of tumor cells This was conducted using flow cytological technology. 2.4 Preparation of the serums induced by fungal polysaccharides with different ratios We used the animals from the in vivo tumor growth tests, which included tests 1 and 2. The ratios of fungal polysaccharide covered (WSNP: ASAP): 100:0, 95:5, 90:10 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 0:100.

UgJcg^.d^xlOd, ig., These animals were killed on the 11th day and blood was taken from the eye sockets, separated serum with sterilization, use filter membrane of 0.45um micropore to filter away bacteria, kept at -20° for later use. 3. Results 3.1 The effects of the preparation with different ratios of fungal polysaccharide complex on the growth of tumor in vivo.

Test 1 The results are presented in Table 1.

Table 1- The effects of the fungal polysaccharide preparation with different ratios of WSNP and ASAP on the growth of Siso tumor in mice (N=10, x±S) Ratios Tumor Inhibition Groups WSNP:ACAP Doses (mg) (%) MS 0 - - 2.02±0.21 - FPC 1 100:0 1.5 g.kg"1 0.97+0.33 51.89 2 90:10 1.5 g.kg"1 0.94+0.36 53.47 3 80:20 1.5 g.kg"1 0.88+0.61 * ** 9 56.44 4 70:30 1.5 g.kg" 0.89±0.61 * #* 9 55.94 60:40 1.5 g.kg"1 0.96±0.72 52.48 6 0:100 1.5 g.kg"1 1.13+0.64 44.06 CY 7 mg.kg"' 0.43±0.45 78.71 * P<0.05(group 3,4 vs group 1,6) **P<0.01(group 3,4 vs group 0) Figure 1 also illustrates the results. It illustrates the effect of the fungal polysaccharide preparation with different ratios of WSNP and ASAP on the growth of Siso tumor in mice (n=10, x±S) and shows that the preferred ratios of WSNP: ASAP fell in the region 80:20, 70:30.

Test 2 The results are presented in Table 2.

Table 2 The effects of FPC with different ratios of WSNP : ASAP on the growth of Siso tumor in mice (N=10, x±S) Ratios Tumor Inhibition Groups WSNP:ACAP Doses (mg) (%> NS 0 - _ 2.12+0.41 - FPC 1 95:5 1.5 g.kg'1 0.96+0.55 54.71 2 85:15 1.5 g.kg1 0.91+0.63 57.06 3 75:25 1.5 g.kg"1 0.76+0.35 ♦ ** 9 64.15 4 65:35 1.5 g-kg"1 0.94+0.71 55.66 CY mg.kg'1 0.41+0.36 80.66 * P<0.05 (group 3 vs group 1,4) **P<0.01 (group 3 vs group 0) 11 The results of Test 2-1 are also illustrated in Figure 2. This illustrates the effect of the fungal polysaccharide preparation with different ratios of WSNP and ASAP on the growth of Sigo tumor in mice (n=10 x±S) and it illustrates that the best ratios of WSNP: ASAP fell on 75:25 Figure 3 further illustrates the effect of the fungal polysaccharide preparation with different ratios of WSAP: ASAP on the growth of Si80 tumor in mice and particularly illustrates that best ratio of the fungal polysaccharide preparation anti-tumor effect in mice fell on 75:25. 3.2 The effects of the serum induced by FPC with different ratios on growth of Siso tumor cells in vitro We used the serum induced by the fungal polysaccharide preparations prepared from Ganoderma Lusidum with the different ratios, and added them into the cultures of Siso tumor cells in vitro to investigate the growth of tumor cells. The negative control group used normal saline, the positive control group used Etoposide (VP-16). The results of these studies are presented in Table 3 and in Figures 4,5.

Table 3: The effects of serum induced by FPC with different ratios on the growth of Siso tumor cells in vitro (N=10, x±S) Ganoderma Lusidum Group Ratios WSNP:ACAP Dose g-kg^.d"1* lOd Density of tumor cells D (570nm) Anti-tumor effect % NS 0 - - 0.452±0.034 - FPC 1 100:0 1.5 0.354+0.041 21.68 2 95:5 1.5 0.367±0.081 18.81 3 90:10 1.5 0.349±0.059 22.79 4 85:15 1.5 0.316+0.028 .09 80:20 1.5 0.301±0.068 * ** 9 33.41 6 75:25 1.5 0.296±0.015 * ** ) 34.51 7 70:30 1.5 0.283+0.027 * ** > 37.39 8 65:35 1.5 0.343±0.032 24.12 9 60:40 1.5 0.356+0.045 21.24 0:100 1.5 0.386±0.084 14.60 VP-16 11 mg.L"1 80ml 0.230+0.089 49.16 , J P'0 nuice of M- * P<0.05 (group 5,6,7 vs group 1,10 ) **P<0.01 (group 5,6,7 vs group 0) 12 Figure 4 illustrates the effects of serum induced by the fungal polysaccharide preparation prepared from Ganoderma Lusidum having different ratios on the growth of Siso tumor cells in vitro. (n=10, x±S), and Figure 5 illustrates the effects of serum induced by FPC with different ratio on inhibition of tumor cells in vitro (N=10, x±S).

Table 3 and figure 4, 5 shows the best ratios of the fungal polysaccharide preparation prepared from Ganoderma Lusidum for anti-tumor in vitro fell on 80:20, 75:25, 70:30, and the ratio of 70:30 was the best. Group 5,6,7 compared with groups 1,10, P<0.05, the differences were significant. 3.3 The effects of serums induced by the Fungal Polysaccharide Preparation with different ratios on apoptosis of Si8o tumor cells in vitro.

Took the serums induced by the Fungal Polysaccharide Preparation with different ratios and Siso tumor cells to be cultivated together 72h, the results showed the best ratios of the Fungal Polysaccharide Preparation on the apoptosis fell on 80: 20, 75: 25, 70:30, see Table 4.

Table 4: The effects of serums induced by the Fungal Polysaccharide Preparation with different ratios on apoptosis of Siso tumor cells (N=10 » x±S) Ganoderma Lusidum Group Ratio WSNP:ACAP Doses g-kg^.d"1* lOd Apoptosis %) NS 0 - - 0.66+1.35 FPC 1 100:0 1.5 29.41±6.36 2 95:5 1.5 29.13+5.93 3 90:10 1.5 .16±6.03 4 85:15 1.5 .55±6.72 80:20 1.5 32.68+5.86 * ** 9 6 75:25 1.5 33.96±6.15 * ** 9 7 70:30 1.5 33.02+7.01 * ** 9 8 65:35 1.5 .11+6.54 9 60:40 1.5 29.63±5.91 0:100 1.5 .43+5.73 VP-16 11 mg.L"1 80ml 69.78±8.62 P<0.05 (group 5,6,7 vs group 1,10 ) **P<0.01 (group 5,6,7 vs group 0) These results are also presented in Figure 6 which shows the effects of serums induced by the fungal polysaccharide preparation with different ratios on apoptosis of Siso tumor cclla^^jt O^- prCpqrty" Oftce of HZ. 1 1 m 2035 4. Experimental Conclusions Our work has shown that a preparation having both the water soluble neutral polysaccharide and the acid soluble alkaline polysaccharide is far more effective in trains of anti tumour efficacy than the traditional preparation which usually incorporates a single water soluble neutral polysaccharide. This is supported by our results: • In growth test of Sigo tumor cells in vivo, the anti-tumor effects of the Fungal Polysaccharide Preparation of the invention, having the preferred ratios of the invention were significantly stronger than that of traditional preparation having only one water soluble neutral polysaccharide.

• In the growth test of Sigo tumor cells in vitro, the anti-tumor effects of serums induced by the Fungal Polysaccharide Preparation of the invention were significantly stronger than that of traditional preparations with one water soluble neutral polysaccharide.

• In the test of apoptosis of Sigo tumor cells, the effects of serums induced by the Fungal Polysaccharide Preparation of the invention were significantly stronger than that of traditional preparation with one water soluble neutral polysaccharide.

Although use of the novel combination of both forms of polysaccharide (WSNP and ASAP) were by far preferable to the conventional polysaccharide preparation, and provided surprisingly good results, we have determined that the best proportion of the two fungal polysaccharides (WSNP and ASAP) is substantially 75:25 w/w.

The serum induced by the Fungal Polysaccharide Preparation of the invention with ratio of 70: 30 was the best effect on anti-tumor in vitro. In the 10 groups of the Fungal Polysaccharide Preparation of the invention with different ratios, the anti-tumor activities increased from 80: 20 to 70: 30, reached the highest (see figure 5).

In the apoptosis effects of tumor cells cultivated in vitro, the serums induced by the Fungal Polysaccharide Preparation of the invention with 80: 20, 75: 25, 70: 30 were stronger than the others, and particularly that the 75:25 was the best.

Without wishing to be bound by any particular theory, the results of our studies suggest that the immure pathway and the effective position of the two polysaccharides, the WSNP and ASAP are different. We believe that the WSNP primarily acts on humoral immunity in the recipient and secondarily on cellular immunity. We believe that the ASAP primarily acts on the recipient's cellular immunity, and secondarily on humoral immunity. Thus we believe that two sorts of these 14 polysaccharides combined together particularly in the preferred ratios of the invention greatly increase both the liquid and cellular immunity.

Where in the foregoing description reference has been made to elements or integers having known equivalents, thai such equivalents are included as if they were individually set forth.

Although the invention has been described by way of example and with reference to particular embodiments, it is to be understood that modifications and/or improvements may be made without departing from the scope or spirit of the invention. * *-tV

Claims (36)

15 WHAT I CLAIM IS:

1. Ail active fungal polysaccharide preparation, comprising or including both: a) a water soluble polysaccharide obtained from a fungus, and b) an acid soluble polysaccharide obtained from a fungus.

A preparation as claimed in claim 1 wherein both a) and b) are extracted from the same fungal species.

3. A preparation as claimed in claim 2 wherein both a) and b) are extracted from the same fruiting body of the fungal species.

4. A preparation as claimed in claim 1 wherein a) and b) are obtained from different fungal species.

A preparation as claimed in any one of the preceding claims wherein the water soluble polysaccharide is a neutral polysaccharide, and the acid soluble polysaccharide is an alkaline polysaccharide.

6. A preparation as claimed in claim 5 wherein the water soluble polysaccharide has a pH substantially in the range 6.5~7.5.

7. A preparation as claimed in claim 5 wherein the acid soluble polysaccharide has a pH substantially in the range 2.0~4.0 lnte;iestual Property Office cf M.Z. 1 1 W 16

8. A preparation as claimed in any one of the preceding claims wherein a) and/or b) are obtained from the Ganoderma Lucidum fungus.

9. A preparation as claimed in claim 8 wherein both a) and b), in isolation, are active polysaccharides.

10. A preparation as claimed in claim 8 wherein only one of a) or b), in isolation, is an active polysaccharide.

11. A preparation as claimed in claim 9 or 10 wherein: a) comprises 70-80% w/w of the preparation and b) comprises 20-30% w/w of the preparation.

12. A preparation as claimed in claim 11 wherein a) comprises substantially 75% w/w and b) comprises substantially 25% w/w of the preparation.

13. A preparation as claimed in any one of the preceding claims wherein the combination of a) and b) is a synergistic combination.

14. A preparation as claimed in any one of the preceding claims wherein the preparation takes the form of a dosage unit.

15. A preparation as claimed in claim 14 wherein the dosage unit is selected from a measured dose in powdered form, a dosage unit in tablet form, or a dosage unit in capsule form.

16. A preparation as claimed in claim 15 wherein the dosage unit is a capsule comprising or including: 17 a) water soluble polysaccharide: 105-120 mg per capsule. b) Acid soluble polysaccharide: 30-45 mg per capsule.

17. A method of preparing an active fungal polysaccharide preparation, comprising or including the steps of: a) extracting a water soluble polysaccharide from a first fungus, and b) extracting an acid soluble polysaccharide from a second fungus (which may or may not be of the same species and the first, and which even may be the same fruiting fungal body as the first), and c) combining the two extracts.

18. A method as claimed in claim 17 wherein the water soluble polysaccharide is a neutral polysaccharide, and the acid soluble polysaccharide is an alkaline polysaccharide.

19. A method as claimed in claim 18 wherein the water soluble polysaccharide has a pH substantially in the range 6.5-7.5.

20. A method as claimed in claim 18 wherein the acid soluble polysaccharide has a pH substantially in the range 2.0-5.0.

21. A method as claimed in any one of claims 17 to 20 wherein the first and/or second fungus is Ganoderma Lucidum.

22. A method as claimed in claim 21 wherein the step c) of combining the two extracts, comprises or includes combining the two extracts in the following proport onsi In* Office cf 18 comprises or includes combining the two extracts in the following proportions: i) water soluble polysaccharide: 70-80% % w/w, and ii) acid soluble polysaccharide: 20-30% w/w.

23. A method as claimed in claim 22 wherein the step c) of combining the two extracts, comprises or includes combining the two extracts in the following proportions: i) water soluble polysaccharide: substantially 75% % w/w, and ii) acid soluble polysaccharide: substantially 25% w/w.

24. A method as claimed in any one of claims 17 to 23 wherein the step a) of extracting the water soluble polysaccharide comprises or includes the steps of: i) adjustment of the pH to substantially 9 ( ±1), and lowering of the pressure; and ii) recovery of the product using water.

25. A method as claimed in any one of claims 17 to 24 wherein the step b) of extracting the acid soluble polysaccharide comprises or includes the steps of: i) application of an acidic pH to extract the acid soluble polysaccharide, and ii) application of an alkaline pH to precipitate the acid soluble polysaccharide.

26. A method as claimed in claim 25 wherein the acidic pH is substantially in the range 1.5 to 2.5, and the alkaline pH is substantially in the range 11 to 12.

27. An active fungal polysaccharide preparation prepared substantially according to the method claimed in any one of claims 17 to 26. Intestec&'ai i-Offlce of 19

28. A use of an active fungal polysaccharide preparation as claimed in any one of claims 1 to 16, in the preparation of a medicament for providing a beneficial effect in a mammalian

29. A use as claimed in claim 28 wherein the beneficial effect is the treatment of tumor cells.

30. A use as claimed in claims 28 or 29 wherein the preparation is formulated for oral administration.

31. A use as claimed in claim 30 wherein the preparation is formulated for administration on one occasion only.

32. A use as claimed in claim 30 wherein the preparation is formulated for administration on a regular basis.

33. A use as claimed in claim 31 or 32 wherein the preparation is formulated for administration to a recipient prior to and/or during and/or following a radiation and/or chemical cancer treatment regime.

34. An active fungal polysaccharide preparation as claimed in any one of claims 1 to 16 and 27 substantially as herein described with reference to any one or more of the examples.

35. A method of preparing an active fungal polysaccharide preparation as claimed in any one of claims 17 to 26 substantially as herein described with reference to any one or more of the examples.

36. A use of an active fungal polysaccharide preparation as claimed in any one of claims 28 to 33 substanitally as herein described with refernce to any one or more of the examples. recipient. END OF CLAIMS Intellectual Property Office of N.Z. 1 8 APR 2005