Molecular basis for Poria cocos mushroom polysaccharide used as an antitumour drug in China

Abstract Poria cocos is an edible medicinal fungus known as “Fuling” in Chinese and has been used as a Chinese traditional medicine for more than two thousand years. Pharmacological studies reveal that polysaccharide is the most abundant substance in Poria cocos and has a wide range of biological activities including antitumour, immunomodulation, anti‐inflammation, antioxidation, anti‐ageing, antihepatitis, antidiabetics and anti‐haemorrhagic fever effects. As a result, “Poria cocos polysaccharide oral solution” was developed and sold as an over‐the‐counter health supplement since 1970s. In 2015, “Polysaccharidum of Poria cocos oral solution” was approved as a drug by Chinese Food and Drug Administration for treating multiple types of cancers, hepatitis and other diseases alone or during chemo‐ or radiation therapy for patients with cancer. In this article, biochemical, preclinical and clinical studies of Poria cocos polysaccharide from 72 independent studies during the past 46 years (1970‐2016) based on PubMed, VIP (Chongqing VIP Chinese Scientific Journals Database), CNKI (China National Knowledge Infrastructure) and Wanfang database searches are summarized. The structure, pharmacological effects, clinical efficacy, immunobalancing molecular mechanism and toxicity of Poria cocos polysaccharide are deliberated to provide a general picture of Poria cocos polysaccharide as a clinically used antitumour drug.

mannose and galactose are detected in PCP. b-Glucan is the major PCP with b-(1?3)-linked glucose backbone and b-(1?6)-linked glucose side chains as shown in Figure 2. 3,6 The b-glucan from Poria cocos has poor water solubility but decent anticancer activity. 7 Chihara et al removed the b-(1?6) glucose in the b-glucan of PCP by periodate oxidation and Smith degradation. The derivative is named "pachymaran," which exhibits better anti-S-180 tumour activities. 8 Hamuro et al further improved the water solubility issue of pachymaran by chemical carboxymethylation. The carboxymethylated pachymaran (CMP) has enhanced antitumour activity compared to that of pachymaran. 9 Subsequently, different chemical modifications, such as sulfation, 10 carboxymethylation plus sulfation, 11 methylation, hydroxyethylation and hydroxpropylation, have been conducted and different types of modified pachymarans are reported. 12 In general, these chemical modified pachymaran derivatives are water-soluble and show improved bioactivities. and 4 will be the major topics discussed in this article. Moreover, the data that show PCPs can overcome immunosuppression and adverse reactions associated with radiation therapy and chemotherapy 13 will also be presented and discussed.

| PCP-based drug in China
2 | PHARMACOLOGICAL ACTIVITIES OF PCP

| Antitumour
Parallel to other reported polysaccharides from fungi, [14][15][16] PCP and its derivatives have more impressive anticancer cell proliferation activities F I G U R E 1 The fruiting body of Mushroom Poria cocos. Poria cocos is an edible medicinal fungus known as "Fuling" in Chinese and has been used as a Chinese traditional medicine for more than two thousand years F I G U R E 2 A schematic diagram of b-glucan structure in Poria cocos. b-Glucan is the major Poria cocos polysaccharide with b-(1? 3)-linked glucose backbone and b-(1?6)-linked glucose side chains. The b-glucan from Poria cocos has poor water solubility but decent anticancer activity F I G U R E 3 Pharmacological activities of Poria cocos polysaccharides (PCPs). Sixty-six articles related to pharmacological activities of PCPs are summarized. Thirty-eight per cent of studies are about antitumour activities of PCPs. Twelve per cent of studies are about antitumour mechanisms. Studies on immunoregulation, antioxidant and toxicity account for 19%, 10% and 7%, respectively. Fourteen per cent of pharmacological activity studies of PCPs are defined as "others" that are further explained in Figure 4 F I G U R E 4 Other pharmacological activities of PCPs. Antihepatitis effects: 30%; antidiabetic effects: 10%; anti-epidemic haemorrhagic fever: 10%; anti-ageing effects: 10%; anti-inflammatory effects: 10%; and anti-acute lymphoblastic leukaemia (ALL): 10%  Table 2, the inhibition rates of PCP or its derivatives on the proliferation of cancer cells are largely concentration-dependent, which resembles the control drug 5-fluoro-2,4(1H,3H) pyrimidinedione or 5-Fu.
In contrast, in cancer cell-injected animal models (Table 3), the inhibition rates of PCP or its derivatives on tumour growth are only partially concentration-dependent in that within a certain range, the higher the concentrations, the higher the inhibition rates are. But beyond the range, the inhibition rates will drop. For example, the experiment conducted by Cheng et al 18 showed that the inhibition rates of cancer cell growth are 0, 69%, 87%, 92% and 89%, respectively, with increasing CMP concentrations.
The inhibition of tumour growth in vivo is measured either by the reduced tumour weight or by ultrasound compared to controls in different animal models and presented as tumour inhibition rates (%). The controls include blank and positive controls where the chemotherapeutic drug, such as 5-fluoro-2, 4 (1H, 3H) pyrimidinedione (5-Fu), is used. PCPs have potent antitumour activities in different animal tumour models when compared to blank or 5-Fu controls ( when PCP is used with 5-Fu compared with the inhibition rates of 41% when 5-Fu is used alone. 29

| Antitumour mechanisms
PCPs exert their antitumour activity via assisting the host to overcome adverse biological stresses, to increase immunity against the tumours and to promote the apoptosis of tumour cells directly. The possible mechanisms reported so far are summarized in Table 5A-D and Figure 5 33-38 and discussed below.

| Enhancing the innate immunity through activating the immune cells
Polysaccharides could activate effector immune cells, such as macrophages, lymphocytes and natural killer (NK) cells to activate the innate immune system to exert antitumour activity by accelerating the host's defence mechanisms. 33 It is reported that the ratio of lymphocytes could increase to 62% in the treatment group compared  increased Bax/Bcl-2 ratio is responsible for the apoptosis of S180 tumour cells in the mouse model. 33

| Antioxidation
Free radicals refer to the dissociative molecules, atoms or ions with unpaired electron that reacting rapidly with other substances. Under certain range, free radicals can help to eliminate microorganisms intruding into body or abnormal cells. However, if free radicals are excessively produced, they would attack the normal cells and tissues.
Therefore, human body constantly produces and removes free radicals to maintain a dynamic balance. Studies showed that PCP has antioxidant activity by scavenging free radicals. As shown in

| Other pharmacological activities
PCPs have other biological effects, such as anti-inflammatory, 56 antiageing, 57 antihepatitis, 58,59 antidiabetic, 60 anti-ALL (acute lymphoblastic leukaemia), 61 anti-nephritic 62 and antihypertensive effects. 63 These pharmacological activities are summarized in Table 7A,B. Hou et al observed that PCP reduces the size of granuloma. 56 As an anti-ageing reagent, PCPs enhance the activities of both T-SOD and Cu-SOD and reduce MAD and MAO (monoamine oxidase) activities. 57 To understand the antihepatitis effects of CMP, it is found that CMP reduces the expression of HBsAg and HBeAg in a concentration-dependent manner. 58      Meanwhile, CMP significantly improves the survival rates of mice suffering acute lymphoblastic leukaemia or ALL. 61

| CLINICAL EFFICACY OF PCP
Seven clinical studies on PCPs were found through literature search. 59,[64][65][66][67][68] The clinical data are summarized in Table 8. Most of studies are related to the antitumour effects of CMP where these studies use IL-2 or IFN-a obtained from human peripheral blood lymphocytes induced by CMP in combination with chemotherapy or radiotherapy. Sheng et al reported that the total effective rate could reach 97% when CMP-induced IL-2 is combined with chemotherapy during cancer treatment compared to that of 27% by chemotherapy alone. 65 The effectiveness is defined as improving the symptoms of the disease by increasing appetite, elevating the levels of cAMP in blood circulation, regulating the ratio of cAMP/cGMP, protecting and restoring damaged liver and reducing the side effects of chemotherapy. 66 In treating epidemic haemorrhagic fever, cure rate could reach 100% when CMP-induced IFN-a is combined with normal therapy compared to that of 63% with normal therapy alone. 64 Chen et al also found that the total effective rate could reach 90% during hepatitis treatment. 66

CONFLI CT OF INTEREST
The authors declare no conflict of interests. All grants and funding agencies play no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication.