WO2010098652A1 - Cola nitida ( cola nut ) as an anticancer agent - Google Patents

Cola nitida ( cola nut ) as an anticancer agent Download PDF

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Publication number
WO2010098652A1
WO2010098652A1 PCT/MY2009/000204 MY2009000204W WO2010098652A1 WO 2010098652 A1 WO2010098652 A1 WO 2010098652A1 MY 2009000204 W MY2009000204 W MY 2009000204W WO 2010098652 A1 WO2010098652 A1 WO 2010098652A1
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cola
cola nitida
cancer
nitida
liver
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PCT/MY2009/000204
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French (fr)
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Fauziah Othman
Asmah Rahmat
Susi Endrini
Suherman Jakfa
Wan Nor I ' Z Zah Wan Mohamad Zain
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Universiti Putra Malaysia
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Publication of WO2010098652A1 publication Critical patent/WO2010098652A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • COLA NITIDA COLA NUT
  • COLA NUT AS AN ANTICANCER AGENT
  • the present invention relates to an anticancer agent. More particularly, the present invention relates to an anticancer agent which is Cola nitida (Cola nuts).
  • Cancer is a group of disease which is characterized by uncontrolled cellular growth with local tissue invasion and or systematic metastasis.
  • the number of new cancer cases has been increasing over the past nine decades. It is the largest single cause of death in both men and women, claiming over 6 million lives each year in the world. Cancer is the most feared of all diseases. Unlike other diseases, cancer usually causes a slow death involving pain, suffering, mental anguish and a feeling of hopelessness.
  • the process starts with an insult to the genetic cellular material (genotoxic events; known as initiation) and then proceeds to the production of abnormal DNA (nongenotoxic events; known as promotion).
  • the transformation of the altered cells may lead to the proliferation of cells with invasive (malignant) or noninvasive (benign) qualities.
  • invasive malignant
  • noninvasive benign
  • carcinogenesis is driven by multiple interactive factors.
  • the external factors include chemicals, radiation and viruses, whereas the internal factors are hormones, immune condition and inherited mutation. Therefore, every one of us is at risk of developing cancer no matter at what age one is.
  • Cola nitida had been screened to be effective towards liver cancer.
  • the present invention is focused on the effect of Cola nitida on hepatocellular carcinoma.
  • Tumors of the liver are classified as being either primary (originating from the liver) or metastatic (spread from another body organ to the liver).
  • Primary liver tumors may be divided into those that are benign (not cancerous and remain in the liver) or malignant (cancerous and may be spread beyond the liver).
  • therapies and therapeutic use of natural products especially from plant derivative and in trend towards the use of natural substances which is also believed to have potential value as cancer chemopreventive or therapeutic agents.
  • Cola nitida is an astringent, bitter-sweet, anti-depressant herb that has a stimulating effect, especially on the heart. It contains alkaloid caffeine and phenolics, such as catechin, epicatechin and procyanidins.
  • the Cola nitida tree is native to West Africa. It has been naturalized to South America, Central America, the West Indies, Sri Lanka, and Malaysia.
  • Cola nitida contains the methylxanthine alkaloids that also occur in coffee, cocoa, and tea.
  • West Africans have been chewing Cola nitida for thousands of years. Its stimulant effects are the predominant application in the United States and Europe.
  • Cola nitida In Africa, however, Cola nitida has been used as an appetite and thirst suppressant, enabling soldiers who chew them to travel long distances without much food. Cola twigs, with an extremely bitter taste, are used to clean the teeth and gums. Cola maintains a strong cultural significance in West Africa, partly due to the fact that cola is a valuable commodity. It has been traded to other countries since at least the fourteenth century and it is used particularly by Islamic people, who, according to their religion, cannot drink alcohol, but desire a "social lubricant”. Today, Cola nitida is exported worldwide. Cola nitida is also used in non-pharmaceutical preparations, including (at least formerly) cola-based beverages such as Coca Cola. It is on the GRAS (generally recognized as safe) list for food additives in the United States.
  • Cola nitida is an anticancer agent provided from its anticancer properties inclusive of antioxidant activities (FTC-TBA, DPPH), antioxidant minerals (oxygen, carbon, potassium, phosphorus and magnesium), total phenolic and total flavonoid content.
  • FIG. 1 shows scanning electroscope (SEM) image of unprocessed (fresh) Cola nitida seed.
  • FIG. 2 shows cytotoxicity effect of Cola nitida on MCF-7, HepG-2 and Chang cell lines.
  • FIG. 3(a) to (c) show flowcytometry analysis on effect of Cola nitida on HepG-2 hepatocellular carcinoma cell lines. Higher (20 ⁇ g/ml) and lower (10 ⁇ g/ml) concentrations were compared with control to quantify the apoptotic cell. C illustrated the quantity of apoptotic cells.
  • FIG. 4 shows body weight profile of rats for the 8-week of bioassay
  • N Normal control
  • NCn Normal + Cola nitida
  • C Cancer control
  • CCn Cancer + Cola nitida
  • CG Cancer + Glycyrrhizin.
  • the present invention relates to an anticancer agent. More particularly, the present invention relates to an anticancer agent which is Cola nitida.
  • an anticancer agent which is Cola nitida.
  • Plants are an important source of essential nutrients and beneficial components that are crucial for healthy living. Knowledge about the beneficial effects of certain plants is mainly transmitted by personal communication; therefore, it remains unknown to the general population. Many studies need to be done to promote the usage and application of native plants.
  • the phytochemical or chemical composition of plants can be characterized as major and minor constituents, depending on their general abundance in the plants.
  • Major constituents include carbohydrates lipids or fats, and proteins which are present at gram level in each 100 g portion of sample.
  • Minor constituents include vitamins, secondary metabolites and minerals. When present, these constituents generally are found in the microgram to milligram range per 100 gram sample.
  • Antioxidant vitamins, including polyphenol, pigments and flavonoids possess antimutagenic properties as well as blood glucose decreasing activities.
  • the antioxidant minerals such as calcium (Ca), magnesium (Mg), zinc (Zn) and copper (Cu) were determined by using atomic absorption spectrophotometer (GBC, Model 908 AA, USA) taken from the ash solution of Cola nitida.
  • the surface morphological structure and biological composition of Cola nitida was viewed and investigated under a variable pressure scanning electron microscope (VPSEM) equipped with energy dispersive x-ray (EDX) microanalysis (VPSEM model LEO VP-1455).
  • VPSEM enables unprocessed samples to be viewed and examined.
  • point and identification (ID) was used which enabled specific part of the sample to be viewed and the minerals content identified.
  • Cola nitida seed Six replicates of unprocessed (fresh) Cola nitida seed were observed for their surface morphological structure under scanning electron microscope (SEM). The seed had to be cut into a number of 1 cm 3 slice and was sticked onto the stub. Samples were then viewed under the VPSEM at 50Ox magnification. Together with the image, elemental analysis using EDX was carried out by using scanning electron microscope (LEO 1455 Variable Pressure-EDX). Numerous crystals can be seen distributed in clusters within the cell wall of Cola nitida. The crystals were variable in shape and size with diameter ranging from 2 to 10 ⁇ m. The crystals were densely packed with limited space in between them (FIG. 1 ).
  • Potassium is important in regulating the transfer of nutrients to the cells, phosphorus plays a role in metabolic reaction in the body, while magnesium plays a significant role in photosynthesis, carbohydrate metabolism, nucleic acids and binding agents of cell walls.
  • the antioxidant properties of Cola nitida were evaluated by using Ferric thiocyanate (FTC)-Thiobarbituric acid (TBA) method, 1 ,1 -diphenyi-2-picrylhydrazyl (DPPH) radical scavenging method, total phenolic and total flavonoid content.
  • FTC evaluates amount of peroxides at the initial stage of lipid peroxidation while TBA shows the amount of peroxides at the final stage of lipid peroxidation.
  • the DPPH radical scavenging activity of Cola nitida methanolic extract was evaluated to be 78.21 ⁇ 0.11 % at the concentration of 4.96 mg/ml (Table 2).
  • Antioxidant assays Percentage of activities (%) Cola nut Vitamin E (standard)
  • the total phenolic content of Cola nitida was expressed as gallic acid equivalents (GAE) which was 68.41 ⁇ 0.01 mg GAE/g sample. While the total flavonoid content of Cola nitida methanolic extract which was expressed as rutin equivalents (RE) was detected to be 30.04 ⁇ 0.04 mg RE/g sample (Table 3).
  • Percentage of cell viability was measured by comparing the optical density (OD) against the control. The antiproliferative activities are presented as percentage of cell viability versus concentration. Percentage of cell viability could be seen decreasing with the increasing of sample's concentrations (FIG. 2). Cola nitida was found to inhibit 50% cell growth of HepG-2 (human hepatocellular carcinoma cell line) at the concentration of 8.01 ⁇ 0.23 ⁇ g/ml and 78.00 ⁇ 0.1 1 ⁇ g/ml on MCF-7 (human mammary carcinoma cell line. No effect was detected on Chang normal cell line.
  • Cola nitida seed possessed the strongest IC 50 value on HepG-2 liver cancer cell lines and the mechanism of the cytotoxic effect was studied through apoptosis pathway using flow cytometry analysis.
  • the elimination of tumor cells by the induction of apoptosis has become an important and a new technique and approach in cancer therapy.
  • From flowcytometry analysis (FIG. 3) Cola nitida was shown to cause apoptosis in HepG-2. The result showed that the quantity of apoptotic cells was higher in the cancer cells treated with Cola nitida compared to control.
  • hepatocarcinogenesis will be further confirmed from the results of histological changes with hematoxylin and eosin (H&E) stained slides, liver enzyme activity (microsomal gamma-glutamyl transpeptidase; GGT, cytosol glutathione s-transf erase; GST), blood alpha feto protein (AFP) concentration and AFP gene expression in tissue by reverse transcription-polymerase chain reaction (RT-PCR) that are still in process.
  • Changes of body weight of rats during 8 weeks of bioassay are shown in FIG. 4. It shows the effect of Cola nitida on weight gains of rats during hepatocarcinogenesis.
  • DEN/AAF rats body weight of the rats increased as the time of experiment increased.
  • Body weights of carcinogenesis induced rats increased more slowly compared to non- cancer rats.
  • the body weights of DEN/AAF induced rats decreased after the injection of DEN.
  • the body weight gain in DEN/AAF induced rats was very small compared to the non-cancerous rats.
  • Rats' starvation at termination of bioassay caused a decrease in body weight in all groups.
  • Table 3 evaluates the final body weight and liver weight as well as the relative liver weight profile of the rats.
  • N normal rats supplemented with Cola nitida
  • NCn Cola nitida
  • CG glycyrrhizin
  • Body weight profile of rats might also be affected by other factors such as age, environment, genetic, health level and diet changes associated with food consumption. The decreased in body weight might be due to low food consumption by the DEN/AAF treated rats at the neoplastic stage.
  • liver weights to body weight ratio were used to investigate the change in the liver size. It was shown that the rats induced with DEN and AAF had higher liver weight to body weight ratios than that of the normal rats.
  • the ratio of liver weight to body weight of the rats induced with DEN and AAF and given Cola nitida was significantly lower than the rats induced with DEN and AAF without Cola nitida.
  • the results of this research showed effect of DEN/AAF on liver in the process of hepatocarcinogenesis and that Cola nitida was able to improve or ameliorate its effect. From the gross results, livers of the rats induced with DEN/AAF exhibited pale reddish colour compared to the normal groups. Liver nodules appeared in the form of whitish nodules in liver rat induced with DEN/AAF without Cola nitida or cancer rat (C).
  • the present invention contributes to the new finding towards anticancer agent from Cola nitida contributing to the effort of treatment and suppressive of the cancer diseases.
  • Cola nitida had been examined for its surface morphology, as well as the element content.
  • For antioxidant activity total antioxidant activity of methanol crude extract from Cola nitida was determined by using Ferric-thiocyanate (FTC)-Thiobarbituric acid (TBA) method.
  • FTC Ferric-thiocyanate
  • TSA Thiobarbituric acid
  • the DPPH radical scavenging activity of Cola nitida methanolic extract was also evaluated, besides total phenolic and total flavonoid content.
  • cytotoxic activity was carried out through the growth of viable cell using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) assay.
  • Cola nitida seed possessed the strongest IC 50 value on HepG-2 liver cancer cell lines and was further assayed with flowcytometry analysis.
  • Cola nitida Effect of Cola nitida on hepatocarcinogenic rats had been studied. From the gross results, differences could be seen in normal rat liver (N), normal rat liver treated with Cola nitida (NCn), cancer rat liver (C), cancer rat liver treated with Cola nitida (CCn) and cancer rat liver treated with control drug; glycyrrhizin (CG). Besides that, the results for the body and liver weight profile exhibited differences as well.
  • Cola nitida had been examined for its surface morphology and also the element content. Numerous crystals can be seen distributed in clusters within the cell wall of Cola nitida. The crystals were variable in shape and size with diameter ranging from 2 to 10 ⁇ m. The crystals were densely packed with limited space in between them. The elemental analysis results showed that the sample consisted of many important minerals like oxygen (49.79 ⁇ 0.36 %), carbon (47.56 ⁇ 0.33 %), potassium (1.93 ⁇ 0.15 %), phosphorus (0.43 ⁇ 0.08 %) and magnesium (0.27 ⁇ 0.05 %).
  • total antioxidant activity of methanol crude extract from Cola nitida was determined by using Ferric-thiocyanate (FTC)-Thiobarbituric acid (TBA) method. Results of ANOVA analysis indicated that antioxidant activity of Cola nitida was significantly higher (p ⁇ 0.05) than positive control, vitamin E. From the FTC method, the total antioxidant activity was evaluated to be 31 .61 ⁇ 0.01 % at the concentration of 1 mg/ml, compared to vitamin E (28.46 ⁇ 0.1 1 %). While from TBA method, Cola nitida exhibited 58.85 ⁇ 0.11 % antioxidant activity compared to standard; ⁇ -tocopherol (58.10 ⁇ 0.69 %).
  • FTC Ferric-thiocyanate
  • TBA Thiobarbituric acid
  • the DPPH radical scavenging activity of Cola nitida methanolic extract was evaluated to be 78.21 ⁇ 0.1 1 % at the concentration of 4.96 mg/ml.
  • the total phenolic content of Cola nitida was expressed as gallic acid equivalents (GAE) which was 68.41 ⁇ 0.01 mg GAE/g sample.
  • GAE gallic acid equivalents
  • RE rutin equivalents
  • cytotoxic activity was evaluated through the growth of viable cell using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay.
  • Cola nitida was found to inhibit 50% cell growth of HepG-2 (human hepatocellular carcinoma cell line) at the concentration of 8.01 ⁇ 0.23 ⁇ g/ml and 78.00 ⁇ 0.11 ⁇ g/ml on MCF-7 (human mammary carcinoma cell line). No effect was detected on Chang normal cell line.
  • Cola nitida seed possessed the strongest IC 50 value on HepG-2 liver cancer cell lines and was further assayed with flowcytometry analysis.

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Abstract

The present invention relates to Cola nitida, an anticancer agent, the anticancer properties include antioxidant activities: FTC (31.61 ± 0.01 %), TBA (58.85 ± 0.11 %) and DPPH (78.21 ± 0.1 1 %); antioxidant minerals: oxygen (49.79 ± 0.36 %), carbon (47.56 ± 0.33 %), potassium (1.93 ± 0.15 %), phosphorus (0.43 ± 0.08 %) and magnesium (0.27 ± 0.05 %); total phenolic (68.41 ± 0.01 mg GAE/g sample) and total flavonoid (30.04 ± 0.04 mg RE/g sample) content.

Description

COLA NITIDA ( COLA NUT ) AS AN ANTICANCER AGENT
FIELD OF INVENTION
The present invention relates to an anticancer agent. More particularly, the present invention relates to an anticancer agent which is Cola nitida (Cola nuts).
BACKGROUND OF INVENTION
Cancer is a group of disease which is characterized by uncontrolled cellular growth with local tissue invasion and or systematic metastasis. The number of new cancer cases has been increasing over the past nine decades. It is the largest single cause of death in both men and women, claiming over 6 million lives each year in the world. Cancer is the most feared of all diseases. Unlike other diseases, cancer usually causes a slow death involving pain, suffering, mental anguish and a feeling of hopelessness.
In the year 2000, it has been estimated that there were over 10 million new cases, 6.2 million deaths and 22.4 million persons living with cancer. In terms of incidence, the most common causes are lung (12.3%), breast (10.4%) and stomach (8.7%). By 2020, the World Health Organization estimated that 20 million new cases of cancer will be diagnosed each year.
Overall, 26089 cancer cases had been diagnosed among Malaysian people in Peninsular Malaysia in the year of 2002. From this amount, 1 1 , 815 were men while the other 14, 274 were women. It was estimated that 10659 cases had not been registered. One in 5.5 Malaysian people were in the risk to get cancer in their lifetime. Nevertheless, if it was observed from the unregistered cases, the risk may be increased to 1 in 4 people. Cancer is the major cause of death in Government Hospitals, which is 2.8 times higher than that of the heart diseases (16%).
There are various steps that have to occur before a tumor is formed. The process starts with an insult to the genetic cellular material (genotoxic events; known as initiation) and then proceeds to the production of abnormal DNA (nongenotoxic events; known as promotion). The transformation of the altered cells may lead to the proliferation of cells with invasive (malignant) or noninvasive (benign) qualities. From a confined region of tissue, cancer spread to other tissue to develop secondary tumors or metastases and result in an ultimate cascading effect of the tumor cells. The process of carcinogenesis is driven by multiple interactive factors. The external factors include chemicals, radiation and viruses, whereas the internal factors are hormones, immune condition and inherited mutation. Therefore, every one of us is at risk of developing cancer no matter at what age one is.
The most common causes of death due to cancer are cancers of the lung (17.8%), stomach (10.4%) and liver (8.8%). In the present invention, Cola nitida had been screened to be effective towards liver cancer. Thus, the present invention is focused on the effect of Cola nitida on hepatocellular carcinoma.
Tumors of the liver are classified as being either primary (originating from the liver) or metastatic (spread from another body organ to the liver). Primary liver tumors may be divided into those that are benign (not cancerous and remain in the liver) or malignant (cancerous and may be spread beyond the liver). In the recent years, there has been growing interest in alternative therapies and therapeutic use of natural products, especially from plant derivative and in trend towards the use of natural substances which is also believed to have potential value as cancer chemopreventive or therapeutic agents.
The use of natural products together with their therapeutic properties is as ancient as human civilisation and, for along time, mineral, plant and animal products were the main source of drugs. However, the potential use of plants as a source of new drugs is still poorly explored. Of the estimated 250000 to 500000 plant species, only a small percentage has been investigated phytochemically and an even smaller percentage has been properly studied in terms of their pharmacological properties.
Traditional medicine is well known for its nutritional value, as well as its ability to cure various ailments. In recent years various constituents have been found to provide protection against any disease including cancer. Any significant role by dietary intervention is encouraging and emerging as an acceptable approach for controlling the cancer incidence worldwide.
Cola nitida is an astringent, bitter-sweet, anti-depressant herb that has a stimulating effect, especially on the heart. It contains alkaloid caffeine and phenolics, such as catechin, epicatechin and procyanidins. The Cola nitida tree is native to West Africa. It has been naturalized to South America, Central America, the West Indies, Sri Lanka, and Malaysia. Related to cocoa, Cola nitida contains the methylxanthine alkaloids that also occur in coffee, cocoa, and tea. West Africans have been chewing Cola nitida for thousands of years. Its stimulant effects are the predominant application in the United States and Europe. In Africa, however, Cola nitida has been used as an appetite and thirst suppressant, enabling soldiers who chew them to travel long distances without much food. Cola twigs, with an extremely bitter taste, are used to clean the teeth and gums. Cola maintains a strong cultural significance in West Africa, partly due to the fact that cola is a valuable commodity. It has been traded to other countries since at least the fourteenth century and it is used particularly by Islamic people, who, according to their religion, cannot drink alcohol, but desire a "social lubricant". Today, Cola nitida is exported worldwide. Cola nitida is also used in non-pharmaceutical preparations, including (at least formerly) cola-based beverages such as Coca Cola. It is on the GRAS (generally recognized as safe) list for food additives in the United States.
SUMMARY OF THE INVENTION
Accordingly, Cola nitida is an anticancer agent provided from its anticancer properties inclusive of antioxidant activities (FTC-TBA, DPPH), antioxidant minerals (oxygen, carbon, potassium, phosphorus and magnesium), total phenolic and total flavonoid content.
The present invention consists of several novel features and a combination of parts hereinafter fully described and illustrated in the accompanying description and drawings, it being understood that various changes in the details may be made without departing from the scope of the invention or sacrificing any of the advantages of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, wherein:
FIG. 1 shows scanning electroscope (SEM) image of unprocessed (fresh) Cola nitida seed.
FIG. 2 shows cytotoxicity effect of Cola nitida on MCF-7, HepG-2 and Chang cell lines.
FIG. 3(a) to (c) show flowcytometry analysis on effect of Cola nitida on HepG-2 hepatocellular carcinoma cell lines. Higher (20 μg/ml) and lower (10 μg/ml) concentrations were compared with control to quantify the apoptotic cell. C illustrated the quantity of apoptotic cells.
FIG. 4 shows body weight profile of rats for the 8-week of bioassay; N: Normal control, NCn: Normal + Cola nitida, C: Cancer control, CCn: Cancer + Cola nitida and CG: Cancer + Glycyrrhizin. "Carcinogens induced only to C, CCn and CG groups. Significance; Nabcde, NCnabcde, Cabcd, CCnabcde and CGabcde; values with different superscript were significantly different at p<0.05.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention relates to an anticancer agent. More particularly, the present invention relates to an anticancer agent which is Cola nitida. Hereinafter, this specification will describe the present invention according to the preferred embodiments of the present invention. However, it is to be understood that limiting the description to the preferred embodiments of the invention is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications and equivalents without departing from the scope of the appended claims.
Plants are an important source of essential nutrients and beneficial components that are crucial for healthy living. Knowledge about the beneficial effects of certain plants is mainly transmitted by personal communication; therefore, it remains unknown to the general population. Many studies need to be done to promote the usage and application of native plants. The phytochemical or chemical composition of plants can be characterized as major and minor constituents, depending on their general abundance in the plants. Major constituents include carbohydrates lipids or fats, and proteins which are present at gram level in each 100 g portion of sample. Minor constituents include vitamins, secondary metabolites and minerals. When present, these constituents generally are found in the microgram to milligram range per 100 gram sample. Antioxidant vitamins, including polyphenol, pigments and flavonoids possess antimutagenic properties as well as blood glucose decreasing activities. In the present invention, the antioxidant minerals such as calcium (Ca), magnesium (Mg), zinc (Zn) and copper (Cu) were determined by using atomic absorption spectrophotometer (GBC, Model 908 AA, USA) taken from the ash solution of Cola nitida. The surface morphological structure and biological composition of Cola nitida was viewed and investigated under a variable pressure scanning electron microscope (VPSEM) equipped with energy dispersive x-ray (EDX) microanalysis (VPSEM model LEO VP-1455). VPSEM enables unprocessed samples to be viewed and examined. In the present invention, point and identification (ID) was used which enabled specific part of the sample to be viewed and the minerals content identified. Six replicates of unprocessed (fresh) Cola nitida seed were observed for their surface morphological structure under scanning electron microscope (SEM). The seed had to be cut into a number of 1 cm3 slice and was sticked onto the stub. Samples were then viewed under the VPSEM at 50Ox magnification. Together with the image, elemental analysis using EDX was carried out by using scanning electron microscope (LEO 1455 Variable Pressure-EDX). Numerous crystals can be seen distributed in clusters within the cell wall of Cola nitida. The crystals were variable in shape and size with diameter ranging from 2 to 10 μm. The crystals were densely packed with limited space in between them (FIG. 1 ). The elemental analysis results (Table 1 ) showed that the sample consisted of many important minerals like oxygen (49.79 ± 0.36 %), carbon (47.56 ± 0.33 %), potassium (1 .93 ± 0.15 %), phosphorus (0.43 ± 0.08 %) and magnesium (0.27 ± 0.05 %).
Sample Symbol Weight (%)
Oxygen O 49.79 ± 0.363
Carbon C 47.56 ± 0.332
Magnesium Mg 0.27 ± 0.054
Potassium K 1.93 ± 0.145
Phosphorus P 0.43 ± 0.080
Table 1 . Quantitative elemental analysis by using EDX-VPSEM
Potassium is important in regulating the transfer of nutrients to the cells, phosphorus plays a role in metabolic reaction in the body, while magnesium plays a significant role in photosynthesis, carbohydrate metabolism, nucleic acids and binding agents of cell walls. The antioxidant properties of Cola nitida were evaluated by using Ferric thiocyanate (FTC)-Thiobarbituric acid (TBA) method, 1 ,1 -diphenyi-2-picrylhydrazyl (DPPH) radical scavenging method, total phenolic and total flavonoid content. FTC evaluates amount of peroxides at the initial stage of lipid peroxidation while TBA shows the amount of peroxides at the final stage of lipid peroxidation. Results of ANOVA analysis indicated that antioxidant activity of Cola nitida was significantly higher (p<0.05) than positive control, vitamin E. From the FTC method, the total antioxidant activity was evaluated to be 31 .61 ± 0.01 % at the concentration of 1 mg/ml, compared to vitamin E (28.46 ± 0.1 1 %). While from TBA method, Cola nitida exhibited 58.85 ± 0.11 % antioxidant activity compared to standard; α-tocopherol (58.10 ± 0.69 %). However, the results were not significantly different (p>0.05). The DPPH radical scavenging activity of Cola nitida methanolic extract was evaluated to be 78.21 ± 0.11 % at the concentration of 4.96 mg/ml (Table 2). Antioxidant assays Percentage of activities (%) Cola nut Vitamin E (standard)
FTC 31.61 ± 0.01 28.46 ± 0.11
TBA 58.85 ± 0.1 1 58.10 ± 0.69 DPPH 78.21 ± 0.1 1 91.47 ± 2.24
Table 2. Total activities of cola nut with different assays. Results were compared with standard (vitamin E). Values are expressed as mean ± standard deviation of six replicate measurements.
The total phenolic content of Cola nitida was expressed as gallic acid equivalents (GAE) which was 68.41 ± 0.01 mg GAE/g sample. While the total flavonoid content of Cola nitida methanolic extract which was expressed as rutin equivalents (RE) was detected to be 30.04 ± 0.04 mg RE/g sample (Table 3).
Sample Total phenolic content Total flavonoid content
(mg GAEs/g sample) (mg REs/g sample) Cola nitida 68.4±0.01 30.04±0.04
Table 3. Values are expressed as mean ± standard deviation of six replicate measurements.
The approach to the discovery of new anticancer drugs has recently evolved from a reliance on empiric cell-based screening for cytotoxic testing, in vitro including the specific molecular lesion thought to be responsible for the development and maintenance of the malignant phenotype in various form of cancer. Current legislation demands that source of drugs such as herbal medicine; food additives or drugs go through extensive cytotoxic testing before they are released to the consumer. Cytotoxic activities of Cola nitida were studied by using MTT (3, 4, 5, dimethylthiazol-2, 5 diphenyl tetrazolium bromide) assay. Micro-culture Tetrazolium Salt (MTT) assay was used in the present invention to measure the amount of cell viability. Cells, grown in a 96 wells tissue culture plate, were incubated with the yellow MTT solution for approximate 4 hours. After this incubation period, purple formazan salts crystals were formed. These salt crystals are insoluble in aqueous solution, but may be solubilized by adding the solubilization solution and incubating the plates overnight in humidified atmosphere (370C, 5% CO2). The solubilized formazan product is spectrophotometrically quantified using an ELISA reader. An increase in number of living cells results in an increase in the total metabolic activity in Cola nitida sample. This increase directly correlated to the amount of purple formazan crystals formed, as monitored by the absorbance. Percentage of cell viability was measured by comparing the optical density (OD) against the control. The antiproliferative activities are presented as percentage of cell viability versus concentration. Percentage of cell viability could be seen decreasing with the increasing of sample's concentrations (FIG. 2). Cola nitida was found to inhibit 50% cell growth of HepG-2 (human hepatocellular carcinoma cell line) at the concentration of 8.01 ± 0.23 μg/ml and 78.00 ± 0.1 1 μg/ml on MCF-7 (human mammary carcinoma cell line. No effect was detected on Chang normal cell line. Cola nitida seed possessed the strongest IC50 value on HepG-2 liver cancer cell lines and the mechanism of the cytotoxic effect was studied through apoptosis pathway using flow cytometry analysis. The elimination of tumor cells by the induction of apoptosis has become an important and a new technique and approach in cancer therapy. From flowcytometry analysis (FIG. 3), Cola nitida was shown to cause apoptosis in HepG-2. The result showed that the quantity of apoptotic cells was higher in the cancer cells treated with Cola nitida compared to control. Thus, it can be concluded that the antioxidant properties inclusive of the useful minerals of Cola nitida might be one of the mechanisms of cytotoxic properties on HepG-2 and MCF-7 cell lines. Further investigation had been carried out to study the anticancer effect of Cola nitida extract towards neoplastic hepatocytes in vivo. This study was based on the hepatocarcinogenesis model of SoIt & Farber (1976) for the duration of 1 1 weeks. Diethylnitrosamine (DEN) and acetylaminofluorene (AAF) were used as the initiator and promoter, respectively, but without partial hepatectomy. Water extract of Cola nitida was used in vivo to create natural conditions as consumed by human. Male Sprague Dawley rats were used to investigate the anticancer properties of Cola nitida. As the liver was the main organ that was affected by carcinogens such as DEN and AAF, this model was appropriate as a liver cancer model. The severity of hepatocarcinogenesis by DEN/AAF with and without Cola nitida was confirmed by evaluating the relative body and liver weight as well as the morphological changes from the gross results. However, the severity of hepatocarcinogenesis will be further confirmed from the results of histological changes with hematoxylin and eosin (H&E) stained slides, liver enzyme activity (microsomal gamma-glutamyl transpeptidase; GGT, cytosol glutathione s-transf erase; GST), blood alpha feto protein (AFP) concentration and AFP gene expression in tissue by reverse transcription-polymerase chain reaction (RT-PCR) that are still in process. Changes of body weight of rats during 8 weeks of bioassay are shown in FIG. 4. It shows the effect of Cola nitida on weight gains of rats during hepatocarcinogenesis. With the exceptional of DEN/AAF rats, body weight of the rats increased as the time of experiment increased. Body weights of carcinogenesis induced rats increased more slowly compared to non- cancer rats. The body weights of DEN/AAF induced rats decreased after the injection of DEN. After the supplementation of 0.02% AAF, the body weight gain in DEN/AAF induced rats was very small compared to the non-cancerous rats. Rats' starvation at termination of bioassay caused a decrease in body weight in all groups. Table 3 evaluates the final body weight and liver weight as well as the relative liver weight profile of the rats.
Cancer rats (C) and cancer rats supplemented with Cola nitida (CCn) had significantly higher (p<0.05) ratio of liver weight to body weight compared to the normal (N), normal rats supplemented with Cola nitida (NCn) and cancer rats supplemented with control drug; glycyrrhizin (CG). There was no significant different of body weight profile of rats in different groups except for the DEN/AAF or cancer (C) group. Body weight profile of rats might also be affected by other factors such as age, environment, genetic, health level and diet changes associated with food consumption. The decreased in body weight might be due to low food consumption by the DEN/AAF treated rats at the neoplastic stage.
There were significant differences (p<0.05) of relative liver weight between the groups.
However, there was no significant different (p>0.05) between normal rats (n) and cancer rats supplemented with Cola nitida (CCn). There was also no significant different (p>0.05) among the normal rats supplemented with Cola nitida (NCn) and cancer rats supplemented with control drug; glycyrrhizin (CG).
Sample Final body weight (g) Liver weight (g) Relative liver weight
(g/10Og body weight)
Normal 363.20±25.23 lO.OO±O.OO 2.75±0.00c Normal + Cola nitida 365.07±21.36 10.44±0.73 2.86±0.20b
Cancer 330.80±6.83 9.67±0.58 2.93±0.17a
Cancer + Cola nitida 335.54±32.31 9.25±0.71 2.76±0.21 c
Cancer + Glycyrrhizin 359.00±28.85 10.22±1.30 2.85±0.36b
Table 3. Final body weight, liver weight and relative weight of rats. Values shown are mean ± standard deviation. Values of different superscript letters showing significant difference at p<0.05
The significant difference of relative liver weight between the normal (N) and cancer (C) rats indicated that combination of DEN and AAF caused severity of cancer incident in a short time even without the stimulating factor that is the partial hepatectomy. Hepatocarcinogenesis inhibits the regenerating of liver cells following partial hepatectomy, and at higher dose it may lead to liver cell death. Focal, slowly proliferating cell population, called hyperplastic nodules, occur in the liver before the appearance of cancer. Proliferation of cells will increase the size of the liver. Therefore, assessment of the liver weights to body weight ratio was used to investigate the change in the liver size. It was shown that the rats induced with DEN and AAF had higher liver weight to body weight ratios than that of the normal rats. In the present invention, the ratio of liver weight to body weight of the rats induced with DEN and AAF and given Cola nitida was significantly lower than the rats induced with DEN and AAF without Cola nitida. The results of this research showed effect of DEN/AAF on liver in the process of hepatocarcinogenesis and that Cola nitida was able to improve or ameliorate its effect. From the gross results, livers of the rats induced with DEN/AAF exhibited pale reddish colour compared to the normal groups. Liver nodules appeared in the form of whitish nodules in liver rat induced with DEN/AAF without Cola nitida or cancer rat (C).
The present invention contributes to the new finding towards anticancer agent from Cola nitida contributing to the effort of treatment and suppressive of the cancer diseases. Cola nitida had been examined for its surface morphology, as well as the element content. For antioxidant activity, total antioxidant activity of methanol crude extract from Cola nitida was determined by using Ferric-thiocyanate (FTC)-Thiobarbituric acid (TBA) method. The DPPH radical scavenging activity of Cola nitida methanolic extract was also evaluated, besides total phenolic and total flavonoid content. The cytotoxic activity was carried out through the growth of viable cell using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) assay. Cola nitida seed possessed the strongest IC50 value on HepG-2 liver cancer cell lines and was further assayed with flowcytometry analysis.
Further investigation had been carried out to investigate more beneficial medicinal properties of Cola nitida. Effect of Cola nitida on hepatocarcinogenic rats had been studied. From the gross results, differences could be seen in normal rat liver (N), normal rat liver treated with Cola nitida (NCn), cancer rat liver (C), cancer rat liver treated with Cola nitida (CCn) and cancer rat liver treated with control drug; glycyrrhizin (CG). Besides that, the results for the body and liver weight profile exhibited differences as well.
Cola nitida had been examined for its surface morphology and also the element content. Numerous crystals can be seen distributed in clusters within the cell wall of Cola nitida. The crystals were variable in shape and size with diameter ranging from 2 to 10 μm. The crystals were densely packed with limited space in between them. The elemental analysis results showed that the sample consisted of many important minerals like oxygen (49.79 ± 0.36 %), carbon (47.56 ± 0.33 %), potassium (1.93 ± 0.15 %), phosphorus (0.43 ± 0.08 %) and magnesium (0.27 ± 0.05 %).
For antioxidant activity, total antioxidant activity of methanol crude extract from Cola nitida was determined by using Ferric-thiocyanate (FTC)-Thiobarbituric acid (TBA) method. Results of ANOVA analysis indicated that antioxidant activity of Cola nitida was significantly higher (p<0.05) than positive control, vitamin E. From the FTC method, the total antioxidant activity was evaluated to be 31 .61 ± 0.01 % at the concentration of 1 mg/ml, compared to vitamin E (28.46 ± 0.1 1 %). While from TBA method, Cola nitida exhibited 58.85 ± 0.11 % antioxidant activity compared to standard; α-tocopherol (58.10 ± 0.69 %). The DPPH radical scavenging activity of Cola nitida methanolic extract was evaluated to be 78.21 ± 0.1 1 % at the concentration of 4.96 mg/ml. The total phenolic content of Cola nitida was expressed as gallic acid equivalents (GAE) which was 68.41 ± 0.01 mg GAE/g sample. While the total flavonoid content of Cola nitida methanolic extract which was expressed as rutin equivalents (RE) was detected to be 30.04 ± 0.04 mg RE/g sample. The cytotoxic activity was evaluated through the growth of viable cell using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Cola nitida was found to inhibit 50% cell growth of HepG-2 (human hepatocellular carcinoma cell line) at the concentration of 8.01 ± 0.23 μg/ml and 78.00 ± 0.11 μg/ml on MCF-7 (human mammary carcinoma cell line). No effect was detected on Chang normal cell line. Cola nitida seed possessed the strongest IC50 value on HepG-2 liver cancer cell lines and was further assayed with flowcytometry analysis. From flowcytometry analysis, Cola nitida was shown to cause apoptosis in HepG-2 The result showed that the quantity of apoptotic cells was higher in the cancer cells treated with Cola nitida compared to control Thus, it can be concluded that the antioxidant properties inclusive of the useful minerals of Cola nitida might be one of the mechanisms of cytotoxic properties on HepG-2 and MCF-7 cell lines Further investigation on effect of Cola nitida on liver cancer was carried out through the hepatocarcinogenesis study Results from the present invention indicates that Cola nitida may have anticancer properties against DEN-mitiated and AAF-promoted hepatocarcinogenesis in rats

Claims

1 Cola nitida is an anticancer agent, the anticancer properties include antioxidant activities (FTC-TBA, DPPH), antioxidant minerals, total phenolic and total flavonoid content
2 The anticancer properties according to claim 1 , wherein the antioxidant activities are in the following values FTC (31 61 ± 0 01 %), TBA (58 85 ± 0 11 %) and DPPH (78 21 ±
0 1 1 %)
3 The anticancer properties according to claim 1 , therein the antioxidant minerals are present in the following ratio oxygen (49 79 ± 0 36 %), carbon (47 56 ± 0 33 %), potassium (1 93 ± 0 15 %), phosphorus (0 43 ± 0 08 %) and magnesium (0 27 ± 0 05 %)
4 The anticancer properties as claimed in claim 1 , whereby the total phenolic and total flavonoid content are present in the following value total phenolic (68 41 ± 0 01 mg GAE/g sample) and total flavonoid (30 04 ± 0 04 mg RE/g sample)
5 The anticancer agent as claimed in claim 1 , wherein the cancer is liver cancer
6 A use of an effective amount of Cola nitida in the manufacture of a medicament for preventing or treating cancer, particularly liver cancer in a patient in need thereof
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