The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma –––– a systematic a systematic a systematic a systematic

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours and the second most frequent cause of cancer-related death worldwide. The aim of this review is to identify whether phytotherapy has an effect over the treatment of HCC or if it is suitable as a combination with chemotherapy. A systematic review was performed in order to offer current information over the use of phytotherapy in HCC. We conducted an electronic search of articles published in English in peer reviewed journals between 2012-2022. After keywords were associated, 302 articles were found. After the exclusion of articles which did not meet the inclusion criteria, there were 77 articles eligible for abstract evaluation. The following were discarded: two case report, one systematic review, 36 in vitro studies and nine studies which discussed other pathologies or with no regard to phytotherapy. The remaining articles encompassed 27 in vivo studies of phytotherapy in hepatocellular carcinoma and two randomized control trials. This selection process is illustrated in the Prisma Flow Diagram. Amongst the evaluated articles, two of them researched the effect of phytotherapy over human subjects in two randomized control trials, while the others illustrated the outcomes of phytotherapy over hepatocellular carcinoma cells and murine specimens. To sum up, phytotherapy has proven its usefulness in hepatocellular carcinoma, especially throughout the following mechanisms: anti-inflammatory effect, suppressing malignant cell proliferation, inhibiting angiogenesis, stimulating apoptosis, and even sensitizing cells to chemotherapy.

The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma The use of phytotherapy in hepatocellular carcinoma ----a systematic a systematic a systematic a systematic review review review review Cristina P. URSU 1 , Emil I. MOIȘ 1,2 *, Luminița E. FURCEA 1,2 , Rodica S. POP 3 , Ștefan URSU 1 , Diana SCHLANGER 1 , Andra CIOCAN 1,2 , Florin V. ZAHARIE 1,2 , Aida PUIA 3 , Nadim AL HAJJAR 1,2 , Florin GRAUR 1,2 Introduction Introduction Introduction Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumours and the second most frequent cause of cancer-related death worldwide. Hepatocellular carcinoma acts as the most common type of primary liver neoplasm, the majority of cases are developing in the context of chronic liver damage and inflammation, especially due to chronic hepatitis B virus or hepatitis C virus infections and chronic alcohol intake. The incidence of nonviral HCC is increasing, notably in developed countries. Non-alcoholic fatty liver disease or metabolic associated fatty liver disease leads to non-alcoholic steatohepatitis (NASH), resulting in liver cirrhosis and lastly, in HCC (Boyer et al., 2012;Tang et al., 2017).
Early-stage clinical symptoms of this pathology are nonspecific; therefore, patients were usually diagnosed at an intermediate or advanced stage. Latter advances in early diagnosis and treatment have improved the short-term prognosis of patients with HCC, but there are still limited treatment options available. At the present moment the main therapies for HCC encompass liver resection (LR), radiofrequency ablation (RFA), liver transplantation (LT), trans arterial chemoembolization (TACE), Sorafenib and also symptomatic treatment for stage D, according to Barcelona Clinic Liver Cancer staging system (BCLC) (Reig et al., 2022). Surgical resection is an optimal option for very early stage or early-stage HCC patients with preserved liver function, whilst patients who present with severe liver dysfunction may be considered for LT. The European Association for the Study of the Liver (EASL) Guidelines provides recommendations regarding liver transplant for BCLC 0, A and even the first subgroup of B stage patients, laying out specific criteria for each category. In spite of these treatment possibilities, early-stage diagnosis of HCC is difficult and it has a poor prognosis, thus explaining the extensive research for new methods of diagnosing and treating this pathology Zamora-Valdes et al., 2017;Reig et al., 2022).
In view of the above-mentioned treatment paths, there is a critical need for searching for alternative approaches, as the use of phytochemicals obtained from dietary sources which offers a preventive and therapeutic approach over HCC Yang et al., 2022). Phytochemicals have antioxidant, anti-inflammatory and anti-proliferative effects, which can combat the oxidative stress and inflammation involved in liver cancer Yang et al., 2022). There are many herbal medicines which have proven their efficiency as anti-inflammatory agents and therefore have the capacity of supressing the development of hepatocellular carcinoma. Clinical trials and reviews are warranted in order to explore the effectiveness of herbal medicine in both prevention and treatment of HCC (Rino et al., 2015;Farazuddin et al., 2019).
Traditional Korean Medicine (TKM) carries out benefits for patients with HCC by improving their quality of life and maintaining tumour size. Lately, TKM has been highly used in cancer treatment. Jang et al. (2018) published a case report describing a 62-years old Korean woman with HCC and lung metastases, who went under associated treatment with TKM and Sorafenib after going under surgery and six cycles of adjuvant chemotherapy and treated afterwards only with Sorafenib, but with no regression and also experiencing most of the side effects of the treatment. Following 8 weeks of TKM treatment, the size of the metastatic nodules decreased and the tolerance for Sorafenib's side effects improved, thus endorsing the advantages of using TKM in combination with standard treatment for HCC and extrahepatic metastasis (Jang et al., 2018).
Newly identified or recurrent HCC patients, who cannot meet criteria for curative therapies, are in need of further adjuvant methods of treatment. Western medicine has its demonstrated advantages over treating 3 subjects who suffer from hepatocellular carcinoma, but still the five-year survival rate is somber. Chinese Herbal Medicine (CHM) for example, has been used in the treatment of HCC patients for many years, and have been proven to be an efficacious and safe option for cirrhosis and chronic hepatitis. Moreover, there is a lot of experience in using CHM in preventing and treating HCC, proven in a series of in vitro studies. For this reason, we propose a systematic review of the existing literature regarding the efficacy of herbal medicine in hepatocellular carcinoma, in both in vitro and in vivo studies Xu et al., 2016;Zamora-Valdes et al., 2017;Yang et al., 2021;Reig et al., 2022).

Aim of the review
The aim of this review is to identify whether herbal medicine has an effect over the treatment of hepatocellular carcinoma or if it is suitable as a combination with the standardized treatment used for this pathology.
Research questions: 1. Which is/are the herbal medication/ herbs used in the treatment of hepatocellular carcinoma? 2. What effects does herbal medicine have over hepatocellular carcinoma cells?

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
A systematic review was performed in order to offer current information over the use of phytotherapy in hepatocellular carcinoma.

Search strategy:
We conducted an electronic research of articles published in English in peer reviewed journals between 2012-2022. The research process took place in October 2022. A combination of the following search terms was used: phytotherapy, hepatocellular carcinoma, herbal medicine, and in vivo studies. The search strategy was limited to the following electronic databases: PubMed, PsycINFO, CINAHL.

Inclusion criteria
This research includes all the articles which show the use of at least one herbal substance/ formulation in hepatocellular carcinoma studies.

Exclusion criteria
The articles excluded from the present research were published earlier than 2012, in other languages than English, not referring to the subject, studies referring to paediatric patients or studies which have not used at least one herbal substance or medicine in order to treat or influence the prognosis of hepatocellular carcinoma. Literature reviews were as well discarded.

Data extraction
The abstracts of these articles were read to detect duplicates and identify articles for full copy retrieval. The articles were then reviewed independently by different members of the team. In addition, the medicinal herbs were categorized according to their clinical or molecular implications. The process for this entailed: identifying the herbs referred to by each article and determining the effects of these in hepatocellular carcinoma. After the initial data collection phase, the reviewers' reports were independently cross-checked and items for clarification were discussed and resolved at a face-to-face meeting.

Results Results Results
After keywords were associated, 302 articles were found. Articles published in other language than English were excluded (n=37), as well regarding articles issued outside a 10-year period (n=129). Afterwards, articles which were not referring to the subject matter (n=58) or concerning paediatric patients (n=1) were excluded. There were 77 articles eligible for abstract evaluation, from which the following were discarded: two case reports, one systematic review, 36 in vitro studies and nine studies which discussed other pathologies or with no regard to phytotherapy. The remaining 29 articles encompassed 27 in vivo studies of phytotherapy in hepatocellular carcinoma and two randomized control trials. These articles were included in the final analysis. This selection process is illustrated in the Prisma Flow Diagram (Figure 1).  The list of articles and associated selected information from the data extraction process is presented in Table 1.  Patients receiving PLQ experienced a relieve regarding to the following symptoms: fever, pain, fatigue, lack of appetite, drowsiness, dry mouth, constipation, but without statistical difference among the 3 groups during 3 days after TACE besides drowsiness and dry mouth  After TACE, both TB and AST/ALT levels were lower in the herbal medicine group Abbreviations: FZJDXJ-Fuzheng Jiedu Xiaoji formulation; AKT-Protein kinase B pathway; OS-one-year overall survival; PFSprogression-free survival; TACE-transcatheter arterial chemoembolization; CE-Calunduloside E; HepG2-; RPR-Rheum palmatum root; TGF-β1-transforming growth factor beta 1; DEN-induced HCC-diethylnitrosamine-induced HCC; PLGA-a nonenzymatically-degradable polymer; TNF-α-tunor necrosis factor-alpha; MOLEE-Moringa oleifera leaf ethanol extract; AFP-Alpha fetoprotein; CEA-Carcinoembryonic antigen; SA-Semecarpus anacardium; AST-aspartate aminotransferase; ALT-alanine aminotransferase; AKP-alkaline phosphatase; GGT-gamma-glutamyl transferase; TGP-Total Glucosides of Paeony; BAFF-B-cell activating factor; DOX-doxorubicin; GAPDH-glyceraldehyde-3-phosphate dehydrogenase; G6PD-glucose-6-phosphate dehydrogenase.

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Amongst the evaluated articles, two of them (6.89%) researched the effect of phytotherapy over human subjects in two randomised control trials, while the other 27 articles (93.10%) illustrated the outcomes of phytotherapy over hepatocellular carcinoma cells and rodents (mice, rats). In 20 of the situations above mentioned the studies used active compounds extracted from one plant, with 9 articles describing the use of a formulation based on more than one plant. Within the latter category there are 6 articles which show the use of specific formulations, which are registered as traditional medicine.
After analysing the articles aforementioned, a series of outcomes was observed. These effects can be divided into two major categories: molecular effects (Table 2) and then both clinical and paraclinical results ( Table 3).
The molecular effects demonstrated through the use of herbal medicine in HCC are: inhibiting tumour cells proliferation, anti-inflammatory effect, induced or enhanced apoptosis and reducing angiogenesis ( Table  2).  As for clinical and paraclinical outcomes, these are as follows: prophylaxy, reducing complications rate, reducing mortality rate, reducing liver enzymes, decreased levels of tumoral markers and raising sensitivity to chemotherapeutic agents (Table 3).
Inhibiting malignant cell proliferation was proven in almost all 29 studies, except for three situations: Moringa oleifera leaf, Black currant skin extract, both possessing an anti-oxidation effect and Livistona chinensis expressing an important inhibitory effect over angiogenesis.
The suppression of cell proliferation was demonstrated in almost all the studies on HepG2 cells, because of an enhanced cytotoxicity against these cells. The herbs which showed such an effect are: Canduloside E, Curcuma longa, Mongolian prescription II. Other type of malignant cells on which the studies were conducted are H22 cell lines, in murine subjects, where GLSP inhibited cell proliferation with the help of activated macrophages and MPII inhibited tumour growth. Moreover, MPII inhibited the proliferation of Huh-7 cells. In addition, cell growth was ceased in BEL-7402 cells by Alpha-pinene.
The anti-inflammatory effect was proven in the studies using the following herbs: Calunduloside E, Ganoderma lucidol, Curcuma longa, Paeonia lactiflora, Coelastrella, alpha-pinene and Salvia chinensis. There is a list of herbs, that have proven their influence in decreasing inflammation through cyclines, inhibiting TNF, reducing cytokines and prostaglandins activity thus reducing the peritumoral edema.
Apoptosis is induced by the following medicinal herbs: Curcuma longa, Coelastrella, Pulsatilla saponin A, Diosmin and Berberine, Ellagic acid Livistona and Salvia chinensis. The same outcome was observed for the formulations containing 12 plants as is Dahuang zhechong or including 5 herbs as Songyou Yin. Rheum palmatum and Paeonia, found in DHZCP have shown their effect in both inducing apoptosis and inhibiting tissular proliferation.
A major advantage for the uptake of medicinal herbs during therapy for hepatocellular carcinoma is reducing the level of transaminase, which was shown while using Curcuma longa, Semercarpus anacardium, Paeonia lactiflora, Coelastrella sp F50, Nigella sativa, Epigallocatechin gallate. Jian Pi Li Qi decoction is a formulation used in the Traditional Chinese Medicine for having positive effects over liver function, especially after chemotherapy, therefore being useful in the treatment of HCC.
There are a few concoctions used in the TCM, such as Fuzheng Jiedu Xiaoji and Zuo-Jin-Wan, which reduce the rate of complications for this pathology. Fuzheng Jiedu Xiaoji also proved it's efficiency in reducing HCC mortality. Other herbs that attested a positive outcome regarding the mortality rate where Curcuma longa and Paeonia lactiflora.
Moringa oleifera leaf, Paeonia lactiflora, Nigella sativa have a favourable effect reducing the level of tumor markers.

Discussion Discussion Discussion
The use of phytotherapy provides multiple benefits for patients diagnosed and treated for hepatocellular carcinoma. Taking into consideration the fact that this illness is still imposing difficulties in early diagnosis thus patients having either treatment option issues or an impaired quality of life, herbal medicine has shown its advantages. Reig et al., 2022) There are several effects of phytotherapy that will be discussed at length: the useful combination between TACE and herbal medication, important for reducing the incidence of complications in HCC, enhancing performance status in B stage patients and inhibiting tumour cell proliferation, or ameliorating postembolisation syndrome; reducing oxidative stress; antimutagenic effects 11 and increasing tumour cells' sensitivity for chemotherapeutic drugs (Yang et al., 2021;Xu et al., 2015;Thoppil et al., 2012;Sadek et al., 2017;El-Saied et al., 2018;Wu et al., 2018).
TACE is one of the standardized treatments of hepatocellular carcinoma, widely used due to its benefits. Most patients with HCC are diagnosed while at an intermediate or advanced stage, with few therapeutic options available, thus being the reason for using TACE, in accordance with BCLC. The chemotherapeutics commonly used for TACE are Cisplatin, for inhibiting DNA replication and transcription and Pirarubicin, which inhibits DNA polymerase, prevents nucleic acid synthesis and as well, cell transition from G2 to M phase. Despite these benefits, recurrence and metastasis after TACE are possible, therefore the combination of this type of therapy and effective drugs, such as TCM, may improve the process of blocking the tumour progression (Galle et al., 2017;Yang et al., 2022). The results of a randomized clinical trial using FZJDXJ treatment were a prolonged overall survival and reduced overall mortality rate in patients, especially those included in BCLC A and B stages. The mortality analysis proved that patients in stages A and B had a reduced incidence of HCC complications, including abdominal infection, encephalopathy, multiple metastases, rupture, or haemorrhage of HCC. This Traditional Chinese concoction had a significant effect on performance status lengthening in B stage patients and inhibited tumour cell proliferation for the same category of individuals. All in all, FZJDXJ inhibited tumour progression (Yang et al., 2021).
Postembolization syndrome (PES) is a complication of TACE which appears in 60-80% of cases and includes the following symptoms: fever, nausea, vomiting, abdominal pain, lack of appetite and impaired liver function. An effective approach to prevent this syndrome is necessary, especially because a combination of antipyretic agents, analgesics, antiemetics and cytoprotective drugs will accentuate the metabolic charge of the liver (Leung et al., 2001;Xu et al., 2015). Jian Pi Li Qi decoction is a frequently used prescription in TCM. A randomized, double-blind, placebo-controlled trial was performed to confirm if this therapy option is capable to prevent and treat TACE related PES, Chen et al found that Jian Pi Bu Qi improves prevention and reduction of PES syndrome. Other herbs as Qingre Jiedu Decoction associated with Western medicine has shown benefits in preventing and treating symptoms after TACE. JPLQ decoction improves the efficacy of chemotherapy and can also improve liver function and relieve the side effects brought on by TACE. (Leung et al., 2001;Xu et al., 2015) This study enrolled 150 patients receiving TACE therapy. The patients were randomly allocated to three groups: A, (subjects received neither herbal medication or placebo) containing in the end 50 patients, group B (placebo treatment group), including 40 patients and C (JPLQ decoction treatment group) with 50 individuals. The number of patients studied was lower than the number enrolled due to their consent withdrawal. The patients were admitted in the hospital for 4 days and supervised during therapy. The study compared the percentage of patients facing severe levels of the most important symptoms (pain, disturbed sleep, distress, fatigue, lack of appetite, drowsiness, dry mouth, nausea, vomiting, bloating, constipation), 1 day before TACE to 3 days after the procedure. The result was that for each symptom, the percentage was higher in the first day after TACE than in the following ones. The instrument to assess postembolization syndrome was M.D. Anderson Symptom Inventory module for use in patients with gastrointestinal cancer (MDASI-GI) (Wang et al., 2010;Xu et al., 2015). On the first day after the procedure there were no statistical differences found among the three groups of subjects. However, the patients in group C faced an attenuation of drowsiness and dry mouth on the first day and they had these symptoms significantly relieved on the second day after TACE: pain, fatigue, lack of appetite, drowsiness, dry mouth, and constipation. It is important to state that although this decoction could provide overall relief for some symptoms, it could not ameliorate severe pain, fatigue, constipation, or the lack of appetite. The post-procedure fever had a peak on the first and second day after TACE and gradually decreased on the third day. The incidence of fever was lower in group C, than in the other groups during the supervision, but the differences were not statistically significant. Regarding liver function after TACE, the results have demonstrated that there is a protective effect over liver function while using JPLQ decoction after this therapy, according to the fact that total bilirubin level (TB) in group C was lower than in group A and B, and the values of aspartate transaminase and alanine transaminase were lower in group C than A, but like the ones expressed by the subjects in group B. The results of this trial also demonstrated that JPLQ can improve the quality of life of patients classified as moderate to advanced stages of HCC. The quality of life and the burden that is expressed by lowering it through symptoms and therapy side effects can be modified using herbal medicine (Leung et al., 2001;Xu et al., 2015;Pop et al., 2022).
Chinese rhubarb, Moringa oleifera, ayuverdic milk extract of Varnish tree, Black currant and an active monomer from green tea gave prophylactic actions in HCC. This effect is due to the attenuation of oxidative stress, the growth of antioxidant enzymes, lowering the lactate dehydrogenase (LDH) activity which interferes with glycolysis therefore protecting the integrity of hepatocytes membrane, having also antimutagenic effects Joseph et al., 2013;Sadek et al., 2017;El-Saied et al., 2018;Tang et al., 2020).
Raising sensitivity of hepatocellular carcinoma cells to chemotherapeutic agents is an important effect that increases the efficacy of this therapy (Wang et al., 2013;Nanda et al., 2017;Wu et al., 2018;Zhong et al., 2018). DHZCP is composed out of 12 plants known for their therapeutic effects in TCM. This formulation inhibits ATP energy metabolism and improves accumulation of Doxorubicin in the tumour tissue, while Ellagic acid increases the sensitivity of tumour cells for the above-mentioned chemotherapeutic drug (Wu et al., 2018;Zhong et al., 2018). Phytotherapy has impressive effects in hepatocellular carcinoma, and even in preventing the occurrence of such a pathology, although there are studies that request caution when it comes to associating phototherapeutics and biliary diseases that have a high risk of malignant degeneration Puia et al., 2013;Sadek et al., 2017;Tang et al., 2020).
Curcumin represents a natural compound, widely investigated and with multiple therapeutic actions, such as anticancer, anti-virus, anti-arthritis, anti-amyloid, anti-oxidative and anti-inflammatory. Regarding liver diseases, curcumin inhibits HBV gene expression and replication via down-regulation of PGC-1α. Studies have demonstrated that curcumin significantly ameliorated non-alcoholic fatty liver disease, while other papers exhibit curcumin's effects over hepatic stellate cells, inactivating them, and protection against liver steatosis and fibrosis (Farazuddin et al., 2019;White et al., 2019;Yang et al., 2022). Moreover, this compound can suppress stromal cell-derived factor-1/CXCR4 signalling, thus reducing the incidence of circulating gastric cancer cells and decreasing the risk of liver metastases. Thereby, curcumin might show advantages in the therapy of HCC. Unluckily, curcumin has a poor bioavailability and hydrophobicity, which imposes a major issue as for using it as a potent anticancer agent (Duvoix et al., 2005;Farazuddin et al., 2019;Yang et al., 2022). To avoid these problems, Farazuddin et al. (2019) developed a novel, dual-core microcell formulation of curcumin by encapsulating this element in microcells, facilitating its kinetics. Analysis revealed that these microcells helped regression of HCC and maintenance of liver tissue architecture. In addition, free curcumin had a modest effect on neoplastic suppression (Farazuddin et al., 2019;Yang et al., 2022). Tang et al. (2020) described the effects of Epigallocatechin gallate over HCC. EGcG is the most rich and bioactive catechin found in green tea. The results of this study proved that eGcG can inhibit the proliferation of HepG2 and Huh7 cells, reduce the expression of cdc25a and elevate the expression of p21wafl/cip1 in HepG2. Moreover, in vivo eGcG reduced tumour volume and increased survival rates of diethylnitrosamine (DEN)-induced HCC in rats. Although, the findings of Tang's et al. (2020) are the first to show that eGcG possesses chemopreventive properties, there was no significant difference regarding this outcome between GTe (Green Tea extract) and eGcG. Previous studies have shown that GTe at an eGcGequivalent biological concentration exhibits a stronger inhibition concerning squamous cell carcinoma and another study described that EGCG and GTE can be immune checkpoint inhibitors in lung cancer evolution. Concerning the study of Tang et al. (2020) the results have demonstrated that EGCG expresses chemopreventive properties, which can be explained by the reduction in CDC25A, as an important liver cancer gene. Moreover, EGCG and GTE reduced tumour volume, improved the survival rates of HCC subjects, and inhibited the proliferation of hepatoma cells (Huang et al., 2009;Liu et al., 2011;Rawangkan et al., 2018;Tang et al., 2020). Altogether, considering its poor stability and low bioavailability, eGcG can be used as an association with other antitumor medication, thus having a synergistic effect of HCC prevention and treatment (Tang et al., 2020).

Conclusions Conclusions Conclusions Conclusions
This review aimed to explore whether herbal medicine has any beneficial effects for hepatocellular carcinoma patients and if so, which are the herbs that provide such outcomes. From the reviewed articles it has been shown that herbal medicine provides a large series of benefits for patients that were diagnosed with HCC, especially concerning the following mechanisms: anti-inflammatory and anti-oxidative effects, suppressing malignant cell proliferation, inhibiting angiogenesis, stimulating apoptosis, and even sensitizing cells to chemotherapy therefore expressing outstanding results. Moreover, phytotherapy expresses favourable outcomes regarding the quality of life of patients included in advanced stages of the disease or that are experiencing side effects of certain therapies, as for patients receiving TACE. To obtain all these results, studies used either single herbs or a mixture of medicinal herbs, for example: FZJDXJ formulation, Jian Pi Li Qi decoction, Curcumin, Black currant, Chinese rhubarb, Ellagic acid, obtained from nut galls or pomegranate, or one bioactive catechin found in green tea and many more, which were successful in obtaining favourable results. All in all, regardless of the use of only one herb or a concoction, phytotherapy has proven its usefulness in hepatocellular carcinoma.
Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable. Ack Ack Ack